An Mpox Multi-Antigen-Tandem Bivalent mRNA Candidate Vaccine Effectively Protects Mice Against the Vaccinia Virus

Abstract

Background: Since the outbreak of mpox in 2022, the disease has spread rapidly worldwide and garnered significant public attention. Vaccination is regarded as an effective measure to prevent the spread of mpox. The success of the COVID-19 mRNA vaccine demonstrates that mRNA-based vaccines represent a rapid and multifunctional platform with considerable potential, and are expected to be a strategy to address mpox spread.

Methods: In this study, we screened an mpox multi-antigen-tandem bivalent mRNA vaccine candidate: a lipid nanoparticle-encapsulated mRNA-1017 and mRNA-1995 (mRNA-3012-LNP). We then evaluated the immunogenicity of the mpox virus (MPXV) bivalent mRNA vaccine candidate and its protective efficacy against the vaccinia virus (VACV) in a mouse model.

Results: Mice vaccinated with two doses of the mRNA-3012-LNP vaccine exhibited robust binding antibody responses and MPXV-specific Th-1-biased cellular immune responses in vivo. Notably, the boosted immunized mice generated potent neutralizing antibodies against the VACV, effectively protecting them from viral challenge. Additionally, serum transfer protection experiments indicated that serum from mice inoculated with mRNA-3012-LNP was effective in protecting nude mice from VACV challenge.

Conclusions: Our results suggest that the mpox bivalent mRNA candidate vaccine mRNA-3012-LNP induces strong immunogenicity and has the potential to serve as a safe and effective vaccine candidate against mpox epidemics.

Keywords: VACV; bivalent; mRNA vaccine; mpox; tandem.

Figure 1

Design, screening, and characterization of MPXV bivalent mRNA vaccine candidates. (A) Sequence design of mRNAs and design of a bivalent mRNA vaccine candidate for mpox. ECD, extracellular domain; 5′ UTR, 5′-untranslated region; 3′ UTR, 3′-untranslated region; GS, GS linker. (B) The expression of mRNA in cells was analyzed using Western blotting. In vitro synthesized mRNA was transfected into HEK-293T cells, and the cells were lysed 24 h later to collect the lysates, which were subsequently validated by Western blotting. (C) The comparison of IgG titers induced by four mRNA vaccines was conducted using ELISA. (D) The comparison of neutralizing antibody titers induced by four mRNA vaccines was performed using an rTV-Fluc-based neutralization assay. The data are shown as medians, with dashed lines indicating detection limits. Significance was calculated using the Mann–Whitney test (ns, not significant; * p < 0.05 and ** p < 0.01).

Figure 2

Systematic evaluation of the effect of mRNA-3012-LNP on humoral immunization in mice in vivo. (A) The experimental schedule for BALB/c mice encompasses immunization, serum collection, spleen collection, and the timing of viral challenges. (B–F) Systematic evaluation of mRNA-3012-LNP-induced IgG titers by ELISA. (G) Systematic evaluation of RNA-3012-LNP-induced neutralizing antibody titers by rTV-Fluc-based neutralization assay. The data are shown as medians, with dashed lines indicating detection limits. Significant differences were calculated by a Kruskal–Wallis test with Dunn’s multiple comparisons (* p < 0.05; ** p < 0.01; and *** p < 0.001).

Figure 3

MPXV-specific cellular immune response in mice vaccinated with mRNA-3012-LNP. BALB/c mice (n = 5 per group) were immunized intramuscularly with two doses of 25 μg mRNA-3012-LNP, with DPBS serving as the placebo control. On day 30 after the initial immunization, spleens were removed from the mice, and splenocytes were isolated. The evaluation of mRNA-3012-LNP-induced cellular immune responses was conducted following the stimulation of splenocytes using an MPXV-specific peptide library. (A–D) MPXV-specific CD4⁺ and CD8⁺ Tem cells in mouse splenocytes were assessed by flow cytometry. (E) ELISPOT was used to evaluate the level of I cytokines secreted by splenocytes. The data (B, D, IL-2, TNF-α, and IL-4) are presented as mean ± SEM. Significance was calculated using an unpaired t-test. The data (IFN-γ and IL-6) are presented as medians. Significance was calculated using the Mann–Whitney test (ns, not significant; ** p < 0.001; *** p < 0.001 and **** p < 0.0001).

Figure 4

Protection efficacy of mRNA-3012-LNP against VACV challenge in mice. The immunoprotective effects of mRNA-3012-LNP were assessed using a firefly luciferase-based rTV-Fluc experimental animal model. BALB/c mice (n = 3) were immunized intramuscularly with two doses of either 10 μg or 25 μg of mRNA-3012-LNP, with the two immunizations administered 14 days apart; DPBS served as a placebo control. Serum was collected from all mice on day 24 after the initial immunization. (A,B) The protective effect of mRNA-3012-LNP was verified by subcutaneous injection of 7 × 10⁶ TCID₅₀ of the rTV-Fluc strain into mice on day 30 post-initial immunization. Bioluminescence measurements were performed on the mice 24 h after the viral challenge. (C,D) The protective effect of in vitro transfer of mRNA-3012-LNP-immunized mouse serum was evaluated. A total of 20 μL of serum was incubated in vitro with a mixture of 4 × 10³ TCID₅₀ of the rTV-Fluc virulent strain for 1 h, followed by injection into 4-week-old nude mice (n = 3) via tail vein and intraperitoneal injection. Bioluminescence measurements were conducted on the nude mice 24 h later. (E,F) Evaluation of in vivo transfer protection of mRNA-3012-LNP-immunized mouse serum was assessed by injecting 50 μL of serum into 4-week-old nude mice (n = 3) via tail vein. One hour later, 4.9 × 10⁶ TCID₅₀ of the rTV-Fluc strain was injected subcutaneously. Bioluminescence measurements were performed on the nude mice 24 h after the viral challenge. Data are shown as mean ± SEM. Significance was calculated by a one-way ANOVA with multiple comparisons (ns, not significant; ** p < 0.01; *** p < 0.001; and **** p < 0.0001).

Figure 5

Acute and immunological toxicity evaluation of the mRNA-3012-LNP vaccine in mice. BALB/c mice were immunized intramuscularly with 25 μg of mRNA-3012-LNP, while DPBS served as a placebo control. Serum and major organ tissues were collected 48 h later. (A) H&E staining of major organ sections. Scale bar = 200 μm. (B–D) Detection of biochemical indices in mouse serum, which included kidney parameters (uric acid (UA), urea, and creatinine (CREA)), liver parameters (alanine aminotransferase (ALT) and alkaline phosphatase (ALP)), and heart parameters (creatine kinase (CK), creatine kinase isoenzyme (CK-MB), and lactate dehydrogenase isoenzyme-1 (LDH-1)). (E) Assessment of cytokine levels in mouse serum. The data (UA, CREA, ALT, ALP, CK-MB, LDH1, and (E)) are presented as mean ± SEM. Significance was calculated using an unpaired t-test. The data (IFN-γ and IL-6) are presented as medians. Significance was calculated using the Mann–Whitney test (ns, not significant).