Intranasal immunization with Ag85B peptide 25 displayed on Lactococcus lactis using the PilVax platform induces...

Mycobacterium tuberculosis (Mtb) remains a global epidemic despite the widespread use of Bacillus Calmette-Guérin (BCG). Consequently, novel vaccines are required to facilitate a reduction in Mtb morbidity and mortality. PilVax is a peptide delivery strategy for the generation of highly specific mucosal immune responses and is based on the food-grade bacterium Lactococcus lactis that is used to express selected peptides engineered within the Streptococcus pyogenes M1T1 pilus, allowing for peptide amplification, stabilization and enhanced immunogenicity. In the present study, the dominant T-cell epitope from the Mtb protein Ag85B was genetically engineered into the pilus backbone subunit and expressed on the surface of L. lactis. Western blot and flow cytometry confirmed formation of pilus containing the peptide DNA sequence. B-cell responses in intranasally vaccinated mice were analyzed by ELISA while T-cell responses were analyzed by flow cytometry. Serum titers of peptide-specific immunoglobulin (Ig) G and IgA were detected, confirming that vaccination produced antibodies against the cognate peptide. Peptide-specific IgA was also detected across several mucosal sites sampled. Peptide-specific CD4⁺ T cells were detected at levels similar to those of mice immunized with BCG. PilVax immunization resulted in an unexpected increase in the numbers of CD3⁺ CD4^- CD8^- [double negative (DN)] T cells in the lungs of vaccinated mice. Analysis of cytokine production following stimulation with the cognate peptide showed the major cytokine producing cells to be CD4⁺ T cells and DN T cells. This study provides insight into the antibody and peptide-specific cellular immune responses generated by PilVax vaccination and demonstrates the suitability of this vaccine for conducting a protection study.