CHIKV mRNA vaccines encoding conserved structural/envelope proteins confer broad cross-lineage protection against infection

Abstract

With the broad spread of the chikungunya virus (CHIKV), there is an increasing demand for more effective and broadly protective vaccines. Here, we designed CHIKV mRNA vaccines containing full-length structural proteins or part of structural proteins (envelope proteins) based on conserved sequences from 769 viral strains encompassing four lineages. The vaccine induced strong cellular and humoral immune responses in BALB/c mice and provided robust protection. Immunization of BALB/c mice with either of the two vaccines induced high levels of neutralizing antibodies against pseudoviruses from four distinct lineages, highlighting their potential for broad cross-lineage protective efficacy. Immunoglobulin repertoire analysis revealed two important BCR V-J gene combinations, IgHV1-4-IgHJ3 and IgHV1-4-IgHJ2, and lineage-specific immunity analysis revealed significant upregulation of TCRs containing V19 and V20. BCR and TCR immunodiversity may be a potential reason for the broad-spectrum protection against CHIKV afforded by the vaccine. In A129 mice, it elicited lower levels of neutralizing antibodies but prevented mouse mortality and cleared chronic infection. In the rhesus macaque model, both vaccines elicited a certain level of humoral and cellular immune responses and protected the rhesus macaques from the CHIKV challenge. In conclusion, the results from both mouse and rhesus macaque models indicate that the vaccine could be a candidate for clinical use against CHIKV.

Fig. 1

Vaccine design, preparation, and in vitro expression. a Vaccine design. A total of 769 strains were identified from the NCBI database, including 14 from the West African lineage, 335 from the Asian lineage, 121 from the East, Central, and South African lineage, and 299 from the Indian Ocean lineage. Conserved sequences were selected for further analysis; b Utilize Alpha-Fold3 for structural prediction and receptor interaction prediction of the E protein in vaccine sequences and viral sequences (MH670649.1); c Dynamic light scattering (DLS) particle size distribution; d Transmission electron microscopy (TEM) images; e E1, E2, C protein, and β-actin Western blot images; (−) indicates the negative control, and M indicates the control transfected with a commercial kit; f Immunofluorescence experiments were conducted to detect the distribution of the E1 protein, using nontransfected cells as the mock condition

Fig. 2

mCV-1 and mCV-2 induced strong immune responses in BALB/c mice. a The vaccination and sample collection timeline, the collection of serum for cytokine quantification at 5 and 24 h after the initial immunization, serum collection on days 7, 14, 21, and 28, spleen collection on day 28, and challenge experiments on day 42; b The changes in relevant cytokine levels at 5 and 24 h post-immunization, normalized to the levels at 0 hours and logarithmically transformed; log₂FC (0 h) means the ratio of cytokine level at 5 or 24 h to that at 0 h; c and d The titers of binding antibodies (c) and neutralizing antibodies against adapted strains (d) at different time points; e-f, GMTs of pseudovirus neutralizing antibodies against various strains at 28-day post-immunization for mCV-1 (e) and mCV-2 (f). The data are presented as the mean ± SEM (n = 5), and each symbol represents a mouse. Statistical analysis was conducted using one-way ANOVA and Tukey’s multiple comparison tests for bar graphs; ***p < 0.001; ****p < 0.0001; ns, not significant

Fig. 3

Cellular immune response assessment. a–f Elispot experiments to enumerate specific spots indicating cytokine secretion by splenocytes stimulated with the E2 protein for IFN-γ (a), IL-2 (c), and IL-4 (e) and stimulated with the inactivated virus for IFN-γ (b), IL-2 (d), and IL-4 (f); g–j Flow cytometry analysis of the percentages of CD4+ T cells producing IL-2 (g), IL-4 (h), IFN-γ (i), and TNF-α (j); k–m The proportions of CD8+ T cells producing IL-2 (k), IFN-γ (l), and TNF-α (m). The data are presented as the mean ± SEM (n = 5 or 3). Statistical analysis was conducted using two-way ANOVA and Tukey’s multiple comparison test for bar graphs; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant

Fig. 4

mCV-1 and mCV-2 protect BALB/c mice from CHIKV challenge. a and b Changes in viremia within 7 days post-immunization with mCV-1 (a) and mCV-2 (b); Tissue viral loads in mice immunized with mCV-1 (c) and mCV-2 (d) on day 7 post-challenge; e Joint tissue pathological sections and pathological scores, (n = 10). The data are shown as the mean ± SEM. Statistical analysis was conducted using two-way ANOVA and Tukey’s multiple comparison test for bar graphs; ***p < 0.001; ****p < 0.0001

Fig. 5

mCV-1 and mCV-2 protect A129 mice from mortality due to CHIKV infection. a The vaccination and sample collection timeline, Serum collection, and determination of binding antibody titers and neutralizing antibody levels were performed on days 21 and 28 after initial immunization, followed by challenge experiments on day 42. Body temperature, body weight, and joint changes were monitored daily after the challenge until all mice in the placebo group died, and survival curves were plotted; b The titers of binding antibodies at days 21 and 28; c Neutralizing antibody titers at 21, 28, and 56 days post-immunization; d Variation in viremia within 21 days post-challenge; e and f Changes in body weight (e) and the degree of swelling in the right hind limb joints (f) of A129 mice post-challenge; g Survival rate of mice post-challenge; h Viral loads in the spleen and joint tissues of mice on day 21 post-challenge; i Elispots experiments to enumerate specific spots indicating cytokine secretion by splenocytes stimulated with the E2 protein for IFN-γ, IL-2, and IL-4; j-k, Joint tissue pathological sections (k) and pathological scores (j). Statistical analysis was conducted using one-way ANOVA and Tukey’s multiple comparison tests for bar graphs; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant

Fig. 6

BCR and TCR immune repertoire analysis in BALB/c. a Abundance of immunoglobulin types in the three groups. b Statistical analysis of immunoglobulin diversity; c Analysis of the gene mutation of IgM, IgD, IgA and IgG; d and e Significant differences in the abundance of the 15 most abundant V-J gene combinations for BCRs for IgM and IgD; f VDJ matrix of TCR sequences for the three samples; g and h Significant differences in the V-J gene combinations containing V19 (g) and V20 (h) for TCRs. Statistical analysis was conducted using one-way ANOVA and Tukey’s multiple comparison tests for bar graphs; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant

Fig. 7

mCV-1 and mCV-2 protect rhesus macaques from CHIKV challenge. a The immunization schedule for rhesus macaques is depicted in the flowchart, with vaccinations administered on Day 0 and Day 21, at a dosage of 300 μg per animal, using physiological saline as the placebo group. Serum samples were collected on Days 7, 14, 21, 28, and 35 post-primary immunization, followed by a challenging experiment on Day 42 (n = 3); b and c The titers of binding antibodies (b) and neutralizing antibodies against adapted strains (c) at different time points; d GMTs of pseudovirus neutralizing antibodies against various strains at 35-day post-immunization for mCV-1 and mCV-2; e Elispot experiments to enumerate specific spots indicating cytokine secretion by splenocytes stimulated with the E2 protein for IFN-γ, IL-2, and IL-4; f Changes in viremia within 7 days post-immunization with mCV-1 and mCV-2; g Tissue viral loads in rhesus macaques immunized with mCV-1, mCV-2 or physiological saline on day 7 post-challenge, LN: Hilar lymph nodes, Kid: Kidney, PA: Pancreas, ILN: Inguinal lymph nodes, MLN: mesenteric lymph nodes, SMG: submandibular lymph node; h and i pathological scores (h) and pathological sections (i). The data are presented as The mean ± SEM (n = 3), and each symbol represents a mouse. Statistical analysis was conducted using one-way ANOVA and Tukey’s multiple comparison tests for bar graphs; ***p < 0.001; ****p < 0.0001; ns not significant