Mouse Model of Chronic Hepatitis B

#### **Chapter 3**

## Establishment of a Mouse Model of Chronic Hepatitis B Virus Infection and Purification of Hepatic Parenchymal and Non-Parenchymal Cells

*Yan Yan and Chantsalmaa Davgadorj*

#### **Abstract**

The use of replication-competent hepatitis B virus (HBV) DNA to construct a mouse model will help explore antiviral treatment strategies for more than 240 million patients infected with HBV worldwide. Eradication of chronic HBV infection can effectively block the adverse consequences of HBV-induced hepatic cirrhosis, failure and carcinoma. The core reason that HBV is difficult to eradicate is that most of infected people develop chronic HBV infection due to the establishment of immune tolerance. Here, we introduce a mouse model of adeno-associated virus (AAV)-HBV transfection, which produces HBV surface antigen (HBsAg) that can be maintained for more than 6 months. During virus replication, intermediates, transcripts, and proteins can be detected in peripheral blood. At the same time, the prerequisite for studying liver disease formation and immunotherapy through *in vitro* experiments is to isolate hepatic subgroup cells. Here, we describe a cell sorting method based on liberase perfusion technology combined with low-speed centrifugation and magnetic bead antibody labeling to purify hepatic parenchymal cells (PCs) and non-parenchymal cells (NPCs) step by step from murine liver, such as hepatic sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs), which will help accelerate the study of the genetic and clearance mechanistic of chronic HBV infection.

**Keywords:** hepatitis B virus, CD8+ T cell, hepatic parenchymal cell (HPC), hepatic sinusoidal endothelial cell (LSEC), Kupffer cell (KC)

#### **1. Introduction**

Hepatitis B virus (HBV) has an extremely narrow host-range, such as humans [1], chimpanzees [2], Mauritian cynomolgus monkey [3], treeshrew [4, 5] and woodchuck [6, 7]. These model thereby can be exploited to investigate viral-host interaction and pathogenesis including acute hepatitis, chronic hepatitis, hepatic fibrosis, hepatic cirrhosis and hepatic carcinoma. There are certain limitations using of these models, due to expensive facilities required, difficult to care and ethics restriction, and the shortage of reagents for measuring host factors and for investigating host immunopathogenesis, such as chimpanzees. Mouse is considered to be a less expensive alternative animal model, but its liver lacks the sodium taurocholate cotransporting polypeptide (NTCP) needed for HBV infection. Although human-NTCP (huNTCP) transgenic mice are considered to be able to construct HBV-infected mice, the results failed to support the original hypothesis by measuring HBV DNA [8].

Studies on the HBV mouse models have gone through several generations, involved in HBV DNA transgenic mice [9], HBV DNA transfected mice [10], HBV rc-cccDNA transfected mice [11], HBV-infected liver chimeric humanized mice and CRISPR/Cas9 technological NRG/Fah/ immune deficient mice [12]. Their advantages and disadvantages are as follows, separately: (1) 1.3-fold HBV genome transgenic mice support the expression of viral RNA and viral proteins in the liver, and can develop complete pgRNA, viral assembly and viral secretion during in the viral cycle, and support endosomal antiviral [13]. Due to the integration of the HBV genome in the host chromosome, the disadvantages of transgenic mice include the undetectable HBV cccDNA in mouse hepatocytes, the innate immune tolerance to HBV antigens (Ags), and the inability to study Ag-related immune activation. (2) The 1.2-fold HBV genomic DNA hydrodynamic injection (H.I.) model is one major breakthrough and first developed to meet the requirements of mouse nonintegrated viral genomes [14]. Different from immunodeficient mice such as liver humanized mice, the mice can express HBV Ags, and the immune system can also recognize the Ags to stimulate virus-specific immune responses. This model system can be used to test HBV cure strategies and study HBV immunology [15, 16]. However, this mode has limitation of mouse category. For example, experiments have shown that male C3H and C57BL/6 J are more effective, and the success rate varies in mouse models [17, 18]. The operation of H.I. also requires a high level of technical proficiency. The AAV-HBV vector has minimal AAV genome, only the essential AAV inverted terminal repeat sequence (ITR), is used to analyze the virus packaging and does not encode any AAV viral proteins. Therefore, this vector provides a clean background when analyzing HBV-specific immune responses. In our chapter, we will introduce how to establish this mouse model, the detectable HBsAg protein persists in blood or liver more than 6 months and has been widely used as chronic infection model [19]. (3) HBV replication is not detectable and HBV cccDNA non-formation is suggested to be restricted in mouse model [20]. HBV cccDNA is a symbol of continuous virus replication and an indispensable component for HBV to cause liver damage and fibrosis [21]. The half-life of HBV cccDNA is long in the human liver, which is a difficult problem for clinical cure of hepatitis B. Construct a transfected recombinant minicircle cccDNA achieving long-term maintenance in C3H mouse modle contributes to investigating HBV cccDNA-related biology and for evaluating anti-HBV drugs [11]. In order to overcome the shortcoming of cccDNA lasting only a few weeks, a mouse model was constructed using adenovirus vectors and the linear HBV cccDNA genome was delivered to Cre transgenic mouse [22]. (4) Humanized immunodeficient mice have been used to generate HBV-infected mice [23]. However, these animals lace a functional immune system, hindering research on immunological issues related to HBV infection and immunotherapy.

At present, AAV-HBV plasmid transfection can obtain an ideal HBV chronic infection mouse model, and it has been applied in a number of studies [14, 24, 25]. In the clinical recovery process of chronic hepatitis B, there are strict rules on the selectivity of anti-viral therapy for patients to ensure that interferon combined with nucleoside analog therapy produces better results. Therefore, it is necessary to further understand the relationship between drugs, HBV and hepatocyte immunity. As our previous studies have shown, TLRs [26, 27] and chemokines [15, 25] can promote the clearance of HBV by regulating the status of hepatic parenchymal cells (PCs) and non-parenchymal cells (NPCs), and also clarifies the cellular mechanism. Hepatic PCs and NPCs play a key role in mediating liver immune tolerance and mediating early

*Establishment of a Mouse Model of Chronic Hepatitis B Virus Infection and Purification… DOI: http://dx.doi.org/10.5772/intechopen.99939*

innate and adaptive immune responses. Small molecule drugs or cytokines activate these cells to promote virus clearance. The specific mechanism will be verified using isolated mouse hepatocytes and the corresponding mechanism will be explained.

### **2. Materials**

#### **2.1 Materials for transformation and extraction of plasmids**


#### **2.2 Materials, reagents and culture media for hepatocyte extraction and culture**



#### **Table 1.**

*Formulas of perfusion and hepatocyte culture media.*


<sup>1</sup> Dilute 5 mg rat tail collagen type II with 1 mL 0.006 mol/L (0.36 g/L) sterile acetic acid solution, and dissolve 240 μL of collagen II (5 mg /mL) to 95 mL sterile acetic acid solution, and finally dilute to 100 mL. Dissolve the rat tail collagen type II solution used for coating at 5–8 μg/cm<sup>2</sup> (cm2 refers to the bottom surface area of the culture well) (12 μg/mL). Add 300 μL to each well of 24 well plate, and add 150 μL to each well of 48 well plate, then dry overnight under ultraviolet light. The dried plates are kept sealed and can be stored stably for several months (>3 months) at 4–25°C.

*Establishment of a Mouse Model of Chronic Hepatitis B Virus Infection and Purification… DOI: http://dx.doi.org/10.5772/intechopen.99939*


### **3. Method**

#### **3.1 Plasmid enlargement culture and extraction from bacteria**


#### **3.2 Mouse model**


<sup>2</sup> Send the diluted 20 μL DNA for sequencing or real-time PCR to verify accuracy.

Serum levels of HBsAg, HBeAg and HBsAb are detected with ELISA according to the reagent instructions (Jingmei Biotechnology).

3.HBV transcription in the liver: The mouse liver is used to preserve in 4% polyformaldehyde for the next immunohistochemical analysis [10, 25] (Another alternative detection method.<sup>3</sup>

#### **3.3 Perfusion and digestion of mouse liver**


<sup>3</sup> Detect the level of nucleocapsid HBV DNA extracted from 60 mg of murine liver by real-time PCR on the ABI PRISM 7500 Sequence Detection System (ABI PRISM™). Use an All-In-One DNA/RNA/Protein Mini-preps kit (Sangon Biotech) to purify total DNA from serum. According to the manufacturer's instructions, use careHBV PCR Assay V3 reagents (QIAGEN, China) for HBV DNA detection. This type of model chronic HBV mice has no liver damage and abnormal serum alanine aminotransferase (ALT). <sup>4</sup> Using tape instead of puncture fixation can reduce the pain of mice and also prevent the mice from waking up from anesthesia as soon as possible.

<sup>5</sup> Before starting priming, carefully check and remove any visible air bubbles in the pump system. Properly activating the pump and piercing the needle into the portal vein to remove any invisible air in the needle tip area may block the microcapillaries and cause insufficient perfusion of certain liver lobes. <sup>6</sup> This is a non-circulatory perfusion, and all liquid will flow out of the inferior vena cava. Therefore, it is necessary to lay some absorbent tissues on the foam workbench where the mouse is fixed.

<sup>7</sup> Perfusion and digestion are very important steps. During the digestion process, digestive enzymes are injected and the severed inferior vena cava is pressed to close the vascular output, so that the liver is fully perfused and expanded, which can prolong the effective digestion time. After ideal digestion, the liver will turn from red to yellow-white, and the tissues will become soft after digestion. The more complete the liver digestion, the higher the ratio of single cells to live hepatic parenchymal cells, and the higher the survival rate in culture.

*Establishment of a Mouse Model of Chronic Hepatitis B Virus Infection and Purification… DOI: http://dx.doi.org/10.5772/intechopen.99939*


#### **3.4 Isolation and culture of hepatocytes**

	- i. Spin the cells at 50 g at 4°C for 5 min, 3 times, and wash with hepatocyte culture medium (HCM); Centrifuge has 9 speeds of acceleration and 9 speeds of decelerates (i.e., acceleration of 9, deceleration of 9).
	- ii. Aspirate the supernatant into a new 50 mL centrifuge tube. The supernatant contains hepatic non-parenchymal cells (NPCs).<sup>9</sup> The precipitated cells are hepatic PCs.
	- iii. Inoculate hepatic PCs on a 24-well plates covered with collagen II, <sup>360</sup> <sup>μ</sup>L/well (Use HCM to dilute PCs to 3.5 <sup>10</sup><sup>6</sup> cells/ml before inoculation).
	- iv. Incubate at 37°C, 5% CO2, and shake at the crossover level every 15 min for 1 h to suspend the hepatocytes with lower viability. Incubate overnight, wash with PBS 3 times, change the medium and continue culturing. The morphology of hepatic PCs is shown in **Figure 1**. 10

#### **4. Hepatic NPCs (LSECs/KCs)**

i. Spin hepatic NPCs at 4°C, 300 g for 10 min; Accelerate by 9, decelerate by 9; Discard the supernatant and gently scrape the bottom to allow the cells to

<sup>8</sup> Do not damage the gallbladder during the entire process of liver separation, as this will reduce the production and viability of hepatic parenchymal cells. After the liver is digested, only a small amount of fibrous tissue remains, which means it is in the best digestion state.

<sup>9</sup> The supernatant of hepatic PCs of the same mouse each time washing may contain target hepatic NPCs, all of which are mixed together for subsequent cell sorting.

<sup>10</sup> Experimental results show that the survival rate of mouse liver parenchymal hepatocytes needs to reach more than 70%, and a better survival rate can be obtained after culture. Mature hepatocytes mostly have two nuclei and three nuclei. In addition, even if washed with PBS, dead cells will still exist.

**Figure 1.** *hepatic PCs cultured for 48 h*

suspend; Wash the NPCs with 50 mL MACS buffer, pick out if there are dead cells or tissue clumps, centrifuge as above, discard the supernatant, and gently scrape the bottom.


*Establishment of a Mouse Model of Chronic Hepatitis B Virus Infection and Purification… DOI: http://dx.doi.org/10.5772/intechopen.99939*

**Figure 2.** *hepatic NPCs cultured for 48 h.*


#### **Acknowledgements**

We thank Professor Pei-Jer Chen from National Taiwan University for presenting the plasmid pAAV/HBV1.2. This work was supported by the National Natural Science Foundation of China (81701550), the Top Talent Support Program for Young and Middle-Aged People of Wuxi Health Committee (BJ2020094), and the Wuxi Key Medical Talents Program (ZDRC024).

<sup>11</sup> The isolated NPCs can be verified by flow cytometry in a small amount: the cell surface markers of NPCs are stained for flow cytometry analysis as described previously [31]. Briefly, LSEC (CD146+ ) and KCs (CD45, IA/IE+ , and F4/80+ ) can be detected by flow cytometry assay. All Abs are purchased from BD biosciences and eBioscience. The flow test results are analyzed using FlowJo 11.0.

*Hepatitis B*

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Yan Yan1,2\* and Chantsalmaa Davgadorj1

1 Laboratory for Infection and Immunity, The Fifth People's Hospital of Wuxi, Affiliated Hospital of Jiangnan University, Wuxi, China

2 Hepatology Institute of Wuxi, The Fifth People's Hospital of Wuxi, Affiliated Hospital of Jiangnan University, Wuxi, China

\*Address all correspondence to: yanyan198134@hotmail.com

© 2021 The Author(s). Licensee IntechOpen. 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.

*Establishment of a Mouse Model of Chronic Hepatitis B Virus Infection and Purification… DOI: http://dx.doi.org/10.5772/intechopen.99939*

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[20] Lucifora J, Salvetti A, Marniquet X, Mailly L, Testoni B, Fusil F, Inchauspe A, Michelet M, Michel ML, Levrero M, Cortez P, Baumert TF, Cosset FL, Challier C, Zoulim F, Durantel D. Mint: Detection of the hepatitis B virus (HBV) covalentlyclosed-circular DNA (cccDNA) in mice transduced with a recombinant AAV-HBV vector. Antiviral Research. 2017; 145: 14-19. DOI: 10.1016/j. antiviral.2017.07.006

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and accelerates HBV clearance. Journal of Gastroenterology. 2021; 56: 769-785. DOI: 10.1007/s00535-021-01799-8

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[28] Lin YJ, Huang LR, Yang HC, Tzeng HT, Hsu PN, Wu HL, Chen PJ, Chen DS. Mint: Hepatitis B virus core antigen determines viral persistence in a C57BL/6 mouse model. Proceedings of the National Academy of Sciences of the United States of America. 2010; 107: 9340-9345. DOI: 10.1073/ pnas.1004762107

[29] MacWilliams MP, Liao M-K (2006) Luria broth (LB) and luria agar (LA) media and their uses protocol.

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### Section 3

## Levels of Antibody Response against HBV

#### **Chapter 4**

## Level of Antibody Response against Hepatitis B Virus after Vaccination and Seroprevalence of HBV in Children Addis Ababa, Ethiopia

*Habtamu Biazin Kebede and Seifegebriel Teshome*

### **Abstract**

Approximately 2 billion people worldwide are infected with HBV and more than 240 million are chronic carriers. The World Health Organization officially launched the introduction of the hepatitis B vaccine for children in 1980. Since then, different countries have determined the level of response to the vaccine. Since the introduction of the vaccine in Ethiopia in 2007, there have been few studies evaluating the antibody response to the HBV vaccine. Therefore, the purpose of this study is to determine the HBV antibody response after hepatitis B vaccination and to evaluate the HBV seroprevalence of children in Addis Ababa, Ethiopia. A crosssectional study was conducted using a multistage probability sampling technique. Four hundred and fifty children between the ages of five and eight living in Addis Ababa were enrolled. Socio-demographic characteristics were obtained through a structured questionnaire and three to four ml of blood was collected from each child. ELISA was performed to determine antibody levels against HBV. The average age is seven + one (SD) years. Anti-HBs were detected in 54.3% (208/450) of children, and girls 98 (54.7%) had a slightly higher level of protection than boys 110 did (53.9%). The overall coverage rate of the vaccine in this study was 85.1%. The proportion of children with protective levels (> 10 mIU / ml of anti-HBs antibodies) decreased with increasing age of the children: 5, 6, 7 and 8 years were 52.6%, 60%, 43.5% and 37.1%, respectively. The seroprevalence rate for HBsAg is 0.4% and the seroprevalence rate for anti-HBc is 5.6%. Age and antibody response level were negatively correlated (p = 0.001), while gender and history of HBV infection were not significantly correlated. Age was also significantly correlated with anti-HBc seropositivity (p = 0.003). HBV vaccine coverage for children is high, but the antibody response to the vaccine appears to be low. The seropositivity rate for the virus is also very low. Low levels of response to the vaccine should be a problem. For unresponsive children, revaccination or booster doses should be considered. More research needs to be done.

**Keywords:** HBV, antibody response level, vaccination status, HBsAg, Anti HBsAg, Anti HBc

#### **1. Introduction**

Hepatitis B virus (HBV) infection is a serious global health concern. Around two billion people have been infected with HBV worldwide, and more than 257 million people are currently living with hepatitis B virus infection [1]. There are an estimated 600,000 deaths annually from complications of HBV-related liver diseases [2]. The highest numbers of HBsAg carriers are found in developing countries with limited medical facilities. Endemicity levels of chronic HBV infection are classified as high (>8%), intermediate (2-8%), or low (<2%). Based on this classification, sub-Saharan African countries including Ethiopia are considered regions of high endemicity [1].

HBV infection can be prevented by using the HBV vaccine. HBV vaccine has been commercially avail- able since the 1980s. In 1991, WHO recommended the integration of the HBV vaccine into the national immunization programs and by the end of 2005, the vaccine coverage was 82.3% globally [3, 4]. Hepatitis B vaccine for infants had been introduced in 183 countries by the end of 2013. In 2007, almost all sub-Saharan African countries had Hepatitis B vaccination in their national program [5]. HBV vaccine was introduced into the Ethiopian Expanded Program of Immunization (EPI) in 2007 and national coverage had reached 86% by 2015 [6].

Immune response to the vaccine can be determined by measuring the concentration of antibodies against the HBsAg. Anti-HBs in vaccinated children decline with time, especially during the first few years of vaccination. Most children produce a high concentration of antibodies following vaccination; however, few children can have low or no response to the vaccine. The reason for this low or no response against the HBV vaccine is not well known. However, the site of injection and modes of administration are thought to be critical factors in achieving an optimal response [7]. Data on the level of immune responses against HBV vaccine in Ethiopia is very limited. This study was conducted to contribute to the base- line data needed for further monitoring of HBV vaccination effectiveness in Ethiopia and provides information on the level of immune responses against HBV vaccine among children in Addis Ababa.

#### **2. Patients and methods**

#### **2.1 Study area and period**

The study was conducted in Addis Ababa, which is the capital city of Ethiopia from April 2016 to May 2017. A total number of 450 children (5-8 years old) were recruited.

#### **2.2 Study design**

A prospective cross-sectional study design was used. Healthy appearing 5-8 years old children were included in the study following their parents' consent. Vaccination status was used to include and exclude participants.

#### **2.3 Sample size determination and sampling technique**

The required samples size for this study was calculated using 50% of prevalence in vaccine response using the following formula:

$$\mathbf{n} = \frac{\left(\frac{Za}{2}\right)^2 \mathbf{pq}}{\mathbf{d}^2} \tag{1}$$

*Level of Antibody Response against Hepatitis B Virus after Vaccination and Seroprevalence… DOI: http://dx.doi.org/10.5772/intechopen.99970*

where, n = sample size q = 1 – p p = proportion Z = confidence interval. Using confidence interval of 95%, Z = 1.96

$$\text{IN} = \frac{\left(1.96\right)^2 \* 0.5 \* 0.5}{\left(0.5\right)^2} = \text{385} \tag{2}$$

The total number of the samples with 10% non- respondents should come to 424. However, just to be safe we collected 450 samples.

#### **2.4 Data collection**

There was a structured questionnaire to collect all relevant information about the study participants. After the participant's family agreed to take part in the study, they signed an in- formed consent form. 3-4 ml of blood sample was collected from each child and the serum was separated and stored at -20°C until further use.

#### **2.5 Ethical clearance**

Ethical clearance was obtained from the AHRI/ALERT Research Ethics Review Committee and the department of ethical research committee. A support letter was obtained from the Addis Ababa Health Bureau and from the health bureaus of each sub city. Written informed consent was obtained from each participant's parent or guardian before enrollment.

#### **2.6 Data analysis and interpretation**

SPSS Software statistical package version 20.0 was used to analyze the data. Association was determined by Chi-square test. P-values less than 0.05 were considered as statistically significant.

#### **2.7 Serological assays**

Serum level HBsAg, anti-HBc, and anti-HBsAg were determined using sandwich ELISA, where antigens/monoclonal antibodies were used both for capture and detection. All ELISA experiments were performed using BIO-RAD, Monolisa ELISA test kits, France. The test kits have a high sensitivity and specificity and each test procedure was undertaken according to the manufacturer's instruction based on standard operating procedures.

#### **2.8 Operational definitions**

**Hepatitis B virus***:* A DNA virus that attacks the liver and can cause both acute and chronic disease.

**HBsAg***:* It is the surface antigen of the hepatitis B virus. It indicates the presence of active hepatitis B infection.

**Anti-HBsAg***:* An antibody that is produced against HBV infection or vaccination.

**Anti-HBsAg***:* An antibody that indicates previous or ongoing infection with the hepatitis B virus in an undefined period.

### **3. Results**

#### **3.1 Socio-demographic characteristics**

The mean age of the study participants was 7 ± 1 (SD) years. Among these, 244 (54.2%) were male and 383 (85.1%) had been vaccinated. Forty-nine (10.9%) children had a history of infectious disease, fifteen (3.3%) had a history of noninfectious disease, while three (0.7%) were HIV positive (**Table 1**).


**Table 1.**

*Socio demographic characteristics of children between the age of 5 and 8 years old in Addis Ababa Ethiopia.*


#### **Table 2.**

*The coverage of hepatitis B vaccination among 5-8 year-old children in Addis Ababa.*

*Level of Antibody Response against Hepatitis B Virus after Vaccination and Seroprevalence… DOI: http://dx.doi.org/10.5772/intechopen.99970*

#### **3.2 Vaccination status**

383 (85.1%) out of 450 children had received HBV vaccination, of which 99.2% (380/383) of them had received the complete three doses of the vaccination. Only two (0.8%) children among the vaccinated had taken just two doses of the vaccine. There was no one who took only a single dose of the vaccine. The proportion of girls vaccinated (86.9%) is slightly higher than of boys (83.6%) as tabulated in **Table 2**.

#### **3.3 Level of anti HBsAg Ab**

Anti-HBsAg concentration > 10mIU/ml was observed in 214 (47.6%) children, six of whom were unvaccinated. Among the 383 vaccinated children, 208 (54.3%) had a protective level of antibody concentration (anti-HBsAg concentration of >10mIU/ml), while the remaining 47.3% did not. Among 67 unvaccinated children, 61 (91%) had anti HBsAg <10mIU/ml. From the three HIV infected participants, two of them were vaccinated. However, they did not have protective level antibody response (anti-HBsAg <10mIU/ml) (**Figure 1**).

#### **Figure 1.**

*Antibody response against hepatitis B vaccine in children between 5 and 8 years, Addis Ababa Ethiopia.*


#### **Table 3.**

*Seroprevalence of anti-HBc in 5-8 years old children in Addis Ababa, 2016-2017.*

Anti-HBsAg concentration by age: A protective level antibody response against HBV vaccine was observed in 52.6%, 60%, 43.5% and 37.1% of children at the ages of 5, 6, 7, and 8, respectively. There was a significant association between age and the concentration of anti-HBsAg (p = 0.001). The levels of antibody concentration decreased as the age of the participant increased.

Seroprevalence of hepatitis B: From 450 children, only two (0.4%) were positive for HBsAg and 25 (5.6%) were positive for anti-HBc. 1 child (0.2%) was positive for both HBsAg and anti-HBc (**Table 3**). The two children who were positive for HBsAg were females, 5 years old, asymptomatic, and vaccinated for HBV.

Among anti-HBc positive children, 15 (6.1%) were male while 10 (4.9%) were female. There was no significant association between sex and anti-HBcAb (p = 0.551). Ten (2.6%) of anti-HBc positive children had received vaccination prior to this study. Among non-vaccinated children in this study, 15 (22.4%) were anti-HBc positive. There was a negative correlation between vaccination status and anti- HBc positivity (p = 0.000).

The seroprevalence of anti-HBc was 4.2%, 2.3%, 4% and 12.9% in 5, 6, 7 and 8 year old children, respectively. Age was significantly associated with anti- HBc seroprevalence (p = 0.03).

#### **4. Discussion**

The primary goal of vaccination against HBV is to generate an effective antibody response against the virus. The efficacy of HBV vaccine has not been determined in Ethiopia since its introduction in 2007. HBV vaccine coverage rate observed (85.1%) in our study is comparable with the estimated (86%) national coverage reported in 2015 [6]. Evidence has shown that the immune response against the vaccine decreases with increasing age. Our study observed a similar association between age and immune response against HBV vaccine.

In this study, 54.3% of vaccinated children had protective antibody response, as well as was obtained with low seroprevalence of HBsAg and anti-HBc of 0.4% and 5.6%, respectively. This result is comparable with a study conducted in Yemen and in Iran, where 54.8% and 56.3% of the children had protective antibody responses, respectively [8, 9].

However, in other areas, higher proportions of children were reported with protective level antibody responses in comparison to our study. Studies con- ducted in different areas of Iran observed that 78% of 5-10 years old children [10], 84% of 5 to 7 years old children [11], and 87.6% of under 7 years old children [12] had a protective level antibody response against HBV. A study that was conducted in Spain also revealed that 85% of children at the age of seven had protective level antibody response to the vaccine [13]. The difference in these countries could be attributed to differences in dose, vaccine type, and vaccination route.

In contrast to our study, a lower proportion of children with protective level antibody responses were observed in other studies. For example, in a study conducted in Egypt, 39.3% of the children (6-12 years old) had protective level antibody responses [14]. In addition, in different areas of Iran, only 47.9% of 10-11 year old children [15], 48% of 7 to 9 year old children [16], and 30% of the 8 months to 15-year-old children [17] had between ages of 8 months and 15 years had protective level antibody responses against HBV vaccine. These differences may be due to sampling difference, type of vaccine, and different age for vaccine administration.

Seroprevalence of HBsAg among vaccinated children varies in different countries ranging from 0 to 2.5%. Seroprevalence of HBsAg was 1.8% in Yemen [8], 0.13% in Nepal [18], 2.3% in Papua New Guinea [19], 0.77% in Eastern China [20], *Level of Antibody Response against Hepatitis B Virus after Vaccination and Seroprevalence… DOI: http://dx.doi.org/10.5772/intechopen.99970*

2.5% in northwest China [21], while it was 0.4% in our study. This difference in seroprevalence of HBV infection could be attributed to the difference in vaccine coverage and difference in vaccination schedule.

In our study, seroprevalence of anti-HBc was 5.6%, which is lower than anti-HBc seroprevalence observed in studies conducted in Gambia (17.7%) [22], China (14.1%) [21] and Iran (7.5%) [23]. These discrepancies in anti-HBc seroprevalence could be attributed to age difference, race, prevalence of HBV, and immune response level.

Nowadays, mutant hepatitis B viruses are spreading globally. Vaccination regime and vaccine type should also be considered when we administer the vaccine to the child. High seroprevalence of anti- HBc in vaccinated children may indicate the presence of an occult HBV infection, which is a concern for everyone that needs to be addressed [24].

#### **4.1 Limitations of the study**

Some children did not come with their vaccination card. Therefore, we had to use the words of their parents/guardians as evidence for vaccination, which is not always reliable. Other serological markers of active HBV infection, like HBeAg, were not examined in this study. Further, there was an unequal number of vaccinated and non-vaccinated children and the study design did not account for occult infections.

#### **5. Conclusion and recommendation**

The vaccine coverage observed in this study is similar to that of the national estimate in 2014. However, less than half of the children had a protective level of anti- body response against HBV vaccine. Further, a negative association between anti-HBsAg antibody concentration and age was observed. Serological markers for hepatitis B virus were low: 0.4% for HBsAg and 5.6% of anti-HBc.

Persistence of anti-HBs antibodies is necessary for the long-term protection against hepatitis B virus infection. Even if different factors can contribute to low antibody response against the vaccine, we need to follow up children after vaccination, in order to see the effect of the vaccine in producing the desired response over time.

Finally, further studies should be undertaken to determine the duration of antibody response against HBV vaccine that may help in which years the vaccine response becomes less and less. For those who did not respond to the vaccine, booster doses should be given to enhance immunological responses to the vaccine. This can be important to elevate the vaccine response. Follow up is needed for those children who are administered with booster doses to evaluate response against the vaccine in those children.

#### **Acknowledgements**

The Ministry of Health through the Clinical Research Capacity Building program at the Armauer Hansen Research Institute (AHRI) funded this study; therefore, we would like to extend our deepest gratitude to both institutions. We would also like to express our deepest appreciation to AHRI laboratory staff, study participants, health extension workers, and all others who supported us in every step of this work.

### **Conflict of interest**

There was no conflict of interest among the authors or with any other parties.

#### **Author details**

Habtamu Biazin Kebede\* and Seifegebriel Teshome Department of Microbiology, Immunology and Parasitology, College of Health Sciences, Addis Ababa University, Ethiopia

\*Address all correspondence to: habtamu.biazin@aau.edu.et

© 2021 The Author(s). Licensee IntechOpen. 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.

*Level of Antibody Response against Hepatitis B Virus after Vaccination and Seroprevalence… DOI: http://dx.doi.org/10.5772/intechopen.99970*

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Section 4
