BCG-Prime and boost with Esx-5 secretion system deletion mutant leads to better protection against clinical strains of Mycobacterium tuberculosis

Abstract

Although vaccination with BCG prevents disseminated forms of childhood tuberculosis (TB), it does not protect against pulmonary infection or Mycobacterium tuberculosis (Mtb) transmission. In this study, we generated a complete deletion mutant of the Mtb Esx-5 type VII secretion system (Mtb Δesx-5). Mtb Δesx-5 was highly attenuated and safe in immunocompromised mice. When tested as a vaccine candidate to boost BCG-primed immunity, Mtb Δesx-5 improved protection against highly virulent Mtb strains in the murine and guinea pig models of TB. Enhanced protection provided by heterologous BCG-prime plus Mtb Δesx-5 boost regimen was associated with increased pulmonary influx of central memory T cells (T_CM), follicular helper T cells (T_FH) and activated monocytes. Conversely, lower numbers of T cells expressing exhaustion markers were observed in vaccinated animals. Our results suggest that boosting BCG-primed immunity with Mtb Δesx-5 is a potential approach to improve protective immunity against Mtb. Further insight into the mechanism of action of this novel prime-boost approach is warranted.

Keywords: Esx secretion system; Live-attenuated vaccine; Mycobacteria; Tuberculosis.

Fig. 1.. Attenuation of Mtb Δesx-5 in immunocompromised mice.

Survival curve of (A) SCID (n=6) or (B) Rag^−/− mice (n=6) infected IV with ~10⁷ CFU of WT Mtb CDC1551 or two Mtb Δesx-5 deletion mutants. One of two independent experiments is shown. Survival was significantly different for Mtb Δesx-5 versus parental CDC1551 in SCID and Rag^−/− mice (P<0.001, log-rank test). Bacterial burdens were determined at the indicated times in the spleens (C) and lungs (D) of Rag^−/− mice (n=4) infected with 10⁷ CFU IV of WT Mtb CDC1551 or Δesx-5 deletion mutants. The results shown are representative of two experiments. Mean ± SD is plotted. # indicates death or killing owing to extreme morbidity of all the mice in a group.

Fig. 2.. Vaccination of C57BL/6 mice with Mtb Δesx-5 alone or in prime-boost combination.

(A) Experimental design for priming with or without boosting with attenuated strains, followed by Mtb HN878 challenge and subsequent analysis. Bacterial load in lungs (B) and spleens (C). Pathology score over time (D). Data represent individual mice with standard deviation (n=5). *p < 0.05, by ANOVA.

Fig. 3.. Comparison of heterologous versus homologous prime-boost vaccination regimens in mice.

Mice were vaccinated as depicted in panels A and D, and infected with Mtb H37Rv (A-C) or HN878 (D-F). CFUs were determined at the indicated times post challenge in lungs (B and E) and spleens (C and F). One of two independent experiments is shown. Data are mean ± standard deviation for n=4 (B-C). CFU counts in lungs and spleen are shown for each individual mouse and geometric mean is indicated for each group (E-F). *p < 0.05, by ANOVA.

Fig. 4.. Analysis of T cell and myeloid cell subsets in lungs of vaccinated mice.

Flow cytometry analysis of lungs from mice vaccinated and challenged as shown in Fig. 2 A, using cells harvested at 12 weeks post challenge. Dot plots (A) and total cell counts (B) showing CD4⁺ or CD8⁺ CD44^hiCD62L^hi T cells. tSNE plots showing T cell populations of lungs (C) and their total cell count (D). Total cell counts of CD4+ T cells expressing KLRG1 and PD1 expression (E). tSNE plots showing changes in myeloid cell populations (F) and their respective total cell count (G). *p < 0.05 by ANOVA.

Fig. 5.. Protective efficacy vaccination with Mtb Δesx-5 in guinea pigs.

Experimental outlines are depicted in panels A and D. Guinea pigs were vaccinated or not with BCG, Mtb Δesx-5 or Mtb Δesx-3 followed by an aerosol infection with Mtb Beijing (A-C). As determined by CFUs (B) and lung pathology (C) at day 30 post-infection, vaccination with Mtb Δesx-5 conferred protection similar to BCG but superior to Mtb Δesx-3. Graph shows mean ± standard deviation of the CFU of Mtb in lungs, spleens and mesenteric lymph node (MLN) of guinea pigs (n=4) at day 30 post-infection. *P <0.05 by ANOVA. Heterologous prime-boost vaccination with BCG prime followed by Mtb Δesx-5 boost improved guinea pig survival (n=7) compared to BCG group (n=7) and saline group (n=8) (D-E) and lung pathology at day 320 after challenge with Mtb Beijing 212 (F). Ranking analysis of BCG and BCG/Mtb Δesx-5 vaccinated animals, show worse overall pulmonary damage in guinea pigs only vaccinated with BCG (G). *p <0.05 by ANOVA.