Introduction: Tuberculosis (TB) remains one of the leading infectious diseases worldwide. The Bacillus Calmette-Guérin (BCG) vaccine, the only currently available TB vaccine, offers limited protection in adults. Therefore, it is critical to develop new effective vaccines. Considering Mycobacterium tuberculosis (Mtb) primarily infects the lungs, mucosal vaccines may be a promising strategy, as they can stimulate both systemic and mucosal immunity. This study explores the development and evaluation of a novel mucosal TB vaccine utilizing TB multi-epitopes by producing a recombinant chimeric protein in the mammary gland of goat, and in vivo mucosal immunization by utilizing the stable properties of the chimeric protein.

Methods: An AAV vector carrying TB multi-epitopes and secretory IgA-J chain were co-transduced into the mammary gland of goat, and milk was collected daily. The chimeric protein containing the TB multi-epitopes antigen and secretory components in milk was detected by Western blot against anti-His and anti-Ag85b. The protein was then purified from the milk using the Akta Prime purification system. The mice were immunized using purified protein intranasally, and their immunogenicity responses were evaluated by flow cytometry.

Results: In milk containing chimeric protein, bands corresponding to murine secretory component (75 kDa) and multi-epitopes Antigen Fc-alpha (75 kDa) could be detected against anti-His and anti-Ag85b, respectively. After the chimeric protein was purified, a purity of 95% of chimeric protein was obtained and detected at 75 kDa. Mice immunized with the vaccine candidate were shown to elicit immune response by producing a higher level of activated and memory T cells compared to the control group.

Conclusion: The protein complex formed by TB multi-epitopes-Fc alpha and the murine secretory component can be obtained, purified and detected in milk samples, provided that the mammary gland acts as a potential bioreactor for the production of the recombinant vaccine candidate. The immune response was shown to be induced by the protein complex in mice by producing more activated and memory T cells.