Characterization of a novel hepatitis B virus mutant: demonstration of mutation‐induced hepatitis B virus surface antigen group specific ‘a’ determinant conformation change and its application in diagnostic assays

Abstract

We report and characterize a novel mutant of the hepatitis B virus surface antigen (HBsAg). The mutant was isolated from a symptomatic patient who was found to be persistently positive for both HBsAg and anti-hepatitis B surface antibody (anti-HBs) and a long-lasting anti-HBc (core) IgM. Due to the unusual immune serological profile, polymerase chain reaction and sequencing were performed and revealed a genotype D mutant HBV (LBN). Aligned with known HBsAg sequences from GenBank, the LBN variant matched to consensus subtype ayw2 and revealed five mutation positions. Interestingly, none of the mutations was found within the group-specific "a" determinant region (124-147) and, specifically, two of the five mutations, T118K and P120Q, were located only a few amino acids adjacent to the 124-147 region. Using a panel of six monoclonal antibodies and vaccine raised human neutralizing antibodies, the recombinant wild-type HBsAg and the novel variant LBN HBsAg were investigated for their immunological reactivity. Vaccine raised human anti-HBs showed less reactivity to the variant LBN HBsAg than to wild-type HBsAg in enzyme immune assays. Furthermore, our observations demonstrate the influence of adjacent residues on the group-specific "a" determinant structural configuration; this resulted in severe antigenic changes of the immunodominant region as well as in the subtype serology. The importance of the variant LBN lies in the observation that mutations close to the "a" determinant can change the immunodominant region structure and therefore alter the group specific determinant antigenicity even though no mutations are present within this region. Hence, the classical definition of the "a" determinant cluster may need to be extended to cover a broader region to include the requirement of adjacent amino acids to support its conformation. In conclusion, by understanding the HBsAg major immunodominant region structure and by using a combination of antibodies with specificity covering all key mutation locations, maximal anti-HBs-based protection, and highly sensitive diagnostic assays can be achieved.

Hepatitis B virus vaccine breakthrough infection: surveillance of S gene mutants of HBV

“…Similar HBsAg escape mutations in 2nd loop of the "a" determinant (S143L; S143 M) were observed in two virus strains causing fulminant hepatitis, but no such mutations were detected in asymptomatic and chronic hepatitis B patients (T. Michler et al, 2014). Luongo et al also reported a male patient with high seroprotective anti-HBs titer (>200 IU/l) and acute HB with three mutations (M125T, T127P and Q129H) in the virus strain (Kfoury et al, 2001). S gene mutants are the dominant HBV variants in immunized population with fulminant and acute HB (Hsu et al, 1997), because these mutations allow the virus to escape neutralization by antibodies and inefficient CTL-mediated control.…”

“…The mechanisms by which mutated virus causes HBV vaccine breakthrough infection include: 1) altered conformational structure and/or T-cell epitope structure allowing mutated virus to evade recognition and clearance by the immune system; 2) altered antigenicity of HBsAg through changes in hydrophobicity profile and putative glycosylation site, which consequently decrease or abolish affinity for anti-HBs, offering lower protection from vaccination (Luongo et al, 2015) and permitting infection with HBV isolates of different genotypes or subtypes in patients with a normal anti-HBs response (Wu et al, 2012); and 3) the accumulation of aa substitutions because of mutations in and around the "a" determinant region may change immunogenicity of HBsAg, producing less effective neutralizing anti-HBs response and enabling the mutated virus evade clearance and survive long terms (Kfoury et al, 2001). S gene mutations of HBV can occur in vaccinated population with fulminant, acute and chronic HB.…”

Hepatitis B virus surface protein mutations clustered mainly in CTL immune epitopes in chronic carriers: results of an Iranian nationwide study

“…These mutations especially include amino acid residues between the positions 19–28, 80–98, 110–120, 148–162 and 206–215 (Table ). It seems that, as an alternative, virus‐neutralizing activity may reside in antibodies related to distantly located amino acid residues in other parts of the protein rendering the viral particle less immunogenic in producing an effective neutralizing anti‐HBs response to clear the virus . Also, changes located outside the ‘a’ determinant region were reported from immunized infants born to HBV carrier mothers .…”

Hepatitis B virus S gene escape mutants

“…Mutations near the MHR can alter group-specific antigenicity even though the mutations are not within the MHR. [899495] Monoclonal antibody binding assays have also shown that residues 178-186 are exposed on the surface of the 20 nm particle. [96] This is the region which specifies the q sub-determinant.…”