The mycobacterial Ag85B protein generates dysfunctional
IFN-γ-secreting T cells associated with decreased protection against M. tuberculosis infection in mice by BCG vaccine.

G. Piccaro1a, G. Aquino2, V. Gigantino2, V. Tirelli1b, M Sanchez1b, G. Matarese3, A. Cassone4 and C. Palma1a*
1 Dep. Infectious Diseasesa, FASTb, Istituto Superiore di Sanità, Rome, Italy; 2 Pathology Unit, IRCCS “Fondazione Pascale", Naples, Italy 3 IEOS-CNR, University of Naples,Italy 4 Polo GGB, University of Siena, Italy * carla.palma@iss.it

Correlates of protection are still poorly defined in Tuberculosis (TB). Although IFNγ-producing CD4+ T cells are critical for protection against M. tuberculosis (Mtb) infection (1), they are also implicated in TB pathogenesis and vaccines ineffectiveness (2-4). Here, we investigate whether the Ag85B, a candidate for TB vaccines, decreases protection induced by BCG vaccine with the aim to understand whether this protein, abundantly secreted by Mtb, may act as a virulence factor.
C57BL/6 mice were vaccinated with BCG and then treated (or not) with 10 µg of Ag85B protein at 14 and 28 days (referred as BCG/Ag85B mice). After 4 weeks from the last Ag85B injection, mice were challenged with Mtb and sacrificed at 32 and 80 days post-infection (p.i.) to evaluate degree of pathology, phenotypical and functional immune responses.
Despite generation of robust Ag85B-specific IFNγ-producing CD4+ T cells, Ag85B immunization reduced the protective efficacy of BCG vaccination and worsened infection by increasing bacterial burden and lung immunopathology. Analysis of spleen cells at the time of Mtb challenge, showed that T cells of BCG/Ag85B mice were less capable to proliferate and differentiate into effector Thelper(h)1 and Th22 cells in basal condition and upon in vitro Mtb infection than cells of BCG-only mice. On the other hand, BCG/Ag85B mice generated CD4 T cells, not appreciably recognized by their cognate antigens during in vitro and in vivo Mtb infection, but greatly responding to exogenous Ag85B protein with an exaggerate production of IFN-γ associated to CCL-4, IL-2 and IL-10. Ag85B also modulated the PD-1/PD-L1 axis. All this resulted in a reduced capacity of spleen cells of BCG/Ag85B mice to control intracellular Mtb growth when infected in vitro unless stimulated by the Ag85B protein. Thus, Ag85B immunization reduced the generation of protective BCG-specific responses and made spleen cells of BCG/Ag85B mice dependent on extracellular Ag85B protein to promote Mtb killing. The reduced ability to control Mtb growth persisted even during in vivo infection, as indicated by mycobacterial growth inhibition assays with spleen cells recovered from infected mice. Interestingly, although cells of BCG/Ag85B mice retained ex vivo the ability to produce IFN-γ in response to Ag85B protein this mechanism was no more effective in controlling pathogen growth and was even deleterious at 80 days p.i. Of note, in vivo Mtb infection “per se” generated, in unvaccinated and BCG-vaccinated mice, IFN-γ secreting T cells responding to exogenous Ag85B protein. Again, these cells resulted to be uncapable to enhance pathogen elimination even if stimulated to produce IFN-γ. Moreover, ex vivo restimulation with Ag85B protein of spleen cells of Mtb-infected mice promoted foam cell degeneration, a hallmark of TB pathology.
In conclusion, our data suggest that Mtb may benefit from T cell activation driven by Ag85B protein trough generation of a robust dysfunctional inflammatory Th1 response incapable to eliminate the pathogen. This could explain why the Ag85B antigen is highly conserved. It also raises some concerns about current TB vaccine strategies employing the immunodominant Mtb antigen Ag85B.
References
1. Green AM, et al. 2013. J Immunol 190:270-7
2. Mittrücker HW, et al. 2007. Proc Natl Acad Sci U S A. 104:12434-9
3. Palma C, et al. 2007. Cell Microbiol. 9:1455-65.
4. Jasenosky LD, et al. 2015. Immunol Rev 264:74-87.

Carla Palma PhD

Istituto Superiore di Sanità

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