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. 2012 Apr;86(8):4658-69.
doi: 10.1128/JVI.06353-11. Epub 2012 Feb 1.

Amino acid substitutions at positions 122 and 145 of hepatitis B virus surface antigen (HBsAg) determine the antigenicity and immunogenicity of HBsAg and influence in vivo HBsAg clearance

Chunchen Wu  1 , Wanyu Deng, Liu Deng, Liang Cao, Bo Qin, Songxia Li, Yun Wang, Rongjuan Pei, Dongliang Yang, Mengji Lu, Xinwen Chen
Affiliations

Amino acid substitutions at positions 122 and 145 of hepatitis B virus surface antigen (HBsAg) determine the antigenicity and immunogenicity of HBsAg and influence in vivo HBsAg clearance

Chunchen Wu et al. J Virol. 2012 Apr.

Abstract

A variety of amino acid substitutions, such as K122I and G145R, have been identified around or within the a determinant of hepatitis B surface antigen (HBsAg), impair HBsAg secretion and antibody binding, and may be responsible for immune escape in patients. In this study, we examined how different substitutions at amino acid positions 122 and 145 of HBsAg influence HBsAg expression, secretion, and recognition by anti-HBs antibodies. The results showed that the hydrophobicity, the presence of the phenyl group, and the charges in the side chain of the amino acid residues at position 145 reduced HBsAg secretion and impaired reactivity with anti-HBs antibodies. Only the substitution K122I at position 122 affected HBsAg secretion and recognition by anti-HBs antibodies. Genetic immunization in mice demonstrated that the priming of anti-HBs antibody response was strongly impaired by the substitutions K122I, G145R, and others, like G145I, G145W, and G145E. Mice preimmunized with wild-type HBsAg (wtHBsAg) or variant HBsAg (vtHBsAg) were challenged by hydrodynamic injection (HI) with a replication-competent hepatitis B virus (HBV) clone. HBsAg persisted in peripheral blood for at least 3 days after HI in mice preimmunized with vtHBsAg but was undetectable in mice preimmunized with wtHBsAg, indicating that vtHBsAgs fail to induce proper immune responses for efficient HBsAg clearance. In conclusion, the biochemical properties of amino acid residues at positions 122 and 145 of HBsAg have a major effect on antigenicity and immunogenicity. In addition, the presence of proper anti-HBs antibodies is indispensable for the neutralization and clearance of HBsAg during HBV infection.

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Figures

Fig 1
Fig 1
Expression of wt- and vtHBsAgs with amino acid substitutions at position 145 or 122. Huh7 cells were transfected with the expression plasmids pHBsAgWT, pHBsAgK122X (including 122G, 122M, 122L, 122I, 122P, 122N, 122Q, 122T, 122D, 122E, 122W, and 122H), and pHBsAgG145X (including 145M, 145L, 145I, 145P, 145N, 145Q, 145T, 145D, 145E, 145W, 145H, and 145R). Culture supernatants and lysates of Huh7 cells were collected at 48 h after transfection. HBsAgs were detected by a monoclonal mouse antibody to the HA tag for the expression analysis. Beta-actin was used as a loading control. CK, cells transfected with empty plasmid as a negative control. The levels of HBsAg expression were quantified by densitometry, normalized against beta-actin, and expressed in arbitrary units. The relative expression levels of vtHBsAg in supernatants and cell lysates of transfected cells were calculated as a percentage of the level of wtHBsAg expression.
Fig 2
Fig 2
Reactivity of vtHBsAgs expressed in transient transfection in commercial HBsAg assays corrected against the expression levels in cell lysates. The culture supernatants and cell lysates of transiently transfected cells expressing wt- and vtHBsAgs were collected at 48 h. wt- and vtHBsAgs were detected by the Architect system and HBsAg CMIA kits (Abbott Laboratories, Wiesbaden-Delkenheim, Germany) (A and B) and an HBsAg enzyme immunoassay (EIA) kit (Kehua, Shanghai, China) (C and D) according to the manufacturer's instructions. The reactivity of vtHBsAgs with the amino acid substitutions at position 145 (A and C) or 122 (B and D) was standardized by the relative expression levels of wt- and vtHBsAg based on the Western blotting result (Fig. 1) and is expressed as the percentage of the OD450 of samples compared to that of the wtHBsAg. The average values from 4 replicates were calculated and given as the final reactivity of wt- or vtHBsAgs. CK, cells transfected with empty plasmid as a negative control.
Fig 3
Fig 3
Reactivity of vtHBsAg in immunoassays based on three monoclonal anti-HBs antibodies. The culture supernatants (A and C) and cell lysates (B and D) of transiently transfected cells expressing wt- and vtHBsAgs were collected at 48 h. wt- and vtHBsAgs were detected by ELISAs based on three monoclonal anti-HBs antibodies, A11, S11, and S1. The reactivity of vtHBsAgs with the amino acid substitutions at position 145 (A and B) or 122 (C and D) was expressed as the percentage of the OD450 of samples compared to that of the wtHBsAg. The average values from 4 replicates were calculated and given as the final reactivity of wt or vtHBsAgs. CK, cells transfected with empty plasmid as a negative control.
Fig 4
Fig 4
IF staining of wt- and vtHBsAgs with amino acid substitutions with anti-HA antibody. HeLa cells were transfected with the expression plasmid pHBsAgWT, pHBsAgG145X, or pHBsAgK122X, respectively. Cells were fixed 48 h after transfection and stained with a monoclonal mouse antibody to the HA tag. The nuclei were stained with Hoechst 33258. Magnification, ×400. CK, cells transfected with empty plasmid as a negative control.
Fig 5
Fig 5
IF staining of wt- and vtHBsAgs with amino acid substitutions with anti-HBs monoclonal antibody S1. HeLa cells were transfected with the expression plasmid pHBsAgWT, pHBsAgG145X, or pHBsAgK122X, respectively. Cells were fixed 48 h after transfection and stained with the anti-HBs antibody S1. The nuclei were stained with Hoechst 33258. Magnification, ×400. CK, cells transfected with empty plasmid as a negative control.
Fig 6
Fig 6
vtHBsAgs with amino acid substitutions at position 145 or 122 had reduced ability to induce anti-HBs antibody response. BALB/c (H-2 Ld) mice were immunized with 30 μg of the expression plasmid pHBsAgWT (n = 8), pHBsAgG145I (G145I) (n = 6), pHBsAgG145E (G145E) (n = 6), pHBsAgG145W (G145W) (n = 6), pHBsAgG145R (G145R) (n = 10), pHBsAgK122L (K122L) (n = 6), pHBsAgK122I (K122I) (n = 6), or with 30 μl of PBS (n = 10) as a control. The immunizations were given three times within 3-week intervals. Sera were collected and tested for anti-HBs antibodies at day 10 after the last immunization. Results are shown as the anti-HBs IgG (mIU/ml) level for each mouse per group. Filled circles represent IgG titers in individual mice. Solid lines represent geometric mean values. The dotted line represents the cutoff, which was assumed to be 2.1-fold the mean value of the negative samples.
Fig 7
Fig 7
Kinetics of HBsAg and HBV DNA clearance and anti-HBs antibody responses in immunized mice after HI. Hydrodynamic injection with pAAV-HBV-GB1.3 was carried out with immunized mice at day 14 after the last immunization. Sera were collected at 1, 3, 5, 7, 10, 14, 17, and 21 days postinfection, and the anti-HBs antibodies (left y axis) or HBsAgs (right y axis) (A) were detected by ELISA. The HBsAg level was presented as the OD450. The anti-HBs antibody level was presented as the OD450 with a cutoff, which was assumed to be 2.1-fold the mean value of the negative samples. (B) HBV DNA concentrations in mouse sera at 5 days postinfection were determined by real-time PCR. The relative levels of HBV DNA were calculated by the average of HBV DNA concentrations to the average of HBV DNA concentrations of control mice. Control mice developed a level of serum HBV DNA concentrations at 3.4 × 105 copies/ml, which was set as 100%. The hydrodynamic injection with pAAV-HBV GB1.3 was performed with 4 to 7 mice per group as indicated.
Fig 8
Fig 8
HBsAg-specific T-cell immune responses in immunized mice before or after hydrodynamic injection. The numbers (A) and mean spot sizes (B) of HBsAg-specific IFN-γ-producing cells in 2 × 105 splenocytes from BALB/c mice immunized with pHBsAgWT, pHBsAgG145R (G145R), and PBS are shown. The IFN-γ ELISpot assays were performed at day 1 before hydrodynamic injection or at day 5 after hydrodynamic injection with pAAV-HBV-GB1.3 in the presence of the major H-2Ld-restricted CTL epitope of HBsAg amino acids 29 to 38 (10 μg/ml) for 18 h. Experiments were performed with three mice per group. The data were analyzed by t test, and the differences were statistically significant (**, P < 0.01 compared to the PBS group).
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