A five-antigen Esx-5a fusion delivered as a prime-boost regimen protects against M.tb challenge

Abstract

The development of tuberculosis (TB) vaccines has been hindered by the complex nature of Mycobacterium tuberculosis (M.tb) and the absence of clearly defined immune markers of protection. While Bacillus Calmette-Guerin (BCG) is currently the only licensed TB vaccine, its effectiveness diminishes in adulthood. In our previous research, we identified that boosting BCG with an intranasally administered chimpanzee adenovirus expressing the PPE15 antigen of M.tb (ChAdOx1.PPE15) improved its protection. To enhance the vaccine's efficacy, we combined PPE15 with the other three members of the Esx-5a secretion system and Ag85A into a multi-antigen construct (5Ag). Leveraging the mucosal administration safety of ChAdOx1, we targeted the site of M.tb infection to induce localized mucosal responses, while employing modified vaccinia virus (MVA) to boost systemic immune responses. The combination of these antigens resulted in enhanced BCG protection in both the lungs and spleens of vaccinated mice. These findings provide support for advancing ChAdOx1.5Ag and MVA.5Ag to the next stages of vaccine development.

Keywords: BCG; Esx; mucosa; protection; subunit; tuberculosis; vaccines; viral-vector.

Figure 1

Immune responses following a vaccination with ChAdOx1 vaccines. CB6F1 mice were intradermally vaccinated with ChAdOx1 expressing three antigens of the Esx-5a system. Splenocytes were recovered 2 weeks later and stimulated with peptide pools. Antigen-specific immune responses were measured using IFNγ-ELISpot. Each symbol represents one animal, and the line denotes the median of each group. SFU, spot-forming units.

Figure 2

Immunogenicity of mucosally administered ChAdOx1 vaccines. ChAdOx1 expressing the four antigens of the Esx-5a system was administered intranasally to CB6F1 mice. Antigen-specific CD4⁺ Th1 and CD8⁺ immune responses in the (A, B) bronchoalveolar lavage (n = 3–5), (C, D) lung, and (E, F) spleen were measured 4 weeks after vaccination. n = 5 per group, each symbol represents one animal and the error bars denote the interquartile range.

Figure 3

Efficacy of intranasal ChAdOx1 expressing Esx-5a antigens. (A) CB6F1 mice were primed with i.d. BCG and boosted with i.n. ChAdOx1 10 weeks later. Unvaccinated (Naïve) and mice vaccinated with BCG were used as controls. Four weeks after the last vaccination, all mice were challenged with aerosol M.tb, and 4 weeks later, (B) lung was collected for CFU enumeration. (C) Lung and (D) spleen CFU from repeat challenge. Each dot represents one animal, and the line denotes the median of each group. Kruskal–Wallis followed by multi-comparison testing, *p < 0.05, **p < 0.01, ***p < 0.001. CFU, colony-forming units; i.d., intradermal; i.n., intranasal.

Figure 4

Immunogenicity of ChAdOx1 and MVA expressing a string of five antigens. (A) Schematic of the 5-antigen construct. CB6F1 were vaccinated with either (B) ChAdOx1.5Ag or (C) MVA.5Ag intradermally and were harvested after 2 and 1 week, respectively, for the evaluation of antigen-specific immune responses using IFN-γ ELISpot. Bar represents the median value with an interquartile range of three mice per group. (D) PPE15 (left graph) and Ag85A-specific (right) IgG responses were measured using ELISA. Each dot represents one animal (n = 3), the line denotes the median value, and the dotted line indicates the background antibody level. SFU, spot-forming units; OD, optical density.

Figure 5

Immune responses following mucosal and prime-boost vaccinations. (A) CB6F1 mice were vaccinated intranasally (i.n.) with ChAdOx1.5Ag, with some animals boosted with intradermal MVA.5Ag, and some animals left unvaccinated as per experimental schema. (B–D) PPE15-specific CD4⁺ IFNγ (B) and CD8⁺ IFNγ and TNFα (C, D) were measured in the lung. Vaccine-specific immune responses were also evaluated in the spleen, (E–G) CD4⁺ IFNγ, TNFα, IL2, and (H, I) CD8⁺ IFNγ and TNFα. (J) Ag85A-specific IgG responses were measured by ELISA. n = 6 per group. Each dot represents one animal, and the line denotes the median of each group. Kruskal-Wallis followed by multi-comparison testing, *p < 0.05, **p <0.01.

Figure 6

Boosting the protective efficacy of BCG with viral vectors. (A) CB6F1 mice were primed with BCG and boosted with intranasal C5Ag or CPPE15, and some animals received a second intradermal boost of MVA.5Ag. All animals were challenged with aerosol M.tb and lungs and spleen were harvested at the end of the experiment for bacterial enumeration. (B) Lung and (C) spleen bacterial load. Each symbol represents one animal, and the line denotes the median value of each group. Statistical differences were calculated using Kruskal–Wallis followed by multi-comparison testing. *p < 0.05, **p < 0.01, ***p < 0.001. CFU, colony-forming units.