Development and evaluation of two subunit vaccine candidates containing antigens of hepatitis E virus, rotavirus, and astrovirus

Abstract

Hepatitis E virus (HEV), rotavirus (RV), and astrovirus (AstV) are important pathogens that transmit through a common fecal-oral route, causing hepatitis (HEV) and gastroenteritis (RV and AstV) respectively in humans. In this study, we developed and evaluated two subunit vaccine candidates that consisted of the same protruding or spike protein antigens of the three viruses in two formats, a fusion of the three antigens into one molecule (fused vaccine) vs. a mixture of the three free antigens together (mixed vaccine). Both vaccines were easily made via E. coli expression system. Mouse immunization experiments showed that the fused vaccine elicited significantly higher antibody responses against the three viral antigens than those induced by the mixed vaccine. In addition, the mouse post-immune antisera of the fused vaccine revealed significantly higher neutralizing titers against HEV infection in cell culture, as well as significantly higher 50% blocking titers (BT50) against RV VP8-HBGA receptor interactions than those of the post-immune antisera after immunization of the mixed vaccine. Thus, the fused vaccine is a promising trivalent vaccine candidate against HEV, RV, and AstV, which is worth for further development.

Figure 1. Production and characterization of the fused and mixed vaccines.

(A) Schematic diagram of the fused vaccine (AstV P-HEV P-RV VP8) containing the dimeric P domains of astrovirus (AstV) and hepatitis E virus (HEV), as well as the monomeric VP8 protein of rotavirus (RV). A linker consisting of 12 glycines (12G) was added between the two P domains, while another one was between the HEV P domain and RV VP8. A histidine x6 tag (H) was linked to the N-terminus of the fused vaccine. (B,C) SDS PAGE of the affinity column-purified individual AstV P domain (AstV P, ~26 kDa), HEV P domain (HEV P, ~18.6 kDa), and RV VP8 protein (RV VP8, ~17.8 kDa) (B), as well as three elutions (lane 1, 2, and 3) of the fused vaccine (~62 kDa) consisting of the three viral antigens (C). (D) The elution curve of a gel filtration chromatography of the fused vaccine through the size-exclusion column Superdex 200 (10/300 GL, GE Healthcare Life Sciences). The gel filtration columns were calibrated by the Gel Filtration Calibration Kit (GE Healthcare Life Sciences) and the purified recombinant P particles, small P particles, and P dimers of norovirus (VA387). The elution positions of blue Dextran 2000 (~2000 kDa, void), P particle (~830 kDa), small P particle (~420 kDa), P dimer (~69 kDa), and aprotinin (~6.5 kDa) were indicated. The proteins of the two major peaks of the gel-filtrations were analyzed by SDS-PAGE that is shown below the elution curves with indications of peak 1 (fractions 23 and 24) and peak 2 (fractions 26 to 28), respectively. The elution ranges of the fused vaccine are indicated by blue bars. M represents pre-stained protein markers.

Figure 2. The three viral antigens of the fused and mixed vaccines elicited different levels of IgG responses.

Mice (n = 6) were immunized with equal amount of the fused (A,C) and mixed (B,D) vaccines intranasally without (A,B) or with (C,D) MPLA adjuvant. The IgG titers specific to each individual viral antigen of astrovirus (AstV P), hepatitis E virus (HEV P), and rotavirus (RV VP8 and PP-VP8) after immunizations were measured using the purified P domains of AstV (AstV P) and HEV (HEV P), as well as free (RV VP8) and P particle-presented (PP-VP8) RV VP8 as capture antigens, respectively. (A,B) Comparisons of the IgG titers to the three viral antigens, respectively, after immunization of the fused (A) or mixed (B) vaccines without adjuvant. (C,D) Comparisons of the IgG titers after immunization of the fused (C) or mixed (D) vaccine with MPLA adjuvant. The differences between the data groups are indicated by folds, while their statistical differences are shown by *symbols (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 3. The fused vaccine increased IgG responses of the viral antigen components.

Mice (n = 6) were immunized with the fused and the mixed vaccines intranasally without (A, C and E) or with (B, D and F) MPLA adjuvant. The resulting IgG titers specific to the individual viral antigens of astrovirus (AstV) P domain, hepatitis E virus (HEV) P domain, and rotavirus (RV) VP8, respectively, were measured using the purified P domains of AstV (AstV P) and HEV (HEV P), as well as free (RV VP8) and P particle-presented (PP-VP8) RV VP8 as capture antigens, respectively. Phosphate buffered saline (PBS) was administered to mice as negative control. (A, C and E) The fused and the mixed vaccine without adjuvant elicited IgG titers specific to AstV P domain (A), HEV P domain (C), and RV VP8 (E) were shown. (B, D and F) The fused and the mixed vaccine with adjuvant elicited IgG titers specific to AstV P domain (B), HEV P domain (D), and RV VP8 (F) were shown. The differences of the IgG titers between the two vaccines are indicated in folds, while their statistical differences are shown by *symbols (**P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 4. MPLA adjuvant increased the IgG responses of both fused and mixed vaccines.

The viral antigen-specific IgG titers after immunization of with the mixed (A) and the fused (B) vaccines without (−) or with (+) MPLA adjuvant were shown and compared side-by-side. The IgG titers specific to each individual viral antigen of astrovirus P domain (AstV P), hepatitis E virus P domain (HEV P), and rotavirus VP8 (RV VP8) were measured using the purified P domains of AstV (AstV P) and HEV (HEV P), as well as free (RV VP8) and P particle-presented (PP-VP8) RV VP8 as capture antigens, respectively. The increased IgG titers in folds by MPLA adjuvant are indicated, while their statistical differences are shown by *symbols (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 5. The post-immune antisera of the fused vaccine neutralized infection of hepatitis E virus (HEV)in cell culture.

(A,B) Neutralization titers against HEV (Kernow P6) infection in HepG2/C3A cells were determined via fluorescent-focus assays using the post-immune antisera of the fused and mixed vaccines, respectively, without (A) or with (B) MPLA adjuvant. (C) The neutralizing titers of the mouse antisera after immunization of the fused and the mixed trivalent vaccines without (−) or with (+) MPLA adjuvant were compared. The differences of the neutralizing titers between the two vaccines are indicated in folds, while their statistical differences are shown by *symbols (*P < 0.05, **P < 0.01).

Figure 6. Serum blocking titers against attachment of rotavirus (RV) VP8 to its host ligands.

The 50% blocking titers (BT₅₀s) of the post-immune sera of the fused and mixed vaccines without (A) or with (B) MPLA adjuvant against the attachment of the P particle-presented RV VP8 (PP-VP8) to Lewis b (Le^b) antigen-positive salivas were determined and compared. (C) The BT₅₀s of the two vaccines without (−) and with (+) MPLA adjuvant were compared side-by-side. The differences of the neutralizing titers between the two vaccines are indicated in folds, while their statistical differences are shown by *symbols (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).