Circular RNA vaccines against monkeypox virus provide potent protection against vaccinia virus infection in mice

Abstract

Since the outbreak of monkeypox (mpox) in 2022, widespread concern has been placed on imposing an urgent demand for specific vaccines that offer safer and more effective protection. Using an efficient and scalable circular RNA (circRNA) platform, we constructed four circRNA vaccines that could induce robust neutralizing antibodies as well as T cell responses by expressing different surface proteins of mpox virus (MPXV), resulting in potent protection against vaccinia virus (VACV) in mice. Strikingly, the combination of the four circular RNA vaccines demonstrated the best protection against VACV challenge among all the tested vaccines. Our study provides a favorable approach for developing MPXV-specific vaccines by using a circular mRNA platform and opens up novel avenues for future vaccine research.

Keywords: circular RNA vaccines; immune responses; mice; monkeypox virus; protection.

Graphical abstract

Figure 1

Design and characterization of circRNA vaccines (A) circRNA design. The circRNA constructed expresses the MPXV-specific antigens A29L, A35R, M1R, and B6R. (B) The expression of the MPXV-specific antigens A29L, A35R, M1R, and B6R in HEK293T cells was detected by western blotting. (C) The particle diameter and encapsulation deficiency of each circRNA component. (D) IFAs were used to measure the expression of the MPXV-specific antigens A29L, A35R, M1R, and B6R in HEK293T cells.

Figure 2

Humoral immune responses of mice vaccinated with circRNA vaccines (A) Schedule of immunization, sample collection and challenge. Female 6-week-old BALB/c mice were randomly divided into six groups (n = 12) and were immunized with circRNA vaccines or placebo on day 0 and day 14. Serum samples were collected on days 13 and 28 after the initial immunization for analysis of MPXV-specific antibody responses. Two weeks after the second immunization, the mice (n = 6) were challenged intranasally with a lethal dose (1 × 10⁶ PFU) of VTT. (B) Groups of mice. (C–F), The IgG antibody titers against the MPXV-specific antigens A29L, A35R, M1R, and B6R were determined via ELISA. (G) The PRNT₅₀ was determined through neutralization antibody assays based on live VACV (2,000 PFU/mL). The dashed lines on the graphs represent the detection limits of the assays. (H) The PRNT₅₀ was determined through neutralization antibody assays based on live MPXV (700 PFU/mL). The dashed lines on the graphs represent the detection limits of the assays. The data are presented as the mean ± SEM. Significance was calculated using one-way ANOVA with multiple comparison tests (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Figure 3

Cellular immune responses in mice vaccinated with the circRNA vaccine (A–D) Splenocytes were collected 14 days after the second immunization, and the numbers of IFN-γ-, TNF-α-, IL-2-, and IL-4-secreting cells stimulated with the MPXV-A29L, A35R, M1R, and B6R peptides were analyzed via an ELISPOT assay. (E and F) Flow cytometry was employed to evaluate the release of IFN-γ, TNF-α, IL-2, and IL-4 from CD4⁺ and CD8⁺ T cells following stimulation with the MPXV-A29L, A35R, M1R, and B6R peptides. The data are presented as the mean ± SEM. Significance was calculated using one-way ANOVA with multiple comparison tests (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Figure 4

The protective effect of the circRNA vaccines against VTT challenge in mice On the 14th day following the second immunization, the mice (n = 6) were intranasally challenged with VTT (1 × 10⁶ PFU/mouse in 50 μL). (A) Weight changes were monitored for 21 days post infection. (B) Survival curve of mice 21 days after VTT challenge; a weight loss exceeding 25% was considered to indicate death. (C) On the seventh day post challenge, the lungs, turbinate, and trachea were collected, and virus loads were determined by tissue virus titration (n = 3). (D–I) On the seventh day post challenge, H&E staining of lungs from challenged mice was performed. The H&E-stained sections shown are representative results from three test mice. Pictures were taken with a 10× objective. Scale bar, 100 μm. The blue arrow shows pulmonary vein congestion, the green arrow shows lymphocyte infiltration, and the yellow arrow shows necrosis in the alveoli and bronchi. The data are presented as the mean ± SEM. Significance was calculated using one-way ANOVA with multiple comparison tests (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).