Immune responses against Mycobacterium tuberculosis-specific proteins PE35 and CFP10 in mice immunized with recombinant Mycobacterium vaccae.

OBJECTIVE To clone and express Mycobacterium tuberculosis (M. tuberculosis) proteins PE35 and culture filtrate protein (CFP)10 in Mycobacterium vaccae (M. vaccae), and subsequently, evaluate the humoral and cellular immunity responses against these recombinant constructs in mice. METHODS The DNA of PE35 and CFP 10 genes were cloned into the shuttle plasmid pDE22, and the recombinant plasmids were electroporated into M. vaccae. The recombinant constructs were then tested for expression of PE35 and CFP10 by Western immunoblotting using rabbit anti-sera. Furthermore, splenocytes and sera from groups of 5 mice immunized with recombinant M. vaccae (rVaccae) were tested for cellular and humoral responses in proliferation, and antibody assays. Experiments were carried out in the laboratory of the Faculty of Medicine, Kuwait University, Safat, Kuwait between 2009 and 2011. RESULTS The results of Western immunoblot suggested the expression of only PE35. However, splenocyte assays showed positive proliferation in response to peptide pools, and 4 and 5 of the 6 overlapping synthetic peptides covering the sequence of PE35 and CFP10. In addition, positive antibody reactivity was detected with PE35 peptide pool and a single peptide, namely, P2. CONCLUSION The expression of PE35 and CFP10 proteins in rVaccae constructs led to the induction of cellular immune responses to multiple epitopes.