**Hepatitis B and C in Kidney Transplantation Hepatitis B and C in Kidney Transplantation**Additional information is available at the end of the chapter**Provisional chapter Hepatitis B and C in Kidney Transplantation**

[42] O'Shea, R.S., et al., *Alcoholic liver disease*. Hepatology, 2010. **51**(1): p. 307–28.

*cirrhotics*. Gastroenterology, 1981. **80**(6): p. 1405–9.

*terns related to natural history*. Gut, 1974. **15**(1): p. 52–8.

*holic cirrhosis*. Gastroenterology, 1997. **112**(4): p. 1284–9.

*decompensated cirrhosis*. J Hepatol, 2010. **52**(2): p. 176–82.

p. 62–72.

216 Advances in Treatment of Hepatitis C and B

[43] Borowsky, S.A., S. Strome, and E. Lott, *Continued heavy drinking and survival in alcoholic* 

[44] Brunt, P.W., et al., *Studies in alcoholic liver disease in Britain. I. Clinical and pathological pat-*

[45] Luca, A., et al., *Effects of ethanol consumption on hepatic hemodynamics in patients with alco-*

[46] Shim, J.H., et al., *Efficacy of entecavir in treatment-naive patients with hepatitis B virus-related* 

[47] Liaw, Y.F., et al., *Tenofovir disoproxil fumarate (TDF), emtricitabine/TDF, and entecavir in patients with decompensated chronic hepatitis B liver disease*. Hepatology, 2011. **53**(1): Smaragdi Marinaki, Konstantinos Drouzas, Smaragdi Marinaki, Konstantinos Drouzas,Chrysanthi Skalioti and John N. Boletis Smaragdi Marinaki, Konstantinos Drouzas, Chrysanthi Skalioti and John N. Boletis

Chrysanthi Skalioti and John N. Boletis

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65381

#### **Abstract**

The prevalence of chronic hepatitis B and C virus infection has declined among the dialysis population during the past decades. However, it still comprises a major health problem with high morbidity and mortality. Renal transplantation is the optimal treatment for patients with end‐stage renal disease and hepatitis B or C, although it is associated to lower patient and allograft survival compared to seronegative kidney recipients. Novel therapeutic strategies with the use of new antiviral agents, especially direct‐acting antiviral agents in hepatitis C, have significantly changed the natural history of both hepatitis B and C not only in the general population but also in renal‐ transplant recipients. We believe that future research should focus on the impact of new antiviral medications in this specific subset of patients.

**Keywords:** hepatitis B, hepatitis C, transplantation, direct‐acting antiviral agents

## **1. Introduction**

Though the prevalence of both hepatitis B and C is decreasing at least in developed countries, it still ranges from 0.1 to 20% for hepatitis B and from 2.5 to 13% for hepatitis C. General hygiene measures as well as specific measures in dialysis units and vaccination programs contributed to the reduction of hepatitis B and C prevalence in the dialysis population. However, hepatitis B and C seropositivity still remain an important clinical problem in this patient population associated with a high risk of morbidity and mortality. Although kidney transplantation is the treatment of choice for hepatitis B‐ and C‐infected dialysis patients, morbidity and mortality are worse compared to seronegative patients. Major causes of death are liver cirrhosis and hepatocellular carcinoma.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

This review focuses on pretransplant and posttransplant evaluation of prospective donors and recipients emphasizing the optimal use of grafts from hepatitis B‐ or C‐seropositive donors and the impact of hepatitis B or C infection in patient and allograft survival. Additionally, it focuses on the role of novel antiviral agents.

## **2. Kidney transplantation and hepatitis B virus infection**

## **2.1. Epidemiology**

The human hepatitis B virus (HBV) is a small enveloped DNA virus causing acute and chronic hepatitis. Although a safe and effective vaccine has been developed and it has been available for the last two decades, HBV infection still represents a major global health problem. It is estimated that approximately 30% of the world's population has had contact with or are carriers of the HBV. An estimated 350 million of them are HBV carriers [1]. Around one million persons die of HBV‐related causes annually. HBV prevalence varies from 0.1% in Western Europe, United States, Canada, Australia, and New Zealand, up to 20% in southern Asia, China, and sub‐Saharan Africa. Intermediate prevalence (3–5%) is the Mediterranean coun‐ tries, Japan, Central Asia, the Middle East, and South America. Acute infection occasionally results in fulminant hepatitis, but more importantly can progress to a chronic state, with decompensation, cirrhosis, and hepatocellular carcinoma being the most serious complica‐ tions. The progression rate is approximately 90% for an infection acquired perinatally, and decreases to 5% for infections acquired during adulthood [2].

Hemodialysis (HD) patients are at an increased risk of acquiring HBV. Reasons include increased exposure to blood products, shared hemodialysis (HD) equipment, breaching of skin, and immunodeficiency. Hemodialysis, which requires access to the bloodstream, also may favor transmission of HBV between patients, and between patients and staff.

The prevalence of hepatitis B virus infection in hemodialysis patients has significantly decreased over the past few years. This is due to the implementation of effective prevention measures, such as general hygiene rules, separation during hemodialysis, and hepatitis B vaccination. The most important measure to prevent HBV infection is immunization against the virus. Chronic dialysis patients should receive vaccination against hepatitis B. Ideally, patients with chronic kidney disease (CKD) should receive vaccination against hepatitis B at earlier stages of the disease, before starting dialysis, since vaccine immunogenicity is low in dialysis patients (70%) compared to 90% in the general population. Intensified vaccination protocols have been used in hemodialysis patients with good responses. The presence of an adequate hepatitis B antibody (anti‐HBs) titer should be checked annually. If the antibody titer is lower than 10 IU/ml, a booster dose of the vaccine should be administrated [3].

Although rates of new infection are decreasing [4], hepatitis B still remains a problem in dialysis populations. According to data from the United States Renal Data System (USRDS) in 2002, 1% of dialysis patients were seropositive for HBsAg [3]. A registry study of Asia‐Pacific countries found the prevalence of hepatitis B surface antigen (HBsAg) positivity ranging from 1.3 to 14.6% [5]. Despite the decrease of HBsAg prevalence in dialysis patients, 350 million people worldwide are chronic HBV carriers and a large number of them will need transplan‐ tation in the future [6].

Hepatitis B virus–infected patients are at risk of exacerbation of the infection, progressive liver disease, and development of hepatocellular carcinoma after kidney transplantation. Renal transplantation offers higher survival and better quality of life compared to hemodialysis, which also applies to HBV patients, providing that they are receiving antiviral prophylaxis, since it is easier to prevent than treat HBV reactivation [7].

## **2.2. Evaluation of HBV‐infected dialysis patients before transplantation**

This review focuses on pretransplant and posttransplant evaluation of prospective donors and recipients emphasizing the optimal use of grafts from hepatitis B‐ or C‐seropositive donors and the impact of hepatitis B or C infection in patient and allograft survival. Additionally, it

The human hepatitis B virus (HBV) is a small enveloped DNA virus causing acute and chronic hepatitis. Although a safe and effective vaccine has been developed and it has been available for the last two decades, HBV infection still represents a major global health problem. It is estimated that approximately 30% of the world's population has had contact with or are carriers of the HBV. An estimated 350 million of them are HBV carriers [1]. Around one million persons die of HBV‐related causes annually. HBV prevalence varies from 0.1% in Western Europe, United States, Canada, Australia, and New Zealand, up to 20% in southern Asia, China, and sub‐Saharan Africa. Intermediate prevalence (3–5%) is the Mediterranean coun‐ tries, Japan, Central Asia, the Middle East, and South America. Acute infection occasionally results in fulminant hepatitis, but more importantly can progress to a chronic state, with decompensation, cirrhosis, and hepatocellular carcinoma being the most serious complica‐ tions. The progression rate is approximately 90% for an infection acquired perinatally, and

Hemodialysis (HD) patients are at an increased risk of acquiring HBV. Reasons include increased exposure to blood products, shared hemodialysis (HD) equipment, breaching of skin, and immunodeficiency. Hemodialysis, which requires access to the bloodstream, also

The prevalence of hepatitis B virus infection in hemodialysis patients has significantly decreased over the past few years. This is due to the implementation of effective prevention measures, such as general hygiene rules, separation during hemodialysis, and hepatitis B vaccination. The most important measure to prevent HBV infection is immunization against the virus. Chronic dialysis patients should receive vaccination against hepatitis B. Ideally, patients with chronic kidney disease (CKD) should receive vaccination against hepatitis B at earlier stages of the disease, before starting dialysis, since vaccine immunogenicity is low in dialysis patients (70%) compared to 90% in the general population. Intensified vaccination protocols have been used in hemodialysis patients with good responses. The presence of an adequate hepatitis B antibody (anti‐HBs) titer should be checked annually. If the antibody titer

may favor transmission of HBV between patients, and between patients and staff.

is lower than 10 IU/ml, a booster dose of the vaccine should be administrated [3].

Although rates of new infection are decreasing [4], hepatitis B still remains a problem in dialysis populations. According to data from the United States Renal Data System (USRDS) in 2002, 1% of dialysis patients were seropositive for HBsAg [3]. A registry study of Asia‐Pacific countries found the prevalence of hepatitis B surface antigen (HBsAg) positivity ranging from

**2. Kidney transplantation and hepatitis B virus infection**

decreases to 5% for infections acquired during adulthood [2].

focuses on the role of novel antiviral agents.

218 Advances in Treatment of Hepatitis C and B

**2.1. Epidemiology**

All dialysis patients should be checked routinely for HBsAg and when indicated with HBV DNA. HBV infected kidney transplant candidates should be tested for hepatitis B e antigen (HBeAg) and serum HBV DNA prior to transplantation. Patients who are HBeAg positive or have high levels of HBV DNA should receive antiviral treatment before transplantation with one of the available agents (lamivudine (LAM), entecavir (ETV), adefovir, tenofovir, and telbivudine (LdT)) until HBeAg becomes negative and viral replication is suppressed.

According to the Kidney Disease Improving Global Outcomes (KDIGO) guidelines, it is recommended that a liver biopsy is performed in HBsAg‐positive hemodialysis patients on the waiting list for transplantation in order to evaluate liver disease status. If there is ongoing viral activity, candidates should repeat liver biopsy every 3–5 years [8]. Noninvasive testing for liver stiffness as fibroscan (elastography), which tends to replace liver biopsy in the general population, has not yet been validated neither in HBV‐positive patients nor in patients on hemodialysis or after transplantation [9]. So, liver biopsy while on the waiting list still remains the "gold standard" for kidney transplant candidates.

Liver cirrhosis has for long been considered as an absolute contraindication for kidney transplantation alone; combined liver kidney transplantation is the treatment of choice in these patients. However, with the use of new nucleos(t)ide analogs, some dialysis patients with compensated cirrhosis achieve sustained viral response (SVR). If a follow‐up biopsy after 12 months reveals partial reversibility of cirrhosis, those patients can be after that included in the waiting list and undergo kidney transplantation alone. This has been reported for HBV‐ and hepatitis C virus (HCV)‐positive patients after SVR with the new antivirals [10].

HBsAg‐positive renal transplant candidates should start antiviral treatment immediately after transplantation, regardless of the HBV DNA status or the findings of liver histology, due to the risk of severe reactivation, fibrosing cholestatic hepatitis, and rapid histological deteriora‐ tion after the induction of immunosuppression.

## **2.3. Transmission of HBV infection from the donor**

Besides HBV reactivation, HBV may be transmitted from the donor to the recipient. Renal transplantation from HBsAg‐positive donors to HBV‐naïve recipients is not recommended because it carries a significant risk of de novo infection, most often with an aggressive course [11]. On the other hand, as shown by Jiang et al., allografts from HBsAg‐positive donors can safely be transplanted into HBsAg‐negative recipients with natural or acquired immunity (anti‐HBs positive, titer above 10 IU/ml), with concurrent administration of hepatitis B hyperimmune globulin (HBIG) with or without booster vaccination [12]. In such cases, although the risk of transmission is relatively low, it is mandatory to inform patients and to obtain written consent in order to proceed with kidney transplantation. In a study by Singh et al., the successful procedure was described in 104 anti‐HBs–positive recipients [13]. Prevention strategies included booster vaccination and concomitant administration of HBIG in combina‐ tion with antiviral agents while vaccination alone was used in 27 patients.

In our center, we perform kidney transplantations from HBsAg‐positive donors to HBsAg negative/HBsAb positive, that is, immunized either from past infection or from vaccination recipients. Those transplantations are performed only if the antibody titer of the recipient, measured immediately before transplantation, is at least 10 IU/ml and with concomitant administration of one dose of booster vaccination in combination with hepatitis B hyperim‐ mune globulin(HBIG) while most of the recipients are started on antiviral prophylaxis postoperatively. The need and the duration of antiviral treatment in this patient population have not been investigated; moreover, data about monitoring long term are lacking. It seems logical to assume that antibody titer should be checked and booster vaccination should be administered when the titer falls below 10 IU/ml, since transplant recipients receiving immunosuppression are at risk for viral reactivation—if it has been transmitted from the donor —for long. However, to the best of our knowledge, current evidence is so sparse that only suggestions can be made.

Another issue regarding donor to recipient HBV transmission is that there is a very low but substantial risk of HBV transmission from HBsAg‐negative, anti‐HBc‐positive, anti‐HBs– negative donors to HBV‐naïve recipients. In a recent review of 1385 HBsAg‐negative kidney recipients from anti‐HBc–positive donors, seroconversion to HbsAg positivity occurred in 0.28% (4/1385) and to anti‐HBc positivity in 2.3% (32/1385) [14–16]. Ideally, those donors should be checked for anti‐HBc IgM presence, indicating a more recent infection rather than ineffective immune response. Unfortunately, in the case of transplantation from deceased donors, this is impossible, due to the shortness of time. Given the organ shortage and the survival advantage of transplantation over remaining on hemodialysis, kidney transplantation could be consid‐ ered in these cases too, since the risk for transmission is even lower than from HBsAg‐positive donors. Again without data supporting the evidence, one may suggest that, in this case too, transplant candidates should be immunized and receive prophylaxis with booster vaccination and HBIG administration, while antiviral prophylaxis may not be indicated in this setting.

#### **2.4. Outcome of HBV‐infected patients after kidney transplantation**

HBV‐infected renal allograft recipients have worse survival compared to their seronegative counterparts. A meta‐analysis of six observational studies, which included 6050 HBsAg‐ positive patients after kidney transplantation, showed that the relative risk of death and graft loss were 2.49 and 1.44, respectively [17].

The widespread use of antiviral agents since 1986 has significantly improved survival of HBV‐ infected kidney transplant recipients. In a small study from Italy, 42 HbsAg‐positive patients who have been transplanted between 1976 and 1982 had a 12‐year survival rate of 67% [18]. In a more recent study from Hong Kong, a 10‐year survival of 63 HbsAg‐positive renal transplant recipients who were treated with nucleoside/nucleotide analogs reached 81% [19]. However, liver failure remains the leading cause of death in this patient population. HBsAg‐seropositive recipients who are HBeAg‐negative have undetectable viral load, and for mild changes in liver biopsy they should receive preventive antiviral therapy immediately posttransplantation, in order to avoid viral reactivation due to immunosuppressive therapy. The only study that evaluated serial biopsies after kidney transplantation found histological deterioration in 85% of HBsAg‐positive patients. No patient had cirrhosis before kidney transplantation while 28% of them had biopsy‐proven liver cirrhosis after transplantation. Among those with cirrhosis, hepatocellular carcinoma was found in 23% [20].

## **2.5. Antiviral agents**

[11]. On the other hand, as shown by Jiang et al., allografts from HBsAg‐positive donors can safely be transplanted into HBsAg‐negative recipients with natural or acquired immunity (anti‐HBs positive, titer above 10 IU/ml), with concurrent administration of hepatitis B hyperimmune globulin (HBIG) with or without booster vaccination [12]. In such cases, although the risk of transmission is relatively low, it is mandatory to inform patients and to obtain written consent in order to proceed with kidney transplantation. In a study by Singh et al., the successful procedure was described in 104 anti‐HBs–positive recipients [13]. Prevention strategies included booster vaccination and concomitant administration of HBIG in combina‐

In our center, we perform kidney transplantations from HBsAg‐positive donors to HBsAg negative/HBsAb positive, that is, immunized either from past infection or from vaccination recipients. Those transplantations are performed only if the antibody titer of the recipient, measured immediately before transplantation, is at least 10 IU/ml and with concomitant administration of one dose of booster vaccination in combination with hepatitis B hyperim‐ mune globulin(HBIG) while most of the recipients are started on antiviral prophylaxis postoperatively. The need and the duration of antiviral treatment in this patient population have not been investigated; moreover, data about monitoring long term are lacking. It seems logical to assume that antibody titer should be checked and booster vaccination should be administered when the titer falls below 10 IU/ml, since transplant recipients receiving immunosuppression are at risk for viral reactivation—if it has been transmitted from the donor —for long. However, to the best of our knowledge, current evidence is so sparse that only

Another issue regarding donor to recipient HBV transmission is that there is a very low but substantial risk of HBV transmission from HBsAg‐negative, anti‐HBc‐positive, anti‐HBs– negative donors to HBV‐naïve recipients. In a recent review of 1385 HBsAg‐negative kidney recipients from anti‐HBc–positive donors, seroconversion to HbsAg positivity occurred in 0.28% (4/1385) and to anti‐HBc positivity in 2.3% (32/1385) [14–16]. Ideally, those donors should be checked for anti‐HBc IgM presence, indicating a more recent infection rather than ineffective immune response. Unfortunately, in the case of transplantation from deceased donors, this is impossible, due to the shortness of time. Given the organ shortage and the survival advantage of transplantation over remaining on hemodialysis, kidney transplantation could be consid‐ ered in these cases too, since the risk for transmission is even lower than from HBsAg‐positive donors. Again without data supporting the evidence, one may suggest that, in this case too, transplant candidates should be immunized and receive prophylaxis with booster vaccination and HBIG administration, while antiviral prophylaxis may not be indicated in this setting.

HBV‐infected renal allograft recipients have worse survival compared to their seronegative counterparts. A meta‐analysis of six observational studies, which included 6050 HBsAg‐ positive patients after kidney transplantation, showed that the relative risk of death and graft

**2.4. Outcome of HBV‐infected patients after kidney transplantation**

loss were 2.49 and 1.44, respectively [17].

tion with antiviral agents while vaccination alone was used in 27 patients.

suggestions can be made.

220 Advances in Treatment of Hepatitis C and B

The goal of treatment is suppression of viral replication and prevention of hepatic fibrosis, while minimizing resistance to the drugs. HBV DNA levels need to be measured systematically to assess response to therapy, because of the poor likelihood of seroconversion to anti‐HBs and because of low reliability of alanine aminotransferase (ALT) as a marker of liver disease activity.

Treatment must be initiated before or immediately after transplantation. In a study of 15 patients, seven were started on lamivudine at the time of kidney transplantation. All patients had normal transaminase levels preoperatively. Half of those who were not treated initially showed transaminase elevations in the first year of follow‐up requiring lamivudine therapy at that time. By contrast, all seven individuals who had received lamivudine at the time of transplantation remained negative for HBV DNA throughout the follow‐up [21].

Currently, there are several medications available for the treatment of hepatitis B: interferon alfa‐2b, pegylated interferon (PEG‐INF) alfa 2a, and the nucleos(t)ide analogs lamivudine, adefovir, tenofovir, telbivudine, and entecavir.

## *2.5.1. Interferon and PEG‐INF*

In the current era of potent antiviral drugs as nucleoside analogs, the use of interferon‐α (IFN) and PEG‐IFN after transplantation is not recommended anymore. IFN‐α has known immu‐ nomodulatory effects and its use in case series of kidney transplant recipients in the past has been associated with increased rates of graft loss due to rejection and with relapse rates approaching 80% after therapy discontinuation [22].

## *2.5.2. Lamivudine (LAM)*

Lamivudine is a cytosine analog that inhibits HBV reverse transcriptase. The prophylactic use of lamivudine posttransplantation has proven efficacy long term. Since LAM was the first nucleoside analog approved for clinical use, most of the available data on the management of HBsAg‐positive renal transplant recipients are with this agent. A meta‐analysis of 14 clinical studies, which included 184 patients, showed that LAM administration results in undetectable viral load in 91% and a normalization of alanine aminotransferase (ALT) in 81% of patients, for a prolonged period of time [23]. Lamivudine has for long been the cornerstone of therapy in HBV‐infected kidney transplant recipients and has increased survival rates. HBsAg‐positive kidney recipients treated with lamivudine reached 10‐year survival rates of 81%, comparable to HBsAg‐negative patients [24].

Since it is eliminated by the kidney, its dose should be adapted to renal function: recommended dose is 100 mg/day in patients with estimated glomerular filtration rate (eGFR) >50 ml/min and 100 mg every other day in patients with less‐preserved renal function.

The major problem with prolonged lamivudine treatment is the development of resistance. The presentation of the resistance varies. Some patients show only reappearance of serum HBA DNA, while others present with elevated liver enzymes. In most cases, resistance occurs due to a mutation in the tyrosine‐methionine‐aspartate‐aspartate (YMDD) locus of the HBV DNA polymerase [25].

In a series of studies, the rates of lamivudine resistance vary from 20 up to 60% [26, 27]. In a study of 29 renal transplant recipients, after a mean follow‐up of 69 months, 14 patients (48%) developed lamivudine resistance. Among them, 79% presented with a hepatitis flare. The YMDD mutation was found in all cases of resistance [25]. A meta‐analysis of 2004 showed that increased duration of lamivudine therapy was positively associated with lamivudine resist‐ ance [22].

Patients with lamivudine resistance should be treated with adefovir, tenofovir, entecavir, or telbivudine.

## *2.5.3. Tenofovir disoproxil fumarate (TDF)*

Tenofovir disoproxil fumarate (TDF) is a nucleotide analog and a potent inhibitor of human immunodeficiency virus type 1 reverse transcriptase and hepatitis B virus polymerase. Tenofovir is a potent antiviral agent for treatment‐naïve patients and for patients with lamivudine resistance [28, 29]. Data for patients who have undergone kidney transplantation are limited and there are concerns for the development of kidney injury. Daude et al. conducted a study, which showed effective suppression of viral replication after 12 months of follow‐up and preservation of stable kidney function in seven hepatitis B virus‐positive solid‐organ transplant recipients, with three renal‐transplant recipients among them [30]. In a study of patients from the general population with HBV infection, tenofovir was effective in lamivu‐ dine‐resistant cases, and did not induce resistance after up to 48 months of treatment [31].

## *2.5.4. Telbivudine (LdT)*

Telbivudine is not effective in kidney transplant recipients with lamivudine‐resistant HBV, because it shows cross‐resistance to lamivudine and entecavir, since the virus develops the same mutations for both medications. Data about the use of telbivudine in renal transplanta‐ tion are lacking.

## *2.5.5. Entecavir (ETV)*

studies, which included 184 patients, showed that LAM administration results in undetectable viral load in 91% and a normalization of alanine aminotransferase (ALT) in 81% of patients, for a prolonged period of time [23]. Lamivudine has for long been the cornerstone of therapy in HBV‐infected kidney transplant recipients and has increased survival rates. HBsAg‐positive kidney recipients treated with lamivudine reached 10‐year survival rates of 81%, comparable

Since it is eliminated by the kidney, its dose should be adapted to renal function: recommended dose is 100 mg/day in patients with estimated glomerular filtration rate (eGFR) >50 ml/min

The major problem with prolonged lamivudine treatment is the development of resistance. The presentation of the resistance varies. Some patients show only reappearance of serum HBA DNA, while others present with elevated liver enzymes. In most cases, resistance occurs due to a mutation in the tyrosine‐methionine‐aspartate‐aspartate (YMDD) locus of the HBV DNA

In a series of studies, the rates of lamivudine resistance vary from 20 up to 60% [26, 27]. In a study of 29 renal transplant recipients, after a mean follow‐up of 69 months, 14 patients (48%) developed lamivudine resistance. Among them, 79% presented with a hepatitis flare. The YMDD mutation was found in all cases of resistance [25]. A meta‐analysis of 2004 showed that increased duration of lamivudine therapy was positively associated with lamivudine resist‐

Patients with lamivudine resistance should be treated with adefovir, tenofovir, entecavir, or

Tenofovir disoproxil fumarate (TDF) is a nucleotide analog and a potent inhibitor of human immunodeficiency virus type 1 reverse transcriptase and hepatitis B virus polymerase. Tenofovir is a potent antiviral agent for treatment‐naïve patients and for patients with lamivudine resistance [28, 29]. Data for patients who have undergone kidney transplantation are limited and there are concerns for the development of kidney injury. Daude et al. conducted a study, which showed effective suppression of viral replication after 12 months of follow‐up and preservation of stable kidney function in seven hepatitis B virus‐positive solid‐organ transplant recipients, with three renal‐transplant recipients among them [30]. In a study of patients from the general population with HBV infection, tenofovir was effective in lamivu‐ dine‐resistant cases, and did not induce resistance after up to 48 months of treatment [31].

Telbivudine is not effective in kidney transplant recipients with lamivudine‐resistant HBV, because it shows cross‐resistance to lamivudine and entecavir, since the virus develops the same mutations for both medications. Data about the use of telbivudine in renal transplanta‐

and 100 mg every other day in patients with less‐preserved renal function.

to HBsAg‐negative patients [24].

222 Advances in Treatment of Hepatitis C and B

polymerase [25].

ance [22].

telbivudine.

*2.5.4. Telbivudine (LdT)*

tion are lacking.

*2.5.3. Tenofovir disoproxil fumarate (TDF)*

Entecavir, a guanosine analog, is 30 times more potent than lamivudine in suppressing viral replication and nowadays it is used as first‐line prophylactic treatment in renal transplant recipients. This drug has a high antiviral potency, a high genetic barrier for resistance, and a good safety profile. There is sufficient evidence that it can effectively clear the viral load for a prolonged period. A recent prospective study included 27 renal transplant recipients, 18 (67%) were treatment naïve and 9 (33%) had been previously treated with LAM but had no resistant mutations. Entecavir cleared HBV DNA in 70, 74, 96, and 100% of patients after 12, 24, 52, and 104 weeks, respectively. Furthermore, entecavir reached higher rates of undetectable HBV DNA compared to lamivudine (32, 37, 63, and 63% at 12, 24, 52, and 104 weeks, respectively; *P* < 0.005) [32]. However, in patients with lamivudine‐resistant HBV, complete response to entecavir can be delayed for more than 6 weeks, or not be achieved at all. The use of entecavir in renal transplant recipients who had developed lamivudine—or adefovir—resistance has been examined in a small study of 10 solid‐organ‐transplant recipients, with 8 kidney‐allograft recipients among them, who were treated with entecavir for 16.5 months. There was a significant decrease in HBV DNA viral load (50%) without any significant adverse events [33]. Resistance to entecavir has not been documented in renal transplant recipients. In the general population, the rate of entecavir resistance is minimal (1.2%) in treatment‐naïve patient after 5 years of therapy. However, in lamivudine‐resistant patients, the probability of entecavir resistance at years 1–5 rises from 6 to 15, 36, 46, and 51%, respectively [34].

#### *2.5.6. Adefovir dipivoxil (ADV)*

Adefovir is an acyclic nucleotide adenosine analog. Adefovir is effective as monotherapy or in combination with entecavir in the general population with HBV infection and lamivudine resistance [35–38]. The problem with this agent is that it is potentially nephrotoxic. Studies in human immunodeficiency virus (HIV) patients show that high daily doses of adefovir (60–120 mg) may cause renal tubular injury. The drug is mainly used in lamivudine‐resistant HBV cases [39]. Fontaine et al. studied the efficacy of adefovir as monotherapy at 11 renal‐transplant recipients with lamivudine resistance. After 12 months, a satisfactory decline in serum HBV DNA and an absence of hepatitis flares were observed. Importantly, there were no significant clinical and laboratory adverse events [35]. In another study of 11 renal‐transplant recipients with lamivudine resistance, adefovir was given at very low doses (10–2.5 mg/day) and it showed good efficacy, without nephrotoxicity [38]. In another study, evidence of nephrotox‐ icity implementing treatment discontinuation despite dosage adjustment was observed in 29% of patients [39].

## **2.6. Treatment duration**

In the general population, the duration of treatment depends on the HBeAg status. HBeAg‐ positive patients should be treated until HBV DNA and HBeAg are cleared and anti‐HBe seroconversion occurs. Additional treatment is needed for at least 6–12 months after anti‐HBe seroconversion to prevent virological reactivation. Patients without HbeAg should be treated until HBsAg clearance. The duration of antiviral therapy for renal transplant recipients remains unclear, because outcomes after nucleos(t)ide analogs withdrawal in immunosup‐ pressed patients allograft recipients are unknown.

One small retrospective study [40] evaluated the course of 6 out of 14 HBsAg(+) kidney‐ transplant recipients, in whom antiviral treatment had been discontinued after a median of 14 months. All of the six patients in whom antiviral agents had been discontinued were on stable, low‐dose maintenance immunosuppression with undetectable HBV DNA and serological negativity for HBeAg. In four out of the six patients (67%), antiviral withdrawal was successful, without any sign of reactivation after a median follow‐up of 60 months. In the remaining two patients, who had reactivated HBV, antiviral therapy was reintroduced immediately, with subsequent HBV clearance. Though the number of patients is indeed small, the study provides promising results for future investigation.

In the absence of robust data, we can suggest that antiviral treatment after kidney transplan‐ tation may be discontinued only in a subset of carefully selected patients who meet the following criteria: stable renal function and low immunological risk for rejection, low‐dose maintenance immunosuppression for at least 6–9 months, no serological or biochemical evidence for HBV activity and previous antiviral treatment without resistance to any antiviral agent for at least 12 months. Close monitoring of HBV DNA every 3–6 months is essential, while antivirals should be reintroduced whenever immunosuppression must be intensified, that is, in the case of anti‐rejection treatment.

## **2.7. Reactivation of HBV after renal transplantation: the role of immunosuppression**

Immunosuppression is associated with hepatitis B virus reactivation not only in HBsAg‐ positive recipients but also in patients seropositive for anti‐HBc/anti‐HBs, usually in low titers, that is, past infection (reverse seroconversion) [41].

The majority of data come from studies in HBV patients treated for solid‐organ or hemato‐ logical malignancies [41, 42].

The main factors associated with HBV reactivation posttransplantation are the immunocom‐ petence of the recipient, the total amount of immunosuppression, and finally the characteristics of the virus.

The status of immunosuppression changes the interaction between the HBV virus and the host, leading to potentially severe liver injury. Liver damage in the setting of immunosuppression may occur through two different mechanisms. The first mechanism is direct hepatotoxicity after the introduction of immunosuppression due to uncontrolled viral replication as a consequence of reduced immunosurveillance of the host. The second mechanism involves indirect, immune‐mediated liver damage occurring after cessation of immunosuppression, during immune reconstitution. The second mechanism has been described in patients with solid‐organ or hematologic malignancies even up to 6–12 months after completion of chemo‐ therapy [43].

Since renal transplant recipients receive lifelong immunosuppression, hepatotoxicity in this setting may mostly be attributable to the first mechanism with the highest risk for viral reactivation being during the induction period, when the total amount of immunosuppression is high or whenever immunosuppression is intensified after that, as, for example, during anti‐ rejection treatment.

## **2.8. Immunosuppressive agents**

Corticosteroids (CSs), calcineurin inhibitors (CNIs) (cyclosporine and tacrolimus), antimeta‐ bolites (mycophenolate mofetil (MMF) or mycophenolic sodium and azathioprine), and mammalian target of rapamycin (mTOR) inhibitors (sirolimus and everolimus) are the main immunosuppressants used in various combinations in kidney transplantation. Monoclonal antibodies (Rituximab, anti‐IL2 Basiliximab) and polyclonal antibodies as antithymocyte globulin (ATG) are also part of the immunosuppressive regimen used for the induction or for the treatment of rejection. All of them are implicated in alterations of viral replication, mostly by inducing increased viral replication and enhance the risk of HBV reactivation. The risk of HBV reactivation according to specific immunosuppressive drug classes has been estimated by the American Gastroenterological Association (AGA) [44].

#### *2.8.1. Rituximab*

remains unclear, because outcomes after nucleos(t)ide analogs withdrawal in immunosup‐

One small retrospective study [40] evaluated the course of 6 out of 14 HBsAg(+) kidney‐ transplant recipients, in whom antiviral treatment had been discontinued after a median of 14 months. All of the six patients in whom antiviral agents had been discontinued were on stable, low‐dose maintenance immunosuppression with undetectable HBV DNA and serological negativity for HBeAg. In four out of the six patients (67%), antiviral withdrawal was successful, without any sign of reactivation after a median follow‐up of 60 months. In the remaining two patients, who had reactivated HBV, antiviral therapy was reintroduced immediately, with subsequent HBV clearance. Though the number of patients is indeed small, the study provides

In the absence of robust data, we can suggest that antiviral treatment after kidney transplan‐ tation may be discontinued only in a subset of carefully selected patients who meet the following criteria: stable renal function and low immunological risk for rejection, low‐dose maintenance immunosuppression for at least 6–9 months, no serological or biochemical evidence for HBV activity and previous antiviral treatment without resistance to any antiviral agent for at least 12 months. Close monitoring of HBV DNA every 3–6 months is essential, while antivirals should be reintroduced whenever immunosuppression must be intensified,

**2.7. Reactivation of HBV after renal transplantation: the role of immunosuppression**

Immunosuppression is associated with hepatitis B virus reactivation not only in HBsAg‐ positive recipients but also in patients seropositive for anti‐HBc/anti‐HBs, usually in low titers,

The majority of data come from studies in HBV patients treated for solid‐organ or hemato‐

The main factors associated with HBV reactivation posttransplantation are the immunocom‐ petence of the recipient, the total amount of immunosuppression, and finally the characteristics

The status of immunosuppression changes the interaction between the HBV virus and the host, leading to potentially severe liver injury. Liver damage in the setting of immunosuppression may occur through two different mechanisms. The first mechanism is direct hepatotoxicity after the introduction of immunosuppression due to uncontrolled viral replication as a consequence of reduced immunosurveillance of the host. The second mechanism involves indirect, immune‐mediated liver damage occurring after cessation of immunosuppression, during immune reconstitution. The second mechanism has been described in patients with solid‐organ or hematologic malignancies even up to 6–12 months after completion of chemo‐

Since renal transplant recipients receive lifelong immunosuppression, hepatotoxicity in this setting may mostly be attributable to the first mechanism with the highest risk for viral

pressed patients allograft recipients are unknown.

224 Advances in Treatment of Hepatitis C and B

promising results for future investigation.

that is, in the case of anti‐rejection treatment.

that is, past infection (reverse seroconversion) [41].

logical malignancies [41, 42].

of the virus.

therapy [43].

According to the AGA guidelines, Rituximab has the highest risk estimate of HBV reactivation (high >10%) from all immunosuppressants used in kidney transplantation. Moreover, the risk of HBV reactivation may persist up to 12 months, since the antibody has a prolonged phase of immune reconstitution.

Rituximab administration has been associated with HBV reactivation not only in HBsAg‐ positive but also in anti‐HBc–positive and anti‐HBs–positive patients (reverse seroconversion). In a prospective study of 314 HBsAg‐negative patients with B‐cell lymphoma treated with Rituximab, 16.2% were HBV carriers. All of them were anti‐HBc positive, whereas half of them were also anti‐HBs positive. Virus reactivation occurred in 12% of patients. HBV DNA clearance with the use of entecavir permitted readministration of Rituximab [45].

## *2.8.2. Polyclonal antibodies (antithymocyte globulin, ATG)*

Increased viral replication following ATG administration has been described for herpes viruses, Epstein‐Barr virus (EBV), and, to a lesser degree, cytomegalovirus (CMV). In those cases, ATG has been administered to patients with severe aplastic anemia concomitantly with cyclosporine [46]. Data about HBV reactivation after treatment with ATG are lacking.

#### *2.8.3. Corticosteroids (CS)*

Corticosteroids are the oldest and commonest immunosuppressants worldwide. Its use is undoubtedly associated with increased viral replication. Since they are used in many dosages, the risk of HBV reactivation depends on the dose and duration of CS administration. High corticosteroid doses increase viral replication, while ALT may be decreased. The opposite is observed during steroid tapering with elevated aminotransferases 4–6 weeks after steroid discontinuation [40]. According to the AGA guidelines, high CS doses (up to 20 mg/d of

prednisone) and/or prolonged (>3 months) administration are considered as high risk for HBV reactivation while rapid tapering may also increase the risk for viral reactivation due to immune reconstitution.

In kidney transplantation, high CS doses are administered during the first weeks post transplantation; thereafter, they are tapered during a period of 3–6 months to a maintenance dose of 5 mg of prednisone daily or every other day. In stable, low‐immunological risk patients they may be avoided completely (steroid‐avoidance protocols) or they may be withdrawn after 4–6 weeks or even later (steroid‐withdrawal protocols) with excellent outcomes. High CS doses, including intravenous pulses of methylprednisolone up to 500 mg/d, are used for the treatment of acute rejection.

In HBV kidney transplant recipients, steroids must be used at the lowest possible doses and preferably be discontinued or even completely avoided in patients with low immunological risk.

## *2.8.4. Calcineurin inhibitors*

Calcineurin inhibitors are still the cornerstone of immunosuppression in kidney transplanta‐ tion. It has been shown that cyclosporine reduces viral replication in vitro. Nowadays, most immunosuppressive regimens are tacrolimus based. Although there are no definite conclu‐ sions or guidelines, some suggest that cyclosporine may be preferable to Tacrolimus in HBV kidney transplant recipients. Nevertheless, since there are no definite conclusions, the choice of one of the two calcineurin inhibitors depends on the center's practice. Some others may argue that it is easier to withdraw steroids from a Tacrolimus‐based regimen and would prefer this choice [47, 48].

## *2.8.5. Antimetabolites*

Azathioprine, though hepatotoxic per se, has not been associated with an increased risk of HBV reactivation when given as monotherapy. Nevertheless, after the introduction of the more selective and more potent antimetabolites as the mycophenolic acids (MPAs), the use of azathioprine in kidney transplantation has been restricted to patients with special indications [49].

## *2.8.5.1. Mycophenolate acid derivates*

Mycophenolate mofetil and the newer mycophenolate sodium have nowadays replaced azathioprine in most immunosuppressive regimens. Data about MPAs and HBV reactivation are lacking, but in general they are considered safe for HBV kidney‐transplant recipients.

#### *2.8.6. Mammalian target of rapamycin (mTOR) inhibitors*

The reactivation of HBV with the use of mTOR inhibitors has not been studied in renal‐ transplant recipients and generally they are considered safe. Some case reports of HBV reactivation related to everolimus when used as a chemotherapeutic agent have been reported but everolimus dosage in this setting is much higher than the usual doses given as maintenance immunosuppression in kidney transplantation [50].

prednisone) and/or prolonged (>3 months) administration are considered as high risk for HBV reactivation while rapid tapering may also increase the risk for viral reactivation due to

In kidney transplantation, high CS doses are administered during the first weeks post transplantation; thereafter, they are tapered during a period of 3–6 months to a maintenance dose of 5 mg of prednisone daily or every other day. In stable, low‐immunological risk patients they may be avoided completely (steroid‐avoidance protocols) or they may be withdrawn after 4–6 weeks or even later (steroid‐withdrawal protocols) with excellent outcomes. High CS doses, including intravenous pulses of methylprednisolone up to 500 mg/d, are used for the

In HBV kidney transplant recipients, steroids must be used at the lowest possible doses and preferably be discontinued or even completely avoided in patients with low immunological

Calcineurin inhibitors are still the cornerstone of immunosuppression in kidney transplanta‐ tion. It has been shown that cyclosporine reduces viral replication in vitro. Nowadays, most immunosuppressive regimens are tacrolimus based. Although there are no definite conclu‐ sions or guidelines, some suggest that cyclosporine may be preferable to Tacrolimus in HBV kidney transplant recipients. Nevertheless, since there are no definite conclusions, the choice of one of the two calcineurin inhibitors depends on the center's practice. Some others may argue that it is easier to withdraw steroids from a Tacrolimus‐based regimen and would prefer

Azathioprine, though hepatotoxic per se, has not been associated with an increased risk of HBV reactivation when given as monotherapy. Nevertheless, after the introduction of the more selective and more potent antimetabolites as the mycophenolic acids (MPAs), the use of azathioprine in kidney transplantation has been restricted to patients with special indications

Mycophenolate mofetil and the newer mycophenolate sodium have nowadays replaced azathioprine in most immunosuppressive regimens. Data about MPAs and HBV reactivation are lacking, but in general they are considered safe for HBV kidney‐transplant recipients.

The reactivation of HBV with the use of mTOR inhibitors has not been studied in renal‐ transplant recipients and generally they are considered safe. Some case reports of HBV reactivation related to everolimus when used as a chemotherapeutic agent have been reported

immune reconstitution.

226 Advances in Treatment of Hepatitis C and B

treatment of acute rejection.

*2.8.4. Calcineurin inhibitors*

this choice [47, 48].

*2.8.5. Antimetabolites*

*2.8.5.1. Mycophenolate acid derivates*

*2.8.6. Mammalian target of rapamycin (mTOR) inhibitors*

risk.

[49].

In conclusion, all immunosuppressants given in kidney transplantation can be used in HBV‐ positive recipients. The most important issue is the total amount of immunosuppression long term. It is crucial to maintain the lowest level of immunosuppression that is necessary to prevent rejection and to closely monitor the HBV status. Prophylactic antiviral treatment should be initiated immediately after transplantation and continued at least for 1 year after stable and low maintenance immunosuppression. In the carefully preselected patients in whom antivirals may be discontinued, close monitoring for HBV reactivation is mandatory.

#### **2.9. In summarizing the existing evidence about kidney transplantation and HBV**


**•** The total amount of immunosuppression must be kept at the lowest possible levels for the given donor/recipient.

In conclusion, with growing knowledge and evolving evidence in both fields, hepatitis B and transplantation, in the era of potent antivirals as nucleoside analogs, HBsAg‐positive kidney‐ transplant candidates and recipients can be successfully treated and monitored and reach survival rates comparable to their HBsAg‐negative counterparts.

## **3. Kidney transplantation and hepatitis C virus infection**

## **3.1. Epidemiology of hepatitis C virus (HCV) infection**

The prevalence of hepatitis C virus (HCV) infection worldwide is 3% and infected people are estimated to be approximately 170 millions. Prevalence rates in Africa, America, Europe, and South‐East Asia are less than 2.5%. In the Western Pacific regions, the prevalence ranges between 2.5 and 4.9% while in some parts of the Middle East, it reaches 13% [51–53].

The prevalence of hepatitis C in patients with end‐stage renal disease (ESRD) presents great variation worldwide. In northern Europe, it is below 5%, whereas in the US and southern Europe, it stands at 10%. In several North African, Asian, and Latin American countries, the relative disease prevalence varies between 10 and 70% [54]. In Greece, a 2003 collaborative study of the Hellenic Center for Infectious Diseases Control and the Hellenic Society of Nephrology showed that the percentage of patients with hepatitis C was 7.5% in a total of 7016 patients on dialysis [55].

Prior to 1990, the main routes of HCV transmission were blood‐product transfusions, intra‐ venous drug use, and unsafe medical procedures. Since the systematic screening of blood products, the risk of HCV infection related to transfusions is extremely low (1/20000000) [56]. Currently, the main routes of HCV infection are intravenous drug use, unsafe medical procedures, mother‐to‐child transmission, and the use of unsterilized materials in activities such as acupuncture and tattooing. Household and sexual transmission is extremely low. The dialysis‐related risk is estimated at 2% per year. With the screening of blood products and the use of erythropoiesis‐stimulating agents, the risk of transfusion‐related HCV infection in dialysis patients has dramatically declined; however, they continue to comprise a "high‐risk" group. In several studies, the prevalence of HCV infection correlated strongly with the time on dialysis, independently of the burden of transfusions and it was higher in HD than in home HD or peritoneal dialysis patients. These data strongly suggest that nosocomial transmission is of major importance [57].

Therefore, the KDIGO workgroup for the prevention of HCV transmission in dialysis patients focused on the implementation of hygienic precautions regarding the staff of HD units and the sterilization of the dialysis machines. Of major importance is the fact that the isolation of HCV‐infected patients does not seem to protect against HCV transmission in HD units and therefore it is not recommended [53].

## **4. Kidney transplantation versus dialysis for HCV‐infected dialysis patients**

**•** The total amount of immunosuppression must be kept at the lowest possible levels for the

In conclusion, with growing knowledge and evolving evidence in both fields, hepatitis B and transplantation, in the era of potent antivirals as nucleoside analogs, HBsAg‐positive kidney‐ transplant candidates and recipients can be successfully treated and monitored and reach

The prevalence of hepatitis C virus (HCV) infection worldwide is 3% and infected people are estimated to be approximately 170 millions. Prevalence rates in Africa, America, Europe, and South‐East Asia are less than 2.5%. In the Western Pacific regions, the prevalence ranges

The prevalence of hepatitis C in patients with end‐stage renal disease (ESRD) presents great variation worldwide. In northern Europe, it is below 5%, whereas in the US and southern Europe, it stands at 10%. In several North African, Asian, and Latin American countries, the relative disease prevalence varies between 10 and 70% [54]. In Greece, a 2003 collaborative study of the Hellenic Center for Infectious Diseases Control and the Hellenic Society of Nephrology showed that the percentage of patients with hepatitis C was 7.5% in a total of 7016

Prior to 1990, the main routes of HCV transmission were blood‐product transfusions, intra‐ venous drug use, and unsafe medical procedures. Since the systematic screening of blood products, the risk of HCV infection related to transfusions is extremely low (1/20000000) [56]. Currently, the main routes of HCV infection are intravenous drug use, unsafe medical procedures, mother‐to‐child transmission, and the use of unsterilized materials in activities such as acupuncture and tattooing. Household and sexual transmission is extremely low. The dialysis‐related risk is estimated at 2% per year. With the screening of blood products and the use of erythropoiesis‐stimulating agents, the risk of transfusion‐related HCV infection in dialysis patients has dramatically declined; however, they continue to comprise a "high‐risk" group. In several studies, the prevalence of HCV infection correlated strongly with the time on dialysis, independently of the burden of transfusions and it was higher in HD than in home HD or peritoneal dialysis patients. These data strongly suggest that nosocomial transmission

Therefore, the KDIGO workgroup for the prevention of HCV transmission in dialysis patients focused on the implementation of hygienic precautions regarding the staff of HD units and the sterilization of the dialysis machines. Of major importance is the fact that the isolation of HCV‐infected patients does not seem to protect against HCV transmission in HD units and

between 2.5 and 4.9% while in some parts of the Middle East, it reaches 13% [51–53].

survival rates comparable to their HBsAg‐negative counterparts.

**3.1. Epidemiology of hepatitis C virus (HCV) infection**

**3. Kidney transplantation and hepatitis C virus infection**

given donor/recipient.

228 Advances in Treatment of Hepatitis C and B

patients on dialysis [55].

is of major importance [57].

therefore it is not recommended [53].

A meta‐analysis of observational studies tried to establish the impact of hepatitis C virus infection on survival in dialysis patients. It showed that HCV‐positive patients on dialysis have an increased risk of mortality compared with their HCV‐negative counterparts, which is mainly attributed to liver‐associated disease and its complications (relative risk, 5.89) [58].

Kidney transplantation is the treatment of choice for HCV‐positive patients with ESRD. Three retrospective studies showed that transplantation offered a survival advantage in HCV‐ seropositive patients compared to those who remained on the waiting list [59–61]. A recent systematic review that included 9 studies with a total number of 2274 HCV‐infected renal‐ transplant candidates and recipients showed that 5 years posttransplantation, anti‐HCV– positive patients who had undergone kidney transplantation had approximately 55% lower risk of death compared to wait‐listed patients [62].

## **5. Diagnosis and assessment of liver disease in HCV‐positive kidney‐ transplant candidates**

The clinical tools that are used for the assessment of liver damage for patients with ESRD do not differ from those used for the general population. Several studies have shown that aminotransferase (AST, ALT) levels are low in patients on dialysis and this reduction appears to occur in patients with advanced chronic kidney disease even before the initiation of renal‐ replacement treatment [63, 64].

All patients on the waiting list for a kidney allograft should be tested for hepatitis C, initially with an anti‐HCV enzyme‐linked immunosorbent assay (ELISA) and after a positive result by polymerase chain reaction assay (PCR) for the quantification of HCV RNA. Identification and classification of HCV genotype should follow. Screening for HCV must be a clinical routine and it must be performed once a year in all dialysis patients, since they are at constant risk of acquiring HCV infection. Dialysis units with a high prevalence of HCV should adopt a more strict protocol by examining their patient population for the presence of viremic activity, regardless of the result of the ELISA test [53].

Liver biopsy is recommended by the KDIGO guidelines as the "gold standard" for assessing the severity of hepatic damage and the prognosis of the disease. Furthermore, it can provide valuable assistance in planning the future treatment strategy [65]. A study on percutaneous liver biopsy in chronic hepatitis C patients found the procedure to be safe without increased risk in patients with ESRD [66]. The necessity of a liver biopsy is underlined by the following factors:

**•** There is no reliable correlation between the fluctuation of aminotransferases levels or the measurements of HCV RNA and the severity of liver injury as shown by histological findings in this group of patients [67].


Novel, noninvasive, simple radiographic and serologic tests are used to validate hepatic fibrosis. Transient elastography (TE) evaluates the severity of fibrosis by liver‐stiffness measurement. It has been used in non‐uremic patients for the staging of fibrosis with satis‐ factory results [69]. In the dialysis population with chronic HCV infection, TE, performed with a Fibroscan machine, seemed to be efficient in estimating fibrosis in one study available [70]. Aspartate aminotransferase‐to‐platelet ratio index (APRI) is a serologic marker of fibrosis, easy to calculate. APRI is useful in diagnosing the degree of fibrosis [71], although it has a lower diagnostic accuracy than TE especially in cases of cirrhosis, in HD as well as in non‐uremic patients with HCV [70]. Larger cohort studies are needed before noninvasive techniques can replace liver biopsy. Nevertheless, they can be useful when the biopsy cannot be performed because of contraindications or patient refusal.

## **6. Kidney donation from HCV‐positive donors**

All prospective donors should be evaluated for the risk of HCV infection based on blood tests, medical history, and lifestyle habits. Prior to transplantation, deceased and living donors should be screened for anti‐HCV antibodies, preferably using ELISA third generation. However, the presence of antibodies against HCV in the donor may indicate a previous cleared infection and nontransmissibility. Thus, conducting PCR for HCV RNA is the next step for anti‐HCV–positive donors. In the setting of cadaveric kidney transplantation, the results of HCV RNA will be available after transplantation. Therefore, the KDIGO guidelines advise against transplantation from HCV‐positive donors to HCV‐negative recipients [53], since it is well established that hepatitis C can be transmitted by solid‐organ transplantation with a high frequency that approaches 100% in some studies [72, 73]. Viral transmission results in the occurrence of liver disease in the immunocompromised recipient, leading eventually to poor clinical outcomes due to infectious complications, development of cholestatic syndrome, and progression to hepatic failure [74].

Allocation of HCV‐positive kidneys is controversial. The strategy of many transplant centers, including ours nowadays, is to accept kidneys from HCV‐positive‐deceased donors for HCV‐ positive‐transplant candidates. According to the latest KDIGO guidelines [53], seropositive recipients should be tested by PCR for HCV RNA and must have an active viremia. This practice is based on the fact that kidney transplantation of HCV‐infected dialysis patients from HCV‐positive donors reduces the time in the transplant waiting list and is associated with superior survival compared to those who remain on the list waiting for a seronegative donor [75]. Additionally, a retrospective study by Morales et al. examined the differences between HCV‐positive recipients who were transplanted either from HCV‐positive donors or from HCV‐negative donors. In terms of decompensated liver disease, no differences were observed between the two groups (10.3 vs. 6.2%). Moreover, 5‐ and 10‐year patient survival were similar in the two groups, namely 84.8 and 72.7% in the subset of recipients from HCV‐positive donors versus 86.6 and 76.5%, respectively, in those who received an HCV‐negative renal allograft. Five‐ and ten‐year graft survivals were decreased in the HCV‐positive donor group (58.9% at 5 years and 34.4% at 10 years) compared to the HCV‐negative donor group (65.5% at 5 years and 47.6% at 10 years, *p*: 0.006). However, this difference was not associated to HCV seropo‐ sitivity in the multivariate regression analysis [76]. Ideally, donors and recipients should be matched for HCV genotype to minimize the risk of super‐infection, even if this procedure is rarely performed during a deceased donor evaluation. However, two retrospective studies showed that the number of HCV genotypes has no significant effect on patient survival [77, 78]. In the new era of HCV treatment with the direct‐acting antiviral agents (DAAs), the knowledge of the donors' genotype will be useful for the assessment of future treatment strategies.

**•** The percentage of HCV‐positive renal transplant recipients that develop liver disease in the

**•** Studies have shown that up to 25% of ESRD patients with chronic hepatitis C infection have

**•** The finding of advanced fibrosis in liver biopsy is a contraindication for renal transplanta‐ tion, because 10‐year survival is lower than 26% [52]. Patients with adequately compensated

Novel, noninvasive, simple radiographic and serologic tests are used to validate hepatic fibrosis. Transient elastography (TE) evaluates the severity of fibrosis by liver‐stiffness measurement. It has been used in non‐uremic patients for the staging of fibrosis with satis‐ factory results [69]. In the dialysis population with chronic HCV infection, TE, performed with a Fibroscan machine, seemed to be efficient in estimating fibrosis in one study available [70]. Aspartate aminotransferase‐to‐platelet ratio index (APRI) is a serologic marker of fibrosis, easy to calculate. APRI is useful in diagnosing the degree of fibrosis [71], although it has a lower diagnostic accuracy than TE especially in cases of cirrhosis, in HD as well as in non‐uremic patients with HCV [70]. Larger cohort studies are needed before noninvasive techniques can replace liver biopsy. Nevertheless, they can be useful when the biopsy cannot be performed

All prospective donors should be evaluated for the risk of HCV infection based on blood tests, medical history, and lifestyle habits. Prior to transplantation, deceased and living donors should be screened for anti‐HCV antibodies, preferably using ELISA third generation. However, the presence of antibodies against HCV in the donor may indicate a previous cleared infection and nontransmissibility. Thus, conducting PCR for HCV RNA is the next step for anti‐HCV–positive donors. In the setting of cadaveric kidney transplantation, the results of HCV RNA will be available after transplantation. Therefore, the KDIGO guidelines advise against transplantation from HCV‐positive donors to HCV‐negative recipients [53], since it is well established that hepatitis C can be transmitted by solid‐organ transplantation with a high frequency that approaches 100% in some studies [72, 73]. Viral transmission results in the occurrence of liver disease in the immunocompromised recipient, leading eventually to poor clinical outcomes due to infectious complications, development of cholestatic syndrome, and

Allocation of HCV‐positive kidneys is controversial. The strategy of many transplant centers, including ours nowadays, is to accept kidneys from HCV‐positive‐deceased donors for HCV‐ positive‐transplant candidates. According to the latest KDIGO guidelines [53], seropositive recipients should be tested by PCR for HCV RNA and must have an active viremia. This practice is based on the fact that kidney transplantation of HCV‐infected dialysis patients from HCV‐positive donors reduces the time in the transplant waiting list and is associated with

course of transplantation varies in different studies between 19 and 64% [59, 60].

hepatic disease should be referred for simultaneous liver‐kidney transplantation.

subclinical pre‐cirrhotic disease in liver biopsy [68].

230 Advances in Treatment of Hepatitis C and B

because of contraindications or patient refusal.

progression to hepatic failure [74].

**6. Kidney donation from HCV‐positive donors**

Living donors with HCV infection and viremia should preferably receive appropriate treat‐ ment prior to donation, since the duration of therapy is short and it leads to sustained SVR [79]. On the other hand, prior to donation the transplant team should carefully consider and explain to the donor the risk for developing HCV‐associated renal disease or diabetes mellitus in the future.

Based on the aforementioned data, the policy of transplanting a kidney from an anti‐HCV– positive donor to an anti‐HCV–positive recipient is considered to be a safe approach with good clinical outcomes in the long term. In any case prior to receiving an allograft, the HCV‐infected‐ transplant candidate should be informed in detail about the HCV status of the donor, the risk of super‐infection or other complications, the data regarding patient and graft survival, as well as the new treatment options.

## **7. Impact of HCV infection on posttransplant outcomes**

Hepatitis C adversely affects the survival of both patients and grafts. Numerous, predomi‐ nantly retrospective cohort studies report inferior 10‐year survival rate of HCV‐positive patients in comparison to uninfected kidney recipients [80–82]. Age at transplantation and the presence of anti‐HCV antibodies were independently associated with patient survival [81]. However, a serious limitation of these studies is that histological data regarding the severity of hepatic disease pretransplantation were not available in the majority of them.

A recent meta‐analysis of 18 observational trials that included 133,530 renal allograft recipients revealed an increased rate of all‐cause mortality in HCV‐positive patients after transplantation, regardless of the year of transplantation and thus the immunosuppressive regimen that was used, the country of origin or the number of patients. The main causes of death were cirrhosis and hepatocellular cancer. It is worth noting that hepatic disease developed late after trans‐ plantation. Cardiovascular mortality and cardiovascular disease were also more prevalent in this study group [83]. Additional extrahepatic causes of morbidity and mortality were new onset diabetes after transplantation (NODAT), de novo and recurrent glomerular diseases (mainly de novo type I membranoproliferative GN), and sepsis [84–86].

The abovementioned studies demonstrated also that graft survival is decreased in seropositive patients posttransplantation. More specifically, the meta‐analysis by Fabrizi et al. showed that the adjusted relative risk of graft loss in these patients compared to those who are not infected was 1.76 [83]. Allograft failure has been attributed to the aforestated morbidity factors, namely diabetes and glomerulonephritides, as well as to the occurrence of transplant glomerulopathy and chronic allograft injury [83–87].

## **8. Therapy**

Treatment of patients infected with HCV comprises the traditional approach with interferon and ribavirin, as well as novel regimens, interferon‐a‐free that consist of the direct‐acting antiviral agents. Therapeutic regimens aim at the elimination of the virus. The viral load, based on HCV RNA quantification in serum, must be undetectable (10–15 IU/ml) 12 weeks after the end of treatment (SVR).

## **8.1. Traditional therapy**

In the past decade, interferon and ribavirin were considered to be the cornerstone of HCV antiviral treatment. Nonetheless, these drugs were associated with considerable toxicity. More specifically, the use of interferon after kidney transplantation induced acute kidney injury, episodes of rejection resistant to steroid therapy, and graft loss [88, 89]. Therefore, before 2013 transplant candidates could only be treated prior to transplantation as the KDIGO guidelines recommended, with the exception of patients with fibrosing cholestatic hepatitis [53]. How‐ ever, the Dialysis Outcomes Practice Patterns Study demonstrated that only a minority of ESRD patients on dialysis were treated for HCV [90]. Among 4589 HCV‐positive HD patients who were observed from 1996 to 2011, only 48 (1%) were treated for HCV, whereas among the subset of patients waiting on the list for transplantation, only 3.7% were treated for HCV. The reasons for this approach were as follows:


Nevertheless, HCV clearance when achieved was maintained posttransplantation in the vast majority of patients despite the use of immunosuppression [93].

## **8.2. Novel therapeutic agents**

and hepatocellular cancer. It is worth noting that hepatic disease developed late after trans‐ plantation. Cardiovascular mortality and cardiovascular disease were also more prevalent in this study group [83]. Additional extrahepatic causes of morbidity and mortality were new onset diabetes after transplantation (NODAT), de novo and recurrent glomerular diseases

The abovementioned studies demonstrated also that graft survival is decreased in seropositive patients posttransplantation. More specifically, the meta‐analysis by Fabrizi et al. showed that the adjusted relative risk of graft loss in these patients compared to those who are not infected was 1.76 [83]. Allograft failure has been attributed to the aforestated morbidity factors, namely diabetes and glomerulonephritides, as well as to the occurrence of transplant glomerulopathy

Treatment of patients infected with HCV comprises the traditional approach with interferon and ribavirin, as well as novel regimens, interferon‐a‐free that consist of the direct‐acting antiviral agents. Therapeutic regimens aim at the elimination of the virus. The viral load, based on HCV RNA quantification in serum, must be undetectable (10–15 IU/ml) 12 weeks after the

In the past decade, interferon and ribavirin were considered to be the cornerstone of HCV antiviral treatment. Nonetheless, these drugs were associated with considerable toxicity. More specifically, the use of interferon after kidney transplantation induced acute kidney injury, episodes of rejection resistant to steroid therapy, and graft loss [88, 89]. Therefore, before 2013 transplant candidates could only be treated prior to transplantation as the KDIGO guidelines recommended, with the exception of patients with fibrosing cholestatic hepatitis [53]. How‐ ever, the Dialysis Outcomes Practice Patterns Study demonstrated that only a minority of ESRD patients on dialysis were treated for HCV [90]. Among 4589 HCV‐positive HD patients who were observed from 1996 to 2011, only 48 (1%) were treated for HCV, whereas among the subset of patients waiting on the list for transplantation, only 3.7% were treated for HCV. The reasons

**•** The use of ribavirin in this patient population aggravated anemia that was already present

**•** Pegylated interferon‐α (PegIFN‐α) as monotherapy resulted in poor outcomes, with SVR

**•** Addition of ribavirin in low doses increased the SVR to 55% after 6 months, but also

**•** A substantial percentage of patients (18–30%) dropped out of therapy.

(mainly de novo type I membranoproliferative GN), and sepsis [84–86].

and chronic allograft injury [83–87].

232 Advances in Treatment of Hepatitis C and B

**8. Therapy**

end of treatment (SVR).

**8.1. Traditional therapy**

for this approach were as follows:

due to chronic kidney disease.

increased side effects [92].

30–35% [91].

Thorough understanding of the HCV structure, replication mechanism, and cell cycle has led to the development of the DAAs. These drugs are small molecules that target nonstructural (NS) viral proteins and inhibit HCV replication. Four classes of DAAs exist, namely NS3/4A protease inhibitors (PIs) simeprevir, paritaprevir, and grazoprevir, NS5B nucleoside polymer‐ ase inhibitors (NPIs) and non‐nucleoside polymerase inhibitors (NNPIs) sofosbuvir and dasabuvir, respectively, and NS5A inhibitors ledipasvir, daclatasvir, ombitasvir, and elbasvir [94].

The introduction of these new agents has modernized the therapeutic ammunition and has radically changed the treatment of patients with HCV infection; ongoing trials are expected to prove the safety and efficacy of DAAs in patients with impaired renal function and ESRD and establish proper dosing regimens. Besides the spectacular effectiveness of these drugs (SVR over 95%) in patients who had not received prior therapy [95], another important issue is the improved tolerance to treatment, due to reduced treatment duration and fewer side effects.


Different combinations of DAAs are administered based on the different HCV genotypes (**Table 1**).

**Table 1.** Treatment recommendation (EASL 2015) for chronic hepatitis C patients without liver cirrhosis.

The first studies that evaluated the effectiveness of DAAs excluded patients with estimated glomerular filtration rate (eGFR) less than 30 ml/min/1.73m2 , patients on dialysis, and renal‐ transplant recipients. It is worth noting that sofosbuvir is contraindicated for patients with eGFR <30 ml/min/1.73m2 and for dialysis patients [94, 95]. Therefore, treatment options for this study group with HCV infection from genotypes 2, 3, 5, and 6 of HCV are limited, because all regimens include sofosbuvir. Severe, urgent cases should receive treatment after careful expert consultation. On the other hand, results are very promising for patients with genotypes 1 and 4 in comparison with the general population. Ruby‐I is a single‐arm multicenter study, in which 20 patients with HCV genotype 1 and CKD stage 4,5 or in dialysis were given ombitasvir co‐ formulated with paritaprevir and ritonavir, administered with dasabuvir for 12 weeks. Patients with HCV genotype 1a infection also received ribavirin (n:13), whereas those with genotype1b infection did not (n:7). The majority of patients, 90%, achieved the primary end point which was SVR 12 weeks after the end of treatment (SVR12). One patient did not achieve an SVR12 because of a relapse and another one died from causes not related to treatment. The most common adverse event was anemia (69%) due to ribavirin treatment, which led to drug discontinuation in nine cases [96]. C‐Surfer is a multicenter, phase 3, randomized study of safety and observational study of efficacy regarding the combination of grazoprevir and elbasvir (both approved by the Food and Drug Administration (FDA) and wait to be approved by the European Medicines Agency (EMA) in 2016) for patients with genotype 1 infection and stage 4–5 CKD. The treatment group consisted of 111 patients, who received grazoprevir and elbasvir for 12 weeks. The results were remarkable. The SVR12 was 99%, with only one patient relapsing, whereas the drugs were well tolerated with minor adverse events that did not lead to drug discontinuation [97].

The use of interferon‐free treatment regimens is of major importance in renal transplantation because it eliminates the risk of acute allograft rejection and subsequent graft loss. An important question that arises is when is the proper timing of treatment, pre‐ or post trans‐ plantation? The introduction of DAAs permits us to exceed the narrow timeframes before transplantation and treat our patients after transplantation. Thus, we have the advantage of using allografts from HCV‐positive donors for recipients willing to accept them. This practice minimizes the time on the waiting list and subsequently the time on dialysis and all its deleterious effects as we have already mentioned, but it cannot be applied in small countries such as Greece with extremely long waiting time on the list. On the other hand, treatment with DAAs prior to transplantation may offer the advantage of increasing the overall survival of patients by diminishing the risk of hepatic and extrahepatic complications especially severe, evolving liver disease, glomerulonephritis and NODAT. Another important issue to consider when deciding the timing of treatment is the virus genotype. Eradication of the virus in patients infected with genotype 1 or 4 is plausible before transplantation, since sofosbuvir‐free regimens are available.

In renal transplantation, the DAAs are used according to the guidelines applied to the general population and the liver‐transplant recipients. Until 2016, there were no data to guide the use of these agents in kidney transplant patients and to demonstrate their efficacy and safety to this subpopulation of patients. The policy of many transplant centers, including ours, is that all kidney‐transplant patients with chronic HCV infection and eGFR >30 ml/min/1.73m2 receive appropriate therapy with a new, interferon‐free antiviral regimen based on the detected genotype (**Table 1**). The dose of DAAs is not adjusted when eGFR is greater than 30 ml/min/ 1.73m2 . Ribavirin is not recommended with eGFR <30 ml/min/1.73m2 although it has been used in patients after renal transplantation with a close monitoring of hemoglobin levels [98].

Of great importance are the drug‐drug interactions between the DAAs and the immunosup‐ pressive agents and the mandatory dose adjustments (**Table 2**).


**Green**: No significant interaction is expected.

**Orange**: Possible interaction which requires close monitoring, changing the dosage, and/or drug‐delivery time.

**Red**: Avoid concomitant use of drugs.

eGFR <30 ml/min/1.73m2

234 Advances in Treatment of Hepatitis C and B

to drug discontinuation [97].

regimens are available.

and for dialysis patients [94, 95]. Therefore, treatment options for this

study group with HCV infection from genotypes 2, 3, 5, and 6 of HCV are limited, because all regimens include sofosbuvir. Severe, urgent cases should receive treatment after careful expert consultation. On the other hand, results are very promising for patients with genotypes 1 and 4 in comparison with the general population. Ruby‐I is a single‐arm multicenter study, in which 20 patients with HCV genotype 1 and CKD stage 4,5 or in dialysis were given ombitasvir co‐ formulated with paritaprevir and ritonavir, administered with dasabuvir for 12 weeks. Patients with HCV genotype 1a infection also received ribavirin (n:13), whereas those with genotype1b infection did not (n:7). The majority of patients, 90%, achieved the primary end point which was SVR 12 weeks after the end of treatment (SVR12). One patient did not achieve an SVR12 because of a relapse and another one died from causes not related to treatment. The most common adverse event was anemia (69%) due to ribavirin treatment, which led to drug discontinuation in nine cases [96]. C‐Surfer is a multicenter, phase 3, randomized study of safety and observational study of efficacy regarding the combination of grazoprevir and elbasvir (both approved by the Food and Drug Administration (FDA) and wait to be approved by the European Medicines Agency (EMA) in 2016) for patients with genotype 1 infection and stage 4–5 CKD. The treatment group consisted of 111 patients, who received grazoprevir and elbasvir for 12 weeks. The results were remarkable. The SVR12 was 99%, with only one patient relapsing, whereas the drugs were well tolerated with minor adverse events that did not lead

The use of interferon‐free treatment regimens is of major importance in renal transplantation because it eliminates the risk of acute allograft rejection and subsequent graft loss. An important question that arises is when is the proper timing of treatment, pre‐ or post trans‐ plantation? The introduction of DAAs permits us to exceed the narrow timeframes before transplantation and treat our patients after transplantation. Thus, we have the advantage of using allografts from HCV‐positive donors for recipients willing to accept them. This practice minimizes the time on the waiting list and subsequently the time on dialysis and all its deleterious effects as we have already mentioned, but it cannot be applied in small countries such as Greece with extremely long waiting time on the list. On the other hand, treatment with DAAs prior to transplantation may offer the advantage of increasing the overall survival of patients by diminishing the risk of hepatic and extrahepatic complications especially severe, evolving liver disease, glomerulonephritis and NODAT. Another important issue to consider when deciding the timing of treatment is the virus genotype. Eradication of the virus in patients infected with genotype 1 or 4 is plausible before transplantation, since sofosbuvir‐free

In renal transplantation, the DAAs are used according to the guidelines applied to the general population and the liver‐transplant recipients. Until 2016, there were no data to guide the use of these agents in kidney transplant patients and to demonstrate their efficacy and safety to this subpopulation of patients. The policy of many transplant centers, including ours, is that

appropriate therapy with a new, interferon‐free antiviral regimen based on the detected genotype (**Table 1**). The dose of DAAs is not adjusted when eGFR is greater than 30 ml/min/

receive

all kidney‐transplant patients with chronic HCV infection and eGFR >30 ml/min/1.73m2

This table is based on data by the University of Liverpool on the site http://www.hep‐druginteractions.org (University of Liverpool). For additional drug‐drug interactions and for a more extensive range of drugs, detailed pharmacokinetic interaction data, and dosage adjustments, refer to the abovementioned website.

**Table 2.** Interactions between immunosuppressive drugs and DAA agents.

Since 2016, several studies have emerged. Kamar et al. tried to assess the efficacy and safety of an interferon‐free regiment based on sofosbuvir. Twenty‐five renal‐transplant recipients with HCV infection (19/25 Genotype 1, 2/25 Genotype 2, 1/25 Genotype 3, 3/25 Genotype 4) received various combinations of sofosbuvir with other agents; ribavirin (n:3), daclatasvir (n:4), simeprevir (n:6), simeprevir and ribavirin (n:1), ledipasvir (n:9), ledipasvir and ribavirin (n:1), pegylated interferon and ribavirin (n:1). At week 12, an impressive SVR of 100% was recorded. During therapy, no significant adjustments in the dose of immunosuppressive drugs were required and kidney function remained stable [109]. However, after virus clearance, trough levels of tacrolimus decreased without any dose change. It is already known that HCV infection alters the pharmacokinetics of CNIs and results in increased drug exposure [99]. Therefore, we must be cautious after HCV clearance and adjust the dose of CNIs accordingly. A case series study of 20 HCV‐infected kidney recipients (85% Genotype 1, 15% Genotype 2) who were treated off‐label with sofosbuvir in combination with simeprevir (n:9), ribavirin (n:3), ledipas‐ vir (n:7), and daclatasvir (n:1) demonstrated a sustained virological response of 100% at week 12 and it was maintained for a short median follow‐up period of 8.6 months [100].

These impressive results show that the efficacy and safety of DAAs in renal transplant recipients is comparable with the general population. It remains to be determined if viral clearance after transplantation will improve long‐term patient and kidney‐allograft outcomes. The optimal timing of HCV therapy (posttransplantation or pretransplantation) has not clearly been determined. Taking into account that based on clinical trials the DAAs will be available for patients with eGFR <30/ml/min/1.73m2 in the near future, treating these patients before transplantation may prevent posttransplantation complications and improve the overall outcomes. For the time being, ESRD patients infected with HCV Genotypes 2, 3, 5, 6 can be treated with DAAs only after transplantation or when it is absolutely obligatory in life‐ threatening conditions.

## **9. Immunosuppression in HCV‐positive kidney transplant recipients**

Immunosuppression may increase hepatitis C viral proliferation after transplantation and thus accelerate the evolution of hepatic damage [101]. Information regarding the use of immuno‐ suppressive drugs in seropositive allograft recipients comes mostly from liver transplantation, as well as from the experience in the field of oncology‐hematology. Large, prospective studies examining the effect of immunosuppressive drugs in HCV‐seropositive recipients are lacking. However, the total amount of induction and maintenance immunosuppression may play an important role in the reactivation of the virus post transplantation.

## **10. Immunosuppressive agents**

## **10.1. Rituximab**

The use of anti‐CD20 monoclonal antibody rituximab has been reported in a small number of seven HCV‐positive patients after kidney transplantation. It was not related to the recurrence of the infection in a follow‐up period of 19 months [102]. Larger studies in the field of hema‐ tology have shown a high incidence of hepatic flares in HCV‐seropositive patients following treatment with Rituximab for lymphoma [103].

## **10.2. Induction therapy**

Data from the Scientific Registry of Transplant Recipients (SRTRs) demonstrated that induction therapy, with polyclonal or monoclonal antibodies, has been associated with a lower risk of death. This finding could probably be attributed to lower rejection rates in patients receiving induction treatment [104]. Anti‐CD3 monoclonal antibody OKT3, however, has been associ‐ ated with recurrence of HCV in liver transplantation [105]. It is therefore avoided in HCV‐ infected patients after transplantation. On the other hand, the administration of the polyclonal antibody antithymocyte globulin (ATG) as induction therapy in 104 HCV‐infected kidney‐ transplant patients did not induce viral replication [106], a finding that was confirmed by subsequent studies [107]. Contradictory data exist regarding monoclonal anti‐IL2 antibodies, such as daclizumab. A single‐center study in a small number of patients showed that therapy with daclizumab is followed by faster progression of liver fibrosis compared to ATG [108]. Large studies based on data from the United Network for Organ‐Sharing UNOS base indicate that liver‐transplant recipients with chronic HCV infection exhibit satisfactory graft and patient survival after receiving induction with daclizumab [109, 110].

## **10.3. Corticosteroids (CS)**

These impressive results show that the efficacy and safety of DAAs in renal transplant recipients is comparable with the general population. It remains to be determined if viral clearance after transplantation will improve long‐term patient and kidney‐allograft outcomes. The optimal timing of HCV therapy (posttransplantation or pretransplantation) has not clearly been determined. Taking into account that based on clinical trials the DAAs will be available

transplantation may prevent posttransplantation complications and improve the overall outcomes. For the time being, ESRD patients infected with HCV Genotypes 2, 3, 5, 6 can be treated with DAAs only after transplantation or when it is absolutely obligatory in life‐

**9. Immunosuppression in HCV‐positive kidney transplant recipients**

important role in the reactivation of the virus post transplantation.

Immunosuppression may increase hepatitis C viral proliferation after transplantation and thus accelerate the evolution of hepatic damage [101]. Information regarding the use of immuno‐ suppressive drugs in seropositive allograft recipients comes mostly from liver transplantation, as well as from the experience in the field of oncology‐hematology. Large, prospective studies examining the effect of immunosuppressive drugs in HCV‐seropositive recipients are lacking. However, the total amount of induction and maintenance immunosuppression may play an

The use of anti‐CD20 monoclonal antibody rituximab has been reported in a small number of seven HCV‐positive patients after kidney transplantation. It was not related to the recurrence of the infection in a follow‐up period of 19 months [102]. Larger studies in the field of hema‐ tology have shown a high incidence of hepatic flares in HCV‐seropositive patients following

Data from the Scientific Registry of Transplant Recipients (SRTRs) demonstrated that induction therapy, with polyclonal or monoclonal antibodies, has been associated with a lower risk of death. This finding could probably be attributed to lower rejection rates in patients receiving induction treatment [104]. Anti‐CD3 monoclonal antibody OKT3, however, has been associ‐ ated with recurrence of HCV in liver transplantation [105]. It is therefore avoided in HCV‐ infected patients after transplantation. On the other hand, the administration of the polyclonal antibody antithymocyte globulin (ATG) as induction therapy in 104 HCV‐infected kidney‐ transplant patients did not induce viral replication [106], a finding that was confirmed by subsequent studies [107]. Contradictory data exist regarding monoclonal anti‐IL2 antibodies,

in the near future, treating these patients before

for patients with eGFR <30/ml/min/1.73m2

**10. Immunosuppressive agents**

treatment with Rituximab for lymphoma [103].

**10.1. Rituximab**

**10.2. Induction therapy**

threatening conditions.

236 Advances in Treatment of Hepatitis C and B

High pulses of corticosteroids can cause up to 100 times increase of the viral load, but this has only been demonstrated in liver transplantation [107]. Although rapid steroid discontinuation leads to lower rates of diabetes and HCV recurrence, it has been associated with worst outcomes in liver transplantation [111, 112]. In the aforementioned study by Luan et al., in a total of 3708 HCV‐positive kidney transplant patients, mortality rates were similar between those who received CS and those who did not [104].

## **10.4. Calcineurin inhibitors (CNIs)**

In vitro studies have shown that cyclosporine may have an antiviral effect by suppressing HCV replication and the expression of proteins [113]. Moreover, cyclosporine is less diabetogenic in comparison with tacrolimus. However, in a cohort of 71 patients with HCV infection posttransplantation, liver fibrosis and viral replication were similar regardless of the CNI used [114]. Additionally, data from the Scientific Registry of Transplant Recipients (SRTR) [104] did not confer a survival advantage of cyclosporine over tacrolimus in renal allograft recipients.

## **10.5. Antimetabolites**

MPAs appear to be safe in HCV‐seropositive individuals after kidney transplantation. Notably, MMF administration was related to a reduced risk of death (hazard ratio (HR): 0.77, *p*: 0.005) in the study by Luan et al., implying a possible advantageous effect of the drug in renal recipients with chronic HCV infection [104].

#### **10.6. Mammalian target of rapamycin (mTOR) inhibitors**

Data regarding the use of mTOR inhibitors in transplant patients with HCV infection are limited. Sirolimus was associated with decreased evolution of hepatic fibrosis and cell proliferation in vitro, in an animal model of hepatic fibrosis [115]. This finding was not confirmed in a small cohort study of HCV‐infected kidney recipients, where switch from CNI to sirolimus was not followed by lower viral load [116].

In conclusion, almost all immunosuppressive agents can be used in HCV‐positive renal recipients. As in the case of HBV, the most important issue is the total level of immunosup‐ pression, which should be kept as low as possible based on the specific conditions of trans‐ plantation and the immunological profile of the recipient. Close monitoring of HCV RNA is mandatory.

## **11. In summarizing the existing evidence about kidney transplantation and HCV**

	- **◦** All patients with eGFR >30 ml/min/1.73 m2 should receive a new, interferon‐free antiviral regimen based on the virus genotype.
	- **◦** Patients with eGFR <30 ml/min/1.73 m2 should not be treated with sofosbuvir. Treatment options for genotypes 2, 3, 5, and 6 of HCV are limited. In severe conditions, treatment should be discussed with experts.
	- **◦** In the case of HCV genotype 1 or 4, the combination grazoprevir‐elbasvir can be admin‐ istered.

In conclusion, the development of direct‐acting antiviral agents (DAAs) may change the natural history of HCV infection in renal allograft recipients. Randomized, prospective trials are expected to prove the safety and efficacy, as well as the optimal dose of DAAs in patients with impaired renal function, ESRD, and kidney transplantation.

## **Author details**

**11. In summarizing the existing evidence about kidney transplantation**

**•** The prevalence of hepatitis C in patients with ESRD presents great variation worldwide and

**•** Mortality is lower among patients who undergo kidney transplantation compared to those

**•** Systematic screening for HCV should be routinely done in all ESRD patients. Dialysis units with a high prevalence of HCV should preferably test all patients for HCV RNA, regardless

**•** Renal transplant recipients with chronic HCV infection have lower patient and allograft survival post transplantation compared with noninfected renal transplant recipients.

**•** Major causes of mortality in HCV‐infected renal transplant recipients are cirrhosis and hepatocellular cancer. Additional causes of morbidity following kidney transplantation are

**•** Transplantation of a renal allograft from an HCV‐infected donor may cause HCV infection

**•** All potential kidney donors, deceased and living, should be evaluated for the risk of HCV

**•** Interferon should not be administered in renal transplant recipients with chronic HCV

**•** We suggest the following approach regarding antiviral treatment in HCV‐infected renal

**◦** Patients with eGFR <30 ml/min/1.73 m2 should not be treated with sofosbuvir. Treatment options for genotypes 2, 3, 5, and 6 of HCV are limited. In severe conditions, treatment

**◦** In the case of HCV genotype 1 or 4, the combination grazoprevir‐elbasvir can be admin‐

should receive a new, interferon‐free antiviral

**•** Kidneys from HCV‐positive donors are donated to anti‐HCV–positive recipients.

**•** Kidney transplantation is the choice of therapy for HCV‐infected patients with ESRD.

**•** Liver biopsy should be performed in all HCV‐infected renal transplant candidates.

**•** Well‐compensated cirrhosis is not a contraindication to kidney transplantation.

de novo and recurrent and glomerular diseases and NODAT.

infection based on blood tests, medical history, and lifestyle habits.

infection because it is associated with rejection episodes and graft loss.

**and HCV**

is correlated with the time on dialysis.

of the presence of anti‐HCV antibodies.

remaining on the waiting list.

238 Advances in Treatment of Hepatitis C and B

to the recipient.

allograft recipients:

istered.

**◦** All patients with eGFR >30 ml/min/1.73 m2

regimen based on the virus genotype.

should be discussed with experts.

Smaragdi Marinaki, Konstantinos Drouzas, Chrysanthi Skalioti\* and John N. Boletis

\*Address all correspondence to: c\_skalioti@yahoo.com

National and Kapodistrian University of Athens, Medical School, Nephrology Department and Renal Transplantation Unit, Laiko Hospital, Athens, Greece

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**Investigational Treatment Strategies**

#### **Strategies to Preclude Hepatitis C Virus Entry** Strategies to Preclude Hepatitis C Virus Entry

Thierry Burnouf, Ching-Hsuan Liu and Liang-Tzung Lin Thierry Burnouf, Ching-Hsuan Liu and Liang-Tzung Lin

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65470

#### Abstract

Without a preventive vaccine, hepatitis C virus (HCV) remains an important pathogen worldwide with millions of carriers at risk of end-stage liver diseases. Despite the introduction of novel direct-acting antivirals (DAAs), resistance problems, challenges with the difficult-to-treat populations and high costs limit the widespread application of these drugs. Antivirals with alternative mechanism(s) of action, such as by restricting viral entry or cell-to-cell spread, could help expand the scope of antiviral strategies for the management of hepatitis C. Transfusion-associated HCV infection remains another issue in endemic and resource-limited areas around the world. This chapter describes some of the latest developments in antiviral strategies to preclude HCV entry, such as through monoclonal antibodies and small molecules, as well as measures to enhance the safety of therapeutic plasma products in blood transfusion.

Keywords: hepatitis C virus, viral entry, antivirals, entry inhibitors, monoclonal antibodies, small molecules, therapeutic plasma products

## 1. Introduction

Hepatitis C virus (HCV) is a major pathogen that predisposes about 170–300 million people worldwide to risks of end-stage liver diseases (ESLD), including cirrhosis and hepatocellular carcinoma (HCC). The hepatotropic virus remains one of the top indications for liver transplantation in treating ESLD [1]. While a preventive vaccine remains unavailable, the recent introduction of direct-acting antivirals (DAAs) has revolutionized the treatment for hepatitis C, phasing out the decade-old interferon (IFN)-based regimens. The majority of DAAs, however, focus on targeting viral replication such as via inhibition of the HCV NS3/4A protease, the NS5A cofactor, and the NS5B polymerase [2]. Although the DAAs have significantly improved the rate of sustained virological response (SVR) in the most prevalent genotype 1 patients, several challenges persist in real-world setting including high cost, drug-drug

© The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

interactions, emergence of drug resistance, hard-to-treat populations (e.g., human immunodeficiency virus [HIV] coinfection, ESLD, and transplant patients), and management of DAA failures [3–5]. With the advent of hepatitis C treatment in larger populations and borrowing from the experience with HIV cocktail therapy, it is becoming clear that developing therapeutic strategies with different modes of action would be necessary to address the various limitations of current DAAs. In addition, HCV transmission due to transfusion of contaminated blood products remains an issue in endemic areas around the world. This is particularly the case in resource-limited countries that face inadequate supply of safe blood products or have poorly controlled blood screening practices, leading to significant risk of transfusionassociated HCV infection [6]. Measures to enhance the safety of therapeutic plasma products such as through the implementation of viral inactivation treatments are therefore a necessity to reduce such risk.

The multistep process of HCV entry makes it an attractive target since it is the foremost fundamental prerequisite in establishing an infection. Following successful entry, the viral life cycle initiates to produce more virions, and with this development the underlying disease begins its progression. Blocking HCV infection by targeting its entry therefore has important implications for both prophylactic and therapeutic purposes since it abolishes the viral life cycle. As a prophylactic treatment, it can be used to prevent infection or reinfection. This is particularly useful in liver transplant setting of hepatitis C wherein the liver allograft is inevitably reinfected [7, 8]. As a therapeutic treatment, precluding HCV entry via de novo infection or cell-to-cell transmission helps to restrict viral spread in an infected person which could slow the progression of the disease. In addition, incorporation of strategies to block HCV entry into existing DAA treatments is expected to maximize the treatment response rate, even producing a synergistic effect [9], as with the experience of using multiple inhibitors in HIV cocktail therapy to concomitantly target various stages of the viral life cycle. Since more steps are being targeted in such a multipronged approach, the inclusion of entry inhibitors to existing DAAs could impose a higher genetic barrier to drug-resistance development. Such tactic not only aids in disrupting persistent HCV infection but could also help to ultimately achieve viral clearance. These aspects therefore make the development of HCV entry blocking strategies highly advantageous in both expanding the scope of antiviral treatments against hepatitis C and providing new insights into antiviral management. This chapter describes some of the latest development of strategies in precluding HCV entry for the management of hepatitis C.

## 2. Overview of HCV entry

Owing to the development of infectious HCV culture systems (e.g., cell-culture-derived HCV, HCVcc) and viral pseudoparticles bearing HCV glycoproteins (e.g., HCV pseudoparticles, HCVpp), a scenario of how HCV entry occurs has slowly emerged over the last decade of research. It is widely recognized that the HCV particle undergoes a series of intimate and wellorchestrated interactions with various receptors/coreceptors on the hepatocyte host cell surface as well as in the tight junctions, which ultimately lead to the attachment, internalization, and fusion of the virion with the cellular membrane. A number of these receptor interactions are thought to be attributed to the highly lipidated nature of the HCV virion. Specifically, HCV exists as a lipo-viro particle (LVP) with a lipid composition that includes the apolipoproteins and resembles that of very low-density lipoproteins (VLDLs) and low-density lipoproteins (LDLs) [10–15]. The association with lipids on the viral particle is thought to contribute to the shielding of HCV glycoproteins from neutralization by the host antibody-mediated response. In addition, the presence of the apolipoproteins on the virion has a large influence on the production of infectious HCV and also its tissue tropism [13, 16–22].

Following circulation in the blood, the HCV viral particles reach the liver and begin the interactions with molecules at the surface of the hepatocytes (Figure 1). The initial contacts are with nonspecific receptor(s) including the glycosaminoglycan (GAG) heparan sulfate moieties [23–25] that can be found on the transmembrane core proteins syndecans [26, 27]. These early interactions facilitate the attachment of the HCV virion and its accumulation on the hepatocytes for subsequent binding to more specific receptors. Although the LDL receptor (LDLR) has also been suggested as a potential initial attachment factor [28–30], recent evidence suggests that it may play a more essential role in viral replication [31, 32].

Figure 1. Overview of HCV entry.

interactions, emergence of drug resistance, hard-to-treat populations (e.g., human immunodeficiency virus [HIV] coinfection, ESLD, and transplant patients), and management of DAA failures [3–5]. With the advent of hepatitis C treatment in larger populations and borrowing from the experience with HIV cocktail therapy, it is becoming clear that developing therapeutic strategies with different modes of action would be necessary to address the various limitations of current DAAs. In addition, HCV transmission due to transfusion of contaminated blood products remains an issue in endemic areas around the world. This is particularly the case in resource-limited countries that face inadequate supply of safe blood products or have poorly controlled blood screening practices, leading to significant risk of transfusionassociated HCV infection [6]. Measures to enhance the safety of therapeutic plasma products such as through the implementation of viral inactivation treatments are therefore a necessity

The multistep process of HCV entry makes it an attractive target since it is the foremost fundamental prerequisite in establishing an infection. Following successful entry, the viral life cycle initiates to produce more virions, and with this development the underlying disease begins its progression. Blocking HCV infection by targeting its entry therefore has important implications for both prophylactic and therapeutic purposes since it abolishes the viral life cycle. As a prophylactic treatment, it can be used to prevent infection or reinfection. This is particularly useful in liver transplant setting of hepatitis C wherein the liver allograft is inevitably reinfected [7, 8]. As a therapeutic treatment, precluding HCV entry via de novo infection or cell-to-cell transmission helps to restrict viral spread in an infected person which could slow the progression of the disease. In addition, incorporation of strategies to block HCV entry into existing DAA treatments is expected to maximize the treatment response rate, even producing a synergistic effect [9], as with the experience of using multiple inhibitors in HIV cocktail therapy to concomitantly target various stages of the viral life cycle. Since more steps are being targeted in such a multipronged approach, the inclusion of entry inhibitors to existing DAAs could impose a higher genetic barrier to drug-resistance development. Such tactic not only aids in disrupting persistent HCV infection but could also help to ultimately achieve viral clearance. These aspects therefore make the development of HCV entry blocking strategies highly advantageous in both expanding the scope of antiviral treatments against hepatitis C and providing new insights into antiviral management. This chapter describes some of the latest development of strategies in precluding HCV entry for

Owing to the development of infectious HCV culture systems (e.g., cell-culture-derived HCV, HCVcc) and viral pseudoparticles bearing HCV glycoproteins (e.g., HCV pseudoparticles, HCVpp), a scenario of how HCV entry occurs has slowly emerged over the last decade of research. It is widely recognized that the HCV particle undergoes a series of intimate and wellorchestrated interactions with various receptors/coreceptors on the hepatocyte host cell surface as well as in the tight junctions, which ultimately lead to the attachment, internalization, and

to reduce such risk.

252 Advances in Treatment of Hepatitis C and B

the management of hepatitis C.

2. Overview of HCV entry

Nevertheless, these initial interactions have been shown to be mediated via apolipoprotein E (apoE) on the virion [29, 33–36]. The capturing process of the HCV particle is finalized by its interaction with the scavenger receptor class B type I (SR-BI) [37, 38], which is able to associate with the virion's lipoproteins [37, 39] as well as the HCV E2 glycoprotein [40, 41]. Binding of HCV with SR-BI induces lipoprotein rearrangements that help prime the virion for subsequent binding to other host cell factors and promote entry. This process is proposed to occur via SR-BI's lipid transfer activity between the viral particle and the plasma membrane [37, 42] and/or by direct interaction with the hypervariable region 1 (HVR1) domain on E2 [37, 43], which ultimately leads to conformational change and the exposure of functional glycoprotein epitopes for additional receptor binding. Closely following this event is the engagement of the HCV particle with the tetraspanin receptor CD81 [44, 45], which is an important entry factor for the virus [41, 46, 47]. HCV binding to CD81 is proposed to induce a dynamic lateral diffusion of virus-receptor complexes toward the tight junction area for further interactions with additional entry factors and viral internalization [22, 48]. Specifically, CD81 forms a coreceptor complex with the tight junction protein claudin-1 (CLDN1) [49, 50] and is engaged in late events of HCV entry [51]. This re-localization and virusreceptor complex association with CLDN1 involves multiple signaling pathways (e.g., Rho GTPases, PI3K/AKT, and ERK/MAPK) [52, 53], includes the activation of host cell kinases such as the epidermal growth factor receptor (EGFR) and ephrin receptor A2 (EphA2) [54, 55], and is influenced by the absence of the CD81-associated partner EWI-2wint on the hepatocytes [56, 57]. The EWI-2wint molecule is normally bound to CD81 on most cell type surfaces and inhibits its diffusion which is required to promote HCV entry; however, it is not expressed in the hepatocytes, and hence its absence has been suggested to contribute to the restricted tropism of the virus [56]. Following interaction with the CD81/CLDN1 complex, the HCV particle is presumed to then interact with the tight junction protein occludin (OCLN) prior to viral internalization [58]. Additional proteins that take part in influencing virion entry into the hepatocyte include the transferrin receptor 1 (TfR1) [59] and the cholesterol transporter Niemann-Pick C1-like 1 (NPC1L1) [60], although their specific role and interplay with other entry factors in the HCV entry process remain to be defined. The HCV particle finally enters the cell via clathrin-mediated endocytosis [61]. The HCV-receptor complexes then migrate to endosomal compartments [62, 63] where acidification occurs to induce membrane fusion, which allows the release of viral RNA into the host cytosol.

The above sequential and multistep entry process consequently yields the successful release of the HCV genome into the host cytoplasm for direct translation and the ensuing launch of viral replication. The roles played by several of these entry factors including SR-BI, CD81, CLDN1, and OCLN not only mediate HCV entry but also presumably help to define tissue and species tropism of the virus [64–67]. The understanding of how HCV achieves viral entry has led to the possibility of antiviral targeting. From docking to virus internalization, essentially all steps are targetable to prevent HCV infection of the host cell. In addition, given the association of HCV with lipoproteins and the viral particle's interaction with lipoprotein and lipid receptors (LDLR, SR-BI, and NPC1L1), the lipidic nature of HCV virion also offers various methods of pharmacological intervention. Finally, many of the entry factors including CD81, SR-BI, CLDN1, OCLN, and NPC1L1 also play a role in mediating HCV cell-to-cell transmission between intercellular junctions [68–71], and therefore targeting these molecules could help restrict both cell-free entry and cell-to-cell spread of HCV.

## 3. Current development in inhibition of HCV entry

#### 3.1. Use of monoclonal antibodies to target host cell receptors or viral antigens

Recent insight into the molecular interactions of HCV at the cellular membrane has significantly enhanced the understanding of the HCV entry paradigm and revealed potential targets for drug intervention, including the use of monoclonal antibodies (mAbs) to mask HCV entry receptors/coreceptors or viral antigens. As described below and summarized in Table 1, the use of mAbs targeting CD81, SR-BI, CLDN1, or the HCV E2 has been shown to have prophylactic/therapeutic effects against HCV infection in both cell culture and animal models.

## 3.1.1. Anti-CD81 monoclonal antibodies

Nevertheless, these initial interactions have been shown to be mediated via apolipoprotein E (apoE) on the virion [29, 33–36]. The capturing process of the HCV particle is finalized by its interaction with the scavenger receptor class B type I (SR-BI) [37, 38], which is able to associate with the virion's lipoproteins [37, 39] as well as the HCV E2 glycoprotein [40, 41]. Binding of HCV with SR-BI induces lipoprotein rearrangements that help prime the virion for subsequent binding to other host cell factors and promote entry. This process is proposed to occur via SR-BI's lipid transfer activity between the viral particle and the plasma membrane [37, 42] and/or by direct interaction with the hypervariable region 1 (HVR1) domain on E2 [37, 43], which ultimately leads to conformational change and the exposure of functional glycoprotein epitopes for additional receptor binding. Closely following this event is the engagement of the HCV particle with the tetraspanin receptor CD81 [44, 45], which is an important entry factor for the virus [41, 46, 47]. HCV binding to CD81 is proposed to induce a dynamic lateral diffusion of virus-receptor complexes toward the tight junction area for further interactions with additional entry factors and viral internalization [22, 48]. Specifically, CD81 forms a coreceptor complex with the tight junction protein claudin-1 (CLDN1) [49, 50] and is engaged in late events of HCV entry [51]. This re-localization and virusreceptor complex association with CLDN1 involves multiple signaling pathways (e.g., Rho GTPases, PI3K/AKT, and ERK/MAPK) [52, 53], includes the activation of host cell kinases such as the epidermal growth factor receptor (EGFR) and ephrin receptor A2 (EphA2) [54, 55], and is influenced by the absence of the CD81-associated partner EWI-2wint on the hepatocytes [56, 57]. The EWI-2wint molecule is normally bound to CD81 on most cell type surfaces and inhibits its diffusion which is required to promote HCV entry; however, it is not expressed in the hepatocytes, and hence its absence has been suggested to contribute to the restricted tropism of the virus [56]. Following interaction with the CD81/CLDN1 complex, the HCV particle is presumed to then interact with the tight junction protein occludin (OCLN) prior to viral internalization [58]. Additional proteins that take part in influencing virion entry into the hepatocyte include the transferrin receptor 1 (TfR1) [59] and the cholesterol transporter Niemann-Pick C1-like 1 (NPC1L1) [60], although their specific role and interplay with other entry factors in the HCV entry process remain to be defined. The HCV particle finally enters the cell via clathrin-mediated endocytosis [61]. The HCV-receptor complexes then migrate to endosomal compartments [62, 63] where acidification occurs to induce membrane fusion, which allows the release of viral RNA into the host cytosol.

254 Advances in Treatment of Hepatitis C and B

The above sequential and multistep entry process consequently yields the successful release of the HCV genome into the host cytoplasm for direct translation and the ensuing launch of viral replication. The roles played by several of these entry factors including SR-BI, CD81, CLDN1, and OCLN not only mediate HCV entry but also presumably help to define tissue and species tropism of the virus [64–67]. The understanding of how HCV achieves viral entry has led to the possibility of antiviral targeting. From docking to virus internalization, essentially all steps are targetable to prevent HCV infection of the host cell. In addition, given the association of HCV with lipoproteins and the viral particle's interaction with lipoprotein and lipid receptors (LDLR, SR-BI, and NPC1L1), the lipidic nature of HCV virion also offers various methods of pharmacological intervention. Finally, many of the entry factors including CD81, SR-BI, CLDN1, OCLN, and NPC1L1 also play a role in mediating HCV cell-to-cell transmission CD81 is the first putative receptor identified for HCV entry [72, 73] and plays an important role in the virus infection. The molecule is a member of the tetraspanin superfamily with four transmembrane domains and two extracellular loops and is expressed in most human tissues [74]. Commercial CD81 mAb JS-81 has been applied in human liver-chimeric mouse model and shown prophylactic protection but no postexposure effect inhibiting HCV infection [75]; nonetheless, this experimental test inspired subsequent studies of anti-CD81 mAbs as antiviral agents. Of the newly generated antibodies, mAb QV-6A8-F2-C4 produced by genetic immunization could efficiently inhibit HCVcc infection and pan-genotypic HCVpp entry in a similar range as mAb JS-81 [76]. The antibody also appeared to block neutralizing antibody-resistant HCV cell-to-cell transmission and viral dissemination in a dose-dependent manner, with a less cytotoxic or antiproliferative property than JS-81 in vitro. In a recent study, another mAb K04 generated with hybridoma technique not only showed inhibitory effect against HCVpp and HCVcc infection in hepatoma cells and primary human hepatocytes (PHH), but also surprisingly blocked HCV infections in both prophylactic setting and postinfection stage in human liver-chimeric mice [77]. This is probably due to the improved intrinsic binding affinity of mAb K04 to CD81 large extracellular loop (LEL) and a different binding epitope as compared to mAb JS81. However, treatment-associated reductions in body weight and human serum albumin levels were observed in this study. Further research will be needed to determine the minimal dose of antibodies needed to provide protection and to evaluate the toxicology of anti-CD81 mAbs for long-term development.

#### 3.1.2. Anti-SR-BI monoclonal antibodies

SR-BI is a member of the CD36 family primarily expressed in liver and non-placental steroidogenic tissues which facilitates selective cholesterol uptake [78]. The molecule has been proposed to be a horseshoe-like glycoprotein with a large extracellular loop anchored to the plasma membrane at both N- and C-termini with short extensions into the cytoplasm [79]. It was first identified as the alternative E2 receptor on HepG2 cells which efficiently recognize



Table 1. Antiviral strategies to preclude HCV entry.

Candidates Effect(s)

256 Advances in Treatment of Hepatitis C and B

Passive Immunotherapy Against HCV

Small Molecule Inhibitors Heparin, heparin-derived

Hydrolysable tannins CHLA

CLDN1-derived peptide

(CL58)

compounds

& PUG

mAbs Against Host Entry Factors

Stage of

Anti-CD81 mAbs Inhibit CD81-E2 interaction Mouse model [75–77] Anti-SR-BI mAbs Inhibit SR-BI-E2 interaction Mouse model [80–82] Anti-CLDN1 mAbs Inhibit E2-CD81-CLDN1 association Mouse model [84–87]

Anti-E2 mAbs Neutralize circulating virion Phase II [89–96] Polyclonal IgG Neutralize circulating virion Phase III [190, 192,

Heparinases Heparan sulfate enzyme Cell culture [25]

Delphinidin Alter viral shape Cell culture [101] SSb2 Inhibit attachment & viral fusion Cell culture [102] GA Inactivate virion Cell culture [103]

LOD Inactivate virion; inhibit attachment Cell culture [105] DHMD Inactivate virion; inhibit attachment Cell culture [106] Curcumin Decrease viral envelope fluidity; inhibit cell-to-cell spread Cell culture [107] CV-N E1/E2 glycan-binding protein Cell culture [109] Griffithsin E1/E2 glycan-binding protein Mouse model [110] MBL E1/E2 glycan-binding protein Cell culture [111] Recombinant L-ficolin E1/E2 glycan-binding protein Cell culture [112] BA-LNCs E2 glycan-binding protein Cell culture [114] Oleanolic acid E2 glycan-binding protein Cell culture [115] CD81-derived peptides Interact with E2 Cell culture [116, 117]

E2-derived peptide Interfere with E1/E2 hetero-dimerization Cell culture [119] Terfenadine CD81 competitor Cell culture [120] ITX 5061 SR-BI inhibitor Phase Ib [121–124] Aspirin Down regulates CLDN1 Cell culture [125] Erlotinib EGFR inhibitor; inhibit cell-to-cell spread Mouse model [54] Dasatinib EphA2 inhibitor; inhibit cell-to-cell spread Cell culture [54]

EGCG Compete with heparan sulfate; alter viral shape; inhibit cell-to-cell spread

Heparan sulfate competitors Cell culture [24, 25]

Inactivate virion; inhibit attachment & cell-to-cell spread Cell culture [104]

Interact with E1 & E2 Cell culture [81]

Development Reference

193]

Cell culture [99–101]

soluble E2 proteins but do not express CD81 on their surface [40]. As described above, both CD81 and SR-BI are considered necessary for HCV entry, since the overexpression of CD81 on HepG2 cells restores HCVpp entry in these originally poorly permissive cells [41]. Monoclonal antibodies targeting SR-BI that inhibited HCV infections include mAb C167, mAb16-71, mAb8, and mAb151. For HCV inhibitory activities in vitro, mAb C167 effectively prevented infection in hepatoma cells with HCVcc and ex vivo virus recovered from HCVcc-infected chimpanzees [80]; mAb16-71 exhibited preventive effect against HCVcc infection in both hepatoma cells and PHH [81]; mAb8 and mAb151 also prevented HCVcc infection in reporter Huh-7 cells [82]. Additionally, mAb16-71, mAb8, and mAb151 all showed their ability in blocking HCV cell-tocell spread in vitro and in vivo. Human liver-chimeric mouse models challenged with serumderived HCV isolates of different genotypes revealed the anti-HCV property in vivo of the three antibodies in both prophylactic and postexposure settings. Specifically, mAb16-71 showed complete blockage of infection and intrahepatic spread of HCV isolates with a prophylactic treatment, but had no effect on chronically infected chimeric mice; mAb151, on the other hand, appeared to be effective against an HCV variant escaped from adaptive immune response in a liver transplant patient and displayed better antiviral activity in inhibiting viral spread and amplification in the postexposure setup.

#### 3.1.3. Anti-CLDN1 monoclonal antibodies

The CLDN1 tight junction protein has four transmembrane domains and is highly expressed in the liver [83]. Its role in HCV entry is proposed to occur in the post-binding steps [64]. Anti-CLDN1 antibodies directed against the CLDN1 extracellular loops were found effective in neutralizing HCV infection in hepatoma cells through disrupting CD81-CLDN1 association and therefore inhibiting E2 binding to the cell surface [84]. A CLDN1 mAb OM-7D3-B3 targeting CLDN1 extracellular loop was found to be effective in inhibiting HCV isolates in vitro [85]. Further experiments in human liver-chimeric mouse models confirmed its potency in preventing HCV infection and eliminating persistent infection in vivo [86]. Pretreatment of another anti-CLDN1 mAb 3A2 targeting CLDN1 extracellular loop also showed protective effect in a chimeric mouse model [87]. Safety profiles of these antibodies were also assessed regarding the levels of human albumin, aspartate transaminase, alanine transaminase and total bilirubin, and potential side effects on the other organs and tight junction integrity. Further studies were suggested to assess potential immune-mediated adverse effects to ensure its relevance for clinical use [86, 87].

#### 3.1.4. Anti-HCV E2 monoclonal antibodies

Another approach to developing entry-inhibiting mAbs is to target the glycoproteins on the HCV virion surface. Albeit HCV glycoproteins exhibit high variability and are protected by glycosylation and lipids on the viral particle, neutralizing mAbs have been designed to target more conserved and accessible regions, specifically on the E2 glycoprotein [88]. Effects of E2 mAbs have been shown in vitro and in vivo [89–94]. Clinical trials have been carried out to assess the protective function of human anti-E2 mAbs HCV-AbXTL68 and MBL-HCV1 in liver transplant settings of HCV-positive patients. With a higher dose and daily infusion of HCV-AbXTL68, HCV RNA in patient serum showed transient reduction in the first week posttransplantation but not yet below the detectable limits [95]. MBL-HCV1, on the other hand, successfully suppressed the viral load from 7 to 28 days after transplantation in genotype 1ainfected patients with multiple infusions. Although the primary endpoint at day 42 was not met, the viral rebound was significantly delayed, and the magnitude of the viral load reduction was greater than the previous HCV-AbXTL68 therapy [96]. The result indicates that mAbs may be a promising class of entry inhibitors that adsorbs circulating virions to protect the new liver from reinfection after transplantation. A study of combination therapy with DAAs to prevent allograft HCV infection is currently underway [96].

Current obstacles to the development of mAbs as therapeutic antiviral agents include the high cost of production, storage, and administration, which can only be done by injection so far [88]. Nevertheless, the associated immune responses such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) may help to clear the viruses and infected cells [88, 97]. Antibodies that directly block host cell entry factors are more likely to be effective for the diverse circulating viral strains; however, due to the distribution and multiple functions of such molecules, the blockage may cause potential adverse side effects [97]. As for antibodies targeting viral antigens, designing suitable candidates may be a challenging issue due to the heterogeneity of the HCV glycoproteins [98], but such antibodies may provide a safer option for the synergistic therapy with other antivirals of different modes of action to suppress the development of resistance, particularly at the early post-transplantation stage [96]. Additional neutralizing antibodies against other entry factors have also been reported to antagonize HCV infection in vitro, such as anti-TfR1 [59] and anti-NPC1L1 [60] antibodies, suggesting they could also be potentially developed for treatments against hepatitis C.

## 3.2. Small-molecule inhibitors of HCV entry

3.1.3. Anti-CLDN1 monoclonal antibodies

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3.1.4. Anti-HCV E2 monoclonal antibodies

adverse effects to ensure its relevance for clinical use [86, 87].

prevent allograft HCV infection is currently underway [96].

The CLDN1 tight junction protein has four transmembrane domains and is highly expressed in the liver [83]. Its role in HCV entry is proposed to occur in the post-binding steps [64]. Anti-CLDN1 antibodies directed against the CLDN1 extracellular loops were found effective in neutralizing HCV infection in hepatoma cells through disrupting CD81-CLDN1 association and therefore inhibiting E2 binding to the cell surface [84]. A CLDN1 mAb OM-7D3-B3 targeting CLDN1 extracellular loop was found to be effective in inhibiting HCV isolates in vitro [85]. Further experiments in human liver-chimeric mouse models confirmed its potency in preventing HCV infection and eliminating persistent infection in vivo [86]. Pretreatment of another anti-CLDN1 mAb 3A2 targeting CLDN1 extracellular loop also showed protective effect in a chimeric mouse model [87]. Safety profiles of these antibodies were also assessed regarding the levels of human albumin, aspartate transaminase, alanine transaminase and total bilirubin, and potential side effects on the other organs and tight junction integrity. Further studies were suggested to assess potential immune-mediated

Another approach to developing entry-inhibiting mAbs is to target the glycoproteins on the HCV virion surface. Albeit HCV glycoproteins exhibit high variability and are protected by glycosylation and lipids on the viral particle, neutralizing mAbs have been designed to target more conserved and accessible regions, specifically on the E2 glycoprotein [88]. Effects of E2 mAbs have been shown in vitro and in vivo [89–94]. Clinical trials have been carried out to assess the protective function of human anti-E2 mAbs HCV-AbXTL68 and MBL-HCV1 in liver transplant settings of HCV-positive patients. With a higher dose and daily infusion of HCV-AbXTL68, HCV RNA in patient serum showed transient reduction in the first week posttransplantation but not yet below the detectable limits [95]. MBL-HCV1, on the other hand, successfully suppressed the viral load from 7 to 28 days after transplantation in genotype 1ainfected patients with multiple infusions. Although the primary endpoint at day 42 was not met, the viral rebound was significantly delayed, and the magnitude of the viral load reduction was greater than the previous HCV-AbXTL68 therapy [96]. The result indicates that mAbs may be a promising class of entry inhibitors that adsorbs circulating virions to protect the new liver from reinfection after transplantation. A study of combination therapy with DAAs to

Current obstacles to the development of mAbs as therapeutic antiviral agents include the high cost of production, storage, and administration, which can only be done by injection so far [88]. Nevertheless, the associated immune responses such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) may help to clear the viruses and infected cells [88, 97]. Antibodies that directly block host cell entry factors are more likely to be effective for the diverse circulating viral strains; however, due to the distribution and multiple functions of such molecules, the blockage may cause potential adverse side effects [97]. As for antibodies targeting viral antigens, designing suitable candidates may be a challenging issue due to the heterogeneity of the HCV glycoproteins [98], but such

In addition to the mAbs, great efforts have been put into identifying small molecules with potent antiviral effects against HCV entry. The source of such entry inhibitors includes clinically approved medications, synthetic molecules, and natural product-based compounds. These small molecules could be further evaluated for development as drug candidates or drug leads. Below is a panel of small molecules that have been investigated with their activities inhibiting HCV entry (Table 1).

## 3.2.1. Small molecules inhibiting viral attachment

The attachment step represents the primary interaction of an HCV virion with its host cell surface. Since the GAG heparan sulfate moieties dominate the capturing of HCV virions, the heparan sulfate homologue heparin and its derivatives as well as the enzyme heparinases which degrade the molecule were all shown to inhibit the viral binding to hepatoma cells [24, 25]. (-)- Epigallocatechin-3-gallate (EGCG), a green tea catechin, was speculated to exert its inhibitory effect on viral attachment [99] by competing with heparan sulfate for HCV binding [100] or altering the viral shape [101]. Delphinidin, an anthocyanidin extracted from plant pigment, was also demonstrated to inactivate HCVpp by altering its shape and was particularly potent when added concurrently with the viral inoculation [101]. The natural terpenoid saikosaponin b2 (SSb2), isolated from the root of Bupleurum kaoi, was observed to specifically block HCV particle binding and early viral entry without affecting other stages of the viral life cycle [102]. SSb2 could inactivate cell-free HCV particles and was suggested to target the glycoprotein E2 in mediating its antiviral effect against HCV infection. Several other natural compounds including the gallic acid (GA) extracted from Limonium sinense [103], the hydrolyzable tannins chebulagic acid (CHLA) and punicalagin (PUG) [104], and the hepatoprotective plant Phyllanthus urinariaderived monolactone loliolide (LOD) [105] and butenolide (4R,6S)-2-dihydromenisdaurilide (DHMD) [106] were also found to efficiently inactivate cell-free HCV viral particles and impede viral attachment. Another natural compound curcumin extracted from turmeric was shown to decrease the fluidity of viral envelope and therefore prevent the binding and fusion [107], possibly by inserting into the membrane in a manner similar to cholesterol [108].

#### 3.2.2. Small molecules blocking viral glycoproteins

A variety of broad-spectrum antiviral agents have exhibited their ability to interact with the glycans on viral glycoproteins. In the case of HCV, glycan-binding proteins interfere with the association between the E1/E2 heterodimer and the host cell receptor CD81. Lectins such as cyanovirin-N (CV-N) and griffithsin, isolated from cyanobacterium Nostoc ellipsosporum and the red alga Griffithsia sp., respectively, were reported to have such effect. CV-N was shown to interact with N-linked glycans of HCV glycoproteins and disrupt E1/E2 binding to CD81 [109]. The inhibitory effect of griffithsin on HCV entry was also quenched when N-linked highmannose oligosaccharides were present, indicating a pattern similar to CV-N of affecting the glycoproteins-CD81 interaction [110]; pretreatment of griffithsin was shown to delay the viral infection in chimeric mouse model. Humoral lectins of the innate immune systems including the mannan-binding lectin (MBL) and L-ficolin were also considered to have analogous effect neutralizing HCV particles. MBL [111] and recombinant oligomeric L-ficolin [112] were found to interact with the glycans on the E1/E2 heterodimer in a calcium-dependent manner, thereby inhibiting the viral entry. Notably, the MBL-associated complement system was activated upon its binding to HCV E1/E2, suggesting the use of humoral lectins as viral entry inhibitors may also help facilitate viral clearance. However, the detailed mechanism and specific target of the humoral lectins remain to be defined. The boronic acid (BA)-modified nanoparticles were also found to suppress HCV entry in a way that acted similar to lectins [113], with the incorporation of lipid nanocapsule (BA-LNC) techniques enhancing their stability and solubility [114]. Chemically modified oleanolic acid, a triterpene compound originally extracted from Dipsacus asperoides, was found able to interrupt the E2-CD81 interaction by binding to E2 [115].

Besides the glycan-binding proteins, molecules imitating HCV host entry factors or viral glycoproteins were also developed in the attempt to block the viral entry. An imidazolebased scaffold presenting CD81 helix D amino acid side chains [116] and stapled peptides based on CD81 LEL [117] were designed to antagonize the E2-CD81 interaction by mimicking the putative E2-binding region of CD81. A CLDN1-derived peptide, CL58, was also found to inhibit HCV entry in the post-attachment stage by interacting with HCV E1 and E2 [118]. As for viral glycoprotein-based molecules, an E2-derived peptide was found able to block E1/E2-mediated fusion by targeting E1 and therefore interfere with the hetero-dimerization of the glycoproteins [119].

## 3.2.3. Small molecules targeting host entry factors and CD81-triggered signaling pathway

In addition to the therapeutic antibodies mentioned in the previous section, several small molecules have been suggested to exert their inhibitory activity of HCV entry by targeting cellular receptors/coreceptors. Terfenadine, an antihistamine, was found able to prevent HCV infection by competing with the CD81 antibody JS81 binding to the LEL of CD81 protein on the hepatoma cell surface [120]. ITX 5061, a clinical stage compound originally characterized as a p38 MAPK inhibitor, was identified with its capability of antagonizing SR-BI [121] and further validated for its potency of inhibiting HCV entry at post-binding step [122]. The anti-HCV effect of ITX 5061 was found additive to synergistic in combination with several standard-of-care therapeutics, and the resistant mutant was defined on the viral glycoprotein E2 [123]. A latest phase 1b clinical trial [124] revealed that the ITX 5061-treated patients, especially the genotype 1-infected patients, had a significant reduction in HCV RNA through the first week after liver transplantation and viral evolution were restricted; however, the viral RNA levels became comparable in both ITX 5061-treated and untreated patients, suggesting the need to incorporate other antiviral agents using different modes of actions to eliminate HCV infection. Aspirin, alternatively, inhibited HCV entry by downregulating CLDN1 [125].

Since the receptor tyrosine kinases are also involved in the HCV entry process, two clinically approved protein kinase inhibitors were evaluated for their ability to abrogate the viral entry. Both erlotinib, an EGFR inhibitor, and dasatinib, an EphA2 inhibitor, could successfully block HCV entry in a dose-dependent manner as well as the cell-to-cell transmission. Specifically, erlotinib was shown to inhibit the membrane fusion of hepatoma cells overexpressing HCV glycoproteins. In vivo treatment of erlotinib resulted in a significant suppression of the viral load in PHH-chimeric mouse model with HCV infection [54]. Furthermore, inhibitors of EGFR downstream kinases Ras (tipifarnib) and Raf (sorafenib) were also assessed and found effective in blocking HCV entry [126].

Inhibitors of other entry factors were also shown to be effective in hampering the viral entry. Pretreatment of ferristatin, a TfR1 inhibitor that binds to the molecule and causes its internalization and degradation, was shown to decrease HCVcc infection in vitro [59]. The NPC1L1 internalization inhibitor ezetimibe, which is also an FDA-approved cholesterol-lowering medication, diminished HCVcc foci formation before and during the viral challenge. Daily oral administration of ezetimibe starting two weeks before infection also delayed the viral growth of a genotype 1 clinical isolate in PHH-chimeric mouse model [60]. PF-429242, an SKI-1/S1P inhibitor, potentially impeded HCV entry by downregulating NPC1L1 and LDLR expression [127]. On the other hand, phenothiazines, a group of synthesized nitrogen- and sulfur-containing tricyclic compounds, inhibited HCV fusion into the cell by modulating the host cell membrane. Insertion of phenothiazines into the cholesterol-rich membrane increased its fluidity, thus possibly decreasing the local inhomogeneity of the cell required for the viral fusion [128].

## 3.2.4. Inhibition of clathrin-mediated endocytosis and viral fusion

the red alga Griffithsia sp., respectively, were reported to have such effect. CV-N was shown to interact with N-linked glycans of HCV glycoproteins and disrupt E1/E2 binding to CD81 [109]. The inhibitory effect of griffithsin on HCV entry was also quenched when N-linked highmannose oligosaccharides were present, indicating a pattern similar to CV-N of affecting the glycoproteins-CD81 interaction [110]; pretreatment of griffithsin was shown to delay the viral infection in chimeric mouse model. Humoral lectins of the innate immune systems including the mannan-binding lectin (MBL) and L-ficolin were also considered to have analogous effect neutralizing HCV particles. MBL [111] and recombinant oligomeric L-ficolin [112] were found to interact with the glycans on the E1/E2 heterodimer in a calcium-dependent manner, thereby inhibiting the viral entry. Notably, the MBL-associated complement system was activated upon its binding to HCV E1/E2, suggesting the use of humoral lectins as viral entry inhibitors may also help facilitate viral clearance. However, the detailed mechanism and specific target of the humoral lectins remain to be defined. The boronic acid (BA)-modified nanoparticles were also found to suppress HCV entry in a way that acted similar to lectins [113], with the incorporation of lipid nanocapsule (BA-LNC) techniques enhancing their stability and solubility [114]. Chemically modified oleanolic acid, a triterpene compound originally extracted from Dipsacus

asperoides, was found able to interrupt the E2-CD81 interaction by binding to E2 [115].

3.2.3. Small molecules targeting host entry factors and CD81-triggered signaling pathway

In addition to the therapeutic antibodies mentioned in the previous section, several small molecules have been suggested to exert their inhibitory activity of HCV entry by targeting cellular receptors/coreceptors. Terfenadine, an antihistamine, was found able to prevent HCV infection by competing with the CD81 antibody JS81 binding to the LEL of CD81 protein on the hepatoma cell surface [120]. ITX 5061, a clinical stage compound originally characterized as a p38 MAPK inhibitor, was identified with its capability of antagonizing SR-BI [121] and further validated for its potency of inhibiting HCV entry at post-binding step [122]. The anti-HCV effect of ITX 5061 was found additive to synergistic in combination with several standard-of-care therapeutics, and the resistant mutant was defined on the viral glycoprotein E2 [123]. A latest phase 1b clinical trial [124] revealed that the ITX 5061-treated patients, especially the genotype 1-infected patients, had a significant reduction in HCV RNA through the first week after liver transplantation and viral evolution were restricted; however, the viral RNA levels became comparable in both ITX 5061-treated and untreated patients, suggesting the need to incorporate other antiviral agents using different modes of actions to eliminate HCV infection. Aspirin, alternatively, inhibited HCV entry by downregulating CLDN1 [125].

ization of the glycoproteins [119].

260 Advances in Treatment of Hepatitis C and B

Besides the glycan-binding proteins, molecules imitating HCV host entry factors or viral glycoproteins were also developed in the attempt to block the viral entry. An imidazolebased scaffold presenting CD81 helix D amino acid side chains [116] and stapled peptides based on CD81 LEL [117] were designed to antagonize the E2-CD81 interaction by mimicking the putative E2-binding region of CD81. A CLDN1-derived peptide, CL58, was also found to inhibit HCV entry in the post-attachment stage by interacting with HCV E1 and E2 [118]. As for viral glycoprotein-based molecules, an E2-derived peptide was found able to block E1/E2-mediated fusion by targeting E1 and therefore interfere with the hetero-dimer-

Since HCV fusion has been discovered to be facilitated by clathrin-mediated endocytosis and requires an acidic environment, several reagents were assessed for their effectiveness in preventing HCV entry through blocking such pathways. Chlorpromazine, an inhibitor of clathrin-coated pit formation, was shown to inhibit both HCVpp and HCVcc infection in vitro in the validation of clathrin-mediated endocytosis pathway of HCV fusion to the host cell membrane [61]. Arbidol, a broad-spectrum antiviral agent that blocks viral entry and has been licensed in some regions for influenza, was described to trap the HCV virion in clathrin-coated vesicles, thereby hindering the release of viral genome and the following infection [129]. It was also suggested that arbidol could generally cause the intracellular accumulation of clathrincoated structures and restrain the formation of clathrin-coated pits on the cell surface [129], possibly due to its tropism for lipid bilayers.

Small molecules disturbing the acidic endosomal compartments were also identified as HCV entry inhibitors in the discovery of the low pH-triggered entry. These include bafilomycin A1 and concanamycin A, which are inhibitors of vacuolar H+-ATPases [130]. Weak bases such as chloroquine and ammonium chloride were also found to inhibit the low pH-dependent conformational change required for the viral fusion, based on their ability to penetrate lysosomes and increase the pH [131]. Finally, dUY11, one of the rigid amphipathic fusion inhibitors (RAFIs), was suggested to inhibit HCV entry by interacting with the hydrophobic structures in virions and preventing the formation of negative curvature required for viral fusion [132]. Curcumin [107] is also able to affect the fusion step as previously mentioned.

#### 3.2.5. Small molecules inhibiting cell-to-cell transmission

Besides inhibiting the HCV entry in de novo infection, blocking cell-to-cell spread of the viral particles is also important as this mode of transmission facilitates efficient spread of the virus in the liver escaping from neutralizing antibodies [68, 69]. Ferroquine was speculated to interact with HCV glycoprotein E1 and abrogate cell-to-cell spread of the virus [133]. Triazine-based compounds indicated to be closely related to the amino acids on the glycoprotein could also selectively inhibit genotype 1 HCV entry at the post-attachment step along with cellto-cell transmission [134, 135]. Several molecules also block cell-to-cell spread in addition to their activities in hindering HCV viral entry. For instance, besides impeding viral attachment, CHLA and PUG exhibit pronounced antiviral effects at the postinfection stage, especially in restricting HCV foci expansion [104]. Others include EGCG [99], curcumin [107], erlotinib, and dasatinib [54]. Silibinin, the major component of Silybum marianum that has been designated as an orphan drug for the prevention of recurrent hepatitis C in liver transplant patients [136], was also suggested to possess a prominent effect blocking transmission of the viral particles between intercellular junctions [137, 138], although other studies have proposed that it may slow down clathrin-mediated endocytosis [139] as well as inhibit viral membrane fusion [140]. This could be useful since DAA-resistant HCV variants have been suggested to escape via cellto-cell transmission route [141]. Therefore, the choice of inhibitors exhibiting mechanistic effect against both HCV cell-to-cell spread and cell-free entry, or a combination of such two types of inhibitors, should facilitate viral clearance.

## 3.2.6. Additional candidate entry inhibitors

Some other molecules were found able to prevent the infection at different steps of HCV entry. The estrogen receptor modulator tamoxifen [142] and HCV infectivity inhibitor 1 (HCV II-1 or GS-563253) [143] were shown to inhibit the HCV infection at both attachment and post-binding steps. HCV II-1 was also found capable of impeding infectious virion propagation [143]. HCV entry inhibitor 1 (EI-1 or BJ486K), a flavonoid ladanein, was shown to interrupt the viral entry at post-attachment stage [144]. The exact mechanisms of these molecules require further investigations. Other compounds such as serum amyloid A [145, 146], p7 ion channel-derived peptide H2-3 [147], amphipathic DNA polymers [148], lactoferrins [149], tellimagrandin I and its derivatives [150], indole derivatives [151], and imidazo[1,2α][1,8]naphthyridine derivatives [152] were found able to inhibit HCV entry with mechanisms that remain to be clarified.

#### 3.3. Control of HCV infection risks in human blood-derived therapeutic products

Many viruses can contaminate human blood. HCV, along with HIV and HBV are a major cause of infectious complications of blood product transfusion therapy. HCV contamination in patients by transfusion of blood components such as red blood cells, platelets or clinical plasma, as well as industrial fractionated plasma products, has been well documented. At the time of the "tainted blood scandal," numerous recipients of blood components and hemophiliacs receiving plasma-derived factor VIII concentrates were contaminated through transfusion of nonvirally inactivated products prepared from blood products that were not HCV-tested. HCV transmission through blood transfusion is a major medical issue, as infection can lead to high risk of liver cirrhosis and eventually cancer complications.

## 3.3.1. HCV safety nets for blood components

3.2.5. Small molecules inhibiting cell-to-cell transmission

262 Advances in Treatment of Hepatitis C and B

inhibitors, should facilitate viral clearance.

3.2.6. Additional candidate entry inhibitors

Besides inhibiting the HCV entry in de novo infection, blocking cell-to-cell spread of the viral particles is also important as this mode of transmission facilitates efficient spread of the virus in the liver escaping from neutralizing antibodies [68, 69]. Ferroquine was speculated to interact with HCV glycoprotein E1 and abrogate cell-to-cell spread of the virus [133]. Triazine-based compounds indicated to be closely related to the amino acids on the glycoprotein could also selectively inhibit genotype 1 HCV entry at the post-attachment step along with cellto-cell transmission [134, 135]. Several molecules also block cell-to-cell spread in addition to their activities in hindering HCV viral entry. For instance, besides impeding viral attachment, CHLA and PUG exhibit pronounced antiviral effects at the postinfection stage, especially in restricting HCV foci expansion [104]. Others include EGCG [99], curcumin [107], erlotinib, and dasatinib [54]. Silibinin, the major component of Silybum marianum that has been designated as an orphan drug for the prevention of recurrent hepatitis C in liver transplant patients [136], was also suggested to possess a prominent effect blocking transmission of the viral particles between intercellular junctions [137, 138], although other studies have proposed that it may slow down clathrin-mediated endocytosis [139] as well as inhibit viral membrane fusion [140]. This could be useful since DAA-resistant HCV variants have been suggested to escape via cellto-cell transmission route [141]. Therefore, the choice of inhibitors exhibiting mechanistic effect against both HCV cell-to-cell spread and cell-free entry, or a combination of such two types of

Some other molecules were found able to prevent the infection at different steps of HCV entry. The estrogen receptor modulator tamoxifen [142] and HCV infectivity inhibitor 1 (HCV II-1 or GS-563253) [143] were shown to inhibit the HCV infection at both attachment and post-binding steps. HCV II-1 was also found capable of impeding infectious virion propagation [143]. HCV entry inhibitor 1 (EI-1 or BJ486K), a flavonoid ladanein, was shown to interrupt the viral entry at post-attachment stage [144]. The exact mechanisms of these molecules require further investigations. Other compounds such as serum amyloid A [145, 146], p7 ion channel-derived peptide H2-3 [147], amphipathic DNA polymers [148], lactoferrins [149], tellimagrandin I and its derivatives [150], indole derivatives [151], and imidazo[1,2α][1,8]naphthyridine derivatives [152] were found able to inhibit HCV entry with mechanisms that remain to be clarified.

3.3. Control of HCV infection risks in human blood-derived therapeutic products

Many viruses can contaminate human blood. HCV, along with HIV and HBV are a major cause of infectious complications of blood product transfusion therapy. HCV contamination in patients by transfusion of blood components such as red blood cells, platelets or clinical plasma, as well as industrial fractionated plasma products, has been well documented. At the time of the "tainted blood scandal," numerous recipients of blood components and hemophiliacs receiving plasma-derived factor VIII concentrates were contaminated through transfusion of nonvirally inactivated products prepared from blood products that were not HCV-tested. There are now over 100 million whole blood donations collected each year in the world. Collected blood is most often separated by "blood establishments" into red blood cell concentrates, platelet concentrates, and plasma that are transfused at nearby hospitals. Plasma, which can be obtained from whole blood collection or drawn by specialized apheresis procedures, can also be used as raw material for the production of "industrial" plasma protein products. These protein drugs include immunoglobulins G (IgGs), various coagulation factors, albumin, and many others. Industrial plasma products are manufactured from pools of plasma of several thousand liters, making them statistically more susceptible to contamination by HCV and other viruses as one highly infectious donation would contaminate the whole plasma pool and potentially the derived products.

Today in developed economies benefiting from strict regulatory oversight, several measures are in place to decrease the possibility for patients to acquire HCV by transfusion. Blood transfusion HCV safety nets for blood components rely on complementary measures encompassing (a) epidemiological control of the population, (b) individual screening of candidate blood donors to defer those identified as presenting potential risk factors, and (c) individual blood donation testing to identify and eliminate donations reactive to anti-HCV antibodies and/or HCV RNA nucleic acid test (NAT) [153]. In technology-advanced countries applying such procedures, this has allowed to decrease the risk of acquiring HCV by transfusion of single blood components down to approximately 1 per 1.8 million. The remaining risk reflects the inevitable presence of "window-phase" donations for which all markers to detect donor infection by HCV, either indirect or direct, are found nonreactive [154, 155]. Understandably, HCV transmission risks are substantially higher in less developed economies (a) lacking a safe blood donor base, (b) relying on paid or "replacement" donors to increase the blood supply, (c) with a deficient blood collection system, and (d) with a lack of reliable viral testing procedures [6]. The ultimate barrier to avoiding HCV transmission risks from blood products collected during the window-phase period relies on the implementation of dedicated viral reduction treatments. Those have been developed for industrial plasma protein products, plasma for transfusion, and platelet concentrates. Until now, however, no treatment is available commercially for whole blood and red blood cell concentrates.

#### 3.3.2. HCV reduction treatment of industrial plasma protein products

Development and implementation of dedicated viral/HCV reduction treatments of industrial plasma protein products took place in the 1980s and early 1990s [156]. In the early 1980s, albumin, a relatively heat-stable protein, was the only plasma product subjected to specific HCV inactivation by heat treatment at 60 °C for 10 h in the liquid state (a process called pasteurization), in the presence of fatty acid stabilizers. From the mid-1980s to the early 1990s, heat treatment of freeze-dried coagulation factors at 60–68 °C for 24–96 h or 80 °C for 72 h were developed to inactivate HIV and HCV concomitantly [156]. Although pasteurization has successfully been adapted to several plasma products (such as antithrombin and alpha 1 antitrypsin), a milestone in the safety of industrial plasma products was the development of the solvent/detergent (S/D) incubation procedure at 20–37 °C [157] designed to dissolve the lipid envelope of viruses, including HCV, without affecting plasma protein functions. This technique is still largely used for a wide range of industrial plasma products owing to wellproven efficacy and a safety profile established by years of industrial and clinical practices [158]. Other HCV viral inactivation treatments include low pH incubation and caprylic acid precipitation/incubation of immunoglobulin products [159]. An additional milestone to enhance plasma protein product safety is nanofiltration, a procedure of filtration of protein solutions on 15–35 nm nanopore membrane devices designed to entrap and remove viruses [160]. This dedicated virus removal methodology is well established, including for HCV, and is currently applied to most plasma products [156]. Thanks to the implementation of such reduction treatments, most often combined in a complementary manner at different stages of the manufacturing process, no case of HCV transmission by industrial plasma products has been reported since 1993 [154].

#### 3.3.3. HCV reduction treatment applied to plasma and platelet concentrates for transfusion

#### 3.3.3.1. Plasma

Several viral inactivation treatments of clinical plasma are licensed in various countries [161]. The S/D technology was adapted to 100–500 l of pooled industrial plasma in the early 1990s [162] and demonstrated, prior to HCV identification, to efficiently inactivate non-A-non-B hepatitis virus [163]. The removal of the S/D agents is typically achieved by oil extraction and column hydrophobic interaction chromatography [162]. A miniaturized version of the S/D process using a different detergent (Triton X-45 instead of Triton X-100) has been developed allowing its implementation in single-use equipment, thereby facilitating its application in developing countries, such as Egypt, currently lacking industrial capacity [164]. The efficacy of such method to inactivate HCV has been specifically demonstrated using an in vitro culture assay [165].

A procedure consisting in adding methylene blue and illuminating acellular plasma was made available in the early 1990s [166]. The method leads to inactivation of free HCV particles through photochemical alteration of nucleic acids and incapacity of replication [154, 167].

Two other photoinactivation procedures of plasma have been licensed more recently. One combines the addition of psoralen S-59 (amotosalen) with ultraviolet light A illumination [168]. The other is based on the addition of riboflavin followed by UV irradiation [169]. These small molecules can penetrate membranes and intercalate with helical regions of HCV nucleic acids. Subsequent UV illumination irreversibly alters nucleic acids, making HCV particles unable to replicate [154, 170].

#### 3.3.3.2. Platelets

Development of HCV inactivation methods in cellular blood products in general, and platelet concentrates in particular, has been more challenging due to the difficulty to inactivate intracellular viruses without affecting cell function for transfusion. The two photoinactivation methods applied to plasma could nevertheless be adapted to the inactivation of HCV and other viruses in platelet concentrates [170–172].

## 3.3.3.3. Cryoprecipitate

has successfully been adapted to several plasma products (such as antithrombin and alpha 1 antitrypsin), a milestone in the safety of industrial plasma products was the development of the solvent/detergent (S/D) incubation procedure at 20–37 °C [157] designed to dissolve the lipid envelope of viruses, including HCV, without affecting plasma protein functions. This technique is still largely used for a wide range of industrial plasma products owing to wellproven efficacy and a safety profile established by years of industrial and clinical practices [158]. Other HCV viral inactivation treatments include low pH incubation and caprylic acid precipitation/incubation of immunoglobulin products [159]. An additional milestone to enhance plasma protein product safety is nanofiltration, a procedure of filtration of protein solutions on 15–35 nm nanopore membrane devices designed to entrap and remove viruses [160]. This dedicated virus removal methodology is well established, including for HCV, and is currently applied to most plasma products [156]. Thanks to the implementation of such reduction treatments, most often combined in a complementary manner at different stages of the manufacturing process, no case of HCV transmission by industrial plasma products has

3.3.3. HCV reduction treatment applied to plasma and platelet concentrates for transfusion

inactivate HCV has been specifically demonstrated using an in vitro culture assay [165].

A procedure consisting in adding methylene blue and illuminating acellular plasma was made available in the early 1990s [166]. The method leads to inactivation of free HCV particles through photochemical alteration of nucleic acids and incapacity of replication

Two other photoinactivation procedures of plasma have been licensed more recently. One combines the addition of psoralen S-59 (amotosalen) with ultraviolet light A illumination [168]. The other is based on the addition of riboflavin followed by UV irradiation [169]. These small molecules can penetrate membranes and intercalate with helical regions of HCV nucleic acids. Subsequent UV illumination irreversibly alters nucleic acids, making HCV particles unable to

Development of HCV inactivation methods in cellular blood products in general, and platelet concentrates in particular, has been more challenging due to the difficulty to inactivate

Several viral inactivation treatments of clinical plasma are licensed in various countries [161]. The S/D technology was adapted to 100–500 l of pooled industrial plasma in the early 1990s [162] and demonstrated, prior to HCV identification, to efficiently inactivate non-A-non-B hepatitis virus [163]. The removal of the S/D agents is typically achieved by oil extraction and column hydrophobic interaction chromatography [162]. A miniaturized version of the S/D process using a different detergent (Triton X-45 instead of Triton X-100) has been developed allowing its implementation in single-use equipment, thereby facilitating its application in developing countries, such as Egypt, currently lacking industrial capacity [164]. The efficacy of such method to

been reported since 1993 [154].

264 Advances in Treatment of Hepatitis C and B

3.3.3.1. Plasma

[154, 167].

replicate [154, 170].

3.3.3.2. Platelets

Cryoprecipitate, obtained by a freeze-thaw process of plasma, is rich in factor VIII, von Willebrand factor, and fibrinogen. This plasma fraction is still largely used in many developing countries for substitution therapy in hemophilia A, von Willebrand factor disease, or fibrinogen deficiency, respectively. The frequency of treatment of patients with congenital deficiency exposes them to a high risk of infection in countries such as Egypt with a close to 10% HCV incidence [173, 174]. Similar mini-pool methods of HCV inactivation used for clinical plasma are applied to cryoprecipitates [164].

## 3.3.3.4. Red blood cell concentrates and whole blood

No methodology is licensed yet for HCV inactivation in red blood cell concentrates or whole blood. However, the riboflavin/UV pathogen reduction technology is being adapted to the treatment of whole blood [175] and has been shown recently to contribute to lower the risk of malaria transmission in a clinical study in Ghana [176]. It is still uncertain whether a pathogen reduction technology can be developed to substantially inactivate HCV in whole blood or red blood cell concentrates without detrimentally affecting their transfusion quality and functionality or immunogenic potential.

## 3.4. Therapeutic apheresis and passive immunotherapy

Additional methods of precluding HCV infection are to remove circulating virus through therapeutic apheresis or attempting to neutralize HCV infectivity by administering plasmaderived anti-HCV immunoglobulins. These strategies are aimed at reducing the infectious viral load and have been explored in clinical trials.

## 3.4.1. Therapeutic apheresis for the removal of HCV virions

Therapeutic apheresis is the process of transiently circulating the blood outside the body and removing the components causing particular diseases by membrane separation and adsorption separation technologies. In the case of HCV, immunoadsorption apheresis was first applied to treat the chronic hepatitis C-related cryoglobulinemia that causes autoimmune symptoms [177]. The technique of heparin-induced extracorporeal LDL precipitation (HELP) apheresis, which could eliminate apolipoprotein B-containing lipoproteins, was then discovered to reduce HCV viral load [178]; however, the decline was found not correlated with LDL reduction in plasma and appeared to be transient due to the high turnover rate of HCV [179]. Studies using combination therapy of antiviral agents and double-filtration plasmapheresis (DFPP) that selectively removes substances with high molecular weight including HCV particles and therefore, happened to display better effects of suppressing the viral kinetics and therefore have been substantially explored during the past decade. Patients who underwent the prophylactic combination treatment of low-dose IFN, ribavirin, and

DFPP had no evidence of HCV recurrence or fibrosing cholestatic hepatitis exacerbation for more than 1 year after liver transplantation [180]. Combination of DFPP and IFN also achieved impressive SVR in difficult-to-treat patients (i.e., relapsed, nonresponder, or HIVcoinfected patients) [181–184] and may also be safe for the elderly population [185]. However, the approach of apheresis for decreasing HCV viral load requires specialty equipment and possesses potential risk of adverse events (e.g., blood pressure lowering, puncture site hematoma, or infection) [181, 185].

## 3.4.2. Passive immunotherapy using plasma-derived polyclonal HCV immunoglobulins

Passive immunotherapy, also known as antibody therapy, is a very well-established treatment based on the administration of polyclonal hyperimmune immunoglobulins extracted from plasma or mAbs prepared by genetic engineering technologies. One application of passive immunity is to prevent or treat infections due to viruses or to reduce the pathologies associated with bacterial or venom toxins. Human immunoglobulins for passive immunotherapy are fractionated from the plasma of immunized donors having high-titer antibodies against a particular organism or antigen. For the fractionation process, plasma donations from hundreds or thousands of donors are pooled and subjected to various purification and viral inactivation steps, as described in this chapter, to isolate an essentially pure Ig preparation [159, 186]. Current human plasma-derived hyperimmune globulin products are used for the prophylaxis and treatment of viral diseases due to hepatitis B virus (HBV), rabies virus, cytomegalovirus, hepatitis A virus, or respiratory syncytial virus [187]. Human plasma-derived polyclonal hepatitis B immunoglobulin for intravenous use has been made available commercially for over 20 years in some countries. These licensed preparations are efficacious to predictably prevent HBV recurrence after liver transplantation and vertical HBV transmission from mother to child and are used as prophylactic treatment to prevent infection following contact with HBV-contaminated body fluids [188].

The possibility to use polyclonal HCV immunoglobulin to treat or prevent HCV infection has been proposed for many years [189], but no commercial preparation is available yet as it is not proven whether such immunoglobulin can prevent HCV infection or control viremia in infected patients. The rationale in polyclonal HCV immunoglobulins made from large pool of plasma units is to have a preparation that contains neutralizing antibodies to various strains of HCV [189]. However, the presence of neutralizing antibodies has been unclear initially as their presence in plasma was just considered to reflect the occurrence of an infection. Data have suggested that HCV-neutralizing antibodies exist in anti-HCV-positive plasma, but the anti-HCV antibody titer does not correlate with neutralizing capacity [190]. In vitro and animal experiments in a mice model have nevertheless suggested the presence of neutralizing antibodies in polyclonal IgG from a patient with a long-standing HCV infection [191]. A clinical study was initiated in the USA to evaluate the capacity of polyclonal plasma-derived HCV immunoglobulins to "prevent post-transplantation HCV infection of the liver graft and related progression of HCV-related liver disease." This clinical trial was "designed to evaluate a polyclonal human hepatitis C immune globulin given during and post liver transplantation for preventing or reducing the impact of recurrent HCV infection" [192]. However the trial was terminated in 2012 after treatment of seven patients (five receiving the immunoglobulins and two standard-of-care treatment alone) and no data reported. A new trial has begun in 2013 and was recently completed [193]. It is unclear whether plasma-derived polyclonal HCV immunoglobulin will be developed. If this occurs, clear donor screening and donation testing criteria should be defined to determine the specifications of the plasma donations suitable for fractionation, as well as the fractionation methodology itself to exclude any infectious risks from the fractionation of plasma donations. It should be noted that several mAbs for clinical use in HCV-infected patients have been proposed and one has undergone a clinical trial [190]. The future will indicate whether any HCV immunoglobulin, either polyclonal or monoclonal, has a role to play in the control of HCV infection.

## 4. Prospects of targeting HCV entry in clinical setting

Treatment options against hepatitis C have significantly improved owing to recent advances in the development of anti-HCV therapeutics. Nevertheless, there is still much room for improvement due to potential drug resistance and possibility of viral rebound, which usually require long periods of monitoring and analysis to uncover. More importantly, there is currently no immunization or prophylactic treatment against hepatitis C. Introducing novel antivirals with a different mode of action, such as targeting viral entry using mAbs or small molecules, not only helps expand the spectrum of anti-HCV drugs but also in developing novel treatment modalities. Many of the mAbs targeting HCV receptors/coreceptors as well as small-molecule inhibitors of HCV entry impede both viral attachment and cell-to-cell transmission; this is useful in providing protection against de novo infection and at the same time in helping restrict viral spread. The inclusion of viral entry inhibitors to current DAAs has already been shown to produce synergistic treatment effect [9]. Furthermore, taking a multistep targeting approach would help elevate the genetic barrier against selection of resistant variants, thus facilitating viral clearance. Finally, the advantage of developing entry inhibitors is its potential prophylactic application against hepatitis C, which is particularly useful in protecting liver allografts from recurrent HCV infection. Other protective measures of hepatitis C transmission in clinical scenarios include implementation of viral inactivation methods for the removal of HCV infectivity in therapeutic plasma products [165]. In addition, therapeutic apheresis [180] and protective anti-HCV immunoglobulins [192, 193] have also been suggested for prevention of HCV reinfection in liver transplant patients. In the absence of an approved hepatitis C vaccine, these approaches could be explored as preventive and prophylactic measures against HCV infection. With the above-described strategies to preclude HCV entry, it is foreseeable, in a not-too-distant future, that these tactics under development will help provide a better management of chronic and recurrent hepatitis C, particularly in liver transplant setting.

## Acknowledgements

DFPP had no evidence of HCV recurrence or fibrosing cholestatic hepatitis exacerbation for more than 1 year after liver transplantation [180]. Combination of DFPP and IFN also achieved impressive SVR in difficult-to-treat patients (i.e., relapsed, nonresponder, or HIVcoinfected patients) [181–184] and may also be safe for the elderly population [185]. However, the approach of apheresis for decreasing HCV viral load requires specialty equipment and possesses potential risk of adverse events (e.g., blood pressure lowering, puncture site

Passive immunotherapy, also known as antibody therapy, is a very well-established treatment based on the administration of polyclonal hyperimmune immunoglobulins extracted from plasma or mAbs prepared by genetic engineering technologies. One application of passive immunity is to prevent or treat infections due to viruses or to reduce the pathologies associated with bacterial or venom toxins. Human immunoglobulins for passive immunotherapy are fractionated from the plasma of immunized donors having high-titer antibodies against a particular organism or antigen. For the fractionation process, plasma donations from hundreds or thousands of donors are pooled and subjected to various purification and viral inactivation steps, as described in this chapter, to isolate an essentially pure Ig preparation [159, 186]. Current human plasma-derived hyperimmune globulin products are used for the prophylaxis and treatment of viral diseases due to hepatitis B virus (HBV), rabies virus, cytomegalovirus, hepatitis A virus, or respiratory syncytial virus [187]. Human plasma-derived polyclonal hepatitis B immunoglobulin for intravenous use has been made available commercially for over 20 years in some countries. These licensed preparations are efficacious to predictably prevent HBV recurrence after liver transplantation and vertical HBV transmission from mother to child and are used as prophylactic treatment to prevent

The possibility to use polyclonal HCV immunoglobulin to treat or prevent HCV infection has been proposed for many years [189], but no commercial preparation is available yet as it is not proven whether such immunoglobulin can prevent HCV infection or control viremia in infected patients. The rationale in polyclonal HCV immunoglobulins made from large pool of plasma units is to have a preparation that contains neutralizing antibodies to various strains of HCV [189]. However, the presence of neutralizing antibodies has been unclear initially as their presence in plasma was just considered to reflect the occurrence of an infection. Data have suggested that HCV-neutralizing antibodies exist in anti-HCV-positive plasma, but the anti-HCV antibody titer does not correlate with neutralizing capacity [190]. In vitro and animal experiments in a mice model have nevertheless suggested the presence of neutralizing antibodies in polyclonal IgG from a patient with a long-standing HCV infection [191]. A clinical study was initiated in the USA to evaluate the capacity of polyclonal plasma-derived HCV immunoglobulins to "prevent post-transplantation HCV infection of the liver graft and related progression of HCV-related liver disease." This clinical trial was "designed to evaluate a polyclonal human hepatitis C immune globulin given during and post liver transplantation for preventing or reducing the impact of recurrent HCV infection" [192]. However the trial was terminated in 2012 after treatment of seven patients (five receiving the immunoglobulins and

3.4.2. Passive immunotherapy using plasma-derived polyclonal HCV immunoglobulins

infection following contact with HBV-contaminated body fluids [188].

hematoma, or infection) [181, 185].

266 Advances in Treatment of Hepatitis C and B

CHL is a recipient of the 2016 CanHepC (Canadian Network on Hepatitis C) Summer Research Scholarship and the MOST 105 College Research Scholarship (105-2815-C-038-018- B). TB (NSC 102-2320-B-038-041-MY3) and LTL (NSC101-2320-B-038-038-MY2; MOST103- 2320-B-038-031-MY3) are supported by funding from the Ministry of Science and Technology of Taiwan.

## Author details

Thierry Burnouf<sup>1</sup> , Ching-Hsuan Liu<sup>2</sup> and Liang-Tzung Lin2,3\*

\*Address all correspondence to: ltlin@tmu.edu.tw

1 Graduate Institute of Biomedical Materials and Tissue Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei, Taiwan

2 Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan

3 Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan

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Author details

268 Advances in Treatment of Hepatitis C and B

Thierry Burnouf<sup>1</sup>

Taipei, Taiwan

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2 Department of Microbiology and Immunology, School of Medicine, College of Medicine,

3 Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University,

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282 Advances in Treatment of Hepatitis C and B


**Provisional chapter**

## **Hepatitis C Virus Infection Treatment: Recent Advances and New Paradigms in the Treatment Strategies Hepatitis C Virus Infection Treatment: Recent Advances and New Paradigms in the Treatment Strategies**

Imran Shahid, Waleed H. AlMalki,

Mohammed W. AlRabia, Muhammad H. Hafeez and Imran Shahid, Waleed H. AlMalki, Mohammed W. AlRabia, Muhammad H. Hafeez

Muhammad Ahmed and Muhammad Ahmed

[192] ClinicalTrials.gov: A service of the U.S. National Institutes of Health. Randomized Phase II Study of Hepatitis C Immune Globulin Intravenous (Human), Civacir(TM), in Liver Transplantation [Internet]. 2007. updated May 7 2012. Available from: https://

[193] ClinicalTrials.gov: A service of the U.S. National Institutes of Health. CivacirW Polyclonal Immune Globulin (IgG) to Prevent Hepatitis C Virus (HCV) Recurrence in Liver Transplant Patients [Internet]. 2013. updated April 20 2016. Available from: https://

clinicaltrials.gov/ct2/show/study/NCT00473824 [Accessed: 2016-08-15]

284 Advances in Treatment of Hepatitis C and B

www.clinicaltrials.gov/ct2/show/NCT01804829 [Accessed: 2016-08-15]

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65873

#### **Abstract**

The advancement in hepatitis C virus (HCV) therapeutics has been profoundly enhanced by an improved understanding of viral life cycle in host cells, development of novel direct-acting antivirals (DAAs), and exploring other emerging treatment paradigms on the horizon. The approvals of first-, second-, and next-wave direct-acting antivirals highlight the swift pace of progress in the successful development of an expanding variety of therapeutic regimens for use in patients with chronic hepatitis C virus infection. Triple or quadruple therapies based on a combination of different direct-acting antivirals with or without pegylated interferon (IFN) and ribavirin (RBV) have raised the hopes to improve the current treatment strategies for other difficult-to-treat individuals. The development of more efficacious, well-tolerated, and cost-effective interferons with a low frequency of adverse events and short treatment durations is also in the pipeline. An experimental protective vaccine against hepatitis C virus demonstrated promise in preliminary human safety trials, and a larger phase II clinical trials are under consideration to further determine the efficacy of the vaccine. This pragmatic book chapter discusses the current state of knowledge in hepatitis C virus therapeutics and provides a conceptual framework of emerging and investigational treatment strategies directed against this silent epidemic.

**Keywords:** HCV medications, direct-acting antivirals, NS3/4A serine protease inhibitors, NS5A inhibitors, polymerase inhibitors, antiviral resistance, all oral interferon-free antivirals, triple or quadruple therapies, interferon lambda, anti-HCV vaccine model

and reproduction in any medium, provided the original work is properly cited.

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

## **1. Introduction**

Afflicting around 170 million people worldwide, hepatitis C virus (HCV) infection represents a disease of significant global impact. The regional prevalence for HCV varies substantially around the world, where the infection presents the state of universal coverage in East and South Asia (e.g., in Egypt, the prevalence is as high as 22 %) to no access at all to others (i.e., some North American and European countries) [1, 2]. The new incidents of chronic HCV infection are increasing 3–4 million every year without previous ascertainment of HCV risk, and it seems tough to determine because many acute HCV cases are not noticed clinically [2, 3]. Analogously, acute HCV infection, a multifaceted disease is often asymptomatic or sometimes linked with nonspecific symptoms that lead to chronic hepatitis C in 80 % of infected individuals [4]. Chronic HCV-infected patients may at high risk of developing HCV-associated liver diseases (fibrosis, cirrhosis, and hepatocellular carcinoma) if not treated timely, and infection persists for an extended time [5]. The morbidity and the mortality rate is rising unexpectedly in developing world and even in resource-replete countries (e.g., the United States and the United Kingdom), where more patients are now dying from HCV and associated hepatic diseases (e.g., hepatocellular carcinoma) than HIV [6].

Pegylated interferon alpha (PEG-IFNα) and weight-based nucleoside analog ribavirin (RBV) were recommended as "gold standard of care" more than a decade and still considered an integral part of some newly developed anti-HCV direct-acting antiviral therapeutic regimens [2]. The therapy is used in combination to attain a sustained virologic response rate (SVR; HCV RNA undetectable after 6-month treatment completion) in acute and chronic HCV-infected individuals [2, 7]. The SVR rates achieve up to 80 % in HCV genotype 3-infected patients and not more than 50–60 % of genotype 1- and 4-infected patients [2, 7]. At present, less than 10 % of patients with chronic HCV have been treated successfully because of the failure of risk-based screening to identify all infected patients and the low efficacy and high rate of side effects from regimens based on IFN and RBV [2, 8]. By this token then, PEG-IFN/RBV has proven an ineffective means of managing the HCV infection burden. It is highly significant then that we stand today, at the cusp of a pharmacological revolution.

The current therapeutic approaches in the pipeline to coup HCV infection are the development of novel direct-acting antivirals (DAAs), which directly target viral genome via covalent or non-covalent interactions and disrupt HCV replication and translation [9]. The most widely studied direct-acting antivirals are protease inhibitors (PIs), NS5A inhibitors, and polymerase inhibitors which inhibit HCV translation and replication, respectively, by achieving higher sustained virologic response rates (SVR) with or without PEG-IFNα/RBV in treated patients (**Table 1**) [10]. Two first-generation protease inhibitors (i.e., telaprevir (TLV) and boceprevir (BOC)) were approved by the United States (US) Food and Drug Administration (FDA) in 2011 to treat chronic HCV genotype 1 infection [11]. Simeprevir classified as second-generation protease inhibitor got approval in 2013 to treat chronic HCV genotype 1 populations, and sofosbuvir (SOF) (a viral RNA-dependent RNA polymerase inhibitor) was also recommended in the same year to treat chronic HCV genotype 1-, 2-, 3 and 4-infected patients [11]. These innovative treatment regimens have revolutionized the field of HCV medicine and **1. Introduction**

286 Advances in Treatment of Hepatitis C and B

Afflicting around 170 million people worldwide, hepatitis C virus (HCV) infection represents a disease of significant global impact. The regional prevalence for HCV varies substantially around the world, where the infection presents the state of universal coverage in East and South Asia (e.g., in Egypt, the prevalence is as high as 22 %) to no access at all to others (i.e., some North American and European countries) [1, 2]. The new incidents of chronic HCV infection are increasing 3–4 million every year without previous ascertainment of HCV risk, and it seems tough to determine because many acute HCV cases are not noticed clinically [2, 3]. Analogously, acute HCV infection, a multifaceted disease is often asymptomatic or sometimes linked with nonspecific symptoms that lead to chronic hepatitis C in 80 % of infected individuals [4]. Chronic HCV-infected patients may at high risk of developing HCV-associated liver diseases (fibrosis, cirrhosis, and hepatocellular carcinoma) if not treated timely, and infection persists for an extended time [5]. The morbidity and the mortality rate is rising unexpectedly in developing world and even in resource-replete countries (e.g., the United States and the United Kingdom), where more patients are now dying from HCV and

Pegylated interferon alpha (PEG-IFNα) and weight-based nucleoside analog ribavirin (RBV) were recommended as "gold standard of care" more than a decade and still considered an integral part of some newly developed anti-HCV direct-acting antiviral therapeutic regimens [2]. The therapy is used in combination to attain a sustained virologic response rate (SVR; HCV RNA undetectable after 6-month treatment completion) in acute and chronic HCV-infected individuals [2, 7]. The SVR rates achieve up to 80 % in HCV genotype 3-infected patients and not more than 50–60 % of genotype 1- and 4-infected patients [2, 7]. At present, less than 10 % of patients with chronic HCV have been treated successfully because of the failure of risk-based screening to identify all infected patients and the low efficacy and high rate of side effects from regimens based on IFN and RBV [2, 8]. By this token then, PEG-IFN/RBV has proven an ineffective means of managing the HCV infection burden. It is highly significant then that we

The current therapeutic approaches in the pipeline to coup HCV infection are the development of novel direct-acting antivirals (DAAs), which directly target viral genome via covalent or non-covalent interactions and disrupt HCV replication and translation [9]. The most widely studied direct-acting antivirals are protease inhibitors (PIs), NS5A inhibitors, and polymerase inhibitors which inhibit HCV translation and replication, respectively, by achieving higher sustained virologic response rates (SVR) with or without PEG-IFNα/RBV in treated patients (**Table 1**) [10]. Two first-generation protease inhibitors (i.e., telaprevir (TLV) and boceprevir (BOC)) were approved by the United States (US) Food and Drug Administration (FDA) in 2011 to treat chronic HCV genotype 1 infection [11]. Simeprevir classified as second-generation protease inhibitor got approval in 2013 to treat chronic HCV genotype 1 populations, and sofosbuvir (SOF) (a viral RNA-dependent RNA polymerase inhibitor) was also recommended in the same year to treat chronic HCV genotype 1-, 2-, 3 and 4-infected patients [11]. These innovative treatment regimens have revolutionized the field of HCV medicine and

associated hepatic diseases (e.g., hepatocellular carcinoma) than HIV [6].

stand today, at the cusp of a pharmacological revolution.



<sup>a</sup> Drug efficacy profile was based on the overall SVR rates achieved in phase II/III clinical trials where SVRs > 95 % = high profile, SVRs > 90 % = average profile, and SVRs > 85–90 % = low profile.

<sup>b</sup> Drug-resistance barrier profile is based upon the clinical data registered to clinicaltrials.gov.

<sup>c</sup> Pan-genotypic coverage was based on the fact that the DAA combination was therapeutically effective against 1–6 genotypes = high profile, two/three genotypes = average profile, and one genotype > = low profile.

<sup>d</sup> Adverse event (AE) profile was accomplished on the basis of percentage occurrence of adverse effects in phase II/III clinical trials which caused treatment discontinuation in treated individuals, where 10 % AEs > high profile, 10→5 % AEs > average profile, and 5→0 % AEs > low profile.

<sup>e</sup> Drug-drug interaction profile was established on the basis of the DAA ability to induce/inhibit hepatic cytochrome P450 system, P-glycoprotein (P-gp), and organic anion-transporting polypeptide (OATP) induction/inhibition. CYP 450, P-gp, and OATP induction/inhibition = high profile, P-gp and OATP induction/inhibition = average profile, and one or none of these CYP 450 or P-gp or OATP inductions/inhibitions = low profile. High profile = +++, average profile = ++, and low profile = +

**Table 1.** The most promising direct-acting antivirals against HCV with their therapeutic activity profile, current stage of development, and targeted active sites.

provided optimism that cure rates in chronically infected HCV patients have much improved with these new drugs.

From 2015, HCV therapy has achieved higher response rates, fewer contraindications, shorter durations, and greater tolerability after the approval of interferon-free antiviral therapies. All oral interferon-free therapeutic regimens directed against hepatitis C virus are shown to be highly effective in the entire spectrum of patient populations, including the previously difficult-to-treat "special" situations (e.g., HCV subtype 1a patients with resistance-associated amino acid variants (RAVs), partial or null responders to first-generation protease and PEG-IFN/RBV-based triple therapies, decompensated cirrhosis, IL28 polymorphism, chronic kidney diseases, and HCV/HIV-coinfected patients). These revolutionary drug strategies now incorporate a cocktail of agents blended to take advantage of the synergistic mechanism of action. With these patient-friendlier attributes, the demand for treatment will conceivably reach unprecedented heights, but will health services be able to match this demand with supply? HCV antiviral therapy is not cheap; the current going rate, which new therapies are likely to exceed, stands at approximately US \$ 80,000–100,000 per treatment course. So with more than 170 million people living with chronic infection around the world, clearly we cannot afford at least immediately to treat everyone.

On the other hand, treatment-emergent adverse events (e.g., risk of developing hepatocellular carcinoma and adverse cardiovascular effects) in treated individuals are also posing some serious challenges to the newly developed DAAs. The developers of prophylactic or protective vaccines have faced the most difficult challenges of rapid mutation rate (10−5 to 10−4 nucleotides per HCV replication cycle) and remarkable genetic heterogeneity of the virus in experimental trials [12,13]. It is beyond the scope of this article to cover every anti-HCV drug studies in details, so we primarily focus on FDA-approved direct-acting antivirals whose clinical efficacies have proven against chronic HCV both in vitro and in vivo (**Table 1**). We also highlight some interferon derivatives and investigational HCV vaccine models which mark the recent trends and new paradigms in the treatment strategies against HCV (**Table 1**).

## **2. Potential active sites for anti-HCV agents**

provided optimism that cure rates in chronically infected HCV patients have much improved

**Table 1.** The most promising direct-acting antivirals against HCV with their therapeutic activity profile, current stage of

<sup>a</sup> Drug efficacy profile was based on the overall SVR rates achieved in phase II/III clinical trials where SVRs > 95 % = high

<sup>c</sup> Pan-genotypic coverage was based on the fact that the DAA combination was therapeutically effective against 1–6

<sup>d</sup> Adverse event (AE) profile was accomplished on the basis of percentage occurrence of adverse effects in phase II/III clinical trials which caused treatment discontinuation in treated individuals, where 10 % AEs > high profile, 10→5 %

<sup>e</sup> Drug-drug interaction profile was established on the basis of the DAA ability to induce/inhibit hepatic cytochrome P450 system, P-glycoprotein (P-gp), and organic anion-transporting polypeptide (OATP) induction/inhibition. CYP 450, P-gp, and OATP induction/inhibition = high profile, P-gp and OATP induction/inhibition = average profile, and

From 2015, HCV therapy has achieved higher response rates, fewer contraindications, shorter durations, and greater tolerability after the approval of interferon-free antiviral therapies. All oral interferon-free therapeutic regimens directed against hepatitis C virus are shown to be highly effective in the entire spectrum of patient populations, including the previously

with these new drugs.

development, and targeted active sites.

**Drug name Drug** 

**(4) Interferon derivatives**

Consensus interferon

Interferon lambda

**(5) HCV vaccines**

**efficacy<sup>a</sup>**

288 Advances in Treatment of Hepatitis C and B

**(3.2) Non-nucleoside analog inhibitors (NNIs)**

**Drug-resistance barrierb**

**Pan-genotype coveragec**

Tegobuvir ++ + ++ ++ + Phase II

Setrobuvir ++ +++ + + + Phase II

Filibuvir ++ ++ + + + Phase II

BMS-791325 ++ ++ + + + Phase II

ChronVac-C − − − − − Phase I/II

GI-5005 − − − − − Phase II

ChAd3/MVA − − − − − Phase I/II

<sup>b</sup> Drug-resistance barrier profile is based upon the clinical data registered to clinicaltrials.gov.

one or none of these CYP 450 or P-gp or OATP inductions/inhibitions = low profile.

genotypes = high profile, two/three genotypes = average profile, and one genotype > = low profile.

profile, SVRs > 90 % = average profile, and SVRs > 85–90 % = low profile.

AEs > average profile, and 5→0 % AEs > low profile.

High profile = +++, average profile = ++, and low profile = +

TG4040 − − − − − Phase I clinical

+++ − − + + Phase II

**Adverse effects<sup>d</sup>**

+ - ++ +++ − Approved Type 1 interferon

**Drug-drug interactionse** **Development phase**

clinical trials

clinical trials

clinical trials

clinical trials

clinical trials

clinical trials

clinical trials

clinical trials

trials

−

−

−

−

**Target site**

NS5B/NNI site 1/ thumb 1

NS5B/NNI site 1/ thumb 1

NS5B/NNI site 2/ thumb 2

NS5B/NNI site 4/ palm 1

Type 1 interferon receptors

> HCV replication and translation into the cytoplasm of host cells facilitate the direct exposure of direct-acting antivirals to their targeted active sites [14]. Moreover, the direct-acting antivirals are structure specific to their targeted active sites (**Figure 1**) [15]. Viral-encoded proteases (e.g., NS3/4A serine protease), HCV replication complex (NS5A), and viral RNA-dependent RNA polymerase (RdRp; NS5B) enzyme are the core targeted sites for the first-, second-, and next-wave direct-acting antivirals (**Figure 1**) [16]. Viral attachment and entry into the host cell, viral assembly, packaging, and virion release are the less specific anti-HCV drug targets but still important to develop novel anti-HCV compounds with promising therapeutic activity (**Figure 1**) [17]. NS3/4A inhibitors inhibit viral translation by disrupting the downstream polyprotein processing of HCV genome. NS5B inhibitors obstruct HCV replication by blocking the addition of further nucleotide in growing mRNA chain (**Figure 1**) [15]. NS5A inhibitors prevent the formation of a membranous web structure which is crucial for HCV replication [16]. Viral assembly and packaging are disturbed by host-targeting agents which target a host-encoded enzyme responsible for viral assembly [18]. Cyclophilin (nonimmune suppressive cyclosporins) inhibitors block the interaction of cyclophilin (a family of a highly conserved cellular peptidylprolyl isomerase involved in protein folding and trafficking) with other HCV proteins to prevent the formation of a functional replication complex (**Figure 1**) [17]. α-Glucosidase inhibitors interrupt the release of newly formed viral particles from the host cells [17]. Some immunoglobulins (i.e., monoclonal and polyclonal antibodies) prevent the viral attachment and entry into host cells, but their therapeutic benefits are not highly significant [11].

**Figure 1.** Potential active sites for direct-acting antivirals against hepatitis C virus. NS3/4A serine protease, NS5A replication complex, and RNA-dependent RNA polymerase are the key targeted sites for anti-HCV drug development. Some other anti-HCV drug targets have also been demonstrated by the investigators including viral attachment and entry into host cells, host-targeting agents, and α-glucosidase. However, these drug targets are less specific but still significant to develop novel anti-HCV agents. Direct-acting antivirals according to their target specificity are also enlisted in and rectangular square boxes in the figure. NIs, nucleoside analog inhibitors; NNIs, non-nucleoside analog inhibitors; CLDN1, claudin 1; OCLN, occludin.

## **3. NS3-4A serine protease inhibitors**

## **3.1. First-generation protease inhibitors**

Telaprevir and boceprevir represent the first in class of direct-acting antivirals or more correctly the first-generation protease inhibitors which were approved by the US FDA in 2011 for the treatment of genotype 1 hepatitis C infection. Telaprevir (TLV, Incivek®) is an orally bioavailable, a peptidomimetic NS3-4A protease inhibitor, which forms a reversible covalent bond with NS3/4A serine protease and impedes downstream HCV polyprotein processing [19]. The therapy was recommended along with PEG-IFN α and RBV for treatment-naïve genotype 1 adult patients with associated liver diseases, in null responders (i.e., HCV RNA decline <2 log10 at week 12 during PEG-IFN α plus RBV therapy), in poor or partial responders (HCV RNA decline ≥2 log10 at week 12 but positive at week 24 during PEG-IFN α plus RBV therapy), and in the relapsers (HCV RNA negative at the end of treatment but recurrence of HCV RNA during the follow-up of 6 months) of PEG-IFN α plus RBV dual therapy [20]. The therapeutic efficacy was evaluated in a series of multicenter phase II and III clinical trials named as PROVE 1, PROVE 2, PROVE 3, ADVANCE, ILLUMINATE, and REALIZE (**Table 2**) [20]. The overall SVR rates were achieved from 60 to 90 % in treated patients. However, TLV monotherapy and suboptimal doses resulted in


**3. NS3-4A serine protease inhibitors**

Telaprevir and boceprevir represent the first in class of direct-acting antivirals or more correctly the first-generation protease inhibitors which were approved by the US FDA in 2011 for the treatment of genotype 1 hepatitis C infection. Telaprevir (TLV, Incivek®) is an orally bioavailable, a peptidomimetic NS3-4A protease inhibitor, which forms a reversible covalent bond with NS3/4A serine protease and impedes downstream HCV polyprotein processing [19]. The therapy was recommended along with PEG-IFN α and RBV for treatment-naïve genotype 1 adult patients with associated liver diseases, in null responders (i.e., HCV RNA decline <2 log10 at week 12 during PEG-IFN α plus RBV therapy), in poor or partial responders (HCV RNA decline ≥2 log10 at week 12 but positive at week 24 during PEG-IFN α plus RBV therapy), and in the relapsers (HCV RNA negative at the end of treatment but recurrence of HCV RNA during the follow-up of 6 months) of PEG-IFN α plus RBV dual therapy [20]. The therapeutic efficacy was evaluated in a series of multicenter phase II and III clinical trials named as PROVE 1, PROVE 2, PROVE 3, ADVANCE, ILLUMINATE, and REALIZE (**Table 2**) [20]. The overall SVR rates were achieved from 60 to 90 % in treated patients. However, TLV monotherapy and suboptimal doses resulted in

**Figure 1.** Potential active sites for direct-acting antivirals against hepatitis C virus. NS3/4A serine protease, NS5A replication complex, and RNA-dependent RNA polymerase are the key targeted sites for anti-HCV drug development. Some other anti-HCV drug targets have also been demonstrated by the investigators including viral attachment and entry into host cells, host-targeting agents, and α-glucosidase. However, these drug targets are less specific but still significant to develop novel anti-HCV agents. Direct-acting antivirals according to their target specificity are also enlisted in and rectangular square boxes in the figure. NIs, nucleoside analog inhibitors; NNIs, non-nucleoside analog

**3.1. First-generation protease inhibitors**

inhibitors; CLDN1, claudin 1; OCLN, occludin.

290 Advances in Treatment of Hepatitis C and B


Poordad *et al.* [29]

UNITY-1

−

+

+

1

NCT01979939

GT1 T.N = 92 %


**Referenced clinical studies**

**(2) NS5A inhibitors**

**Daclatasvir**

Hezode *et al.* [24] Hezode *et al.* [24] **Daclatasvir and sofosbuvir combination**

Poordad *et al.* [25] Sulkowski *et al.,* [110]

Wyles *et al.* [26] Nelson *et al.* [27]

Leroy *et al.* [28]

ALLY-3+ **Daclatasvir asunaprevir and beclabuvir combination**

UNITY-1

−

+

+

1

NCT01979939

GT1 T.N = 92 %

Poordad *et al.* [29]

±RBV

+

+

3

−

ALLY-3

−

+

+

3

NCT02032901

GT3 T. N = 90 %

GT3 T. E = 86 % GT3 T. N (12 weeks) = 88 %

GT3 T. E (16 weeks) = 92 %

ALLY-2

−

+

+

1/4

NCT02032888

GT1/4 T.N (12 weeks) = 97 %

GT1/4 T.N (8 weeks) = 76 %

GT1/4 T.E (12 weeks) = 98 %

A1444040

±RBV

+

+

1/3

NCT01359644

GT3 T.N = 89 % GT2 T.N = 92 % GT1 T. N = 98 %

GT1T. E = 98 %

ALLY-1

±RBV

+

+

1/3

NCT02032875

GT1 (cirrhotics)

 = 83 %

GT1(posttransplant)

GT3 = 88%

 = 95 %

COMMAND-4

+

+

−

4

NCT01448044

COMMAND-4

 = 82–86 %

Phase III COMMAND-1

+

+

−

1/4

NCT01125189

GT1 (20 mg) = 65 %

GT1 (60 mg) = 90 %

GT4 (20 mg) = 75 %

GT4 (60 mg) = 100 %

+

+

+

1-6

−

Overall SVRs =

59–100 %

292 Advances in Treatment of Hepatitis C and B

**Trials name/phase**

**±PEG-IFN/RBV**

**Treatmentnaïve patient**

**Treatment- experienced**

**Genotype coverage**

**NCT#**

**Overall SVR**


**Table 2.** The most promising phase II and III clinical trials for novel direct-acting antivirals with overall SVRs in HCV-infected patientsa. the rapid emergence of viral escape mutants followed by viral breakthrough (HCV RNA remains lower limit of quantification but increased to ≥100 IU/ml or ≥1 log10 during telaprevir therapy) in some patients during therapy and significantly in all the patients after treatment completion [48]. The addition of PEG-IFN α plus RBV reduced the frequency of viral escape mutants and achieved higher SVR rates in both treatment-naive and treatmentexperienced patients in phase II and III clinical trials than PEG-IFN α plus RBV therapy alone [49]. Similarly, some studies also describe that telaprevir is not equally useful for difficult-to-treat populations (i.e., patients with decompensated liver diseases, IL28B polymorphism, HCV genotype 3, 4, 5, and 6 infections) as compared with HCV genotype 1 infection [48]. For such HCV-infected patients, interferon-free antiviral combination therapies are the best treatment options. Boceprevir (BOC; Victrelis®) another first-generation protease inhibitor shares the similar pharmacokinetics and pharmacodynamics like telaprevir to confer anti-HCV activity. The clinical efficacy of boceprevir-based triple therapy (along with PEG-IFN α plus RBV) was evaluated in phase III clinical trials both in treatment-naïve (SPRINT-2 clinical trial) and poor responders to PEG-IFN α and RBV therapy (RESPOND-2 clinical trial) patients (**Table 2**) [50–52]. The overall cure rates were almost equivalent to telaprevir-based triple therapy. However, the treatment-naïve patients achieved higher SVR rates than treatment-experienced patients (**Table 2**). Telaprevir or boceprevir monotherapy seems effective in treatment-naïve HCV genotype 1-infected patients. However, administration of telaprevir or boceprevir as monotherapy in infected individuals is not a suitable option because of the early emergence of viral escape mutants during therapy and followed by viral breakthrough after treatment completion [53]. The minor resistant populations exist at baseline in all HCV-infected individuals and are selected rapidly with telaprevir or boceprevir monotherapy (**Table 3**) [51]. Consequently, the first-generation DAAs/PIs still require a platform of PEG-IFN α and RBV to prevent the emergence of viral escape mutants and also to achieve significantly higher SVR rates [54]. Similarly, notable drug-drug interactions with many HIV antiretrovirals and calcineurin inhibitors also decrease the therapeutic activity of telaprevir and boceprevir monotherapy [54]. Drug toxicities and unusual adverse event profile (anemia, rashes, dysgeusia, and depression) of first-generation protease inhibitors also limit their clinical efficacy in treated patients [54]. These adverse effects result in treatment withdrawal in the majority of treated individuals, and this ratio is 10 % much higher in telaprevir- or boceprevir-based triple therapy vs. PEG-IFNα and RBV therapy alone [50, 52]. The physicians and hepatologists do not recommend telaprevir or boceprevir monotherapy or even in combination with PEG-IFN/RBV due to the harsh adverse event profile and the emergence of viral escape mutants. Similarly, premature treatment discontinuation and mandatory intake of food have heightened the adherence concern related to first-generation protease inhibitors. The treatment has already discontinued in the United States and expected to be stopped from other parts of the world after the approval of interferon-free therapeutic regimens.

#### **3.2. Second-generation protease inhibitors**

**Referenced clinical studies**

Rockstroh *et al.* [38]

Kwo *et al.* [39]

Zeuzem *et al.* [40]

Roth *et al.* [41]

Feld *et al.* [42]

Zeuzem *et al.* [43] Sulkowski *et al.* [44]

Poordad *et al.* [45] Andreone *et al.* [46]

Ferenci *et al.* [47] Ferenci *et al.* [47]

C-EDGE T. N

C-SURFER **Paritaprevir-ombitasvir-ritonavir and dasabuvir combination**

SAPPHIRE I SAPPHIRE II TURQUOISE I

TURQUOISE II

PEARL II PEARL III PEARL IV **Paritaprevir-ombitasvir-and ritonavir combination**

Hezode *et al.* [24] Asselah *et al.,* [129]

Waked *et al.,* [128]

clinicaltrials.gov websites. +

SVR =

**Table 2.**

 = sustained virologic response

included, - =

not included, ±

 =

with or without, T.N

The most promising phase II and III clinical trials for novel direct-acting antivirals with overall SVRs in HCV-infected patientsa

 =

treatment-naïve, T.E

 =

treatment-experienced, RBV

 =

.

ribavirin, GT =

genotype,

PEARL-I AGATE-I AGATE-II

+RBV

+

+ a Only those clinical studies/trials were referenced which were considered by the US FDA for the approval of anti-HCV compounds and which were registered to

clinicaltrials.gov. Similarly, the clinical trial data regarding the inclusion of patients, genotype coverage, and SVR rates were extracted only from FDA, NCBI, and

4

NCT02247401

Overall SVR = 93–97 %

+RBV

+

+

4

NCT02265237

Overall SVR = 97–98 %

±RBV

+

+

4

NCT01685203

Overall SVR =

91–100 %

±RBV

+

−

1a

NCT01833533

Overall SVR = 90–97 %

±RBV

+

−

1b

NCT01767116

Overall SVR =

99–99.5 %

±RBV

−

+

1b

NCT01674725

Overall SVR =

96–100 %

+RBV

+

+

1

NCT01939197

Overall SVR = 92–96 %

+RBV

+

+

1/HIV

NCT01704755

Overall SVR = 91–94 %

+RBV

−

+

1

NCT01715415

Overall SVR = 96 %

+RBV

+

−

1

NCT01716585

Overall SVR = 96 %

−

+

−

1

NCT02092350

C-SURFER = 99 %

−

+

−

1/4/6

NCT02105467

C-EDGE T. N = 95 %

C-EDGE

−

+

−

1/4/6

NCT02105662

Overall SVR = 96 %

coinfection

C-EDGE T. E

±RBV

−

+

1/4/6

NCT02105701

C-EDGE +

C-EDGE-RBV(12)

C-EDGE + C-EDGE-RBV(16)

 = 92 %

RBV(16) = 97 %

 = 92 %

RBV(12) = 94 %

294 Advances in Treatment of Hepatitis C and B

**Trials name/phase**

**±PEG-IFN/RBV**

**Treatmentnaïve patient**

**Treatment- experienced**

**Genotype coverage**

**NCT#**

**Overall SVR**

Telaprevir and boceprevir were found more effective in treatment-naive HCV genotype 1 patients and had to be administered three times daily [54]. The situation demanded to


**Table 3.** Resistance amino acid variants (RAVs) associated with antiviral drug resistance to direct-acting antivirals.

develop the regimens, which must be effective against other difficult-to-treat HCV populations with an improved dosing schedule. Simeprevir (Olysio®) was approved by the US FDA to treat chronic HCV genotype 1 infection in 2013 (www.hcvonline.org/page/treatment/drugs/simeprevir-drug) [55, 56]. The drug was approved along with PEG-IFN α-2a or 2b and RBV for treatment-naive HCV genotype 1-infected patients [35], for patients with compensated liver diseases (fibrosis and cirrhosis), and for those who are relapsers and nonresponders to interferon-based therapy [32,57]. Simeprevir 150 mg once daily along with PEG-IFN/RBV achieved overall SVR rates approximately 80–90 % in treatment-naïve and relapsers [56] and 67–80 % of previously treated and null responders to interferonbased therapy (**Table 2**) (QUEST-1 and QUEST-2 clinical trials). The therapy seems to be more efficient than telaprevir/boceprevir-based triple therapies; however, there are certain disadvantages. For example, SVR rates were lower at the start of antiviral therapy in patients with advanced hepatic fibrosis as well as in patients with Q80R/K polymorphism of NS3 protein [58]. It is a naturally occurring amino acid substitution at NS3 codon 80, where glutamine is replaced to lysine. The Q80K polymorphism exists naturally in HCV genotype 1a (30–50 %)- and 1b (0.5 %)-infected patients [56]. It substantially reduces the efficacy of simeprevir-based triple therapy in HCV genotype 1a-infected patients. In QUEST-1 clinical studies, SVR rates were demonstrated 20 % lower in HCV genotype 1a-infected individuals than 1b [21, 56]. Furthermore, approximately one-third HCV genotype 1a patients were found with Q80K NS3 protein mutation at baseline [21]. Interestingly, the mutation was infrequent, was nonexistent, and did not impact the SVR rates significantly in HCV genotype 1b patients [56, 59]. HCV genotype 1a-infected patients are strongly recommended for Q80K polymorphism screening and NS3 genotype test before the start of treatment and to avoid any complications during therapy. The mutations associated with NS3 protease inhibitors may detect by reverse transcribing HCV RNA, followed by PCR amplification and then DNA sequencing of NS3 gene [21, 60]. Some institute/laboratories in the United States have launched the tests for Q80K polymorphism (Quest Diagnostics®) and HCV drug-resistance test for NS3/4A protease inhibitors (LabCorp®). Treatment withdrawal and alternative treatment strategies may adopt for the patients found with a Q80K polymorphism in NS3 genotype analysis. Skin rashes, itching, nausea, and photosensitivity reactions are the most commonly observed adverse events with simeprevir-based triple therapies [56]. Sunscreen creams and lotions are highly recommended to use while taking the drug [61].

In 2014, simeprevir was approved in combination with sofosbuvir to treat chronic HCV genotype 1 patients with or without cirrhosis. The combination regimen (150/400 mg q.d.) without PEG-IFN/RBV was found almost equally effective in both patient arms; however, relatively better in treatment-naïve than treatment-experienced patients (OPTIMIST-1 and OPTIMIST-2 clinical trials) (**Table 2**) [22, 23]. Similarly, the therapeutic outcome was much significant in subtype 1a patients without Q80K polymorphism and in noncirrhotics as compared to those with cirrhosis and Q80K polymorphism (**Table 2**). The frequency of treatment-emergent adverse events was similar in both clinical trials, and the drug combination was well tolerated and safe even in patients with Q80K polymorphism as compared to simeprevir and PEG-IFN/ RBV-based triple therapies.

## **4. NS5A inhibitor-based direct-acting antivirals**

develop the regimens, which must be effective against other difficult-to-treat HCV populations with an improved dosing schedule. Simeprevir (Olysio®) was approved by the US FDA to treat chronic HCV genotype 1 infection in 2013 (www.hcvonline.org/page/treatment/drugs/simeprevir-drug) [55, 56]. The drug was approved along with PEG-IFN α-2a or 2b and RBV for treatment-naive HCV genotype 1-infected patients [35], for patients with compensated liver diseases (fibrosis and cirrhosis), and for those who are relapsers and nonresponders to interferon-based therapy [32,57]. Simeprevir 150 mg once daily along with PEG-IFN/RBV achieved overall SVR rates approximately 80–90 % in treatment-naïve and relapsers [56] and 67–80 % of previously treated and null responders to interferonbased therapy (**Table 2**) (QUEST-1 and QUEST-2 clinical trials). The therapy seems to be more efficient than telaprevir/boceprevir-based triple therapies; however, there are certain disadvantages. For example, SVR rates were lower at the start of antiviral therapy in patients with advanced hepatic fibrosis as well as in patients with Q80R/K polymorphism of NS3 protein [58]. It is a naturally occurring amino acid substitution at NS3 codon 80, where glutamine is replaced to lysine. The Q80K polymorphism exists naturally in HCV genotype 1a (30–50 %)- and 1b (0.5 %)-infected patients [56]. It substantially reduces the efficacy of simeprevir-based triple therapy in HCV genotype 1a-infected patients. In QUEST-1 clinical

Paritaprevir V36A/M/T, F43L, V55I, Y56H, Q80L, I132V, R155K, A156G

Y93C/H/N

Ombitasvir K24R, M28A/T/V, Q30E/K/R, H/Q54Y, H58D/P/R,

Dasabuvir G307R, C316Y, M414I/T, E446K/Q, A450V, Y561H

**Table 3.** Resistance amino acid variants (RAVs) associated with antiviral drug resistance to direct-acting antivirals.

**Drug name Resistance amino acid variants**

Faldaprevir R155K/T/Q; D168A/V/T/H Danoprevir R155K/T/Q; D168A/V/T/H

ABT-450 R155K/T/Q; D168A/V/T/H Daclatasvir M28; Q30; L31; Y93Y/H

Velpatasvir Q30R, Y93H/N, L31M Elbasvir A156T, D168A/Y, R155K Grazoprevir M28, Q30, L31, Y93

Sofosbuvir 282 T

296 Advances in Treatment of Hepatitis C and B

Asunaprevir Q80R/K; R155K/T/Q; D168A/V/T/H

Telaprevir V36A/M; T54S/A; R155K/T/Q; A156S; B156T/V

Simeprevir Q80R/K; R155K/T/Q; B156T/V; D168A/V/T/H

Ledipasvir K24R, M28T/V, Q30R/H/K/L, L31M, Y93H/N

Boceprevir V36A/M; T54S/A; V55A; R155K/T/Q; A156S; B156T/V;

V170A/T

Daclatasvir (Daklinza®) is the first in a new class of direct-acting antivirals, which inhibit the action of NS5A, a protein essential to play a diverse role in HCV replication, assembly, and release [62]. The US FDA approved initially daclatasvir and sofosbuvir combination to treat chronic HCV genotype 3-infected patients in July 2015. However, the indications were modified and expanded to treat HCV genotype 1- and 3-infected patients, patients with decompensated cirrhosis, patients with post-liver transplantation, and HCV/HIVcoinfected patients in February 2016 [63]. Daclatasvir monotherapy along with PEG-IFN/ RBV leads initially to higher SVR rates in treated patients, but viral escape mutants occur rapidly indicating its relatively lower genetic barrier to resistance (COMMAND-1 and COMMAND-4 clinical trials) (**Table 2**) [64]. To overcome the emergence of viral escape mutants and to achieve high SVR rates, the treatment is recommended along with sofosbuvir and with or without ribavirin. Higher SVR rates were documented in HCV genotype 1 and 3 treatment-naive patients when administered to daclatasvir and sofosbuvir combination [24]. Other multi-series clinical trials also demonstrate high clinical efficacy and well tolerability of daclatasvir-sofosbuvir with or without RBV against HCV genotype 1–6 infected patients (ALLY 1–3 clinical trial) (**Table 2**) [25–28]. Similarly, higher SVR rates (87–98 %) were achieved in HCV genotype 1-infected patients treated with a combination of daclatasvir, asunaprevir, and beclabuvir for 12 weeks (UNITY 1–2 clinical trial) (**Table 2**) [29, 30]. Headache, nausea, and vomiting were the most commonly noticed adverse effects, and the adverse event profile was almost similar in all genotype-treated patients and managed during or after the treatment completion [62]. The viral-resistant mutants were found commonly at residue M28, Q30, L31, and Y93 of NS5A region in HCV genotype 1a patients (**Table 3**) [25, 28]. However, viral-resistant mutants were reported less frequently at position L31 and Y93 in HCV genotype 1b patients [64]. Some experimental studies demonstrate that these mutations are responsible for increasing the EC50 (i.e., the concentration of a drug which produces therapeutic response halfway between the baseline and maximum after a certain period of time) of daclatasvir in treated patients [64]. In contrast to NS3 protease, viral-resistant mutants against NS5A inhibitors do not impair replication fitness and do not disappear during follow-up examinations at the end of treatment. One-year follow-up studies predict the persistence of NS5A-resistance mutants, but no cross-resistance has reported between daclatasvir and other direct-acting antivirals as yet [65]. The approval of daclatasvir monotherapy and daclatasvir with other different regimens is expected soon.

## **5. HCV polymerase inhibitor-based therapeutic regimens**

Sofosbuvir (Sovaldi®; SOF) was approved by the US FDA in 2013 to treat treatment-naïve and treatment-experienced HCV genotype 1–6-infected patients [66]. SOF is a uridine nucleotide analog inhibitor, which exerts its antiviral activity by competing with endogenous uridine triphosphate of the growing HCV mRNA chain incorporated by HCV polymerase enzyme [66,67]. After incorporation into growing mRNA chain, no further nucleotides can be added, and mRNA chain is terminated [67]. The drug is active against all HCV genotypes (1–6) as well as difficult-to-treat HCV populations [68]. The approval was momentous and based on the results of four registration studies in phase III clinical trials (FISSION, POSITRON, FUSION, and NEUTRINO clinical trials) where the therapy endpoint was to achieve a sustained virologic response rate at week 12 after stopping the active treatment (i.e., SVR12) (**Table 2**) [69]. Sofosbuvir in combination with ribavirin presented very promising clinical efficacy data against HCV genotype 1–4-treated patients (**Table 2**) [33, 34]. The relapse rate was only 9 % among all the four phase III drug registration studies [34]. No major side effects or severe cardiac adverse events were reported during SOF plus RBV dual therapy. However, the adverse event profiles of sofosbuvir monotherapy and even in combination with RBV are not sufficiently addressed because of the lack of controlled trials [66]. Meaningful historical controls, as well as ribavirin monotherapy arm as a comparator, are not available due to which we may not draw conclusions about the eventual adverse effects of sofosbuvir [33, 34]. However, adverse events associated with sofosbuvir-related therapies are not frequent and severe in nature. Antiviral resistance was only observed in a single patient after sofosbuvir monotherapy, which was successfully re-treated with SOF plus RBV dual therapy [69].

1 and 3 treatment-naive patients when administered to daclatasvir and sofosbuvir combination [24]. Other multi-series clinical trials also demonstrate high clinical efficacy and well tolerability of daclatasvir-sofosbuvir with or without RBV against HCV genotype 1–6 infected patients (ALLY 1–3 clinical trial) (**Table 2**) [25–28]. Similarly, higher SVR rates (87–98 %) were achieved in HCV genotype 1-infected patients treated with a combination of daclatasvir, asunaprevir, and beclabuvir for 12 weeks (UNITY 1–2 clinical trial) (**Table 2**) [29, 30]. Headache, nausea, and vomiting were the most commonly noticed adverse effects, and the adverse event profile was almost similar in all genotype-treated patients and managed during or after the treatment completion [62]. The viral-resistant mutants were found commonly at residue M28, Q30, L31, and Y93 of NS5A region in HCV genotype 1a patients (**Table 3**) [25, 28]. However, viral-resistant mutants were reported less frequently at position L31 and Y93 in HCV genotype 1b patients [64]. Some experimental studies demonstrate that these mutations are responsible for increasing the EC50 (i.e., the concentration of a drug which produces therapeutic response halfway between the baseline and maximum after a certain period of time) of daclatasvir in treated patients [64]. In contrast to NS3 protease, viral-resistant mutants against NS5A inhibitors do not impair replication fitness and do not disappear during follow-up examinations at the end of treatment. One-year follow-up studies predict the persistence of NS5A-resistance mutants, but no cross-resistance has reported between daclatasvir and other direct-acting antivirals as yet [65]. The approval of daclatasvir monotherapy and daclatasvir with other

different regimens is expected soon.

298 Advances in Treatment of Hepatitis C and B

**5. HCV polymerase inhibitor-based therapeutic regimens**

Sofosbuvir (Sovaldi®; SOF) was approved by the US FDA in 2013 to treat treatment-naïve and treatment-experienced HCV genotype 1–6-infected patients [66]. SOF is a uridine nucleotide analog inhibitor, which exerts its antiviral activity by competing with endogenous uridine triphosphate of the growing HCV mRNA chain incorporated by HCV polymerase enzyme [66,67]. After incorporation into growing mRNA chain, no further nucleotides can be added, and mRNA chain is terminated [67]. The drug is active against all HCV genotypes (1–6) as well as difficult-to-treat HCV populations [68]. The approval was momentous and based on the results of four registration studies in phase III clinical trials (FISSION, POSITRON, FUSION, and NEUTRINO clinical trials) where the therapy endpoint was to achieve a sustained virologic response rate at week 12 after stopping the active treatment (i.e., SVR12) (**Table 2**) [69]. Sofosbuvir in combination with ribavirin presented very promising clinical efficacy data against HCV genotype 1–4-treated patients (**Table 2**) [33, 34]. The relapse rate was only 9 % among all the four phase III drug registration studies [34]. No major side effects or severe cardiac adverse events were reported during SOF plus RBV dual therapy. However, the adverse event profiles of sofosbuvir monotherapy and even in combination with RBV are not sufficiently addressed because of the lack of controlled trials [66]. Meaningful historical controls, as well as ribavirin monotherapy arm as a comparator, are not available due to which we may not draw conclusions about the eventual adverse effects of sofosbuvir [33, 34]. However, adverse events associated with sofosbuvir-related therapies are not frequent and Sofosbuvir in combination with ledipasvir (Harvoni®; the first fixed-dose drug combination claims to "two firsts") with or without RBV was approved in 2014 for HCV genotype 1 infection treatment. Later on, the approval was expanded to treat HCV genotype 4-, 5-, 6-, and HCV/HIV-coinfected patients in 2015. Recently, the US FDA has extended the treatment recommendation to liver transplant genotype 1 and 4 patients with compensated cirrhosis and in genotype 1 patients with decompensated cirrhosis (http://www.hepatitisc.uw.edu/page/ treatment/drugs/ledipasvir-sofosbuvir#drug-summary) [31, 70]. Ledipasvir (90 mg) and sofosbuvir (400 mg) have formulated as a single coformulated pill [31]. However, ledipasvir (an NS5A inhibitor) monotherapy is not approved by the US FDA to treat the infection as yet. This combo drug seems very efficient to achieve SVR rates more than 90 % of HCV genotype 1- and 4-treated patients without cirrhosis when administered to 8, 12, or 24 weeks (**Table 2**) [31, 71]. Cirrhotic patients may also achieve higher SVR rates with 12- or 24-week treatment schedule [72]. Severe adverse events are rare and manageable during or after the therapy. The inclusion of ribavirin does not affect the therapeutic efficacy and achievement of higher SVR rates. Consequently, the addition of RBV does not seem prerequisite in every all-oral DAA combinations against HCV. RBV results in hemolytic anemia and is highly teratogenic [11], and for this reason, RBV-sparing regimens are considerably advantageous and eagerly awaited in the future.

## **6. Treatment paradigms for difficult-to-treat populations**

As the treatment success in HCV-infected patients mainly depends on HCV genotype, previous treatment history, cirrhosis or fibrosis score, and the high barrier to antiviral drug resistance, the combination of experimental drug velpatasvir and sofosbuvir might simplify the treatment strategies in "difficult-to-treat subgroups" in the future. Velpatasvir (An NS5A inhibitor) and sofosbuvir in a fixed-dose combination (100 mg/400 mg) exhibit the pan-genotypic coverage with a simple 12-week regimen in treatment-naïve, treatment-experienced, as well as in cirrhotic patients as determined in multicentered clinical trials (i.e., ASTRAL 1–5) conducted at 81 sites including the United States, Canada, and Europe in 2014 (**Table 2**) [36]. The adverse event profile was almost similar to each clinical trials, and headache, nausea, and vomiting were the most common ones. However, overall the drug combination was well tolerated and safe in treated patients. The US FDA has approved the fixed-dose combination of velpatasvir and sofosbuvir (Epclusa®) in June 2016 to treat chronic HCV genotype 1–6-infected adult patients.

The discovery and development of elbasvir/grazoprevir fixed-dose combination have shifted the treatment paradigm for genotype 1 and 4 patients with stage 4–5 chronic kidney disease (CKD) and HCV/HIV coinfection [40, 41]. The US FDA has approved elbasvir/grazoprevir (50/100 mg q.d.) (Zepatier®) combination for HCV genotype 1 and 4 infection with chronic kidney diseases and HCV/HIV coinfection with some specific clinical requirements (i.e., viral genotype, prior treatment experience, NS5A-associated RAVs at position M28, Q30, L31, or Y93) (http://www.hepatitisc.uw.edu/page/treatment/dugs/elbasvir-grazoprevir#drugsummary) [73, 74]. Similarly, the treatment regimen is prescribed with many precautions in subtype 1a patient with prior testing of NS5A-associated RAVs, because it determines the overall treatment duration and the inclusion of ribavirin to therapy (**Table 3**) [38, 40, 41]. The FDA approval was granted on the findings of a series of multicenter phase III clinical trials (C-EDGE and C-SURFER) in treatment-naïve, treatment-experienced, and other difficult-tocure populations (i.e., HCV/HIV coinfection, stage 4/5 CKD including hemodialysis patients) where overall SVR rates were highly promising (i.e., 92–99 %) (**Table 2**) [37–41]. The adverse event profile was not serious in the treatment groups, and the renal system adverse effects were comparable without significant changes in estimated glomerular filtration rate (eGFR) value and creatinine levels [38, 40, 41]. Headache, nausea, and fatigue were the most commonly observed adverse events with an elevation in alanine aminotransferase (ALT) levels five times more than the normal one [38, 40, 41]. However, most of the adverse effects resolved at or after the treatment completion.

The first 3D regimen, "Viekira Pak®" (i.e., a combination of three direct-acting antivirals: ombitasvir, paritaprevir, and dasabuvir) along with ritonavir, to treat chronic HCV genotype 1 infection was approved by the US FDA in 2014 (http://www.hepatitisc.uw.edu/page/treatment/drugs/3d#drug-summary) [75]. The drug combination is prescribed to genotype 1-compensated cirrhotic patients, however, still contraindicated to decompensated cirrhotics [42]. The approval was based on phase II/III multicenter clinical trials (**Table 2**) involving more than 2300 patients with chronic HCV 1 infection, some of whom had cirrhosis [43–47, 76]. Cure rates across the various groups were ranged from 91 % to 100 % (**Table 2**). The therapeutic outcomes demonstrated that the drug combination was safe with no significant adverse effects in a population with compensated cirrhosis [47]; however, the drug combination is forbidden in decompensated cirrhotic patients.

A recent ongoing clinical trial conducted in HCV genotype 4 noncirrhotic patients showed the promising therapeutic activity of ombitasvir, paritaprevir, and ritonavir with or without RBV for a 12-week course (PEARL-I clinical studies) [24]. Similarly, the therapeutic regimen along with RBV was also found highly effective to treat genotype 4, cirrhotic patients, as shown by the results of AGATE-I and AGATE-II ongoing clinical studies. PEARL-I, phase 2 open-label, randomized, and multicentre clinical trials were conducted in Europe, Turkey, and the United States [24]. The treatment-naïve patients included in the study received the ombitasvir, paritaprevir, and ritonavir combination with or without RBV; however, all treatment-experienced patients (i.e., previously treated with PEG-IFN/RBV) received the active treatment with RBV. Interestingly, dasabuvir was not included in the treatment regimen as it does not show therapeutic activity against HCV genotype 4. The overall SVR rates were achieved 91 % (40/44) in treatment-naïve patients who received active treatment without RBV and 100 % (42/42) in those who took RBV along with the regimen. All treatment-experienced patients achieved SVR12 at the treatment completion (49/49, 100 %) (**Table 2**). Viral relapse occurred in two patients, and the virologic breakthrough was experienced in one patient. The adverse event profile was almost negligible including headache and decreased hemoglobin level (i.e., 100 g/L, anemic state), but no treatment discontinuation was attributed to active regimen except the dose modification of RBV to nullify anemic state [24].

The AGATE-I open-label phase 3 multicenter and randomized clinical trial revealed the therapeutic outcome of ombitasvir, paritaprevir, and ritonavir plus RBV in HCV genotype 4 patients with compensated cirrhosis [77]. The study was conducted in both treatment-naïve and treatment-experienced patients (PEG-IFN/RBV treated) at different locations in Europe and the United States. Overall, 120 adult patients were enrolled in the study of which 59 patients received 12-week treatment (i.e., ombitasvir, paritaprevir, and ritonavir plus RBV) and 61 patients were assigned to take 16 weeks of the same treatment regimen. The overall SVR rates were achieved 97 % (57/59) in the 12-week group and 98 % (60/61) in the 16-week group. The adverse event frequency was significantly higher, mixed in both patient arms, and noticed in more than 10 % of all patients including asthenia, fatigue, headache, anemia, pruritus, nausea, and dizziness more common ones. However, virologic breakthrough was reported in one patient, and one patient discontinued the treatment at day 1 in the 12-week treatment group, and one missed the posttreatment week 12 visit in the 16-week group [77].

The AGATE-II open label and partly randomized clinical studies were conducted in native Egyptian population infected with HCV genotype 4 [77]. Overall 182 patients both treatmentnaïve and treatment-experienced (previously treated to PEG-IFN/RBV-based dual therapies) anticipated in the clinical trial of whom 160 were eligible for inclusion criteria. 100 patients had no cirrhosis and received active regimen and RBV for 12 weeks. The remaining 60 having cirrhosis were randomly assigned to 12-week active treatment (n = 31) or 24-week treatment group (n = 29). The SVR rates were achieved 94 % in the noncirrhotic 12-week patient arm (94/100), 97 % in cirrhotic patients (30/31) enrolled in the 12-week treatment group, and 93 % (27/29) in cirrhotic patients administered for 24-week active treatment (**Table 2**). The adverse event profile was significantly higher particularly in noncirrhotic patients including headache and fatigue the most common (i.e., 41 % and 35 %, respectively) ones than the cirrhotic ones (29–38 %, respectively). However, no treatment discontinuation was related to drug side effects or active treatment itself [77].

The findings of the ongoing PEARL-I, AGATE-I, and AGATE-II clinical trials are promising while treating HCV genotype 4 cirrhotic and noncirrhotic patients; however, no additional benefits were reported in terms of higher SVR, when treatment duration was extended for cirrhotic patients from 12 weeks to 16 weeks or even 24 weeks in native Egyptian population. Similarly, a small number of patients enrolled in the clinical trials limit the comprehensive determination of the drug side effects. Further studies are eagerly awaited in this prospect, and as a precaution, any HCV cirrhotic patients using such combination therapeutic regimens should be hepatically intact for any hepatic complications/comorbidities because the patients in clinical trials are often very carefully monitored than patients in real-world clinical practice [40].

## **7. Interferon derivatives**

L31, or Y93) (http://www.hepatitisc.uw.edu/page/treatment/dugs/elbasvir-grazoprevir#drugsummary) [73, 74]. Similarly, the treatment regimen is prescribed with many precautions in subtype 1a patient with prior testing of NS5A-associated RAVs, because it determines the overall treatment duration and the inclusion of ribavirin to therapy (**Table 3**) [38, 40, 41]. The FDA approval was granted on the findings of a series of multicenter phase III clinical trials (C-EDGE and C-SURFER) in treatment-naïve, treatment-experienced, and other difficult-tocure populations (i.e., HCV/HIV coinfection, stage 4/5 CKD including hemodialysis patients) where overall SVR rates were highly promising (i.e., 92–99 %) (**Table 2**) [37–41]. The adverse event profile was not serious in the treatment groups, and the renal system adverse effects were comparable without significant changes in estimated glomerular filtration rate (eGFR) value and creatinine levels [38, 40, 41]. Headache, nausea, and fatigue were the most commonly observed adverse events with an elevation in alanine aminotransferase (ALT) levels five times more than the normal one [38, 40, 41]. However, most of the adverse effects resolved

The first 3D regimen, "Viekira Pak®" (i.e., a combination of three direct-acting antivirals: ombitasvir, paritaprevir, and dasabuvir) along with ritonavir, to treat chronic HCV genotype 1 infection was approved by the US FDA in 2014 (http://www.hepatitisc.uw.edu/page/treatment/drugs/3d#drug-summary) [75]. The drug combination is prescribed to genotype 1-compensated cirrhotic patients, however, still contraindicated to decompensated cirrhotics [42]. The approval was based on phase II/III multicenter clinical trials (**Table 2**) involving more than 2300 patients with chronic HCV 1 infection, some of whom had cirrhosis [43–47, 76]. Cure rates across the various groups were ranged from 91 % to 100 % (**Table 2**). The therapeutic outcomes demonstrated that the drug combination was safe with no significant adverse effects in a population with compensated cirrhosis [47]; however, the drug combination is

A recent ongoing clinical trial conducted in HCV genotype 4 noncirrhotic patients showed the promising therapeutic activity of ombitasvir, paritaprevir, and ritonavir with or without RBV for a 12-week course (PEARL-I clinical studies) [24]. Similarly, the therapeutic regimen along with RBV was also found highly effective to treat genotype 4, cirrhotic patients, as shown by the results of AGATE-I and AGATE-II ongoing clinical studies. PEARL-I, phase 2 open-label, randomized, and multicentre clinical trials were conducted in Europe, Turkey, and the United States [24]. The treatment-naïve patients included in the study received the ombitasvir, paritaprevir, and ritonavir combination with or without RBV; however, all treatment-experienced patients (i.e., previously treated with PEG-IFN/RBV) received the active treatment with RBV. Interestingly, dasabuvir was not included in the treatment regimen as it does not show therapeutic activity against HCV genotype 4. The overall SVR rates were achieved 91 % (40/44) in treatment-naïve patients who received active treatment without RBV and 100 % (42/42) in those who took RBV along with the regimen. All treatment-experienced patients achieved SVR12 at the treatment completion (49/49, 100 %) (**Table 2**). Viral relapse occurred in two patients, and the virologic breakthrough was experienced in one patient. The adverse event profile was almost negligible including headache and decreased hemoglobin level (i.e., 100 g/L, anemic state), but no treatment discontinuation was attributed to active

regimen except the dose modification of RBV to nullify anemic state [24].

at or after the treatment completion.

300 Advances in Treatment of Hepatitis C and B

forbidden in decompensated cirrhotic patients.

Over the years, the principal objectives to develop novel interferon formulations include the replacement of conventional interferon with the new ones, to reduce the adverse effects of current interferon and ease of administration with an improved dosing schedule [78]. The treatment success for HCV is primarily dependent on the patient adherence to therapy so that the development of unique IFN formulations with improved pharmacokinetics is a prime objective of HCV therapeutics nowadays. The main advantage of this approach may seem to maintain viral suppression across the longer dose interval, avoid of inter-dose trough, and reduce dosage frequencies (twice or even once per month as compared to once per week for the current PEG-IFN and consensus interferons). Although the development of new interferon formulations primarily focuses on HCV genotype 1 patients, their administration can also be prized for HCV genotype 2- and 3-infected individuals [79]. Similarly, the duration of treatment can also be reduced in easy-to-treat populations (HCV genotype 2- and 3-infected patients) up to 12 weeks if a rapid virologic response achieves earlier [79]. This approach may suggest a very convenient therapeutic regimen of only three injections if long-acting IFNs are used in treated patients.

## **7.1. Interferon lambda**

Interferon lambda (IFN-λ) is still promising and may be beneficial as an adjuvant therapy to treat HCV infection in combination with other DAAs in the near future. It has also raised the hopes to replace the conventional interferons (IFN-α 2a, IFN-α 2b, PEG-IFN α) to reduce the frequency of side effects and treatment discomfort during and after the treatment completion in infected individuals. The discovery of three interferon-λ cytokines (i.e., interferons λ1, λ2, and λ3 encoded by IL29, IL28A, and IL28B, respectively) in 2003 has suggested their plausible role in suppression of HCV replication [80]. This fact was supported by the identification of common genetic variants in IL28B genome, which are highly associated with responses to PEG-IFN α/RBV treatment for chronic HCV infection [81]. Similarly, genome-wide association studies and in vitro studies also demonstrate an interactive and complementary relationship between IFN-α and IFN-λ for suppressing HCV replication [82]. IFN-λ like another type 3 interferons binds to different host cell receptors than type 1 interferons (e.g., IFN-α 2a, 2b) to trigger the JAK-STAT antiviral pathways (**Figure 2**). However, the downstream cell signaling pathways are largely comparable to interferon-α which upregulate several hundreds of interferon-stimulated genes to initiate antiviral activity [80]. IFN-λ binds mainly to IL28 receptors, which are positioned only to hepatocytes, plasmacytoid dendritic cells, peripheral B cells, and epithelial cells [83]. This restricted distribution of IL28 receptors for interferon-λ facilitates its targeted hepatic delivery, better tolerability, and increase safety profile than the conventional interferons [83]. IFN-λ may also escalate the subsaturating levels of IFN-α and increase its antiviral efficacy. In vitro studies reveal that IFN-α induces the expression of IFN-λ genes that upregulate a distinct pattern of signal transduction and interferon-stimulated genes than IFN-α to abort HCV replication [84]. Consequently, the combination of IFN-λ and IFN-α may provide additive therapeutic effects due to the complementary roles of two types of cytokines. Interferon-λ has been pegylated and phase I clinical trials with or without ribavirin have been completed [85]. Subsequent phase II clinical trials demonstrated that the administration of PEG-IFN λ (240 μg, 180 μg, or 120 μg once weekly) showed 10 % higher rapid virologic response (RVR; HCV RNA negative after 4 weeks of therapy) rates and 20 % higher extended rapid virologic response (eRVR; HCV RNA negative at the lower limit of detection but not the lower limit of quantification between week 4 and week 12 during PEG-IFN therapy) rates

**Figure 2.** Interferon-lambda (λ) cell signaling pathways to induce anti-HCV activity. Interferon lambda binds to different cell receptors than IFN alpha to activate JAK-STAT pathways and initiate the antiviral activity by upregulating a distinct pattern of signal transduction. IFN, interferon; IL, interleukin; R, receptor; JAK, Janus kinase; TYK, tyrosine kinase; STAT, signal transducer and activator of transcription; IRF, interferon regulatory factor; P, phosphate; ISRE, interferonstimulated response element.

than PEG-IFN α-2a in treatment-naïve patients [86]. IFN-λ is associated with less adverse event profile, including less hematologic toxicity, flu-like symptoms, and muscular pain, but increased aminotransferase and bilirubin levels in treated patients [86]. Now, full phase III clinical trials of interferon-λ with other DAAs (i.e., in combination with daclatasvir and asunaprevir plus RBV) are under consideration [87].

## **8. HCV vaccine technology**

The treatment success for HCV is primarily dependent on the patient adherence to therapy so that the development of unique IFN formulations with improved pharmacokinetics is a prime objective of HCV therapeutics nowadays. The main advantage of this approach may seem to maintain viral suppression across the longer dose interval, avoid of inter-dose trough, and reduce dosage frequencies (twice or even once per month as compared to once per week for the current PEG-IFN and consensus interferons). Although the development of new interferon formulations primarily focuses on HCV genotype 1 patients, their administration can also be prized for HCV genotype 2- and 3-infected individuals [79]. Similarly, the duration of treatment can also be reduced in easy-to-treat populations (HCV genotype 2- and 3-infected patients) up to 12 weeks if a rapid virologic response achieves earlier [79]. This approach may suggest a very convenient therapeutic regimen of only three injections if long-acting IFNs are

Interferon lambda (IFN-λ) is still promising and may be beneficial as an adjuvant therapy to treat HCV infection in combination with other DAAs in the near future. It has also raised the hopes to replace the conventional interferons (IFN-α 2a, IFN-α 2b, PEG-IFN α) to reduce the frequency of side effects and treatment discomfort during and after the treatment completion in infected individuals. The discovery of three interferon-λ cytokines (i.e., interferons λ1, λ2, and λ3 encoded by IL29, IL28A, and IL28B, respectively) in 2003 has suggested their plausible role in suppression of HCV replication [80]. This fact was supported by the identification of common genetic variants in IL28B genome, which are highly associated with responses to PEG-IFN α/RBV treatment for chronic HCV infection [81]. Similarly, genome-wide association studies and in vitro studies also demonstrate an interactive and complementary relationship between IFN-α and IFN-λ for suppressing HCV replication [82]. IFN-λ like another type 3 interferons binds to different host cell receptors than type 1 interferons (e.g., IFN-α 2a, 2b) to trigger the JAK-STAT antiviral pathways (**Figure 2**). However, the downstream cell signaling pathways are largely comparable to interferon-α which upregulate several hundreds of interferon-stimulated genes to initiate antiviral activity [80]. IFN-λ binds mainly to IL28 receptors, which are positioned only to hepatocytes, plasmacytoid dendritic cells, peripheral B cells, and epithelial cells [83]. This restricted distribution of IL28 receptors for interferon-λ facilitates its targeted hepatic delivery, better tolerability, and increase safety profile than the conventional interferons [83]. IFN-λ may also escalate the subsaturating levels of IFN-α and increase its antiviral efficacy. In vitro studies reveal that IFN-α induces the expression of IFN-λ genes that upregulate a distinct pattern of signal transduction and interferon-stimulated genes than IFN-α to abort HCV replication [84]. Consequently, the combination of IFN-λ and IFN-α may provide additive therapeutic effects due to the complementary roles of two types of cytokines. Interferon-λ has been pegylated and phase I clinical trials with or without ribavirin have been completed [85]. Subsequent phase II clinical trials demonstrated that the administration of PEG-IFN λ (240 μg, 180 μg, or 120 μg once weekly) showed 10 % higher rapid virologic response (RVR; HCV RNA negative after 4 weeks of therapy) rates and 20 % higher extended rapid virologic response (eRVR; HCV RNA negative at the lower limit of detection but not the lower limit of quantification between week 4 and week 12 during PEG-IFN therapy) rates

used in treated patients.

302 Advances in Treatment of Hepatitis C and B

**7.1. Interferon lambda**

## **8.1. Barriers to developing prophylactic and protective HCV vaccines**

The tendency of acute HCV infection to develop into chronic infection and optimal outcomes of the current therapies in the majority of treated patients underscores an urgent need to search and develop potential anti-HCV vaccine molecules. Interestingly, the efforts to develop HCV vaccines are facing real challenges due to some reasons. First, despite the consistent efforts by the researchers, still now there is no permissive cell culture system available where HCV can replicate persistently at high enough levels to evaluate antibodies which may neutralize [88]. Second, there is no authenticated and handy animal model available which is susceptible to HCV and can tackle the candidate vaccine challenge studies [88]. Although the chimpanzee model is the first choice for the investigators in HCV replication and candidate vaccine studies, it is expensive, endangered, and difficult to handle [89]. Third, genome variations in HCV genotypes, subtypes, and quasispecies nature of HCV may require the construction of polyvalent vaccines, which protect against a significant number of closely related epitopes [90]. In fact, the genetic heterogeneity of HCV in an infected individual and immune responses selected for neutralization of escape mutants within the hypervariable regions (HVR1) of E1 envelope glycoprotein as well as cytotoxic T lymphocytes (CTL) escape mutants may limit the effectiveness and utility of any HCV vaccine model [91]. Fourth, it is unclear that either HCV envelope glycoproteins contain all the antigenic determinants require for effective neutralization or not [92]. Fifth, acute HCV infection persists in the majority of infected individuals, even though innate and acquired immune responses accelerate against nearly all of the HCVencoded polyproteins [93]. T-cell responses immediately accelerate to clear HCV in acutely infected individuals; therefore, a successful anti-HCV vaccine has to elicit both CD4+ and CD8+ T-cell responses in infected individuals [94]. Sixth, HCV may associate with immunoglobulin or β-lipoprotein in the blood, which may "mask" the virus and reduce the efficiency of neutralization [95].

## **8.2. Current anti-HCV vaccine models**

Due to the above-mentioned qualms, the development of prophylactic or protective HCV vaccine is a highly challenging task and fraught with barriers. Anyhow, the advancement in vaccinology has inspired the researchers to work out different models of protective HCV candidate vaccines (**Table 1**). In this vein, the principle goal of an anti-HCV vaccine design should be to wipe out the chronic infection in exposed individuals or remove the virus from already infected individuals by boosting the innate and acquired host immune responses, which also seems an uphill task [94]. In this context, the strategy paradigms have been shifted from the production of traditional recombinant envelope proteins to the engineering of complex viral vectors directing the expression of multiple hepatitis C viral antigens (i.e., Core, NS3, NS4, and NS5B) [94]. We briefly describe here the recent advancement in HCV vaccine technology, which may consider as the role models for the successful development of an effective preventive/protective vaccine in the future.

An HCV immunoglobulin (Civacir) has studied for its therapeutic effects on recurrent hepatitis C following liver transplantation in phase II clinical trials, but the SVR data from these trials are not yet available [96]. Similarly, the high-dose monoclonal antibodies were evaluated against HCV glycoprotein E2 in genotype 1a patients undergoing liver transplantation in phase II clinical trials [97]. Although the strategy was not effective to prevent the recurrence of HCV infection, the reappearance of viremia in liver transplant patients significantly delayed as compared to the placebo control population [97]. Now, this regimen along with a directacting antiviral is under consideration for further clinical trials.

Currently, a DNA vector-based vaccine (ChronVac-C) is in clinical development against chronic HCV genotype 1 infection [98]. By using DNA electroporation, the gene-encoding HCV NS3/4A protein was introduced into the patient skeletal muscle. The skeletal muscle expressed NS3/4A protein, which in turn stimulated the particular host innate and acquired immune responses against HCV. The clinical efficacy of ChronVac-C was evaluated in 12 treatment-naïve HCV genotype 1-infected patients with four different doses given monthly for four months in phase I/II clinical trials [98]. T-cell responses were detected in one patient, and viral load reduction up to 1.2 log10–2.4 log10 was reported in two out of three patients with the highest dose [98]. Further clinical trials of this candidate vaccine are under consideration.

## **8.3. T-cell-based vaccines**

selected for neutralization of escape mutants within the hypervariable regions (HVR1) of E1 envelope glycoprotein as well as cytotoxic T lymphocytes (CTL) escape mutants may limit the effectiveness and utility of any HCV vaccine model [91]. Fourth, it is unclear that either HCV envelope glycoproteins contain all the antigenic determinants require for effective neutralization or not [92]. Fifth, acute HCV infection persists in the majority of infected individuals, even though innate and acquired immune responses accelerate against nearly all of the HCVencoded polyproteins [93]. T-cell responses immediately accelerate to clear HCV in acutely infected individuals; therefore, a successful anti-HCV vaccine has to elicit both CD4+ and CD8+ T-cell responses in infected individuals [94]. Sixth, HCV may associate with immunoglobulin or β-lipoprotein in the blood, which may "mask" the virus and reduce the efficiency of neu-

Due to the above-mentioned qualms, the development of prophylactic or protective HCV vaccine is a highly challenging task and fraught with barriers. Anyhow, the advancement in vaccinology has inspired the researchers to work out different models of protective HCV candidate vaccines (**Table 1**). In this vein, the principle goal of an anti-HCV vaccine design should be to wipe out the chronic infection in exposed individuals or remove the virus from already infected individuals by boosting the innate and acquired host immune responses, which also seems an uphill task [94]. In this context, the strategy paradigms have been shifted from the production of traditional recombinant envelope proteins to the engineering of complex viral vectors directing the expression of multiple hepatitis C viral antigens (i.e., Core, NS3, NS4, and NS5B) [94]. We briefly describe here the recent advancement in HCV vaccine technology, which may consider as the role models for the successful development of an

An HCV immunoglobulin (Civacir) has studied for its therapeutic effects on recurrent hepatitis C following liver transplantation in phase II clinical trials, but the SVR data from these trials are not yet available [96]. Similarly, the high-dose monoclonal antibodies were evaluated against HCV glycoprotein E2 in genotype 1a patients undergoing liver transplantation in phase II clinical trials [97]. Although the strategy was not effective to prevent the recurrence of HCV infection, the reappearance of viremia in liver transplant patients significantly delayed as compared to the placebo control population [97]. Now, this regimen along with a direct-

Currently, a DNA vector-based vaccine (ChronVac-C) is in clinical development against chronic HCV genotype 1 infection [98]. By using DNA electroporation, the gene-encoding HCV NS3/4A protein was introduced into the patient skeletal muscle. The skeletal muscle expressed NS3/4A protein, which in turn stimulated the particular host innate and acquired immune responses against HCV. The clinical efficacy of ChronVac-C was evaluated in 12 treatment-naïve HCV genotype 1-infected patients with four different doses given monthly for four months in phase I/II clinical trials [98]. T-cell responses were detected in one patient, and viral load reduction up to 1.2 log10–2.4 log10 was reported in two out of three patients with the highest dose [98]. Further clinical trials of this candidate vaccine are under consideration.

tralization [95].

**8.2. Current anti-HCV vaccine models**

304 Advances in Treatment of Hepatitis C and B

effective preventive/protective vaccine in the future.

acting antiviral is under consideration for further clinical trials.

Vaccines based on robust T-cell responses are vital and crucial because such vaccine triggers both antibodies and cytotoxic T-cell responses against an insidious virus [11]. The HCVinfected cells display the viral surface and internal particles/molecules (i.e., HCV genome) to the immune system of the body, especially to CD4+ and CD8+ killer cells, which induce the host innate and acquired immune responses against the virus surface particles as well as the HCV genome [11]. CD8+ T-cell immunity induced by CD4+ T cells is mainly responsible for HCV viral infection control in human as well as in chimpanzee challenge studies [11]. Recently researchers have evaluated heterologous T-cell vaccine (ChAd3/MVA) targeting HCV nonstructural proteins in HCV genotype 1b-infected individuals [13]. The vaccine is a combination of replication-defective chimpanzee adenovirus (ChAd3) and modified vaccinia Ankara (MVA) vectors so named as ChAd3/MVA [13]. The vaccine induced higher magnitude of T-cell responses in most infected individuals against all six nonstructural (from NS2 to NS5B) antigenic pools in phase I clinical studies [13]. CD8+ memory T cells were also generated, and CD8+ T-cell polyfunctionality was also increased in vaccinated individuals [13]. There were no signs of regulatory T-cell induction which might suppress an anti-HCV immune response [13]. The viral heterogeneity and high mutation rate of HCV are always potential biological barriers to developing protective HCV vaccine. ChAd3/MVA vaccine generates cross-reactive T-cell responses between heterologous viral genotypes so may be tested in diverse HCV populations [13]. Now, this vaccine is under consideration for a larger phase II clinical trials to further determine the efficacy of the vaccine.

Another therapeutic T-cell-based vaccine (GI-5005) is in clinical development which contains a fusion of HCV structural "core" and nonstructural "NS3" protein in yeast vector. The therapeutic efficacy of the vaccine was tested in both treatment-naïve and prior null responders to PEG-IFNα/RBV treatment in phase IIb clinical trials [99, 100]. Overall, 133 HCV genotype 1-infected patients (96 treatment-naïve patients) were administered to once monthly dose of vaccine along with PEG-IFNα plus RBV vs. placebo (PEG-IFNα/RBV) for 48 weeks [100]. SVR rates were reported slightly higher in vaccinated patient's arm than the patients in placebo (47 % vs. 35 %, respectively) [100]. However, the achieved SVR rates were not statistically significant. The same trends in SVR rates were reported for the patients who were stratified by their prior treatment status. In that case, the previous null responders of PEG-IFNα plus RBV therapy achieved SVR rates only 17 % upon vaccination as compared to control studies who received PEG-IFN/RBV (SVR 5 % only) [101]. At subtype levels, treatment-naïve patients with unfavorable IL28B TT polymorphism met SVR24 in vaccinated patient's arm as compared to those who treated with PEG- IFNα plus RBV alone [100]. The study was further expanded to 17 additional treatment-naïve patients with IL28B TT polymorphism, and the results were compared to the original 10 IL28B TT patients from the first cohort [100]. An undetectable HCV RNA level at the end of treatment was reported in 10 out of 16 patients (63 %) who received GI-5005 as compared to 3 out of 11 patients (27 %) who were treated with PEG-IFNα plus RBV alone [100].

Another recombinant poxvirus vaccine (TG-4040) expressing HCV nonstructural proteins (NS3, NS4, and NS5B) is in phase I clinical trials and demonstrated significantly higher SVR rates in treated patients [102]. Some other potential anti-HCV vaccine models are also in the pipeline, including T-cell-based peptide vaccines, recombinant HCV subunit vaccines, and pseudo-viral particles expressing HCV glycoproteins (E1 and E2) [103, 104]. Preclinical studies of some of these vaccine models have shown spectacular results, but a lot of further clinical studies are required to evaluate their therapeutic outcomes.

## **8.4. Clinical issues regarding HCV vaccine trials**

No doubt, much work is being done on the development of an effective anti-HCV vaccine model, but all the efforts revolve around HCV genotype 1 infection. We know very well that more than 100 million people worldwide are infected with HCV genotype 3, 4, 5, and 6 infections, and in some countries like Egypt, the infection is almost an endemic [11]. It may indicate that the cross-reactive protective immunity induced by a proposed HCV vaccine model against one genotype would not be sufficient for selecting the best candidate HCV vaccine for the whole world. The other potential challenge is the lack of vaccine clinical trials in a high-risk population where some other factors are responsible for HCV transmission. In the United States and the Western world, where only 1–2 % general population are infected with HCV, more than 100,000 infected individuals have to be enrolled in clinical trials to determine vaccine efficacy[94]. The situation is different in developing world where HCV prevalence depends on various predisposing factors including injection drug users (IDUs), blood transfusion without screening anti-HCV antibodies, unsterilized medical instruments use, piercing of ears and nose, unprotected sexual act, and healthcare workers. Such regions represent an alternative place to conduct HCV vaccine clinical trials. Similarly, in a highly endemic area (e.g., Egypt, where almost 22 % of the country's population have afflicted with HCV), preventive or protective vaccine trials may initiate. Unfortunately, the test results would be genotype specific in that area (i.e., HCV genotype 4 is the most prevalent genotype in Egypt and the Middle East) and may not apply to the other parts of the world. The lack of basic health infrastructure and intrinsic ethical and drug regulatory issues are also potential challenges in developing countries to initiate vaccine clinical trials.

Designing of vaccine clinical trials in some concrete and high-risk populations also depends on certain crucial factors including HCV prevalence, exposure frequency of the infection, viral infectivity, and infection chronicity. The infection rate should be reduced up to 50 % in the appropriate population size for the acceptable vaccine efficacy. By assigning a certain value to the above mentioned parameters in a high-risk population, at least, 500–10,000 individuals are required in vaccine clinical trials [105]. Although this number can be managed easily with the high-risk population and in HCV endemic area, but the testing and handling of large candidate vaccines pose particular challenges in this context. The ultimate success and realistic goal of HCV vaccines must be to prevent the chronic infection in infected individuals. To achieve this objective, the term "chronic HCV infection" must be clearly defined and should not rely on the classical definition based on chronic HBV infection (i.e., the persistence of HCV viral replication more than 6 months detected quantitatively by polymerase chain reaction). Some studies have demonstrated that during acute HCV infection, viral RNA fluctuates markedly from undetectable limit to a higher level of quantification (106 IU/ml) [106]. In such acutely infected individuals, the viral clearance may not occur until 1 year later. Consequently, the clinical trials for HCV vaccines need longer test duration (approximately 2 years). Withholding therapy is another ethical issue while treating acutely infected patients with PEG-IFNα and RBV after a close follow-up of 6–9 months [107]. Development of surrogate biomarkers and their validation to evaluate vaccine efficacy is also challenging. In all clinical trials, a standard methodology should be applied to compare the vaccine efficacy results with each other especially in high-risk populations.

One of the big concerns which is always debatable among doctors, researchers, public healthcare workers, policy makers, and patient groups is how to implement a successful vaccine program when once effective vaccines would be available in the future. In this scenario, costeffective analysis, careful monitoring of the adverse effects, and compliance with futility rules must be extensively scrutinized before issuing public health policies regarding mass vaccination program [108]. Implementation of a universal HCV vaccination program instead of targeting high-risk populations would be more appropriate and have a profound impact in some developing countries where HCV is highly prevalent.

## **9. Conclusions**

rates in treated patients [102]. Some other potential anti-HCV vaccine models are also in the pipeline, including T-cell-based peptide vaccines, recombinant HCV subunit vaccines, and pseudo-viral particles expressing HCV glycoproteins (E1 and E2) [103, 104]. Preclinical studies of some of these vaccine models have shown spectacular results, but a lot of further clinical

No doubt, much work is being done on the development of an effective anti-HCV vaccine model, but all the efforts revolve around HCV genotype 1 infection. We know very well that more than 100 million people worldwide are infected with HCV genotype 3, 4, 5, and 6 infections, and in some countries like Egypt, the infection is almost an endemic [11]. It may indicate that the cross-reactive protective immunity induced by a proposed HCV vaccine model against one genotype would not be sufficient for selecting the best candidate HCV vaccine for the whole world. The other potential challenge is the lack of vaccine clinical trials in a high-risk population where some other factors are responsible for HCV transmission. In the United States and the Western world, where only 1–2 % general population are infected with HCV, more than 100,000 infected individuals have to be enrolled in clinical trials to determine vaccine efficacy[94]. The situation is different in developing world where HCV prevalence depends on various predisposing factors including injection drug users (IDUs), blood transfusion without screening anti-HCV antibodies, unsterilized medical instruments use, piercing of ears and nose, unprotected sexual act, and healthcare workers. Such regions represent an alternative place to conduct HCV vaccine clinical trials. Similarly, in a highly endemic area (e.g., Egypt, where almost 22 % of the country's population have afflicted with HCV), preventive or protective vaccine trials may initiate. Unfortunately, the test results would be genotype specific in that area (i.e., HCV genotype 4 is the most prevalent genotype in Egypt and the Middle East) and may not apply to the other parts of the world. The lack of basic health infrastructure and intrinsic ethical and drug regulatory issues are also potential challenges in

Designing of vaccine clinical trials in some concrete and high-risk populations also depends on certain crucial factors including HCV prevalence, exposure frequency of the infection, viral infectivity, and infection chronicity. The infection rate should be reduced up to 50 % in the appropriate population size for the acceptable vaccine efficacy. By assigning a certain value to the above mentioned parameters in a high-risk population, at least, 500–10,000 individuals are required in vaccine clinical trials [105]. Although this number can be managed easily with the high-risk population and in HCV endemic area, but the testing and handling of large candidate vaccines pose particular challenges in this context. The ultimate success and realistic goal of HCV vaccines must be to prevent the chronic infection in infected individuals. To achieve this objective, the term "chronic HCV infection" must be clearly defined and should not rely on the classical definition based on chronic HBV infection (i.e., the persistence of HCV viral replication more than 6 months detected quantitatively by polymerase chain reaction). Some studies have demonstrated that during acute HCV infection, viral RNA fluctuates markedly from undetectable limit to a higher level of quantification (106 IU/ml) [106]. In such acutely infected individuals, the viral clearance may not occur until 1 year later. Consequently, the clinical trials for HCV

studies are required to evaluate their therapeutic outcomes.

**8.4. Clinical issues regarding HCV vaccine trials**

306 Advances in Treatment of Hepatitis C and B

developing countries to initiate vaccine clinical trials.

The development of novel direct-acting antivirals has simplified the treatment paradigm for chronically infected and difficult-to-treat HCV genotype populations around the globe. These game changer regimens have revolutionized the HCV therapeutics regarding pan-genotypic coverage, low pill burden, fewer drug-drug interactions, improved adverse event profile, and high barrier to drug resistance. However, the treatment cost and accessibility of the drugs to infected patients are major issues which must be resolved to get full therapeutic benefits of such therapeutic regimens. Interferon lambda is promising as more efficacious, well tolerated, and short treatment of duration and has been entered into phase III clinical trials with several direct-acting antivirals. It may be beneficial as an adjuvant therapy in IFN-α-intolerant patients as well as in individuals where IL28B genetic polymorphism is highly associated to lower SVR rates. The steadily improved DAAs are playing a frontline role to surmount the burden of HCV around the globe, but the development and implementation of a successful HCV vaccine program would be mandatory to win an uphill battle against this silent epidemic. In this context, the core knowledge of intricate interplays between molecular and cellular immune responses toward HCV, viral clearance and persistence, and long-lasting immune responses would play a significant role to develop an effective protective HCV vaccine model. Adenovirus-based vector vaccines have shown promising results while generating durable, broad, sustained, and balanced innate and acquired immune response in chimpanzees and humans. A heterologous T-cell vaccine (ChAd3/MVA) has also shown very high levels of T-cell responses against multiple HCV antigens in HCV genotype 1b patients. If HCV vaccines are available in the near future, then mass vaccination program in high-risk populations would probably have a profound impact on eradicating HCV infection. The technology and scientific innovation definitely play its part, but the role of scientific community, implementation of controlled HCV healthcare policies, applications of risk prediction tools, collective will, and public health notes will galvanize the efforts to a proper ending of this silent epidemic from the world. Thus, this two-pronged attack on HCV—a variety of novel direct-acting antivirals and the possibility of a vaccine—suggests the global eradication of HCV. Overall, the achievements and improvements in the field of HCV medicine predict that the future of HCV therapeutics is bright and becoming brighter every day.

## **Author details**

Imran Shahid1, 2 \*, Waleed H. AlMalki1 , Mohammed W. AlRabia3 , Muhammad H. Hafeez<sup>4</sup> and Muhammad Ahmed<sup>1</sup>

\*Address all correspondence to: iyshahid@uqu.edu.sa

1 Department of Pharmacology and Toxicology, College of Pharmacy, Umm Al-Qura University, Makkah, Saudi Arabia

2 Applied and Functional Genomics Laboratory, Center of Excellence in Molecular Biology (CEMB), University of the Punjab, Lahore, Pakistan

3 Department of Medical Microbiology, College of Medicine, King Abdul Aziz University, Jeddah, Saudi Arabia

4 Department of Gastroenterology and Hepatology, Fatima Memorial College of Medicine and Dentistry, Shadman, Lahore, Pakistan

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direct-acting antivirals and the possibility of a vaccine—suggests the global eradication of HCV. Overall, the achievements and improvements in the field of HCV medicine predict that

, Mohammed W. AlRabia3

Department of Pharmacology and Toxicology, College of Pharmacy, Umm Al-Qura

Applied and Functional Genomics Laboratory, Center of Excellence in Molecular Biology

Department of Medical Microbiology, College of Medicine, King Abdul Aziz University,

Department of Gastroenterology and Hepatology, Fatima Memorial College of Medicine

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**Author details**

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Muhammad Ahmed<sup>1</sup>

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**Provisional chapter**

#### **Importance of MicroRNAs in Hepatitis B and C Diagnostics and Treatment Importance of MicroRNAs in Hepatitis B and C Diagnostics and TreatmentImportance of MicroRNAs in Hepatitis B and C Diagnostics and Treatment**

Mateja M. Jelen and Damjan Glavač Mateja Damjan GlavačMateja M. Jelen and Damjan Glavač

Additional information is available at the end of the chapter Additional information is available at the end of the Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66498

#### **Abstract**

MicroRNAs (miRNAs) are small‐sized RNAs with ability to regulate gene expression and have been recently discovered as promising diagnostic and therapeutic biomarkers in the field of clinical medicine and microbiology, specifically in viral diseases. Infections with hepatitis B virus (HBV) or hepatitis C virus (HCV) often lead to chronic infections and development of liver hepatocellular carcinoma (HCC). Challenges in early diag‐ nosis of HCC and rapid development of novel HCV antivirals call for identification of novel miRNA biomarkers. An extensive selection of single miRNAs and miRNA pan‐ els has been provided by accumulating studies, discovering miRNA potentials in HBV and HCV diagnostics and treatment. Currently, the diagnostic potential of miRNAs in HBV and HCV has not been established yet. However, a promising HCV treatment drug Miravirsen, a locked nucleic acid, complementary to miRNA‐122, has entered a human clinical trial recently. In this review, we outline the role of miRNAs in HBV and HCV pathogenesis and differences in up‐ and downregulation of miRNAs upon HBV and HCV infection and HCC development.

**Keywords:** microRNA, hepatitis B virus, hepatitis C virus, diagnosis, treatment

## **1. Introduction**

Hepatitis B and C viruses (HBV and HCV) are globally spread hepatotropic pathogens and major etiological factors of liver cirrhosis and hepatocellular carcinoma (HCC), infecting mil‐ lions of people worldwide. HBV and HCV significantly differ in structure, genomic charac‐ teristics, and pathogenesis [1, 2].

and reproduction in any medium, provided the original work is properly cited.

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed the terms of the Creative License (http://creativecommons.org/licenses/by/3.0), unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, MicroRNAs (miRNAs) are small noncoding RNAs that control gene expression and par‐ ticipate in complex cellular pathways and pathogenesis of various viral infections and cancers [3–5].

Despite the availability of prophylactic HBV vaccine and recently improved HCV therapy strategies, early diagnosis of HBV/HCV‐related HCC, viral reactivation, resistance, drug interactions, and viral interferences in HBV/HCV co‐infected patients remain major obstacles in currently available HBV and HCV diagnostics and treatments [6, 7].

Multiple studies have proposed specific miRNA or miRNA panels to be used as biomarkers for HBV/HCV‐related liver disease, staging of liver disease progression, and anti‐HBV/HCV therapeutic options.

In this chapter, we briefly outline the HBV and HCV biology and basis of miRNA expression in liver and HCC. Subsequently, we summarize recently described and proposed miRNAs for HBV‐ and HCV‐associated diagnostics, particularly HBV/HCV‐related HCC.

## **2. Hepatitis B virus**

Hepatitis B virus is an enveloped, partially double‐stranded DNA virus, classified in the *Hepadnaviridae* family. HBV genome is approximately 3.2 kb long and contains four RNA transcripts (P, S, C, and X), of which the S transcript encodes the surface antigen HBsAg, the main indicator of HBV infection [1]. Upon infection, cellular polymerase converts HBV DNA into covalently closed circular DNA (cccDNA), which represents a constant template for pregenomic RNA and mRNA transcription. Due to the stable state of HBV cccDNA in hepatocytes, HBV can reactivate after immunosuppression [1, 8].

HBV is classified into eight genotypes (designated A through H). Most chronic infections are related to infection with HBV genotypes B and C; however, in Europe, genotype D has been shown to be more often associated with active liver disease [1].

Globally, two billion people are infected with HBV, with majority of HBV infections occur‐ ring in South‐Eastern Asia and sub‐Saharan Africa [9, 10]. Approximately, one‐fourth of HBV‐infected individuals suffer from liver cirrhosis and 70–90% of HCC develop from cir‐ rhotic liver [1, 10]. In developing countries, HBV infection accounts for about 60% of the total liver cancer and in developed countries for about 23% [11]. In endemic areas, HBV infections occur mostly by vertical and perinatal transmission and in more than 90% lead to chronic HBV infections. In low prevalence countries, HBV infections occur mostly through a horizontal transfer (sexual transmission), which in more than 90% lead to acute HBV infec‐ tions with spontaneous viral clearance [10]. Despite improved HBV antiviral therapy over the past two decades, and the prophylactic anti‐HBV vaccine, available since 1981, prena‐ tal maternal HBeAg seropositivity in endemic countries remains significantly connected to HCC and elimination of cccDNA remains the major challenge in HBV‐related treatment strategies [1, 12, 13].

## **3. Hepatitis C virus**

MicroRNAs (miRNAs) are small noncoding RNAs that control gene expression and par‐ ticipate in complex cellular pathways and pathogenesis of various viral infections and

Despite the availability of prophylactic HBV vaccine and recently improved HCV therapy strategies, early diagnosis of HBV/HCV‐related HCC, viral reactivation, resistance, drug interactions, and viral interferences in HBV/HCV co‐infected patients remain major obstacles

Multiple studies have proposed specific miRNA or miRNA panels to be used as biomarkers for HBV/HCV‐related liver disease, staging of liver disease progression, and anti‐HBV/HCV

In this chapter, we briefly outline the HBV and HCV biology and basis of miRNA expression in liver and HCC. Subsequently, we summarize recently described and proposed miRNAs for

Hepatitis B virus is an enveloped, partially double‐stranded DNA virus, classified in the *Hepadnaviridae* family. HBV genome is approximately 3.2 kb long and contains four RNA transcripts (P, S, C, and X), of which the S transcript encodes the surface antigen HBsAg, the main indicator of HBV infection [1]. Upon infection, cellular polymerase converts HBV DNA into covalently closed circular DNA (cccDNA), which represents a constant template for pregenomic RNA and mRNA transcription. Due to the stable state of HBV cccDNA in

HBV is classified into eight genotypes (designated A through H). Most chronic infections are related to infection with HBV genotypes B and C; however, in Europe, genotype D has been

Globally, two billion people are infected with HBV, with majority of HBV infections occur‐ ring in South‐Eastern Asia and sub‐Saharan Africa [9, 10]. Approximately, one‐fourth of HBV‐infected individuals suffer from liver cirrhosis and 70–90% of HCC develop from cir‐ rhotic liver [1, 10]. In developing countries, HBV infection accounts for about 60% of the total liver cancer and in developed countries for about 23% [11]. In endemic areas, HBV infections occur mostly by vertical and perinatal transmission and in more than 90% lead to chronic HBV infections. In low prevalence countries, HBV infections occur mostly through a horizontal transfer (sexual transmission), which in more than 90% lead to acute HBV infec‐ tions with spontaneous viral clearance [10]. Despite improved HBV antiviral therapy over the past two decades, and the prophylactic anti‐HBV vaccine, available since 1981, prena‐ tal maternal HBeAg seropositivity in endemic countries remains significantly connected to HCC and elimination of cccDNA remains the major challenge in HBV‐related treatment

in currently available HBV and HCV diagnostics and treatments [6, 7].

HBV‐ and HCV‐associated diagnostics, particularly HBV/HCV‐related HCC.

hepatocytes, HBV can reactivate after immunosuppression [1, 8].

shown to be more often associated with active liver disease [1].

cancers [3–5].

322 Advances in Treatment of Hepatitis C and B

therapeutic options.

**2. Hepatitis B virus**

strategies [1, 12, 13].

Hepatitis C virus has a positive‐sense single‐stranded RNA genome of approximate length of 9.6 kb and is classified in the family *Flaviviridae*. The HCV genome encodes a polyprotein that is cleaved into three structural and seven nonstructural proteins. Based on HCV genomic sequence diversity, HCV is classified into seven genotypes and more than 60 subtypes are identified [2, 14]. Genotypes 1, 2, and 3 are globally distributed and cause most of HCV infec‐ tions in North America and Europe. In Middle East and North and Central Africa, genotype 4 prevails generally, in South Africa genotype 5, in Asia genotype 6, while a recently discovered genotype 7 originates from Central Africa [2, 15].

Currently, no vaccine is available for the prevention of HCV infection. Most HCV transmis‐ sions occur by intravenous drug abuse, sexual transmission, and occupational exposure to HCV‐infected blood [2]. Potential long‐term outcomes in chronically HCV‐infected people are liver cirrhosis and HCC, which remain the leading cause for liver transplantation. HCV is globally infecting over 150 million individuals [16–18]. Recent estimate on global anti‐HCV prevalence is 115 million‐infected individuals, of whom 80 million are actively viremic [19].

A recent study by Sibley et al. [20] forecasted changes in HCV‐related disease up to 2030 and concluded that HCV‐related morbidity and mortality are estimated to increase due to an aging of the HCV‐infected population and currently available treatment will be inadequate if reductions in HCV‐related disease of this magnitude are to be achieved [20].

## **4. MicroRNAs**

Micro‐ribonucleic acids (microRNAs/miRNAs) are noncoding RNAs of 18–25 nucleotides in length that complementarily target the 3′‐untranslated regions (3′ UTRs), or less commonly 5′‐untranslated regions (5′ UTRs) of messenger RNAs (mRNAs) [3]. Genes encoding miRNAs are located in intragenic regions or introns of mRNAs or noncoding RNAs [21]. MiRNAs are transcribed from the genome by the RNA polymerase II into primary‐miRNA (pri‐miRNA) hairpins, which are processed by Drosha (class III RNase) into pre‐miRNAs. Pre‐miRNAs are exported from the nucleus to the cytoplasm, where they are processed by a second RNaseIII Dicer into short double‐stranded mature miRNAs, consisting of 5' and 3' arms. Finally, single‐ stranded miRNAs are assembled with specific proteins and form a RNA‐induced‐silencing complex (RISC). At least two to seven nucleotide complementarities with the target sequence are required for RISC‐mediated target silencing [22–24].

The binding of miRNAs in posttranscriptional or translational level provides a rapid and sensitive mechanism of gene expression regulation, either by suppressing the translation of mRNA or by promoting mRNA degradation [25]. Gene silencing by a full complementary miRNA sequence directs cleavage of the target mRNA, while partial complementary miRNA sequence suppresses mRNA translation [26, 27]. Currently, more than 2,588 mature miRNAs are reported in a human genome [28, 29] and due to sufficient partial complementarity to the target sequence it has been shown that one type of miRNA could affect up to 200 genes, and over a 100 different targets can be involved in approximately 100 different biochemical pathways [30, 31].

## **4.1. MiRNAs mechanism**

MiRNAs are participating in various cellular processes such as cell development, differen‐ tiation, proliferation, metabolism, immune responses, apoptosis, and oncogenesis [32, 33]. Estimates in humans suggest that 60–70% of all genes are regulated by miRNAs [4]. Being involved in numerous biological pathways, their expression and regulation reflect in various diseases, stages of the particular disease, especially in cancer development [34, 35].

MiRNAs are cell‐free‐circulating molecules that can be detected in almost every body fluid. Their high stability and accessibility make them ideal noninvasive markers for the early diagnosis of different pathophysiological processes. Indeed, a large amount of evidence sug‐ gests that miRNA profiles could provide a classification system for various tumors, as well as an important tool for the diagnosis and treatment of cancer and viral diseases [36–41]. Accordingly, cellular miRNAs have an ability to regulate pathogenesis of viral infections, and at the same time viruses manipulate with host cellular machinery, including miRNAs [42, 43].

Increased interest in hepatitis B and hepatitis C disease pathogenesis and diagnostics has lead to the emergence of various studies over the last 15 years that have tried to evaluate plasma and tissue levels of miRNAs in order to provide or improve the diagnosis of HBV and HCV infections as well as HBV‐ and HCV‐related HCC [5].

## **5. MiRNAs in normal liver**

Liver consists of various cell types, mainly divided in the parenchymal cells (hepatocytes) and non‐parenchymal cells (biliary epithelial cells and lymphoid cells, etc.). Each cell type expresses its unique miRNA profile. While miRNAs are up‐ or downregulated in almost every stage of hepatic development, they accelerate or inhibit liver proliferation and play the major role in regulation of diverse liver functions [44]. It has been shown that a total of 277 miRNAs are expressed in the liver, with miR‐122 being one of the most abundant and liver‐ specific miRNAs [45, 46]. Besides miR‐192, miR‐199a/b‐3p, miR‐101, miR‐99a, and let‐7a/b/c/f (let‐7 family), which account for 80% of the total miRNA in liver, miR‐122 accounts for 70% of the total liver miRNAs [45, 47]. Expression of miRNAs in the normal liver has been estab‐ lished by microarray systems and library sequencing [47–49].

## **5.1. Function of miRNAs in normal liver**

The function of miR‐122 has been explored in a variety of *in vivo* studies, including the miR‐122 gene knockdown or silencing of miR‐122 with antagomirs [50–52]. In the miR‐122 gene knock‐ down mice, it has been shown that the deletion of the miR‐122 gene resulted in hepatosteato‐ sis, hepatitis, and the development of liver tumors [52]. Beyond that, studies implicate miR‐122 as a key regulator of cholesterol and fatty‐acid metabolism [50]. However, results of studies evaluating up‐ and downregulated miRNA profiles in differentiating liver cells are not con‐ sistent. Besides various technical issues, including differences in clinical samples and different miRNA matrices in miRNA assays, different degrees of miRNA expression among studies suggest that the miRNA profile is also influenced by the origin of the progenitor cell and that it is difficult to compare miRNA profiles in different cellular developmental stages [53].

However, it has been shown in two studies by Liu et al. [54] and Tzur et al. [55] that embry‐ onic liver mainly contains miRNAs‐122, ‐192, ‐194, ‐451, and ‐483‐3p, whereas miR‐122 can be detected in embryonic stem cells as well as in hepatocytes and continues to be expressed in the adulthood. Studies of hepatic malignancy pathologies have shown that miRNAs have specific targets in specific disease states [44, 53, 56]. Interestingly, a biphasic pattern of miRNA expres‐ sion was observed in rats after liver surgical resection [57]. In the first 18 h after hepatectomy, about 40% of miRNAs were upregulated, whereas by 24 h there was a negative feedback mechanism which downregulated about 70% of all miRNAs [57]. These negative feedback loops are postulated to play an important role in liver regeneration processes, required for recovery of liver cells after injury; however, the abundance of miRNAs does not directly cor‐ relate with the predictable role of specific cells.

Since essential knowledge on liver regeneration processes has been delivered from rodent model studies, further studies are warranted to confirm post‐hepatectomy miRNA level changes in humans [4, 57]. Investigation of miRNA levels in specific stages of liver organo‐ genesis may reveal potential biomarkers for liver disease states.

## **6. miRNA and HBV‐ and HCV‐related liver disease**

the target sequence it has been shown that one type of miRNA could affect up to 200 genes, and over a 100 different targets can be involved in approximately 100 different biochemical

MiRNAs are participating in various cellular processes such as cell development, differen‐ tiation, proliferation, metabolism, immune responses, apoptosis, and oncogenesis [32, 33]. Estimates in humans suggest that 60–70% of all genes are regulated by miRNAs [4]. Being involved in numerous biological pathways, their expression and regulation reflect in various

MiRNAs are cell‐free‐circulating molecules that can be detected in almost every body fluid. Their high stability and accessibility make them ideal noninvasive markers for the early diagnosis of different pathophysiological processes. Indeed, a large amount of evidence sug‐ gests that miRNA profiles could provide a classification system for various tumors, as well as an important tool for the diagnosis and treatment of cancer and viral diseases [36–41]. Accordingly, cellular miRNAs have an ability to regulate pathogenesis of viral infections, and at the same time viruses manipulate with host cellular machinery, including miRNAs [42, 43]. Increased interest in hepatitis B and hepatitis C disease pathogenesis and diagnostics has lead to the emergence of various studies over the last 15 years that have tried to evaluate plasma and tissue levels of miRNAs in order to provide or improve the diagnosis of HBV and HCV

Liver consists of various cell types, mainly divided in the parenchymal cells (hepatocytes) and non‐parenchymal cells (biliary epithelial cells and lymphoid cells, etc.). Each cell type expresses its unique miRNA profile. While miRNAs are up‐ or downregulated in almost every stage of hepatic development, they accelerate or inhibit liver proliferation and play the major role in regulation of diverse liver functions [44]. It has been shown that a total of 277 miRNAs are expressed in the liver, with miR‐122 being one of the most abundant and liver‐ specific miRNAs [45, 46]. Besides miR‐192, miR‐199a/b‐3p, miR‐101, miR‐99a, and let‐7a/b/c/f (let‐7 family), which account for 80% of the total miRNA in liver, miR‐122 accounts for 70% of the total liver miRNAs [45, 47]. Expression of miRNAs in the normal liver has been estab‐

The function of miR‐122 has been explored in a variety of *in vivo* studies, including the miR‐122 gene knockdown or silencing of miR‐122 with antagomirs [50–52]. In the miR‐122 gene knock‐ down mice, it has been shown that the deletion of the miR‐122 gene resulted in hepatosteato‐ sis, hepatitis, and the development of liver tumors [52]. Beyond that, studies implicate miR‐122

diseases, stages of the particular disease, especially in cancer development [34, 35].

infections as well as HBV‐ and HCV‐related HCC [5].

lished by microarray systems and library sequencing [47–49].

**5.1. Function of miRNAs in normal liver**

**5. MiRNAs in normal liver**

pathways [30, 31].

**4.1. MiRNAs mechanism**

324 Advances in Treatment of Hepatitis C and B

HCC is the fifth most common cancer worldwide. Deregulation of miRNA expression could be one of the key factors in the development of liver pathology, including viral hepatitis and HCC [11]. Evidence is rapidly growing that specific miRNAs could be used as potential bio‐ markers for HCC, tumor progression, and response to therapeutic targets [4].

Infection with HBV and HCV can lead to chronic hepatitis, liver cirrhosis, or even HCC. Approximately 50–80% of HCC cases are associated with chronic HBV or HCV infection [10]. In the past several years, the involvement of miRNA in the pathogenesis of HBV‐/HCV‐ related liver diseases has been well documented [53]. Since miRNAs can be directly involved in antiviral immune‐pathological events, it is inevitable that miRNA target sequences in viral populations remained conserved, providing relevant evidence of the biological significance of potential miRNA‐based antiviral interventions [58]. For the time being, no HBV‐ or HCV‐ encoded miRNAs have been reported. Using computational approaches, one candidate HBV miRNA has been found but its function remains undetermined [59].

It should be noted that miRNA dysregulation has been studied in various experimental settings, mainly involving *in vitro* systems, HBV‐/HCV‐replication‐supporting cell lines, transgenic mice, cultured hepatocytes (mouse/rat/human), circulating blood cells or serum of HBV‐/HCV‐infected individuals, and liver tissue samples. MiRNA expression in model systems has been measured mainly with qualitative real time‐polymerase chain reactions (RT‐PCRs) and/or miRNA microarrays and less frequently with next‐generation sequencing (NGS) (for review, see [23, 60]).

## **6.1. MiRNAs and HBV infection**

It is well known that numerous cellular miRNAs are able to promote or repress the HBV lifecycle, either by directly targeting HBV transcripts or by indirectly targeting cellular mediators, involved in the HBV pathogenesis. Alternatively, HBV infection dysregulates cellular miRNAs and in this manner controls the host gene expression to promote its rep‐ lication [60].

A variety of miRNAs have been reported in regulation of HBV replication namely miR‐122, let‐7 family, miR‐199 family, miR‐15 family, miR‐125 family, and miR‐17‐92 cluster (exten‐ sively reviewed in [60]). Reported effects of the most abundant liver miRNA, miR‐122, on HBV lifecycle remain mixed. Whereas some studies reported miRNAs as inhibitors of HBV replication [61–64], others have failed to even identify miR‐122 as a regulator of the HBV life‐ cycle [65]. For example, in a study by Qiu et al. [63], in comparison to the control system, co‐ transfection of Huh7 cells with miR‐122 inhibitor, and a plasmid encoding the HBV genome, the production of HBsAg and HBeAg increased, suggesting a negative regulatory effect of miR‐122 on HBV lifecycle.

The study of Wang et al. [64] reported downregulation of miR‐122 expression in liver of patients with HBV infection, in comparison to healthy controls, and showed that the miR‐122 levels were negatively correlated with intrahepatic viral load and hepatic inflammation. Researchers concluded that HBV‐induced miR‐122 downregulation enhances HBV replica‐ tion through cyclin G1‐modulated P53 activity and that HBV mRNAs harboring complemen‐ tary sites to miR‐122 sequester miR‐122 and contribute to viral persistence and carcinogenesis [61, 64].

Guo et al. [66] showed that members of the miRs‐371‐372‐373 (miRs‐371‐3) gene cluster were co‐upregulated in HBV‐producing HepG2.2.15 cells and revealed that miRs‐372/373 pro‐ motes HBV expression by targeting the transcription factor NFIB. Furthermore, Zhang et al. [65] reported that miR‐1 promotes HBV replication, transcription, and antigen expression by indirect modulation of host genes expression in HCC cell line. In addition, they have shown that miR‐1 arrested cell cycle, inhibited proliferation, and therefore reversed the cancer cell phenotype, which is in contradiction with HBV‐induced carcinogenesis, since HBV infection promotes hepatocellular proliferation [65].

Jin et al. [67] have shown that downregulation of miR‐501 in HepG2.2.15 cells could signifi‐ cantly inhibit HBV replication, thus representing a potential therapeutic target. They sug‐ gested that miR‐501 promotes HBV replication through inhibition of the HBXIP, which interacts with HBx protein and normally represses HBV replication [67]. In a mouse model, Dai et al. [68] reported that miR‐15b promoted HBV replication by direct inhibition of hepa‐ tocyte nuclear factor HNF1α.

On the other hand, miR‐125 family members, miR‐125a and miR‐125b, have been reported to suppress the HBV lifecycle. In the PLC/PRF/5 cell line, miR‐125a‐5p was identified as a down‐ regulator of HBsAg expression by directly targeting HBV RNAs [69], while miR‐125b inhibited HBV in HepG2.2.15 cells [70]. The miR‐22 has been reported as a regulatory molecule which inhibits HBV infection [71]. Furthermore, miR‐199a‐3p and miR‐210 were shown to repress the HBV replication in HepG2.2.15 cells by directly targeting the HBV S protein‐coding region [72], while inhibition of miR‐20 and miR‐92a‐1 increased levels of HBV RNAs in HepAD38 hepa‐ toma cells [73]. Hu et al. [74] demonstrated that miR‐141 suppresses HBV replication by reduc‐ ing HBV promoter activities and two separate studies suggested upregulation of miR‐181a in HBV‐infected hepatoma cells, implying an important role in the development of HCC [75, 76].

## **6.2. MiRNAs and HCV infection**

of HBV‐/HCV‐infected individuals, and liver tissue samples. MiRNA expression in model systems has been measured mainly with qualitative real time‐polymerase chain reactions (RT‐PCRs) and/or miRNA microarrays and less frequently with next‐generation sequencing

It is well known that numerous cellular miRNAs are able to promote or repress the HBV lifecycle, either by directly targeting HBV transcripts or by indirectly targeting cellular mediators, involved in the HBV pathogenesis. Alternatively, HBV infection dysregulates cellular miRNAs and in this manner controls the host gene expression to promote its rep‐

A variety of miRNAs have been reported in regulation of HBV replication namely miR‐122, let‐7 family, miR‐199 family, miR‐15 family, miR‐125 family, and miR‐17‐92 cluster (exten‐ sively reviewed in [60]). Reported effects of the most abundant liver miRNA, miR‐122, on HBV lifecycle remain mixed. Whereas some studies reported miRNAs as inhibitors of HBV replication [61–64], others have failed to even identify miR‐122 as a regulator of the HBV life‐ cycle [65]. For example, in a study by Qiu et al. [63], in comparison to the control system, co‐ transfection of Huh7 cells with miR‐122 inhibitor, and a plasmid encoding the HBV genome, the production of HBsAg and HBeAg increased, suggesting a negative regulatory effect of

The study of Wang et al. [64] reported downregulation of miR‐122 expression in liver of patients with HBV infection, in comparison to healthy controls, and showed that the miR‐122 levels were negatively correlated with intrahepatic viral load and hepatic inflammation. Researchers concluded that HBV‐induced miR‐122 downregulation enhances HBV replica‐ tion through cyclin G1‐modulated P53 activity and that HBV mRNAs harboring complemen‐ tary sites to miR‐122 sequester miR‐122 and contribute to viral persistence and carcinogenesis

Guo et al. [66] showed that members of the miRs‐371‐372‐373 (miRs‐371‐3) gene cluster were co‐upregulated in HBV‐producing HepG2.2.15 cells and revealed that miRs‐372/373 pro‐ motes HBV expression by targeting the transcription factor NFIB. Furthermore, Zhang et al. [65] reported that miR‐1 promotes HBV replication, transcription, and antigen expression by indirect modulation of host genes expression in HCC cell line. In addition, they have shown that miR‐1 arrested cell cycle, inhibited proliferation, and therefore reversed the cancer cell phenotype, which is in contradiction with HBV‐induced carcinogenesis, since HBV infection

Jin et al. [67] have shown that downregulation of miR‐501 in HepG2.2.15 cells could signifi‐ cantly inhibit HBV replication, thus representing a potential therapeutic target. They sug‐ gested that miR‐501 promotes HBV replication through inhibition of the HBXIP, which interacts with HBx protein and normally represses HBV replication [67]. In a mouse model, Dai et al. [68] reported that miR‐15b promoted HBV replication by direct inhibition of hepa‐

(NGS) (for review, see [23, 60]).

326 Advances in Treatment of Hepatitis C and B

**6.1. MiRNAs and HBV infection**

lication [60].

[61, 64].

miR‐122 on HBV lifecycle.

promotes hepatocellular proliferation [65].

tocyte nuclear factor HNF1α.

HCV lifecycle is influenced by host miRNAs in all stages: entry, translation, replication, and assembly [43]. As the HCV genome is single‐stranded RNA, it serves as a template for its rep‐ lication and direct binding site for host miRNAs. Among high number of miRNAs reported to be involved in the regulation of HCV infection and replication, most miRNAs have been documented to directly target the HCV genome: miR‐1, miR‐30, miR‐122, miR‐128, miR‐196, miR‐296, miR‐351, miR‐431, and miR‐448 [77, 78].

Microarray analysis on human hepatoma cells has revealed changed expression profiles of 108 human miRNAs after HCV infection [79]. Furthermore, Liu et al. [79] showed that after acute HCV infection, miR‐122 was downregulated, whereas miR‐296 and miR‐351 were significantly upregulated. In addition, it has been shown that HCV infection upregulated the expression of miR‐192, miR‐194, and miR‐215, whereas the expression of miR‐320 and miR‐491 was downregulated [80]. It was reported that miR‐192/miR‐215 and miR‐491 could enhance HCV replication [80].

For the most abundant miRNA in the liver, miR‐122, it has been demonstrated that it pro‐ motes HCV replication by direct binding to the less commonly used UTR‐binding site, the 5′ UTR site of the HCV RNA, which leads to Argonaute (Ago) protein complex recruitment, stabilization of the viral RNA, and activation of the RNA translation [77, 81, 82]. *In vitro* stud‐ ies have shown that miR‐122 is essential for HCV replication [81].

On the other hand, it has been shown that miR‐122 exhibits anti‐inflammatory and anti‐ tumorigenic properties in mice knockdown studies [52]. Mixed results exist on expression levels of miR‐122 and development of HCC‐ or HCV‐induced HCC. Coulouarn et al. [83] have shown that the loss of miR‐122 expression in liver cancer correlated with HCC progression, whereas in another study, the upregulation of miR‐122 promoted the HCV‐related HCC [84].

The increased expression of miR‐155 in HCV‐infected patients promotes hepatocarcinogenesis and inhibits apoptosis of hepatocytes [85]. Furthermore, the direct effect on HCV replication cycle has been determined in the cell culture system for the miR‐196b, which is complementary to the NS5A region of the HCV genome and is downregulated in HCV‐infected patients. MiR‐196b inhibits HCV replication directly by targeting HCV RNA or indirectly by increasing the expres‐ sion of HMOX1. It has anti‐inflammatory, antioxygenic, and hepatoprotective properties [86].

Some miRNAs can facilitate HCV lifecycle by targeting host proteins involved in innate immunity‐signaling pathways. For example, HCV induced upregulation of miR‐130 blocks expression of interferon stimulatory gene IFITM1, which promotes HCV entry into host cells [87]. Furthermore, miR‐491 promotes HCV replication through inhibition of the PI3 kinase/ Akt pathway, one of the pathways leading to cancerous properties [80]. Studies analyzing circulating miRNA profiles in serum provide novel insights on miRNA expression in HCV pathogenesis [88, 89]. In a study by Shwetha et al. [89], it has been shown that the expression of miR‐134, miR‐198, miR‐320c, and miR‐483‐5p was upregulated in patients infected with HCV 1 and HCV 3 genotypes.

Complex correlation between hepatic expression of abundant liver miRNAs, miR‐122, miR‐126, miR‐136, and miR‐181a, and histopathological and clinical characteristic of HCV‐ infected patients has been reported by Boštjančič et al. [31]. The study included liver biopsies of patients infected with different genotypes (1, 1a, 1b, 3, and 4) and provided an important insight into miRNA expression patterns in different stages and grades of liver disease and revealed association among specific miRNA deregulation and patient gender, serum HCV viral load, presence of steatosis, and mode of HCV transmission [31].

By contrast, another study by Elhelw et al. [90] has demonstrated upregulation of miR‐181a in serum samples of HCV genotype 4‐infected individuals and downregulation in HCV‐infected Huh7 cells. In addition, the inverse correlation between miR‐181a serum levels, viral load, and liver enzymes was observed. Due to complexity of viral and host factors involved in HCV infection and progression to HCV‐induced HCC, multiple clinical parameters should be con‐ sidered and controlled for in the future studies. A systematic approach was recently reported by Oliveira et al. [91]. The study evaluated liver and serum expression of miR‐122 in patients infected with HCV genotypes 1 and 3 to identify possible associations between miR‐122 expression and lipid profiles, HCV viral load, apolipoproteins, and liver enzymes [91].

## **7. MiRNAs as biomarkers in diagnostics and treatment of HBV and HCV**

Early diagnosis and treatment of HCC remain challenging due to the lack of early detection meth‐ ods, limited access to diagnosis, expensive medications and the presence of comorbidities, coin‐ fections, and contraindications due to different host and viral factors. Most of HBV‐/HCV‐infected individuals remain undiagnosed before they seek medical help due to advanced HCC [10, 92].

The gold standard for the etiology of liver diseases is liver biopsy, an invasive method being replaced recently by serological methods and imaging technologies. Current diagnostic tech‐ niques for HCC, which are generally divided into radiological (first‐line diagnostic method is ultrasound) and serological methods (serological marker alpha‐fetoprotein, AFP, and des‐ gamma‐carboxy prothrombin), provide limited reliability [93, 94].

## **7.1. MiRNAs as biomarkers in body fluids**

MiRNAs can be detected in various body fluids, such as plasma, serum, urine, and infected or diseased tissue and may exhibit host responses to the pathogen or other inflammatory processes; however, miRNA levels in body fluids may not necessarily reflect the miRNA level in the infected/diseased tissue, and second, the same miRNA may be upregulated in one state of disease and downregulated in another. To provide optimal management of HBV‐ and HCV‐related diseases, novel surrogate miRNA biomarkers should be considered [41]. A great amount of studies provide promising information on miRNAs as potential diagnostic biomarkers of HBV or HCV infection as well as potential diagnostic and treatment tool in HBV‐/HCV‐related HCC. According to specific miRNA targets and up‐ or downregulation of miRNA expression, potential imitating or antagonistic characteristics of miRNAs could be used in HBV‐/HCV‐related therapy.

## **7.2. HBV miRNA biomarkers**

Some miRNAs can facilitate HCV lifecycle by targeting host proteins involved in innate immunity‐signaling pathways. For example, HCV induced upregulation of miR‐130 blocks expression of interferon stimulatory gene IFITM1, which promotes HCV entry into host cells [87]. Furthermore, miR‐491 promotes HCV replication through inhibition of the PI3 kinase/ Akt pathway, one of the pathways leading to cancerous properties [80]. Studies analyzing circulating miRNA profiles in serum provide novel insights on miRNA expression in HCV pathogenesis [88, 89]. In a study by Shwetha et al. [89], it has been shown that the expression of miR‐134, miR‐198, miR‐320c, and miR‐483‐5p was upregulated in patients infected with

Complex correlation between hepatic expression of abundant liver miRNAs, miR‐122, miR‐126, miR‐136, and miR‐181a, and histopathological and clinical characteristic of HCV‐ infected patients has been reported by Boštjančič et al. [31]. The study included liver biopsies of patients infected with different genotypes (1, 1a, 1b, 3, and 4) and provided an important insight into miRNA expression patterns in different stages and grades of liver disease and revealed association among specific miRNA deregulation and patient gender, serum HCV

By contrast, another study by Elhelw et al. [90] has demonstrated upregulation of miR‐181a in serum samples of HCV genotype 4‐infected individuals and downregulation in HCV‐infected Huh7 cells. In addition, the inverse correlation between miR‐181a serum levels, viral load, and liver enzymes was observed. Due to complexity of viral and host factors involved in HCV infection and progression to HCV‐induced HCC, multiple clinical parameters should be con‐ sidered and controlled for in the future studies. A systematic approach was recently reported by Oliveira et al. [91]. The study evaluated liver and serum expression of miR‐122 in patients infected with HCV genotypes 1 and 3 to identify possible associations between miR‐122 expression and lipid profiles, HCV viral load, apolipoproteins, and liver enzymes [91].

**7. MiRNAs as biomarkers in diagnostics and treatment of HBV and HCV**

Early diagnosis and treatment of HCC remain challenging due to the lack of early detection meth‐ ods, limited access to diagnosis, expensive medications and the presence of comorbidities, coin‐ fections, and contraindications due to different host and viral factors. Most of HBV‐/HCV‐infected individuals remain undiagnosed before they seek medical help due to advanced HCC [10, 92].

The gold standard for the etiology of liver diseases is liver biopsy, an invasive method being replaced recently by serological methods and imaging technologies. Current diagnostic tech‐ niques for HCC, which are generally divided into radiological (first‐line diagnostic method is ultrasound) and serological methods (serological marker alpha‐fetoprotein, AFP, and des‐

MiRNAs can be detected in various body fluids, such as plasma, serum, urine, and infected or diseased tissue and may exhibit host responses to the pathogen or other inflammatory

viral load, presence of steatosis, and mode of HCV transmission [31].

gamma‐carboxy prothrombin), provide limited reliability [93, 94].

**7.1. MiRNAs as biomarkers in body fluids**

HCV 1 and HCV 3 genotypes.

328 Advances in Treatment of Hepatitis C and B

Currently used markers in the diagnostics of HBV can serve as indicators of specific HBV infec‐ tion phases; however, none of them can be used to predict the HBV infection outcome [41].

Several studies suggest that the use of miRNA panels in serum or liver tissue could improve the specificity of HBV diagnostics. For example, Li et al. [41] have reported that their 13‐ miRNA‐based biomarker panel could accurately discriminate between HBV cases from healthy controls and HCV cases, as well as HBV‐positive HCC cases from healthy controls and HBV‐infected patients. The panel of 13 miRNA consisted of the following miRNAs: miR‐375, miR‐92a, miR‐10a, miR‐223, miR‐423, miR‐23b/a, miR‐342‐3p, miR‐99a, miR‐122a, miR‐125b, miR‐150, and let‐7c [41] (**Table 1**).

In order to reliably differentiate HCC from chronic HBV infection, cirrhosis, and healthy sub‐ jects, plasma panel of seven miRNAs (miR‐122, miR‐192, miR‐21, miR‐223, miR‐26a, miR‐27a, and miR‐801) has been investigated by Zhou et al. [104]. Mizuguchi et al. [107] employed sequencing‐based miRNA clustering and showed that the panel of miRNAs was more effec‐ tive for the detection of high‐risk patients for HBV‐related HCC recurrence after liver surgery, in comparison to investigation of a single miRNA. MiR‐122, miR‐21, and miR‐34a were identi‐ fied as potential biomarkers for the prediction of HBV‐related HCC.

The miR‐34a is a direct target of the P53 and it has been shown that the expression of miR‐34a is downregulated in several human cancers, and when overexpressed, miR‐34a can repress several oncogenes and induces apoptosis and arrest of the cell cycle. Deletion of gene region encoding miR‐34a has been well detected in breast, lung, cervical, and prostate cancers (reviewed by Agostini and Knight [111]). A mimic miR‐34a (MRX34) became a promising therapeutic tool for HCC and has reached a clinical trial, phase 1 in 2013 [111].

Individual miRNAs or combination of miRNA and serological markers for HCC have been examined and proposed. While an upregulated oncogenic miRNA‐27a has been suggested as a therapeutic target in HBV‐related HCC patients [108] and miR‐101 as a potential noninva‐ sive biomarker to differentiate HBV‐related HCC from HBV liver cirrhosis [97, 112], a combi‐ nation of circulating miR‐126 and AFP has been proposed as a promising noninvasive‐specific diagnostic biomarker for HBV‐related HCC. Furthermore, combinations of miR‐126/AFP AFP and miR‐142‐3p/AFP showed higher efficiency rather than AFP alone in discriminating HCC from non‐HCC patients [99].



**miRNA Deregulation Type of sample Type of method Clinical relevance Reference**

Microarray, qRT‐PCR

Microarray, qRT‐PCR,

Microarray and Northern blotting

Microarray, qRT‐PCR, NGS

Microarray, Northern blotting, qRT‐PCR

qRT‐PCR, microarray Biomarker for monitoring the progression of tumor development in HBV‐ related HCC

Decrease in survival time, increase in the recurrence rate and HCC differentiation in HBV‐related HCC

from healthy, chronic hepatitis and cirrhosis

HBV‐related HCC

HBV‐related HCC

HBV‐related HCC diagnostics

HBV‐related HCC

Potential research interest in chronic HBV infections and HBV‐ induced HCC

in human hepatoma

Important role in HBV‐induced HCC development

HBV‐related HCC

HCC differentiation from healthy, chronic hepatitis and cirrhosis

cells

qRT‐PCR Potential biomarker for

Involved in chronicity of HBV infection

qRT‐PCR, NGS HCC differentiation

Microarray Potential biomarker for

qRT‐PCR Potential biomarker for

Microarray Potential biomarker for

NGS, qRT‐PCR Clinical value in

Zhang et al. [95], Wei et al. [96], Fu et al. [97]

Yen et al. [98]

Li et al. [41]

Ghosh et al. [99]

Wen et al. [100]

Tan et al. [101]

Ghosh et al. [99]

Liu et al. [75], Gui et al. [102], Zhang et al. [95]

Hou et al. [47], Zhang et al. [95]

Su et al. [103]

Liu et al. [75], Zou et al. [76]

Wen et al. [100]

Zhou et al. [104]

HepG2.2.15, HBV‐ HCC tissue, Hep3B, and L02, HBVHCC‐ related serum

serum, liver tissue of HBV‐related HCC

HCC, liver tissue of HCC

related HCC patients

related HCC patients

tissue of HBV‐ related HCC

HepG2.2.15, HBV‐ related HCC and

HepG2.2.15, HBV‐ related HCC, HBV‐ infected serum

HepG2.2.15

related HCC patients

plasma

miR‐155 Up HepG2, H7402 qRT‐PCR Inhibits HBV infection

serum

Up HBV‐infected HCC patients

miR‐101 Up/down HepG2 and

330 Advances in Treatment of Hepatitis C and B

miR‐122a Up HBV‐infected

miR‐126 Up Plasma of HBV

miR‐132‐3p Up Plasma of HBV‐

miR‐141‐3p Up Serum of HBV‐

miR‐142‐3p Up Plasma and liver

miR‐146a Up/down HepG2 and

miR‐146b‐5p Up/down HepG2 and

miR‐181a/b Up HepG2 and

miR‐185‐5p Up Plasma of HBV‐

miR‐192 Up HBV‐related HCC

miR‐106b‐25 cluster (miR‐106b, miR‐93, miR‐25)



**miRNA Deregulation Type of sample Type of method Clinical relevance Reference**

NGS, clone count

qRT‐PCR Potential biomarker for

qRT‐PCR Potential biomarker for

qRT‐PCR Potential biomarker for

controls

controls

controls

qRT‐PCR, NGS Biomarker for

qRT‐PCR, NGS Biomarker for

qRT‐PCR, NGS Biomarker for

NGS, qRT‐PCR Clinical value in

qRT‐PCR, NGS Biomarker for

controls

qRT‐PCR, microarray

qRT‐PCR, microarray, NGS

Microarray and Northern blotting

Microarray, NGS, Northern blotting, qRT‐PCR

HBV‐related HCC

HBV‐related HCC

HBV‐related HCC

differentiation of HBV‐ positive HCC from

differentiation of HBV‐ positive HCC from

differentiation of HBV‐ positive HCC from

HBV‐related HCC diagnostics

HCC differentiation from healthy, chronic hepatitis and cirrhosis

Potential biomarker for HBV‐related HCC

differentiation of HBV‐ positive HCC from

Involved in chronicity of HBV infection

Potential research interest in chronic HBV infections and HBV‐ induced HCC

miRNA in HCC

interest in chronic HBV infections and HBV‐ induced HCC

Microarray Potential research

liver cancer

Expressed aberrantly in

Wen et al. [100]

Mizuguchi et al.

Wen et al. [100]

Wen et al. [100]

Li et al. [41]

Li et al. [41]

Li et al. [41]

Tan et al. [101]

Zhou et al. [104]

Wen et al. [100], Hou et al. [47], Li et al. [41]

Li et al. [41]

Liu et al. [75], Ghosh et al. [99]

Zhang et al. [95], Li et al. [109]

Hou et al. [47]

Zhang et al. [95]

[107]

related HCC

samples

HCC

HCC

serum

serum

serum

related HCC patients

serum, plasma of HBV HCC

HepG2.2.15, plasma of HBV‐related HCC, liver tissue of HCC

HepG2.2.15, HBV‐ related HCC serum

HepG2.2.15

miR‐100 Down HBV‐related HCC qRT‐PCR Important deregulated

plasma

serum

miR‐30a‐5p Up Plasma of HBV‐

332 Advances in Treatment of Hepatitis C and B

miR‐34a Up HBV‐related liver

miR‐320a Up Plasma of HBV

miR‐324‐3p Up Plasma of HBV

miR‐342‐3p Up HBV‐infected

miR‐375 Up HBV‐infected

miR‐423 Up HBV‐infected

miR‐433‐3p Up Serum of HBV‐

miR‐801 Up HBV‐related HCC

miR‐92a/92a‐3p Up HBV‐infected

let‐7f Up HBV‐infected

miR‐15a Up/down HepG2 and

miR‐18a/b Up/down HepG2 and

miR‐106a Down HepG2 and

*Abbreviations*: HCC, hepatocellular carcinoma; NGS, next‐generation sequencing; ND, no data available; qRT‐PCR, quantitative real‐time polymerase chain reaction.

**Table 1.** Studies reporting on miRNA deregulation in HBV‐infected patients with HCC or in HBV‐expressing cell lines.

Serum miRNAs could serve as biomarkers for the detection of liver pathologies [102]. Serum HBV‐related miRNAs for HBV‐related HCC diagnosis have been investigated by Tan et al. [101]. The study identified eight miRNAs (miR‐206, miR‐141‐3p, miR‐433‐3p, miR‐1228‐5p, miR‐199a‐5p, miR‐122‐5p, miR‐192‐5p, and miR‐26a‐5p) and constructed a miRNA set that provided high diagnostic accuracy for HBV‐related HCC [101]. The study published by Winther et al. [113] presented a panel of circulating plasma miRNAs that are differentially expressed in immunological phases of chronically HBV‐infected children and positively cor‐ related with the quantity of HBsAg.

A multicenter study was conducted by Wen et al. [100] to discover a panel of plasma miRNAs to discriminate HBV‐related HCC patients from healthy controls. The study revealed that four miRNAs (miR‐20a‐5p, miR‐320a, miR‐324‐3p, and miR‐375) (alone or combined with AFP) could be used as preclinical biomarkers in HCC screening, while the expression profile of eight miRNAs (miR‐20a‐5p, miR‐25‐3p, miR‐30a‐5p, miR‐92a‐3p, miR‐132‐3p, miR‐185‐5p, miR‐ 320a, and miR‐324‐3p) can discriminate HCC patients from noncancerous controls [100]. Some of the described miRNAs were studied as well by Zhang et al. [95]. MiR‐18a, miR‐125b‐5p, and miR‐223‐3p were well described as potential biomarkers for HBV‐related HCC [109, 110].

Hou et al. [47] performed an extensive study of miRNomes in human normal liver, hepati‐ tis liver, and HCC. Researchers presented 15 deregulated miRNAs in 40 HBV‐related HCC samples in comparison to healthy controls. Additionally, the consistent decline of miR‐ 199a/b‐3p in HCC and its significant correlation with poor prognosis of HCC patients has been elucidated, suggesting miR‐199a/b‐3p as a potential HBV therapeutic target. Gao et al. [105] investigated the expression of cancer‐related miRNA profiles in early stages of HBV‐ related HCC development and observed altered miRNA expression at various pre‐malignant stages of HCC and persistent downregulation of miR‐145 and miR‐199b and upregulation of miR‐244 throughout the HCC development. The miR‐145 has been suggested as a candidate tumor‐suppressive miRNA due to suppression of cell proliferation caused by overexpression of miR‐145 precursor in HepG2 cell lines and abundant expression of miR‐145 in non‐tumor‐ ous livers as well as pre‐malignant low‐grade dysplastic nodules.

Several studies have suggested the role of HBx protein in miRNA expression during HBV infec‐ tion [67, 96, 98, 106]. For example, the recent study, conducted on 120 patients with HBV‐related HCC, has shown that the expression of miR‐106b was significantly higher in HBV‐related HCC patients in comparison to non‐HBV/non‐HCV‐related HCC patients and suggested that HBx enhances miR‐106b transcription and therefore promotes tumor progression in HBV‐related HCC [98]. Transfection with the HBx expression plasmid has been recently used in an addi‐ tional study by Yu et al. [114] to investigate HBx‐related regulation of miR‐19a, miR‐122, and miR‐223 in malignant hepatocytes. The study has shown that the expression of miRNAs was regulated by HBx protein, which enhanced the proliferation of HBx‐transfected HepG2 cells.

## **7.3. HCV miRNA biomarkers**

Apart from a variety of published studies focusing on the identification of HCV infec‐ tion miRNA biomarkers [88], our review focused more on difference in miRNAs profiles between HCV‐infected cancerous liver cells and HCV‐infected cells without progression to HCC. Despite the fact that treatment for most common HCV genotype 1 has been evolving rapidly in the past 10 years, no effective and safe anti‐HCV vaccine is available and only approximately 50% of patients, infected with HCV genotype 1 (the more common geno‐ type in USA and Western Europe), reach sustained viral response (SVR) while treated with most accessible treatment, the interferon. Therefore, the management of HCV‐induced liver disease remains problematic [115].

Serum miRNAs could serve as biomarkers for the detection of liver pathologies [102]. Serum HBV‐related miRNAs for HBV‐related HCC diagnosis have been investigated by Tan et al. [101]. The study identified eight miRNAs (miR‐206, miR‐141‐3p, miR‐433‐3p, miR‐1228‐5p, miR‐199a‐5p, miR‐122‐5p, miR‐192‐5p, and miR‐26a‐5p) and constructed a miRNA set that provided high diagnostic accuracy for HBV‐related HCC [101]. The study published by Winther et al. [113] presented a panel of circulating plasma miRNAs that are differentially expressed in immunological phases of chronically HBV‐infected children and positively cor‐

A multicenter study was conducted by Wen et al. [100] to discover a panel of plasma miRNAs to discriminate HBV‐related HCC patients from healthy controls. The study revealed that four miRNAs (miR‐20a‐5p, miR‐320a, miR‐324‐3p, and miR‐375) (alone or combined with AFP) could be used as preclinical biomarkers in HCC screening, while the expression profile of eight miRNAs (miR‐20a‐5p, miR‐25‐3p, miR‐30a‐5p, miR‐92a‐3p, miR‐132‐3p, miR‐185‐5p, miR‐ 320a, and miR‐324‐3p) can discriminate HCC patients from noncancerous controls [100]. Some of the described miRNAs were studied as well by Zhang et al. [95]. MiR‐18a, miR‐125b‐5p, and miR‐223‐3p were well described as potential biomarkers for HBV‐related HCC [109, 110]. Hou et al. [47] performed an extensive study of miRNomes in human normal liver, hepati‐ tis liver, and HCC. Researchers presented 15 deregulated miRNAs in 40 HBV‐related HCC samples in comparison to healthy controls. Additionally, the consistent decline of miR‐ 199a/b‐3p in HCC and its significant correlation with poor prognosis of HCC patients has been elucidated, suggesting miR‐199a/b‐3p as a potential HBV therapeutic target. Gao et al. [105] investigated the expression of cancer‐related miRNA profiles in early stages of HBV‐ related HCC development and observed altered miRNA expression at various pre‐malignant stages of HCC and persistent downregulation of miR‐145 and miR‐199b and upregulation of miR‐244 throughout the HCC development. The miR‐145 has been suggested as a candidate tumor‐suppressive miRNA due to suppression of cell proliferation caused by overexpression of miR‐145 precursor in HepG2 cell lines and abundant expression of miR‐145 in non‐tumor‐

Several studies have suggested the role of HBx protein in miRNA expression during HBV infec‐ tion [67, 96, 98, 106]. For example, the recent study, conducted on 120 patients with HBV‐related HCC, has shown that the expression of miR‐106b was significantly higher in HBV‐related HCC patients in comparison to non‐HBV/non‐HCV‐related HCC patients and suggested that HBx enhances miR‐106b transcription and therefore promotes tumor progression in HBV‐related HCC [98]. Transfection with the HBx expression plasmid has been recently used in an addi‐ tional study by Yu et al. [114] to investigate HBx‐related regulation of miR‐19a, miR‐122, and miR‐223 in malignant hepatocytes. The study has shown that the expression of miRNAs was regulated by HBx protein, which enhanced the proliferation of HBx‐transfected HepG2 cells.

Apart from a variety of published studies focusing on the identification of HCV infec‐ tion miRNA biomarkers [88], our review focused more on difference in miRNAs profiles

ous livers as well as pre‐malignant low‐grade dysplastic nodules.

related with the quantity of HBsAg.

334 Advances in Treatment of Hepatitis C and B

**7.3. HCV miRNA biomarkers**

Antagonism of miR‐122 by locked nucleic acid is a promising tool for the treatment of HCV. The current most advanced research on miR‐122 antagonist Miravirsen is discussed in the following section. Likewise, the therapeutic potential of mimic miR‐196b is presented by Kaluzna [30], reviewing studies which confirmed the ability of miR‐196b to inhibit HCV rep‐ lication and revealed that interferon‐induced overexpression of miR‐196b decreases inflam‐ mation and leads to a better response in interferon‐based HCV therapy.

Circulating serum levels of miR‐122 and miR‐222 have been shown to be useful potential diagnostic biomarkers for chronic HCV infection in Egyptian patients [116–118], whereas in another study miR‐122, miR‐199a, and miR‐16 have been implicated as potential early diagnostic biomarkers for HCC in Egyptian patients, chronically infected with HCV [118] (**Table 2**).

Of note, miR‐222 and miR‐224 have been reported to be upregulated in both, HBV and HCV infections [95, 105, 116, 121]. However, Bandopadhyay et al. [106] and Zhang et al. [95] reported downregulation of miR‐222 and miR‐224 in HBV‐infected patients, respectively. As expres‐ sion profiles of circulating serum biomarkers became a subject of interest in different disease studies, serum miRNAs possibly involved in HCV‐related HCC have been investigated in several studies. Oksuz et al. [119] examined HCV‐infected patients with chronic infection, cirrhosis, and HCC and compared them with control group samples. When all groups of samples were compared, the study revealed deregulation of miR‐30c‐5p, miR‐223‐3p, miR‐ 302c‐3p, and miR‐17‐5p in cirrhosis and HCC, suggesting possible novel noninvasive bio‐ markers for HCC.

Using whole‐genome expression profiling, Abdalla and Haj‐Ahmad [120] identified 10 potential HCV‐induced HCC biomarker candidates in urine; five of which were upregu‐ lated in HCC: miR‐335, miR‐618, miR‐625, miR‐532, and miR‐7, and five downregulated: miR‐323, miR‐449, miR‐520d, miR‐516‐5p, and miR‐650. The proposed tandem signature of downregulated miR‐650 and upregulated miR‐618 showed improved sensitivity and specificity for HCC detection, in comparison to the traditional AFP‐level‐based detection method.

Increased expression of miR‐155 in hepatocytes of patients infected with HCV has been con‐ firmed *in vitro* and *in vivo* by Zhang et al. [85]. In addition, the study revealed that overexpres‐ sion of miR‐155 inhibits apoptosis, and promotes hepatocyte proliferation and tumorigenesis, which suggested miR‐155 could be a negative prognostic biomarker for HCC (reviewed by Kaluzna [30]). In addition, miR‐155 has been shown to be upregulated in HBV‐infected human hepatoma cells, where it inhibits HBV infection [103] (**Figure 1**).



**MiRNA** miR‐10a miR‐15a

miR‐16 miR‐17‐5p

miR‐100 miR‐122 miR‐125b

miR‐155 miR‐192 miR‐194 miR‐200a

miR‐215 miR‐221 miR‐222‐3p miR‐224‐3p

Up

ND

HCV‐related HCC

Microarray

Up

ND

HCV‐related HCC

Microarray

Up

ND

Up

Chimera of 1a/2

Huh7, Huh‐RepSI

HCV‐related HCC

Microarray

Microarray

Relevant to carcinogenesis

Exclusively expressed in

HCV‐associated HCC

Exclusively expressed in

Diaz et al. [121]

HCV‐associated HCC

Exclusively expressed in

Diaz et al. [121]

HCV‐associated HCC

Ishida et al. [80]

Diaz et al. [121]

Up

4

Up

Up

Chimera of 1a/2

Chimera of 1a/2

Huh7, Huh‐RepSI

Urine of HCV‐related

Microarray, qRT‐PCR

HCC

Microarray

Huh7, Huh‐RepSI

Microarray

Up

ND

HCV‐infected patients

Stem loop qRT‐PCR

Promotes

Zhang et al. [85]

hepatocarcinogenesis

Relevant to carcinogenesis

Relevant to carcinogenesis

Potential HCC biomarker

Abdalla et al. [120]

Ishida et al. [80]

Ishida et al. [80]

Up

ND

Up/down

ND

Serum of HCV patients

RT‐PCR, qRT‐PCR,

Detection of HCC in HCV‐

El‐Garem et al. [117],

Motawi et al. [116],

El‐Abd et al. [118],

Varnholt et al. [84]

Varnholt et al. [84]

chronic patients

stem loop qRT‐PCR,

microarray

serum of patients with

chronic HCV infection,

HCV‐related HCC

HCV‐related HCC

qRT‐PCR

Increased expression in HCV‐

related HCC

Up

ND

HCV‐related HCC

qRT‐PCR

Up

ND

Up/down

ND

HCV‐related HCC,

qRT‐PCR, stem loop

Associated with progression

Varnholt et al. [84],

336 Advances in Treatment of Hepatitis C and B

El‐Abd et al. [118]

of HCC

qRT‐PCR

serum of HCV chronic

infections

Plasma of HCV‐related

qRT‐PCR

Noninvasive biomarker of

Oksuz et al. [119]

HCV‐positive HCC

Increased expression in HCV‐

Varnholt et al. [84]

related HCC

HCC

Up

ND

HCV‐related HCC

qRT‐PCR

Up

ND

**Deregulation**

**HCV genotype**

**Type of sample**

HCV‐related HCC

qRT‐PCR

**Method**

**Clinical relevance**

Increased expression in HCV‐

related HCC

Increased expression in HCV‐

Varnholt et al. [84]

related HCC

**Reference**

Varnholt et al. [84]



**MiRNA** miR‐640

miR‐7 miR‐765

miR‐9 miR‐1269

let‐7g miR‐16‐5p

miR‐104 miR‐106a miR‐122a miR‐125a‐5p miR‐125b‐5p

miR‐130a

miR‐134 miR‐137

Down

ND

Down

ND

HCV‐related HCC

qRT‐PCR

tumors

HCV‐related HCC

qRT‐PCR

tumors

Down

ND

HCV‐related HCC

Microarray

Down

ND

HCV‐related HCC

Microarray

Down

ND

HCV‐related HCC

Microarray

Exclusively expressed in

Diaz et al. [121]

HCV‐associated HCC

Exclusively expressed in

Diaz et al. [121]

HCV‐associated HCC

Exclusively expressed in

Diaz et al. [121]

HCV‐associated HCC

Decreased expression in

Varnholt et al. [84]

HCV‐related HCC

Decreased expression in

Varnholt et al. [84]

HCV‐related HCC

Down

ND

Down

ND

Down

ND

HCV‐related HCC

qRT‐PCR

tumors

HCV‐related HCC

qRT‐PCR

tumors

Mainly HCV‐related

Microarray

HCC

Down

ND

HCV‐related HCC

Microarray

Up

ND

HCV‐related HCC

qRT‐PCR

Up

ND

HCV‐related HCC

Microarray

Up

ND

Up

4

Up

4

Up

4

**Deregulation**

**HCV genotype**

**Type of sample**

Urine of HCV‐related

Microarray, qRT‐PCR

HCC patients

Urine of HCV‐related

Microarray, qRT‐PCR

Potential HCC biomarker

Abdalla et al. [120]

HCC patients

Urine of HCV‐related

Microarray, qRT‐PCR

Potential HCC biomarker

Abdalla et al. [120]

338 Advances in Treatment of Hepatitis C and B

HCC patients

HCV‐related HCC

qRT‐PCR

Increased expression in HCV‐

Varnholt et al. [84]

related HCC

Exclusively expressed in

Diaz et al. [121]

HCV‐associated HCC

Increased expression in HCV‐

Varnholt et al. [84]

related HCC

Exclusively expressed in

Diaz et al. [121]

HCV‐associated HCC

Decreased expression in

Varnholt et al. [84]

HCV‐related HCC

Decreased expression in

Varnholt et al. [84]

HCV‐related HCC

Deregulated in HCV‐related

Gramantieri et al.

[122]

HCC

**Method**

**Clinical relevance**

Potential HCC biomarker

Abdalla et al. [120]

**Reference**

#### Importance of MicroRNAs in Hepatitis B and C Diagnostics and Treatment http://dx.doi.org/10.5772/66498 339



**MiRNA** miR‐29c miR‐204 miR‐214 miR‐218 miR‐221 miR‐223‐3p miR‐301a‐3p

miR‐302b

miR‐320 miR‐323 miR‐330 miR‐368 miR‐424‐3p

miR‐449 miR‐491

Down

chimera of 1a/2

Huh7, Huh‐RepSI

Microarray

Relevant to carcinogenesis

Ishida et al. [80]

Down

4

Urine of HCV‐related

Microarray, qRT‐PCR

Potential HCC biomarker

Abdalla et al. [120]

HCC patients

Down

ND

HCV‐related HCC

Microarray

Down

ND

HCV‐related HCC

qRT‐PCR

Down

ND

Down

4

Down

Chimera of 1a/2

Huh7, Huh‐RepSI

Urine of HCV‐related

Microarray, qRT‐PCR

HCC patients

HCV‐related HCC

qRT‐PCR

Decreased expression in

Varnholt et al. [84]

HCV‐related HCC

Decreased expression in

Varnholt et al. [84]

HCV‐related HCC

Exclusively expressed in

Diaz et al. [121]

HCV‐associated HCC

Microarray

Down

ND

HCV‐related HCC

qRT‐PCR

Down

ND

HCV‐related HCC

Microarray

Down

ND

Plasma of HCV‐related

qRT‐PCR

HCC

Down

ND

Serum chronic HCV

RT‐PCR, qRT‐PCR

Potential noninvasive

El‐Garem et al. [117]

biomarker for HCV‐related

HCC

Noninvasive biomarker of

Oksuz et al. [119]

HCV‐positive HCC

Exclusively expressed in

Diaz et al. [121]

HCV‐associated HCC

Decreased expression in

Varnholt et al. [84]

HCV‐related HCC

Relevant to carcinogenesis

Potential HCC biomarker

Abdalla et al. [120]

Ishida et al. [80]

patients

Down

ND

HCV‐related HCC

qRT‐PCR

Down

ND

HCV‐related HCC

Microarray

Down

ND

HCV‐related HCC

qRT‐PCR

Down

ND

**Deregulation**

**HCV genotype**

**Type of sample**

HCV‐related HCC

qRT‐PCR

**Method**

**Clinical relevance**

Decreased expression in

HCV‐related HCC

Decreased expression in

Varnholt et al. [84]

HCV‐related HCC

Exclusively expressed in

Diaz et al. [121]

340 Advances in Treatment of Hepatitis C and B

HCV‐associated HCC

Decreased expression in

Varnholt et al. [84])

HCV‐related HCC

**Reference**

Varnholt et al. [84]


**Figure 1.** miRNAs deregulated in HBV‐ and HCV‐related HCC or HBV/HCV‐expression cell lines. miRNAs upregulated in HBV and HCV infections are presented in red color, miRNAs downregulated in HBV and HCV infections are presented in blue color, while miRNAs that were shown to be up‐ and downregulated in different studies are presented in violet color. MiRNAs reported to be up‐ or downregulated in HBV and HCV infections are presented in green color.

Despite the fact that several studies examined the expression of miRNAs in HCC, discrep‐ ancies exist among published data. Diaz et al. [121] assumed that non‐concordance may be the result of differences in selection of noncancerous control samples or commonly included HCC samples without prior confirmation of possible HBV or HCV infection. In some previ‐ ous studies, Diaz et al. [121] investigated the expression of miRNAs in HCV‐induced HCC, in comparison to a wide range of liver samples and identified 18 miRNAs exclusively expressed in HCV‐induced HCC. Several other studies have as well examined subsets of miRNAs potentially involved in hepatocellular changes, advancing to HCC [80, 84, 122].

According to an increasing amount of miRNAs identified and examined throughout various stages Of HBV/HCV infection and HCC development (**Tables 1** and **2**), miRNAs not specifically presented in this review should be as well included as a subject of interest in future studies.

## **7.4. MiRNA‐122 in HBV and HCV**

Despite the fact that several studies examined the expression of miRNAs in HCC, discrep‐ ancies exist among published data. Diaz et al. [121] assumed that non‐concordance may be the result of differences in selection of noncancerous control samples or commonly included HCC samples without prior confirmation of possible HBV or HCV infection. In some previ‐ ous studies, Diaz et al. [121] investigated the expression of miRNAs in HCV‐induced HCC, in comparison to a wide range of liver samples and identified 18 miRNAs exclusively expressed in HCV‐induced HCC. Several other studies have as well examined subsets of miRNAs

**Figure 1.** miRNAs deregulated in HBV‐ and HCV‐related HCC or HBV/HCV‐expression cell lines. miRNAs upregulated in HBV and HCV infections are presented in red color, miRNAs downregulated in HBV and HCV infections are presented in blue color, while miRNAs that were shown to be up‐ and downregulated in different studies are presented in violet color. MiRNAs reported to be up‐ or downregulated in HBV and HCV infections are presented in green color.

342 Advances in Treatment of Hepatitis C and B

According to an increasing amount of miRNAs identified and examined throughout various stages Of HBV/HCV infection and HCC development (**Tables 1** and **2**), miRNAs not specifically presented in this review should be as well included as a subject of interest in future studies.

potentially involved in hepatocellular changes, advancing to HCC [80, 84, 122].

The levels of liver‐specific miRNA‐122 (miR‐122) are down‐ and upregulated in HBV and HCV, respectively. The miR‐122 promotes the replication of HCV and blocks the replication of HBV. It has been shown recently that HBV inhibits the miR‐122 expression, suggesting a possibility of miR‐122 replacement therapy in HBV‐infected individuals [61, 64]. The study by Fan et al. [62] showed that miR‐122 inhibited the expression of the NDRG3 protein, which subsequently inhibited malignant cell transformation and presented the miR‐122 and its tar‐ get NDRG3 as key diagnostic markers and potential therapeutic targets in HBV‐related HCC. On the other hand, in HCV infections, it has been shown *in vitro* and *in vivo* that antagonistic utility of miR‐122 inhibits HCV replication cycle and reduces viral load, and thus represents an effective treatment of HCV infection. A model of Miravirsen interaction with miR‐122 is shown in **Figure 2** [77, 123].

**Figure 2.** Miravirsen interaction with miR‐122 inhibits binding between miR‐122 and the 5′ UTR of the HCV RNA. The most abundant liver miRNA‐miRNA‐122 binds with two seed sites in the 5′ UTR of the HCV genome. Miravirsen sequesters mature miR‐122 and suppresses HCV.

Recently, the safety and efficacy of the Miravirsen, a locked nucleic acid form of antisense‐ miR‐122 that sequesters miR‐122, has been evaluated in a phase 2a clinical study, which included 36 patients with chronic HCV genotype 1 infection from seven international sites [40]. The study observed that treatment with Miravirsen prolonged dose‐dependent reduc‐ tions in HCV RNA, without evidence of viral resistance [40]. Moreover, a recent study, con‐ ducted on 51 HCV‐infected Japanese patients, treated with interferon, presented miR‐122 as an independent predictor of SVR [124]. Unfortunately, no single miRNA representing a promising treatment option in HBV infections has been pointed out as yet.

Nevertheless, therapies that silence HBV RNAs are emerging. Multiple cell‐line‐based stud‐ ies and *in vivo* studies on mouse models have evaluated synthetically engineered or chemi‐ cally modified small RNAs that complementarily target HBV transcripts and lead to RNA degradation and thus to the inhibition of HBV replication. However, due to short duration of their activity, the lack of applicable animal model for testing in clinical trials, and subsequent pharmacokinetic difficulties, further investigations are warranted to evaluate RNA‐interfer‐ ence‐based approaches to clinical practice (reviewed by Ivacik et al. [125]).

HBV/HCV dual infection is not an uncommon event, occurring in approximately 2–10% of chronically infected HCV patients and in 5–20% of chronically infected HBV patients [126]. Dual HBV/HCV infection has prognosis of a more aggressive clinical course of liver disease than either mono‐infection. Despite the fact that compiling evidence exists on reciprocal inhi‐ bition between HBV and HCV and that miR‐122 represents a crucial host gene involved in pathogenesis of both viruses, the role of miR‐122 in HBV/HCV dual infection has not been defined so far [127].

## **8. Conclusion**

Cellular miRNAs contribute to HBV and HCV pathogenesis by direct or indirect interac‐ tions with viral genome or proteins and molecules critical for regulation of the cell cycle. Regulation of miRNAs expression upon HBV and HCV infection significantly differs between both viruses. Reports summarized in this chapter indicate that miRNAs represent an effective, noninvasive biomarker tools for early diagnosis of HBV and HCV infection, early diagnosis of liver disease and its progressive stages, particularly HCC. Mimic and antagonistic effects of cellular miRNAs have been considered in diagnostic and treatment of HBV/HCV‐related liver disease, with miR‐122 representing a promising treatment option for chronic infection with HCV genotype 1. Because most studies identified and validated miRNAs in heterogenic tumors and because miRNA targets were validated mostly in the already‐transformed cell culture systems, transfected with plasmids encoding HBV or HCV genome or parts of their genome, discrepancies exist in candidate biomarker miRNAs across published studies. Due to an extensive number of miRNA targets and other clinical factors considered in significant number of studies published in the last 10 years, efforts should be made to establish a spe‐ cific, repetitive, and easy‐to‐operate method to identify reliable panels of miRNA biomarkers for early diagnosis and treatment of HBV‐HCV‐related diseases. Suitable reference miRNA targets and positive and negative controls should be included in such profiling applications. The application of novel techniques such as next‐generation sequencing, development of syn‐ thetic small RNAs, and hepatoma cell lines will impact the subsequent advances in miRNA studies related to HBV and HCV pathogenesis as well as miRNA deregulation in other patho‐ logical conditions.

## **Author details**

Mateja M. Jelen and Damjan Glavač\*

\*Address all correspondence to: damjan.glavac@mf.uni‐lj.si

Department of Molecular Genetics, Institute of Pathology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia

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pharmacokinetic difficulties, further investigations are warranted to evaluate RNA‐interfer‐

HBV/HCV dual infection is not an uncommon event, occurring in approximately 2–10% of chronically infected HCV patients and in 5–20% of chronically infected HBV patients [126]. Dual HBV/HCV infection has prognosis of a more aggressive clinical course of liver disease than either mono‐infection. Despite the fact that compiling evidence exists on reciprocal inhi‐ bition between HBV and HCV and that miR‐122 represents a crucial host gene involved in pathogenesis of both viruses, the role of miR‐122 in HBV/HCV dual infection has not been

Cellular miRNAs contribute to HBV and HCV pathogenesis by direct or indirect interac‐ tions with viral genome or proteins and molecules critical for regulation of the cell cycle. Regulation of miRNAs expression upon HBV and HCV infection significantly differs between both viruses. Reports summarized in this chapter indicate that miRNAs represent an effective, noninvasive biomarker tools for early diagnosis of HBV and HCV infection, early diagnosis of liver disease and its progressive stages, particularly HCC. Mimic and antagonistic effects of cellular miRNAs have been considered in diagnostic and treatment of HBV/HCV‐related liver disease, with miR‐122 representing a promising treatment option for chronic infection with HCV genotype 1. Because most studies identified and validated miRNAs in heterogenic tumors and because miRNA targets were validated mostly in the already‐transformed cell culture systems, transfected with plasmids encoding HBV or HCV genome or parts of their genome, discrepancies exist in candidate biomarker miRNAs across published studies. Due to an extensive number of miRNA targets and other clinical factors considered in significant number of studies published in the last 10 years, efforts should be made to establish a spe‐ cific, repetitive, and easy‐to‐operate method to identify reliable panels of miRNA biomarkers for early diagnosis and treatment of HBV‐HCV‐related diseases. Suitable reference miRNA targets and positive and negative controls should be included in such profiling applications. The application of novel techniques such as next‐generation sequencing, development of syn‐ thetic small RNAs, and hepatoma cell lines will impact the subsequent advances in miRNA studies related to HBV and HCV pathogenesis as well as miRNA deregulation in other patho‐

Department of Molecular Genetics, Institute of Pathology, Faculty of Medicine, University of

ence‐based approaches to clinical practice (reviewed by Ivacik et al. [125]).

defined so far [127].

344 Advances in Treatment of Hepatitis C and B

**8. Conclusion**

logical conditions.

**Author details**

Mateja M. Jelen and Damjan Glavač\*

Ljubljana, Ljubljana, Slovenia

\*Address all correspondence to: damjan.glavac@mf.uni‐lj.si


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#### **Can Proteomic Profiling Identify Biomarkers and/or Therapeutic Targets for Liver Fibrosis? Can Proteomic Profiling Identify Biomarkers and/or Therapeutic Targets for Liver Fibrosis?**

Seyma Katrinli, H. Levent Doganay, Kamil Ozdil and Gizem Dinler-Doganay Seyma Katrinli, H. Levent Doganay, Kamil Ozdil and Gizem Dinler-Doganay

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65608

#### **Abstract**

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Liver fibrosis is a serious disease that affects around 350–400 million people worldwide. The main approach for fibrosis staging is liver biopsy, which is an invasive procedure that is not endured pretty well by patients. Currently, some serum-based biomarker panels are available for diagnosis and staging of liver fibrosis. Recent high-throughput proteomic studies are also very promising for identification of novel biomarkers for diagnosis and/or treatment of liver fibrosis. We hereby review the application of proteomic profiling studies for identification of fibrosis biomarkers with their advantages and drawbacks.

**Keywords:** proteome profiling, liver fibrosis, biomarkers, therapeutic markers

## **1. Liver fibrosis**

Liver fibrosis results from chronic damage to the liver and causes accumulation of excessive matrix or scar. This scar tissue may inhibit blood flow due to the contraction of liver that results progressive liver damage and cirrhosis (the most advanced stage of liver fibrosis) or even hepatocellular carcinoma (HCC) [1]. Liver fibrosis is prominently observed in chronic liver diseases such as viral hepatitis, alcoholic steatohepatitis, nonalcoholic fatty liver disease (NAFLD), toxic liver injury, auto-immune diseases, and some genetic diseases [2]. From these chronic liver diseases, chronic hepatitis B (CHB) and chronic hepatitis C are major global health problems, and despite national vaccination programs, around 350–400 million people are infected with hepatitis B virus (HBV) and 130–150 million people are infected with hepatitis C

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

virus (HCV) worldwide [3, 4]. Chronic HBV (CHB) infection results in liver fibrosis that can further develop into cirrhosis or HCC, both being the major causes of liver-related death [5]. The annual incidence of cirrhosis in patients infected with HBV has been evaluated at 1.3–2.4% [6], and although the cumulative 5-year-old survival rate for patients with compensated cirrhosis is 84% [7], in patients with decompensated cirrhosis, this survival rate decreases to 14–35% [7, 8].

Regeneration of liver is an extremely complex process, but recent studies in human and animal models have indicated that liver fibrosis could be reversible in specific cases [9, 10]. It is hoped that deeper understanding of the etiology of liver fibrosis will contribute to improved diagnostic tools and potential therapeutic approaches for liver fibrosis and cirrhosis. Even though curing the underlying disease may reverse fibrosis progression, currently, the most effective treatment that prolongs survival in advanced cirrhotic patients is liver transplantation [11]. However, this approach is limited because of the shortages of organs, the presence of concurrent disease affecting other tissues, and recurrence of the original disease in transplant patients [12]. Despite the advancement in noninvasive tests, liver biopsy still remains as the gold standard test for evaluation of liver disease severity [13–16]. However, it has several disadvantages such as invasive character, sampling errors and limitations for effective surveillance, and follow-up [17–19]. Upon antiviral treatment, HCV-infected patients may clear HCV RNA from their bloodstream [5]. For the treatment of CHB, current therapies do not accomplish complete eradication of HBV infection. HBV remains in infected hepatocytes in the form of covalently closed circular DNA (cccDNA) even if the patient clears HBsAg, and this cccDNA can possibly be reactivated with the right stimulus [20]. Hence, the therapeutic strategy for CHB is to prevent liver fibrosis and the other complications of advanced liver disease that can further develop cirrhosis and HCC. Therefore, recent studies focus on the search of biomarkers for noninvasive diagnosis and staging of liver fibrosis and for discovery of new therapeutic targets to prevent HBV-related liver fibrosis.

Proteomics, which studies the complex protein mixtures in a biological system, is a valuable tool to investigate cellular pathways, protein–protein interactions, and identify target proteins [21]. No requirement of a priori knowledge of protein identities present in a biological system makes proteomic profiling an ideal tool for screening the most discerning set of biomarkers [22].

In this review, we will focus on the advances in the proteomic research concerning liver fibrosis and evaluate whether proteomic profiling studies are applicable in the search of protein biomarkers and/or therapeutic targets for this condition with a focus on HBV and HCV infection.

## **2. Pathogenesis and staging of liver fibrosis**

Hepatic fibrosis develops as a result of wound healing response of the liver to chronic injury in conjunction with the deposition of extracellular matrix (ECM) proteins [23]. Deposition of ECM proteins forms a fibrous scar that alters hepatic architecture, and subsequent formation of nodules of regenerating hepatocytes results in cirrhosis [24]. After an acute liver damage (e.g., HBV and HCV infection), parenchymal cells regenerate and substitute the necrotic and apoptotic cells. This process is accompanied with an inflammatory response and minor accumulation of ECM. Following persistent damage, eventually liver regeneration declines, and hepatocytes are replaced with abundant ECM, including fibrillar collagen. Origin of liver injury determines the distribution of this fibrous material. While in chronic hepatitis and chronic cholestatic disorders, the localization of fibrotic tissue is around portal tracts, in alcohol-induced liver diseases, its localization is in pericentral and perisinusoidal areas [25].

virus (HCV) worldwide [3, 4]. Chronic HBV (CHB) infection results in liver fibrosis that can further develop into cirrhosis or HCC, both being the major causes of liver-related death [5]. The annual incidence of cirrhosis in patients infected with HBV has been evaluated at 1.3–2.4% [6], and although the cumulative 5-year-old survival rate for patients with compensated cirrhosis is 84% [7], in patients with decompensated cirrhosis, this survival rate decreases to 14–35% [7, 8]. Regeneration of liver is an extremely complex process, but recent studies in human and animal models have indicated that liver fibrosis could be reversible in specific cases [9, 10]. It is hoped that deeper understanding of the etiology of liver fibrosis will contribute to improved diagnostic tools and potential therapeutic approaches for liver fibrosis and cirrhosis. Even though curing the underlying disease may reverse fibrosis progression, currently, the most effective treatment that prolongs survival in advanced cirrhotic patients is liver transplantation [11]. However, this approach is limited because of the shortages of organs, the presence of concurrent disease affecting other tissues, and recurrence of the original disease in transplant patients [12]. Despite the advancement in noninvasive tests, liver biopsy still remains as the gold standard test for evaluation of liver disease severity [13–16]. However, it has several disadvantages such as invasive character, sampling errors and limitations for effective surveillance, and follow-up [17–19]. Upon antiviral treatment, HCV-infected patients may clear HCV RNA from their bloodstream [5]. For the treatment of CHB, current therapies do not accomplish complete eradication of HBV infection. HBV remains in infected hepatocytes in the form of covalently closed circular DNA (cccDNA) even if the patient clears HBsAg, and this cccDNA can possibly be reactivated with the right stimulus [20]. Hence, the therapeutic strategy for CHB is to prevent liver fibrosis and the other complications of advanced liver disease that can further develop cirrhosis and HCC. Therefore, recent studies focus on the search of biomarkers for noninvasive diagnosis and staging of liver fibrosis and for discovery

of new therapeutic targets to prevent HBV-related liver fibrosis.

**2. Pathogenesis and staging of liver fibrosis**

biomarkers [22].

356 Advances in Treatment of Hepatitis C and B

infection.

Proteomics, which studies the complex protein mixtures in a biological system, is a valuable tool to investigate cellular pathways, protein–protein interactions, and identify target proteins [21]. No requirement of a priori knowledge of protein identities present in a biological system makes proteomic profiling an ideal tool for screening the most discerning set of

In this review, we will focus on the advances in the proteomic research concerning liver fibrosis and evaluate whether proteomic profiling studies are applicable in the search of protein biomarkers and/or therapeutic targets for this condition with a focus on HBV and HCV

Hepatic fibrosis develops as a result of wound healing response of the liver to chronic injury in conjunction with the deposition of extracellular matrix (ECM) proteins [23]. Deposition of ECM proteins forms a fibrous scar that alters hepatic architecture, and subsequent formation of nodules of regenerating hepatocytes results in cirrhosis [24]. After an acute liver damage In the fibrotic liver, the main ECM-producing cells are hepatic stellate cells (HSCs) [26]. In the healthy liver, HSCs are found in the space of Disse and act as the major repository sites of vitamin A. Following sustained injury, HSCs activate or transdifferentiate into myofibroblastlike cells that have contractile, proinflammatory, and fibrogenic characteristics [27, 28]. Activated HSCs, which migrate and accumulate at the wound repair locations, secrete bulk amounts of ECM and mediate ECM degradation [29].

Some other hepatic cells, besides HSCs, may show fibrogenic properties. One of them is myofibroblasts derived from small portal vessels which reproduce around biliary tracts in cholestatis-induced liver fibrosis to induce collagen accumulation [30, 31]. The origin of the liver injury may determine the relative significance of each cell type in liver fibrogenesis. For instance, while HSCs exert the main fibrogenic activity in alcohol-induced liver fibrosis, portal myofibroblasts may be the most crucial fibrogenic cell types in viral hepatitis or chronic cholestatic disorders [1]. Thus, origin of liver injury may determine the molecular pathway differentiation in the formation of each liver disease, affecting the final proteomic outcome.

During fibrosis development, a complex interaction occurs between different hepatic cell types [32]. Most of the hepatoxic agents such as hepatitis viruses, alcohol metabolites, and bile acids target hepatocytes [33]. Injured hepatocytes secrete reactive oxygen species (ROS) and fibrogenic mediators, which triggers the activation of lymphocytes by inflammatory cells. Apoptosis of these injured hepatocytes further induces the fibrogenic actions of liver myofibroblasts [34]. Inflammatory cells such as lymphocytes and polymorphonuclear cells stimulate HSCs for collagen synthesis [35]. Activated HSCs also release inflammatory chemocines, secrete cell adhesion molecules, and mediate activation of lymphocytes [36]. Thus, a fierce cycle in which inflammatory and fibrogenic cells induce each other likely appears [37]. Kupffer cells, which are the local macrophages of liver, also greatly participate in liver inflammation by secreting ROS and cytokines [38, 39]. In conclusion, fibrogenesis is directly activated by alterations in the ECM composition and this altered ECM can serve as a repository for MMPs, growth factors, and inflammatory cytokines [1, 40].

Fibrosis progression is generally evaluated by two different accepted scoring systems: Ishak (modified Knodell score) and METAVIR scores. While in METAVIR, only interface hepatitis and lobular necrosis are used to determine the grade of activity, in Ishak, portal infiltrate and confluent necrosis are included with the two previous parameters [41]. Generally, fibrosis begins to develop as expansion of portal tracts occurring with interface hepatitis. As fibrosis advances, portal-portal linkage develops in conjunction with septa formation. At the end, fibrous tissue completely surrounds hepatocyte nodules. While complete cirrhosis develops generally in several years in some circumstances such as in the case of viral hepatitis, following liver transplantation cirrhosis may develop much more rapidly. Parenchymal fibrosis can also be observed in the presence of lobular inflammation, especially in areas of bridging necrosis [42]. This may be the cause of portal-central septa formation, which has been considered as more crucial process in the development of cirrhosis than portal-portal linkages [43]. In the terminology of liver fibrosis, septa indicate expansion of portal tract edges without formation of bridges or actual connection between portal areas or portal area and central vein. On the other hand, the term bridge is used to assess actual fibrous connection between two portal areas or portal area and central vein [44]. It is important to consider these mentioned staging systems in a descriptive sense that a patient with stage 2 fibrosis cannot be assumed to have sustained twice as much liver damage as one with stage 1 fibrosis, nor half as much as one with stage 4 fibrosis because numerical stages are not evenly distributed along the progression of fibrosis, and also transition from one stage to the next one is not linear. Nonetheless, pathologists' interobserver agreement in fibrosis staging among one stage is approximately 90% [45, 46].

## **3. Biomarkers of liver fibrosis**

An optimal biomarker of liver fibrosis would not get affected by functional distress in liver or kidneys and only be specific to liver, also be easily observed with simple, inexpensive, and noninvasive assays [13]. Liver enzymes that are routinely measured in serum such as alanine transaminase (ALT) and aspartate transaminase (AST) are not suitable biomarkers of liver fibrosis as they have poor correlation with liver fibrosis. Studies demonstrated that 20% of the biopsy-proven cirrhotic patients' ALT levels are in normal range [47]. Unfortunately, canonical markers of liver synthetic dysfunction [e.g., albumin, platelet count (PLT), prothrombin time (PT)] are shown to be unsuccessful in the detection of early fibrotic stages [48]. Currently, novel serum proteins have been observed with altered expression in progressing liver fibrosis such as apolipoprotein A1 (ApoA1), serum transferrin, and alpha 2 macroglobulin [49–51]. Biomarker panels that incorporate combination of these individual markers are also applicable for improved accuracy of fibrotic stage assessment [46]. The most currently used biomarker panels are AST to platelet ratio index [52], FibroTest that includes apolipoprotein A1 (ApoA1), haptoglobin (HPT), gamma-glutamyl transpeptidase (γGT), γ-globin, total bilirubin, and alanine aminotransferase as biomarkers [53], and FibroIndex that combines PLT, AST, and γGT [54]. These noninvasive biomarker panels have shown to achieve good negative predictive scores in patients with low fibrosis stages and good positive predictive scores in those with advanced stages. However, intermediate fibrotic stages are not successfully interpreted by these combined biomarkers [53]. Unfortunately, this setback limits the use of current available biomarker panels for routine clinical assessments of liver fibrosis [55].

## **4. Current proteomic profiling methodologies**

Proteomics, which is a swiftly developing area, is currently preferred in discovery of novel disease biomarkers due to its potential to surpass the drawbacks of traditional screening methods. The first step of the proteomic biomarker screening research is to separate and profile whole proteome of the biological fluid (e.g., serum, whole blood, saliva) or tissue of interest. Then, protein profile of the diseased sample is compared with a relevant control to identify the differentially expressed proteins related to that disease. Several different techniques based on in-gel separation and/or mass spectrometry are currently used for protein separation.

Mass spectrometry (MS) is the common technique in proteomic profiling methodologies. The basic concept of mass spectrometry is to evaluate the mass-to-charge (m/z) ratio for determination of the exact mass of the protein. The components of a mass spectrometry are an ion source, a mass analyzer, and a mass detector. Ionization of proteins is done either with matrixassisted laser desorption/ionization (MALDI) or electrospray ionization (ESI). Following ionization, proteins pass through one or two mass analyzers that measure their m/z ratio (MS or versus tandem MS/MS). Time-of-flight (TOF) that measures the time spent by the protein through the vacuum tube in an electric field can be coupled with one or two quadrupoles (Q-TOF or Q-Q-TOF) with oscillating electric field that enables molecules with specific m/z ratios to travel without collision [56, 57].

#### **4.1. Two-dimensional gel electrophoresis (2D-PAGE)**

liver transplantation cirrhosis may develop much more rapidly. Parenchymal fibrosis can also be observed in the presence of lobular inflammation, especially in areas of bridging necrosis [42]. This may be the cause of portal-central septa formation, which has been considered as more crucial process in the development of cirrhosis than portal-portal linkages [43]. In the terminology of liver fibrosis, septa indicate expansion of portal tract edges without formation of bridges or actual connection between portal areas or portal area and central vein. On the other hand, the term bridge is used to assess actual fibrous connection between two portal areas or portal area and central vein [44]. It is important to consider these mentioned staging systems in a descriptive sense that a patient with stage 2 fibrosis cannot be assumed to have sustained twice as much liver damage as one with stage 1 fibrosis, nor half as much as one with stage 4 fibrosis because numerical stages are not evenly distributed along the progression of fibrosis, and also transition from one stage to the next one is not linear. Nonetheless, pathologists' interobserver agreement in fibrosis staging among one stage is approximately

An optimal biomarker of liver fibrosis would not get affected by functional distress in liver or kidneys and only be specific to liver, also be easily observed with simple, inexpensive, and noninvasive assays [13]. Liver enzymes that are routinely measured in serum such as alanine transaminase (ALT) and aspartate transaminase (AST) are not suitable biomarkers of liver fibrosis as they have poor correlation with liver fibrosis. Studies demonstrated that 20% of the biopsy-proven cirrhotic patients' ALT levels are in normal range [47]. Unfortunately, canonical markers of liver synthetic dysfunction [e.g., albumin, platelet count (PLT), prothrombin time (PT)] are shown to be unsuccessful in the detection of early fibrotic stages [48]. Currently, novel serum proteins have been observed with altered expression in progressing liver fibrosis such as apolipoprotein A1 (ApoA1), serum transferrin, and alpha 2 macroglobulin [49–51]. Biomarker panels that incorporate combination of these individual markers are also applicable for improved accuracy of fibrotic stage assessment [46]. The most currently used biomarker panels are AST to platelet ratio index [52], FibroTest that includes apolipoprotein A1 (ApoA1), haptoglobin (HPT), gamma-glutamyl transpeptidase (γGT), γ-globin, total bilirubin, and alanine aminotransferase as biomarkers [53], and FibroIndex that combines PLT, AST, and γGT [54]. These noninvasive biomarker panels have shown to achieve good negative predictive scores in patients with low fibrosis stages and good positive predictive scores in those with advanced stages. However, intermediate fibrotic stages are not successfully interpreted by these combined biomarkers [53]. Unfortunately, this setback limits the use of current available

biomarker panels for routine clinical assessments of liver fibrosis [55].

Proteomics, which is a swiftly developing area, is currently preferred in discovery of novel disease biomarkers due to its potential to surpass the drawbacks of traditional screening

**4. Current proteomic profiling methodologies**

90% [45, 46].

**3. Biomarkers of liver fibrosis**

358 Advances in Treatment of Hepatitis C and B

The 2D-PAGE technique separates protein according to two independent parameters, isoelectric point and molecular weight, and therefore provides the best resolution possible in protein separation currently [58, 59]. Following staining and digitalization with specific softwares, protein quantitation is performed by evaluation of spot intensities. 2D-PAGE also enables detection of posttranslational modifications, such as phosphorylation, or presence of different protein isoforms due to the emerging shifts in protein mass or isoelectric point [46]. In addition, two-dimensional difference gel electrophoresis (2D-DIGE) presents various advances including reproducibility, detection sensitivity, and credibility of analysis [60–62]. In 2D-DIGE, different samples are labeled with charge- and mass-matched fluorescent cyanine dyes, Cy3 and Cy5. The internal standard prepared by mixing equal amounts of all samples is labeled by Cy2. The Cy3 and Cy5 labeled samples and Cy2 labeled internal standard are then mixed and co-separated on the same 2-DE gel, providing accurate spot detection and intra-gel matching with reduced experimental variations. Running internal standard within all gels also improves gel-to-gel spot matching and enables for statistically strong comparisons between protein samples [63]. At the end, protein spots cut from 2D gels were identified by mass spectrometry [64].

## **4.2. Liquid chromatography coupled mass spectrometry (LC-MS)**

Gel-based techniques such as 2D-PAGE are not very successful and reliable for profiling of small (>10 kDa) or hydrophobic proteins; besides, the evaluation of large numbers of samples is time-consuming and expensive. LC-MS, which couples a prefractionation stage with different types of mass spectrometry, is a relatively new gel-free proteomic methodology for proteomic profiling. One of the highly used MS methods is MALDI-TOF. In this technique, first, protein mixtures are fractionated by their physicochemical characteristics such as hydrophobicity or isoelectric point by liquid chromatography. Then, bound proteins are vaporized and ionized by a laser. Finally, peptide mass is computed from the time spend to reach the detector ("time-of-flight"). Another frequently applied method is LC-MS/MS which efficiently profiles large numbers of samples with the analysis of extremely small volume samples (i.e., <75 µl) by evaluating proteins with masses ranging from 2 to 200 kDa with tremendous efficiency and reasonable reproducibility [65]. In addition, SELDI-TOF MS, which couples a prefractination stage with MALDI-TOF, is currently used for proteomic profiling studies. In SELDI, protein mixtures that selectively bind to an array with a specified characteristic are analyzed. This methodology requires very low amount of crude sample, such as serum or needle biopsy samples, and it is very efficient in analysis of low molecular weight proteins. Considering the minimal labor required for SELDI application, this technique is very useful for high-throughput screening. However, higher cost of SELDI still limits its large clinical scale usage [66–68].

## **5. Proteomic profiling studies in search of biomarkers for liver fibrosis**

Proteomic studies on liver fibrosis mainly focus on cirrhosis and HCC, which are the very end and morbid stage of liver fibrosis. One of the earlier studies has compared tumor tissue and surrounding nontumor tissue from eight HCC patients and has showed overexpression of 14-3-3γ protein in HCC [69]. Another study has investigated the proteomic differences between tumor and adjacent nontumor tissue samples of 12 HBV-associated HCC patients and has found out upregulation of members of the heat shock protein 70 and 90 families and downregulation of metabolism-associated mitochondrial and peroxisomal proteins in HCC [70]. A recent study has analyzed sera of 40 HCC patients and 47 healthy controls and has discovered leucine-rich α2-glycoprotein (LRG) and haptoglobin (HPT) between HCV- and HBV-related HCC [71]. Molleken and Sitek (72) also have analyzed cirrhotic septa and liver parenchyma of seven cirrhotic patients and discovered an increase in cell structure-associated proteins, which are actin, prolyl 4-hydroxylase, tropomyosin, calponin, transgelin, and human microfibrilassociated protein 4 (MFAP-4). However, all these studies investigate the alterations occurring at the very end stage of fibrosis and did not give information about the proteomic changes during fibrosis progression.

To identify therapeutic targets and their involved pathways in fibrosis, the proteomic changes between different fibrotic stages should be investigated. There are several studies that focus on proteomic changes between different fibrotic stages. One of these studies has investigated serum protein profiles of HCV-infected patients and has showed that Mac-2 binding protein, α-2-macroglobin, and hemopexin were increased in cirrhosis, and α-1-antitrypsin, LRG, and fetuin-A (also named as alpha-2-HS-glycoprotein) were decreased in cirrhosis [73]. A recent research, which has enrolled sera of 16 healthy controls and 45 HCV patients with different fibrotic stages graded due to METAVIR, has found out that α-2-macroglobin (A2M) was increased, while vitamin D-binding protein (VDBP) and apolipoprotein A1 (ApoA1) were decreased in late fibrosis [51]. One of the studies examining serum samples of seven healthy controls and 27 HBV-infected patients with different stages of fibrosis has shown that fibrinogen, collagen, A2M, hemopexin, α-1-antitrypsin, transthyretin, and thiredoxin peroxidase were upregulated, while HPT, serotransferrin, CD5 antigen-like protein, clusterin, ApoA1, and LRG were downregulated along with fibrogenesis [74]. A recent study has analyzed sera of 19 CHB, six HBV-related cirrhotic patients, and five healthy controls and observed increased plasma myeloperoxidase levels in cirrhotic patients and decreased transthyretin, ceruloplasmin, and α-1-antitrypsin levels in both CHB- and HBVrelated cirrhosis patients and downregulation of ApoA1 in HBV-related cirrhosis [75]. These studies about liver fibrosis have revealed the proteomic changes of serum samples throughout fibrogenesis. There are few studies that investigated proteomic changes in HCV-associated fibrogenesis. Diamond et al. demonstrated the effect of oxidative stress proteins to fibrosis progression in biopsy samples of HCV-infected patients [76]. The same group recently analyzed proteomic mechanisms of HCV-mediated liver fibrosis in posttransplant recipients by LC-MS (liquid chromatography coupled mass spectrometry) and demonstrated once again the important role of enhanced oxidative stress in the rapid fibrosis progression observed in HCV-infected liver transplant patients [77]. Ferrin et al. studied liver biopsies of HCV-infected alcoholic patients with cirrhosis for altered proteins in the progression of HCC and observed deregulation of ceruloplasmin (CP), paraoxanase (PON1), complement component 4a (CD4a), and fibrinogen-α (FGA) expression [78]. Another study investigated the differences in the protein profiles between liver samples from HBV-infected transgenic mouse and nontransgenic mouse and demonstrated increased aldehyde dehydrogenase 2 (ALDH2), protein disulfide isomerase precursor (PRDX1), actin, 78 kDa glucose-regulated protein (GRP78), tumor rejection antigen (GRP94), keratin 18 (KRT18), and decreased glutamate dehydrogenease 1 (GLUD1) and high mobility group 1 (HMGB1) protein levels [79]. An extensive list of potential biomarkers emerging from these studies is listed in **Table 1**.

vaporized and ionized by a laser. Finally, peptide mass is computed from the time spend to reach the detector ("time-of-flight"). Another frequently applied method is LC-MS/MS which efficiently profiles large numbers of samples with the analysis of extremely small volume samples (i.e., <75 µl) by evaluating proteins with masses ranging from 2 to 200 kDa with tremendous efficiency and reasonable reproducibility [65]. In addition, SELDI-TOF MS, which couples a prefractination stage with MALDI-TOF, is currently used for proteomic profiling studies. In SELDI, protein mixtures that selectively bind to an array with a specified characteristic are analyzed. This methodology requires very low amount of crude sample, such as serum or needle biopsy samples, and it is very efficient in analysis of low molecular weight proteins. Considering the minimal labor required for SELDI application, this technique is very useful for high-throughput screening. However, higher cost of SELDI still limits its large

**5. Proteomic profiling studies in search of biomarkers for liver fibrosis**

Proteomic studies on liver fibrosis mainly focus on cirrhosis and HCC, which are the very end and morbid stage of liver fibrosis. One of the earlier studies has compared tumor tissue and surrounding nontumor tissue from eight HCC patients and has showed overexpression of 14-3-3γ protein in HCC [69]. Another study has investigated the proteomic differences between tumor and adjacent nontumor tissue samples of 12 HBV-associated HCC patients and has found out upregulation of members of the heat shock protein 70 and 90 families and downregulation of metabolism-associated mitochondrial and peroxisomal proteins in HCC [70]. A recent study has analyzed sera of 40 HCC patients and 47 healthy controls and has discovered leucine-rich α2-glycoprotein (LRG) and haptoglobin (HPT) between HCV- and HBV-related HCC [71]. Molleken and Sitek (72) also have analyzed cirrhotic septa and liver parenchyma of seven cirrhotic patients and discovered an increase in cell structure-associated proteins, which are actin, prolyl 4-hydroxylase, tropomyosin, calponin, transgelin, and human microfibrilassociated protein 4 (MFAP-4). However, all these studies investigate the alterations occurring at the very end stage of fibrosis and did not give information about the proteomic changes

To identify therapeutic targets and their involved pathways in fibrosis, the proteomic changes between different fibrotic stages should be investigated. There are several studies that focus on proteomic changes between different fibrotic stages. One of these studies has investigated serum protein profiles of HCV-infected patients and has showed that Mac-2 binding protein, α-2-macroglobin, and hemopexin were increased in cirrhosis, and α-1-antitrypsin, LRG, and fetuin-A (also named as alpha-2-HS-glycoprotein) were decreased in cirrhosis [73]. A recent research, which has enrolled sera of 16 healthy controls and 45 HCV patients with different fibrotic stages graded due to METAVIR, has found out that α-2-macroglobin (A2M) was increased, while vitamin D-binding protein (VDBP) and apolipoprotein A1 (ApoA1) were decreased in late fibrosis [51]. One of the studies examining serum samples of seven healthy controls and 27 HBV-infected patients with different stages of fibrosis has shown that fibrinogen, collagen, A2M, hemopexin, α-1-antitrypsin, transthyretin, and

clinical scale usage [66–68].

360 Advances in Treatment of Hepatitis C and B

during fibrosis progression.

Currently, studies also focused on understanding whether proteomic alterations may predict the treatment response in chronic hepatitis C. Hence, the effect of pegylated interferon (PegIFN) plus ribavirin (RBV) therapy, which is the common HCV treatment, may be understood better. When the serum samples from patients with chronic hepatitis C were subjected to metabolomics analysis to investigate the pretreatment and posttreatment characteristics of their metabolites by using capillary electrophoresis and liquid chromatography coupled mass spectrometry, tryptophan has been found to be associated with response to PegIFN/RBV therapy [82]. Moreover, identification of factors that predict virological response to antiviral therapy may improve treatment response through patient-specific treatment strategy. Recent studies revealed significant variances in proteome profiles throughout longitudinal serum samples in virological responders, in patients with mild fibrosis, and in those with mild necroinflammation [83]. In the current phase 2 studies (PROVE1, PROVE2, and PROVE3) of the direct-acting antiviral drug telaprevir, serum samples from responders and nonresponders to HCV treatment were analyzed by proteomic profiling and 15 differentially expressed proteins, with seven of them belonging to focal adhesion proteins or other macromolecular assemblies that constitute structural links between integrins and the actin cytoskeleton, were observed [84]. The ultimate goal of performing pretreatment serum proteome profiling prior to treatment is to predict sustained virological response (SVR) and nonresponse (NR) to antiviral drugs in chronic HCV infection and design suitable treatments for each patient.



**Protein Proteomic Analysis Sample Disease Positive or**

78 kDa glucose regulated protein,

362 Advances in Treatment of Hepatitis C and B

Carboxymethylenebutenolisade

homologue

GRP78

5'-3' exoribonuclease 1 LC-MS Liver biopsy HCV + [77]

A-1-antitrypsin 2D-PAGE Serum HCV - [73]

Actin 2D-PAGE Liver tissue HCV + [72]

Aldehyde dehydrogenase 2 2D-DIGE Mouse liver tissue HBV + [79] Apolipoprotein A1 2D-PAGE Serum CHB - [75]

Aryl sulfotransferase 1A3 LC-MS Liver biopsy HCV + [77] Bone martr stromal cell antigen 2 LC-MS Liver biopsy HCV + [77] Calponin LC-MS Liver biopsy HCV + [80]

CD44 antigen LC-MS Liver biopsy HCV + [77] CD5 antigen like protein 2D-DIGE Serum HBV - [74] Ceruloplasmin 2D-DIGE Serum HCV + [78] Clusterin 2D-DIGE Serum HBV - [74] Collagen 2D-DIGE Serum HBV + [74]

Complement component 4a 2D-DIGE Serum HCV + [78] Cystathione beta synthase LC-MS Liver biopsy HCV - [77] Cysteine and glycine rich protein 2LC-MS Liver biopsy HCV + [80] Cytochrome b-245 beta LC-MS Liver biopsy HCV + [77] Cytochrome c LC-MS Liver biopsy HCV + [76] Fetuin A 2D-PAGE Serum HCV - [73] Fibrinogen 2D-DIGE Serum HCV + [78]

Fibulin-5 LC-MS Liver biopsy HCV + [80] FK506 binding protein 14 LC-MS Liver biopsy HCV - [77] Gelsolin 2D-PAGE Serum HBV - [81] Glutamate dehydrogenase 1 2D-DIGE Mouse liver tissue HBV - [79] Gluthatione-S-transferases LC-MS Liver biopsy HCV - [77] Haptoglobin 2D-PAGE Serum HCV - [73]

Hemopexin 2D-PAGE Serum HCV + [73]

High mobility group 1 2D-DIGE Mouse liver tissue HBV - [79]

2D-DIGE Mouse liver tissue HBV + [79]

2D-DIGE Serum HBV + [74]

2D-DIGE Mouse liver tissue HBV + [79]

2D-DIGE Serum HBV - [74] 2D-DIGE Serum HCV - [51]

2D-PAGE liver tissue HCV + [72]

LC-MS Liver biopsy HCV - [77]

LC-MS Liver biopsy HCV + [80]

2D-DIGE Serum HBV + [74]

2D-DIGE Serum HBV - [74]

2D-DIGE Serum HBV + [74]

**Nagative Markera**

**Reference**

a Proteins up- (+) or downregulated (S) in liver fibrosis, as detected in proteomic studies.

b When multiple comparisons have been performed between individual fibrosis stages certain proteins might have been reported as positive and negative markers.

**Table 1.** Candidate biomarkers of liver fibrosis identified from proteomic studies.

## **6. Limitations of proteomics**

Proteomics have been shown as a promising tool in the evaluation of the molecular insights of liver fibrosis and in complementing previously known fibrosis biomarkers. Proteomic research is prone to unexpected and sometimes unpredictable biases [85]. Especially in analysis with multiple testing, extensive care should be given to assure that alterations observed are biologically significant and associated with the target disease [86]. Moreover, the unstable nature of biological samples makes them prone to degradation and alteration during sample processing [87]. Low-abundant proteins such as some stress expressed proteins and transcription factors are quite hard to be detected by proteomic screening.

Over 90% of the total serum protein concentration is constituted by some abundant proteins such as albumin and immunoglobins. Therefore, these abundant proteins may prevent detection of low-abundant proteins [88]. Depletion of serum from high-abundant proteins may increase the resolution and detection of low-abundance proteins [89]. However, while depleting serum from albumin, some potentially important proteins may bind to albumin and be lost for the upcoming analysis [90].

For the tissue samples, the diagnostic quality of biopsied tissue is limited for the evaluation of liver fibrosis. Presentation of only a very small part of the liver (approximately 1/50,000) by needle biopsy causes high sampling variability [91, 92]. Especially since fibrotic tissue is not distributed homogeneously inside the liver, sampling errors form 10% of false-negative diagnoses [91]. Moreover, interobserver agreement is not very high for particularly intermediate fibrosis stages. By considering these facts altogether, proteomic studies of liver fibrosis carry a robust characteristic.

## **7. Future directions and concluding remarks**

Future studies in search of biomarkers for liver fibrosis should involve an adequate reference standard. Moreover, it is fairly possible that each chronic liver disease (CLD) could have its etiology-specific biomarkers, and further research should cover the identification of optimal biomarker sets for each cause of CLD (such as HBV, HCV, NASH, alcohol abuse). Serum proteomic studies might be combined with imaging techniques such as MALDI imaging to improve the performance of noninvasive techniques [93].

In summary, proteomic studies offer a great insight into differentially expressed proteins in plasma and hepatic tissue of patients with liver fibrosis. The results of this proteomic knowledge present researchers a better understanding about the pathobiology of liver fibrosis and lead to the discovery of the best set of biomarkers for the noninvasive assessment of the clinical stage of patients.

## **Author details**

**6. Limitations of proteomics**

364 Advances in Treatment of Hepatitis C and B

be lost for the upcoming analysis [90].

**7. Future directions and concluding remarks**

improve the performance of noninvasive techniques [93].

a robust characteristic.

stage of patients.

Proteomics have been shown as a promising tool in the evaluation of the molecular insights of liver fibrosis and in complementing previously known fibrosis biomarkers. Proteomic research is prone to unexpected and sometimes unpredictable biases [85]. Especially in analysis with multiple testing, extensive care should be given to assure that alterations observed are biologically significant and associated with the target disease [86]. Moreover, the unstable nature of biological samples makes them prone to degradation and alteration during sample processing [87]. Low-abundant proteins such as some stress expressed proteins and transcrip-

Over 90% of the total serum protein concentration is constituted by some abundant proteins such as albumin and immunoglobins. Therefore, these abundant proteins may prevent detection of low-abundant proteins [88]. Depletion of serum from high-abundant proteins may increase the resolution and detection of low-abundance proteins [89]. However, while depleting serum from albumin, some potentially important proteins may bind to albumin and

For the tissue samples, the diagnostic quality of biopsied tissue is limited for the evaluation of liver fibrosis. Presentation of only a very small part of the liver (approximately 1/50,000) by needle biopsy causes high sampling variability [91, 92]. Especially since fibrotic tissue is not distributed homogeneously inside the liver, sampling errors form 10% of false-negative diagnoses [91]. Moreover, interobserver agreement is not very high for particularly intermediate fibrosis stages. By considering these facts altogether, proteomic studies of liver fibrosis carry

Future studies in search of biomarkers for liver fibrosis should involve an adequate reference standard. Moreover, it is fairly possible that each chronic liver disease (CLD) could have its etiology-specific biomarkers, and further research should cover the identification of optimal biomarker sets for each cause of CLD (such as HBV, HCV, NASH, alcohol abuse). Serum proteomic studies might be combined with imaging techniques such as MALDI imaging to

In summary, proteomic studies offer a great insight into differentially expressed proteins in plasma and hepatic tissue of patients with liver fibrosis. The results of this proteomic knowledge present researchers a better understanding about the pathobiology of liver fibrosis and lead to the discovery of the best set of biomarkers for the noninvasive assessment of the clinical

tion factors are quite hard to be detected by proteomic screening.

Seyma Katrinli1 , H. Levent Doganay2 , Kamil Ozdil2 and Gizem Dinler-Doganay1\*

\*Address all correspondence to: gddoganay@itu.edu.tr

1 Molecular Biology and Genetics Department, Istanbul Technical University, Maslak, Istanbul, Turkey

2 Department of Gastroenterology, Umraniye Teaching and Research Hospital, Umraniye, Istanbul, Turkey

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## **New Strategy Treating Hepatitis B Virus (HBV) Infection: A Review of HBV Infection Biology New Strategy Treating Hepatitis B Virus (HBV) Infection: A Review of HBV Infection Biology**

Yong-Yuan Zhang Yong-Yuan Zhang

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Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66083

#### **Abstract**

Chronic hepatitis B virus (HBV) infection affects 240 million people worldwide and represents a significant burden on public health. Current antiviral treatment of chronic hepatitis B mainly focuses on inhibiting viral replication. A main deficiency of the current treatment is unable to protect uninfected liver cells or hepatocytes that cleared HBV from next rounds of infection. HBV infection biology shows that natural clearance of HBV cccDNA from infected cells frequently occurs, HBV infection including chronic HBV infection is established and maintained by multiround infection, and the course of HBV infection is largely determined by the number of round of infection. Thus, an effective treatment of HBV infection must block new rounds of infection. A proposed new strategy for treating chronic HBV infection aims to immediately interrupt infection course and to achieve HbsAg seroconversion as early as possible. Under this strategy, a main target of antiviral treatment is extracellular viruses, and an effective therapeutics is specific neutralizing (anti-HBs) antibodies. A difference in tempo and efficiency of treating HBV infection between current antivirals and neutralizing antibody is that the antivirals inhibit viral infection only after cells are virus infected while the neutralizing antibody clears viruses before the infection of cells takes place.

**Keywords:** hepatitis B virus, acute hepatitis B, chronic HBV infection, HBV infection biology, antivirals, neutralizing antibody, anti-HBs

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction: why we need a better understanding of HBV infection biology?**

Chronic hepatitis B virus (HBV) infection affects 240 million people worldwide [1]. Estimated 4.5 million of new HBV infection occurs each year [2]. A significant portion of new HBV infections occurs in infants borne to HBV-positive mothers despite a fully scheduled HBV immunization. More than 90% of HBV-infected infants will become chronic [3–5], resulting in constant expansion of chronic HBV-infected population.

Chronic HBV infection can induce severe or repeated liver injury, which can lead to advanced liver diseases including cirrhosis [6, 7] and hepatocellular carcinoma [8]. The disease burden of HBV infection is enormous, and WHO reports that 780,000 people die of HBV-related liver diseases or complications annually [9]. In addition, as many as 70% of chronic HBV-infected patients who have persistently normal ALT already experienced significant alterations in liver histology [10–13]. We need to provide more effective treatment to chronic HBV-infected patients.

Current antiviral treatment is recommended for chronic HBV-infected patients who have evidence of liver injury related to HBV infection [14, 15]. Antivirals that mainly consist of nucleos/tide analogues can potently inhibit HBV replication, mitigate liver injury and slowdown progression of necroinflammation in the liver [16, 17]. However, the current treatment rarely clears chronic HBV infection. Majority of chronic HBV-infected patients are not suitable for current antiviral treatment. Untreated patients, despite normal ALT history, can experience unpredictable flare-ups of liver injury [18, 19]. Exacerbation insults, if occurred in patients with chronic liver injury or significant alterations in liver histology despite normal ALT, can trigger acute chronic liver failure with up to 70% mortality [20, 21].

Clearly, the current antiviral treatment strategy and available antivirals do not meet clinical needs.

Here, we briefly discuss a number of limitations of current antiviral strategy and antivirals.

## **1.1. Current treatment strategy does not protect virus-cleared cells or uninfected cells from new rounds of infection**

Current antiviral therapy is guided by belief and strategy that a viral infection can be cleared by directly inhibiting viral replication. This approach certainly mitigates viral diseases associated with viral replication, even clears viral infection under certain circumstances, for instance, a significant portion of chronic HCV infection can be cleared with a relatively short course of direct-acting antiviral agents [22, 23]. However, it does not take consideration of viral infection biology, which is featured with multiround infection (see below). This is why current antiviral strategy cannot clear chronic HBV infection. The NA-based antiviral therapy for chronic HBV infection, even with early generation of NAs like lamivudine, can clear HBV from HBV-infected cells, evidence includes wild-type (WT) virus, or early viral population was eliminated and replaced with drug-related mutant (MT) virus [24, 25]. Additional evidence consists of 5–6 logs reduction of serum HBV DNA level [26, 27–29] and 100-fold reduction of

intracellular total HBV DNA and cccDNA levels in treated animals and patients [30–32]. However, during the same period, serum HBsAg level is only reduced by two- to three fold [33], suggesting HBsAg level remains constantly high. The enduring high level of HBsAg keeps depleting the limited amount of endogenous neutralizing antibodies and leaves HBV virions that are produced by infected cells in the same livers, unneutralized and infectious, which continue to cause new rounds of infection. Under the current antiviral strategy, virus-cleared cells can immediately become infected again, gains in clearing viral infection are continuously reversed, and it is extremely difficult to establish permanent and complete viral clearance under current antiviral strategy because virus-cleared cells and uninfected cells are not protected.

**1. Introduction: why we need a better understanding of HBV infection**

Chronic hepatitis B virus (HBV) infection affects 240 million people worldwide [1]. Estimated 4.5 million of new HBV infection occurs each year [2]. A significant portion of new HBV infections occurs in infants borne to HBV-positive mothers despite a fully scheduled HBV immunization. More than 90% of HBV-infected infants will become chronic [3–5], resulting in constant expansion

Chronic HBV infection can induce severe or repeated liver injury, which can lead to advanced liver diseases including cirrhosis [6, 7] and hepatocellular carcinoma [8]. The disease burden of HBV infection is enormous, and WHO reports that 780,000 people die of HBV-related liver diseases or complications annually [9]. In addition, as many as 70% of chronic HBV-infected patients who have persistently normal ALT already experienced significant alterations in liver histology [10–13]. We need to provide more effective treatment to chronic HBV-infected

Current antiviral treatment is recommended for chronic HBV-infected patients who have evidence of liver injury related to HBV infection [14, 15]. Antivirals that mainly consist of nucleos/tide analogues can potently inhibit HBV replication, mitigate liver injury and slowdown progression of necroinflammation in the liver [16, 17]. However, the current treatment rarely clears chronic HBV infection. Majority of chronic HBV-infected patients are not suitable for current antiviral treatment. Untreated patients, despite normal ALT history, can experience unpredictable flare-ups of liver injury [18, 19]. Exacerbation insults, if occurred in patients with chronic liver injury or significant alterations in liver histology despite normal ALT, can trigger

Clearly, the current antiviral treatment strategy and available antivirals do not meet clinical

Here, we briefly discuss a number of limitations of current antiviral strategy and antivirals.

**1.1. Current treatment strategy does not protect virus-cleared cells or uninfected cells from**

Current antiviral therapy is guided by belief and strategy that a viral infection can be cleared by directly inhibiting viral replication. This approach certainly mitigates viral diseases associated with viral replication, even clears viral infection under certain circumstances, for instance, a significant portion of chronic HCV infection can be cleared with a relatively short course of direct-acting antiviral agents [22, 23]. However, it does not take consideration of viral infection biology, which is featured with multiround infection (see below). This is why current antiviral strategy cannot clear chronic HBV infection. The NA-based antiviral therapy for chronic HBV infection, even with early generation of NAs like lamivudine, can clear HBV from HBV-infected cells, evidence includes wild-type (WT) virus, or early viral population was eliminated and replaced with drug-related mutant (MT) virus [24, 25]. Additional evidence consists of 5–6 logs reduction of serum HBV DNA level [26, 27–29] and 100-fold reduction of

acute chronic liver failure with up to 70% mortality [20, 21].

**biology?**

patients.

needs.

**new rounds of infection**

of chronic HBV-infected population.

374 Advances in Treatment of Hepatitis C and B

## **1.2. Antivirals target viral replication in infected cells and do not directly act against extracellular viruses**

Two events occur simultaneously during antiviral therapy. One is viral replication is inhibited intracellularly, and the other is new rounds of infection continue as long as there are unneutralized extracellular viruses and susceptible cells that are not protected. Antivirals only suppress viral replication in infected cells, reducing production of new viruses that will lower level of extracellular viruses, which eventually lead to reducing new rounds of infection. Once no more viruses available for new rounds of infection, the infection course is actually interrupted, which brings the infection course to the end. Thus, the direct impact of treatment with antivirals is to reduce viral replication while an indirect impact is leading to limiting spread of infection, which really matters in containing and clearing viral infection. However, the antivirals do not directly act against extracellular viruses that may continuously cause new rounds of infection and compromise antiviral efficacy. This feature determines relatively ineffectiveness of direct antivirals in treating chronic HBV infection.

## **1.3. Effectiveness of antivirals depends on HBV replication efficiency**

The third issue is that antivirals do not inhibit or kill viruses that were already produced. They only inhibit producing new viruses upon replication. Antivirals will not function if there was no active viral replication. Actual effectiveness of an antiviral in treating hepatitis B is largely determined by HBV replication efficiency. In clinic, a link between HBV replication level and antiviral response has been indicated. For instance, in patients with long-term entecavir therapy, a full virological response rate was 59, 84, 90, 93, and 95% after 48, 96, 144, 192, and 240 weeks of therapy, respectively [27]. Clearly, 59% of treated patients achieved the full virological response to entecavir in the first 48 weeks, but only 16, 6, 3 and 2% of net increased response were, respectively, achieved when the therapy was extended from 48 to 96, 144, 192 and 240 weeks. The net increased response rate was progressively declined along with extending therapy period since the viral replication was increasingly depressed to very low level. The replication-dependent effectiveness determines the direct antivirals are not effective against HBV infection with inactive replication, for instance in anti-HBe–positive patients with low HBV DNA level or only HBsAg positive patients.

## **1.4. Ineffective against severe acute HBV diseases**

Direct antivirals would not be effective in treating severe acute hepatitis B since they take a few days or longer to significantly reduce viral replication or serum viral load (a log or greater) [34–37]. They would take much longer time to mitigate pathologic injury and to improve clinical manifestations [38]. Relatively slow action and no direct inhibition of extracellular viruses highlight potential ineffectiveness of antivirals in treating fulminant hepatitis B and acute severe exacerbation of chronic HBV infection-induced liver injury, both of which induce rapidly deteriorated clinic course and high mortality [39–41].

## **1.5. Antivirals alone cannot completely clear viral infection**

Antivirals may be no longer effective once the viral replication was inhibited to low level. Residual viruses can be secreted out, or released by turnovers of infected cells, leading to clearing infection or infected cells. The released residual viruses are small in quantity and can be completely neutralized if there is relatively sufficient amount of endogenous neutralizing antibodies, which lead to stopping the course of viral infection by blocking new rounds of infection. This scenario likely happens to clearing chronic hepatitis C virus infection with direct antivirals.

The released residual viruses following potent inhibition of virus production will still be capable of causing new rounds of infection if there were no sufficient neutralizing antibodies, prolonging the course of chronic infection. This scenario likely happens to antivirals-treated HBV-infected patients.

Clearly, antivirals alone cannot complete clearing viral infection,which requires the presence of sufficient neutralizing antibodies.

What we need is new treatment strategies and new therapeutics that can improve current treatment approach and antivirals in treating chronic HBV infection.

We believe we can find effective solutions to current problems in treating chronic HBV infection through a better understanding of HBV infection biology, which will also accelerate developing new prophylactics and therapeutics.

## **2. Understanding HBV infection biology**

## **2.1. Natural course of HBV infection**

Infection biology is science illustrating fundamentals of infection, which is centered on understanding how a productive infection is established and maintained. Understanding of infection biology starts with understanding natural course of infection. In this review, we only focus on the natural course of HBV infection that causes hepatitis. The natural course of hepatitis B consists of initial infection, incubation period and clinical phase (**Figure 1**).

**Figure 1.** Schematic illustration of a typical course of infection that consists of initial infection, incubation and clinical phases. Progression of infection course is directly driven by new rounds of infections. It also suggests the infection course can be ended at any stage as long as extracellular pathogens are completely neutralized.

## *2.1.1. Initial HBV infection*

**1.4. Ineffective against severe acute HBV diseases**

376 Advances in Treatment of Hepatitis C and B

rapidly deteriorated clinic course and high mortality [39–41].

**1.5. Antivirals alone cannot completely clear viral infection**

antivirals.

HBV-infected patients.

of sufficient neutralizing antibodies.

new prophylactics and therapeutics.

**2.1. Natural course of HBV infection**

**2. Understanding HBV infection biology**

Direct antivirals would not be effective in treating severe acute hepatitis B since they take a few days or longer to significantly reduce viral replication or serum viral load (a log or greater) [34–37]. They would take much longer time to mitigate pathologic injury and to improve clinical manifestations [38]. Relatively slow action and no direct inhibition of extracellular viruses highlight potential ineffectiveness of antivirals in treating fulminant hepatitis B and acute severe exacerbation of chronic HBV infection-induced liver injury, both of which induce

Antivirals may be no longer effective once the viral replication was inhibited to low level. Residual viruses can be secreted out, or released by turnovers of infected cells, leading to clearing infection or infected cells. The released residual viruses are small in quantity and can be completely neutralized if there is relatively sufficient amount of endogenous neutralizing antibodies, which lead to stopping the course of viral infection by blocking new rounds of infection. This scenario likely happens to clearing chronic hepatitis C virus infection with direct

The released residual viruses following potent inhibition of virus production will still be capable of causing new rounds of infection if there were no sufficient neutralizing antibodies, prolonging the course of chronic infection. This scenario likely happens to antivirals-treated

Clearly, antivirals alone cannot complete clearing viral infection,which requires the presence

What we need is new treatment strategies and new therapeutics that can improve current

We believe we can find effective solutions to current problems in treating chronic HBV infection through a better understanding of HBV infection biology, which will also accelerate developing

Infection biology is science illustrating fundamentals of infection, which is centered on understanding how a productive infection is established and maintained. Understanding of infection biology starts with understanding natural course of infection. In this review, we only focus on the natural course of HBV infection that causes hepatitis. The natural course of hepatitis B consists of initial infection, incubation period and clinical phase (**Figure 1**).

treatment approach and antivirals in treating chronic HBV infection.

Initial infection represents beginning of an infection and it provides seed of infection and usually consists of a few or small clusters of infected cells in the liver. The initial infection is important, but it cannot become a full-blown infection without incubation period.

#### *2.1.2. Incubation period*

The incubation refers to a period between initial infection and appearance of clinical symptoms. Length of the incubation varies considerably from individual to individual, but it is required for every HBV infection that causes hepatitis, which suggests the number of virus involved in the initial infection is so small that cannot immediately cause significant injury or clinical manifestations. Likely sequential events during the incubation include that infected HBV replicates in initial infected cells, progeny viruses are released through secretion or/and cytopathic destruction of infected cells, resulting in new rounds of infection of more cells, most likely in neighboring areas because short distances between viruses and susceptible cells favor higher efficiency of infection. Continuously released viruses keep initiating new rounds of infections, infecting more cells and extending the scope of infection. When the number of destructed cells reached an extent that causes clinical symptoms, the incubation is progressing to clinical phase.

Requirement of incubation period in full-blown HBV infection provides evidence that more than one round infection is required to establish and maintain a full-blown infection or hepatitis B (**Figure 1**). Thus, acute hepatitis B is caused by multiround of infection.

A main factor that allows multiround of infection is that there is no sufficient amount of endogenous neutralizing antibodies to neutralize the released viruses.

New rounds of infections inevitably occur during the infection course as long as there are unneutralized extracellular viruses and susceptible cells.

#### *2.1.3. Clinical phase of acute HBV infection*

Acute HBV infection in adults is a self-limiting disease, and 95% of them will be recovered without treatment [42]. A unique kinetics of serum HBV DNA level during acute HBV infection includes a rapid fall of HBV DNA level after the peak (**Figure 2**), suggesting HBV infection is being progressively cleared from the liver, as evidenced by rising ALT level (a result of destructing the infected cells) at the same time frame. Critically, it also suggests no new rounds of HBV infection, implying a block of new rounds of infection is required for clearing HBV infection. A mechanism behind these changes is called "HBsAg seroconversion," a hallmark for resolving HBV infection.

**Figure 2.** Kinetics of serum HBV DNA and ALT levels during acute hepatitis B. HBV DNA quickly falls after the peak, which coincides with rising ALT level. The rapid fall of HBV DNA suggests no new round infection.

When destruction of the infected cells is started during acute HBV infection, it will initially cause transit elevation of serum HBsAg and HBV DNA levels by releasing viral particles. However, the serum HBsAg and HBV DNA levels will then keep falling because the number of infected cells that supply and replenish serum HBsAg and HBV virion is progressively reduced by the liver injury (the supply reduced) while the removal or the half-life of viral particles from/in circulation should be at a constant rate, contributing to rapidly significant reduction of serum HBsAg and HBV levels. At certain point, the relative ratio between amounts of HBV particles (both viral and subviral particles) and anti-HBs antibodies will be reversed from the former greatly exceeding the latter, to the latter exceeding the former (HBsAg seroconversion). This conversion creates a sufficient anti-HBs capacity to clear serum HBsAg and to block new round infection, which allows uninfected cells and cells that cleared HBV infection permanently stay infection free. This process highlights an indispensable requirement of anti-HBs antibodies in blocking new rounds of HBV infection and resolving HBV infection.

New rounds of infections inevitably occur during the infection course as long as there are

Acute HBV infection in adults is a self-limiting disease, and 95% of them will be recovered without treatment [42]. A unique kinetics of serum HBV DNA level during acute HBV infection includes a rapid fall of HBV DNA level after the peak (**Figure 2**), suggesting HBV infection is being progressively cleared from the liver, as evidenced by rising ALT level (a result of destructing the infected cells) at the same time frame. Critically, it also suggests no new rounds of HBV infection, implying a block of new rounds of infection is required for clearing HBV infection. A mechanism behind these changes is called "HBsAg seroconversion," a hallmark

**Figure 2.** Kinetics of serum HBV DNA and ALT levels during acute hepatitis B. HBV DNA quickly falls after the peak,

When destruction of the infected cells is started during acute HBV infection, it will initially cause transit elevation of serum HBsAg and HBV DNA levels by releasing viral particles. However, the serum HBsAg and HBV DNA levels will then keep falling because the number of infected cells that supply and replenish serum HBsAg and HBV virion is progressively reduced by the liver injury (the supply reduced) while the removal or the half-life of viral particles from/in circulation should be at a constant rate, contributing to rapidly significant reduction of serum HBsAg and HBV levels. At certain point, the relative ratio between amounts of HBV particles (both viral and subviral particles) and anti-HBs antibodies will be reversed from the former greatly exceeding the latter, to the latter exceeding the former (HBsAg seroconversion). This conversion creates a sufficient anti-HBs capacity to clear serum HBsAg and to block new round infection, which allows uninfected cells and cells that cleared HBV infection permanently stay infection free. This process highlights an indispensable require-

which coincides with rising ALT level. The rapid fall of HBV DNA suggests no new round infection.

unneutralized extracellular viruses and susceptible cells.

*2.1.3. Clinical phase of acute HBV infection*

378 Advances in Treatment of Hepatitis C and B

for resolving HBV infection.

Different impacts of liver injury on outcomes of infection are expected if occurred at different frequencies. For instance, continuous or progressive liver injury at moderate or medium scale, as is in acute hepatitis B, or at massive scale as is in fulminant hepatitis can lead to complete clearance of HBV infection because of successful reversal of the ratio of serum HBsAg and anti-HBs. On the other hand, if occurred intermittently, it unlikely leads to a complete viral clearance as is in chronic hepatitis B because of no reversal of the ratio of HBsAg and anti-HBs. The different outcomes once again emphasize that HBsAg seroconversion is critically required for completely clearing HBV infection.

A HBV infection can be ended at any stage if subsequently released viruses were completely neutralized by endogenous neutralizing antibodies. Most of HBV infections are aborted during the incubation period without developing into a full-blown infection because relative amount of infected viruses is still low. Those individuals who ended the HBV infection before clinical stage only show detectable anti-HBs and anti-HBc antibodies without clinical manifestations and noticeable HBV infection history [43, 44].

Thus, a natural strategy to clear viral infection or end infection course utilized by the host is to include producing neutralizing antibodies to block new rounds of infection with newly released viruses [45]. However, in minority cases, the infection will advance to clinical stage or viral disease will be aggravated or clinical stage will be prolonged if the amount of endogenous neutralizing antibodies produced in the host is not high enough to neutralize all specific viruses during the incubation or clinical stage of infection. This is how acute and chronic infections are established.

Cellular immunity consists of two major functional mechanisms in controlling viral infection [46–48], one is to kill infected cells by cytotoxic T cells that not only contribute to pathological changes of viral disease, but also release viruses for new rounds of infection if not neutralized. The impacts of killing infected cells by the specific immunity are similar to cytopathic effects of viruses. The other is to inhibit replication of viruses in the infected cells by cytokines, which will reduce the number of released viruses. From controlling infection point of view, the cellular immunity can be counterproductive or helpful dependent on net effect of two actions (the amount of virions released) as well as the level of specific neutralizing antibodies. A difference in tempo of clearing viral infection between cellular and humoral immunity is that the cellular immunity clears viral infection only after cells are virus infected while the neutralizing antibody clears viruses before the infection takes place, one step ahead of the cellular immunity (**Figure 3**). Humoral immunity of neutralizing antibody is direct, decisively effective and required for controlling viral infection. Once new rounds of infection were completely blocked by sufficient amount of neutralizing antibodies, the viral infection in the already infected cells will be cleared by virus secretion, cytopathic effects of viral replication, cell turnover or the cellular immunity. Alternatively, the viral infection will be restricted to those already infected cells if the infected cells were long-lived, implying that viral infection has been brought under control with the neutralizing antibody.

**Figure 3.** Differences in tempo and efficiency of clearing viral infection between neutralizing antibody and antivirals. (A) Administrated neutralizing antibody immediately neutralizes extracellular viruses and protect uninfected cells; (B) administrated small molecule inhibitor first allows virus to infect cells then starts inhibiting viral replication in infected cells. It cannot immediately and completely remove viremia.

A chronic HBV infection is usually maintained by new rounds of infection. The chronic infection can be spontaneously cleared if the level of endogenous neutralizing antibodies is high and can neutralize all released viruses to stop new rounds of infection. For instance, spontaneous clearance of chronic HBV infection occurs at annual rates of 1–2% and is featured with seroconversion of HBsAg to anti-HBs antibody [49, 50].

## *2.1.4. A main risk that will prolong HBV infection course is extracellular viruses, and a main target in treating viral diseases is extracellular viruses*

Clearly, to cure HBV infection, it is essential and also efficient to interrupt infection course that consists of repeated rounds of infection.

There are three consequent scenarios during the course of hepatitis B: one is the infected cells become lost, resulting in releasing more virions as a consequence of cytopathic effects of infected HBV, cell turnover or destruction by cellular immunity; the second one is the infection is spontaneously cleared from infected cells by virus secretion or/and cellular immunity that reduces production and release of viruses, a same outcome as inhibiting viral replication by antivirals, and the third one is the infected cells are relatively long-lived (non-cytopathic infection) and producing and releasing low or high level of viruses. A shared main consequence produced by each scenario is releasing viruses. Thus, a main target in treating HBV infection should be the extracellular viruses, which cause new rounds of infection. Direct antivirals like NAs are not agents that can directly counter the extracellular viruses. Overall effectiveness of treating chronic HBV infection will be significantly improved if neutralizing antibodies are employed.

## **2.2. HBV infection course and persistence**

**Figure 3.** Differences in tempo and efficiency of clearing viral infection between neutralizing antibody and antivirals. (A) Administrated neutralizing antibody immediately neutralizes extracellular viruses and protect uninfected cells; (B) administrated small molecule inhibitor first allows virus to infect cells then starts inhibiting viral replication in infected

A chronic HBV infection is usually maintained by new rounds of infection. The chronic infection can be spontaneously cleared if the level of endogenous neutralizing antibodies is high and can neutralize all released viruses to stop new rounds of infection. For instance, spontaneous clearance of chronic HBV infection occurs at annual rates of 1–2% and is featured

*2.1.4. A main risk that will prolong HBV infection course is extracellular viruses, and a main target in*

Clearly, to cure HBV infection, it is essential and also efficient to interrupt infection course that

There are three consequent scenarios during the course of hepatitis B: one is the infected cells become lost, resulting in releasing more virions as a consequence of cytopathic effects of infected HBV, cell turnover or destruction by cellular immunity; the second one is the infection is spontaneously cleared from infected cells by virus secretion or/and cellular immunity that reduces production and release of viruses, a same outcome as inhibiting viral replication by antivirals, and the third one is the infected cells are relatively long-lived (non-cytopathic infection) and producing and releasing low or high level of viruses. A shared main consequence produced by each scenario is releasing viruses. Thus, a main target in treating HBV infection should be the extracellular viruses, which cause new rounds of infection. Direct antivirals like NAs are not agents that can directly counter the extracellular viruses. Overall effectiveness of treating chronic HBV infection will be significantly improved if neutralizing

cells. It cannot immediately and completely remove viremia.

380 Advances in Treatment of Hepatitis C and B

*treating viral diseases is extracellular viruses*

consists of repeated rounds of infection.

antibodies are employed.

with seroconversion of HBsAg to anti-HBs antibody [49, 50].

HBV infection is known for long incubation period that may last up to 6 months (average 2–3 months) [51, 52] before occurrence of clinical symptoms of acute HBV infection. We believed that multiround infection occurs during the incubation period. Duck hepatitis B virus (DHBV) experimental infection was utilized to verify this understanding of HBV infection dynamics during the incubation period. Ducklings were inoculated with high DHBV inoculation dose, and three animals were daily sacrificed for 7 days after inoculation. Viral core protein was stained on liver sections, and DHBV DNA was detected in liver tissues [45]. As shown in **Figure 4A** and **B**, DHBV infection rapidly expanded from a few clusters of initially infected cells in the infected livers and reached a full-blown infection in 7 days, showing there were repeatedly new rounds of infection that expanded the infection scope in the livers, even under the circumstances of experimental infection that used a very large inoculum.

**Figure 4.** Multiround infection during the incubation period of DHBV infection. Viral replicative intermediates (A) and antigen-staining cells (B) were detected in livers of DHBV-infected ducks. Three ducks were sacrificed at the indicated days postinfection, and replicative intermediates were extracted from the liver. The viral DNA was detected by hybridization. The percent antigen-staining hepatocytes in some of the samples is indicated at the top of the lanes.

Are there continuously new rounds of infections after the full-blown HBV infection was established?

Current theory views that chronic HBV infection is a consequence of the host's insufficient T-cell immunity that cannot kill all HBV-infected cells or cannot clear HBV infection from infected cells [53, 54], implying chronic HBV infection is a simple extension of the initial or early HBV infection; the hepatocytes, once infected with HBV, are long-lived and constantly infected with the same initial viruses during chronic course.

However, such view is not supported by experimental and clinical evidence.

To investigate whether DHBV infection and DHBV-infected cell populations are stable after liver was fully infected, 3-day-old ducklings were inoculated with a large inoculum containing both DHBV 3 and 16 viruses at 1:1 ratio. The first biopsy was conducted at day 11 by which the liver was fully infected, and then, series of liver biopsies were performed every 3 weeks. Distinct genetic markers in two viral genomes allowed us to monitor kinetic changes in DHBV 3 and 16 singly and dually infected populations by determining cccDNA genotypes at single nucleus level. It was found that majority of hepatocytes were singly infected and nearly 20% of cells exhibited dual infection with both viruses at day 11 postinfection (p.i.) (**Figure 5**). We detected new rounds of infections at each of 5 time points after day 11 because the fraction of DHBV3-infected cells was expanding, but the expansion occurred in only singly infected fashion, suggesting that already infected cells resisted superinfection while the new rounds of infection were occurring. It also suggested occurrence of viral clearance in DHBV-infected liver, which generated uninfected cells for new rounds of infection. This viral clearance conclusion is consistent with data showing that the fraction of DHBV16-infected cells was decreased from 80% at day 32, to about 40% at day 131 p.i., and at least 40% of DHBV16-infected cells either cleared the infection or were eliminated, which triggered regeneration of hepatocytes. Either of two scenarios would produce uninfected cells targeted by new rounds of infection. The results suggest DHBV infection even after the full infection was established in the liver remains dynamic and is featured with new rounds of infections.

**Figure 5.** Repeated new rounds of infection after a liver was fully infected. Three-day-old ducklings (n = 20) were infected with an inoculum containing both DHBV3 and 16. Six biopsies were performed on one animal at day 11, 32, 66, 88, 109 and 131 days postinfection (shown in this figure). cccDNA genotypes were determined by sequencing PCR products amplified from cccDNA released from single individual nuclei. DHBV3-infected cells were expanding, while DHBV16-infected cells were decreasing during the period of six times of biopsies, suggesting ongoing viral clearance and new rounds of infection. As a quality control for our procedure, the same number of nuclei isolated from DHBV3 and DHBV16 singly infected livers was mixed and single individual nucleus was sorted into individual well of 96-well plates for PCR amplification and sequencing. No dual infections were detected in 128 mixed nuclei, suggesting that our procedure for detection is valid.

The results on clearing of DHBV16 cccDNA and new establishing of DHBV3 cccDNA pool from new rounds of infection are consistent with the early reports, in which a replication defective pre-core mutant or revertants successfully spread infection and became the predominant viral population following elimination of wild type (WT) or the initially inoculated viruses (**Figure 6**) [55, 56]. Taken together, all results suggest that a chronic DHBV infection is not a simple extension of the initial infection because the initial infection was cleared and the early cccDNA genotypes were replaced. Rather chronic DHBV infection course is dynamic and consists of viral clearance and new rounds of infection. Thus, repeatedly new rounds of infection maintain the persistence of viral cccDNA and prolong the course of chronic infection.

To investigate whether DHBV infection and DHBV-infected cell populations are stable after liver was fully infected, 3-day-old ducklings were inoculated with a large inoculum containing both DHBV 3 and 16 viruses at 1:1 ratio. The first biopsy was conducted at day 11 by which the liver was fully infected, and then, series of liver biopsies were performed every 3 weeks. Distinct genetic markers in two viral genomes allowed us to monitor kinetic changes in DHBV 3 and 16 singly and dually infected populations by determining cccDNA genotypes at single nucleus level. It was found that majority of hepatocytes were singly infected and nearly 20% of cells exhibited dual infection with both viruses at day 11 postinfection (p.i.) (**Figure 5**). We detected new rounds of infections at each of 5 time points after day 11 because the fraction of DHBV3-infected cells was expanding, but the expansion occurred in only singly infected fashion, suggesting that already infected cells resisted superinfection while the new rounds of infection were occurring. It also suggested occurrence of viral clearance in DHBV-infected liver, which generated uninfected cells for new rounds of infection. This viral clearance conclusion is consistent with data showing that the fraction of DHBV16-infected cells was decreased from 80% at day 32, to about 40% at day 131 p.i., and at least 40% of DHBV16-infected cells either cleared the infection or were eliminated, which triggered regeneration of hepatocytes. Either of two scenarios would produce uninfected cells targeted by new rounds of infection. The results suggest DHBV infection even after the full infection was established in the liver remains

**Figure 5.** Repeated new rounds of infection after a liver was fully infected. Three-day-old ducklings (n = 20) were infected with an inoculum containing both DHBV3 and 16. Six biopsies were performed on one animal at day 11, 32, 66, 88, 109 and 131 days postinfection (shown in this figure). cccDNA genotypes were determined by sequencing PCR products amplified from cccDNA released from single individual nuclei. DHBV3-infected cells were expanding, while DHBV16-infected cells were decreasing during the period of six times of biopsies, suggesting ongoing viral clearance and new rounds of infection. As a quality control for our procedure, the same number of nuclei isolated from DHBV3 and DHBV16 singly infected livers was mixed and single individual nucleus was sorted into individual well of 96-well plates for PCR amplification and sequencing. No dual infections were detected in 128 mixed nuclei, suggesting that

dynamic and is featured with new rounds of infections.

382 Advances in Treatment of Hepatitis C and B

our procedure for detection is valid.

**Figure 6.** WT DHBV was being replaced over time. The fraction of WT DHBV DNA in serum of four DHBV-infected ducks was determined by PCR sequencing assay and plotted against the time postinfection. The kinetics of replacing WT DHBV infection suggests DHBV infection in fully infected livers remains dynamic and featured with clearing WT and new rounds of infection with MT.

These conclusions are supported by clinical HBV infection data.

It is well known that WT HBV infection is frequently replaced with mutant (MT) infection [25, 57–82] in untreated chronic HBV-infected patients. For instance, naturally occurring pre-core, core, pre-S and S mutants became only or dominant viral population following the WT was being eliminated from infected livers during natural course of chronic HBV infection. The data strongly imply that WT or early HBV infection is frequently cleared at cccDNA level during chronic HBV infection. It is notable that such cccDNA clearance naturally occurs without intervention. This understanding is consistent with cccDNA clearance in adult patients with acute HBV infection in which HBV infection is naturally resolved, and no antiviral treatment is needed.

Available data also suggest that HBV WT is replaced with HBV MT in NAs-treated chronic HBV-infected patients as evidenced by emergence of drug resistant mutant infection [25, 82]. The drug mutant infection that may not bear drug resistant phenotypes was also frequently detected in new generation of NAs-treated patients. A recent report shows that approximately 50% of patients treated with tenofovir (TDF) who remained viremic, developed pol/RT mutant infection though none of patients showed the drug resistance phenotypes [83]. The frequency of mutant infection in this report may be still underestimated because only the pol/RT sequence was analyzed. The observation that drug-related mutants spread following elimination of WT during NAs treatment suggests frequent viral clearance at cccDNA level and new rounds of infection with mutants.

## **3. New strategy and therapeutics for treating HBV infection guided by the viral infection biology**

We propose a new strategy for treating HBV infection. This strategy directly aims to establish HBsAg seroconversion as early as possible through administrating sufficient amount of specific neutralizing antibodies, which will constantly and completely neutralize extracellular viruses to block repeated rounds of infection. This new strategy represents a paradigm shift in treating HBV infection, which has been treated primarily by inhibiting viral replication.

## **3.1. Directly dealing with the huge pool of HBsAg in chronic HBV infection**

As the evidence points out, chronic HBV infection is not a simple extension of the initial infection, but is established and maintained by new rounds of infection. A unique situation in HBV infection is that it produces a huge pool of subviral particles (HBsAg) that are 1000–10,000 fold higher than virions [84]. The HBsAg primarily depletes the limited amount of endogenous neutralizing antibodies and leaves virions unneutralized and infectious. Current treatment strategy and approved antivirals are not designed to deal with new rounds of infection, almost impossibly deliver HBsAg seroconversion, and this is why current treatment rarely cures chronic HBV infection. The key to curing chronic HBV infection is to establish HBsAg seroconversion and to interrupt the infection course as early as possible by providing and maintaining high level of HBV neutralizing antibodies until the amount of endogenous HBV neutralizing antibodies exceeds the amount of HBV particles or all infected cells cleared HBV.

Advantages of the new strategy:


## **4. Conclusions**

intervention. This understanding is consistent with cccDNA clearance in adult patients with acute HBV infection in which HBV infection is naturally resolved, and no antiviral treatment

Available data also suggest that HBV WT is replaced with HBV MT in NAs-treated chronic HBV-infected patients as evidenced by emergence of drug resistant mutant infection [25, 82]. The drug mutant infection that may not bear drug resistant phenotypes was also frequently detected in new generation of NAs-treated patients. A recent report shows that approximately 50% of patients treated with tenofovir (TDF) who remained viremic, developed pol/RT mutant infection though none of patients showed the drug resistance phenotypes [83]. The frequency of mutant infection in this report may be still underestimated because only the pol/RT sequence was analyzed. The observation that drug-related mutants spread following elimination of WT during NAs treatment suggests frequent viral clearance at cccDNA level and new rounds of

**3. New strategy and therapeutics for treating HBV infection guided by the**

We propose a new strategy for treating HBV infection. This strategy directly aims to establish HBsAg seroconversion as early as possible through administrating sufficient amount of specific neutralizing antibodies, which will constantly and completely neutralize extracellular viruses to block repeated rounds of infection. This new strategy represents a paradigm shift in treating HBV infection, which has been treated primarily by inhibiting viral replication.

As the evidence points out, chronic HBV infection is not a simple extension of the initial infection, but is established and maintained by new rounds of infection. A unique situation in HBV infection is that it produces a huge pool of subviral particles (HBsAg) that are 1000–10,000 fold higher than virions [84]. The HBsAg primarily depletes the limited amount of endogenous neutralizing antibodies and leaves virions unneutralized and infectious. Current treatment strategy and approved antivirals are not designed to deal with new rounds of infection, almost impossibly deliver HBsAg seroconversion, and this is why current treatment rarely cures chronic HBV infection. The key to curing chronic HBV infection is to establish HBsAg seroconversion and to interrupt the infection course as early as possible by providing and maintaining high level of HBV neutralizing antibodies until the amount of endogenous HBV neutralizing antibodies exceeds the amount of HBV particles or all infected cells cleared HBV.

**1.** It directly immediately targets extracellular viruses and blocks the spread of infection;

**2.** It facilitates permanent and complete viral clearance in the liver;

**3.1. Directly dealing with the huge pool of HBsAg in chronic HBV infection**

is needed.

infection with mutants.

384 Advances in Treatment of Hepatitis C and B

**viral infection biology**

Advantages of the new strategy:

In this paper, we analyze the deficiencies of current HBV treatment strategy and antivirals and the reason why they cannot cure chronic HBV infection. We also review the viral infection biology to fresh our understanding of general phases and natural course of HBV infection. We conclude that a full-blown HBV infection is established and maintained through multiround infection. We propose a new strategy for treating HBV infection. The core of this new strategy is that we must achieve HBsAg seroconversion naturally or interventionally to effectively clear HBV infection. Under the proposed strategy, a main target of the treatment is extracellular viruses, and an effective therapeutics is specific neutralizing antibodies.

## **Author details**

Yong-Yuan Zhang

Address all correspondence to: yongyuanzhang@hbvtech.com

HBVtech, Frederick Innovative Technology Center, Frederick, MD, USA

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394 Advances in Treatment of Hepatitis C and B

## *Edited by Naglaa Allam*

As in many areas of medicine, treatment of viral hepatitis has seen an acceleration of change driven by new therapies and evolving technology. Thanks to the direct-acting antiviral agents (DAAs), the era of HCV eradication and cure has begun. As regards to hepatitis B therapy, potent antiviral drugs for suppression of viral replication are available, new research activities to enhance eradication are visible, and these may influence clinical practice in the coming years. This book covers the latest advances in hepatitis C and hepatitis B therapeutics as well as the emerging and investigational treatment strategies. "Advances in Treatment of Hepatitis C and Hepatitis B" book is an up-to-date source of information for physicians, residents, and advanced medical students seeking a broader understanding of treatment of viral hepatitis. The authors of the chapters come from many eminent centers around the world and are experts in their respective fields.

Advances in Treatment of Hepatitis C and B

Advances in Treatment of

Hepatitis C and B

*Edited by Naglaa Allam*

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