Reactogenicity to major tuberculosis antigens absent in BCG is linked to improved protection against Mycobacterium tuberculosis

Abstract

MTBVAC is a live-attenuated Mycobacterium tuberculosis vaccine, currently under clinical development, that contains the major antigens ESAT6 and CFP10. These antigens are absent from the current tuberculosis vaccine, BCG. Here we compare the protection induced by BCG and MTBVAC in several mouse strains that naturally express different MHC haplotypes differentially recognizing ESAT6 and CFP10. MTBVAC induces improved protection in C3H mice, the only of the three tested strains reactive to both ESAT6 and CFP10. Deletion of both antigens in MTBVAC reduces its efficacy to BCG levels, supporting a link between greater efficacy and CFP10- and ESAT6-specific reactogenicity. In addition, MTBVAC (but not BCG) triggers a specific response in human vaccinees against ESAT6 and CFP10. Our results warrant further exploration of this response as potential biomarker of protection in MTBVAC clinical trials.

Conflict of interest statement

C.M. and J.G.-A. are co-inventors in a patent application entitled “Tuberculosis vaccine” filled by the University of Zaragoza (application number: PCT/ES 2007/070051). The remaining authors declare no competing financial interests.

Figures

Figure 1. Expression and secretion of ESAT6 and CFP10 by MTBVAC.

(a) Normalized expression (using sigA as housekeeping gene) of fbpB, esat6 and cfp10 genes in log-phase broth-cultured BCG and MTBVAC. Data in the graph are represented as the relative quantity (RQ) using MTBVAC as comparator. Results are the average from triplicate experiments. Data are mean±s.d. (b,c) Immunoblot analysis of GroEL2, Ag85A, ESAT6 and CFP10 in BCG, MTBVAC and M. tuberculosis protein extracts. (b) Intracellular (left) and supernatant (right) fractions of BCG and MTBVAC were assessed. (c) Comparison between M. tuberculosis and MTBVAC-secreted fractions. A BCG cell lysate sample was used as positive control for GroEL2 detection. Four independent protein extractions of each strain were included in the analysis. Full blots are shown in Supplementary Fig. 7a–c.

Figure 2. Improved protection of MTBVAC compared to BCG is dependent on the host genetics.

(a,b,c) Antigen-specific IFNγ production following stimulation with PPD (5 μg ml−1), ESAT6 (2 μg ml−1), CFP10 (2 μg ml−1) and Ag85B (2 μg ml−1) during 48 h of splenocytes from mock-, BCG- and MTBVAC-vaccinated C57BL/6, BALB/c and C3H/HeNRj mice. (d–i) Lung (d,e,f) and spleen (g,h,i) bacterial load 4 weeks post low-dose H37Rv intranasal challenge. C57BL/6 (d,g), BALB/c (e,h) and C3H/HeNRj (f,i) were vaccinated with BCG, MTBVAC or unvaccinated eight weeks before challenge. (a,b,c) Data are representative from one of two independent experiments (n=5 mice per group per experiment). (d–i) Data in the graphs represent a pool of two independent experiments (n=12 mice per group). All data are mean±s.e.m. (a,b,c) NS, non-significant; ***P<0.001 by unpaired t-student test. (d–i) NS, non-significant; *P<0.05; **P<0.01; ***P<0.001 by one-way ANOVA and Bonferroni post-test.

Figure 3. MTBVAC-induced immune response specific to ESAT6 and CFP10 is protective.

(a) Immunoblot analysis of GroEL2, ESAT6 and CFP10 in MTBVAC and MTBVACΔE6C10 lysate samples. Full blot is shown in Supplementary Fig. 7d. (b,c) Antigen-specific IFNγ production following stimulation with PPD (5 μg ml−1), ESAT6 (2 μg ml−1), CFP10 (2 μg ml−1) and Ag85B (2 μg ml−1) during 48 h of splenocytes from mock-, MTBVAC- and MTBVACΔE6C10-vaccinated C57BL/6 (left) and C3H/HeNRj (right) mice. (d–i) Lung (d,e,f) and spleen (g,h,i) bacterial load 4 weeks post low-dose H37Rv intranasal challenge. C57BL/6 (d,g), BALB/c (e,h) and C3H/HeNRj (f,i) were vaccinated with BCG, MTBVAC, MTBVACΔE6C10 or unvaccinated 8 weeks before challenge. (b,c) Data are representative from one of two independent experiments (n=5 mice per group per experiment). (d,e,g,h) Data are derived from n=6. (f,i) Data represent a pool of two independent experiments (n=12 mice per group). All data are mean±s.e.m. (b,c) NS, non-significant; **P<0.01; ***P<0.001 by unpaired t-student test. (d–i) NS, non-significant; *P<0.05; **P<0.01; ***P<0.001 by one-way ANOVA and Bonferroni post-test.

Figure 4. Ag85B-specific immunogenicity induced by MTBVAC is not protective.

(a,b) Antigen-specific IFNγ production following stimulation with PPD (5 μg ml), ESAT6 (2 μg ml−1), CFP10 (2 μg ml−1) and Ag85B (2 μg ml−1) during 48 h of splenocytes from mock-, MTBVAC- and MTBVACΔAg85B-vaccinated C57BL/6 (left) and C3H/HeNRj (right) mice. (c–e) Lung bacterial load 4 weeks post low-dose H37Rv intranasal challenge. C57BL/6 (c), BALB/c (d) and C3H/HeNRj (e) were vaccinated with BCG, MTBVAC, MTBVACΔAg85B or unvaccinated 8 weeks before challenge. (a,b) Data are representative from one of two independent experiments (n=5 mice per group per experiment). (c–e) Data are derived from n=6. All data are mean±s.e.m. (a,b) NS, non-significant; **P<0.01; ***P<0.001 by unpaired t-student test. (c–e) *P<0.05; **P<0.01; ***P<0.001 by one-way ANOVA and Bonferroni post-test.

Figure 5. Enriched in vivo expression of genes from ESX-1 secretion system.

(a,b) Expression of esat6, cfp10, espA, espC, fbpA and fbpB genes (normalized with 16s gene expression) from H37Rv isolated from lungs from C3H/HeNRj (a) and C57BL/6 (b) 4 weeks after high-dose (103 CFU) intranasal challenge, in comparison to expression obtained under 7H9-culture in vitro conditions. Normalized results are represented for each gene and experimental condition as the fold-change induction in comparison to fbpB expression level. In vivo data are derived from n=4 mice (a) and n=6 mice (b). In vitro data represent a pool of four independent RNA extractions. All data are mean±s.e.m. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 by unpaired t-student test.

Figure 6. Vaccination with MTBVAC induces a CFP10-specific immune response in humans.

Specific IFNγ ELISPOT results from the first-in-human MTBVAC clinical trial are shown for CFP10 (a,b) and ESAT6 (c,d), comparing for each volunteer the number of spots pre- and post-BCG (b,d) and MTBVAC (a,c) vaccination with 5 × 105 CFU. Wilcoxon matched-pairs signed rank test was used to compare pre- and post-vaccination status for each vaccine and antigen. P value is indicated for MTBVAC vaccinees.