Recombinant proteins A29L, M1R, A35R, and B6R vaccination protects mice from mpox virus challenge

Abstract

Since May 2022, mutant strains of mpox (formerly monkeypox) virus (MPXV) have been rapidly spreading among individuals who have not traveled to endemic areas in multiple locations, including Europe and the United States. Both intracellular and extracellular forms of mpox virus have multiple outer membrane proteins that can stimulate immune response. Here, we investigated the immunogenicity of MPXV structural proteins such as A29L, M1R, A35R, and B6R as a combination vaccine, and the protective effect against the 2022 mpox mutant strain was also evaluated in BALB/c mice. After mixed 15 μg QS-21 adjuvant, all four virus structural proteins were administered subcutaneously to mice. Antibody titers in mouse sera rose sharply after the initial boost, along with an increased capacity of immune cells to produce IFN-γ alongside an elevated level of cellular immunity mediated by Th1 cells. The vaccine-induced neutralizing antibodies significantly inhibited the replication of MPXV in mice and reduced the pathological damage of organs. This study demonstrates the feasibility of a multiple recombinant vaccine for MPXV variant strains.

Keywords: antibody response; mpox virus; recombinant protein; vaccination; virus challenge.

Figure 1

Characterization of multiple recombinant MPXV vaccines from outer membrane proteins constructs and immunization in mice. (A) Detection of MPXV A29L, A35R, M1R, and B6R proteins using western blot. (B) Experimental groups and immunisation regimens. (C) Schematic diagram of immunization regimens. Mice were injected subcutaneously on days 0, 21 and 42, n=8/group. Two weeks after the third immunization, mice were inoculated intranasally and intraperitoneally with MPXV, n=5/group. (D) Immunofluorescence staining with mouse serum to verify the expression of binding antibody. The primary antibody for the positive control was 6x His Tag Monoclonal Antibody, and for the rest of the group, mouse serum was collected after the second boost (D56). (E) HE staining of spleen sections from mice after the second boost (D56) to observe enlargement of the germinal centers induced by the immune response to vaccination.

Figure 2

Humoral immune responses in multiple recombinant vaccine immunization in mice. (A–C) Measurement of serum on the day of post-prime, post-initial boost, post-second boost, and post-challenge (D21, D42, D56, and D61) binding antibodies against MPXV A29L, A35R, M1R, and B6R proteins by ELISA. Data shown represent mean OD 450 nm values (mean + SD) for each group of eight mice, n = 8/group. (D–F) Endpoint IgG titers by ELISA, n = 8/group. (G) Inhibition rate of MPXV virus by neutralizing antibodies in serum as measured by PRNT, n = 8/group at post-prime, post-initial boost, post-second boost, n = 5/group at post-challenge. (H) Plaque reduction assay for MPXV by serum at a dilution of 1:20. (I–L) PRNT of neutralizing antibodies against MPXV live viruses on the day of post-prime, post-initial boost, post-second boost, and post-challenge (D21, D42, D56, and D61). Data shown are geometric mean titers and are mean plus SD. Data were analyzed by one-way ANOVA with a multiple comparison test, n = 8/group at post-prime, post-initial boost, post-second boost, n = 5/group at post-challenge. P < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3

Cellular immune responses and protection against challenges of MPXV in multiple recombinant vaccine immunization in mice. (A, B) T cell responses against MPXV proteins A29L, M1R, A35R, and B6R as measured by IFN-γ ELISpot. Three mice from each group were euthanized 2 weeks after the second boost, before splenocytes were harvested, and a single-cell suspension was stimulated for 30 h with recombinant proteins of A29L, M1R, A35R, and B6R, n = 3/group, each sample contained three replicates. (C) Body weight changes following virus challenges, n = 5/group. (D) qPCR detection of viral loads in the lungs, ovaries, and spleen. Data shown are mean plus SD. Data were analyzed by one-way ANOVA with a multiple comparison test. P < 0.05 was considered statistically significant, n = 5/group, each sample contained three replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 4

HE staining of lung sections from mice after virus challenge to visualize lung lesions. (A) Lung sections of mice in PBS group. (B) Lung sections of mice in QS-21 group. (C) Lung sections of mice in vaccine group. Scale bars from left to right are 200 μm, 100 μm, 50 μm.