Heat-inactivated Mycobacterium bovis and P22PI protein immunocomplex: Two candidates for use as immunostimulants of innate immune response

Tuberculosis (TB), caused by members of the Mycobacterium tuberculosis complex, remains a critical global health challenge, affecting humans and a wide range of domestic and wild animals. Despite the availability of anti-TB drugs, cure rates remain suboptimal, exacerbated by the rise of multidrug-resistant TB strains. The Bacille Calmette-Guérin (BCG) vaccine, the only licensed vaccine against TB, has demonstrated efficacy in reducing lesion severity and bacterial burden in animals, as well as lowering TB-related and all-cause mortality in infants. However, BCG presents several safety concerns inherent to live vaccines. To overcome these limitations, exploring alternative vaccine candidates that do not incorporate live mycobacteria is crucial. This study aimed to evaluate and compare the immunostimulatory potential of two candidates based in mycobacteria inactivated or their derivatives, heat-inactivated Mycobacterium bovis (HIMB) and P22PI protein immunocomplex (P22PI), in bovine foetal lung cells. To assess the expression of innate immune components, including Toll-like receptors (TLRs), cathelicidins, and cytokines, bovine foetal lung were exposed to different concentrations of HIMB and P22PI immunostimulants, starting at 7.8 × 10⁶ CFU/ml and 10 µg/ml, respectively. These initial concentrations were subsequently diluted to 1/2 and 1/10 to evaluate dose-dependent effects. Our findings reveal that both HIMB and P22PI significantly stimulate innate immune mechanisms, as evidenced by the upregulation of TLR2 and TLR4, alongside the induction of BMAP28 cathelicidin, tumour necrosis factor alpha (TNFA) and interferons (IFNs). These results suggest their potential to orchestrate a robust innate immune response providing valuable insights into the immunological mechanisms underlying the protective effects of these immunostimulants. This underscores their potential role in in vivo studies as vaccine candidates. Furthermore, their ability to enhance antigen recognition via TLR and induce pro-inflammatory cytokines also indicates broader applications in immune modulation, potentially extending protection against heterologous pathogens through trained immunity.