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. 2017 Jan 3;12(1):e0167871.
doi: 10.1371/journal.pone.0167871. eCollection 2017.

Investigation of a Novel Hepatitis B Virus Surface Antigen (HBsAg) Escape Mutant Affecting Immunogenicity

Md Golzar Hossain  1 , Keiji Ueda  1
Affiliations

Investigation of a Novel Hepatitis B Virus Surface Antigen (HBsAg) Escape Mutant Affecting Immunogenicity

Md Golzar Hossain et al. PLoS One. .

Abstract

Mutation in the hepatitis B virus surface antigen (HBsAg) may affect the efficiency of diagnostic immunoassays or success of vaccinations using HBsAg. Thus, antigenicity and immunogenicity analyses of the mutated HBsAg are necessary to develop novel diagnostic tools and efficient vaccinations. Here, the in vitro antigenicity of three wild-type HBsAg open reading frames (ORFs) (adr4, W1S [subtype adr] and W3S [subtype adr]) isolated from clinically infected patients and nineteen synthesized single/double/multiple amino acid-substituted mutants were tested with commercial ELISA kits. Immunofluorescence staining of transfected cells and Western blot analysis confirmed that these ORFs were expressed at comparable levels in HEK-293 cells. W1S and adr4 were clearly detected, whereas W3S could not be detected. Using the same commercial immunoassay kit, we found that the single mutants, K120P and D123T, were marginally reactive, whereas W3S-aW1S and the double mutant, K120P/D123T, exhibited antigenicity roughly equivalent to the wild-type wako1S. On the other hand, the single mutants of W1S, P120K and T123D, significantly impaired the reactivity, while W1S-aW3S and the double mutant of W1S, P120K/T123D, resulted in a complete loss of antigenicity. In addition, ELISA revealed reduced HBs antigenicity of two mutants, W1S N146G and W1S Q129R/G145R. These commercial ELISA-based antigenic reactivities of HBsAg were also strongly correlated with the predicted Ai alterations of affected amino acids due to the specific mutation. In conclusion, this study showed for the first time that lysine (K120) and aspartate (D123) simultaneously affected HBsAg antigenicity, leading to diagnostic failure. These findings will improve diagnostic assays and vaccine development.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Amino acid sequence comparison of wild-type and mutated HBsAg.
(A) Amino acid alignment of the wild-type HBsAgs (W1S, W3S and adr4 [X01587]) with other published sequences (adw [AF100308], adw [AY220698.1], ayr [AY38845.1] and ayw [DQ336692.1]). Only the different amino acids at the positions are represented. The dot represents the same amino acid as the first line. (B) The amino acid (aa) sequence alignment of the wild-type (W1S and W3S) and different mutants of HBsAg. The amino acid residues identical to those of HBsAg (W1S) are indicated by dots.
Fig 2
Fig 2. Expression of the wild-type HBsAg.
(A) Western blotting analysis. HEK293-T cells were transfected with plasmid vectors expressing the wild-type HBsAgs (W1S, W3S and adr4). The expressed HBsAgs were purified from the culture supernatants and the cell lysates 72 h after transfection and an equal volume of each sample was run on SDS-PAGE. An anti-Xpress mAb against the Xpress tag was used to detect each HBsAg. (B) Immunofluorescence assay. Cells on the culture slides were fixed 72 h after transfection and stained with an anti-Xpress mAb as for the Western blot followed by an anti-mouse IgG conjugated with Alexa Fluor 488. Cell nuclei were stained with DAPI. Non-transfected cells were used as a negative control (NC). Each experiment was performed independently three times and one representative result is shown. wt: wild-type.
Fig 3
Fig 3. Reactivity of HBsAg (W1S, W3S and adr4) with a commercial ELISA and an anti-tag Ab.
All the wild-type HBsAgs (W1S, W3S and adr4) were expressed by transient transfection in HEK 293-T cells. The culture supernatants and the cells were collected 72 h after transfection. (A) Reactivity of HBsAg from the culture supernatants and the cell lysates with a commercial ELISA kit. (B) Detection and reactivity of the wild-type HBsAg of PEG-precipitated culture supernatants and cell lysates against anti-Xpress mAb. The expressed HBsAgs were captured on a Ni-coated plate and reacted with an anti-Xpress Ab followed by anti-mouse Ab conjugated with HRP. The reactivity of HBsAgs was presented as the percentage of the OD450-630 of samples compared to W1S. The OD values of HBsAg ELISA and the anti-tag reactivity were normalized by the ratios of densitometric intensity of the Western blotting compared to W1S (Fig 2A). Non-transfected cells served as a negative control (NC). The mean value of three independent experiments is shown as the final reactivity of HBsAg. Error bars are shown as the standard error of means (SEM). wt: wild-type.
Fig 4
Fig 4. Detection of the wild-type and mutated HBsAg with Western blot and immunofluorescence analysis.
(A) Western blot analysis. The HEK 293-T cells were transfected with plasmid vectors expressing the wild-type and mutants of W3S and W1S HBsAg. The expressed HBsAg was purified from the culture supernatant and the cell lysate 72 h after transfection and subjected to Western blot with an anti-Xpress mAb against the Xpress tag to detect the HBsAg. The upper panels shows blots from the culture supernatants and the lower one blots from cell lysates. (B) Immunofluorescence assay. The cells on the culture slides were fixed 72 h after transfection and stained with an anti-Xpress mAb against the Xpress tag followed by an anti-mouse IgG conjugated with Alexa Fluor 488. The cell nuclei were stained with DAPI. Non-transfected cells were used as a negative control (NC). Each experiment was performed independently three times and one representative result is shown. wt: wild-type.
Fig 5
Fig 5. Reactivity of the wild-type HBsAg in a commercial ELISA assay and against an anti-tag mAb.
All the mutants of W3S and wild-type HBsAgs (W1S and W3S) were expressed by transient transfection in HEK 293-T cells. The culture supernatants and the cells were collected 72 h after transfection. (A and B) Reactivity of the wild-type and mtHBsAg from culture supernatants and cell lysates on a commercial ELISA kit. (C and D) Detection and reactivity of the wild-type and mtHBsAg from PEG-precipitated culture supernatants and cell lysates against an anti-Xpress mAb. The reactivity of HBsAgs was presented as the percentage of the OD450-630 of samples compared to W1S in all cases (A, B, C and D). The OD values of HBsAg ELISA and anti-tag reactivity (except W1S N146G from the culture supernatant) were normalized by the ratios of densitometric intensity of Western blotting compared to W1S, except W1S Q129R/G145R from the supernatants, since this mutant exhibited a remarkable loss in secretion ability (Fig 4A). Non-transfected cells served as negative control (NC). The mean value of three independent experiments is shown as the final reactivity of HBsAg. Error bars are shown as the standard errors of means (SEM). wt: wild-type; mt: mutant.
Fig 6
Fig 6. Hypothesis of the mechanism of HBV diagnostic- and immune-escape induced by the HBsAg amino acid mutations.
The lysine and aspartate might form a salt bridge with their side chains and thereby change the conformation of HBsAg, leading to a loss of the binding capacity with anti-HBs Abs.
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Grants and funding

This work was funded by the Wako Pure Chemicals Research Fund to K.U. (J120701101, J1607799316); the Research Program on Hepatitis from the Japan Agency for Medical Research and Development (AMED) to K.U. (15fk0310006h0004). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.