Vaccinia Virus Vector Bivalent Norovirus Vaccine

Abstract

Norovirus is a major etiological agent of nonbacterial gastroenteritis around the world. Due to its in vitro culture complexity, high genome diversity, and the lack of cross-reactive immunity between genogroups, there is an unmet urgent need for polyvalent norovirus vaccines that provide broad-spectrum protection, and no vaccine has gained global approval to date. In this study, we constructed a bivalent norovirus vaccine, based on the highly attenuated poxvirus [strain VG9] vector, expressing the major capsid protein VP1 from genotypes GII.4 and GII.17. VG9-NOR exhibited a comparable replication ability to the authentic virus while preserving good safety. After the intramuscular and intranasal immunization of mice, VG9-NOR induced high IgG- and IgA-binding antibody (Ab) titers against GII.4 and GII.17, increased the secretion of GII.4 and GII.17-specific HGBA-blocking antibodies, and enhanced GII.17-specific mucosal immunity. Furthermore, VG9-NOR also induced a Th1-mediated cellular response. These results demonstrate that the polyvalent poxvirus vector vaccine expressing VP1 variants from different subtypes is able to elicit effective protection. Our study highlights the VG9 vector as a highly promising candidate for the development of polyvalent norovirus vaccines.

Keywords: bivalent vaccine; immune response; norovirus; poxvirus vector.

Figure 1

Construction and validation of vaccinia vector bivalent norovirus vaccine: (A) The gene segment that was inserted into VG9 and shuttle plasmid construction. Different domains were labelled with capital letters. Terminal regions were located on both ends of the gene segment. The VP1 protein sequences of the GII.4 and GII.17 strains were expressed using the ELO160 and PE/L promoters, respectively. The sequences of the VG9 TK site [J2R] were used as the homologous arm. An EGFP expression cassette driven by the P11 promoter was inserted between the CRE-recognition sites d. (B) VG9-NOR-infected Vero cells before and after EGFP knockout, observed using an inverted fluorescence microscope. (C) Validation of VP1 gene insertion in VG9-NOR by PCR. (D,E) Evaluation ofVP1 protein expression by Western blot analysis. BHK21 cells were infected with 5 MOI of VG9-NOR and VG9 for 24 h and then lysed. Protein samples were separated using 10% polyacrylamide SDS-PAGE and the specific bands detected using rabbit polyclonal antibodies against GII.4 and GII.17 VP1, respectively.

Figure 2

Genetic stability of VG9-NOR: (A) Growth curve of VG9-NOR and VG9 in different cells. Vero, BHK-21, and HeLa cells were infected with 0.05 MOI of VG9-NOR. Viral titers were determined using the plaque assay at 12 h, 24 h, 36 h, 48 h, and 72 h post-infection (the third generation to establish the growth curve). (B) Chick embryo chorioallantoic membrane (CAM) assay. The CAM of chick embryos was inoculated with 1 × 10⁶ PFU of VG9 or VG9-NOR. The upper images show the VG9-induced pocks and the lower images show the VG9-NOR-induced pocks (n = 4). (C,D) Western blot analysis of VP1 expression in different generations. BHK-21 cells were infected with 0.05 MOI of P3 generation VG9-VOR. Following cell lysis, the virus was passaged for 6 generations. The protein samples of each generation were separated by 10% acrylamide SDS-PAGE and detected using rabbit polyclonal antibodies against GII.4 and GII.17 VP1, combined with mouse polyclonal antibodies against VG9-E3 and GAPDH. The E3 protein was used as loading control. Black arrows indicate the location of GII.4 and GII.17 VP1, GAPDH, and VG9 E3. The approximate molecular weight is indicated on the left.

Figure 3

Safety evaluation of VG9-NOR bivalent norovirus vaccine: (A) Pathological analysis of the lungs and spleen of mice following intramuscular vaccination. (B) Skin condition monitoring following the administration of different doses of VG9/VG9-NOR in rabbits. Concentration from top to below: 10⁷, 10⁶, 10⁵, 10⁴, and 10³ PFU. VG9-NOR was injected at the left side of the spinal cord, and the right side was injected with VG9. (C) Red pock lesion size monitoring in the injected rabbits. (D) Weight changes after VG9-NOR injection [days 2–13], where day 1 represents the pre-infection weight. Control-group mice received 100 μL of VG9 suspension, intranasally or intramuscularly in high dose [1 × 10⁷ PFU] or low dose [1 × 10⁶ PFU].

Figure 4

Induction of humoral immunity by VG9-NOR bivalent vaccine: (A) Mouse immunization procedure. (B) Serum IgG binding Ab titer induced by VG9-NOR immunization. (C) Serum IgA Ab titer induced by VG9-NOR immunization. (D) HGBA-blocking antibody production in response to the vaccine. The IgA- and IgG-binding Abs titers were determined according to the colorimetric absorbance (A₄₅₀) value of the maximum antibody dilution in ELISA, which was 2.1-fold higher than the blank control. The reciprocal of this dilution was used as the binding antibody titer. BT50 values were calculated based on the reciprocal of the highest dilution of the A₄₅₀ value just below the median A₄₅₀ of the positive and negative control groups. The bars indicate means ± SEM. Each dot represents a single mouse. (E) Fecal IgA Abs in the VG9-NOR immunized mice. The number of fecal IgA-Abs-positive mice/total number of mice is labelled above each bar. The baseline titer value was calculated based on the mean A₄₅₀ value of five unimmunized mice +3 standard deviations. The A₄₅₀ of immunized mice ≥ baseline titer value was considered positive, and vice versa [ns, not significant; * p < 0.05, ** p < 0.005, *** p < 0.0001, and **** p < 0.0001; each dot represents a mouse, n = 5].

Figure 5

Cellular immunity induced by the VG9-NOR bivalent vaccine. (A) Percentage of IFN-γ-, IL-2-, IL-4-, and TNF-α-secreting CD4⁺ cells against GII.4 (B) and GII.17 (C). Percentage of IFN-γ-and TNF-α-secreting CD8+ cells against GII.4 and GII.17 [ns, not significant; * p < 0.05, ** p < 0.005; each dot represents a mouse, n = 5].