CHAPTER 2: LITERATURE REVIEW
2.7 Genetic variability of HBV
2.7.2 HBV gene specific mutants
2.7.2.4 Pre-surface (pre-S) and surface (S) gene mutants
The pre-S domain is located upstream of the S domain and is divided into pre-S1 and pre-S2 regions (Lüsbrink et al., 2009). The pre-S1 domain bears the epitope for HBV-specific host cell receptor, NTCP, and as such is responsible for binding of the virus to host cells (Neurath et al., 1986; Yan et al., 2012). The pre-S2 together with the pre-S1 and S domains encode the large surface protein (LHBs). The LHBs is essential to virion assembly, and its intracellular levels are linked with the release of viral particles from the infected cell. The medium (MHBs) and small (SHBs) surface proteins on the other hand are encoded by the pre-S2/S and S domains respectively and appear to be uninvolved in virion assembly and release. Mutations in the pre-S2 start codon result in a lack of synthesis of the pre-S2 protein (Yokosuka and Arai, 2006). Deletions in the entire pre-S region or its binding site have also been associated with reduced synthesis of SHBs (typically the most abundant of the three surface proteins) while increasing LHBs levels proportionally. This results in accumulation of LHBs at the host cell ER where the viral envelope is synthesized, and leads to stressing of the ER and consequently the infected hepatocytes as well. This is commonly observed as the development of „ground glass‟ hepatocytes in HBV-related HCC and chronically infected individuals (Wang et al., 2006; Yokosuka and Arai, 2006).
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The viral envelope protein (encoded by pre-S1/pre-S2/S domains) comprises of 389 or 400 amino acid (aa) residues, depending on the HBV variant. A total of 226aa of this protein constitute the HBsAg. The main antigenic determinant, „α‟, is a component of the HBsAg and serves to prime the host immune system to elicit neutralizing antibodies during an infection.
These protective antibodies bind to the Major Hydrophilic Region (MHR), a conformational epitope of the HBsAg. This immune response makes neutralization of the virus possible and effects immune clearance of an HBV infection (Echevarría and Avellόn, 2006).
Amino acid substitutions in the S domain are the main cause of development of HBV surface gene mutants. This particularly occurs within the „α‟ antigenic determinant, resulting in a conformational change to the epitopes and thus altering the antigenic properties of the HBsAg. The host neutralizing antibodies may then be unable to bind to MHR due to the conformational changes and as such the mutations that arise may confer immune escape properties to the virus (Weber, 2005; Echevarría and Avellόn, 2006). Immune escape mutants may be selected for by immune pressure from the host immune response to infection or immunomodulators, such as hepatitis B immunoglobulin (HBIG), which mainly contain antibodies against HBsAg. The virus thus persists and in the absence of effective host immune protection or immune therapy, causes a heightened infection (Zuckerman and Zuckerman, 2003).
Diagnostic escape is another property of HBV S gene mutants. Manufacturers of diagnostic assays that screen for HBsAg usually target different regions of the viral surface protein using conjugated antibodies. These monoclonal and polyclonal antibodies may be unable to detect mutant variants of HBV and thus mutations in the regions targeted by these assays may allow the virus to remain undetected, leading to under-diagnoses of infection (Echevarría and Avellόn, 2006; Yokosuka and Arai, 2006; Alavian et al., 2012). This presents with a major public health problem as these assays are used in routine diagnosis as well as screening of blood donations, organ donors and pregnant women. Diagnostic escape mutants are also responsible for under-diagnosis of occult HBV infections. This is as a result of reduced immunoreactivity of the mutant surface protein with commercially available assays leading to undetectable HBsAg despite the presence of viral DNA (Alavian et al., 2012). Impaired production of HBsAg leading to diagnosis of occult HBV infection is also linked with mutations in the pre-S region. Occult HBV infections are a medical and public health concern as asymptomatic carriers usually remain undiagnosed and untreated,
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and serve as a major source of spread of HBV infection (Torbenson and Thomas, 2002;
Purdy, 2007).
Commercially available hepatitis B vaccines contain subunit preparations of HBsAg and so may select for HBV surface gene mutants with vaccine escape properties. Administration of HBIG therapy has also been implicated in the selection of HBV vaccine escape mutants (Carman, 1997; Yokosuka and Arai, 2006). The vaccine escape mutants are not neutralized by the antibodies induced by the current vaccines as they do not cover for HBV mutant strains. For this reason, vaccine escape mutants persist and cause HBV infection in fully vaccinated individuals, referred to as breakthrough HBV infections (Carman et al., 1990;
Carman, 1997; Zuckerman and Zuckerman, 2003). Vaccine escape mutants are stable and thus may be transmitted to uninfected individuals. This creates a major concern as vaccination is currently the most effective preventive measure available (Weber, 2005). The most notable mutation responsible for HBV vaccine escape is a point mutation (G→A) at nucleotide position 587 of the S gene, consequently replacing glycine (G) with arginine (R) at amino acid residue 145 within the „α‟ antigenic determinant (G145R). Other common mutations involved in HBV vaccine escape include D144A, P142S, Q129H, I/T126N/A and M133L (Carman et al., 1990; Carman, 1997; Torresi, 2002; Yokosuka and Arai, 2006).
In addition to the primary pre-S/S gene mutations, mutations in the P gene can further select for S gene mutants and vice versa due to the overlapping orientation of the two genes. A triple lamivudine resistance mutation; rtV173L/rtL180M/rtM204V for example, has been reported to induce amino acid substitutions within the surface protein (E164D/I195M) which consequently results in enhanced viral replication as well as reduced antigenicity of HBsAg (Torresi et al., 2002). Thus immune, vaccine as well as diagnostic escape mutants may be selected for by mutations in the overlapping P gene. Diagnostic escape mutants selected for in this manner cause patients who were previously treated with Lamivudine (and developed resistance to it) to appear to have cleared the infection as HBsAg is undetectable (Sheldon and Soriano, 2008). Mutations in the surface protein (viral envelope protein) which are induced by P gene mutations, such as insertion of stop codons, may also impair viral fitness.
However compensatory mutations within the polymerase domain help to counteract these deleterious mutations and restore viral replication and viral fitness (Torresi, 2002; Torresi et al., 2002).
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