**Targeting Norovirus: Strategies for the Discovery of New Antiviral Drugs**

Joana Rocha-Pereira and Maria São José Nascimento *Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal* 

#### **1. Introduction**

120 Antiviral Drugs – Aspects of Clinical Use and Recent Advances

Wang, H.; Feng, Z.; Shu, Y.; Yu, H.; Zhou, L.; Zu, R.; Huai, Y.; Dong, J.; Bao, C.; Wen, L.;

Weber, F.; Kochs, G.; Gruber, S. & Haller, O. (1998). A classical bipartite nuclear localization

Webster, R.G.; Bean, W.J.; Gorman, O.T.; Chambers, T.M. & Kawaoka, Y. (1992). Evolution

White, J, Kartenbeck, J. & Helenius, A. (1982). Membrane fusion activity of influenza virus.

Wingfiel, W.L.; Pollack, D. & Grunert, R.R. (1969). Therapeutic efficacy of amantadine HCl

Wise, H.M.; Foeglein, A.; Sun, J.; Dalton, R.M.; Patel, S.; Howard, W.; Anderson, E.C.;

World Health Organization. (1980). A revision of the system of nomenclature for influenza

Yamashita, M. (2011). Laninamivir and its prodrug, CS-8958: long-acting neuraminidase

Ye, Q.; Krug, R.M. & Tao, Y.J. (2006). The mechanism by which influenza A virus

*The EMBO journal*, Vol.1, No.2, pp. 217-222, ISSN 0021-9525

*Virology*, Vol.83, No.16, pp. 8021-8031, ISSN 0022-538X

1427-1434, ISSN 0140-6736

pp. 9-18, ISSN 0042-6822

179, ISSN 0146-0749

No.4, pp. 585-591

1082, ISSN 0028-0836

No.2, pp. 71-84, ISSN 0956-3202

4793

Wang, H.; Yang, P.; Zhao, W.; Dong, L.; Zhou, M.; Liao, Q.; Yang, H.; Wang, M.; Lu, X.; Shi, Z.; Wang, W.; Gu, L.; Zhu, F.; Li, Q.; Yin, W.; Yang, W.; Li, D.; Uyeki, T.M. & Wang, Y. (2008). Probable limited person-to-person transmission of highly pathogenic avian influenza A (H5N1) virus in China. *Lancet*, Vol.371, No.9622, pp.

signal on Thogoto and influenza A virus nucleoproteins. *Virology*, Vol.250, No.1,

and ecology of influenza A viruses. *Microbiological reviews*, Vol.56, No.1, pp. 152-

and rimantadine HCl in naturally occurring influenza A2 respiratory illness in man. *The New England journal of medicine*, Vol.281, No.11, pp. 579-584, ISSN 0028-

Barclay, W.S. & Digard, P. (2009). A complicated message: Identification of a novel PB1-related protein translated from influenza A virus segment 2 mRNA. *Journal of* 

viruses: a WHO memorandum. *Bulletin of the World Health Organization*, Vol.58,

inhibitors for the treatment of influenza. *Antiviral chemistry & chemotherapy*, Vol.21,

nucleoprotein forms oligomers and binds RNA. *Nature*, Vol.444, No.7122, pp. 1078-

Gastroenteritis is globally responsible for great morbidity and mortality among all ages. In the developing countries, it still represents one of the top causes of death for children <5 years of age, resulting in 1,8 million fatalities every year (Boschi-Pinto et al., 2008; Bryce et al., 2005). Viruses are responsible for the majority of cases of gastroenteritis with rotaviruses and noroviruses being the major pathogens.

Noroviruses are today recognized as the leading cause of foodborne outbreaks and sporadic cases of gastroenteritis worldwide (Glass et al., 2009; Patel et al., 2009). Nowadays, they are even considered the second most important agent of severe childhood diarrhea after rotavirus (Koopmans, 2008; Patel et al., 2008; Ramani & Kang, 2009) but the importance of norovirus in this age group is expected to increase in the upcoming years, as a consequence of the implementation of routine rotavirus vaccination (Koo et al., 2010).

The recognition of the clinical importance of norovirus only began in the late 1990s when sensitive routine diagnostic methods became available. In fact, the first norovirus (the *Norwalk* virus) was discovered in 1972 (Kapikian et al., 1972) but more than twenty years were necessary to disclose the important role of these viruses as human pathogens. Noroviruses are now on the upswing and a fundamental question is being raised: are norovirus really emerging? (Widdowson et al., 2005)

Despite the increasing attention given to norovirus today and the significant morbidity and mortality associated with norovirus gastroenteritis, no specific antiviral drugs or vaccines are yet available for treatment or prevention of norovirus illness. Only recently, a recombinant intranasal vaccine has entered a phase I clinical trial (El-Kamary et al., 2010; Vinje, 2010)

This chapter will highlight the importance of finding specific antiviral therapy for norovirus infection and explore biological features of norovirus, pointing out directions to stop or control this important human pathogen.

#### **1.1 Clinical disease, transmission and epidemiology**

Norovirus gastroenteritis is generally acute and self-limited, but in infants, elderly, and immunocompromised individuals it may be more severe and prolonged since they are more

Targeting Norovirus: Strategies for the Discovery of New Antiviral Drugs 123

of norovirus causing worldwide epidemics suggests a pattern of epochal evolution

Susceptibility to norovirus infection involves both acquired immunity and genetic resistance (Parrino et al., 1977). Volunteer studies found that some individuals were repeatedly susceptible to norovirus infection whereas others were repeatedly resistant (Parrino et al., 1977). Although it was initially unclear why some subjects did not develop illness, current research suggests that host genotype is a prominent factor in the development of norovirus infection since it depends on the presence of specific human histo-blood group antigen (HBGA) receptors in the gut of susceptible hosts (Lindesmith et al., 2003). Infection by norovirus relies on the recognition of HBGAs in the initial viral attachment and this key event most likely controls host susceptibility and resistance to norovirus (Hutson et al., 2004;

HBGAs are complex carbohydrates linked to proteins or lipids on the surface of red blood cells and mucosal epithelia of the respiratory, genitourinary and digestive tracts, or present as free oligosaccharide in biological fluids such as milk and saliva. These antigens provide diversity within the human population and their biosynthesis is controlled by the enzyme products of alleles at the ABH, fucosyltransferase (FUT) 2, and FUT 3 loci (Hutson et al.,

A number of distinct binding patterns of noroviruses to HBGAs have been described according to the ABO, Lewis and secretor types of the human HBGAs (Huang et al., 2003; Huang et al., 2005). This explains the correlation between secretor status and susceptibility to Norwalk virus infection, where secretor individuals with a wild-type *FUT2* gene (~80% of the population), who express HBGAs on gut epithelial cells and in body fluids, are susceptible to Norwalk virus infection, while nonsecretors, with a null *FUT2* allele, are

The binding patterns of noroviruses to HBGAs are currently sorted into three major groups, the H, the A/B, and the Lewis binding groups (Tan & Jiang, 2011). While norovirus strains display distinct HBGA binding properties, collectively they can infect nearly all individuals due to their high genetic variability (Le Pendu et al., 2006). This highlights the highly adaptive nature of noroviruses and the likelihood of a long co-evolution of human

Noroviruses are a genetically diverse group of viruses belonging to the genus *Norovirus* of the family *Caliciviridae* (Green, 2007)*.* The Norwalk virus was the first norovirus to be discovered and associated to a gastroenteritis outbreak in an elementary school in Norwalk, Ohio, USA in 1968 and is today considered the prototype of the genus *Norovirus* (Kapikian

The family *Caliciviridae* comprises, besides *Norovirus*, four accepted (*Sapovirus, Lagovirus*, *Vesivirus, Nebovirus)* and two tentative genera (*Recovirus, Valovirus*), that include human and non-human pathogenic viruses (Farkas et al., 2008; Green, 2007; Green et al., 2000; L'Homme

resembling that of influenza (Glass et al., 2009).

**1.2 Host susceptibility and virus-host interaction** 

Marionneau et al., 2002; Tan & Jiang, 2005, 2011).

completely resistant (Lindesmith et al., 2003).

**1.3 Classification and genome organization** 

noroviruses with their human host.

2004).

et al., 1972).

susceptible to complications due to dehydration (Green, 2007; Patel et al., 2008). After an incubation period of 24–48 h, there is an acute onset of symptoms of nausea, vomiting, abdominal cramps, myalgias, and intense non-bloody diarrhea which usually resolves in 2– 3 days (Green, 2007). The median duration of illness can be longer, lasting up to six weeks in infants and young children (Kirkwood & Streitberg, 2008; Murata et al., 2007; Patel et al., 2009). Prolonged shedding is also documented in transplant patients and other immunosuppressed individuals, with symptoms lasting over two years (Hutson et al., 2004; Widdowson et al., 2005). Deaths have been reported in elderly during outbreaks in nursing homes, hospitals and cruise ships (Gotz et al., 2002; Lopman et al., 2004; Patel et al., 2008). Repeated associations of norovirus infections with clinical outcomes other than gastroenteritis have been reported (CDC, 2002; Chen et al., 2009; Kawano et al., 2007; Marshall et al., 2007; Turcios-Ruiz et al., 2008). Moreover, norovirus RNA has been detected in the blood of children with norovirus gastroenteritis and in the cerebrospinal fluid of a child with encephalopathy (Ito et al., 2006; Takanashi et al., 2009). This data suggests that norovirus infection is probably not limited to the intestine and could disseminate to systemic sites.

Noroviruses are transmitted by the fecal-oral route either directly from person-to-person or indirectly through consumption of contaminated food (fresh fruit, vegetables, shellfish and bakery products), water (drinking, ice or swimming) or following exposure to contaminated environmental surfaces and to airborne vomitus droplets (Green, 2007). Concerns about a potential zoonotic transmission have been raised given the close genetic relatedness between norovirus found in humans and animals and the presence of antibodies to animal strains in humans (Bank-Wolf et al., 2010).

Norovirus outbreaks are notably extensive and often occur in semi-closed environments (nursing homes, hospitals, day-care centers, schools, cruise ships and restaurants) that favor person-to-person transmission (Glass et al., 2009; Patel et al., 2009). Moreover, modern lifestyles make people more vulnerable to norovirus. More elderly people and infants live in communal settings, people eat more food outside the household (handled by potentially infected workers), consume more imported fresh fruit and vegetables from countries where crops are still irrigated with sewage-contaminated water and also more people are travelling and being exposed to norovirus in hotels, airplanes and cruise ships (Widdowson et al., 2005).

The very low infectious dose of norovirus (≈ 17 virus particles), their long persistence in the environment, withstanding sanitary measures effective against other microorganisms (freezing, heating and chlorination) combined with prolonged asymptomatic viral shedding make norovirus extremely infectious and explain the extensiveness of outbreaks (Duizer et al., 2004a; Siebenga et al., 2008; Teunis et al., 2008; Widdowson et al., 2005). Additionally, repeated infections can occur throughout life with re-exposure, likely due to the lack of lasting immunity and the lack of complete cross-protection against the diverse norovirus strains. (Donaldson et al., 2010; Patel et al., 2009).

Over the past years, a global increase in the number of norovirus outbreaks was noticed, with the GII.4 variants being accountable for the vast majority of cases. These GII.4 variants have become globally predominant and were responsible for four pandemics in the last two decades (Lindesmith et al., 2008; Siebenga et al., 2008). This emergence of dominants strains of norovirus causing worldwide epidemics suggests a pattern of epochal evolution resembling that of influenza (Glass et al., 2009).

#### **1.2 Host susceptibility and virus-host interaction**

122 Antiviral Drugs – Aspects of Clinical Use and Recent Advances

susceptible to complications due to dehydration (Green, 2007; Patel et al., 2008). After an incubation period of 24–48 h, there is an acute onset of symptoms of nausea, vomiting, abdominal cramps, myalgias, and intense non-bloody diarrhea which usually resolves in 2– 3 days (Green, 2007). The median duration of illness can be longer, lasting up to six weeks in infants and young children (Kirkwood & Streitberg, 2008; Murata et al., 2007; Patel et al., 2009). Prolonged shedding is also documented in transplant patients and other immunosuppressed individuals, with symptoms lasting over two years (Hutson et al., 2004; Widdowson et al., 2005). Deaths have been reported in elderly during outbreaks in nursing homes, hospitals and cruise ships (Gotz et al., 2002; Lopman et al., 2004; Patel et al., 2008). Repeated associations of norovirus infections with clinical outcomes other than gastroenteritis have been reported (CDC, 2002; Chen et al., 2009; Kawano et al., 2007; Marshall et al., 2007; Turcios-Ruiz et al., 2008). Moreover, norovirus RNA has been detected in the blood of children with norovirus gastroenteritis and in the cerebrospinal fluid of a child with encephalopathy (Ito et al., 2006; Takanashi et al., 2009). This data suggests that norovirus infection is probably not limited to the intestine and could disseminate to

Noroviruses are transmitted by the fecal-oral route either directly from person-to-person or indirectly through consumption of contaminated food (fresh fruit, vegetables, shellfish and bakery products), water (drinking, ice or swimming) or following exposure to contaminated environmental surfaces and to airborne vomitus droplets (Green, 2007). Concerns about a potential zoonotic transmission have been raised given the close genetic relatedness between norovirus found in humans and animals and the presence of antibodies to animal

Norovirus outbreaks are notably extensive and often occur in semi-closed environments (nursing homes, hospitals, day-care centers, schools, cruise ships and restaurants) that favor person-to-person transmission (Glass et al., 2009; Patel et al., 2009). Moreover, modern lifestyles make people more vulnerable to norovirus. More elderly people and infants live in communal settings, people eat more food outside the household (handled by potentially infected workers), consume more imported fresh fruit and vegetables from countries where crops are still irrigated with sewage-contaminated water and also more people are travelling and being exposed to norovirus in hotels, airplanes and cruise ships (Widdowson et al.,

The very low infectious dose of norovirus (≈ 17 virus particles), their long persistence in the environment, withstanding sanitary measures effective against other microorganisms (freezing, heating and chlorination) combined with prolonged asymptomatic viral shedding make norovirus extremely infectious and explain the extensiveness of outbreaks (Duizer et al., 2004a; Siebenga et al., 2008; Teunis et al., 2008; Widdowson et al., 2005). Additionally, repeated infections can occur throughout life with re-exposure, likely due to the lack of lasting immunity and the lack of complete cross-protection against the diverse norovirus

Over the past years, a global increase in the number of norovirus outbreaks was noticed, with the GII.4 variants being accountable for the vast majority of cases. These GII.4 variants have become globally predominant and were responsible for four pandemics in the last two decades (Lindesmith et al., 2008; Siebenga et al., 2008). This emergence of dominants strains

systemic sites.

2005).

strains in humans (Bank-Wolf et al., 2010).

strains. (Donaldson et al., 2010; Patel et al., 2009).

Susceptibility to norovirus infection involves both acquired immunity and genetic resistance (Parrino et al., 1977). Volunteer studies found that some individuals were repeatedly susceptible to norovirus infection whereas others were repeatedly resistant (Parrino et al., 1977). Although it was initially unclear why some subjects did not develop illness, current research suggests that host genotype is a prominent factor in the development of norovirus infection since it depends on the presence of specific human histo-blood group antigen (HBGA) receptors in the gut of susceptible hosts (Lindesmith et al., 2003). Infection by norovirus relies on the recognition of HBGAs in the initial viral attachment and this key event most likely controls host susceptibility and resistance to norovirus (Hutson et al., 2004; Marionneau et al., 2002; Tan & Jiang, 2005, 2011).

HBGAs are complex carbohydrates linked to proteins or lipids on the surface of red blood cells and mucosal epithelia of the respiratory, genitourinary and digestive tracts, or present as free oligosaccharide in biological fluids such as milk and saliva. These antigens provide diversity within the human population and their biosynthesis is controlled by the enzyme products of alleles at the ABH, fucosyltransferase (FUT) 2, and FUT 3 loci (Hutson et al., 2004).

A number of distinct binding patterns of noroviruses to HBGAs have been described according to the ABO, Lewis and secretor types of the human HBGAs (Huang et al., 2003; Huang et al., 2005). This explains the correlation between secretor status and susceptibility to Norwalk virus infection, where secretor individuals with a wild-type *FUT2* gene (~80% of the population), who express HBGAs on gut epithelial cells and in body fluids, are susceptible to Norwalk virus infection, while nonsecretors, with a null *FUT2* allele, are completely resistant (Lindesmith et al., 2003).

The binding patterns of noroviruses to HBGAs are currently sorted into three major groups, the H, the A/B, and the Lewis binding groups (Tan & Jiang, 2011). While norovirus strains display distinct HBGA binding properties, collectively they can infect nearly all individuals due to their high genetic variability (Le Pendu et al., 2006). This highlights the highly adaptive nature of noroviruses and the likelihood of a long co-evolution of human noroviruses with their human host.

#### **1.3 Classification and genome organization**

Noroviruses are a genetically diverse group of viruses belonging to the genus *Norovirus* of the family *Caliciviridae* (Green, 2007)*.* The Norwalk virus was the first norovirus to be discovered and associated to a gastroenteritis outbreak in an elementary school in Norwalk, Ohio, USA in 1968 and is today considered the prototype of the genus *Norovirus* (Kapikian et al., 1972).

The family *Caliciviridae* comprises, besides *Norovirus*, four accepted (*Sapovirus, Lagovirus*, *Vesivirus, Nebovirus)* and two tentative genera (*Recovirus, Valovirus*), that include human and non-human pathogenic viruses (Farkas et al., 2008; Green, 2007; Green et al., 2000; L'Homme

Targeting Norovirus: Strategies for the Discovery of New Antiviral Drugs 125

Fig. 1. Schematic representation of a viral particle and genome organization of norovirus. Genomic and subgenomic RNA of norovirus with the genome linked protein VPg at the 5' end and the poly(A) tail at 3' end is shown along with the nonstructural proteins (NS1-7) encoded by ORF1 as well as the structural proteins (VP1 and VP2), encoded by ORF2 and 3.

the outmost surface of the viral capsid and comprises a hypervariable region, where resides the binding interface for HBGA association with norovirus (Bu et al., 2008; Cao et al., 2007; Choi et al., 2008; Tan et al., 2003). The VP2 is a small, basic structural protein encoded by the ORF3 which is present in one or two copies per virion (Glass et al., 2000; Hardy, 2005). Its function in viral replication is currently undefined but there is evidence that it increases the level of expression of VP1 and stabilizes virus-like particles (VLPs, generated through expression of the VP1) (Bertolotti-Ciarlet et al., 2003). The basic charge of VP2 suggests that

The replication of norovirus has not been yet fully elucidated and most of the current knowledge is drawn by analogy with other (+)ssRNA viruses and studies with related animal caliciviruses. Norovirus is one of the positive-sense ssRNA viruses whose genome functions directly as the mRNA, beginning the infectious cycle with the synthesis of a precursor that only gives rise to the nonstructural proteins, including the RdRp enzyme that transcribes then one subgenomic mRNA encoding the structural proteins (VP1 and VP2) (Green, 2007). Like the other (+)ssRNA viruses , the replication of norovirus occurs in the

it may function in encapsidation of the viral genome (Karst, 2010).

cytoplasm. In Fig 2 is represented a scheme of the replication of norovirus.

See text for further details

**1.4 Replication of norovirus** 

et al., 2009). The two genera *Norovirus* (NoV) and *Sapovirus* (SaV) are the only that comprise human pathogenic agents, both causing acute gastroenteritis. The other genera include important veterinary pathogens such as the rabbit hemorrhagic disease virus (RHDV) which causes an often fatal hemorrhagic disease in rabbits in the genera *Lagovirus*, the feline calicivirus (FCV) which causes a respiratory disease in domestic and wild cat species in the genera *Vesivirus*, and the Newbury-1 virus which infect bovines in the genera *Nebovirus.* The tentative genera *Recovirus,* comprises the Tulane virus (TV) isolated from stool samples of rhesus macaques whose pathogenicity remains to be elucidated (Farkas et al., 2008). The other tentative genera *Valovirus* comprises the St-Valérien-like viruses isolated from pig feces (L'Homme et al., 2009).

Noroviruses are today classified into five genogroups (GI-V) divided into at least 31 genetic clusters or genotypes based on sequence diversity in the complete capsid protein VP 1 (Zheng et al., 2006). Genogroups share > 60% amino acid identity in the VP 1 and each genetic cluster or genotype shares > 80% identity in amino acid sequence of VP 1 (Green et al., 2000; Zheng et al., 2006). Human noroviruses have been associated with GI, GII and GIV and bovine and murine noroviruses belong to GIII and GV, respectively (Wobus et al., 2004; Zheng et al., 2006). GII also contains swine strains and GIV comprises the feline (lion) and canine strains (Martella et al., 2007; Martella et al., 2008; Mesquita et al., 2010).

Noroviruses are small icosahedric non-enveloped viruses of 27-32 nm with a positive-sense single stranded (ss) RNA genome of 7.4–7.7 kb, organized into three open reading frames (ORF1-3) (Fig 1). The 5' proximal region of the norovirus genome encodes a polyprotein of six/seven nonstructural protein products in a single ORF (ORF1). ORF2 encodes the major structural capsid protein VP1 and ORF3 the minor structural protein VP2. Norovirus genome is covalently linked, at the 5' end, to a viral protein called VPg (virion protein, genome-linked) and is polyadenlyated at the 3' end (Green, 2007).

The ORF1 of norovirus encodes the six/seven nonstructural proteins in the following order: the p48/ N-terminal protein (or NS1-2), the NTPase (NS3), the p22 (NS4), the VPg (NS5), the viral protease (Pro, NS6), and the viral RNA-dependent RNA polymerase (RdRp, NS7). Some of these proteins have defined activities such as NS3, an NTPase (nucleoside triphosphatase) (Pfister & Wimmer, 2001), NS6, a protease (Liu et al., 1996), and NS7, an RNA-dependent RNA polymerase (RdRp) (Fukushi et al., 2004; Rohayem et al., 2006a). Also, NS5 known as VPg, is a protein that is covalently attached to the 5' ends of viral genomes in place of a typical 5' cap and that can function as a primer in viral RNA replication (Burroughs & Brown, 1978; Rohayem et al., 2006b). The role of the remaining nonstructural proteins (NS1-2 and NS4) in norovirus replication is not yet well defined (Green, 2007). Available data suggests that NS1-2 and NS4, also known as N-term/p48 and p22, respectively, both contribute to norovirus replication complex formation on intracellular membranes, including that of the Golgi apparatus, and disrupt intracellular host protein trafficking (Ettayebi & Hardy, 2003; Fernandez-Vega et al., 2004; Hyde et al., 2009).

The major structural protein of norovirus, the VP1, is encoded by ORF2. Virions contain 180 copies or 90 dimers of VP1 that assemble into icosahedral particles (Prasad et al., 1999; Prasad et al., 1994). The VP1 protein is divided into a conserved internal shell domain (S) and a more variable protruding domain, the P domain, that forms the arch-like protrusions and is further subdivided into P1 and P2 domains (Fig. 1). The P2 subdomain is located at

et al., 2009). The two genera *Norovirus* (NoV) and *Sapovirus* (SaV) are the only that comprise human pathogenic agents, both causing acute gastroenteritis. The other genera include important veterinary pathogens such as the rabbit hemorrhagic disease virus (RHDV) which causes an often fatal hemorrhagic disease in rabbits in the genera *Lagovirus*, the feline calicivirus (FCV) which causes a respiratory disease in domestic and wild cat species in the genera *Vesivirus*, and the Newbury-1 virus which infect bovines in the genera *Nebovirus.* The tentative genera *Recovirus,* comprises the Tulane virus (TV) isolated from stool samples of rhesus macaques whose pathogenicity remains to be elucidated (Farkas et al., 2008). The other tentative genera *Valovirus* comprises the St-Valérien-like viruses isolated from pig

Noroviruses are today classified into five genogroups (GI-V) divided into at least 31 genetic clusters or genotypes based on sequence diversity in the complete capsid protein VP 1 (Zheng et al., 2006). Genogroups share > 60% amino acid identity in the VP 1 and each genetic cluster or genotype shares > 80% identity in amino acid sequence of VP 1 (Green et al., 2000; Zheng et al., 2006). Human noroviruses have been associated with GI, GII and GIV and bovine and murine noroviruses belong to GIII and GV, respectively (Wobus et al., 2004; Zheng et al., 2006). GII also contains swine strains and GIV comprises the feline (lion) and

Noroviruses are small icosahedric non-enveloped viruses of 27-32 nm with a positive-sense single stranded (ss) RNA genome of 7.4–7.7 kb, organized into three open reading frames (ORF1-3) (Fig 1). The 5' proximal region of the norovirus genome encodes a polyprotein of six/seven nonstructural protein products in a single ORF (ORF1). ORF2 encodes the major structural capsid protein VP1 and ORF3 the minor structural protein VP2. Norovirus genome is covalently linked, at the 5' end, to a viral protein called VPg (virion protein,

The ORF1 of norovirus encodes the six/seven nonstructural proteins in the following order: the p48/ N-terminal protein (or NS1-2), the NTPase (NS3), the p22 (NS4), the VPg (NS5), the viral protease (Pro, NS6), and the viral RNA-dependent RNA polymerase (RdRp, NS7). Some of these proteins have defined activities such as NS3, an NTPase (nucleoside triphosphatase) (Pfister & Wimmer, 2001), NS6, a protease (Liu et al., 1996), and NS7, an RNA-dependent RNA polymerase (RdRp) (Fukushi et al., 2004; Rohayem et al., 2006a). Also, NS5 known as VPg, is a protein that is covalently attached to the 5' ends of viral genomes in place of a typical 5' cap and that can function as a primer in viral RNA replication (Burroughs & Brown, 1978; Rohayem et al., 2006b). The role of the remaining nonstructural proteins (NS1-2 and NS4) in norovirus replication is not yet well defined (Green, 2007). Available data suggests that NS1-2 and NS4, also known as N-term/p48 and p22, respectively, both contribute to norovirus replication complex formation on intracellular membranes, including that of the Golgi apparatus, and disrupt intracellular host protein trafficking (Ettayebi & Hardy, 2003; Fernandez-Vega et al., 2004; Hyde et al., 2009). The major structural protein of norovirus, the VP1, is encoded by ORF2. Virions contain 180 copies or 90 dimers of VP1 that assemble into icosahedral particles (Prasad et al., 1999; Prasad et al., 1994). The VP1 protein is divided into a conserved internal shell domain (S) and a more variable protruding domain, the P domain, that forms the arch-like protrusions and is further subdivided into P1 and P2 domains (Fig. 1). The P2 subdomain is located at

canine strains (Martella et al., 2007; Martella et al., 2008; Mesquita et al., 2010).

genome-linked) and is polyadenlyated at the 3' end (Green, 2007).

feces (L'Homme et al., 2009).

Fig. 1. Schematic representation of a viral particle and genome organization of norovirus. Genomic and subgenomic RNA of norovirus with the genome linked protein VPg at the 5' end and the poly(A) tail at 3' end is shown along with the nonstructural proteins (NS1-7) encoded by ORF1 as well as the structural proteins (VP1 and VP2), encoded by ORF2 and 3. See text for further details

the outmost surface of the viral capsid and comprises a hypervariable region, where resides the binding interface for HBGA association with norovirus (Bu et al., 2008; Cao et al., 2007; Choi et al., 2008; Tan et al., 2003). The VP2 is a small, basic structural protein encoded by the ORF3 which is present in one or two copies per virion (Glass et al., 2000; Hardy, 2005). Its function in viral replication is currently undefined but there is evidence that it increases the level of expression of VP1 and stabilizes virus-like particles (VLPs, generated through expression of the VP1) (Bertolotti-Ciarlet et al., 2003). The basic charge of VP2 suggests that it may function in encapsidation of the viral genome (Karst, 2010).

#### **1.4 Replication of norovirus**

The replication of norovirus has not been yet fully elucidated and most of the current knowledge is drawn by analogy with other (+)ssRNA viruses and studies with related animal caliciviruses. Norovirus is one of the positive-sense ssRNA viruses whose genome functions directly as the mRNA, beginning the infectious cycle with the synthesis of a precursor that only gives rise to the nonstructural proteins, including the RdRp enzyme that transcribes then one subgenomic mRNA encoding the structural proteins (VP1 and VP2) (Green, 2007). Like the other (+)ssRNA viruses , the replication of norovirus occurs in the cytoplasm. In Fig 2 is represented a scheme of the replication of norovirus.

Targeting Norovirus: Strategies for the Discovery of New Antiviral Drugs 127

Fig. 2. Replication scheme of norovirus and key events.

In the first step, the viral attachment of the virion to the cell receptor, the P2 subdomain of the VP1 binds to a sugar residue, mostly to the HBGA carbohydrates in the case of human noroviruses but also to sialic acid or heparan sulfate (Hutson et al., 2002; Marionneau et al., 2002; Rydell et al., 2009; Stuart & Brown, 2007; Tamura et al., 2004; Taube et al., 2009). This interaction between VP1 and HBGA seems not to be enough for the entry of norovirus in host cells and the involvement of a membrane protein as a receptor or co-receptor for subsequent penetration/entry is suspected (Tan & Jiang, 2010).

Norovirus enters the cell using a non-clathrin-, non-caveolin-mediated endocytic pathway but dependent on dynamin II and cholesterol (Gerondopoulos et al., 2010; Perry & Wobus, 2010). Moreover, this entry step is pH-independent and no conformational changes in the capsid required for viral uncoating are observed with acidic intracellular pH (Perry et al., 2009).

After the internalization in the cell and uncoating of viral genome, the translation of ORF1 of the viral genomic RNA produces a large protein, the so called nonstructural polyprotein. The initiation of translation is dependent on the interaction of the VPg with the cellular translation initiation machinery (Daughenbaugh et al., 2003; Daughenbaugh et al., 2006). A co-translational processing releases the nonstructural proteins and their precursors (Sosnovtsev, 2010). The proteolytic processing is mediated by the viral protease (NS6) which is autocatalytically released from the polyprotein precursor (Putics et al., 2010).

The replication of norovirus is believed to occur in a replication complex (RC) formed by intracellular membranous structures which contain all the viral nonstructural proteins along with host proteins that will help viral replication as well as the viral RNA intermediate, ssRNA and double stranded RNA (dsRNA) (Hyde & Mackenzie, 2010; Hyde et al., 2009). The recruitment of host membranes (RE, Golgi, endossomes) necessary for the formation of the RC, is induced by the viral non structural proteins p48 (NS1-2) and p22 (NS4), through a modulation of the host cell secretory pathway (Denison, 2008; Hyde & Mackenzie, 2010; Sharp et al., 2010).

Once the RC is assembled, the RdRp (NS7) starts the synthesis of the antigenomic RNA (negative sense) from the genomic (positive sense) RNA template. The initiation of antigenomic RNA synthesis by the RdRp is dependent upon uridylylation of VPg that serves as a primer in the presence of the polyadenylated genomic RNA (Rohayem et al., 2006a; Rohayem et al., 2006b). This antigenomic RNA is then used as a template for synthesis of the new genomic RNA and of the subgenomic RNA.

The newly synthesized genomic RNA is either translated as a polyprotein precursor or used for packaging in the assembled viral protein core. The subgenomic RNA (positive sense) is translated as structural proteins, VP1 and VP2. Finally, the structural proteins are assembled and the genomic RNA packaged, followed by release of the mature virion from the cell. This late stages of replication are, however, poorly understood.

#### **1.5 Surrogate models for the study of human norovirus**

Human noroviruses are not cultivable in routine laboratory cell culture or primary tissue cultures (Duizer et al., 2004b). There has been a single report using a 3-D cell culture system demonstrated for the first time successful passage of both GI and GII norovirus *in vitro*

In the first step, the viral attachment of the virion to the cell receptor, the P2 subdomain of the VP1 binds to a sugar residue, mostly to the HBGA carbohydrates in the case of human noroviruses but also to sialic acid or heparan sulfate (Hutson et al., 2002; Marionneau et al., 2002; Rydell et al., 2009; Stuart & Brown, 2007; Tamura et al., 2004; Taube et al., 2009). This interaction between VP1 and HBGA seems not to be enough for the entry of norovirus in host cells and the involvement of a membrane protein as a receptor or co-receptor for

Norovirus enters the cell using a non-clathrin-, non-caveolin-mediated endocytic pathway but dependent on dynamin II and cholesterol (Gerondopoulos et al., 2010; Perry & Wobus, 2010). Moreover, this entry step is pH-independent and no conformational changes in the capsid required for viral uncoating are observed with acidic intracellular pH (Perry et al.,

After the internalization in the cell and uncoating of viral genome, the translation of ORF1 of the viral genomic RNA produces a large protein, the so called nonstructural polyprotein. The initiation of translation is dependent on the interaction of the VPg with the cellular translation initiation machinery (Daughenbaugh et al., 2003; Daughenbaugh et al., 2006). A co-translational processing releases the nonstructural proteins and their precursors (Sosnovtsev, 2010). The proteolytic processing is mediated by the viral protease (NS6) which

The replication of norovirus is believed to occur in a replication complex (RC) formed by intracellular membranous structures which contain all the viral nonstructural proteins along with host proteins that will help viral replication as well as the viral RNA intermediate, ssRNA and double stranded RNA (dsRNA) (Hyde & Mackenzie, 2010; Hyde et al., 2009). The recruitment of host membranes (RE, Golgi, endossomes) necessary for the formation of the RC, is induced by the viral non structural proteins p48 (NS1-2) and p22 (NS4), through a modulation of the host cell secretory pathway (Denison, 2008; Hyde & Mackenzie, 2010;

Once the RC is assembled, the RdRp (NS7) starts the synthesis of the antigenomic RNA (negative sense) from the genomic (positive sense) RNA template. The initiation of antigenomic RNA synthesis by the RdRp is dependent upon uridylylation of VPg that serves as a primer in the presence of the polyadenylated genomic RNA (Rohayem et al., 2006a; Rohayem et al., 2006b). This antigenomic RNA is then used as a template for

The newly synthesized genomic RNA is either translated as a polyprotein precursor or used for packaging in the assembled viral protein core. The subgenomic RNA (positive sense) is translated as structural proteins, VP1 and VP2. Finally, the structural proteins are assembled and the genomic RNA packaged, followed by release of the mature virion from the cell. This

Human noroviruses are not cultivable in routine laboratory cell culture or primary tissue cultures (Duizer et al., 2004b). There has been a single report using a 3-D cell culture system demonstrated for the first time successful passage of both GI and GII norovirus *in vitro*

is autocatalytically released from the polyprotein precursor (Putics et al., 2010).

synthesis of the new genomic RNA and of the subgenomic RNA.

late stages of replication are, however, poorly understood.

**1.5 Surrogate models for the study of human norovirus** 

subsequent penetration/entry is suspected (Tan & Jiang, 2010).

2009).

Sharp et al., 2010).

Fig. 2. Replication scheme of norovirus and key events.

Targeting Norovirus: Strategies for the Discovery of New Antiviral Drugs 129

frame (Thackray et al., 2007). ORF1 of MNV also encodes the nonstructural proteins and ORFs 2 and 3 encode the two proteins of the the viral capsid (Sosnovtsev et al., 2006). The conserved molecular features of MNV and human norovirus genomes suggest that many fundamental mechanisms of replication are conserved between murine and human noroviruses (Wobus et al., 2006). Although MNV infection seems to be asymptomatic in immunocompetent mice, it causes diarrhea and lethality in mice deficient in components of innate immunity (Karst et al., 2003; Mumphrey et al., 2007). Hence, the clinical presentation of MNV is different from the one of human norovirus, and for this reason MNV does not

The recent discovery of the Tulane virus (TV), a novel calicivirus isolated from stool samples of rhesus macaques, together with the likelihood of this virus causing intestinal infection and the availability of a tissue culture system could make TV a valuable surrogate for

An alternative approach to the study of norovirus RNA replication was established when a human norovirus replicon-bearing cell line was created by transfecting a plasmid containing most of the Norwalk virus genome into mammalian cell lines (Chang et al., 2006). These cell lines are capable of constitutively expressing the replicative enzymes and other nonstructural proteins, allowing the study of RNA replication and providing a platform for

Reverse genetics systems have been developed for some caliciviruses, namely for PEC, FCV, MNV and TV (Chang et al., 2005; Chaudhry et al., 2007; Sosnovtsev & Green, 1995; Ward et al., 2007; Wei et al., 2008; Wobus et al., 2004). These systems have helped to elucidate fundamental aspects of caliciviruses replication through the introduction of deliberate changes in certain genes and the analysis of the resultant effect in the virus phenotype. Reverse genetics systems represent also an important tool for the development of antivirals

Virus-like particles (VLPs) which result of the independent expression and self-assembly of the VP1 are also valuable tools that have been used in many research areas of norovirus (El-Kamary et al., 2010; Jiang et al., 1992). These VLPs are morphologically and antigenically indistinguishable from the native forms of viruses found in human stools, and retain the binding properties of native norovirus virions at least in terms of carbohydrate association (Jiang et al., 1992). VLPs have been used for the development of immunological assays, the study of virus-host interaction, structural studies of the norovirus capsid, investigation of antigenic relationships and as potential vaccine candidates (Green, 2007; Tan & Jiang, 2005).

The antiviral research of norovirus is still in its infancy and there are only few reports of antivirals for norovirus. Recently, a new chemical scaffold, the 2-styrylchromones, has shown anti-norovirus activity in the MNV surrogate model, opening the door to the search of anti-norovirus drugs among a wider range of novel compounds from different chemical families (Rocha-Pereira et al., 2010). Additionally, nitoxanide, a prodrug used to treat protozoal gastroenteritis has been reported to reduce the duration of norovirus gastroenteritis in a clinical trial although the mechanism of action remains unclear

constitute yet the ideal model for studying human norovirus.

human norovirus (Farkas et al., 2008).

screening antiviral compounds.

against norovirus (Putics et al., 2010).

(Rossignol & El-Gohary, 2006).

**2. Strategies to inhibit the replication of norovirus** 

(Straub et al., 2007) but several independent laboratories failed to reproduce such results (Papafragkou et al., 2009). For this reason, the study of human norovirus has been made using surrogate viruses.

Initially, surrogate models for norovirus infectivity included viruses from other genera within the family *Caliciviridae,* namely porcine enteric calicivirus, a *Sapovirus*, and the feline calicivirus (FCV), a *Vesivirus* (Doultree et al., 1999; Duizer et al., 2004a) (Fig.3)*.*

Fig. 3. Characteristics of the different surrogates models for human norovirus within the genera of the family C*aliciviridae*

FCV was then frequently used as a surrogate model for survival, persistence and inactivation studies of human noroviruses until the more recent discovery of murine norovirus (MNV) (Bae & Schwab, 2008; Cannon et al., 2006; Wobus et al., 2004; Wobus et al., 2006). MNV is a genogroup V norovirus and is to date the only *Norovirus* able to replicate both in cell culture and in a small animal model. Moreover, like human norovirus MNV is an enteric pathogen that spreads through the fecal-oral route and is shed at high levels in the feces (Karst et al., 2003; Wobus et al., 2004). On the other hand, the use of FCV was criticized because: (i) it does not belong to the genus *Norovirus*; (ii) it is a respiratory virus; (iii) it is not shed in feces and (iv) it cannot survive at low pH, a necessary characteristic of enteric viruses that must survive passage through the stomach (Bae & Schwab, 2008; Cannon et al., 2006; Duizer et al., 2004b; Wobus et al., 2006). Therefore, MNV is considered today the best surrogate model for human norovirus (Wobus et al., 2004; Wobus et al., 2006).

MNV has the size, shape, and buoyant density characteristic of human norovirus (Green, 2007; Karst et al., 2003). MNV genome has also the three ORFs characteristic of noroviruses (Wobus et al., 2006), but it has an additional ORF4 that overlaps ORF2 in a different reading

(Straub et al., 2007) but several independent laboratories failed to reproduce such results (Papafragkou et al., 2009). For this reason, the study of human norovirus has been made

Initially, surrogate models for norovirus infectivity included viruses from other genera within the family *Caliciviridae,* namely porcine enteric calicivirus, a *Sapovirus*, and the feline

Fig. 3. Characteristics of the different surrogates models for human norovirus within the

FCV was then frequently used as a surrogate model for survival, persistence and inactivation studies of human noroviruses until the more recent discovery of murine norovirus (MNV) (Bae & Schwab, 2008; Cannon et al., 2006; Wobus et al., 2004; Wobus et al., 2006). MNV is a genogroup V norovirus and is to date the only *Norovirus* able to replicate both in cell culture and in a small animal model. Moreover, like human norovirus MNV is an enteric pathogen that spreads through the fecal-oral route and is shed at high levels in the feces (Karst et al., 2003; Wobus et al., 2004). On the other hand, the use of FCV was criticized because: (i) it does not belong to the genus *Norovirus*; (ii) it is a respiratory virus; (iii) it is not shed in feces and (iv) it cannot survive at low pH, a necessary characteristic of enteric viruses that must survive passage through the stomach (Bae & Schwab, 2008; Cannon et al., 2006; Duizer et al., 2004b; Wobus et al., 2006). Therefore, MNV is considered today the best surrogate model for human norovirus (Wobus et al., 2004; Wobus et al., 2006). MNV has the size, shape, and buoyant density characteristic of human norovirus (Green, 2007; Karst et al., 2003). MNV genome has also the three ORFs characteristic of noroviruses (Wobus et al., 2006), but it has an additional ORF4 that overlaps ORF2 in a different reading

calicivirus (FCV), a *Vesivirus* (Doultree et al., 1999; Duizer et al., 2004a) (Fig.3)*.*

using surrogate viruses.

genera of the family C*aliciviridae*

frame (Thackray et al., 2007). ORF1 of MNV also encodes the nonstructural proteins and ORFs 2 and 3 encode the two proteins of the the viral capsid (Sosnovtsev et al., 2006). The conserved molecular features of MNV and human norovirus genomes suggest that many fundamental mechanisms of replication are conserved between murine and human noroviruses (Wobus et al., 2006). Although MNV infection seems to be asymptomatic in immunocompetent mice, it causes diarrhea and lethality in mice deficient in components of innate immunity (Karst et al., 2003; Mumphrey et al., 2007). Hence, the clinical presentation of MNV is different from the one of human norovirus, and for this reason MNV does not constitute yet the ideal model for studying human norovirus.

The recent discovery of the Tulane virus (TV), a novel calicivirus isolated from stool samples of rhesus macaques, together with the likelihood of this virus causing intestinal infection and the availability of a tissue culture system could make TV a valuable surrogate for human norovirus (Farkas et al., 2008).

An alternative approach to the study of norovirus RNA replication was established when a human norovirus replicon-bearing cell line was created by transfecting a plasmid containing most of the Norwalk virus genome into mammalian cell lines (Chang et al., 2006). These cell lines are capable of constitutively expressing the replicative enzymes and other nonstructural proteins, allowing the study of RNA replication and providing a platform for screening antiviral compounds.

Reverse genetics systems have been developed for some caliciviruses, namely for PEC, FCV, MNV and TV (Chang et al., 2005; Chaudhry et al., 2007; Sosnovtsev & Green, 1995; Ward et al., 2007; Wei et al., 2008; Wobus et al., 2004). These systems have helped to elucidate fundamental aspects of caliciviruses replication through the introduction of deliberate changes in certain genes and the analysis of the resultant effect in the virus phenotype. Reverse genetics systems represent also an important tool for the development of antivirals against norovirus (Putics et al., 2010).

Virus-like particles (VLPs) which result of the independent expression and self-assembly of the VP1 are also valuable tools that have been used in many research areas of norovirus (El-Kamary et al., 2010; Jiang et al., 1992). These VLPs are morphologically and antigenically indistinguishable from the native forms of viruses found in human stools, and retain the binding properties of native norovirus virions at least in terms of carbohydrate association (Jiang et al., 1992). VLPs have been used for the development of immunological assays, the study of virus-host interaction, structural studies of the norovirus capsid, investigation of antigenic relationships and as potential vaccine candidates (Green, 2007; Tan & Jiang, 2005).

#### **2. Strategies to inhibit the replication of norovirus**

The antiviral research of norovirus is still in its infancy and there are only few reports of antivirals for norovirus. Recently, a new chemical scaffold, the 2-styrylchromones, has shown anti-norovirus activity in the MNV surrogate model, opening the door to the search of anti-norovirus drugs among a wider range of novel compounds from different chemical families (Rocha-Pereira et al., 2010). Additionally, nitoxanide, a prodrug used to treat protozoal gastroenteritis has been reported to reduce the duration of norovirus gastroenteritis in a clinical trial although the mechanism of action remains unclear (Rossignol & El-Gohary, 2006).

Targeting Norovirus: Strategies for the Discovery of New Antiviral Drugs 131

Lewis binding groups can be found in both two major genogroups of human noroviruses

The existence of only three HBGA-binding interfaces makes possible the design of antivirals against these targets in noroviruses. Any compound that targets a given HBGA binding interface may be capable of blocking infection of all strains that bind that same type of HBGA. Thus, only three different HBGA-binding interfaces would need to be targeted by compounds to block nearly all noroviruses in the GI and GII genogroups (Tan et al., 2009). The strategy of targeting this first step of virus-receptor interaction could be of great interest to use as prophylactic therapy since it would be effective in preventing infections of individuals in high risk settings or that were in contact with an index case during an outbreak (Tan & Jiang, 2008). Furthermore, it would be expected that such compounds would be able to reduce the severity of symptoms in already infected individuals and likely reduce virus excretion, limiting its propagation to higher numbers of individuals (Tan & Jiang, 2008). Citrate, and other glycomimetics, showed to have the potential to block human noroviruses from binding

to HBGAs (Hansman et al., 2011) providing a starting point for norovirus inhibitors.

that viruses within the same family use similar cellular receptors (Tan & Jiang, 2010).

of the best target for chemical compounds to block or interfere with virus attachment.

also occur after the binding of virus to cellular receptors (Tsai, 2007).

**2.2 Targeting entry and uncoating of norovirus** 

Much remains to be unraveled in this field, therefore there are still doubts about the selection

In order to enter host cells, viruses take advantage of cellular processes entering by an endocytic pathway, most commonly a clathrin-mediated endocytosis. Viral entry can also occur via caveolin-mediated endocytosis, clathrin/caveolin-independent endocytosis, macropinocytosis, or phagocytosis (Marsh & Helenius, 2006). After entrance in the host cell, the uncoating of virus must occur in order to deliver the viral genome into the host cytoplasm. This event is often triggered by the acidic environment of endosomes but it can

Studies with FCV showed this virus enters cells by clathrin-mediated endocytosis in a pHdependent manner (Stuart & Brown, 2007). However, it was demonstrated that the entry of MNV is clathrin/caveolin-independent but mediated by dynamin II and cholesterol (Gerondopoulos et al., 2010; Perry & Wobus, 2010). In addition, studies with MNV show this

However, there are predictable difficulties to this strategy, one of which being the fact that the early steps of norovirus life cycle are not yet fully disclosed. Recent findings of FCV and MNV binding to sialic acid (Rydell et al., 2009; Stuart & Brown, 2007; Taube et al., 2009) or heparan sulfate (Tamura et al., 2004) broadens the spectrum of sugar residues that interact with these viruses. Moreover, the identification of a membrane protein, the junctional adhesion molecule A (JAM-A), as a receptor for FCV (Makino et al., 2006) raises the hypothesis of this protein or other members of the Ig superfamily being also cellular receptors for caliciviruses, like they are for reoviruses and picornaviruses (Tan & Jiang, 2010). According to the proposed model for reoviruses, the virus interact firstly with a sugar residue like sialic acid, as a determinant of tropism that is responsible for initial virus attachment, and later use a membrane protein (JAM-1) to enter the host cell (Barton et al., 2001). The role of these two molecules in viral attachment and entry is likely to occur also in FCV, and one could even speculate that it could be extended to noroviruses since it is usual

(GI and GII).

In the following section, potential targets and strategies to inhibit norovirus life cycle are presented, based on the information available today on norovirus genome organization, functions of structural and nonstructural proteins, replication and virus-host interaction (Fig. 4). The predictable advantages and/or disadvantages of these strategies are discussed and some antiviral drugs previously identified as active against similar molecular targets in other plus stranded RNA viruses are suggested as a starting point.

Fig. 4. Targeting the various steps of the replication of norovirus

#### **2.1 Targeting cellular receptors of norovirus**

Human noroviruses recognize HBGA carbohydrates present on cell surface, which are key players in the initial viral attachment, acting most likely as cellular receptors or co-receptors of norovirus (Hutson et al., 2002; Marionneau et al., 2002; Tan & Jiang, 2005, 2011)

Noroviruses present diverse binding patterns to HBGAs, being currently sorted into three major binding groups, the H, the A/B, and the Lewis binding group (Tan & Jiang, 2011). The interaction between noroviruses and HBGA is highly strain-specific rather than genogroup- or genotype-specific (Tan & Jiang, 2010). Hence, strains of the H, A/B and

In the following section, potential targets and strategies to inhibit norovirus life cycle are presented, based on the information available today on norovirus genome organization, functions of structural and nonstructural proteins, replication and virus-host interaction (Fig. 4). The predictable advantages and/or disadvantages of these strategies are discussed and some antiviral drugs previously identified as active against similar molecular targets in

other plus stranded RNA viruses are suggested as a starting point.

Fig. 4. Targeting the various steps of the replication of norovirus

Human noroviruses recognize HBGA carbohydrates present on cell surface, which are key players in the initial viral attachment, acting most likely as cellular receptors or co-receptors

Noroviruses present diverse binding patterns to HBGAs, being currently sorted into three major binding groups, the H, the A/B, and the Lewis binding group (Tan & Jiang, 2011). The interaction between noroviruses and HBGA is highly strain-specific rather than genogroup- or genotype-specific (Tan & Jiang, 2010). Hence, strains of the H, A/B and

of norovirus (Hutson et al., 2002; Marionneau et al., 2002; Tan & Jiang, 2005, 2011)

**2.1 Targeting cellular receptors of norovirus** 

Lewis binding groups can be found in both two major genogroups of human noroviruses (GI and GII).

The existence of only three HBGA-binding interfaces makes possible the design of antivirals against these targets in noroviruses. Any compound that targets a given HBGA binding interface may be capable of blocking infection of all strains that bind that same type of HBGA. Thus, only three different HBGA-binding interfaces would need to be targeted by compounds to block nearly all noroviruses in the GI and GII genogroups (Tan et al., 2009).

The strategy of targeting this first step of virus-receptor interaction could be of great interest to use as prophylactic therapy since it would be effective in preventing infections of individuals in high risk settings or that were in contact with an index case during an outbreak (Tan & Jiang, 2008). Furthermore, it would be expected that such compounds would be able to reduce the severity of symptoms in already infected individuals and likely reduce virus excretion, limiting its propagation to higher numbers of individuals (Tan & Jiang, 2008). Citrate, and other glycomimetics, showed to have the potential to block human noroviruses from binding to HBGAs (Hansman et al., 2011) providing a starting point for norovirus inhibitors.

However, there are predictable difficulties to this strategy, one of which being the fact that the early steps of norovirus life cycle are not yet fully disclosed. Recent findings of FCV and MNV binding to sialic acid (Rydell et al., 2009; Stuart & Brown, 2007; Taube et al., 2009) or heparan sulfate (Tamura et al., 2004) broadens the spectrum of sugar residues that interact with these viruses. Moreover, the identification of a membrane protein, the junctional adhesion molecule A (JAM-A), as a receptor for FCV (Makino et al., 2006) raises the hypothesis of this protein or other members of the Ig superfamily being also cellular receptors for caliciviruses, like they are for reoviruses and picornaviruses (Tan & Jiang, 2010). According to the proposed model for reoviruses, the virus interact firstly with a sugar residue like sialic acid, as a determinant of tropism that is responsible for initial virus attachment, and later use a membrane protein (JAM-1) to enter the host cell (Barton et al., 2001). The role of these two molecules in viral attachment and entry is likely to occur also in FCV, and one could even speculate that it could be extended to noroviruses since it is usual that viruses within the same family use similar cellular receptors (Tan & Jiang, 2010).

Much remains to be unraveled in this field, therefore there are still doubts about the selection of the best target for chemical compounds to block or interfere with virus attachment.

#### **2.2 Targeting entry and uncoating of norovirus**

In order to enter host cells, viruses take advantage of cellular processes entering by an endocytic pathway, most commonly a clathrin-mediated endocytosis. Viral entry can also occur via caveolin-mediated endocytosis, clathrin/caveolin-independent endocytosis, macropinocytosis, or phagocytosis (Marsh & Helenius, 2006). After entrance in the host cell, the uncoating of virus must occur in order to deliver the viral genome into the host cytoplasm. This event is often triggered by the acidic environment of endosomes but it can also occur after the binding of virus to cellular receptors (Tsai, 2007).

Studies with FCV showed this virus enters cells by clathrin-mediated endocytosis in a pHdependent manner (Stuart & Brown, 2007). However, it was demonstrated that the entry of MNV is clathrin/caveolin-independent but mediated by dynamin II and cholesterol (Gerondopoulos et al., 2010; Perry & Wobus, 2010). In addition, studies with MNV show this

Targeting Norovirus: Strategies for the Discovery of New Antiviral Drugs 133

Although a detailed knowledge of the role of nonstructural proteins is still not available, some important clues have raised from studies with MNV which showed that all these proteins play a role in norovirus replication and are associated with the replication complex (RC) and the viral RNA intermediate dsRNA (Hyde et al., 2009). There is also evidence that the replication complex of MNV is associated with host membranes, namely of the endoplasmic reticulum (ER), the Golgi apparatus and endossomes which suffer virusinduced rearrangements (Hyde et al., 2009; Wobus et al., 2004). These and other features of each nonstructural protein are described below, as well as how these could be used for the

The role of p48 or NS1-2 is not yet well defined but it is thought to be comparable to that of the analogous picornavirus 2B protein, which participates in intracellular membrane changes that occur during virus replication (Sosnovtsev, 2010). Studies with Norwalk virus indicated that the p48 may interfere with disassembly of the Golgi complex and cellular protein trafficking (Ettayebi & Hardy, 2003; Fernandez-Vega et al., 2004) while studies with MNV and FCV point out that p48 is associated with the recruitment of ER membranes to the RC (Bailey et al., 2009; Hyde & Mackenzie, 2010). This recruitment of membranes is vital to the synthesis of new viral proteins by the actively replicating noroviruses (Hyde et al., 2009; Wobus et al., 2004). Hence, the impairment of the mechanisms controlled by p48 in the membrane rearrangements could be a good strategy

The NTPase or NS3 shares sequence motifs with other viral NTPases, namely the picornaviruses 2C protein and the flaviviruses NS3 helicase/NTPase. It has also been classified in the superfamily 3 of RNA helicases (Hardy, 2005; Pfister & Wimmer, 2001). The role of these enzymes consists in catalyzing the hydrolysis reaction of nucleoside triphosphates and using the released energy to unwind the viral nucleic acids (Kwong et al., 2005). However, until now there is only experimental data confirming the NTPase but not

In recent years, substantial efforts have been made to identify inhibitors of such proteins, since they are well conserved and essential for viral replication. The thiazolobenzimidazoles were identified as potent inhibitors of picornaviruses, showing one of these compounds, TBZE-029 [1-(2,6-difluorophenyl)-6-trifluoromethyl-1*H*,3*H*-thiazolo[3,4-a]benzimidazole] to target the protein 2C of coxsackievirus B3 (De Palma et al., 2008a; De Palma et al., 2008c). These and other picornavirus 2C-targeting compounds, such as MRL-1237 [1-(4 fluorophenyl)-2-(4-imino-1,4-dihydropyridin-1-yl) methylbenzimidazole hydrochloride] and HBB [2-(α-hydroxybenzyl)-benzimidazole] (De Palma et al., 2008a; De Palma et al., 2008c; Norder et al., 2011) could as well be inhibitors of the NTPase of norovirus. The multiple mechanisms through which compounds could inhibit the helicase/NTPase have been extensively described elsewhere (Rohayem et al., 2010). The search of norovirus NTPase inhibitors could result in the development of an innovative antiviral strategy and at

the helicase activity of this nonstructural protein of norovirus (Hardy, 2005).

the same time reveal functional details of this enzyme of norovirus.

discovery of antiviral drugs against norovirus.

to stop the norovirus life cycle.

**2.4.2 NTPase** 

**2.4.1 p48** 

virus is pH-independent and that a low intracellular pH does not trigger conformational changes in the capsid required for MNV uncoating (Perry et al., 2009). This difference in sensitivity to low pH between FCV and MNV was suggested to be related with the different routes of infection of these viruses (Perry et al., 2009). While MNV is an enteric virus that infects its host by the small intestine and retains infectivity for hours at a pH of 2 (similarly to the human Norwalk virus), FCV is a respiratory virus that significantly decreases infectivity at low pH (Cannon et al., 2006; Dolin et al., 1972).

Further understanding of the cellular mechanisms of norovirus entry and uncoating would bring out new antiviral targets.

#### **2.3 Targeting structural proteins of norovirus**

The VP1 is the major structural protein of norovirus and its P2 subdomain, which is located at the outmost surface of the viral capsid, comprises the binding surface for HBGA (Bu et al., 2008; Cao et al., 2007; Choi et al., 2008; Tan et al., 2003). The function of VP2 is currently undefined and there is not sufficient information to address this minor protein as an antiviral target.

The search of compounds targeting VP1 of norovirus was explored through a saliva-based enzyme immunoassay (EIA) that measures their capacity to block the binding of norovirus VLPs to HBGAs present in such biological fluid (Feng & Jiang, 2007). Different chemical compounds were found to be strong inhibitors of this binding being potential candidates for further development as antivirals for norovirus (Feng & Jiang, 2007), but one should be aware that a saliva-based EIA was used instead of a cellular system or animal model and this could be regarded as an handicap.

Antiviral drugs that target viral surface proteins of other RNA virus have been described, such as pleconaril, a picornavirus capsid-binding compound, but resulted in a not entirely successfully strategy (Field & Vere Hodge, 2008). The problem with this kind of drugs relies on their low genetic barrier since the viral surface proteins can undergo variations without compromising viral fitness, easily allowing resistant strains to emerge (Field & Vere Hodge, 2008). The same problem would most likely take place with norovirus given its well known antigenic variation. A strategy could be the use of combination therapy of this class of compounds together with drugs against well conserved nonstructural proteins with critical functions. This would avoid or at least delay the development of resistance.

#### **2.4 Targeting nonstructural proteins**

The six/seven nonstructural proteins of norovirus are the p48/ N-terminal protein (NS1-2), the NTPase (NS3), the p22 (NS4), the VPg (NS5), the viral protease (Pro, NS6), and the viral RNA-dependent RNA polymerase (RdRp, NS7). Since noroviruses share some similarities with picornaviruses, the nomenclature and functions of these ORF1-encoded proteins of norovirus was initially predicted through comparative sequence analysis of their picornavirus counterparts. Hence, the norovirus RdRp was called 3D-like, the protease was called 3C-like, the p22 was called 3A-like, the NTPase was named 2C-like and the p48/Nterm was related to the 2B protein (Green, 2007). This resemblance could be important for the search of antivirals against norovirus since the known strategies and targets for inhibiting the replication of picornavirus might be also effective for norovirus.

Although a detailed knowledge of the role of nonstructural proteins is still not available, some important clues have raised from studies with MNV which showed that all these proteins play a role in norovirus replication and are associated with the replication complex (RC) and the viral RNA intermediate dsRNA (Hyde et al., 2009). There is also evidence that the replication complex of MNV is associated with host membranes, namely of the endoplasmic reticulum (ER), the Golgi apparatus and endossomes which suffer virusinduced rearrangements (Hyde et al., 2009; Wobus et al., 2004). These and other features of each nonstructural protein are described below, as well as how these could be used for the discovery of antiviral drugs against norovirus.

#### **2.4.1 p48**

132 Antiviral Drugs – Aspects of Clinical Use and Recent Advances

virus is pH-independent and that a low intracellular pH does not trigger conformational changes in the capsid required for MNV uncoating (Perry et al., 2009). This difference in sensitivity to low pH between FCV and MNV was suggested to be related with the different routes of infection of these viruses (Perry et al., 2009). While MNV is an enteric virus that infects its host by the small intestine and retains infectivity for hours at a pH of 2 (similarly to the human Norwalk virus), FCV is a respiratory virus that significantly decreases

Further understanding of the cellular mechanisms of norovirus entry and uncoating would

The VP1 is the major structural protein of norovirus and its P2 subdomain, which is located at the outmost surface of the viral capsid, comprises the binding surface for HBGA (Bu et al., 2008; Cao et al., 2007; Choi et al., 2008; Tan et al., 2003). The function of VP2 is currently undefined and there is not sufficient information to address this minor protein as an

The search of compounds targeting VP1 of norovirus was explored through a saliva-based enzyme immunoassay (EIA) that measures their capacity to block the binding of norovirus VLPs to HBGAs present in such biological fluid (Feng & Jiang, 2007). Different chemical compounds were found to be strong inhibitors of this binding being potential candidates for further development as antivirals for norovirus (Feng & Jiang, 2007), but one should be aware that a saliva-based EIA was used instead of a cellular system or animal model and

Antiviral drugs that target viral surface proteins of other RNA virus have been described, such as pleconaril, a picornavirus capsid-binding compound, but resulted in a not entirely successfully strategy (Field & Vere Hodge, 2008). The problem with this kind of drugs relies on their low genetic barrier since the viral surface proteins can undergo variations without compromising viral fitness, easily allowing resistant strains to emerge (Field & Vere Hodge, 2008). The same problem would most likely take place with norovirus given its well known antigenic variation. A strategy could be the use of combination therapy of this class of compounds together with drugs against well conserved nonstructural proteins with critical

The six/seven nonstructural proteins of norovirus are the p48/ N-terminal protein (NS1-2), the NTPase (NS3), the p22 (NS4), the VPg (NS5), the viral protease (Pro, NS6), and the viral RNA-dependent RNA polymerase (RdRp, NS7). Since noroviruses share some similarities with picornaviruses, the nomenclature and functions of these ORF1-encoded proteins of norovirus was initially predicted through comparative sequence analysis of their picornavirus counterparts. Hence, the norovirus RdRp was called 3D-like, the protease was called 3C-like, the p22 was called 3A-like, the NTPase was named 2C-like and the p48/Nterm was related to the 2B protein (Green, 2007). This resemblance could be important for the search of antivirals against norovirus since the known strategies and targets for

functions. This would avoid or at least delay the development of resistance.

inhibiting the replication of picornavirus might be also effective for norovirus.

infectivity at low pH (Cannon et al., 2006; Dolin et al., 1972).

**2.3 Targeting structural proteins of norovirus** 

this could be regarded as an handicap.

**2.4 Targeting nonstructural proteins** 

bring out new antiviral targets.

antiviral target.

The role of p48 or NS1-2 is not yet well defined but it is thought to be comparable to that of the analogous picornavirus 2B protein, which participates in intracellular membrane changes that occur during virus replication (Sosnovtsev, 2010). Studies with Norwalk virus indicated that the p48 may interfere with disassembly of the Golgi complex and cellular protein trafficking (Ettayebi & Hardy, 2003; Fernandez-Vega et al., 2004) while studies with MNV and FCV point out that p48 is associated with the recruitment of ER membranes to the RC (Bailey et al., 2009; Hyde & Mackenzie, 2010). This recruitment of membranes is vital to the synthesis of new viral proteins by the actively replicating noroviruses (Hyde et al., 2009; Wobus et al., 2004). Hence, the impairment of the mechanisms controlled by p48 in the membrane rearrangements could be a good strategy to stop the norovirus life cycle.

#### **2.4.2 NTPase**

The NTPase or NS3 shares sequence motifs with other viral NTPases, namely the picornaviruses 2C protein and the flaviviruses NS3 helicase/NTPase. It has also been classified in the superfamily 3 of RNA helicases (Hardy, 2005; Pfister & Wimmer, 2001). The role of these enzymes consists in catalyzing the hydrolysis reaction of nucleoside triphosphates and using the released energy to unwind the viral nucleic acids (Kwong et al., 2005). However, until now there is only experimental data confirming the NTPase but not the helicase activity of this nonstructural protein of norovirus (Hardy, 2005).

In recent years, substantial efforts have been made to identify inhibitors of such proteins, since they are well conserved and essential for viral replication. The thiazolobenzimidazoles were identified as potent inhibitors of picornaviruses, showing one of these compounds, TBZE-029 [1-(2,6-difluorophenyl)-6-trifluoromethyl-1*H*,3*H*-thiazolo[3,4-a]benzimidazole] to target the protein 2C of coxsackievirus B3 (De Palma et al., 2008a; De Palma et al., 2008c). These and other picornavirus 2C-targeting compounds, such as MRL-1237 [1-(4 fluorophenyl)-2-(4-imino-1,4-dihydropyridin-1-yl) methylbenzimidazole hydrochloride] and HBB [2-(α-hydroxybenzyl)-benzimidazole] (De Palma et al., 2008a; De Palma et al., 2008c; Norder et al., 2011) could as well be inhibitors of the NTPase of norovirus. The multiple mechanisms through which compounds could inhibit the helicase/NTPase have been extensively described elsewhere (Rohayem et al., 2010). The search of norovirus NTPase inhibitors could result in the development of an innovative antiviral strategy and at the same time reveal functional details of this enzyme of norovirus.

Targeting Norovirus: Strategies for the Discovery of New Antiviral Drugs 135

RNA translation was shown to be the interaction with another component of the complex, the eIF4A (a RNA helicase) (Chaudhry et al., 2006). This was demonstrated by the inhibitor of eIF4A hippuristanol which blocked the translation of both FCV and MNV viral proteins *in vitro* (Chaudhry et al., 2006). However, the potential use of hippuristanol in therapy is limited by the fact that this compound is also an inhibitor of cellular protein synthesis, therefore expected to be cytotoxic. Panteamine is another compound that has the potential of interfering with VPg/ eIF4F complex, since it stimulates the helicase/NTPase activity of eIF4A, dysregulating its function within the eIF4F complex (Bordeleau et al., 2006). Therefore, these classes of compounds could potentially be explored as antivirals for

The norovirus protease or NS6 is a cysteine protease that is responsible for processing the nonstructural polyprotein precursor into the six/seven nonstructural proteins (Green, 2007). The atomic structure of the norovirus protease has been resolved (Nakamura et al., 2005). A two-domain structure was identified as being similar to other viral cysteine proteases, like those of picornavirus 3C proteases that show a chymotrypsin-like fold with a cysteine as the

Similarly to what happens with the picornavirus 3C protease, the scissile bonds of the calicivirus proteases are restricted to a few number of dipeptides, revealing a very high specificity of substrate recognition in the cleavage sites of these enzymes which have also shown to be highly conserved among all caliciviruses (Sosnovtsev, 2010). Hence, the development of substrate-like peptides that block the protease active site constitutes an interesting strategy to inhibit the protease of norovirus. The inhibitors of the 3C protease of picornavirus, such as ruprintrivir, may potentially inhibit the norovirus protease given the overall similarity between the proteases of these viruses. (Binford et al., 2005; De Palma et

The detailed structural information available for norovirus protease constitutes a good basis for the rational design of protease inhibitors and a valuable tool for the *in silico* screening of large compound libraries (Tan & Jiang, 2008). The recent development of functional assays for the detection of protease activity of calicivirus, using fluorogenic substrate peptides (Zeitler et al., 2006) or bioluminescence technologies (Oka et al., 2011) can constitute useful

However, the role of norovirus protease appears to be more extensive. An association of the protease of MNV with mitochondria was suggested since they were found to co-localize within the cell (Hyde & Mackenzie, 2010). In this case, the protease could be implicated in an up-regulation of apoptosis of norovirus, which is known to occur via a mitochondrialmediated apoptotic pathway involving caspase-9 (Bok et al., 2009). The involvement of viral protease with apoptotic pathways has been described for their picornavirus counterparts (Drahos & Racaniello, 2009). An association of proteases with mitochondria was linked, in hepatitis A and C viruses, to a cleavage of the mitochondrial antiviral signaling protein (MAVS) (Yang et al., 2007). Since MAVS induces antiviral responses of interferon and NFkB, one may implicate viral proteases in prevention of immune signaling (Hyde &

norovirus but with less cytotoxic derivatives.

active-site nucleophile (Nakamura et al., 2005; Zeitler et al., 2006).

**2.4.5 Protease** 

al., 2008c; Leyssen et al., 2008).

Mackenzie, 2010).

tools for the screening of protease inhibitors.

#### **2.4.3 p22**

The function of p22 or NS4 in norovirus life cycle is also not yet fully understood. This protein occupies in norovirus genome a position similar to that of the 3A protein in picornavirus genomes but they share only limited sequence similarity (Green, 2007; Hardy, 2005). The 3A protein is known to inhibit the protein trafficking from the ER to Golgi apparatus, a function central to the cellular homeostasis (Wessels et al., 2006). A similar function has been recently described for Norwalk virus p22 (Sharp et al., 2010). It was reported that this protein contains a well-conserved motif that mimics the normal ER export signal, leading to the inhibition of protein trafficking to Golgi, inducing its disassembly and ultimately inhibiting cellular protein secretion (Sharp et al., 2010). Since this motif is highly conserved in human noroviruses, it may constitute a new target for the design of antinorovirus drugs (Sharp et al., 2010).

Besides, based on what has been described for picornavirus 3A protein, one of the consequences of the inhibition of cellular protein secretion by p22 could be a more severe infection due to the decrease of surface MHC class I proteins and normal cytokine release that leads to a reduction of clearance in infected cells (Sharp et al., 2010; Wessels et al., 2006).

Additionally, there is evidence suggesting that p22 is part of the norovirus RC participating in the recruitment of membranes from the late secretory pathway (Golgi and endossomes) to the RC, being the main viral protein known to be responsible for the recruitment of endossome membranes (Hyde & Mackenzie, 2010).

Two compounds, enviroxime and TTP-8307, have shown to inhibit the *in vitro* replication of enteroviruses and rhinoviruses by targeting the nonstructural protein 3A (De Palma et al., 2009; Heinz & Vance, 1995). Therefore, these compounds are good candidates to be evaluated for their ability to inhibit p22 and the norovirus replication.

#### **2.4.4 VPg**

The VPg or NS5 is covalently linked to the 5'-end of the norovirus RNA genome and plays an important role in the replication of norovirus. Initiation of RNA synthesis by viral RdRp is dependent upon uridylylation of VPg that serves as a primer in the presence of the polyadenylated genomic RNA (Rohayem et al., 2006a; Rohayem et al., 2006b). This "proteinprimed" initiation occurs after the annealing of the elongated VPg-poly(U) to the poly(A) tail of the viral genome (Rohayem et al., 2006a; Rohayem et al., 2006b).

The inhibition of this uridylylation step or prevention of annealing to the poly(A) tail through a disruption of interaction between VPg and the viral genome or the RdRp could be an effective manner to stop the norovirus life cycle and therefore a good antiviral strategy (Rohayem et al., 2010).

It is known that the initiation of translation of norovirus proteins is dependent on the interaction of the VPg with eIF4F complex (eukaryotic initiation factor 4F), a key component of the cellular translation initiation machinery (Daughenbaugh et al., 2003; Daughenbaugh et al., 2006). The interaction of the VPg with individual eIF4F components has been described. A direct interaction with eIF4E (cap binding protein) was firstly demonstrated with the VPg of Norwalk virus (Goodfellow et al., 2005). A similar interaction with this component was also seen with FCV and MNV but, in these viruses, the key requirement for RNA translation was shown to be the interaction with another component of the complex, the eIF4A (a RNA helicase) (Chaudhry et al., 2006). This was demonstrated by the inhibitor of eIF4A hippuristanol which blocked the translation of both FCV and MNV viral proteins *in vitro* (Chaudhry et al., 2006). However, the potential use of hippuristanol in therapy is limited by the fact that this compound is also an inhibitor of cellular protein synthesis, therefore expected to be cytotoxic. Panteamine is another compound that has the potential of interfering with VPg/ eIF4F complex, since it stimulates the helicase/NTPase activity of eIF4A, dysregulating its function within the eIF4F complex (Bordeleau et al., 2006). Therefore, these classes of compounds could potentially be explored as antivirals for norovirus but with less cytotoxic derivatives.

#### **2.4.5 Protease**

134 Antiviral Drugs – Aspects of Clinical Use and Recent Advances

The function of p22 or NS4 in norovirus life cycle is also not yet fully understood. This protein occupies in norovirus genome a position similar to that of the 3A protein in picornavirus genomes but they share only limited sequence similarity (Green, 2007; Hardy, 2005). The 3A protein is known to inhibit the protein trafficking from the ER to Golgi apparatus, a function central to the cellular homeostasis (Wessels et al., 2006). A similar function has been recently described for Norwalk virus p22 (Sharp et al., 2010). It was reported that this protein contains a well-conserved motif that mimics the normal ER export signal, leading to the inhibition of protein trafficking to Golgi, inducing its disassembly and ultimately inhibiting cellular protein secretion (Sharp et al., 2010). Since this motif is highly conserved in human noroviruses, it may constitute a new target for the design of anti-

Besides, based on what has been described for picornavirus 3A protein, one of the consequences of the inhibition of cellular protein secretion by p22 could be a more severe infection due to the decrease of surface MHC class I proteins and normal cytokine release that leads to a reduction of clearance in infected cells (Sharp et al., 2010; Wessels et al., 2006). Additionally, there is evidence suggesting that p22 is part of the norovirus RC participating in the recruitment of membranes from the late secretory pathway (Golgi and endossomes) to the RC, being the main viral protein known to be responsible for the recruitment of

Two compounds, enviroxime and TTP-8307, have shown to inhibit the *in vitro* replication of enteroviruses and rhinoviruses by targeting the nonstructural protein 3A (De Palma et al., 2009; Heinz & Vance, 1995). Therefore, these compounds are good candidates to be

The VPg or NS5 is covalently linked to the 5'-end of the norovirus RNA genome and plays an important role in the replication of norovirus. Initiation of RNA synthesis by viral RdRp is dependent upon uridylylation of VPg that serves as a primer in the presence of the polyadenylated genomic RNA (Rohayem et al., 2006a; Rohayem et al., 2006b). This "proteinprimed" initiation occurs after the annealing of the elongated VPg-poly(U) to the poly(A)

The inhibition of this uridylylation step or prevention of annealing to the poly(A) tail through a disruption of interaction between VPg and the viral genome or the RdRp could be an effective manner to stop the norovirus life cycle and therefore a good antiviral strategy

It is known that the initiation of translation of norovirus proteins is dependent on the interaction of the VPg with eIF4F complex (eukaryotic initiation factor 4F), a key component of the cellular translation initiation machinery (Daughenbaugh et al., 2003; Daughenbaugh et al., 2006). The interaction of the VPg with individual eIF4F components has been described. A direct interaction with eIF4E (cap binding protein) was firstly demonstrated with the VPg of Norwalk virus (Goodfellow et al., 2005). A similar interaction with this component was also seen with FCV and MNV but, in these viruses, the key requirement for

**2.4.3 p22** 

**2.4.4 VPg** 

(Rohayem et al., 2010).

norovirus drugs (Sharp et al., 2010).

endossome membranes (Hyde & Mackenzie, 2010).

evaluated for their ability to inhibit p22 and the norovirus replication.

tail of the viral genome (Rohayem et al., 2006a; Rohayem et al., 2006b).

The norovirus protease or NS6 is a cysteine protease that is responsible for processing the nonstructural polyprotein precursor into the six/seven nonstructural proteins (Green, 2007). The atomic structure of the norovirus protease has been resolved (Nakamura et al., 2005). A two-domain structure was identified as being similar to other viral cysteine proteases, like those of picornavirus 3C proteases that show a chymotrypsin-like fold with a cysteine as the active-site nucleophile (Nakamura et al., 2005; Zeitler et al., 2006).

Similarly to what happens with the picornavirus 3C protease, the scissile bonds of the calicivirus proteases are restricted to a few number of dipeptides, revealing a very high specificity of substrate recognition in the cleavage sites of these enzymes which have also shown to be highly conserved among all caliciviruses (Sosnovtsev, 2010). Hence, the development of substrate-like peptides that block the protease active site constitutes an interesting strategy to inhibit the protease of norovirus. The inhibitors of the 3C protease of picornavirus, such as ruprintrivir, may potentially inhibit the norovirus protease given the overall similarity between the proteases of these viruses. (Binford et al., 2005; De Palma et al., 2008c; Leyssen et al., 2008).

The detailed structural information available for norovirus protease constitutes a good basis for the rational design of protease inhibitors and a valuable tool for the *in silico* screening of large compound libraries (Tan & Jiang, 2008). The recent development of functional assays for the detection of protease activity of calicivirus, using fluorogenic substrate peptides (Zeitler et al., 2006) or bioluminescence technologies (Oka et al., 2011) can constitute useful tools for the screening of protease inhibitors.

However, the role of norovirus protease appears to be more extensive. An association of the protease of MNV with mitochondria was suggested since they were found to co-localize within the cell (Hyde & Mackenzie, 2010). In this case, the protease could be implicated in an up-regulation of apoptosis of norovirus, which is known to occur via a mitochondrialmediated apoptotic pathway involving caspase-9 (Bok et al., 2009). The involvement of viral protease with apoptotic pathways has been described for their picornavirus counterparts (Drahos & Racaniello, 2009). An association of proteases with mitochondria was linked, in hepatitis A and C viruses, to a cleavage of the mitochondrial antiviral signaling protein (MAVS) (Yang et al., 2007). Since MAVS induces antiviral responses of interferon and NFkB, one may implicate viral proteases in prevention of immune signaling (Hyde & Mackenzie, 2010).

Targeting Norovirus: Strategies for the Discovery of New Antiviral Drugs 137

syncytial virus (RSV) infections (Graci & Cameron, 2006). It has also been used against a number of other viruses such as Lassa fever virus, Crimean Congo hemorrhagic fever, hantaviruses and severe acute respiratory syndrome coronavirus (SARS-CoV) (Haagmans & Osterhaus, 2006; Leyssen et al., 2008). Ribavirin has shown to be active against norovirus in the Norwalk replicon model and the MNV model (Chang & George, 2007). The main mechanism of action in the Norwalk replicon model was associated with the depletion of GTP in the cells since the addition of guanosine moderately reversed the antiviral effect of ribavirin (Chang & George, 2007). This depletion of intracellular GTP could be due to the inhibition of the cellular inosine monophosphate dehydrogenase (IMPDH) by ribavirin (Graci & Cameron, 2006; Leyssen et al., 2005). Mycophenolic acid (MPA), a potent noncompetitive inhibitor of IMPDH, also showed to inhibit Norwalk virus replication

Another of the proposed mechanisms of ribavirin is an increased mutation frequency via incorporation of ribavirin into newly synthesized genomes leading to error catastrophe (Graci & Cameron, 2006). However, ribavirin seemed not to induce catastrophic mutations in norovirus since there was no increase in the mutation rates in the ribavirin-treated

Overall, this data indicates that ribavirin or its analogs, such as EICAR (5-ethynyl-1-β-Dribofuranosylimidazole-4-carboxamide) and viramidine, could constitute a promising

The norovirus RNA genome or viral transcripts also constitute an important target to inhibit the replication of norovirus. Antisense oligonucleotides and siRNAs (small interfering RNA) can be designed to target conserved regions of the norovirus genome with the aim of disrupting viral replication. For that, these oligonucleotides need to fulfill some requisites, namely the target sequence has to be involved in viral replication, accessible for oligonucleotide hybridization and conserved among different viral strains (Spurgers et al.,

The existence of conserved secondary structures among calicivirus genomes opens the possibility of designing an oligonucleotide that would present a broad spectrum activity among noroviruses or troughout the family *Caliciviridae*. These conserved structures include 5' terminal stem-loops, 3' terminal hairpins, a stem-loop just upstream of the ORF1/2 junction in the antigenomic strand and a stem-loop at the 5' end of the polymerase coding region with a motif characteristic of picornavirus *cis*-acting replication elements (*cre* elements) that dictate VPg uridylylation (Simmonds et al., 2008; Victoria et al., 2009). Some of these structures were found to be critical for the replication of MNV and for its infectivity (Simmonds et al., 2008). By using MNV reverse genetics system it was demonstrated that the disruption of the 5'-stem loops or the 3'-hairpins strongly impaired MNV replication *in vitro* (Simmonds et al., 2008). Moreover, a polypyrimidine tract located at the 3'-end of the genome has been incriminated in regulating viral fitness and virulence of MNV *in vivo* (Bailey et al., 2010). The important roles played by these conserved RNA structures in norovirus replication and virulence makes them potentially

(Chang & George, 2007).

2008).

good antiviral targets.

Norwalk replicon-bearing cells (Chang & George, 2007).

**2.5 Targeting the norovirus genome** 

starting point in the development of inhibitors of norovirus replication.

Whether the protease of MNV plays a role in prevention of immune signaling through the cleavage of MAVS is currently under investigation (Hyde & Mackenzie, 2010). It is known so far that the innate immune response against MNV starts with the recognition of viral RNA by MDA-5 (McCartney et al., 2008), the cytosolic receptor which initiates the antiviral signaling cascade by interacting with MAVS, its downstream partner.

In conclusion, targeting protease may be one of the most promising approaches to inhibit norovirus replication given the important function of this protein.

#### **2.4.6 RNA-dependent RNA polymerase**

The RdRp or NS7 is the central enzyme of replication, being responsible for the synthesis of the genomic, subgenomic and antigenomic RNA of norovirus. Given its critical role in norovirus replication, the RdRp constitutes one of the most important antiviral targets.

The crystal structure of the norovirus RdRp in an enzymatically active form has been resolved and overall showed catalytic and structural elements characteristic of RdRp of other positive stranded RNA viruses (Ferrer-Orta et al., 2006; Nakamura et al., 2005; Ng et al., 2004). However, its carboxyl terminus folds into the active site cleft of the enzyme which constitutes a distinctive aspect of the norovirus RdRp deserving special attention when designing an RdRp inhibitor (Hardy, 2005; Ng et al., 2004). It would be expected that a compound that could interrupt the interaction between the carboxyl-terminus and the active site cleft of the RdRp would hamper norovirus replication (Tan & Jiang, 2008).

A more accurate picture of how the RdRp of Norwalk virus functions was provided recently when the crystal structure of the Norwalk virus polymerase was resolved bound to the RNA primer template and to its natural substrate, a nucleoside triphosphate (NTP) (Zamyatkin et al., 2008). This ternary RdRp·RNA·NTP complex revealed details underlying the nucleotidyl transfer reaction, which is thought to be highly conserved among viral polymerases. This complex was also resolved bound to a potent inhibitor of picornavirus polymerases, the 5 nitrocytidine triphosphate (NCT), revealing differences between NCT and NTP complexes, which indicate a novel mechanism of inhibition of the RdRp that should be explored in the design of anti-norovirus compounds mimicking natural nucleosides and nucleotides (Zamyatkin et al., 2008).

A functional assay was developed for the detection of norovirus RdRp activity (Rohayem et al., 2006a; Rohayem et al., 2006b) and the inhibitory activity of some nucleoside analogs such as 2'-arauridine-5'-triphosphate and 3'-deoxyuridine-5'-triphosphate has been reported (Rohayem et al., 2010). Nucleoside analogs display similar mechanisms of inhibition of RdRps and some, like 2'-*C*-methylcytidine, 2'-*C*-methyladenosine and 4'-azidocytidine have shown a broad spectrum of activity against plus stranded RNA viruses, such as picornavirus and HCV (De Palma et al., 2008b; Goris et al., 2007; Klumpp et al., 2006). Hence, this class of compounds has great potential to be inhibitors of the norovirus RdRp and ought to be studied as antiviral drugs.

Ribavirin is a guanosine analogue with broad-spectrum activity against RNA virus. Although it was discovered over 30 years ago, its mechanism of action still remains controversial. Ribavirin was formally approved to treat chronic HCV infections in combination with pegylated interferon and in aerosol form to treat pediatric respiratory

Whether the protease of MNV plays a role in prevention of immune signaling through the cleavage of MAVS is currently under investigation (Hyde & Mackenzie, 2010). It is known so far that the innate immune response against MNV starts with the recognition of viral RNA by MDA-5 (McCartney et al., 2008), the cytosolic receptor which initiates the antiviral

In conclusion, targeting protease may be one of the most promising approaches to inhibit

The RdRp or NS7 is the central enzyme of replication, being responsible for the synthesis of the genomic, subgenomic and antigenomic RNA of norovirus. Given its critical role in norovirus replication, the RdRp constitutes one of the most important antiviral targets.

The crystal structure of the norovirus RdRp in an enzymatically active form has been resolved and overall showed catalytic and structural elements characteristic of RdRp of other positive stranded RNA viruses (Ferrer-Orta et al., 2006; Nakamura et al., 2005; Ng et al., 2004). However, its carboxyl terminus folds into the active site cleft of the enzyme which constitutes a distinctive aspect of the norovirus RdRp deserving special attention when designing an RdRp inhibitor (Hardy, 2005; Ng et al., 2004). It would be expected that a compound that could interrupt the interaction between the carboxyl-terminus and the active

A more accurate picture of how the RdRp of Norwalk virus functions was provided recently when the crystal structure of the Norwalk virus polymerase was resolved bound to the RNA primer template and to its natural substrate, a nucleoside triphosphate (NTP) (Zamyatkin et al., 2008). This ternary RdRp·RNA·NTP complex revealed details underlying the nucleotidyl transfer reaction, which is thought to be highly conserved among viral polymerases. This complex was also resolved bound to a potent inhibitor of picornavirus polymerases, the 5 nitrocytidine triphosphate (NCT), revealing differences between NCT and NTP complexes, which indicate a novel mechanism of inhibition of the RdRp that should be explored in the design of anti-norovirus compounds mimicking natural nucleosides and nucleotides

A functional assay was developed for the detection of norovirus RdRp activity (Rohayem et al., 2006a; Rohayem et al., 2006b) and the inhibitory activity of some nucleoside analogs such as 2'-arauridine-5'-triphosphate and 3'-deoxyuridine-5'-triphosphate has been reported (Rohayem et al., 2010). Nucleoside analogs display similar mechanisms of inhibition of RdRps and some, like 2'-*C*-methylcytidine, 2'-*C*-methyladenosine and 4'-azidocytidine have shown a broad spectrum of activity against plus stranded RNA viruses, such as picornavirus and HCV (De Palma et al., 2008b; Goris et al., 2007; Klumpp et al., 2006). Hence, this class of compounds has great potential to be inhibitors of the norovirus RdRp

Ribavirin is a guanosine analogue with broad-spectrum activity against RNA virus. Although it was discovered over 30 years ago, its mechanism of action still remains controversial. Ribavirin was formally approved to treat chronic HCV infections in combination with pegylated interferon and in aerosol form to treat pediatric respiratory

site cleft of the RdRp would hamper norovirus replication (Tan & Jiang, 2008).

signaling cascade by interacting with MAVS, its downstream partner.

norovirus replication given the important function of this protein.

**2.4.6 RNA-dependent RNA polymerase** 

(Zamyatkin et al., 2008).

and ought to be studied as antiviral drugs.

syncytial virus (RSV) infections (Graci & Cameron, 2006). It has also been used against a number of other viruses such as Lassa fever virus, Crimean Congo hemorrhagic fever, hantaviruses and severe acute respiratory syndrome coronavirus (SARS-CoV) (Haagmans & Osterhaus, 2006; Leyssen et al., 2008). Ribavirin has shown to be active against norovirus in the Norwalk replicon model and the MNV model (Chang & George, 2007). The main mechanism of action in the Norwalk replicon model was associated with the depletion of GTP in the cells since the addition of guanosine moderately reversed the antiviral effect of ribavirin (Chang & George, 2007). This depletion of intracellular GTP could be due to the inhibition of the cellular inosine monophosphate dehydrogenase (IMPDH) by ribavirin (Graci & Cameron, 2006; Leyssen et al., 2005). Mycophenolic acid (MPA), a potent noncompetitive inhibitor of IMPDH, also showed to inhibit Norwalk virus replication (Chang & George, 2007).

Another of the proposed mechanisms of ribavirin is an increased mutation frequency via incorporation of ribavirin into newly synthesized genomes leading to error catastrophe (Graci & Cameron, 2006). However, ribavirin seemed not to induce catastrophic mutations in norovirus since there was no increase in the mutation rates in the ribavirin-treated Norwalk replicon-bearing cells (Chang & George, 2007).

Overall, this data indicates that ribavirin or its analogs, such as EICAR (5-ethynyl-1-β-Dribofuranosylimidazole-4-carboxamide) and viramidine, could constitute a promising starting point in the development of inhibitors of norovirus replication.

#### **2.5 Targeting the norovirus genome**

The norovirus RNA genome or viral transcripts also constitute an important target to inhibit the replication of norovirus. Antisense oligonucleotides and siRNAs (small interfering RNA) can be designed to target conserved regions of the norovirus genome with the aim of disrupting viral replication. For that, these oligonucleotides need to fulfill some requisites, namely the target sequence has to be involved in viral replication, accessible for oligonucleotide hybridization and conserved among different viral strains (Spurgers et al., 2008).

The existence of conserved secondary structures among calicivirus genomes opens the possibility of designing an oligonucleotide that would present a broad spectrum activity among noroviruses or troughout the family *Caliciviridae*. These conserved structures include 5' terminal stem-loops, 3' terminal hairpins, a stem-loop just upstream of the ORF1/2 junction in the antigenomic strand and a stem-loop at the 5' end of the polymerase coding region with a motif characteristic of picornavirus *cis*-acting replication elements (*cre* elements) that dictate VPg uridylylation (Simmonds et al., 2008; Victoria et al., 2009). Some of these structures were found to be critical for the replication of MNV and for its infectivity (Simmonds et al., 2008). By using MNV reverse genetics system it was demonstrated that the disruption of the 5'-stem loops or the 3'-hairpins strongly impaired MNV replication *in vitro* (Simmonds et al., 2008). Moreover, a polypyrimidine tract located at the 3'-end of the genome has been incriminated in regulating viral fitness and virulence of MNV *in vivo* (Bailey et al., 2010). The important roles played by these conserved RNA structures in norovirus replication and virulence makes them potentially good antiviral targets.

Targeting Norovirus: Strategies for the Discovery of New Antiviral Drugs 139

simvastatin and lovastatin, are well known drugs that interfere with cholesterol pathways by inhibiting *de novo* synthesis of cholesterol through inhibition of HMG-CoA (3-hydroxy-3 methyl glutaryl-coenzyme A), reducing plasma cholesterol levels by upregulating low density lipoprotein receptor (LDLR) and promoting the uptake of LDL bound cholesterol to cells. It has been demonstrated that the use of statins resulted in a reduction of HCV replication through blockage of protein geranylgeranylation and the proper formation of viral replicase complexes (Ye, 2007; Ye et al., 2003). On the contrary, the inhibition of cholesterol biosynthesis using statins significantly enhanced the replication of Norwalk virus which was correlated with an increased expression of LDLR (Chang, 2009). It was postulated that LDLR could play an important direct role in virus replication such as

participating in viral replication complexes as an essential cofactor (Chang, 2009).

be further developed as anti-norovirus drugs.

**3. Final remarks** 

that helped this giant pursuit.

human pathogen in a near future.

**4. Acknowledgments** 

**5. References** 

The activity of acyl-CoA:cholesterol acyltransferase (ACAT) is also an important factor for cholesterol biosynthesis. Unlike statins, treatment with ACAT inhibitors such as Cl-976, Sandoz 58-035, YIC-C8-434 and pyripyropene resulted in reduced levels of Norwalk replication and interestingly, in reduced levels of LDLR (Chang, 2009). This data indicate that ACAT may be a novel target for inhibiting norovirus replication and its inhibitors could

Antiviral therapy is still not available today for norovirus and certainly a long road still lies ahead. A significant progress has been made in the elucidation of the replication strategy of norovirus, for which the use of surrogate viruses, the generation of a Norwalk replicon model, the available crystal structures of norovirus proteins were landmark developments

In this chapter, we review and speculate about potential targets and antiviral strategies. Many were the targets suggested, however we believe that priority should be given to viral enzymes of replication, such as the RdRp and protease. Besides being key enzymes in norovirus life cycle, they are conserved across this genetically diverse group of viruses and divergent enough from cellular enzymes for their inhibitors to have good selectivity and minimal toxicity. Moreover, viral enzymes of replication are in general less prone to

As the understanding of norovirus replication deepens, one could look forward to new opportunities for the development of innovative antiviral strategies targeting this important

We thank Joana Macedo (Faculdade de Farmácia, Universidade do Porto) for designing the figures of this chapter. Thanks are due to FCT – Fundação para a Ciência e a Tecnologia for

Bae, J. & Schwab, K.J. (2008). Evaluation of murine norovirus, feline calicivirus,

poliovirus, and MS2 as surrogates for human norovirus in a model of viral

variation than structural proteins, minimizing the emergence of drug resistance.

the PhD grant of J. Rocha-Pereira (SFRH/BD/48156/2008).

The 5′-UTR (untranslated region) sequence of the norovirus genome has already been successfully targeted by an antisense strategy. A panel of peptide-conjugated phosphorodiamidiate morpholino oligomers (PPMOs) specific for the 5′-UTR of MNV proved to be effective in inhibiting its replication *in vitro* (Bok et al., 2008). Also, a consensus PPMO (designated *Noro 1.1*), designed to target the corresponding region of several diverse human norovirus genotypes, inhibited Norwalk virus protein expression in replicon-bearing cells (Bok et al., 2008). Moreover, PMOs targeting the 5′-UTR of the FCV genome were used successfully in three clinical trials during FCV outbreaks in kittens (Smith et al., 2008). Overall, these studies suggests that PPMOs directed against the relatively conserved 5′-end of the norovirus genome may show broad antiviral activity against this genetically diverse group of viruses and might translate into a successful clinical application but only further studies in animal models will say.

An alternative nucleic acid-based strategy is the use of RNA interference (RNAi) for silencing the viral genome. A wide range of viruses have been inhibited with RNAi (Leonard & Schaffer, 2006) and concerning calicivirus the first preliminary results have been published (Bergmann & Rohayem, 2010; Rohayem et al., 2010). In this study, siRNAs targeting the 5'-UTR and the subgenomic region of the FCV genome were successful in inhibiting FCV replication *in vitro*, namely by reducing infectivity, reducing the levels of viral genomic RNA and inhibiting viral translation.

#### **2.6 Other strategies to stop norovirus replication**

The enhancement of the host cell's antiviral mechanisms may constitute a valuable strategy to inhibit norovirus replication. Interferons (IFNs) are critical components of the innate immune response which establish an antiviral state of cells through interactions with IFN receptors expressed in nearly all nucleated cells. The binding of IFNs to their receptors triggers activation of STATs (signal transducer and activator of transcription) and cascade events which results in the induction of various antiviral proteins, such as RNA-dependent protein kinase (PKR) and RNase L (Samuel, 2001). However, many viruses are armed with anti-IFN mechanisms, such as those counteracting the STATs and inhibiting IFN synthesis (Samuel, 2001).

Interferons, both type I (IFN-α) and type II (IFN-γ), showed to inhibit the replication of norovirus in the Norwalk replicon model (Chang & George, 2007; Chang et al., 2006). Norwalk virus did not present a strong anti-IFN mechanism in the replicon-bearing cells, and this may be a reason for its high sensitivity to IFNs (Chang & George, 2007; Chang et al., 2006). It was also demonstrated that IFN responses were critical to control MNV infection *in vivo* and inhibited viral replication *in vitro* (Karst et al., 2003; Wobus et al., 2004). Both type I and type II IFNs block the translation of viral proteins of MNV but while type II IFN-mediated inhibition is dependent on the well-characterized interferon-induced antiviral molecule PKR, type I IFNmediated inhibition occur through a PKR-independent process (Changotra et al., 2009). This data suggests that IFN may be a good therapeutic option for norovirus gastroenteritis.

The cellular pathway of cholesterol biosynthesis has been shown to be upregulated during the replication of several viruses, including HIV and HCV (Giguere & Tremblay, 2004; Ye, 2007). A study using Norwalk replicon system demonstrated that cholesterol pathways were also important in the replication of norovirus (Chang, 2009). Statins, such as simvastatin and lovastatin, are well known drugs that interfere with cholesterol pathways by inhibiting *de novo* synthesis of cholesterol through inhibition of HMG-CoA (3-hydroxy-3 methyl glutaryl-coenzyme A), reducing plasma cholesterol levels by upregulating low density lipoprotein receptor (LDLR) and promoting the uptake of LDL bound cholesterol to cells. It has been demonstrated that the use of statins resulted in a reduction of HCV replication through blockage of protein geranylgeranylation and the proper formation of viral replicase complexes (Ye, 2007; Ye et al., 2003). On the contrary, the inhibition of cholesterol biosynthesis using statins significantly enhanced the replication of Norwalk virus which was correlated with an increased expression of LDLR (Chang, 2009). It was postulated that LDLR could play an important direct role in virus replication such as participating in viral replication complexes as an essential cofactor (Chang, 2009).

The activity of acyl-CoA:cholesterol acyltransferase (ACAT) is also an important factor for cholesterol biosynthesis. Unlike statins, treatment with ACAT inhibitors such as Cl-976, Sandoz 58-035, YIC-C8-434 and pyripyropene resulted in reduced levels of Norwalk replication and interestingly, in reduced levels of LDLR (Chang, 2009). This data indicate that ACAT may be a novel target for inhibiting norovirus replication and its inhibitors could be further developed as anti-norovirus drugs.

#### **3. Final remarks**

138 Antiviral Drugs – Aspects of Clinical Use and Recent Advances

The 5′-UTR (untranslated region) sequence of the norovirus genome has already been successfully targeted by an antisense strategy. A panel of peptide-conjugated phosphorodiamidiate morpholino oligomers (PPMOs) specific for the 5′-UTR of MNV proved to be effective in inhibiting its replication *in vitro* (Bok et al., 2008). Also, a consensus PPMO (designated *Noro 1.1*), designed to target the corresponding region of several diverse human norovirus genotypes, inhibited Norwalk virus protein expression in replicon-bearing cells (Bok et al., 2008). Moreover, PMOs targeting the 5′-UTR of the FCV genome were used successfully in three clinical trials during FCV outbreaks in kittens (Smith et al., 2008). Overall, these studies suggests that PPMOs directed against the relatively conserved 5′-end of the norovirus genome may show broad antiviral activity against this genetically diverse group of viruses and might translate into a successful clinical application but only further

An alternative nucleic acid-based strategy is the use of RNA interference (RNAi) for silencing the viral genome. A wide range of viruses have been inhibited with RNAi (Leonard & Schaffer, 2006) and concerning calicivirus the first preliminary results have been published (Bergmann & Rohayem, 2010; Rohayem et al., 2010). In this study, siRNAs targeting the 5'-UTR and the subgenomic region of the FCV genome were successful in inhibiting FCV replication *in vitro*, namely by reducing infectivity, reducing the levels of

The enhancement of the host cell's antiviral mechanisms may constitute a valuable strategy to inhibit norovirus replication. Interferons (IFNs) are critical components of the innate immune response which establish an antiviral state of cells through interactions with IFN receptors expressed in nearly all nucleated cells. The binding of IFNs to their receptors triggers activation of STATs (signal transducer and activator of transcription) and cascade events which results in the induction of various antiviral proteins, such as RNA-dependent protein kinase (PKR) and RNase L (Samuel, 2001). However, many viruses are armed with anti-IFN mechanisms, such as those counteracting the STATs and inhibiting IFN synthesis

Interferons, both type I (IFN-α) and type II (IFN-γ), showed to inhibit the replication of norovirus in the Norwalk replicon model (Chang & George, 2007; Chang et al., 2006). Norwalk virus did not present a strong anti-IFN mechanism in the replicon-bearing cells, and this may be a reason for its high sensitivity to IFNs (Chang & George, 2007; Chang et al., 2006). It was also demonstrated that IFN responses were critical to control MNV infection *in vivo* and inhibited viral replication *in vitro* (Karst et al., 2003; Wobus et al., 2004). Both type I and type II IFNs block the translation of viral proteins of MNV but while type II IFN-mediated inhibition is dependent on the well-characterized interferon-induced antiviral molecule PKR, type I IFNmediated inhibition occur through a PKR-independent process (Changotra et al., 2009). This

data suggests that IFN may be a good therapeutic option for norovirus gastroenteritis.

The cellular pathway of cholesterol biosynthesis has been shown to be upregulated during the replication of several viruses, including HIV and HCV (Giguere & Tremblay, 2004; Ye, 2007). A study using Norwalk replicon system demonstrated that cholesterol pathways were also important in the replication of norovirus (Chang, 2009). Statins, such as

studies in animal models will say.

(Samuel, 2001).

viral genomic RNA and inhibiting viral translation.

**2.6 Other strategies to stop norovirus replication** 

Antiviral therapy is still not available today for norovirus and certainly a long road still lies ahead. A significant progress has been made in the elucidation of the replication strategy of norovirus, for which the use of surrogate viruses, the generation of a Norwalk replicon model, the available crystal structures of norovirus proteins were landmark developments that helped this giant pursuit.

In this chapter, we review and speculate about potential targets and antiviral strategies. Many were the targets suggested, however we believe that priority should be given to viral enzymes of replication, such as the RdRp and protease. Besides being key enzymes in norovirus life cycle, they are conserved across this genetically diverse group of viruses and divergent enough from cellular enzymes for their inhibitors to have good selectivity and minimal toxicity. Moreover, viral enzymes of replication are in general less prone to variation than structural proteins, minimizing the emergence of drug resistance.

As the understanding of norovirus replication deepens, one could look forward to new opportunities for the development of innovative antiviral strategies targeting this important human pathogen in a near future.

#### **4. Acknowledgments**

We thank Joana Macedo (Faculdade de Farmácia, Universidade do Porto) for designing the figures of this chapter. Thanks are due to FCT – Fundação para a Ciência e a Tecnologia for the PhD grant of J. Rocha-Pereira (SFRH/BD/48156/2008).

#### **5. References**

Bae, J. & Schwab, K.J. (2008). Evaluation of murine norovirus, feline calicivirus, poliovirus, and MS2 as surrogates for human norovirus in a model of viral

Targeting Norovirus: Strategies for the Discovery of New Antiviral Drugs 141

Cannon, J.L., Papafragkou, E., Park, G.W., Osborne, J., Jaykus, L.A. & Vinje, J. (2006).

Cao, S., Lou, Z., Tan, M., Chen, Y., Liu, Y., Zhang, Z., Zhang, X.C., Jiang, X., Li, X. & Rao, Z.

CDC (2002). Outbreak of acute gastroenteritis associated with Norwalk-like viruses among

Chang, K.O. (2009). Role of cholesterol pathways in norovirus replication. *J Virol* Vol.No. 17

Chang, K.O. & George, D.W. (2007). Interferons and ribavirin effectively inhibit Norwalk virus replication in replicon-bearing cells. *J Virol* Vol.No. 22 pp. (12111-12118). Chang, K.O., Sosnovtsev, S.S., Belliot, G., Wang, Q., Saif, L.J. & Green, K.Y. (2005). Reverse

Chang, K.O., Sosnovtsev, S.V., Belliot, G., King, A.D. & Green, K.Y. (2006). Stable expression

Changotra, H., Jia, Y., Moore, T.N., Liu, G., Kahan, S.M., Sosnovtsev, S.V. & Karst, S.M.

Chaudhry, Y., Nayak, A., Bordeleau, M.E., Tanaka, J., Pelletier, J., Belsham, G.J., Roberts,

Chaudhry, Y., Skinner, M.A. & Goodfellow, I.G. (2007). Recovery of genetically defined

Chen, S.Y., Tsai, C.N., Lai, M.W., Chen, C.Y., Lin, K.L., Lin, T.Y. & Chiu, C.H. (2009).

Choi, J.M., Hutson, A.M., Estes, M.K. & Prasad, B.V. (2008). Atomic resolution structural

Daughenbaugh, K.F., Fraser, C.S., Hershey, J.W. & Hardy, M.E. (2003). The genome-linked

Daughenbaugh, K.F., Wobus, C.E. & Hardy, M.E. (2006). VPg of murine norovirus binds translation initiation factors in infected cells. *Virol J* Vol.No. pp. (33). De Palma, A.M., Heggermont, W., Lanke, K., Coutard, B., Bergmann, M., Monforte, A.M.,

initiation complex recruitment. *EMBO J* Vol.No. 11 pp. (2852-2859).

the nonstructural protein 2C. *J Virol* Vol.No. 10 pp. (4720-4730).

for eIF4F components. *J Biol Chem* Vol.No. 35 pp. (25315-25325).

polymerase. *J Gen Virol* Vol.No. Pt 8 pp. (2091-2100).

*Proc Natl Acad Sci U S A* Vol.No. 27 pp. (9175-9180).

norovirus. *J Virol* Vol.No. 11 pp. (5949-5957).

Caliciviridae. *J Virol* Vol.No. 3 pp. (1409-1416).

proteins. *J Virol* Vol.No. 11 pp. (5683-5692).

*Infect Dis* Vol.No. 7 pp. (849-855).

(2761-2765).

Vol.No. 22 pp. (477-479).

pp. (8587-8595).

pp. (463-473).

Surrogates for the study of norovirus stability and inactivation in the environment: A comparison of murine norovirus and feline calicivirus. *J Food Prot* Vol.No. 11 pp.

(2007). Structural basis for the recognition of blood group trisaccharides by

British military personnel--Afghanistan, May 2002. *MMWR Morb Mortal Wkly Rep*

genetics system for porcine enteric calicivirus, a prototype sapovirus in the

of a Norwalk virus RNA replicon in a human hepatoma cell line. *Virology* Vol.No. 2

(2009). Type I and type II interferons inhibit the translation of murine norovirus

L.O. & Goodfellow, I.G. (2006). Caliciviruses differ in their functional requirements

murine norovirus in tissue culture by using a fowlpox virus expressing T7 RNA

Norovirus infection as a cause of diarrhea-associated benign infantile seizures. *Clin* 

characterization of recognition of histo-blood group antigens by Norwalk virus.

protein VPg of the Norwalk virus binds eIF3, suggesting its role in translation

Canard, B., De Clercq, E., Chimirri, A., Purstinger, G., Rohayem, J., van Kuppeveld, F. & Neyts, J. (2008a). The thiazolobenzimidazole TBZE-029 inhibits enterovirus replication by targeting a short region immediately downstream from motif C in

persistence in surface water and groundwater. *Appl Environ Microbiol* Vol.No. 2 pp. (477-484).


Bailey, D., Kaiser, W.J., Hollinshead, M., Moffat, K., Chaudhry, Y., Wileman, T., Sosnovtsev,

Bailey, D., Karakasiliotis, I., Vashist, S., Chung, L.M., Reese, J., McFadden, N., Benson, A.,

Bank-Wolf, B.R., Konig, M. & Thiel, H.J. (2010). Zoonotic aspects of infections with noroviruses and sapoviruses. *Vet Microbiol* Vol.No. 3-4 pp. (204-212). Barton, E.S., Forrest, J.C., Connolly, J.L., Chappell, J.D., Liu, Y., Schnell, F.J., Nusrat, A.,

Bergmann, M. & Rohayem, J. (2010). Inhibition of Calicivirus Replication in Mammalian

Bertolotti-Ciarlet, A., Crawford, S.E., Hutson, A.M. & Estes, M.K. (2003). The 3' end of

Binford, S.L., Maldonado, F., Brothers, M.A., Weady, P.T., Zalman, L.S., Meador, J.W., 3rd,

Bok, K., Cavanaugh, V.J., Matson, D.O., Gonzalez-Molleda, L., Chang, K.O., Zintz, C., Smith,

Bok, K., Prikhodko, V.G., Green, K.Y. & Sosnovtsev, S.V. (2009). Apoptosis in murine

Bordeleau, M.E., Cencic, R., Lindqvist, L., Oberer, M., Northcote, P., Wagner, G. & Pelletier,

Boschi-Pinto, C., Velebit, L. & Shibuya, K. (2008). Estimating child mortality due to

Bryce, J., Boschi-Pinto, C., Shibuya, K. & Black, R.E. (2005). WHO estimates of the causes of

Bu, W., Mamedova, A., Tan, M., Xia, M., Jiang, X. & Hegde, R.S. (2008). Structural basis for

Burroughs, J.N. & Brown, F. (1978). Presence of a covalently linked protein on calicivirus

inhibits translation initiation. *Chem Biol* Vol.No. 12 pp. (1287-1295).

death in children. *Lancet* Vol.No. 9465 pp. (1147-1152).

RNA. *J Gen Virol* Vol.No. 2 pp. (443-446).

pp. (477-484).

2870).

Vol.No. Pt 3 pp. (739-749).

reovirus. *Cell* Vol.No. 3 pp. (441-451).

Vol.No. 21 pp. (11603-11615).

*Chemother* Vol.No. 2 pp. (619-626).

*Virol* Vol.No. 8 pp. (3647-3656).

Vol.No. 2 pp. (328-337).

Cells by RNAi. *Antiviral Research* Vol.No. 1 pp. (A53).

persistence in surface water and groundwater. *Appl Environ Microbiol* Vol.No. 2

S.V. & Goodfellow, I.G. (2009). Feline calicivirus p32, p39 and p30 proteins localize to the endoplasmic reticulum to initiate replication complex formation. *J Gen Virol*

Yarovinsky, F., Simmonds, P. & Goodfellow, I. (2010). Functional analysis of RNA structures present at the 3' extremity of the murine norovirus genome: the variable polypyrimidine tract plays a role in viral virulence. *J Virol* Vol.No. 6 pp. (2859-

Parkos, C.A. & Dermody, T.S. (2001). Junction adhesion molecule is a receptor for

Norwalk virus mRNA contains determinants that regulate the expression and stability of the viral capsid protein VP1: a novel function for the VP2 protein. *J Virol*

Matthews, D.A. & Patick, A.K. (2005). Conservation of amino acids in human rhinovirus 3C protease correlates with broad-spectrum antiviral activity of rupintrivir, a novel human rhinovirus 3C protease inhibitor. *Antimicrob Agents* 

A.W., Iversen, P., Green, K.Y. & Campbell, A.E. (2008). Inhibition of norovirus replication by morpholino oligomers targeting the 5'-end of the genome. *Virology*

norovirus-infected RAW264.7 cells is associated with downregulation of survivin. *J* 

J. (2006). RNA-mediated sequestration of the RNA helicase eIF4A by Pateamine A

diarrhoea in developing countries. *Bull World Health Organ* Vol.No. 9 pp. (710-717).

the receptor binding specificity of Norwalk virus. *J Virol* Vol.No. 11 pp. (5340-5347).


Targeting Norovirus: Strategies for the Discovery of New Antiviral Drugs 143

Field, H.J. & Vere Hodge, R.A. (2008). Antiviral Agents. In: *Desk Encyclopedia of General Virology*, B.W.J. Mahy & M.H.V. Van Regenmortel, pp. 292-304, Elsevier Ltd. Fukushi, S., Kojima, S., Takai, R., Hoshino, F.B., Oka, T., Takeda, N., Katayama, K. &

Gerondopoulos, A., Jackson, T., Monaghan, P., Doyle, N. & Roberts, L.O. (2010). Murine

Giguere, J.F. & Tremblay, M.J. (2004). Statin compounds reduce human immunodeficiency

Glass, P.J., White, L.J., Ball, J.M., Leparc-Goffart, I., Hardy, M.E. & Estes, M.K. (2000).

Glass, R.I., Parashar, U.D. & Estes, M.K. (2009). Norovirus gastroenteritis. *N Engl J Med*

Goodfellow, I., Chaudhry, Y., Gioldasi, I., Gerondopoulos, A., Natoni, A., Labrie, L.,

interaction between VPg and eIF4E. *Embo Reports* Vol.No. 10 pp. (968-972). Goris, N., De Palma, A., Toussaint, J.F., Musch, I., Neyts, J. & De Clercq, K. (2007). 2'-C-

Gotz, H., de, J.B., Lindback, J., Parment, P.A., Hedlund, K.O., Torven, M. & Ekdahl, K.

Graci, J.D. & Cameron, C.E. (2006). Mechanisms of action of ribavirin against distinct

Green, K.Y. (2007). Caliciviridae: The Noroviruses. In: *Fields Virology*, D.M. Knipe & P.M.

Green, K.Y., Ando, T., Balayan, M.S., Berke, T., Clarke, I.N., Estes, M.K., Matson, D.O.,

Haagmans, B.L. & Osterhaus, A.D. (2006). Coronaviruses and their therapy. *Antiviral Res* 

Hansman, G.S., Shahzad-ul-Hussan, S., McLellan, J.S., Chuang G.-Y., Georgiev, I., Shimoike

Hardy, M.E. (2005). Norovirus protein structure and function. *FEMS Microbiol Lett* Vol.No. 1

Heinz, B.A. & Vance, L.M. (1995). The antiviral compound enviroxime targets the 3A coding region of rhinovirus and poliovirus. *J Virol* Vol.No. 7 pp. (4189-4197).

mouth disease virus. *Antiviral Res* Vol.No. 3 pp. (161-168).

viruses. *Rev Med Virol* Vol.No. 1 pp. (37-48).

Howley, pp. 949-979, Lippincott Williams & Wilkins

caliciviruses. *J Infect Dis* Vol.No. pp. (S322-330).

Norovirus. *J Virol* Vol.No. 8 pp. (3889-3896).

*Virol* Vol.No. 21 pp. (12062-12065).

Vol.No. 14 pp. (6581-6591).

Vol.No. 18 pp. (1776-1785).

Vol.No. 2-3 pp. (397-403).

1438).

(115-121).

Oct 2011)

39 pp. (1-8).

Kageyama, T. (2004). Poly(A)- and primer-independent RNA polymerase of

norovirus-1 cell entry is mediated through a non-clathrin-, non-caveolae-, dynamin- and cholesterol-dependent pathway. *J Gen Virol* Vol.No. Pt 6 pp. (1428-

virus type 1 replication by preventing the interaction between virion-associated host intercellular adhesion molecule 1 and its natural cell surface ligand LFA-1. *J* 

Norwalk virus open reading frame 3 encodes a minor structural protein. *J Virol*

Laliberte, J.F. & Roberts, L. (2005). Calicivirus translation initiation requires an

methylcytidine as a potent and selective inhibitor of the replication of foot-and-

(2002). Epidemiological investigation of a food-borne gastroenteritis outbreak caused by Norwalk-like virus in 30 day-care centres. *Scand J Infect Dis* Vol.No. 2 pp.

Nakata, S., Neill, J.D., Studdert, M.J. & Thiel, H.J. (2000). Taxonomy of the

T., Katayama, K., Bewley, C. A. & Kwong. P. D. (2011) Structural basis for norovirus inhibition and fucose mimicry by citrate, *J Virol* (Epub ahead of print 26


De Palma, A.M., Purstinger, G., Wimmer, E., Patick, A.K., Andries, K., Rombaut, B., De

De Palma, A.M., Thibaut, H.J., van der Linden, L., Lanke, K., Heggermont, W., Ireland, S.,

De Palma, A.M., Vliegen, I., De Clercq, E. & Neyts, J. (2008c). Selective inhibitors of

Denison, M.R. (2008). Seeking membranes: positive-strand RNA virus replication

Dolin, R., Blacklow, N.R., DuPont, H., Buscho, R.F., Wyatt, R.G., Kasel, J.A., Hornick, R. &

nonbacterial gastroenteritis. *Proc Soc Exp Biol Med* Vol.No. 2 pp. (578-583). Donaldson, E.F., Lindesmith, L.C., Lobue, A.D. & Baric, R.S. (2010). Viral shape-shifting:

Doultree, J.C., Druce, J.D., Birch, C.J., Bowden, D.S. & Marshall, J.A. (1999). Inactivation of feline calicivirus, a Norwalk virus surrogate. *J Hosp Infect* Vol.No. 1 pp. (51-57). Drahos, J. & Racaniello, V.R. (2009). Cleavage of IPS-1 in cells infected with human

Duizer, E., Bijkerk, P., Rockx, B., De Groot, A., Twisk, F. & Koopmans, M. (2004a). Inactivation of caliciviruses. *Appl Environ Microbiol* Vol.No. 8 pp. (4538-4543). Duizer, E., Schwab, K.J., Neill, F.H., Atmar, R.L., Koopmans, M.P. & Estes, M.K. (2004b). Laboratory efforts to cultivate noroviruses. *J Gen Virol* Vol.No. Pt 1 pp. (79-87). El-Kamary, S.S., Pasetti, M.F., Mendelman, P.M., Frey, S.E., Bernstein, D.I., Treanor, J.J.,

Chanock, R.M. (1972). Biological properties of Norwalk agent of acute infectious

norovirus evasion of the human immune system. *Nat Rev Microbiol* Vol.No. 3 pp.

Ferreira, J., Chen, W.H., Sublett, R., Richardson, C., Bargatze, R.F., Sztein, M.B. & Tacket, C.O. (2010). Adjuvanted intranasal Norwalk virus-like particle vaccine elicits antibodies and antibody-secreting cells that express homing receptors for mucosal and peripheral lymphoid tissues. *J Infect Dis* Vol.No. 11 pp. (1649-1658). Ettayebi, K. & Hardy, M.E. (2003). Norwalk virus nonstructural protein p48 forms a complex

with the SNARE regulator VAP-A and prevents cell surface expression of vesicular

calicivirus representing a new genus of Caliciviridae. *J Virol* Vol.No. 11 pp. (5408-

blood group antigen receptors. *Antimicrob Agents Chemother* Vol.No. 1 pp. (324-331).

Green, K.Y. (2004). Norwalk virus N-terminal nonstructural protein is associated with disassembly of the Golgi complex in transfected cells. *J Virol* Vol.No. 9 pp.

Farkas, T., Sestak, K., Wei, C. & Jiang, X. (2008). Characterization of a rhesus monkey

Feng, X. & Jiang, X. (2007). Library screen for inhibitors targeting norovirus binding to histo-

Fernandez-Vega, V., Sosnovtsev, S.V., Belliot, G., King, A.D., Mitra, T., Gorbalenya, A. &

Ferrer-Orta, C., Arias, A., Escarmis, C. & Verdaguer, N. (2006). A comparison of viral RNAdependent RNA polymerases. *Curr Opin Struct Biol* Vol.No. 1 pp. (27-34).

stomatitis virus G protein. *J Virol* Vol.No. 21 pp. (11790-11797).

picornavirus replication. *Med Res Rev* Vol.No. 6 pp. (823-884).

*Emerg Infect Dis* Vol.No. 4 pp. (545-551).

complexes. *PLoS Biol* Vol.No. 10 pp. (e270).

rhinovirus. *J Virol* Vol.No. 22 pp. (11581-11587).

pp. (1850-1857).

(231-241).

5416).

(4827-4837).

Clercq, E. & Neyts, J. (2008b). Potential use of antiviral agents in polio eradication.

Andrews, R., Arimilli, M., Al-Tel, T.H., De Clercq, E., van Kuppeveld, F. & Neyts, J. (2009). Mutations in the nonstructural protein 3A confer resistance to the novel enterovirus replication inhibitor TTP-8307. *Antimicrob Agents Chemother* Vol.No. 5


Targeting Norovirus: Strategies for the Discovery of New Antiviral Drugs 145

Koopmans, M. (2008). Progress in understanding norovirus epidemiology. *Curr Opin Infect* 

Kwong, A.D., Rao, B.G. & Jeang, K.T. (2005). Viral and cellular RNA helicases as antiviral

L'Homme, Y., Sansregret, R., Plante-Fortier, E., Lamontagne, A.M., Ouardani, M., Lacroix,

representing a new genus of Caliciviridae. *Virus Genes* Vol.No. 1 pp. (66-75). Le Pendu, J., Ruvoen-Clouet, N., Kindberg, E. & Svensson, L. (2006). Mendelian resistance to human norovirus infections. *Semin Immunol* Vol.No. 6 pp. (375-386). Leonard, J.N. & Schaffer, D.V. (2006). Antiviral RNAi therapy: emerging approaches for

Leyssen, P., Balzarini, J., De Clercq, E. & Neyts, J. (2005). The predominant mechanism by

Leyssen, P., De Clercq, E. & Neyts, J. (2008). Molecular strategies to inhibit the replication of

Lindesmith, L., Moe, C., Marionneau, S., Ruvoen, N., Jiang, X., Lindblad, L., Stewart, P.,

Lindesmith, L.C., Donaldson, E.F., Lobue, A.D., Cannon, J.L., Zheng, D.P., Vinje, J. & Baric,

Liu, B., Clarke, I.N. & Lambden, P.R. (1996). Polyprotein processing in Southampton virus:

Lopman, B.A., Reacher, M.H., Vipond, I.B., Sarangi, J. & Brown, D.W. (2004). Clinical

Makino, A., Shimojima, M., Miyazawa, T., Kato, K., Tohya, Y. & Akashi, H. (2006).

Marionneau, S., Ruvoen, N., Le Moullac-Vaidye, B., Clement, M., Cailleau-Thomas, A., Ruiz-

Martella, V., Campolo, M., Lorusso, E., Cavicchio, P., Camero, M., Bellacicco, A.L., Decaro,

Martella, V., Lorusso, E., Decaro, N., Elia, G., Radogna, A., D'Abramo, M., Desario, C.,

Marsh, M. & Helenius, A. (2006). Virus entry: open sesame. *Cell* Vol.No. 4 pp. (729-740). Marshall, J.K., Thabane, M., Borgaonkar, M.R. & James, C. (2007). Postinfectious irritable

viral pathogen. *Clin Gastroenterol Hepatol* Vol.No. 4 pp. (457-460).

individuals. *Gastroenterology* Vol.No. 7 pp. (1967-1977).

G. & Simard, C. (2009). Genomic characterization of swine caliciviruses

which ribavirin exerts its antiviral activity in vitro against flaviviruses and paramyxoviruses is mediated by inhibition of IMP dehydrogenase. *J Virol* Vol.No. 3

LePendu, J. & Baric, R. (2003). Human susceptibility and resistance to Norwalk

R.S. (2008). Mechanisms of GII.4 norovirus persistence in human populations. *PLoS* 

identification of 3C-like protease cleavage sites by in vitro mutagenesis. *J Virol*

manifestation of norovirus gastroenteritis in health care settings. *Clin Infect Dis*

Junctional adhesion molecule 1 is a functional receptor for feline calicivirus. *J Virol*

Palacois, G., Huang, P., Jiang, X. & Le Pendu, J. (2002). Norwalk virus binds to histo-blood group antigens present on gastroduodenal epithelial cells of secretor

bowel syndrome after a food-borne outbreak of acute gastroenteritis attributed to a

N., Elia, G., Greco, G., Corrente, M., Desario, C., Arista, S., Banyai, K., Koopmans, M. & Buonavoglia, C. (2007). Norovirus in captive lion cub (Panthera leo). *Emerg* 

Cavalli, A., Corrente, M., Camero, M., Germinario, C.A., Banyai, K., Di Martino, B.,

targets. *Nat Rev Drug Discov* Vol.No. 10 pp. (845-853).

hitting a moving target. *Gene Ther* Vol.No. 6 pp. (532-540).

RNA viruses. *Antiviral Res* Vol.No. 1 pp. (9-25).

virus infection. *Nat Med* Vol.No. 5 pp. (548-553).

*Dis* Vol.No. 5 pp. (544-552).

pp. (1943-1947).

*Med* Vol.No. 2 pp. (e31).

Vol.No. 4 pp. (2605-2610).

Vol.No. 3 pp. (318-324).

Vol.No. 9 pp. (4482-4490).

*Infect Dis* Vol.No. 7 pp. (1071-1073).


Huang, P., Farkas, T., Marionneau, S., Zhong, W., Ruvoen-Clouet, N., Morrow, A.L., Altaye,

Huang, P., Farkas, T., Zhong, W., Tan, M., Thornton, S., Morrow, A.L. & Jiang, X. (2005).

Hutson, A.M., Atmar, R.L. & Estes, M.K. (2004). Norovirus disease: changing epidemiology and host susceptibility factors. *Trends Microbiol* Vol.No. 6 pp. (279-287). Hutson, A.M., Atmar, R.L., Graham, D.Y. & Estes, M.K. (2002). Norwalk virus infection and

Hyde, J.L. & Mackenzie, J.M. (2010). Subcellular localization of the MNV-1 ORF1 proteins

Hyde, J.L., Sosnovtsev, S.V., Green, K.Y., Wobus, C., Virgin, H.W. & Mackenzie, J.M. (2009).

Ito, S., Takeshita, S., Nezu, A., Aihara, Y., Usuku, S., Noguchi, Y. & Yokota, S. (2006). Norovirus-associated encephalopathy. *Pediatr Infect Dis J* Vol.No. 7 pp. (651-652). Jiang, X., Wang, M., Graham, D.Y. & Estes, M.K. (1992). Expression, self-assembly, and

Kapikian, A.Z., Wyatt, R.G., Dolin, R., Thornhill, T.S., Kalica, A.R. & Chanock, R.M. (1972).

Karst, S.M., Wobus, C.E., Lay, M., Davidson, J. & Virgin, H.W.t. (2003). STAT1-dependent innate immunity to a Norwalk-like virus. *Science* Vol.No. 5612 pp. (1575-1578). Kawano, G., Oshige, K., Syutou, S., Koteda, Y., Yokoyama, T., Kim, B.G., Mizuochi, T.,

Kirkwood, C.D. & Streitberg, R. (2008). Calicivirus shedding in children after recovery from

Klumpp, K., Leveque, V., Le Pogam, S., Ma, H., Jiang, W.R., Kang, H., Granycome, C.,

Koo, H.L., Ajami, N., Atmar, R.L. & DuPont, H.L. (2010). Noroviruses: The leading cause of

gastroenteritis worldwide. *Discov Med* Vol.No. 50 pp. (61-70).

diarrhoeal disease. *J Clin Virol* Vol.No. 3 pp. (346-348).

*Chem* Vol.No. 7 pp. (3793-3799).

distinct strain-specific patterns. *J Infect Dis* Vol.No. 1 pp. (19-31).

binding patterns. *J Virol* Vol.No. 11 pp. (6714-6722).

(1335-1337).

19 pp. (9709-9719).

pp. (748-781).

(617-622).

*Virology* Vol.No. 1 pp. (138-148).

M., Pickering, L.K., Newburg, D.S., LePendu, J. & Jiang, X. (2003). Noroviruses bind to human ABO, Lewis, and secretor histo-blood group antigens: identification of 4

Norovirus and histo-blood group antigens: demonstration of a wide spectrum of strain specificities and classification of two major binding groups among multiple

disease is associated with ABO histo-blood group type. *J Infect Dis* Vol.No. 9 pp.

and their potential roles in the formation of the MNV-1 replication complex.

Mouse norovirus replication is associated with virus-induced vesicle clusters originating from membranes derived from the secretory pathway. *J Virol* Vol.No.

antigenicity of the Norwalk virus capsid protein. *J Virol* Vol.No. 11 pp. (6527-6532).

Visualization by immune electron microscopy of a 27-nm particle associated with acute infectious nonbacterial gastroenteritis. *J Virol* Vol.No. 5 pp. (1075-1081). Karst, S.M. (2010). Pathogenesis of Noroviruses, Emerging RNA Viruses. *Viruses* Vol.No. 3

Nagai, K., Matsuda, K., Ohbu, K. & Matsuishi, T. (2007). Benign infantile convulsions associated with mild gastroenteritis: a retrospective study of 39 cases including virological tests and efficacy of anticonvulsants. *Brain Dev* Vol.No. 10 pp.

Singer, M., Laxton, C., Hang, J.Q., Sarma, K., Smith, D.B., Heindl, D., Hobbs, C.J., Merrett, J.H., Symons, J., Cammack, N., Martin, J.A., Devos, R. & Najera, I. (2006). The novel nucleoside analog R1479 (4'-azidocytidine) is a potent inhibitor of NS5Bdependent RNA synthesis and hepatitis C virus replication in cell culture. *J Biol* 


Targeting Norovirus: Strategies for the Discovery of New Antiviral Drugs 147

Pfister, T. & Wimmer, E. (2001). Polypeptide p41 of a Norwalk-like virus is a nucleic acidindependent nucleoside triphosphatase. *J Virol* Vol.No. 4 pp. (1611-1619). Prasad, B.V., Hardy, M.E., Dokland, T., Bella, J., Rossmann, M.G. & Estes, M.K. (1999). X-ray

Prasad, B.V., Rothnagel, R., Jiang, X. & Estes, M.K. (1994). Three-dimensional structure of baculovirus-expressed Norwalk virus capsids. *J Virol* Vol.No. 8 pp. (5117-5125). Putics, A., Vashist, S., Bailey, D. & Goodfellow, I. (2010). Murine norovirus translation,

Hansman, X.J. Jiang & K.Y. Green, pp. 205-222, Caister Academic Press Ramani, S. & Kang, G. (2009). Viruses causing childhood diarrhoea in the developing world.

Rocha-Pereira, J., Cunha, R., Pinto, D.C.G.A., Silva, A.M.S. & Nascimento, M.S.J. (2010). (E)-

Rohayem, J., Bergmann, M., Gebhardt, J., Gould, E., Tucker, P., Mattevi, A., Unge, T.,

Rohayem, J., Jager, K., Robel, I., Scheffler, U., Temme, A. & Rudolph, W. (2006a).

Rossignol, J.F. & El-Gohary, Y.M. (2006). Nitazoxanide in the treatment of viral

Rydell, G.E., Nilsson, J., Rodriguez-Diaz, J., Ruvoen-Clouet, N., Svensson, L., Le Pendu, J. &

Samuel, C.E. (2001). Antiviral actions of interferons. *Clin Microbiol Rev* Vol.No. 4 pp. (778-

Sharp, T.M., Guix, S., Katayama, K., Crawford, S.E. & Estes, M.K. (2010). Inhibition of

Siebenga, J.J., Beersma, M.F., Vennema, H., van Biezen, P., Hartwig, N.J. & Koopmans, M.

Simmonds, P., Karakasiliotis, I., Bailey, D., Chaudhry, Y., Evans, D.J. & Goodfellow, I.G.

Smith, A.W., Iversen, P.L., O'Hanley, P.D., Skilling, D.E., Christensen, J.R., Weaver, S.S.,

and initiation of RNA synthesis. *J Gen Virol* Vol.No. Pt 9 pp. (2621-2630). Rohayem, J., Robel, I., Jager, K., Scheffler, U. & Rudolph, W. (2006b). Protein-primed and de

*Curr Opin Infect Dis* Vol.No. 5 pp. (477-482).

infections. *Antiviral Res* Vol.No. 2 pp. (162-178).

*Pharmacol Ther* Vol.No. 10 pp. (1423-1430).

*Glycobiology* Vol.No. 3 pp. (309-320).

*Chemistry* Vol.No. 12 pp. (4195-4201).

(287-290).

7069).

809).

(e13130).

pp. (994-1001).

Vol.No. 8 pp. (2530-2546).

crystallographic structure of the Norwalk virus capsid. *Science* Vol.No. 5438 pp.

replication and reverse genetics In: *Caliciviruses: Molecular and Cellular Virology*, G.S.

2-Styrylchromones as potential anti-norovirus agents. *Bioorganic & Medicinal* 

Hilgenfeld, R. & Neyts, J. (2010). Antiviral strategies to control calicivirus

Characterization of norovirus 3Dpol RNA-dependent RNA polymerase activity

novo initiation of RNA synthesis by norovirus 3Dpol. *J Virol* Vol.No. 14 pp. (7060-

gastroenteritis: a randomized double-blind placebo-controlled clinical trial. *Aliment* 

Larson, G. (2009). Human noroviruses recognize sialyl Lewis x neoglycoprotein.

cellular protein secretion by norwalk virus nonstructural protein p22 requires a mimic of an endoplasmic reticulum export signal. *PLoS One* Vol.No. 10 pp.

(2008). High prevalence of prolonged norovirus shedding and illness among hospitalized patients: a model for in vivo molecular evolution. *J Infect Dis* Vol.No. 7

(2008). Bioinformatic and functional analysis of RNA secondary structure elements among different genera of human and animal caliciviruses. *Nucleic Acids Res*

Longley, K., Stone, M.A., Poet, S.E. & Matson, D.O. (2008). Virus-specific antiviral

Marsilio, F., Carmichael, L.E. & Buonavoglia, C. (2008). Detection and molecular characterization of a canine norovirus. *Emerg Infect Dis* Vol.No. 8 pp. (1306-1308).


Mumphrey, S.M., Changotra, H., Moore, T.N., Heimann-Nichols, E.R., Wobus, C.E., Reilly,

Murata, T., Katsushima, N., Mizuta, K., Muraki, Y., Hongo, S. & Matsuzaki, Y. (2007).

Nakamura, K., Someya, Y., Kumasaka, T., Ueno, G., Yamamoto, M., Sato, T., Takeda, N.,

Ng, K.K., Pendas-Franco, N., Rojo, J., Boga, J.A., Machin, A., Alonso, J.M. & Parra, F. (2004).

Norder, H., De Palma, A.M., Selisko, B., Costenaro, L., Papageorgiou, N., Arnan, C.,

Oka, T., Takagi, H., Tohya, Y., Murakami, K., Takeda, N., Wakita, T. & Katayama, K. (2011).

Papafragkou, E., Hewitt, J., Park, G., Straub, T., Greening, G. & Vinje, J. (2009). Challenges of

Patel, M.M., Hall, A.J., Vinje, J. & Parashar, U.D. (2009). Noroviruses: a comprehensive

Patel, M.M., Widdowson, M.A., Glass, R.I., Akazawa, K., Vinje, J. & Parashar, U.D. (2008).

Perry, J.W., Taube, S. & Wobus, C.E. (2009). Murine norovirus-1 entry into permissive

Perry, J.W. & Wobus, C.E. (2010). Endocytosis of murine norovirus 1 into murine

with diarrhea. *Emerg Infect Dis* Vol.No. 6 pp. (980-982).

active site cleft. *J Biol Chem* Vol.No. 16 pp. (16638-16645).

system and in infected cells. *Antiviral Res* Vol.No. 1 pp. (9-16).

Vol.No. 7 pp. (3251-3263).

(13685-13693).

pp. (86-89).

129).

(6163-6176).

Vol.No. 3 pp. (204-218).

review. *J Clin Virol* Vol.No. 1 pp. (1-8).

*Emerg Infect Dis* Vol.No. 8 pp. (1224-1231).

*Pediatr Infect Dis J* Vol.No. 1 pp. (46-49).

Marsilio, F., Carmichael, L.E. & Buonavoglia, C. (2008). Detection and molecular characterization of a canine norovirus. *Emerg Infect Dis* Vol.No. 8 pp. (1306-1308). McCartney, S.A., Thackray, L.B., Gitlin, L., Gilfillan, S., Virgin, H.W. & Colonna, M. (2008). MDA-5 recognition of a murine norovirus. *PLoS Pathog* Vol.No. 7 pp. (e1000108). Mesquita, J.R., Barclay, L., Jose Nascimento, M.S. & Vinje, J. (2010). Novel norovirus in dogs

M.J., Moghadamfalahi, M., Shukla, D. & Karst, S.M. (2007). Murine norovirus 1 infection is associated with histopathological changes in immunocompetent hosts, but clinical disease is prevented by STAT1-dependent interferon responses. *J Virol*

Prolonged norovirus shedding in infants <or=6 months of age with gastroenteritis.

Miyamura, T. & Tanaka, N. (2005). A norovirus protease structure provides insights into active and substrate binding site integrity. *J Virol* Vol.No. 21 pp.

Crystal structure of norwalk virus polymerase reveals the carboxyl terminus in the

Coutard, B., Lantez, V., De Lamballerie, X., Baronti, C., Sola, M., Tan, J., Neyts, J., Canard, B., Coll, M., Gorbalenya, A.E. & Hilgenfeld, R. (2011). Picornavirus nonstructural proteins as targets for new anti-virals with broad activity. *Antiviral Res*

Bioluminescence technologies to detect calicivirus protease activity in cell-free

Culturing Human Norovirus in a 3-D Organoid Cell Culture Model. In Proceedings of *ASM*.109th General Meeting. Philadephia, PA, USA: 09-GM-A-2871-ASM. Parrino, T.A., Schreiber, D.S., Trier, J.S., Kapikian, A.Z. & Blacklow, N.R. (1977). Clinical

immunity in acute gastroenteritis caused by Norwalk agent. *N Engl J Med* Vol.No. 2

Systematic literature review of role of noroviruses in sporadic gastroenteritis.

macrophages and dendritic cells is pH-independent. *Virus Res* Vol.No. 1 pp. (125-

macrophages is dependent on dynamin II and cholesterol. *J Virol* Vol.No. 12 pp.


Targeting Norovirus: Strategies for the Discovery of New Antiviral Drugs 149

Teunis, P.F., Moe, C.L., Liu, P., Miller, S.E., Lindesmith, L., Baric, R.S., Le Pendu, J. &

Thackray, L.B., Wobus, C.E., Chachu, K.A., Liu, B., Alegre, E.R., Henderson, K.S., Kelley,

Tsai, B. (2007). Penetration of nonenveloped viruses into the cytoplasm. *Annu Rev Cell Dev* 

Turcios-Ruiz, R.M., Axelrod, P., St John, K., Bullitt, E., Donahue, J., Robinson, N. & Friss,

Victoria, M., Colina, R., Miagostovich, M.P., Leite, J.P. & Cristina, J. (2009). Phylogenetic

Vinje, J. (2010). A norovirus vaccine on the horizon? *J Infect Dis* Vol.No. 11 pp. (1623-1625). Ward, V.K., McCormick, C.J., Clarke, I.N., Salim, O., Wobus, C.E., Thackray, L.B., Virgin,

Wei, C., Farkas, T., Sestak, K. & Jiang, X. (2008). Recovery of infectious virus by transfection

Wessels, E., Duijsings, D., Lanke, K.H., van Dooren, S.H., Jackson, C.L., Melchers, W.J. & van

Wobus, C.E., Karst, S.M., Thackray, L.B., Chang, K.O., Sosnovtsev, S.V., Belliot, G., Krug, A.,

Wobus, C.E., Thackray, L.B. & Virgin, H.W. (2006). Murine norovirus: a model system to study norovirus biology and pathogenesis. *J Virol* Vol.No. 11 pp. (5104-5112). Yang, Y., Liang, Y.Q., Qu, L., Chen, Z.M., Yi, M.K., Li, K. & Lemon, S.M. (2007). Disruption

Ye, J. (2007). Reliance of host cholesterol metabolic pathways for the life cycle of hepatitis C

Ye, J., Wang, C., Sumpter, R., Jr., Brown, M.S., Goldstein, J.L. & Gale, M., Jr. (2003).

geranylgeranylation. *Proc Natl Acad Sci U S A* Vol.No. 26 pp. (15865-15870).

murine noroviruses. *J Virol* Vol.No. 9 pp. (4092-4101).

intensive care unit. *J Pediatr* Vol.No. 3 pp. (339-344).

(1468-1476).

pp. (10460-10473).

(11050-11055).

(11429-11436).

pp. (e432).

*Biol* Vol.No. pp. (23-43).

*Res Notes* Vol.No. pp. (176).

*Dis* Vol.No. 5 pp. (735-737).

*America* Vol.No. 17 pp. (7253-7258).

virus. *PLoS Pathog* Vol.No. 8 pp. (e108).

sialic acid moieties on murine macrophages function as attachment receptors for

Calderon, R.L. (2008). Norwalk virus: how infectious is it? *J Med Virol* Vol.No. 8 pp.

S.T. & Virgin, H.W.t. (2007). Murine noroviruses comprising a single genogroup exhibit biological diversity despite limited sequence divergence. *J Virol* Vol.No. 19

H.E. (2008). Outbreak of necrotizing enterocolitis caused by norovirus in a neonatal

prediction of cis-acting elements: a cre-like sequence in Norovirus genome? *BMC* 

H.W.t. & Lambden, P.R. (2007). Recovery of infectious murine norovirus using pol II-driven expression of full-length cDNA. *Proc Natl Acad Sci U S A* Vol.No. 26 pp.

of in vitro-generated RNA from tulane calicivirus cDNA. *J Virol* Vol.No. 22 pp.

Kuppeveld, F.J. (2006). Effects of picornavirus 3A Proteins on Protein Transport and GBF1-dependent COP-I recruitment. *J Virol* Vol.No. 23 pp. (11852-11860). Widdowson, M.A., Monroe, S.S. & Glass, R.I. (2005). Are noroviruses emerging? *Emerg Infect* 

Mackenzie, J.M., Green, K.Y. & Virgin, H.W. (2004). Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages. *PLoS Biol* Vol.No. 12

of innate immunity due to mitochondrial targeting of a picornaviral protease precursor. *Proceedings of the National Academy of Sciences of the United States of* 

Disruption of hepatitis C virus RNA replication through inhibition of host protein

treatment for controlling severe and fatal outbreaks of feline calicivirus infection. *Am J Vet Res* Vol.No. 1 pp. (23-32).


Sosnovtsev, S. & Green, K.Y. (1995). RNA transcripts derived from a cloned full-length copy

Sosnovtsev, S.V. (2010). Proteolytic cleavage and viral proteins In: *Caliciviruses: Molecular and* 

Sosnovtsev, S.V., Belliot, G., Chang, K.O., Prikhodko, V.G., Thackray, L.B., Wobus, C.E.,

Spurgers, K.B., Sharkey, C.M., Warfield, K.L. & Bavari, S. (2008). Oligonucleotide antiviral

Straub, T.M., Honer zu Bentrup, K., Orosz-Coghlan, P., Dohnalkova, A., Mayer, B.K.,

Stuart, A.D. & Brown, T.D. (2007). Alpha2,6-linked sialic acid acts as a receptor for Feline

Takanashi, S., Hashira, S., Matsunaga, T., Yoshida, A., Shiota, T., Tung, P.G., Khamrin, P.,

Tamura, M., Natori, K., Kobayashi, M., Miyamura, T. & Takeda, N. (2004). Genogroup II

Tan, M., Huang, P., Meller, J., Zhong, W., Farkas, T. & Jiang, X. (2003). Mutations within the

Tan, M. & Jiang, X. (2005). Norovirus and its histo-blood group antigen receptors: an answer

Tan, M. & Jiang, X. (2008). Norovirus gastroenteritis, increased understanding and future

Tan, M. & Jiang, X. (2010). Virus-Host Interaction and Cellular Receptors of Caliciviruses. In:

Tan, M. & Jiang, X. (2011). Norovirus-host interaction: Multi-selections by human histo-

Tan, M., Xia, M., Chen, Y., Bu, W., Hegde, R.S., Meller, J., Li, X. & Jiang, X. (2009).

Taube, S., Perry, J.W., Yetming, K., Patel, S.P., Auble, H., Shu, L., Nawar, H.F., Lee, C.H.,

evidence for a binding pocket. *J Virol* Vol.No. 23 pp. (12562-12571).

antiviral options. *Curr Opin Investig Drugs* Vol.No. 2 pp. (146-151).

to a historical puzzle. *Trends Microbiol* Vol.No. 6 pp. (285-293).

blood group antigens. *Trends Microbiol* Vol.No. 8 pp. (382-388).

selection in norovirus evolution. *PLoS One* Vol.No. 4 pp. (e5058).

*Am J Vet Res* Vol.No. 1 pp. (23-32).

*Virol* Vol.No. 16 pp. (7816-7831).

*Antiviral Res* Vol.No. 1 pp. (26-36).

111-130, Caister Academic Press

noroviruses. *Emerg Infect Dis* Vol.No. 3 pp. (396-403).

calicivirus. *J Gen Virol* Vol.No. Pt 1 pp. (177-186).

gastroenteritis. *J Clin Virol* Vol.No. 2 pp. (161-163).

cellular membrane. *J Virol* Vol.No. 8 pp. (3817-3826).

2 pp. (383-390).

Academic Press

treatment for controlling severe and fatal outbreaks of feline calicivirus infection.

of the feline calicivirus genome do not require VpG for infectivity. *Virology* Vol.No.

*Cellular Virology*, G.S. Hansman, X.J. Jiang & K.Y. Green, pp. 65-94, Caister

Karst, S.M., Virgin, H.W. & Green, K.Y. (2006). Cleavage map and proteolytic processing of the murine norovirus nonstructural polyprotein in infected cells. *J* 

therapeutics: antisense and RNA interference for highly pathogenic RNA viruses.

Bartholomew, R.A., Valdez, C.O., Bruckner-Lea, C.J., Gerba, C.P., Abbaszadegan, M. & Nickerson, C.A. (2007). In vitro cell culture infectivity assay for human

Okitsu, S., Mizuguchi, M., Igarashi, T. & Ushijima, H. (2009). Detection, genetic characterization, and quantification of norovirus RNA from sera of children with

noroviruses efficiently bind to heparan sulfate proteoglycan associated with the

P2 domain of norovirus capsid affect binding to human histo-blood group antigens:

*Caliciviruses: Molecular and Cellular Virology*, X.J.J.a.K.Y.G. Grant S. Hansman, pp.

Conservation of carbohydrate binding interfaces: evidence of human HBGA

Connell, T.D., Shayman, J.A. & Wobus, C.E. (2009). Ganglioside-linked terminal

sialic acid moieties on murine macrophages function as attachment receptors for murine noroviruses. *J Virol* Vol.No. 9 pp. (4092-4101).


**8** 

*Belgium* 

**Single Domain Camelid Antibodies that** 

*Department of Biomedical Molecular Biology, Ghent University, Ghent,* 

Recombinant antibodies (Abs) are widely regarded as one of the main, if not the most promising tools against cancer and auto-immune, inflammatory, neurodegenerative and infectious diseases (Stiehm *et al.*, 2008). Conventional antibodies are complex molecules consisting of pairs of heavy and light chains, whose N-terminal domain is more variable than the rest of the protein sequence. The antibody heavy chain usually consists of three constant domains (CH1, CH2 and CH3) and a variable domain (VH). The light chain has only two domains, the constant light (CL) and the variable light (VL). Important Glycosylations on the CH2 domain are necessary for antibody effector functions, such as Antibody-Dependent Cellular Cytotoxicity (ADCC) and Complement–Dependent Cytolysis (CDC), and for regulating antibody half time in serum (Fig. 1, A). Antigen-binding is determined by the three hypervariable Complementary Determining Regions (CDR1, CDR2 and CDR3) present in both the VH and VL domains. These regions are located in juxtaposed loops, creating a continuous surface of ~ 1000 Å2 that specifically binds to the epitope in an antigen. Although all CDRs can potentially make contact with the antigen, CDR3 contacts with the epitope are generally more extensive. The structural diversity of the antigen-binding sites of a conventional antibody depends on the size of the CDR3 in the VH and the conjunction with the VL at different angles and distances. These are grouped in three different classes, according to the size and type of antigen: cavities (fitting haptens), grooves (fitting peptides) and planar sites (fitting surface patches of proteins)

In 1993 a surprising observation was made in members of the Artiodactyl Tylopoda family (camelids). Next to conventional IgG antibodies, camelids also naturally produce Heavy Chain antibodies (HCAbs) that lack the light chain (Hamers-Casterman *et al.*, 1993). Two years later, similar single chain antibodies were discovered in cartilaginous fish (sharks) (Greenberg *et al.*, 1995). Although the CH2 and CH3 of the HCAbs and the conventional Abs

**1. Introduction** 

(Johnson *et al.*, 2010).

**1.2 The single variable domain of the heavy chain antibodies** 

**1.1 Conventional antibodies** 

**Neutralize Negative Strand Viruses** 

Francisco Miguel Lopez Cardoso, Lorena Itatí Ibañez,

*Department for Molecular Biomedical Research, Ghent,* 

Bert Schepens and Xavier Saelens


### **Single Domain Camelid Antibodies that Neutralize Negative Strand Viruses**

Francisco Miguel Lopez Cardoso, Lorena Itatí Ibañez, Bert Schepens and Xavier Saelens *Department for Molecular Biomedical Research, Ghent, Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium* 

#### **1. Introduction**

150 Antiviral Drugs – Aspects of Clinical Use and Recent Advances

Zamyatkin, D.F., Parra, F., Alonso, J.M., Harki, D.A., Peterson, B.R., Grochulski, P. & Ng,

Zeitler, C.E., Estes, M.K. & Venkataram Prasad, B.V. (2006). X-ray crystallographic structure

Zheng, D.P., Ando, T., Fankhauser, R.L., Beard, R.S., Glass, R.I. & Monroe, S.S. (2006).

Norwalk virus polymerase. *J Biol Chem* Vol.No. 12 pp. (7705-7712).

(312-323).

K.K. (2008). Structural insights into mechanisms of catalysis and inhibition in

of the Norwalk virus protease at 1.5-A resolution. *J Virol* Vol.No. 10 pp. (5050-5058).

Norovirus classification and proposed strain nomenclature. *Virology* Vol.No. 2 pp.

#### **1.1 Conventional antibodies**

Recombinant antibodies (Abs) are widely regarded as one of the main, if not the most promising tools against cancer and auto-immune, inflammatory, neurodegenerative and infectious diseases (Stiehm *et al.*, 2008). Conventional antibodies are complex molecules consisting of pairs of heavy and light chains, whose N-terminal domain is more variable than the rest of the protein sequence. The antibody heavy chain usually consists of three constant domains (CH1, CH2 and CH3) and a variable domain (VH). The light chain has only two domains, the constant light (CL) and the variable light (VL). Important Glycosylations on the CH2 domain are necessary for antibody effector functions, such as Antibody-Dependent Cellular Cytotoxicity (ADCC) and Complement–Dependent Cytolysis (CDC), and for regulating antibody half time in serum (Fig. 1, A). Antigen-binding is determined by the three hypervariable Complementary Determining Regions (CDR1, CDR2 and CDR3) present in both the VH and VL domains. These regions are located in juxtaposed loops, creating a continuous surface of ~ 1000 Å2 that specifically binds to the epitope in an antigen. Although all CDRs can potentially make contact with the antigen, CDR3 contacts with the epitope are generally more extensive. The structural diversity of the antigen-binding sites of a conventional antibody depends on the size of the CDR3 in the VH and the conjunction with the VL at different angles and distances. These are grouped in three different classes, according to the size and type of antigen: cavities (fitting haptens), grooves (fitting peptides) and planar sites (fitting surface patches of proteins) (Johnson *et al.*, 2010).

#### **1.2 The single variable domain of the heavy chain antibodies**

In 1993 a surprising observation was made in members of the Artiodactyl Tylopoda family (camelids). Next to conventional IgG antibodies, camelids also naturally produce Heavy Chain antibodies (HCAbs) that lack the light chain (Hamers-Casterman *et al.*, 1993). Two years later, similar single chain antibodies were discovered in cartilaginous fish (sharks) (Greenberg *et al.*, 1995). Although the CH2 and CH3 of the HCAbs and the conventional Abs

Single Domain Camelid Antibodies that Neutralize Negative Strand Viruses 153

AA sequence of the VHH FRs is highly similar to those of conventional VHs it was not surprising that the overall architecture of VHHs closely resembles that of VHs (Muyldermans *et al.*, 1994). Both VHH and VH domains fold into two β-sheets with the three CDRs that link these two sheets at one end of the barrel (or domain) (De Genst *et al.*, 2006; Desmyter *et al.*, 1996) (Fig. 1, C,E). However, there are striking structural differences between VHHs and conventional VH. Evidently, VHHs lack an interacting VL domain. Because of this, the hydrophobic amino acids present at the VH surface that is normally interacting with the VL, are substituted by hydrophilic AA (Fig.1, D). This enhances the solubility of VHH single domain proteins compared to engineered VH single domain

The absence of the additional CDRs in VHHs is likely compensated by structural features. First, the CDR3 regions of camelid VHHs are generally longer (13-17 amino acids) than the CDR3 regions of mouse and human VHs (9-12 and 9-17 AA respectively) (Wu *et al.*, 1993). In contrast to conventional Abs, in which the antigen binding surface is often a flat surface, a cavity or a groove, the long CDR3 loop may extend from the antigen binding surface (Desmyter *et al.*, 1996). This enlarges the paratope surface and hence the potential affinity and repertoire of camelid HCAbs. In addition, especially in dromedaries, the CDR1 and CDR3 regions contain a cysteine, which allows formation of a second disulfide bridge next to the single disulfide bridge in conventional VHs (Muyldermans *et al.*, 1994). This extra bridge likely stabilizes the CDR loops, thereby reducing their flexibility. This probably also contributes to the affinity (less entropy is lost upon antigen binding) and structural diversity of VHHs. Long extending CDR3 loops that are stabilized by an extra disulfide bridge can explain the tendency of VHHs to bind to clefts and concaves surfaces more readily than conventional antibodies do (Fig. 1, F) (De Genst *et al.*, 2006). Indeed comparison of multiple structures of hen egg white lysozyme interacting with either several conventional human antibodies or several camelid VHHs clearly illustrated that VHHs tends to bind to the concave substrate-binding pocket, whereas conventional antibodies favor epitopes on the "flat" surface of the antigen (Fig. 1, C). In addition, whereas each of the three CDRs of conventional VHs contributes considerably to the interaction with antigen, VHHs depended mainly on the CDR 3 loop for this interaction. Other antigens that are hard to target by conventional antibodies, but can be targeted by camelid VHHs are ion channels, GPCRs,

haptens and enzymatic sites (Lauwereys *et al.*, 1998; Rasmussen *et al.*, 2011).

Next to an extended CDR3, the AA sequence of the H1 loop that precedes and comprises CDR1 appears to be particularly more variable in camelid VHHs than in conventional VHs. This might be interpreted as an extension of the VHH CDR1 (Vu *et al.*, 1997). Associated with this high variability in camelid VHHs, CH1 loops adopt conformations that deviate from the canonical H1 structures of conventional VHs (Barre *et al.*, 1994; Decanniere *et al.*, 1999; Decanniere *et al.*, 2000). Camelid VHH CH1 loops appear to fold into a more diverse repertoire of structures. The high variability in the AA sequence and conformations of the CH1 loop contribute to the VHH paratope size (850-1150 Å2), which approaches that of conventional antibodies (VH + VL) (Desmyter *et al.*, 2002). Clearly, different biochemical and structural features of camelid VHHs compensate for the lack of a VL domain, thus allowing a broad repertoire of specific high affinity antigen interactions. In addition, due to their small size and typical extruding CDR3 regions, camelid VHH tend to bind in cavities that are not readily accessible for conventional antibodies. Next to these particular features,

proteins.

are highly homologous, there is no CH1 domain in the camelid HCAbs. The single variable domain, called VHH, is the only domain of HCAbs that makes contact with the antigen. Surprisingly, although the VHH have only three CDR regions, their affinity for antigens reaches the low nanomolar to even picomolar range, matching the best affinities of classical antibodies. When expressed as single domains (often referred to as nanobodies, Nb), the VHHs retain their strong epitope specificity and affinity, a feature that might be explained by the VHH architecture (Fig. 1, B). Just like the VHs of conventional antibodies, the amino acid (AA) sequence of VHHs is organised in three hypervariable regions (CDR1, CDR2 and CDR3) separated by four Framework regions (FR1-FR4) (Muyldermans *et al.*, 1994). As the

Fig. 1. Representative diagrams of a conventional antibody, an HCAb, and a VHH. (A) A conventional IgG antibody is a dimeric molecule, and each monomer comprises a heavy chain and a light chain. The heavy chain consists of the constant domains (CH1, CH2 and CH3) and the variable domain (VH). The light chain has only one conserved domain (CL) and a variable domain (VL). Important glycosylation sites (orange stars) are present in CH2, which are responsible for effector functions and the flexibility of the molecule. (B) The HCAb devoid of the light chain and CH1 contains the paratope (yellow box) present only in the single variable domain (VHH). (C) The VHH can be expressed as a prolate-shaped, soluble molecule of ~15 kDa. The yellow box shows the antigen binding site. (D) The VHH sequence is made of four Framework Regions (FR1, light gray; FR2, cyan; FR3, magenta and FR4, yellow), and three Complementary Determining Regions (CDR1, green; CDR2, blue and CDR3, red). Residues F37, E44, G47and R45 (orange) are located in the FR2 and mask a hydrophobic patch. C, C- terminal; N, N-terminal. The dotted red line represents a disulfide bond between the FR2 and the CDR3; this bond stabilizes the molecule and is present in dromedaries. (E) A three-dimensional structure of an anti lysozyme VHH, showing the Ig folding of β sheets, five strands in the front (roman numerals: I – V) and four strands in the back (VI – IX). The enlarged yellow box shows the antigen binding site, formed by juxtaposition of three CDRs. (F) The VHH shown in (F) is drawn in complex with lysozyme (light blue). A protruding paratope consisting mainly of CDR3 (red) recognizes and binds the catalytic cleft of lysozyme, inhibiting its activity.

are highly homologous, there is no CH1 domain in the camelid HCAbs. The single variable domain, called VHH, is the only domain of HCAbs that makes contact with the antigen. Surprisingly, although the VHH have only three CDR regions, their affinity for antigens reaches the low nanomolar to even picomolar range, matching the best affinities of classical antibodies. When expressed as single domains (often referred to as nanobodies, Nb), the VHHs retain their strong epitope specificity and affinity, a feature that might be explained by the VHH architecture (Fig. 1, B). Just like the VHs of conventional antibodies, the amino acid (AA) sequence of VHHs is organised in three hypervariable regions (CDR1, CDR2 and CDR3) separated by four Framework regions (FR1-FR4) (Muyldermans *et al.*, 1994). As the

Fig. 1. Representative diagrams of a conventional antibody, an HCAb, and a VHH. (A) A conventional IgG antibody is a dimeric molecule, and each monomer comprises a heavy chain and a light chain. The heavy chain consists of the constant domains (CH1, CH2 and CH3) and the variable domain (VH). The light chain has only one conserved domain (CL) and a variable domain (VL). Important glycosylation sites (orange stars) are present in CH2, which are responsible for effector functions and the flexibility of the molecule. (B) The HCAb devoid of the light chain and CH1 contains the paratope (yellow box) present only in the single variable domain (VHH). (C) The VHH can be expressed as a prolate-shaped, soluble molecule of ~15 kDa. The yellow box shows the antigen binding site. (D) The VHH sequence is made of four Framework Regions (FR1, light gray; FR2, cyan; FR3, magenta and FR4, yellow), and three Complementary Determining Regions (CDR1, green; CDR2, blue and CDR3, red). Residues F37, E44, G47and R45 (orange) are located in the FR2 and mask a hydrophobic patch. C, C- terminal; N, N-terminal. The dotted red line represents a disulfide bond between the FR2 and the CDR3; this bond stabilizes the molecule and is present in dromedaries. (E) A three-dimensional structure of an anti lysozyme VHH, showing the Ig folding of β sheets, five strands in the front (roman numerals: I – V) and four strands in the

back (VI – IX). The enlarged yellow box shows the antigen binding site, formed by

the catalytic cleft of lysozyme, inhibiting its activity.

juxtaposition of three CDRs. (F) The VHH shown in (F) is drawn in complex with lysozyme (light blue). A protruding paratope consisting mainly of CDR3 (red) recognizes and binds

AA sequence of the VHH FRs is highly similar to those of conventional VHs it was not surprising that the overall architecture of VHHs closely resembles that of VHs (Muyldermans *et al.*, 1994). Both VHH and VH domains fold into two β-sheets with the three CDRs that link these two sheets at one end of the barrel (or domain) (De Genst *et al.*, 2006; Desmyter *et al.*, 1996) (Fig. 1, C,E). However, there are striking structural differences between VHHs and conventional VH. Evidently, VHHs lack an interacting VL domain. Because of this, the hydrophobic amino acids present at the VH surface that is normally interacting with the VL, are substituted by hydrophilic AA (Fig.1, D). This enhances the solubility of VHH single domain proteins compared to engineered VH single domain proteins.

The absence of the additional CDRs in VHHs is likely compensated by structural features. First, the CDR3 regions of camelid VHHs are generally longer (13-17 amino acids) than the CDR3 regions of mouse and human VHs (9-12 and 9-17 AA respectively) (Wu *et al.*, 1993). In contrast to conventional Abs, in which the antigen binding surface is often a flat surface, a cavity or a groove, the long CDR3 loop may extend from the antigen binding surface (Desmyter *et al.*, 1996). This enlarges the paratope surface and hence the potential affinity and repertoire of camelid HCAbs. In addition, especially in dromedaries, the CDR1 and CDR3 regions contain a cysteine, which allows formation of a second disulfide bridge next to the single disulfide bridge in conventional VHs (Muyldermans *et al.*, 1994). This extra bridge likely stabilizes the CDR loops, thereby reducing their flexibility. This probably also contributes to the affinity (less entropy is lost upon antigen binding) and structural diversity of VHHs. Long extending CDR3 loops that are stabilized by an extra disulfide bridge can explain the tendency of VHHs to bind to clefts and concaves surfaces more readily than conventional antibodies do (Fig. 1, F) (De Genst *et al.*, 2006). Indeed comparison of multiple structures of hen egg white lysozyme interacting with either several conventional human antibodies or several camelid VHHs clearly illustrated that VHHs tends to bind to the concave substrate-binding pocket, whereas conventional antibodies favor epitopes on the "flat" surface of the antigen (Fig. 1, C). In addition, whereas each of the three CDRs of conventional VHs contributes considerably to the interaction with antigen, VHHs depended mainly on the CDR 3 loop for this interaction. Other antigens that are hard to target by conventional antibodies, but can be targeted by camelid VHHs are ion channels, GPCRs, haptens and enzymatic sites (Lauwereys *et al.*, 1998; Rasmussen *et al.*, 2011).

Next to an extended CDR3, the AA sequence of the H1 loop that precedes and comprises CDR1 appears to be particularly more variable in camelid VHHs than in conventional VHs. This might be interpreted as an extension of the VHH CDR1 (Vu *et al.*, 1997). Associated with this high variability in camelid VHHs, CH1 loops adopt conformations that deviate from the canonical H1 structures of conventional VHs (Barre *et al.*, 1994; Decanniere *et al.*, 1999; Decanniere *et al.*, 2000). Camelid VHH CH1 loops appear to fold into a more diverse repertoire of structures. The high variability in the AA sequence and conformations of the CH1 loop contribute to the VHH paratope size (850-1150 Å2), which approaches that of conventional antibodies (VH + VL) (Desmyter *et al.*, 2002). Clearly, different biochemical and structural features of camelid VHHs compensate for the lack of a VL domain, thus allowing a broad repertoire of specific high affinity antigen interactions. In addition, due to their small size and typical extruding CDR3 regions, camelid VHH tend to bind in cavities that are not readily accessible for conventional antibodies. Next to these particular features,

Single Domain Camelid Antibodies that Neutralize Negative Strand Viruses 155

(resulting mainly from the H274Y mutation, Wang *et al*., 2002) is evidence of the urgent need for new anti-influenza drugs. It is also urgent to develop new and better antiviral tools against the zoonotic influenza virus, including HPAV. The characteristics of Nbs mentioned above makes them a potentially effective antiviral approach. Several attempts have been made to target conserved epitopes of proteins in the surface proteins of influenza viruses. The main antigenic target in influenza virus is the HA protein. However, the genetic shift of this viral protein, especially in its antigenic regions, complicates this approach. Even though this strategy has been successful in current seasonal vaccines, it is costly and far from optimal: it is not suitable for emerging pandemic viruses, as has been proven not suitable as

The work of Hultberg and colleagues (Hultberg *et al.*, 2011) is the first report of the use of Nb technology as an antiviral tool against influenza. That study proved the binding of Nbs to an influenza protein and the neutralization of the binding of the virion to its cellular receptor in mammalian cells. These results are the proof of principle of the use of Nbs as antivirals. We discuss the most relevant results in scope of the potential further use of Nbs. To obtain Nbs directed against H5N1 viruses, llamas were immunized with recombinant H5N1 HA (H5, A/Vietnam/1203/04). The nanobody repertoire of the hyperimmune animals was cloned into a phage display library, and two promising HA-binding VHHs were isolated. The VHH of the HCab or Nb was cloned, produced as monovalent molecules, purified and screened for specific binding to the antigen, using as competitor the HA surrogate receptor fetuin. Two of the specific binders (B12 and C8) had high affinity to HA (KD = 9.91 and 30.1 nM) as determined by surface plasmon resonance. In addition, in a MLV (H5) pseudotyped neutralization assay both Nbs neutralized the parental virus A/Vietnam/1203/04 and also another clade 1 virus (A/Vietnam/1194/04) with a minimal inhibition concentration (IC50) of 75 nM. The possibility of cross reactivity among different H5N1 clades was also tested. The Nbs efficiency in neutralizing other clades of influenza virus decreased proportionally with the antigenic distance from the virus A/Vietnam/1203/04. Three viruses from clade 2.2 were inhibited by the monovalent Nbs in a similar range as clade 1 (IC50 = 50–150 nM). On the other hand, one virus of clade 2.3.4 and one virus from clade 2.5 showed little or no inhibition. As mentioned above, Nbs are potentially good building blocks for multivalent molecules due their small size, high affinity, and efficacy as a production platform. Bivalent and trivalent constructs were made, based on Nb C-8, using Gly4/Ser linkers (GS) of different lengths. The neutralization potential of the bivalent and trivalent constructs was greatly enhanced against the A/Vietnam/1194/04 virus (IC50 ≤ 1 pM). Inhibition of this clade 1 virus was confirmed by a micronetralization assay in NIBRG-14 infected cells. NIBRG-14 is an engineered recombinant virus whose HA and NA are derived from the A/Vietnam/1194/04 virus. Surprisingly, in the bivalent and trivalent Nbs the IC50 neutralization activity (9 and 3 pM, respectively) decreased by more than 3 logs, compared to the monovalent Nb. These results show that the multimeric molecules outperformed a previously developed monoclonal antibody CR 261, against NIBRG-14 (Throsby *et al.*, 2008). These results were also confirmed in a hemagglutination inhibition assay, which showed an IC50 of 2 nM for the bivalent and trivalent construction, compared

an immediately available vaccine against the Mexican flu in 2009.

**2.1 Targeting influenza HA: the Nb approach** 

to 156 nm of the monovalent.

VHH single domain protein is exceptionally stable and soluble, even under stringent conditions. As VHH are small and naturally monomeric, they can be easily formatted. In addition, the small size of VHHs allows them penetrate deeper into tissue (e.g. tumor tissue) and to occasionally cross the blood-brain barrier. On the down side, the small size of single domain VHH contributes to their rapid clearance from circulation.

Using display technologies, it is possible to select VHHs from large, synthetic or naive libraries (Verheesen *et al.*, 2006). The phage display generated from an immune VHH repertoire is the most widely and powerful technique used nowadays to rapidly select VHHs with the desired specificity (Arbabi Ghahroudi *et al.*, 1997). VHH are easily produced in bacterial or yeast systems in miligram quantities per liter of culture. Their stability, solubility, ease of production and small size make them excellent candidates for multivalent formatting. Tailor-made constructions using VHHs as building blocks enhance the avidity of the molecule even in a 3 log scale, and several constructions are being tested in clinical trials (Els Conrath *et al.*, 2001; Hmila *et al.*, 2010). Their high potential as therapeutics has prompted the creation in Belgium of the company Ablynx in 2001. Because of the publicity surrounding nanotechnology and the small size of the VHH, Ablynx named the VHH as "Nanobody (Nb)", and retains full intellectual property rights of the use of Nbs in therapeutics and diagnosis. The combined features of VHHs makes them ideal tools for many applications. In this chapter, we focus on the development and use of VHHs for antiviral therapy. It is interesting to point out that only one monoclonal antibody is used today (Synagis) as a therapeutic against infectious disease (Groothuis & Simoes, 1993).

#### **2. Influenza virus**

The main prophylactic measure against influenza is vaccination. Therapeutic options for influenza are small molecule drugs targeting the viral proteins Neuraminidase (NA) or matrix protein 2 (M2). Influenza virus poses a great and continuous threat to humans and zoonotic infections also pose a dangerous challenge to human. In the last decade, two important viruses have emerged as pandemic or potentially pandemic outbreaks: the recent pandemic outbreak in the 2009 by the swine-derived H1N1 influenza virus (also called the Mexican Flu) and Highly Pathogenic Avian influenza (HPAV) viruses of the H5N1 subtype, mainly in Asiatic countries. The 2009 H1N1 pandemic presents an interesting case. It was a zoonotic infection that could be transmitted between humans, but had a low mortality rate. On the other hand, the HPAV H5N1 virus infections present a high replication efficiency, broader cell tropism and possible systemic spread in patients. Fulminant pneumonia, multiorgan failure caused by a high viral load and an intense inflammatory response (cytokine storm) are responsible of a mortality rate of 60 % (de Jong *et al.*, 2006). Vaccines to prevent HPAV infection are not available, but NA inhibitors (osetalmivir) are used as antiviral drugs. A combination of antiviral drugs and immunomodulators was used to control infection by HPAV H5N1 in patients, but its use was considered as a risk. On the other hand, passive immunization has been a successful alternative. Immunoglobulins in immune sera derived from animals or humans exposed to a homologous virus had been used to treat HPAV-infected humans (Luke & Subbarao, 2006; Zhou *et al.*, 2007). The genetic shift and drift of the influenza virus underline the need for new antiviral approaches. In addition, the emergence of drug resistant strains poses an extra concern. The Tamiflu Resistant strain (resulting mainly from the H274Y mutation, Wang *et al*., 2002) is evidence of the urgent need for new anti-influenza drugs. It is also urgent to develop new and better antiviral tools against the zoonotic influenza virus, including HPAV. The characteristics of Nbs mentioned above makes them a potentially effective antiviral approach. Several attempts have been made to target conserved epitopes of proteins in the surface proteins of influenza viruses. The main antigenic target in influenza virus is the HA protein. However, the genetic shift of this viral protein, especially in its antigenic regions, complicates this approach. Even though this strategy has been successful in current seasonal vaccines, it is costly and far from optimal: it is not suitable for emerging pandemic viruses, as has been proven not suitable as an immediately available vaccine against the Mexican flu in 2009.

#### **2.1 Targeting influenza HA: the Nb approach**

154 Antiviral Drugs – Aspects of Clinical Use and Recent Advances

VHH single domain protein is exceptionally stable and soluble, even under stringent conditions. As VHH are small and naturally monomeric, they can be easily formatted. In addition, the small size of VHHs allows them penetrate deeper into tissue (e.g. tumor tissue) and to occasionally cross the blood-brain barrier. On the down side, the small size of single

Using display technologies, it is possible to select VHHs from large, synthetic or naive libraries (Verheesen *et al.*, 2006). The phage display generated from an immune VHH repertoire is the most widely and powerful technique used nowadays to rapidly select VHHs with the desired specificity (Arbabi Ghahroudi *et al.*, 1997). VHH are easily produced in bacterial or yeast systems in miligram quantities per liter of culture. Their stability, solubility, ease of production and small size make them excellent candidates for multivalent formatting. Tailor-made constructions using VHHs as building blocks enhance the avidity of the molecule even in a 3 log scale, and several constructions are being tested in clinical trials (Els Conrath *et al.*, 2001; Hmila *et al.*, 2010). Their high potential as therapeutics has prompted the creation in Belgium of the company Ablynx in 2001. Because of the publicity surrounding nanotechnology and the small size of the VHH, Ablynx named the VHH as "Nanobody (Nb)", and retains full intellectual property rights of the use of Nbs in therapeutics and diagnosis. The combined features of VHHs makes them ideal tools for many applications. In this chapter, we focus on the development and use of VHHs for antiviral therapy. It is interesting to point out that only one monoclonal antibody is used today

(Synagis) as a therapeutic against infectious disease (Groothuis & Simoes, 1993).

The main prophylactic measure against influenza is vaccination. Therapeutic options for influenza are small molecule drugs targeting the viral proteins Neuraminidase (NA) or matrix protein 2 (M2). Influenza virus poses a great and continuous threat to humans and zoonotic infections also pose a dangerous challenge to human. In the last decade, two important viruses have emerged as pandemic or potentially pandemic outbreaks: the recent pandemic outbreak in the 2009 by the swine-derived H1N1 influenza virus (also called the Mexican Flu) and Highly Pathogenic Avian influenza (HPAV) viruses of the H5N1 subtype, mainly in Asiatic countries. The 2009 H1N1 pandemic presents an interesting case. It was a zoonotic infection that could be transmitted between humans, but had a low mortality rate. On the other hand, the HPAV H5N1 virus infections present a high replication efficiency, broader cell tropism and possible systemic spread in patients. Fulminant pneumonia, multiorgan failure caused by a high viral load and an intense inflammatory response (cytokine storm) are responsible of a mortality rate of 60 % (de Jong *et al.*, 2006). Vaccines to prevent HPAV infection are not available, but NA inhibitors (osetalmivir) are used as antiviral drugs. A combination of antiviral drugs and immunomodulators was used to control infection by HPAV H5N1 in patients, but its use was considered as a risk. On the other hand, passive immunization has been a successful alternative. Immunoglobulins in immune sera derived from animals or humans exposed to a homologous virus had been used to treat HPAV-infected humans (Luke & Subbarao, 2006; Zhou *et al.*, 2007). The genetic shift and drift of the influenza virus underline the need for new antiviral approaches. In addition, the emergence of drug resistant strains poses an extra concern. The Tamiflu Resistant strain

**2. Influenza virus** 

domain VHH contributes to their rapid clearance from circulation.

The work of Hultberg and colleagues (Hultberg *et al.*, 2011) is the first report of the use of Nb technology as an antiviral tool against influenza. That study proved the binding of Nbs to an influenza protein and the neutralization of the binding of the virion to its cellular receptor in mammalian cells. These results are the proof of principle of the use of Nbs as antivirals. We discuss the most relevant results in scope of the potential further use of Nbs. To obtain Nbs directed against H5N1 viruses, llamas were immunized with recombinant H5N1 HA (H5, A/Vietnam/1203/04). The nanobody repertoire of the hyperimmune animals was cloned into a phage display library, and two promising HA-binding VHHs were isolated. The VHH of the HCab or Nb was cloned, produced as monovalent molecules, purified and screened for specific binding to the antigen, using as competitor the HA surrogate receptor fetuin. Two of the specific binders (B12 and C8) had high affinity to HA (KD = 9.91 and 30.1 nM) as determined by surface plasmon resonance. In addition, in a MLV (H5) pseudotyped neutralization assay both Nbs neutralized the parental virus A/Vietnam/1203/04 and also another clade 1 virus (A/Vietnam/1194/04) with a minimal inhibition concentration (IC50) of 75 nM. The possibility of cross reactivity among different H5N1 clades was also tested. The Nbs efficiency in neutralizing other clades of influenza virus decreased proportionally with the antigenic distance from the virus A/Vietnam/1203/04. Three viruses from clade 2.2 were inhibited by the monovalent Nbs in a similar range as clade 1 (IC50 = 50–150 nM). On the other hand, one virus of clade 2.3.4 and one virus from clade 2.5 showed little or no inhibition. As mentioned above, Nbs are potentially good building blocks for multivalent molecules due their small size, high affinity, and efficacy as a production platform. Bivalent and trivalent constructs were made, based on Nb C-8, using Gly4/Ser linkers (GS) of different lengths. The neutralization potential of the bivalent and trivalent constructs was greatly enhanced against the A/Vietnam/1194/04 virus (IC50 ≤ 1 pM). Inhibition of this clade 1 virus was confirmed by a micronetralization assay in NIBRG-14 infected cells. NIBRG-14 is an engineered recombinant virus whose HA and NA are derived from the A/Vietnam/1194/04 virus. Surprisingly, in the bivalent and trivalent Nbs the IC50 neutralization activity (9 and 3 pM, respectively) decreased by more than 3 logs, compared to the monovalent Nb. These results show that the multimeric molecules outperformed a previously developed monoclonal antibody CR 261, against NIBRG-14 (Throsby *et al.*, 2008). These results were also confirmed in a hemagglutination inhibition assay, which showed an IC50 of 2 nM for the bivalent and trivalent construction, compared to 156 nm of the monovalent.

Single Domain Camelid Antibodies that Neutralize Negative Strand Viruses 157

addition, 48 h after challenge of mice (treated with this dose of bivalent Nb) with 4 LD50 of NIBRG-14 ma, weight loss was observed and also a delay in mortality compared with the

The antigenic site of the HA was mapped by selecting escape mutants in the presence of the monovalent or bivalent Nb. Three escape mutants were selected in the presence of monovalent Nb, K189E/N and N154D/S mutations were found, they are contiguous in the antigenic B site of the HA (Wiley *et al.*, 1981; Yamada *et al.*, 2006). It is noteworthy to mention that N154D/S removes an N-glycosylation site, a possible adaptation to mask an antigenic site (Fig. 2). The escape mutants selected in presence of the bivalent Nb presented not only the K189E/N mutation, but an additional D145N mutation located in the stalk of HA2, 40 residues upstream of the membrane anchor. The results of the hemaglutination assays and microneutralization experiments suggest that mutation K189N/E is necessary and sufficient to abolish binding to the Nb in a monovalent or bivalent conformation, indicating a close proximity between the antigenic B site and the receptor binding domain. Those results are the first one reported of the potential antiviral activity of a Nb against the influenza virus.

Fig. 2. Ribbon representation of the H5N1 HA trimeric protein. Two mutations in the head of the trimer confer resistance to the monovalent and bivalent VHH C-8. The mutation K189N/E was necessary and sufficient to prevent binding of both mono and bivalent VHHs.

The Nb viral neutralization activity against a trimeric HA molecule (HA) was greatly enhanced when presented as bivalent and trimeric molecule, but the dynamics and details

controls.

(PDB : 2IBX)

The multivalency format also resulted in the potential for neutralization of influenza virus of different clades. For three clade 2.2 viruses, two bivalent constructions of the Nb C-8 (9 GS and 15 GS) did not show any decrease in the IC50. On the other hand, the 10 GS linker trivalent molecule showed a 10 to 40-fold increase in the neutralization potential, but the 20 GS linker trivalent showed only two-fold decrease in the IC50, or none at all. Nevertheless, using the monovalent Nb the neutralization of virus from clades 2.3.4 or 2.5 was in the high nM range or absent, respectively. This result confirms the previous result showing that both bivalent and one trivalent (10GS) constructions decrease the IC50 to a low nM range. It is worth mentioning that the retroviral pseudovirus A/Vietnam/1194/04 and the influenza virus NIBRG-14 share the same HA, but different results were obtained using the MLV pseudotyped neutralization assay and the infected cells microneutralization. Using microneutralization, the reported IC50 of the monovalent, bivalent and trivalent molecules was reduced ten-fold as compared to the IC50 obtained by the pseudotyped neutralization. The difference in sensitivity of the assays emphasizes the need to confirm the neutralization results of the different influenza clades in infected cells based assays. The validation of the anti HA in an *in vivo* model was performed in a mouse model by our group (Ibañez *et al.*, 2011).

To confirm the *in vivo* efficacy of the Nbs, Ibañez and colleagues used an H5N1 NIBRG-14 mouse adapted virus strain (NIBRG-14 ma). It is important to point out that the Nbs were administered intranasally in all mouse experiments, in order to enhance penetration in the respiratory tract. Initially, to evaluate the antiviral potential using the bivalent Nb (C-8, 15 GS) *in vivo*, a dose of 5 mg/kg (100 µg) was used in mice. This dose completely prevented loss of body weight at 4, 24 and 48 h before a challenge with 1 LD50 of NIBRG-14 ma, compared to the controls after 4 days of monitoring. Using the same set up, on day 4 after challenge, no detectable lung virus titers were observed when mice had been treated at 4 and 24 hrs before challenge, and at 48 hrs the titer was 50-fold lower than in controls. These results suggested that the bivalent Nb provide strong protection against 1 LD50, but it is important to consider the half life of the molecule. In previous *in vitro* results, the bivalent Nb neutralization activity was even 3 logs higher than that of the monovalent Nb, but *in vivo* there was also a significant improvement using the bivalent. The difference in virus neutralization capacity between the monovalent and bivalent Nbs and the minimal protective dose was assessed by administration of Nbs at different doses at 24 h before challenge with 1 LD50 NIBRG-14. The doses of Nbs ranged from 3 to 0.025 mg/kg, and complete neutralization was confirmed for the highest doses of both constructs. In addition, administration of the highest dose (60 µg, 3 mg/kg) of bivalent Nb 24 h before challenge with 4 LD50 also resulted in complete protection. The monovalent neutralization activity was dependent on the amount of Nb, but it was also statistically significant for doses of 6 or 1.2 µg of Nb per mouse. Remarkably, very low or no lung virus titer was detected in mice treated with the bivalent Nb, even for the lowest doses used (2.5–0.5 µg). These results strongly confirmed the neutralization efficacy of the bivalent Nb when used as prophylactic tool against a NIBRG-14 ma, a highly pathogenic influenza virus model.

The therapeutic efficacy of the bivalent Nb was also tested in the same model. The administration of 60 µg of bivalent Nb prevented the drop in body weight and showed a reduction in the lung viral titers when administered 4, 24 and 48 h after 1 LD50 challenge. On the other hand, 72 h after challenge, the drop in body weight was similar to that of the controls, but statistically significant reduction in lung viral titers was observed. The decrease in viral titers was also confirmed by measuring the amount of viral RNA by RT-PCR. In

The multivalency format also resulted in the potential for neutralization of influenza virus of different clades. For three clade 2.2 viruses, two bivalent constructions of the Nb C-8 (9 GS and 15 GS) did not show any decrease in the IC50. On the other hand, the 10 GS linker trivalent molecule showed a 10 to 40-fold increase in the neutralization potential, but the 20 GS linker trivalent showed only two-fold decrease in the IC50, or none at all. Nevertheless, using the monovalent Nb the neutralization of virus from clades 2.3.4 or 2.5 was in the high nM range or absent, respectively. This result confirms the previous result showing that both bivalent and one trivalent (10GS) constructions decrease the IC50 to a low nM range. It is worth mentioning that the retroviral pseudovirus A/Vietnam/1194/04 and the influenza virus NIBRG-14 share the same HA, but different results were obtained using the MLV pseudotyped neutralization assay and the infected cells microneutralization. Using microneutralization, the reported IC50 of the monovalent, bivalent and trivalent molecules was reduced ten-fold as compared to the IC50 obtained by the pseudotyped neutralization. The difference in sensitivity of the assays emphasizes the need to confirm the neutralization results of the different influenza clades in infected cells based assays. The validation of the anti HA in

an *in vivo* model was performed in a mouse model by our group (Ibañez *et al.*, 2011).

tool against a NIBRG-14 ma, a highly pathogenic influenza virus model.

The therapeutic efficacy of the bivalent Nb was also tested in the same model. The administration of 60 µg of bivalent Nb prevented the drop in body weight and showed a reduction in the lung viral titers when administered 4, 24 and 48 h after 1 LD50 challenge. On the other hand, 72 h after challenge, the drop in body weight was similar to that of the controls, but statistically significant reduction in lung viral titers was observed. The decrease in viral titers was also confirmed by measuring the amount of viral RNA by RT-PCR. In

To confirm the *in vivo* efficacy of the Nbs, Ibañez and colleagues used an H5N1 NIBRG-14 mouse adapted virus strain (NIBRG-14 ma). It is important to point out that the Nbs were administered intranasally in all mouse experiments, in order to enhance penetration in the respiratory tract. Initially, to evaluate the antiviral potential using the bivalent Nb (C-8, 15 GS) *in vivo*, a dose of 5 mg/kg (100 µg) was used in mice. This dose completely prevented loss of body weight at 4, 24 and 48 h before a challenge with 1 LD50 of NIBRG-14 ma, compared to the controls after 4 days of monitoring. Using the same set up, on day 4 after challenge, no detectable lung virus titers were observed when mice had been treated at 4 and 24 hrs before challenge, and at 48 hrs the titer was 50-fold lower than in controls. These results suggested that the bivalent Nb provide strong protection against 1 LD50, but it is important to consider the half life of the molecule. In previous *in vitro* results, the bivalent Nb neutralization activity was even 3 logs higher than that of the monovalent Nb, but *in vivo* there was also a significant improvement using the bivalent. The difference in virus neutralization capacity between the monovalent and bivalent Nbs and the minimal protective dose was assessed by administration of Nbs at different doses at 24 h before challenge with 1 LD50 NIBRG-14. The doses of Nbs ranged from 3 to 0.025 mg/kg, and complete neutralization was confirmed for the highest doses of both constructs. In addition, administration of the highest dose (60 µg, 3 mg/kg) of bivalent Nb 24 h before challenge with 4 LD50 also resulted in complete protection. The monovalent neutralization activity was dependent on the amount of Nb, but it was also statistically significant for doses of 6 or 1.2 µg of Nb per mouse. Remarkably, very low or no lung virus titer was detected in mice treated with the bivalent Nb, even for the lowest doses used (2.5–0.5 µg). These results strongly confirmed the neutralization efficacy of the bivalent Nb when used as prophylactic addition, 48 h after challenge of mice (treated with this dose of bivalent Nb) with 4 LD50 of NIBRG-14 ma, weight loss was observed and also a delay in mortality compared with the controls.

The antigenic site of the HA was mapped by selecting escape mutants in the presence of the monovalent or bivalent Nb. Three escape mutants were selected in the presence of monovalent Nb, K189E/N and N154D/S mutations were found, they are contiguous in the antigenic B site of the HA (Wiley *et al.*, 1981; Yamada *et al.*, 2006). It is noteworthy to mention that N154D/S removes an N-glycosylation site, a possible adaptation to mask an antigenic site (Fig. 2). The escape mutants selected in presence of the bivalent Nb presented not only the K189E/N mutation, but an additional D145N mutation located in the stalk of HA2, 40 residues upstream of the membrane anchor. The results of the hemaglutination assays and microneutralization experiments suggest that mutation K189N/E is necessary and sufficient to abolish binding to the Nb in a monovalent or bivalent conformation, indicating a close proximity between the antigenic B site and the receptor binding domain. Those results are the first one reported of the potential antiviral activity of a Nb against the influenza virus.

Fig. 2. Ribbon representation of the H5N1 HA trimeric protein. Two mutations in the head of the trimer confer resistance to the monovalent and bivalent VHH C-8. The mutation K189N/E was necessary and sufficient to prevent binding of both mono and bivalent VHHs. (PDB : 2IBX)

The Nb viral neutralization activity against a trimeric HA molecule (HA) was greatly enhanced when presented as bivalent and trimeric molecule, but the dynamics and details

Single Domain Camelid Antibodies that Neutralize Negative Strand Viruses 159

subtype virus *in vitro*. On the contrary, another VHH (RSV-E4) could neutralize RSV B

The epitopes of different VHHs were determined by antibody competition assays and diverse antibody escape RSV mutants. Whereas RSV-C4 and RSV-D3 VHHs readily competed with palivizumab for binding to recombinant RSV FTM- or inactivated RSV virions, RSV-E4 competed with 101 Fab, which is known to bind to the antigenic region IV-VI (Wu *et al.*, 2007). These data are in line with the observation that AA substitutions within antigenic regions II and IV-VI, respectively, affected the binding of both RSV-D4 and RSV-C3 VHHs and RSV-E4 VHH. These data strongly suggest that both RSV-C3 and RSV-D4 bind to antigenic region II (palivizumab epitope) (Crowe *et al.*, 1998) whereas RSV-E4 VHH binds to antigenic regions IV-VI, explaining the observed differences in neutralization.

The affinity of the three VHHs, Synagis Mab and Synagis Fab was determined by Surface Plasmon Resonance using recombinant RSV F TM- as bait. The KD of RSV-D3, RSV-E4 and RSV-E4 were in the low nanomolar range: 9.24 nM, 1.78 nM and 0.45 nM, respectively. Although RSV-D3 was more effective than RSV-C4 at neutralizing RSV A, it had a lower affinity for FTM- than RSV-C4. However, the efficient binding of RSV-E4 VHH to a neutralizing epitope (antigenic region IV-VI) was not associated with neutralization of RSV A. This suggests that the affinity of VHHs for the recombinant RSV FTM-, which likely represents the F protein in its post-fusion conformation, does not correlate directly with

\*Obtained from two different cell based assays, microneutralization and plaque assay

Table 1. Inhibition and protection of the RSV virus A binding by Nb RSV-D3. ND = not

The avidity of a binding molecule can be increased by using a multivalent format (Rudge *et al.*, 2007; Wang & Yang, 2010). To increase the antiviral potential of RSV-D3 we formatted it into a bivalent molecule, by using a flexible linker, Gly4/Ser (GS). Surprisingly, bivalent RSV-D3 VHHs with GC linkers of different sizes neutralized RSV A Long virus between 2421 and 4181 times more efficient than monovalent RSV-D3 VHHs, reaching picomolar

infection to some extent.

neutralization of living RSV (Table 1.)

determined.

of the binding are not clear. It has been demonstrated that during intramolecular binding, a multivalent molecule has greater avidity than its monovalent counterpart. But a very interesting question is whether intermolecular binding occurs during Nb binding to the HA. In recent reports, the existence of intermolecular binding was proved to enhance an antiviral effect (Wang & Yang, 2010). Intermolecular binding could explain the increase in the neutralization activity: sterically, the hindrance of the HA for its cellular receptor is enhanced, and the flexibility of the HA is decreased.

#### **3. RSV virus**

Respiratory Syncytial Virus (RSV) infections are the leading cause of acute lower respiratory tract infections (ALRI) in children and associated hospitalizations world wide (Falsey *et al.*, 2005; Nair *et al.*, 2010). There is no specific antiviral therapy for RSV infection available. Each year 66, 000 – 199,000 children die worldwide due to RSV ALRI. Most pediatric cases of fatal RSV infections occur in developing countries. As RSV infections do not evoke protective immunity, infections occur throughout life, causing severe morbidity in young infants, the elderly, and immune-comprised adults (Boyce *et al.*, 2000; Falsey *et al.*, 2005).

Although high levels of RSV neutralizing antibodies correlate with lower frequencies of RSV-associated ALRI, no RSV vaccine is available (Glezen *et al.*, 1981). However, monthly administration of large amounts of a humanized RSV neutralizing antibody, palivizumab (Synagis), reduces RSV-associated hospitalization of high risk infants by about 78-39% (Groothuis & Simoes, 1993). Palivizumab is currently the sole monoclonal antibody that is approved for preventing viral infection. Palivizumab blocks fusion of the RSV membrane with the membrane of the target cell by binding to the RSV fusion protein (F) (Huang *et al.*, 2010). However, due to the high cost of palivizumab, there is an urgent need for new antivirals that can prevent or treat RSV infections. RSV neutralizing Nbs have been developed as an alternative to existing antibodies (Hultberg *et al.*, 2011).

#### **3.1 RSV binding VHHs antiviral effect: comparison with Synagis Mab.**

To investigate if Nbs could be used for antiviral therapy, Nbs that bind to the palivizumab epitope were developed. For this purpose, two llamas were immunized with recombinant RSV A F protein (RSV FTM-) lacking the transmembrane region (Hultberg *et al.*, 2011). This protein folds into trimers that resemble the native RSV F protein in its post-fusion conformation (Ruiz-Arguello *et al.*, 2004). Remarkably, RSV FTM- proteins can be readily recognized by RSV F neutralizing antibodies that, just like palivizumab, bind to the antigenic site II (McLellan *et al.*, 2011; Swanson *et al.*, 2011). In this way, RSV FTMimmunization can potentially induce RSV F antigenic site II specific camelid HCAbs. HCAbs that specifically bind to the RSV F antigenic region II were enriched by biopanning using RSV FTM- protein and competitive elution in the presence of excess of palivizumab antibody. From these HCAbs, VHHs (or Nbs) were produced and tested for binding to the RSV FTMprotein. Twelve VHHs that bound to the RSV F protein were tested for neutralization of RSV Long strain (RSV A subtype) virus in a micro-neutralization assay. Two VHHs (RSV-C4 and the RSV-D3) could neutralize RSV in the high nanomolar range (IC50: 640 nM and 300 nM, respectively), which is similar to the neutralization activity as the Synagis Fab (IC50: 549.2 nM) and about 100-fold less effective than the Synagis Mab (IC50: 3.02 nM). However, in contrast to palivizumab, neither RSV-C4 nor RSV-D3 VHHs could neutralize RSV B

of the binding are not clear. It has been demonstrated that during intramolecular binding, a multivalent molecule has greater avidity than its monovalent counterpart. But a very interesting question is whether intermolecular binding occurs during Nb binding to the HA. In recent reports, the existence of intermolecular binding was proved to enhance an antiviral effect (Wang & Yang, 2010). Intermolecular binding could explain the increase in the neutralization activity: sterically, the hindrance of the HA for its cellular receptor is

Respiratory Syncytial Virus (RSV) infections are the leading cause of acute lower respiratory tract infections (ALRI) in children and associated hospitalizations world wide (Falsey *et al.*, 2005; Nair *et al.*, 2010). There is no specific antiviral therapy for RSV infection available. Each year 66, 000 – 199,000 children die worldwide due to RSV ALRI. Most pediatric cases of fatal RSV infections occur in developing countries. As RSV infections do not evoke protective immunity, infections occur throughout life, causing severe morbidity in young infants, the

Although high levels of RSV neutralizing antibodies correlate with lower frequencies of RSV-associated ALRI, no RSV vaccine is available (Glezen *et al.*, 1981). However, monthly administration of large amounts of a humanized RSV neutralizing antibody, palivizumab (Synagis), reduces RSV-associated hospitalization of high risk infants by about 78-39% (Groothuis & Simoes, 1993). Palivizumab is currently the sole monoclonal antibody that is approved for preventing viral infection. Palivizumab blocks fusion of the RSV membrane with the membrane of the target cell by binding to the RSV fusion protein (F) (Huang *et al.*, 2010). However, due to the high cost of palivizumab, there is an urgent need for new antivirals that can prevent or treat RSV infections. RSV neutralizing Nbs have been developed

To investigate if Nbs could be used for antiviral therapy, Nbs that bind to the palivizumab epitope were developed. For this purpose, two llamas were immunized with recombinant RSV A F protein (RSV FTM-) lacking the transmembrane region (Hultberg *et al.*, 2011). This protein folds into trimers that resemble the native RSV F protein in its post-fusion conformation (Ruiz-Arguello *et al.*, 2004). Remarkably, RSV FTM- proteins can be readily recognized by RSV F neutralizing antibodies that, just like palivizumab, bind to the antigenic site II (McLellan *et al.*, 2011; Swanson *et al.*, 2011). In this way, RSV FTMimmunization can potentially induce RSV F antigenic site II specific camelid HCAbs. HCAbs that specifically bind to the RSV F antigenic region II were enriched by biopanning using RSV FTM- protein and competitive elution in the presence of excess of palivizumab antibody. From these HCAbs, VHHs (or Nbs) were produced and tested for binding to the RSV FTMprotein. Twelve VHHs that bound to the RSV F protein were tested for neutralization of RSV Long strain (RSV A subtype) virus in a micro-neutralization assay. Two VHHs (RSV-C4 and the RSV-D3) could neutralize RSV in the high nanomolar range (IC50: 640 nM and 300 nM, respectively), which is similar to the neutralization activity as the Synagis Fab (IC50: 549.2 nM) and about 100-fold less effective than the Synagis Mab (IC50: 3.02 nM). However, in contrast to palivizumab, neither RSV-C4 nor RSV-D3 VHHs could neutralize RSV B

elderly, and immune-comprised adults (Boyce *et al.*, 2000; Falsey *et al.*, 2005).

as an alternative to existing antibodies (Hultberg *et al.*, 2011).

**3.1 RSV binding VHHs antiviral effect: comparison with Synagis Mab.** 

enhanced, and the flexibility of the HA is decreased.

**3. RSV virus** 

subtype virus *in vitro*. On the contrary, another VHH (RSV-E4) could neutralize RSV B infection to some extent.

The epitopes of different VHHs were determined by antibody competition assays and diverse antibody escape RSV mutants. Whereas RSV-C4 and RSV-D3 VHHs readily competed with palivizumab for binding to recombinant RSV FTM- or inactivated RSV virions, RSV-E4 competed with 101 Fab, which is known to bind to the antigenic region IV-VI (Wu *et al.*, 2007). These data are in line with the observation that AA substitutions within antigenic regions II and IV-VI, respectively, affected the binding of both RSV-D4 and RSV-C3 VHHs and RSV-E4 VHH. These data strongly suggest that both RSV-C3 and RSV-D4 bind to antigenic region II (palivizumab epitope) (Crowe *et al.*, 1998) whereas RSV-E4 VHH binds to antigenic regions IV-VI, explaining the observed differences in neutralization.

The affinity of the three VHHs, Synagis Mab and Synagis Fab was determined by Surface Plasmon Resonance using recombinant RSV F TM- as bait. The KD of RSV-D3, RSV-E4 and RSV-E4 were in the low nanomolar range: 9.24 nM, 1.78 nM and 0.45 nM, respectively. Although RSV-D3 was more effective than RSV-C4 at neutralizing RSV A, it had a lower affinity for FTM- than RSV-C4. However, the efficient binding of RSV-E4 VHH to a neutralizing epitope (antigenic region IV-VI) was not associated with neutralization of RSV A. This suggests that the affinity of VHHs for the recombinant RSV FTM-, which likely represents the F protein in its post-fusion conformation, does not correlate directly with neutralization of living RSV (Table 1.)


\*Obtained from two different cell based assays, microneutralization and plaque assay

Table 1. Inhibition and protection of the RSV virus A binding by Nb RSV-D3. ND = not determined.

The avidity of a binding molecule can be increased by using a multivalent format (Rudge *et al.*, 2007; Wang & Yang, 2010). To increase the antiviral potential of RSV-D3 we formatted it into a bivalent molecule, by using a flexible linker, Gly4/Ser (GS). Surprisingly, bivalent RSV-D3 VHHs with GC linkers of different sizes neutralized RSV A Long virus between 2421 and 4181 times more efficient than monovalent RSV-D3 VHHs, reaching picomolar

Single Domain Camelid Antibodies that Neutralize Negative Strand Viruses 161

reduced RSV replication *in vivo*. The potential of bivalent VHHs for preventing morbidity and pulmonary inflammation upon RSV infection was assessed in a non immunocompromised mouse model. Prophylactic administration of bivalent RSV-D3 VHHs (1 mg/kg) completely prevented body weight loss and pulmonary cell infiltration that was observed in mice treated with control VHHs. Therapeutic treatment with bivalent RSV-D3 VHHs 24 h after infection partially reduced body weight loss and pulmonary cell infiltration. These observations confirm

Fig. 3. Ribbon representation of the structure of the RSV F protein trimer in its post-fusion form. The head and stalk of this recombinant protein are depicted, lacking the fusion peptide, transmembrane region and cytoplasmic domain. The immunogenic epitopes recognized by Mab 101F (site II) and palimuzab (site IV-VI) are in red and blue, respectively, and the rest of the F protein is in green. The RSV-D3 and RSV-C4 resistant *in vitro* escape mutants are shown in yellow. Mutations I432T, K433T and S436F in site II disrupt the binding of the RSV-C4. The K262Y, N268I and K272E in the site IV-IV result in loss of

Currently Ablynx is preparing a phase I clinical trial to evaluate the safety of a trivalent RSV neutralizing VHH format consisting of three identical VHHs. Preclinical evaluation of this lead candidate can readily neutralize a broad spectrum of clinical RSV A and RSV B subtype

the *in vivo* antiviral potential of neutralizing VHHs.

binding of the RSV-D3 molecule. (PDB: 3RKI).

range (IC50: 190-110 pM). In contrast, Synagis Mab was only 200 times more efficient in neutralizing RSV A virus (IC50: 6.5 nM) than its corresponding Fab fragment. In this way, bivalent RSV F specific VHHs outperform the Synagis antibody in RSV neutralization. Moreover, in contrast to its monovalent format, bivalent RSV-D3 could also neutralize RSV B1 strain virus. Also, neutralization was notably boosted against RSV A and B virus subtypes by linking two different VHHs (RSV-D3 and RSV-E4) which target different epitopes. The enhancement of the activity by linking two VHHs is likely due to the flexibility of the linker. Experiments aiming to characterize the binding dynamics of the RSV-D3 to the F protein are necessary for characterizing intra- or intermolecular binding.

The RSV F is responsible for fusion of the viral lipid membrane with the host membrane, but also participates in attachment of the RSV virions to target cells. In addition, it was recently demonstrated that RSV F protein can bind to nucleolin expressed at the surface of target cells, and that this interaction is crucial for RSV infection *in vitro* and *in vivo* (Tayyari *et al.*, 2011). After viral attachment, the RSV F protein mediates fusion of the viral membrane with the plasma membrane of the target cell, thereby releasing the viral genome into the cytoplasm of the host cell. This process involves a series of conformational changes in the F protein from a metastable pre-fusion to a stable post-fusion conformation. We recently demonstrated that bivalent RSV-D3 VHHs can prevent RSV infection both before and after viral attachment and can inhibit syncytia formation, but cannot hamper RSV attachment (Schepens *et al.*, 2011). Together, these observations constantly indicate that, by a similar mechanism as palivizumab, bivalent RSV-D3 VHHs prevent RSV infection by blocking fusion. Although the conformations of the RSV F antigenic regions II and IV-VI are maintained in the post-fusion form, it is more plausible that the RSV VHHs block viral fusion and syncytia formation by binding to the RSV F protein in either its pre-fusion or intermediate conformations (Fig. 3). Possibly, binding of the VHHs to the antigenic region II interferes with the conformational changes of the F protein that are required for fusion.

Immune compromised Balb/c mice (cyclophosphamide treatment) were used to test whether bivalent RSV-D3 VHHs can protect against RSV infection *in vivo* (Schepens *et al.*, 2011). As VHHs are known to remain active in the respiratory tract after nebulisation, bivalent RSV-D3 and control VHHs were administered intranasally (patent application WO 2009/147248). Prophylactic treatment of mice with 5 mg/kg of bivalent RSV-D3 VHH or palivizumab reduced RSV pulmonary titers below the detection limit of the RSV plaque assay. This strong reduction was confirmed by qPCR analysis. Remarkably, as low as 0.6 mg/kg bivalent RSV-D3 could prevent or strongly reduce (at least 100-fold) pulmonary RSV replication. In comparison, monovalent RSV-D3 VHH protected against pulmonary RSV replication about 25 times less efficiently than its bivalent counterpart. For prophylactic treatment to be valuable, even if is easy to administer, its effect should be long lasting. We demonstrate that intranasal administration of bivalent RSV-D3 VHHs can protect against RSV infection for at least 48 hours. Prophylactic treatment with palivizumab in high risk infants reduces RSV associated hospitalization, but no effective therapeutic is available. Therefore, RSV-D3 VHHs were also evaluated as therapeutic treatments. Intranasal administration of RSV-D3 VHHs 4 or 24 hours after infection strongly reduced pulmonary RSV replication (at least 100-fold). Plaque assays also indicated that administration of bivalent RSV-D3 VHHs 72 hours after RSV treatment can reduce pulmonary RSV replication. However, the lung homogenates used to quantify the pulmonary RSV titer in mice that were treated 72 hours after infection still contained neutralizing RSV VHHs. Therefore, it is not clear to which extent treatment at this time point

range (IC50: 190-110 pM). In contrast, Synagis Mab was only 200 times more efficient in neutralizing RSV A virus (IC50: 6.5 nM) than its corresponding Fab fragment. In this way, bivalent RSV F specific VHHs outperform the Synagis antibody in RSV neutralization. Moreover, in contrast to its monovalent format, bivalent RSV-D3 could also neutralize RSV B1 strain virus. Also, neutralization was notably boosted against RSV A and B virus subtypes by linking two different VHHs (RSV-D3 and RSV-E4) which target different epitopes. The enhancement of the activity by linking two VHHs is likely due to the flexibility of the linker. Experiments aiming to characterize the binding dynamics of the RSV-D3 to the F protein are necessary for characterizing intra- or intermolecular binding. The RSV F is responsible for fusion of the viral lipid membrane with the host membrane, but also participates in attachment of the RSV virions to target cells. In addition, it was recently demonstrated that RSV F protein can bind to nucleolin expressed at the surface of target cells, and that this interaction is crucial for RSV infection *in vitro* and *in vivo* (Tayyari *et al.*, 2011). After viral attachment, the RSV F protein mediates fusion of the viral membrane with the plasma membrane of the target cell, thereby releasing the viral genome into the cytoplasm of the host cell. This process involves a series of conformational changes in the F protein from a metastable pre-fusion to a stable post-fusion conformation. We recently demonstrated that bivalent RSV-D3 VHHs can prevent RSV infection both before and after viral attachment and can inhibit syncytia formation, but cannot hamper RSV attachment (Schepens *et al.*, 2011). Together, these observations constantly indicate that, by a similar mechanism as palivizumab, bivalent RSV-D3 VHHs prevent RSV infection by blocking fusion. Although the conformations of the RSV F antigenic regions II and IV-VI are maintained in the post-fusion form, it is more plausible that the RSV VHHs block viral fusion and syncytia formation by binding to the RSV F protein in either its pre-fusion or intermediate conformations (Fig. 3). Possibly, binding of the VHHs to the antigenic region II interferes with the conformational changes of the F protein that are required for fusion.

Immune compromised Balb/c mice (cyclophosphamide treatment) were used to test whether bivalent RSV-D3 VHHs can protect against RSV infection *in vivo* (Schepens *et al.*, 2011). As VHHs are known to remain active in the respiratory tract after nebulisation, bivalent RSV-D3 and control VHHs were administered intranasally (patent application WO 2009/147248). Prophylactic treatment of mice with 5 mg/kg of bivalent RSV-D3 VHH or palivizumab reduced RSV pulmonary titers below the detection limit of the RSV plaque assay. This strong reduction was confirmed by qPCR analysis. Remarkably, as low as 0.6 mg/kg bivalent RSV-D3 could prevent or strongly reduce (at least 100-fold) pulmonary RSV replication. In comparison, monovalent RSV-D3 VHH protected against pulmonary RSV replication about 25 times less efficiently than its bivalent counterpart. For prophylactic treatment to be valuable, even if is easy to administer, its effect should be long lasting. We demonstrate that intranasal administration of bivalent RSV-D3 VHHs can protect against RSV infection for at least 48 hours. Prophylactic treatment with palivizumab in high risk infants reduces RSV associated hospitalization, but no effective therapeutic is available. Therefore, RSV-D3 VHHs were also evaluated as therapeutic treatments. Intranasal administration of RSV-D3 VHHs 4 or 24 hours after infection strongly reduced pulmonary RSV replication (at least 100-fold). Plaque assays also indicated that administration of bivalent RSV-D3 VHHs 72 hours after RSV treatment can reduce pulmonary RSV replication. However, the lung homogenates used to quantify the pulmonary RSV titer in mice that were treated 72 hours after infection still contained neutralizing RSV VHHs. Therefore, it is not clear to which extent treatment at this time point reduced RSV replication *in vivo*. The potential of bivalent VHHs for preventing morbidity and pulmonary inflammation upon RSV infection was assessed in a non immunocompromised mouse model. Prophylactic administration of bivalent RSV-D3 VHHs (1 mg/kg) completely prevented body weight loss and pulmonary cell infiltration that was observed in mice treated with control VHHs. Therapeutic treatment with bivalent RSV-D3 VHHs 24 h after infection partially reduced body weight loss and pulmonary cell infiltration. These observations confirm the *in vivo* antiviral potential of neutralizing VHHs.

Fig. 3. Ribbon representation of the structure of the RSV F protein trimer in its post-fusion form. The head and stalk of this recombinant protein are depicted, lacking the fusion peptide, transmembrane region and cytoplasmic domain. The immunogenic epitopes recognized by Mab 101F (site II) and palimuzab (site IV-VI) are in red and blue, respectively, and the rest of the F protein is in green. The RSV-D3 and RSV-C4 resistant *in vitro* escape mutants are shown in yellow. Mutations I432T, K433T and S436F in site II disrupt the binding of the RSV-C4. The K262Y, N268I and K272E in the site IV-IV result in loss of binding of the RSV-D3 molecule. (PDB: 3RKI).

Currently Ablynx is preparing a phase I clinical trial to evaluate the safety of a trivalent RSV neutralizing VHH format consisting of three identical VHHs. Preclinical evaluation of this lead candidate can readily neutralize a broad spectrum of clinical RSV A and RSV B subtype

Single Domain Camelid Antibodies that Neutralize Negative Strand Viruses 163

characterized, but their contribution to antigenicity is minor. Antigenic site III extends from 330 to 340 amino acids and is linear (Seif *et al.*, 1985). Mutations in this site affect virulence and the host range of the virus. On the contrary, antigenic site II is conformational and discontinuous and is determined by two regions, amino acids 34-42 and 198-200. Site II is responsible for about 70 % of the known Mabs against RVG (Benmansour *et al.*, 1991).

RGV is an interesting target for the VHH platform because alternative cost effective antirabies tools are needed. By using an approach similar to those previously discussed for influenza and RSV, a llama was immunized with recombinant RVG and five VHHs were obtained (Rab – E8, H7, F8, E6 and C12). The neutralizing activity of those VHHs was validated against 10 Rabies genotype 1 viruses: 3 laboratory strains (CVS-11 as prototype, ERA, CB-1) and 7 field isolates and one rabies genotype 5 virus (EBL-V1) was included to validate broad cross neutralization. A cell based assay was used, the Rapid Fluorescent Focus Inhibition Test (RFFIT) (Vene *et al.*, 1998). This assay has been internationally recognized as the *in vitro* standard for testing virus neutralizing antibodies. Mab 8-32, which recognizes antigenic site II of RVG was also included as positive control (Montano-Hirose *et al.*, 1993). VHHs F8, E6, H7 and C12 neutralized the genotype 1 strains: CB-1 and ERA with an IC50 in the low nanomolar range and the CVS-11 strain in the low to high nanomolar range. They could also neutralize several RV field isolates. On the other hand, E8 efficiently neutralized only CB-1 and CVS-11, in the low and high nanomolar range, respectively. C12 and E6 had better neutralization activity than Mab 8-2 against the ERA and CB-1 strains. Using a similar approach as described above for influeza and RSV, the authors also generated the bivalent against the Rabies genotype 1 CVS -11 and the genotype 5 EBLV-1. Bivalent monoparatopic VHHs were constructed using a Gly4/Ser linker, using the 12, H7, E8 and F8 VHHs. The neutralization IC50 of these constructions was reduced from two to 180-fold relative to the monovalent protein, indicating enhancement of the neutralization. Nevertheless, the best results were obtained when biparatopic molecules were used. The E6/H7 and the H7/F8 molecules increased the neutralization potency by a 2 log factor, while the E8/H7 increased 3 log-fold, compared with the monovalents. E8/H7 even outperformed Mab 8 -2 against the CVS-11. On the contrary, in the case of the genotype 5 strain EBLV-1, the monovalent molecules showed modest neutralization or none at all. The enhanced neutralization of biparatopic molecules was confirmed by E8/C12, which presented an increase in the neutralization potential of 147-fold (IC50 = 3.76 nM) relative to the monovalent moiety, but not as low as Mab 8 – 2 (IC50 = 0.12 nM). The results of competition assays of the 5 VHHs against the Mab 8- 2 showed that E6, E8, F8 and H7 compete for the same epitope. On the other hand, C12 did not compete which indicates that it recognizes a different epitope. The difference in epitope recognition could be one of the causes of the strong and broad effect of biparatopic molecules, especially for E8/C12. Experiments using VHHs against Rabies mutant virus, carrying substitutions in the known residues in the antigenic site II could localize the exact binding sites of these new antibodies. For example, it has been reported that substitution K198E of the glycoprotein abolish the binding of the Mab 8–2 (Montano-Hirose *et al.*, 1993). Unfortunately, the crystallographic structure of the RVG protein has not been reported. The use of vesicular stomatitis virus glycoprotein is accepted as a modeling reference and as a surrogate template for RVG structure (Cibulski *et al.*, 2009; Tomar *et al.*, 2010). We used the alignment of this protein with the RVG as reference to show the possible structure of antigenic site II (Fig. 4). The purpose of this estimate is to

viruses more efficiently than Synagis (abstract, 7th international RSV symposium, Rotterdam, 2010). *In vivo* studies demonstrated that both prophylactic and therapeutic treatment with this RSV neutralizing VHH can readily reduce RSV replication in the upper and lower respiratory tract of cotton rats (abstract, 7th international RSV symposium, Rotterdam, 2010).

In summary, neutralizing RSV VHHs are promising new candidates as anti-RSV therapeutics for different reasons. First, VHHs allow versatile formatting including the creation of multivalent formats by the use of flexible linkers. This feature enabled the creation of bivalent and trivalent VHH which can neutralize RSV at picomolar range, more than 1000-fold more efficient than their monovalent counterpart. Second, by linking two different VHHs which neutralize different virus strains (such as the RSV A versus the RSV B subtype strains) cross-reactive VHH constructs can be obtained. Moreover, as a result of avidity effects, cross linking VHH with different specificity can considerably improve the neutralizing activity. Third, the VHHs small size and protruding paratopes can contribute to its neutralization activity. As structural models and electron microscopic analysis indicate that the antigenic region II is located at the side of the RSV F protein trimer, at the dense surface of RSV virions, this region is likely more accessible for small and flexible VHH formats than for large and more rigid antibodies (McLellan *et al.*, 2011; Ruiz-Arguello *et al.*, 2004) (Fig. 3). Fourth, due to their high stability at stringent conditions, VHHs can be administered via nebulisation, which allows a rapid accumulation of high amounts of neutralizing VHH at the site of respiratory viral infections. In addition, due to the high stability of VHHs and the ease of intranasal or pulmonary administration, VHH therapy could potentially be applied more generally, even in developing countries.

#### **4. Rabies virus**

Rabies virus (RV) is a single stranded RNA virus of the *Rhabdoviridae* family, genus *Lyssavirus.* Infection with RV in humans causes acute encephalitis, with a mortality rate of almost 100%. It is transmitted to humans by bites from a carnivore or a quiroptera vector and most cases occur in Asia or Africa. The long incubation period following infection by RV presents a paradox, because of the absence or very weak antiviral immune response (Johnson *et al.*, 2010). The small amount of virus inoculated after infection and the neurotropism of RV are believed to contribute to the absence of effective antibodies in the patient. After the bite, wound cleaning can reduce the chances of a productive infection in humans. Passive immunization and vaccination promptly after exposure is the only effective therapeutic tool available now. Modern vaccines are inactivated virus produced from continuous cell cultures, like the vaccine by Aventis Pasteur (human diploid cells). Nevertheless, in underdeveloped countries, the established RV therapy (attenuated virus, Mab anti RV) is too expensive for most of the population. RV has a genome of 12 kDa coding for 5 proteins: nucleoprotein, phosphoprotein, matrix, RNA-dependent RNA polymerase and the Glycoprotein (RVG). In the virus particle, the RVG is the only viral protein exposed as a trimeric spike, and it is responsible for recognition of cellular receptors, virulence and antigenicity.

#### **4.1 Nbs present a broad protection against Rabies virus**

For more than 25 years, two well-defined antigenic sites in the RVG have been characterized by Mabs: antigenic sites II and III (Lafon *et al.*, 1990). Other epitopes have also been

viruses more efficiently than Synagis (abstract, 7th international RSV symposium, Rotterdam, 2010). *In vivo* studies demonstrated that both prophylactic and therapeutic treatment with this RSV neutralizing VHH can readily reduce RSV replication in the upper and lower respiratory

In summary, neutralizing RSV VHHs are promising new candidates as anti-RSV therapeutics for different reasons. First, VHHs allow versatile formatting including the creation of multivalent formats by the use of flexible linkers. This feature enabled the creation of bivalent and trivalent VHH which can neutralize RSV at picomolar range, more than 1000-fold more efficient than their monovalent counterpart. Second, by linking two different VHHs which neutralize different virus strains (such as the RSV A versus the RSV B subtype strains) cross-reactive VHH constructs can be obtained. Moreover, as a result of avidity effects, cross linking VHH with different specificity can considerably improve the neutralizing activity. Third, the VHHs small size and protruding paratopes can contribute to its neutralization activity. As structural models and electron microscopic analysis indicate that the antigenic region II is located at the side of the RSV F protein trimer, at the dense surface of RSV virions, this region is likely more accessible for small and flexible VHH formats than for large and more rigid antibodies (McLellan *et al.*, 2011; Ruiz-Arguello *et al.*, 2004) (Fig. 3). Fourth, due to their high stability at stringent conditions, VHHs can be administered via nebulisation, which allows a rapid accumulation of high amounts of neutralizing VHH at the site of respiratory viral infections. In addition, due to the high stability of VHHs and the ease of intranasal or pulmonary administration, VHH therapy

tract of cotton rats (abstract, 7th international RSV symposium, Rotterdam, 2010).

could potentially be applied more generally, even in developing countries.

Rabies virus (RV) is a single stranded RNA virus of the *Rhabdoviridae* family, genus *Lyssavirus.* Infection with RV in humans causes acute encephalitis, with a mortality rate of almost 100%. It is transmitted to humans by bites from a carnivore or a quiroptera vector and most cases occur in Asia or Africa. The long incubation period following infection by RV presents a paradox, because of the absence or very weak antiviral immune response (Johnson *et al.*, 2010). The small amount of virus inoculated after infection and the neurotropism of RV are believed to contribute to the absence of effective antibodies in the patient. After the bite, wound cleaning can reduce the chances of a productive infection in humans. Passive immunization and vaccination promptly after exposure is the only effective therapeutic tool available now. Modern vaccines are inactivated virus produced from continuous cell cultures, like the vaccine by Aventis Pasteur (human diploid cells). Nevertheless, in underdeveloped countries, the established RV therapy (attenuated virus, Mab anti RV) is too expensive for most of the population. RV has a genome of 12 kDa coding for 5 proteins: nucleoprotein, phosphoprotein, matrix, RNA-dependent RNA polymerase and the Glycoprotein (RVG). In the virus particle, the RVG is the only viral protein exposed as a trimeric spike, and it is responsible for recognition of cellular receptors,

For more than 25 years, two well-defined antigenic sites in the RVG have been characterized by Mabs: antigenic sites II and III (Lafon *et al.*, 1990). Other epitopes have also been

**4. Rabies virus** 

virulence and antigenicity.

**4.1 Nbs present a broad protection against Rabies virus** 

characterized, but their contribution to antigenicity is minor. Antigenic site III extends from 330 to 340 amino acids and is linear (Seif *et al.*, 1985). Mutations in this site affect virulence and the host range of the virus. On the contrary, antigenic site II is conformational and discontinuous and is determined by two regions, amino acids 34-42 and 198-200. Site II is responsible for about 70 % of the known Mabs against RVG (Benmansour *et al.*, 1991).

RGV is an interesting target for the VHH platform because alternative cost effective antirabies tools are needed. By using an approach similar to those previously discussed for influenza and RSV, a llama was immunized with recombinant RVG and five VHHs were obtained (Rab – E8, H7, F8, E6 and C12). The neutralizing activity of those VHHs was validated against 10 Rabies genotype 1 viruses: 3 laboratory strains (CVS-11 as prototype, ERA, CB-1) and 7 field isolates and one rabies genotype 5 virus (EBL-V1) was included to validate broad cross neutralization. A cell based assay was used, the Rapid Fluorescent Focus Inhibition Test (RFFIT) (Vene *et al.*, 1998). This assay has been internationally recognized as the *in vitro* standard for testing virus neutralizing antibodies. Mab 8-32, which recognizes antigenic site II of RVG was also included as positive control (Montano-Hirose *et al.*, 1993). VHHs F8, E6, H7 and C12 neutralized the genotype 1 strains: CB-1 and ERA with an IC50 in the low nanomolar range and the CVS-11 strain in the low to high nanomolar range. They could also neutralize several RV field isolates. On the other hand, E8 efficiently neutralized only CB-1 and CVS-11, in the low and high nanomolar range, respectively. C12 and E6 had better neutralization activity than Mab 8-2 against the ERA and CB-1 strains. Using a similar approach as described above for influeza and RSV, the authors also generated the bivalent against the Rabies genotype 1 CVS -11 and the genotype 5 EBLV-1. Bivalent monoparatopic VHHs were constructed using a Gly4/Ser linker, using the 12, H7, E8 and F8 VHHs. The neutralization IC50 of these constructions was reduced from two to 180-fold relative to the monovalent protein, indicating enhancement of the neutralization. Nevertheless, the best results were obtained when biparatopic molecules were used. The E6/H7 and the H7/F8 molecules increased the neutralization potency by a 2 log factor, while the E8/H7 increased 3 log-fold, compared with the monovalents. E8/H7 even outperformed Mab 8 -2 against the CVS-11. On the contrary, in the case of the genotype 5 strain EBLV-1, the monovalent molecules showed modest neutralization or none at all. The enhanced neutralization of biparatopic molecules was confirmed by E8/C12, which presented an increase in the neutralization potential of 147-fold (IC50 = 3.76 nM) relative to the monovalent moiety, but not as low as Mab 8 – 2 (IC50 = 0.12 nM). The results of competition assays of the 5 VHHs against the Mab 8- 2 showed that E6, E8, F8 and H7 compete for the same epitope. On the other hand, C12 did not compete which indicates that it recognizes a different epitope. The difference in epitope recognition could be one of the causes of the strong and broad effect of biparatopic molecules, especially for E8/C12. Experiments using VHHs against Rabies mutant virus, carrying substitutions in the known residues in the antigenic site II could localize the exact binding sites of these new antibodies. For example, it has been reported that substitution K198E of the glycoprotein abolish the binding of the Mab 8–2 (Montano-Hirose *et al.*, 1993). Unfortunately, the crystallographic structure of the RVG protein has not been reported. The use of vesicular stomatitis virus glycoprotein is accepted as a modeling reference and as a surrogate template for RVG structure (Cibulski *et al.*, 2009; Tomar *et al.*, 2010). We used the alignment of this protein with the RVG as reference to show the possible structure of antigenic site II (Fig. 4). The purpose of this estimate is to

Single Domain Camelid Antibodies that Neutralize Negative Strand Viruses 165

2011); as well as the *in vivo* validation of the influenza HA binding VHH (Ibañez *et al.*, 2011) and RSV (Schepens *et al.*, 2011). In the case of protection against influenza infection the bivalent format of the Nbs proved superior *in vitro* and *in vivo*. But, as indicated by the successful *in vivo* validation of one of the H5N1 strains, it is imperative to extend this validation to other influenza strains. Furthermore, it would be worthwhile to isolate and characterize Nbs that recognize conserved domains in HA, such as the stalk. In line with the influenza results, the activity of RSV and RVG neutralizing Nbs was significantly higher for the bivalent than the monovalent format: both the cross neutralization activity and potency were higher. Those results manifest the advantages of using a multimeric format against multimeric viral targets. The enhanced antiviral potential of the multimeric format could be due to increased avidity and/or the intra- or intermolecular binding could contribute in the enhancement. Experiments to assess the binding mechanism could lead to further improvements. The results overall confirm two important points: the high potential of the Nbs as prophylactic and therapeutic tools, and the possibility of using Nbs directed against other infectious diseases. There is limited function and sequence similarity among the three proteins used as antigens (HA, RSVF and RVG) other than their trimeric architecture and antigenicity. Nevertheless, the HA and the RVG are functionally similar and both are involved in the cellular receptor binding, whereas RSVF participates also in virion receptor binding, it s main function is in membrane fusion. In all three cases, showed capacity to neutralize the viral target by blocking binding or hampering necessary conformational changes, indicating the great versatility and efficiency of the antiviral discussed here. In competition assays, the recognition of non conventional epitopes by these antiviral was not observed, but could be the focus of research. Nbs recognized well known antigenic sites that are also targeted by Mabs. The HA, RSV F and RVG are not enzymes, and lack extensive antigenic clefts. This could be one reason why Nbs showed preference for recognition of "classical" epitopes in these viral proteins. If the presence of antigenic clefts could lead to recognition of non "classical" epitopes in the viral proteins, targeting viral enzymes could be an interesting approach. Enzymes such as the influenza Neuraminidase are potential targets. This viral sialidase presents a catalytic cleft, in which the framework and substrate contact residues are conserved in most of the influenza strains. The coming years will probably bring potent novel anti-viral Nbs directed against different viruses and it is likely

We are grateful to Dr. Amin Bredan for editing the text. M.C. holds a VIB international PhD fellowship. L.I.I. was a beneficiary of the Belgian Federal Sciences Administration (Federale Wetenschapsbeleid, BELSPO) and was supported by Ghent University IOF grant Stepstone

Arbabi Ghahroudi, M., Desmyter, A., Wyns, L., Hamers, R. & Muyldermans, S. (1997).

Selection and identification of single domain antibody fragments from camel

that some of these Nbs will reach clinical trials.

IOF08/STEP/001. B.S. is a postdoctoral fellow of FWO-Flanders.

heavy-chain antibodies. *FEBS letters* 414, 521-526.

**6. Acknowledgement** 

**7. References** 

show the tendency of the VHHs to recognize conformational rather than linear epitopes. In line with the results of the neutralizing VHHs against influenza and RSV, the results of the broadness and the strong potency against both RV genotypes indicate the RV neutralizing VHHs as a promising. Nevertheless, in contrast with the previous cases of the influenza and RSV VHHs, there is not *in vivo* validation of the RV neutralizing VHHs available.

Fig. 4. Schematic representation of the vesicular stomatitis virus protein G trimer. This protein is taken as reference to depict the amino acids corresponding to the antigenic site II of the Rabies Virus glycoprotein (residues 34-42, and 198-200, in blue). The localization of the K198E mutation that prevents the binding of the Mab 8-2 is shown in red. The VHHs E8, F8, E6 and H7 compete with the Mab 8- 2 for the binding, which means that their epitopes might be within antigenic site II. (PDB: 2CMZ).

#### **5. Conclusion**

The Nb platform is a new and promising antiviral tool. The ease of producing Nbs in bacterial and lower eukaryotic cells, and the possibility of producing tailor-made constructions makes them an attractive and cost effective alternative to some established antiviral drugs. This approach may be useful for the treatment of infectious orphan diseases (including viral) and in developing countries, where the "standard" prophylaxis or therapy is prohibitively expensive or not available. In this work we have discussed findings on recently developed Nbs directed against three viruses affecting humans: the generation and *in vitro* validation of the Nbs or VHHs against influenza H5N1, RSV and RV (Hultberg *et al.*, 2011); as well as the *in vivo* validation of the influenza HA binding VHH (Ibañez *et al.*, 2011) and RSV (Schepens *et al.*, 2011). In the case of protection against influenza infection the bivalent format of the Nbs proved superior *in vitro* and *in vivo*. But, as indicated by the successful *in vivo* validation of one of the H5N1 strains, it is imperative to extend this validation to other influenza strains. Furthermore, it would be worthwhile to isolate and characterize Nbs that recognize conserved domains in HA, such as the stalk. In line with the influenza results, the activity of RSV and RVG neutralizing Nbs was significantly higher for the bivalent than the monovalent format: both the cross neutralization activity and potency were higher. Those results manifest the advantages of using a multimeric format against multimeric viral targets. The enhanced antiviral potential of the multimeric format could be due to increased avidity and/or the intra- or intermolecular binding could contribute in the enhancement. Experiments to assess the binding mechanism could lead to further improvements. The results overall confirm two important points: the high potential of the Nbs as prophylactic and therapeutic tools, and the possibility of using Nbs directed against other infectious diseases. There is limited function and sequence similarity among the three proteins used as antigens (HA, RSVF and RVG) other than their trimeric architecture and antigenicity. Nevertheless, the HA and the RVG are functionally similar and both are involved in the cellular receptor binding, whereas RSVF participates also in virion receptor binding, it s main function is in membrane fusion. In all three cases, showed capacity to neutralize the viral target by blocking binding or hampering necessary conformational changes, indicating the great versatility and efficiency of the antiviral discussed here. In competition assays, the recognition of non conventional epitopes by these antiviral was not observed, but could be the focus of research. Nbs recognized well known antigenic sites that are also targeted by Mabs. The HA, RSV F and RVG are not enzymes, and lack extensive antigenic clefts. This could be one reason why Nbs showed preference for recognition of "classical" epitopes in these viral proteins. If the presence of antigenic clefts could lead to recognition of non "classical" epitopes in the viral proteins, targeting viral enzymes could be an interesting approach. Enzymes such as the influenza Neuraminidase are potential targets. This viral sialidase presents a catalytic cleft, in which the framework and substrate contact residues are conserved in most of the influenza strains. The coming years will probably bring potent novel anti-viral Nbs directed against different viruses and it is likely that some of these Nbs will reach clinical trials.

#### **6. Acknowledgement**

We are grateful to Dr. Amin Bredan for editing the text. M.C. holds a VIB international PhD fellowship. L.I.I. was a beneficiary of the Belgian Federal Sciences Administration (Federale Wetenschapsbeleid, BELSPO) and was supported by Ghent University IOF grant Stepstone IOF08/STEP/001. B.S. is a postdoctoral fellow of FWO-Flanders.

#### **7. References**

164 Antiviral Drugs – Aspects of Clinical Use and Recent Advances

show the tendency of the VHHs to recognize conformational rather than linear epitopes. In line with the results of the neutralizing VHHs against influenza and RSV, the results of the broadness and the strong potency against both RV genotypes indicate the RV neutralizing VHHs as a promising. Nevertheless, in contrast with the previous cases of the influenza and

RSV VHHs, there is not *in vivo* validation of the RV neutralizing VHHs available.

Fig. 4. Schematic representation of the vesicular stomatitis virus protein G trimer. This protein is taken as reference to depict the amino acids corresponding to the antigenic site II of the Rabies Virus glycoprotein (residues 34-42, and 198-200, in blue). The localization of the K198E mutation that prevents the binding of the Mab 8-2 is shown in red. The VHHs E8, F8, E6 and H7 compete with the Mab 8- 2 for the binding, which means that their epitopes

The Nb platform is a new and promising antiviral tool. The ease of producing Nbs in bacterial and lower eukaryotic cells, and the possibility of producing tailor-made constructions makes them an attractive and cost effective alternative to some established antiviral drugs. This approach may be useful for the treatment of infectious orphan diseases (including viral) and in developing countries, where the "standard" prophylaxis or therapy is prohibitively expensive or not available. In this work we have discussed findings on recently developed Nbs directed against three viruses affecting humans: the generation and *in vitro* validation of the Nbs or VHHs against influenza H5N1, RSV and RV (Hultberg *et al.*,

might be within antigenic site II. (PDB: 2CMZ).

**5. Conclusion** 

Arbabi Ghahroudi, M., Desmyter, A., Wyns, L., Hamers, R. & Muyldermans, S. (1997). Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. *FEBS letters* 414, 521-526.

Single Domain Camelid Antibodies that Neutralize Negative Strand Viruses 167

Falsey, A. R., Hennessey, P. A., Formica, M. A., Cox, C. & Walsh, E. E. (2005). Respiratory

Glezen, W. P., Paredes, A., Allison, J. E., Taber, L. H. & Frank, A. L. (1981). Risk of

Greenberg, A. S., Avila, D., Hughes, M., Hughes, A., McKinney, E. C. & Flajnik, M. F. (1995).

Groothuis, J. R. & Simoes, E. A. (1993). Immunoprophylaxis and immunotherapy: role in the

Hamers-Casterman, C., Atarhouch, T., Muyldermans, S., Robinson, G., Hamers, C., Songa,

Hmila, I., Saerens, D., Ben Abderrazek, R., Vincke, C., Abidi, N., Benlasfar, Z., Govaert, J.,

Huang, K., Incognito, L., Cheng, X., Ulbrandt, N. D. & Wu, H. (2010). Respiratory syncytial

Hultberg, A., Temperton, N. J., Rosseels, V., Koenders, M., Gonzalez-Pajuelo, M., Schepens,

Ibañez, L. I., De Filette, M., Hultberg, A., Verrips, T., Temperton, N., Weiss, R. A.,

Johnson, N., Cunningham, A. & Fooks, A. R. (2010). The immune response to the rabies

Lafon, M., Edelman, L., Bouvet, J. P., Lafage, M. & Montchatre, E. (1990). Human

Lauwereys, M., Arbadi Ghahroudi, M., Desmyter, A., Kinne, J., Holzer, W., De Genst, E.,

Luke, C. J. & Subbarao, K. (2006). Vaccines for pandemic influenza. *Emerging infectious* 

dromedary heavy-chain antibodies. In *EMBO J*, pp. 3512-3520.

broadened neutralizing anti-viral molecules. *PloS one* 6, e17665.

infection. *The Journal of infectious diseases* 203, 1063-1072.

infection and vaccination. In *Vaccine*, pp. 3896-3901.

*The Journal of general virology*, pp. 1689-1696.

somatic diversification in sharks. *Nature* 374, 168-173.

*of medicine*, pp. 1749-1759.

of light chains. *Nature* 363, 446-448.

fusion. In *J Virol*, pp. 8132-8140.

15 edn.

pp. 97-103.

24, 3479-3489.

*diseases* 12, 66-72.

syncytial virus infection in elderly and high-risk adults. In *The New England journal* 

respiratory syncytial virus infection for infants from low-income families in relationship to age, sex, ethnic group, and maternal antibody level. In *J Pediatr*, 708-

A new antigen receptor gene family that undergoes rearrangement and extensive

prevention and treatment of repiratory syncytial virus. In *Int J Antimicrob Agents*,

E. B., Bendahman, N. & Hamers, R. (1993). Naturally occurring antibodies devoid

El Ayeb, M., Bouhaouala-Zahar, B. & Muyldermans, S. (2010). A bispecific nanobody to provide full protection against lethal scorpion envenoming. *Faseb J*

virus-neutralizing monoclonal antibodies motavizumab and palivizumab inhibit

B., Ibañez, L. I., Vanlandschoot, P., Schillemans, J., Saunders, M., Weiss, R. A., Saelens, X., Melero, J. A., Verrips, C. T., Van Gucht, S. & de Haard, H. J. (2011). Llama-derived single domain antibodies to build multivalent, superpotent and

Vandevelde, W., Schepens, B., Vanlandschoot, P. & Saelens, X. (2011). Nanobodies with in vitro neutralizing activity protect mice against H5N1 influenza virus

monoclonal antibodies specific for the rabies virus glycoprotein and N protein. In

Wyns, L. & Muyldermans, S. (1998). Potent enzyme inhibitors derived from


Barre, S., Greenberg, A. S., Flajnik, M. F. & Chothia, C. (1994). Structural conservation of

Benmansour, A., Leblois, H., Coulon, P., Tuffereau, C., Gaudin, Y., Flamand, A. & Lafay, F. (1991). Antigenicity of rabies virus glycoprotein. In *J Virol*, pp. 4198-4203. Boyce, T. G., Mellen, B. G., Mitchel, E. F., Wright, P. F. & Griffin, M. R. (2000). Rates of

Cibulski, S. P., Sinigaglia, M., Rigo, M. M., Antunes, D. A., Vieira, G. F., Fulber, C. C., Chies,

Crowe, J. E., Firestone, C. Y., Crim, R., Beeler, J. A., Coelingh, K. L., Barbas, C. F., Burton, D.

De Genst, E., Silence, K., Decanniere, K., Conrath, K., Loris, R., Kinne, J., Muyldermans, S. &

de Jong, M. D., Simmons, C. P., Thanh, T. T., Hien, V. M., Smith, G. J., Chau, T. N., Hoang,

Decanniere, K., Desmyter, A., Lauwereys, M., Ghahroudi, M. A., Muyldermans, S. & Wyns,

Decanniere, K., Muyldermans, S. & Wyns, L. (2000). Canonical antigen-binding loop

Desmyter, A., Spinelli, S., Payan, F., Lauwereys, M., Wyns, L., Muyldermans, S. &

Desmyter, A., Transue, T. R., Ghahroudi, M. A., Thi, M. H., Poortmans, F., Hamers, R.,

*Bioinformatics and Computational Biology*. Rio de Janeiro.

heavy-chain antibodies. In *Proc Natl Acad Sci*, pp. 4586-4591.

hypercytokinemia. In *Nature medicine*, pp. 1203-1207.

*Journal of biological chemistry*, pp. 23645-23650.

*The Journal of biological chemistry* 276, 7346-7350.

920.

252, 373-375.

*Structure*, pp. 361-370.

*Biol*, pp. 83-91.

medicaid. In *J Pediatr*, pp. 865-870.

hypervariable regions in immunoglobulins evolution. In *Nat Struct Biol*, pp. 915-

hospitalization for respiratory syncytial virus infection among children in

J. A. B., Franco, A. C. & Roehe, P. M. (2009). Structure modelling of Rabies Virus Glycoprotein. In *5th International Conference of the Brazilian Association for* 

R., Chanock, R. M. & Murphy, B. R. (1998). Monoclonal antibody-resistant mutants selected with a respiratory syncytial virus-neutralizing human antibody fab fragment (Fab 19) define a unique epitope on the fusion (F) glycoprotein. *Virology*

Wyns, L. (2006). Molecular basis for the preferential cleft recognition by dromedary

D. M., Chau, N. V., Khanh, T. H., Dong, V. C., Qui, P. T., Cam, B. V., Ha do, Q., Guan, Y., Peiris, J. S., Chinh, N. T., Hien, T. T. & Farrar, J. (2006). Fatal outcome of human influenza A (H5N1) is associated with high viral load and

L. (1999). A single-domain antibody fragment in complex with RNase A: noncanonical loop structures and nanomolar affinity using two CDR loops. In

structures in immunoglobulins: more structures, more canonical classes? In *J Mol* 

Cambillau, C. (2002). Three camelid VHH domains in complex with porcine pancreatic alpha-amylase. Inhibition and versatility of binding topology. In *The* 

Muyldermans, S. & Wyns, L. (1996). Crystal structure of a camel single-domain VH antibody fragment in complex with lysozyme. In *Nat Struct Biol*, pp. 803-811. Els Conrath, K., Lauwereys, M., Wyns, L. & Muyldermans, S. (2001). Camel single-domain

antibodies as modular building units in bispecific and bivalent antibody constructs.


Single Domain Camelid Antibodies that Neutralize Negative Strand Viruses 169

Tayyari, F., Marchant, D., Moraes, T. J., Duan, W., Mastrangelo, P. & Hegele, R. G. (2011).

Throsby, M., van den Brink, E., Jongeneelen, M., Poon, L. L., Alard, P., Cornelissen, L.,

Tomar, N. R., Singh, V., Marla, S. S., Chandra, R., Kumar, R. & Kumar, A. (2010). Molecular

Vene, S., Haglund, M., Vapalahti, O. & Lundkvist, A. (1998). A rapid fluorescent focus

Verheesen, P., Roussis, A., de Haard, H. J., Groot, A. J., Stam, J. C., den Dunnen, J. T., Frants,

Vu, K. B., Ghahroudi, M. A., Wyns, L. & Muyldermans, S. (1997). Comparison of llama VH

Wang, M. Z., Tai, C. Y. & Mendel, D. B. (2002). Mechanism by which mutations at his274

Wang, P. & Yang, X. (2010). Neutralization efficiency is greatly enhanced by bivalent

Wiley, D. C., Wilson, I. A. & Skehel, J. J. (1981). Structural identification of the antibody-

Wu, S. J., Schmidt, A., Beil, E. J., Day, N. D., Branigan, P. J., Liu, C., Gutshall, L. L., Palomo,

Wu, T. T., Johnson, G. & Kabat, E. A. (1993). Length distribution of CDRH3 in antibodies. In

Yamada, S., Suzuki, Y., Suzuki, T., Le, M. Q., Nidom, C. A., Sakai-Tagawa, Y., Muramoto, Y.,

H5N1 influenza A viruses to human-type receptors. *Nature* 444, 378-382.

and zanamivir. *Antimicrobial agents and chemotherapy* 46, 3809-3816.

recovered from human IgM+ memory B cells. *PloS one* 3, e3942.

target validation. *Biochimica et biophysica acta* 1764, 1307-1319.

*journal of pharmaceutical sciences* 72, 486-490.

virus. In *J Virol Methods*, pp. 71 - 75.

glycoproteins. In *J Virol*, pp. 7114-7123.

antigenic variation. *Nature* 289, 373-378.

approaches. *The Journal of general virology* 88, 2719-2723.

virus. *Nature medicine*.

1121-1131.

*Proteins*, pp. 1-7.

Identification of nucleolin as a cellular receptor for human respiratory syncytial

Bakker, A., Cox, F., van Deventer, E., Guan, Y., Cinatl, J., ter Meulen, J., Lasters, I., Carsetti, R., Peiris, M., de Kruif, J. & Goudsmit, J. (2008). Heterosubtypic neutralizing monoclonal antibodies cross-protective against H5N1 and H1N1

docking studies with rabies virus glycoprotein to design viral therapeutics. *Indian* 

inhibition test for detection of neutralizing antibodies to tick-borne encephalitis

R. R., Verkleij, A. J., Theo Verrips, C. & van der Maarel, S. M. (2006). Reliable and controllable antibody fragment selections from Camelid non-immune libraries for

sequences from conventional and heavy chain antibodies. *Molecular immunology* 34,

alter sensitivity of influenza a virus n1 neuraminidase to oseltamivir carboxylate

binding of an antibody to epitopes in the V4 region and the membrane-proximal external region within one trimer of human immunodeficiency virus type 1

binding sites of Hong Kong influenza haemagglutinin and their involvement in

C., Furze, J., Taylor, G., Melero, J. A., Tsui, P., Del Vecchio, A. M. & Kruszynski, M. (2007). Characterization of the epitope for anti-human respiratory syncytial virus F protein monoclonal antibody 101F using synthetic peptides and genetic

Ito, M., Kiso, M., Horimoto, T., Shinya, K., Sawada, T., Kiso, M., Usui, T., Murata, T., Lin, Y., Hay, A., Haire, L. F., Stevens, D. J., Russell, R. J., Gamblin, S. J., Skehel, J. J. & Kawaoka, Y. (2006). Haemagglutinin mutations responsible for the binding of


McLellan, J. S., Yang, Y., Graham, B. S. & Kwong, P. D. (2011). Structure of respiratory

preservation of neutralizing epitopes. In *J Virol*, pp. 7788-7796., 17(9):1132-5. Montano-Hirose, J. A., Lafage, M., Weber, P., Badrane, H., Tordo, N. & Lafon, M. (1993).

Muyldermans, S., Atarhouch, T., Saldanha, J., Barbosa, J. A. & Hamers, R. (1994). Sequence

Rasmussen, S. G., Choi, H. J., Fung, J. J., Pardon, E., Casarosa, P., Chae, P. S., Devree, B. T.,

Rudge, J. S., Holash, J., Hylton, D., Russell, M., Jiang, S., Leidich, R., Papadopoulos, N.,

Schepens, B., Ibañez, L. I., Hultberg, A., Bogaert, P., De Bleser, P., De Baets, S., Vervalle, F.,

Stiehm, E. R., Keller, M. A. & Vyas, G. N. (2008). Preparation and use of therapeutic

Swanson, K. A., Settembre, E. C., Shaw, C. A., Dey, A. K., Rappuoli, R., Mandl, C. W.,

antigenic site III of the glycoprotein. In *J Virol*, pp. 926-934.

antibodies primarily of human origin. *Biologicals* 36, 363-374.

*United States of America* 108, 9619-9624.

immunoglobulins lacking light chains. In *Protein Eng*, pp. 1129-1135. Nair, H., Nokes, D. J., Gessner, B. D., Dherani, M., Madhi, S. A., Singleton, R. J., O'Brien, K.

lyssavirus 1 (EBL1) infection in mice. In *Vaccine*, pp. 1259-1266.

systematic review and meta-analysis. *Lancet* 375, 1545-1555.

469, 175-180.

3677-3687.

syncytial virus fusion glycoprotein in the postfusion conformation reveals

Protective activity of a murine monoclonal antibody against European bat

and structure of VH domain from naturally occurring camel heavy chain

L., Roca, A., Wright, P. F., Bruce, N., Chandran, A., Theodoratou, E., Sutanto, A., Sedyaningsih, E. R., Ngama, M., Munywoki, P. K., Kartasasmita, C., Simoes, E. A., Rudan, I., Weber, M. W. & Campbell, H. (2010). Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a

Rosenbaum, D. M., Thian, F. S., Kobilka, T. S., Schnapp, A., Konetzki, I., Sunahara, R. K., Gellman, S. H., Pautsch, A., Steyaert, J., Weis, W. I. & Kobilka, B. K. (2011). Structure of a nanobody-stabilized active state of the beta(2) adrenoceptor. *Nature*

Pyles, E. A., Torri, A., Wiegand, S. J., Thurston, G., Stahl, N. & Yancopoulos, G. D. (2007). VEGF Trap complex formation measures production rates of VEGF, providing a biomarker for predicting efficacious angiogenic blockade. *Proceedings of the National Academy of Sciences of the United States of America* 104, 18363-18370. Ruiz-Arguello, M. B., Martin, D., Wharton, S. A., Calder, L. J., Martin, S. R., Cano, O., Calero,

M., Garcia-Barreno, B., Skehel, J. J. & Melero, J. A. (2004). Thermostability of the human respiratory syncytial virus fusion protein before and after activation: implications for the membrane-fusion mechanism. *The Journal of general virology* 85,

Verrips, T., Melero, J., Vandevelde, W., Vanlandschoot, P. & Saelens, X. (2011). Nanobodies specific for Respiratory Syncytial Virus Fusion protein protect against infection by inhibition of fusion. *The Journal of infectious diseases*, 204, 1692-1701. Seif, I., Coulon, P., Rollin, P. E. & Flamand, A. (1985). Rabies virulence: effect on

pathogenicity and sequence characterization of rabies virus mutations affecting

Dormitzer, P. R. & Carfi, A. (2011). Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers. *Proceedings of the National Academy of Sciences of the* 


**Treatment of Herpes Simplex Virus** 

**an Alternative Therapeutic Formula** 

*1Laboratorio de Patología Bucal, Facultad de Odontología,* 

*Universidad Autónoma del Estado de México,* 

*2Maruzen Pharmaceuticals Co., Ltd., 3Meikai University School of Dentistry,* 

> *1México 2,3Japan*

 **with Lignin-Carbohydrate Complex Tablet,** 

Blanca Silvia González López1, Masaji Yamamoto2 and Hiroshi Sakagami3

Herpes simplex virus type 1 (HSV-1) commonly infects the mucosa and skin epithelial cells, and the virus remains latent in sensory neurons mainly in the trigeminal ganglia. Once a patient has been infected, the infection continues for life (Hunt, 2011a). Differences in HSV-1 prevalence have been reported around the world. According to Smith & Robinson (2002), the incidence in lower socioeconomic countries is higher. Primary infection, occur mainly in infants and young children, infections are usually mild or subclinical. Acute gingivoestomatitis is characterized by the appearance of multiple vesicular and ulcerative painful lesions in oral mucosa, with inflammation and bleeding of the gums, may also be associated with systemic symptoms (Arduino & Porter, 2008). Once the clinical infection concludes, the virus reaches peripheral nerves which supply sensation to the skin, migrating along the nerve axon to the dorsal root ganglia of the trigeminal or facial nerves and goes

Recurrences of HSV-1 can be triggered by internal and external factors. The reactivation mechanism is unknown, the virus begins to replicate within the ganglion and grows down the nerves and out into the skin or mucous membranes (Koelle & Corey, 2008). After a prodromal of tingling, warmth or itching, the clinical lesion appear (Fatahzadeh & Schwartz 2007). The recurrence of oral HSV- 1 is developed almost always in the vermilion border of

Prevention of infection can be achieved by avoiding the physical contact, kissing when the lesions are present, touching or using the articles that the patient has used (eating or drinking utensils, glasses, or straws). However, in order to prevent the recurrences, the control of external factors is recommended; avoiding the exposure to wind burn and ultraviolet radiation, using labial protectants and controlling the emotional stress (Paterson

the lips but lesions can appear elsewhere around perioral skin (Siegel, 2002).

**1. Introduction** 

& Kwong, 2008).

into latency stage (Esmann, 2001)

Zhou, B., Zhong, N. & Guan, Y. (2007). Treatment with convalescent plasma for influenza A (H5N1) infection. *The New England journal of medicine* 357, 1450-1451. **9** 
