Biological significance of amino acid substitutions in hepatitis B surface antigen (HBsAg) for glycosylation, secretion, antigenicity and immunogenicity of HBsAg and hepatitis B virus replication

Abstract

Amino acid substitutions of hepatitis B surface antigen (HBsAg) may affect the antigenicity and immunogenicity of HBsAg, leading to immune escape and diagnostic failure. The amino acid positions 122 and 160 are known as determinants for HBsAg subtypes d/y and w/r, respectively. The substitution K122I has been shown to strongly affect HBsAg antigenicity. In this study, we investigated the significance of naturally occurring amino acid substitutions K122I, T123N, A159G and K160N. Both T123N and K160N substitutions resulted in additional N-glycosylated forms of HBsAg, while the other mutations produced more glycosylated HBsAg compared with the wild type (wt). Detection of HBsAg by ELISA and immunofluorescence staining indicated that variant HBsAg (vtHBsAg) with K122I was not recognized by HBsAg immunoassays, while vtHBsAg with T123N, A159G, K160N and A159G/K160N had reduced antigenicity. DNA immunization in BALB/c mice revealed that wtHBsAg and vtHBsAg with T123N and K160N are able to induce antibodies to HBsAg (anti-HBs), whereas K122I and A159G greatly impair the ability of HBsAg to trigger anti-HBs responses. The cellular immune response to the HBsAg aa 29-38 epitope was enhanced by the K160N substitution. Using replication competent clones of hepatitis B virus (HBV), T123N and A159G substitutions were shown to strongly reduce virion assembly. The amino acid substitution K160N appeared to compensate for the negative effect of A159G on virion production. These results reveal complex effects of amino acid substitutions on biochemical properties of HBsAg, on antigenicity and immunogenicity, and on the replication of HBV.

Productive HBV infection of well-differentiated, hNTCP-expressing human hepatoma-derived (Huh7) cells

“…HBV total DNA was extracted by a method described by Hirt (Hirt, 1967), and core-associated HBV DNA was extracted by a method described elsewhere (Wu et al, 2010). Southern blot was performed as described previously www.virosin.org DECEMBER 2017 VOLUME 32 ISSUE 6 467 (Zhou et al, 2014a).…”

Amino Acid Substitutions at Positions 122 and 145 of Hepatitis B Virus Surface Antigen (HBsAg) Determine the Antigenicity and Immunogenicity of HBsAg and InfluenceIn VivoHBsAg Clearance

“…The mutations were introduced into the wtHBsAg sequence (HBV genotype B2, HBsAg subtype adw2) in the backbone vector pHBsAg-WT by PCRbased mutagenesis using the primer pairs SP1/T145 or T122 and SP2/ G145X-U or K122X-U (X denotes the amino acid replacing G145 or K122), which are listed in Table S2 in the supplemental material, as described previously (28). The plasmids containing the mutated sequences were subjected to sequence analysis to exclude the presence of unintended mutations.…”

“…The expression of HBsAg in Huh7 cells by transient transfection and detection of HBsAg by Western blotting and enzyme-linked immunosorbent assay (ELISA) were performed as described previously (26,28,29). Human hepatoma cell line Huh7 (provided by American Type Culture Collection, Manassas, VA) was used for transient transfection to express recombinant proteins.…”

“…Immunofluorescence (IF) staining of transfected cells was performed as described previously (28). In brief, transfected cells were fixed with 3.7% paraformaldehyde.…”

“…The K122M and K122N substitutions were also found in very few cases. Our previous study demonstrated that vtHBsAg with K122I has a significantly reduced ability to bind anti-HBs antibodies and to induce anti-HBs responses in mice (26,28). Among all of the HBsAg mutants studied, the K122I mutant has the most severe impairment in reactivity with anti-HBs antibodies in immunoassays.…”