Novel mRNA vaccines induce potent immunogenicity and afford protection against tuberculosis

Abstract

Introduction: Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), a disease with a severe global burden. The intractability of Mtb has prevented the identification of clear correlates of protection against TB and hindered the development of novel TB vaccines that are urgently required. Lipid nanoparticle (LNP)-formulated mRNA is a highly promising vaccine platform that has yet to be thoroughly applied to TB.

Methods: We selected five Mtb antigens (PPE15, ESAT6, EspC, EsxI, MetE) and evaluated their potential as LNP-formulated mRNA vaccines, both when each antigen was delivered individually, and when all five antigens were combined in a mix regimen (m-Mix).

Results: Each mRNA construct demonstrated unique cellular and humoral immunogenicity, and both m-Mix, as well as the single antigen EsxI, conferred significant protection in a murine Mtb challenge model. Whilst the potent immune responses of each mRNA were maintained when applied as a boost to BCG, there was no additional increase to the efficacy of BCG. Combination of m-Mix with a recombinant, replication-deficient chimpanzee adenovirus (ChAdOx1), in a heterologous prime-boost delivery (C-m-Mix), appeared to result in increased protection upon murine Mtb infection, than either regimen alone.

Discussion: This work warrants further investigation of LNP-formulated mRNA vaccines for TB, whilst indicating the potential of m-Mix and C-m-Mix to progress to further stages of vaccine development.

Keywords: BCG; mRNA; tuberculosis; vaccines; viral-vector.

Figure 1

Antigen-specific immunogenicity of novel TB mRNA vaccines. (A) Immunisation schedule, created with BioRender.com. Groups of CB6F1 mice were vaccinated twice with one of five single antigen mRNA vaccines (m-Single) administered at 5 μg per dose, or an equal mix of all 5 antigens (m-Mix) for the same total dose (1 μg each antigen). Immune responses in the spleen and blood were quantified four weeks post-boost. (B, C) Flow cytometric analysis of IFNγ expression by (B) CD4+ T cells or (C) CD8+ T cells in the spleen, in response to stimulation by relevant antigens listed on x-axis. For clarity, only statistically significant comparisons are shown. (D, E) Heatmaps demonstrate the proportion of triple cytokine-secreting IFNγ+ TNFα+ IL-2+ (D) CD4+ or (E) CD8+ T cells, in response to stimulation by antigens listed on horizontal axis. (F-J) Sera was analysed by ELISA for endpoint IgG titres to (F) PPE15, (G) ESAT6, (H) EspC, (I) EsxI, or (J) MetE. L.O.D. indicates “limit of detection” for minimum calculable endpoint titre; values under L.O.D. were arbitrarily assigned half the L.O.D. value. Each symbol represents response from 1 animal, n=6 per group. (B, C, F-J) Horizontal bars, or (D, E) colour intensity, indicate median. Statistical significance was determined via Kruskal-Wallis ANOVA with Dunn’s test for multiple comparisons, selected comparisons displayed only.

Figure 2

Impact of mRNA dose on PPE15- and EsxI-specific responses in m-Single compared to m-Mix regimen. Groups of CB6F1 mice were vaccinated twice with 1 μg or 5 μg of m-PPE15 or m-EsxI, or an equal mix of all 5 antigens (m-Mix) (5 μg total). Immune responses were quantified in the spleen and blood four weeks post-boost. (A, B) Flow cytometric analysis of IFNγ expression by (A) CD4+ or (B) CD8+ T cells, in response to PPE15 stimulation. (D, E) As for (A, B), except EsxI. (C, F) Sera was analysed by ELISA for endpoint IgG titres to (C) PPE15 or (F) EsxI. L.O.D. indicates “limit of detection” for minimum calculable endpoint titre; values under L.O.D. were arbitrarily assigned half the L.O.D. value. Each symbol represents response from 1 animal, n=6 per group. Horizontal bars indicate median. Statistical significance determined via Kruskal-Wallis ANOVA with Dunn’s test for multiple comparisons, selected comparisons displayed only.

Figure 3

Protective efficacy of novel TB mRNA vaccines against aerosol Mtb infection. (A) Immunisation schedule and experimental schematic, created with BioRender.com. Mice vaccinated with m-Single (5 μg), or m-Mix (5 μg total), were infected 4 weeks post-boost, whilst BCG-vaccinated mice were infected 7 weeks post-vaccination. (B, C) Mtb colony forming units (CFU) in the (B) lungs and (C) spleen of CB6F1 mice, 4 weeks after infection with low-dose aerosol Mtb. Each symbol represents the bacterial load in 1 animal, n=8 per group. Horizontal bars indicate median. Statistical significance determined via Kruskal-Wallis ANOVA with Dunn’s test for multiple comparisons, selected comparisons displayed only.

Figure 4

Evaluation of TB mRNA vaccine immunogenicity when applied as a boost to BCG. Groups of CB6F1 mice were vaccinated with BCG, and after 10 weeks rest, relevant groups were vaccinated twice with single antigen mRNA vaccines (m-Single) or an equal mix of all 5 antigens (m-Mix). Four weeks post-boost, immune responses in the spleen and blood of all animals were quantified. (A, B) Flow cytometric analysis of IFNγ expression by (A) CD4+ or (B) CD8+ T cells in the spleen, in response to stimulation by relevant antigens listed on x-axis. For clarity, only significant statistical comparisons between the naïve group and m-Single groups, or m-Mix, are shown. (C, D) Heatmaps demonstrate the proportion of triple polypositive IFNγ+ TNFα+ IL-2+ (C) CD4+ or (D) CD8+ T cells, in response to stimulation by antigens listed on horizontal axis. (E-I) Sera was analysed by ELISA for endpoint IgG titres to (E) PPE15, (F) ESAT6, (G) EspC, (H) EsxI, or (I) MetE. L.O.D. indicates “limit of detection” for minimum calculable endpoint titre; values under L.O.D. were arbitrarily assigned half the L.O.D. value. Each symbol represents response from 1 animal, n=6 per group. (A, B, E-I) Horizontal bars, or (C, D) colour intensity, indicate median. Statistical significance determined via Kruskal-Wallis ANOVA with Dunn’s test for multiple comparisons, selected comparisons displayed only.

Figure 5

Protective efficacy of novel TB mRNA vaccines, when delivered as a boost to BCG. (A) Immunisation schedule and experimental schematic, created with BioRender.com. BCG-vaccinated mice were allowed 10 weeks rest, before boosting with two doses of m-Single (5 μg), or m-Mix (5 μg total). Infections were performed 4 weeks post-boost, or 17 weeks post-BCG vaccination. (B, C) Mtb colony forming units (CFU) in the (B) lungs and (C) spleen of CB6F1 mice, 4 weeks after challenge with low-dose aerosol Mtb. For clarity, only the statistical comparison between naïve and BCG groups are shown, although all mRNA vaccine groups were significantly reduced relative to naïve. Each symbol represents response from 1 animal, n=8 per group. Horizontal bars indicate median. Statistical significance determined via Kruskal-Wallis ANOVA with Dunn’s test for multiple comparisons.

Figure 6

Systemic immune responses following heterologous ChAdOx-mRNA prime-boost. (A) Immunisation schedule, created with BioRender.com. Groups of CB6F1 mice were vaccinated twice with mRNA (‘m’), once with ChAdOx1 (‘C’), or with one dose of ChAdOx followed by a booster dose of mRNA (‘C-m’), in either a Mix or EsxI experimental arm. Four weeks post-boost, immune responses in the spleen and blood of all animals were quantified. (B, C) Flow cytometric analysis of IFNγ expression by (B) CD4+ or (C) CD8+ T cells in the spleens of animals in Mix groups, in response to stimulation by relevant antigens listed on x-axis. For clarity, only significant comparisons between the naïve and m-Mix or C-m-Mix groups are shown. (D-H) Sera from groups in the Mix category was analysed by ELISA for endpoint IgG titres to (D) PPE15, (E) ESAT6, (F) EspC, (G) EsxI, or (H) MetE. L.O.D. indicates “limit of detection” for minimum calculable endpoint titre; values under L.O.D. were arbitrarily assigned half the L.O.D. value. (I-K) Flow cytometric analysis of IFNγ expression by (I) CD4+ T cells or (J) CD8+ T cells in the spleens of animals in EsxI groups, in response to stimulation by EsxI peptide pool. (K) Sera from groups in the EsxI category was analysed by ELISA for endpoint IgG titres to EsxI. Each symbol represents the response from 1 animal, n=6 per group. Horizontal bars indicate median. Statistical significance determined via Kruskal-Wallis ANOVA with Dunn’s test for multiple comparisons, selected comparisons displayed only.

Figure 7

Lung immune responses following vaccination with the C-m-Mix regimen. Groups of CB6F1 mice were vaccinated twice with m-Mix, once with C-Mix, or with one dose of C-Mix and a booster of m-Mix. Four weeks post-boost, immune responses in the lungs of all animals were quantified. (A, B) Flow cytometric analysis of PPE15-specific CD8+ T cell (A) IFNγ, (B) TNFα, and (C) IL-2 production in the lungs, or EspC-specific CD8+ T cell (D) IFNγ and (E) TNFα production in the lungs. Each symbol represents response from 1 animal, n=6 per group. Horizontal bars indicate median. Statistical significance determined via Kruskal-Wallis ANOVA with Dunn’s test for multiple comparisons, selected comparisons displayed only.

Figure 8

Protective efficacy of heterologous administration of viral vector and mRNA vaccines against TB. (A) Immunisation schedule and experimental schematic, created with BioRender.com. BCG-vaccinated mice were infected 7 weeks post-vaccination, whilst all other vaccine groups were infected 4 weeks post-boost. (B, C) Mtb colony forming units (CFU) in the (B) lungs and (C) spleen of CB6F1 mice, 4 weeks after challenge with low-dose aerosol Mtb. Each symbol represents response from 1 animal, n=8 per group. Horizontal bars indicate median. Statistical significance determined via Kruskal-Wallis ANOVA with Dunn’s test for multiple comparisons, selected comparisons displayed only.