Mining the CD4 antigen repertoire for next-generation tuberculosis vaccines

Abstract

Tuberculosis (TB) is the leading cause of death from infectious disease worldwide, and Bacillus Calmette-Guérin (BCG) remains the only clinically approved vaccine. An enduring challenge in TB vaccine development is systematic antigen selection from a large repertoire of potential candidates. We performed an efficacy screen in mice of antigens that are targets of CD4 T cells in humans. We found striking heterogeneity in protective efficacy, and most of the top protective antigens are not currently in clinical development. We observed immunologic cross-reactivity among phylogenetically clustered antigens, reflecting common CD4 epitopes. We developed a trivalent mRNA vaccine consisting of PPE20 (Rv1387), EsxG (Rv0287), and PE18 (Rv1788), which augmented and exceeded BCG protection in multiple mouse models. Finally, we observed cellular immune responses to these antigens in 84% of humans exposed to M. tuberculosis. These data advance our understanding of TB vaccine immunology and define a vaccine concept for clinical development.

Keywords: Bacillus Calmette-Guérin; CD4; antigen; mRNA; screen; tuberculosis; vaccine.

Fig. 1.. Large-scale in vivo screening of LTB CD4 and selected clinical antigens.

(A) Protective efficacy of LTB CD4 and selected clinical antigens. Groups of CB6F1 mice were primed and boosted with 50 μg of DNA followed by 100 CFU H37Rv aerosol challenge, lung harvest, and bacterial load quantification. Dots represent individual mice and histograms represent the median fold reduction in lung CFU per vaccine antigen group relative to the median of an internal naïve control group. LTB CD4 antigens are shown in order of decreasing immunodominance. (B) Aggregate protective efficacy of screened antigens. Dots represent the median fold reduction in lung CFU for individual antigens relative to naïve from (A). P value represents a Mann-Whitney U test. ** represents P<0.01. (C) Splenocyte Th1 responses to 8 protective antigens from (A) after 50 μg DNA prime-boost immunization in CB6F1 mice and ex vivo stimulation with overlapping peptide pools. (D) Splenocyte CD8 IFN-γ responses to 8 protective antigens from (A). (E) Boolean analysis of Th1 responses from (C). (F) Splenocyte Th1 responses to selected minimally protective antigens from (A).

Fig. 2.. Phylogenetic and CD4 epitope mapping analysis of protective antigens.

(A) Phylogenetic analysis by Tamura-Nei genetic distance model of screened antigens showing clustering of protective outliers. (B-E) Splenocyte CD4 Th1 (IFN-γ, TNF, and IL-2) responses after 50 μg DNA prime-boost immunization in CB6F1 mice among four protective vaccine antigen phylogenetic clusters following ex vivo stimulation with overlapping peptide pool from autologous and heterologous antigen from each cluster in (A). Graph titles represent the DNA vaccine antigen and x-axes represent the stimulation peptide pools. (F-I) Selected sequence alignments of the two most immunodominant and conserved regions from the four phylogenetic clusters in (A). Black boxes represent amino acid positions with 100% sequence homology, grey boxes represent positons with partial homology, and white boxes represent positions with 0% homology. Colored bars below sequence alignments represent the position of the most immunodominant CD4 16mer peptide for each antigen region.

Fig. 3.. T cell phenotypes and protective efficacy of CD4 antigens mRNA-LNP vaccines.

(A) Splenocyte CD4 Th1 responses after 5 μg mRNA-LNP prime-boost immunization in CB6F1 mice and ex vivo splenocyte overlapping peptide stimulation for the most protective member of each antigen family in Fig. 2. (B) Boolean analysis of Th1 responses from (A). (C) Splenocyte CD8 IFN-γ responses for the same mice from (A). (D) Comparison of the CD4 non-IFN-γ⁺/IFN-γ⁺ ratio after DNA or mRNA-LNP vaccine delivery of CD4 antigens. P values represent Mann-Whitney U tests. ** represents P<0.01. (E) Comparison of the IL-2⁺ fraction after DNA or mRNA-LNP vaccine delivery of CD4 antigens. P values represent Mann-Whitney U tests. ** represents P<0.01. (F) Protective efficacy of monovalent CD4 antigens after 5 μg mRNA-LNP prime-boost immunization in CB6F1 mice, 100 CFU H37Rv aerosol challenge, and lung harvest for bacterial load quantification. P values represent Mann-Whitney U tests for comparisons relative to the naïve group. *** represents P<0.001 and **** represents P<0.0001. (G) Comparison of the protective efficacy of eight CD4 antigens delivered with the DNA or mRNA-LNP vaccine platform. P value represents a Wilcoxon matched-pairs rank test. ** represents P<0.01. (H) Splenocyte CD4 IFN-γ⁺ response after prime-boost trivalent or tetravalent mRNA-LNP immunization (aggregate 20 μg dose divided evenly among antigens). P values represent Mann-Whitney U tests for comparisons relative to the matched antigen in the naïve group. ** represents P<0.01. (I) Protective efficacy of CD4 antigens after prime-boost trivalent or tetravalent mRNA-LNP immunization (aggregate 20 μg dose divided evenly among antigens), 100 CFU H37Rv aerosol challenge, and lung harvest for bacterial load quantification. P values represent Mann-Whitney U tests for comparisons relative to the naïve group. **** represents P<0.0001. (J) Durable protective efficacy after of 15 μg prime-boost trivalent mRNA-LNP immunization, 12 week interval, 100 CFU H37Rv aerosol challenge, and lung harvest for bacterial load quantification. P value represents Mann-Whitney U test relative to naïve. ** represents P<0.01.

Fig. 4.. Immunogenicity and protective efficacy of trivalent mRNA-LNP vaccine compared to BCG in the 100 CFU challenge model.

(A)-(B) Antigen-specific lung CD4 and CD8 IFN-γ⁺ response after BCG prime with or without delayed 15 μg trivalent mRNA-LNP prime-boost in CB6F1 mice. P values represent Mann-Whitney U tests for antigen-specific comparisons (except PPD) between the BCG and mRNA-LNP groups. ** represents P<0.01. (C) Boolean analysis of lung CD4 T cell responses from (A) in the trivalent mRNA-LNP prime-boost group. (D)-(E) Antigen-specific spleen CD4 and CD8 IFN-γ⁺ responses in the same mice from (A). P values represent Mann-Whitney U tests for antigen-specific comparisons (except PPD) between the BCG and mRNA-LNP groups. ** represents P<0.01. (F) Protective efficacy of BCG prime with or without 15 μg trivalent mRNA-LNP delayed prime-boost followed by 100 CFU H37Rv aerosol challenge and lung harvest for bacterial load quantification. P values represent Mann-Whitney U tests. * represents P<0.05 and **** represents P<0.0001. (G)-(H) Antigen-specific lung CD4 and CD8 IFN-γ⁺ response after BCG prime with or without co-administered 15 μg trivalent mRNA-LNP prime-boost in CB6F1 mice. P values represent Mann-Whitney U tests for antigen-specific comparisons (except PPD) between the BCG and mRNA-LNP groups. ** represents P<0.01. (I) Boolean analysis of lung CD4 T cell responses from (G) in the trivalent mRNA-LNP prime-boost group. (J)-(K) Antigen-specific spleen CD4 and CD8 IFN-γ⁺ responses in the same mice from (G). P values represent Mann-Whitney U tests for antigen-specific comparisons (except PPD) between the BCG and mRNA-LNP groups. ** represents P<0.01. (L) Protective efficacy of BCG prime with or without co-administered 15 μg trivalent mRNA-LNP prime-boost followed by100 CFU H37Rv aerosol challenge and lung harvest for bacterial load quantification. P values represent Mann-Whitney U tests. *** represents P<0.001 and **** represents P<0.0001.

Fig. 5.. Immunogenicity and protective efficacy of trivalent mRNA-LNP vaccine compared to BCG in the low dose challenge model.

(A)-(C) Left and right lung lobe bacterial loads after BCG prime with or without 15 μg trivalent mRNA-LNP co-administration in CB6F1 mice followed by 1 MID50 H37Rv aerosol challenge and lung lobe harvest for bacterial load quantification. (D) Comparison of infection rates between the vaccine groups in (A)-(C). P value represents a two-sided Fisher’s exact test. * represents P<0.05. (E) Comparison of bilateral lung lobe dissemination rates among infected mice between the vaccine groups in (A)-(C). P value represents a two-sided Fisher’s exact test. * represents P<0.05. (F) Composite outcome of infection and dissemination between vaccine groups in (A)-(C). P values represent two-sided Fisher’s exact tests. * represents P<0.05. (G) Comparison of bacterial loads between the vaccine groups in (A)-(C). P values represent mixed effects negative binomial models for each vaccine group relative to the naïve group. **** represents P<0.0001. (H)-(J) Left and right lung lobe bacterial loads after BCG prime with or without 15 μg trivalent mRNA-LNP co-administration in C3HeB/Fe mice followed by four weekly 0.3 MID50 H37Rv aerosol challenges and lung lobe harvest for bacterial load quantification. (K)-(L) Comparison of infection and dissemination rates between the vaccine groups in (H)-(J). (M) Composite outcome of infection and dissemination between vaccine groups in (H)-(J). P values represent two-sided Fisher’s exact tests. * represents P<0.05. (N) Pre-challenge PBMC CD4 IFN-γ responses for the vaccine groups in (H)-(J) after ex vivo stimulation with an aggregate peptide pool of Rv1387, Rv0287, and Rv1788. P values represents a Mann-Whitney U tests. ** represents P<0.01 and **** represents P<0.0001.

Fig. 6.. Trivalent vaccine antigen responses during natural infection in humans.

(A) IFN-γ ELISPOT responses among IGRA⁺ ACS participant PMBCs after overnight stimulation with the trivalent vaccine antigens as well as the M72/AS01_E antigens (MTB32A and MTB39A) or a combined ESAT6 and CFP10 positive control. Bottom dotted line represents assay LOD. Top gray line presents media adjusted cutoff for population reactivity calculations. (B) Calculations of population reactivity to individual antigens or antigen combinations representing the trivalent and M72/AS01_E vaccines.