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. 2023 Aug 25;11(9):1420.
doi: 10.3390/vaccines11091420.

A Subunit Vaccine Candidate Composed of Mpox Virus A29L, M1R, A35R, and B6R Elicits Robust Immune Response in Mice

Xuetao Yang  1   2 , Xidan Yang  1   2 , Shouwen Du  3 , Congxia Hu  1 , Xiu Yang  1   2 , Xingyun Wang  4 , Xing Hu  4 , Nino Rcheulishvili  4   5 , Peng George Wang  2   4 , Jihui Lin  1   2   3
Affiliations

A Subunit Vaccine Candidate Composed of Mpox Virus A29L, M1R, A35R, and B6R Elicits Robust Immune Response in Mice

Xuetao Yang et al. Vaccines (Basel). .

Abstract

With no specific antiviral drugs and preventive vaccines against Mpox virus (MPXV), the epidemic has led to the declaration of a Public Health Emergency of International Concern. As a developmental direction for new vaccines, studies of subunit vaccines based upon MPXV antigen proteins are lacking. In this study, A29L, M1R, A35R, and B6R of MPXV were expressed and purified from a prokaryotic system. The four MPXV antigen proteins in combination were mixed with aluminum hydroxide or CpG7909 as adjuvant, and subsequently used to inoculate mice. The results of enzyme-linked immunosorbent assay (ELISA), flow cytometry analyses, and enzyme-linked immunospot (ELISPOT) assays indicated that A29L, M1R, A35R, and B6R elicited high-level antigen-specific antibodies and CD4+ T cells-based cellular immune response in mice. Moreover, the results of virus neutralization assays suggested that sera from the mice immunized with four proteins elicited high neutralizing activities against the vaccinia virus. Notably, the results of ELISA, ELISPOT, and virus neutralization assays also showed that the CpG7909 adjuvant was more effective in inducing an immune response compared with the aluminum adjuvant. In summary, this study offers valuable insights for further studies of subunit vaccine candidates for the prevention of MPXV and other orthomyxoviruses.

Keywords: CpG7909 adjuvant; Mpox virus; immune response; prokaryotic expression; subunit vaccine.

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Conflict of interest statement

None of the authors have any actual or potential conflict of interest.

Figures

Figure 1
Figure 1
Purification and characterization of MPXV A29L, M1R, B6R, and A35R. (A) Construction of prokaryotic vectors expressing MPXV A29L, A35R, M1R, or B6R. The coding sequences of MPXV A29L, M1R, B6R, and A35R were transmembrane/cytoplasmic tails deleted, added with the His-tag at C terminal, and cloned into pET-9a plasmid, respectively. (B) Identification of purified A29L, A35R, M1R, and B6R of MPXV via SDS-PAGE. (C) Activity of purified MPXV A29L, A35R, M1R, and B6R was analyzed via WB assays. (D) Activity of purified MPXV A29L, A35R, M1R, and B6R was analyzed via ELISA assays. Data are presented as means ± SEM (n = 3).
Figure 2
Figure 2
Antigen-specific antibody titers in mice immunized with the mixture of A29L, M1R, B6R, and A35R of MPXV. A29L, M1R, B6R, and A35R in combination were formulated with or without adjuvant and used to immunize mice intramuscularly. Sera of mice were sampled at days 0, 14, 28, and 90. Antigen-specific IgG antibody titers were evaluated via ELISA assays. (A) Schematic diagram of immunization and sera collection. (B) Anti-A27L antibody titers. (C) Anti-M1R antibody titers. (D) Anti-A35R antibody titers. (E) Anti-B6R antibody titers. Data are presented as means ± SEM (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significance (p > 0.05).
Figure 3
Figure 3
Cellular response in BALB/c mice immunized with the mixture of A29L, M1R, B6R, and A35R of MPXV. A29L, M1R, B6R, and A35R in combination were formulated with or without adjuvant and used to immunize mice intramuscularly. At day 90, splenocytes were isolated and stimulated a with mixture of A29L, M1R, B6R, and A35R, and T cell immune response was analyzed via flow cytometry analysis and ELISPOT assays. (A–C) IFN-γ, TNF-α, and IL-4 of CD4+ T cells were analyzed via flow cytometry assay. (D–F) IFN-γ, TNF-α, and IL-4 of CD8+ T cells were analyzed via flow cytometry assay. (G) IFN -γ-producing T cells were analyzed via ELISPOT assays. Data are presented as means ± SEM (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significance (p > 0.05).
Figure 4
Figure 4
Antibody titers of neutralizing antibodies against VACV in mice immunized with A29L, M1R, B6R, and A35R of MPXV. Neutralizing antibody levels against VACV were determined via virus neutralization assays. Serum samples were heat-treated to remove complement and other potential neutralizing agents, three-fold serially diluted from 1:10 to 1:2430, incubated with VACV-EGFP, and used to infect BHK21 cells. Serum dilution that completely inactivated VACV-EGFP with no fluorescence signal in cells was designated as neutralizing antibody titer. Data are presented as means ± SEM (n = 3). ***, p < 0.001.
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