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While Baccillus Calmette-Guerin (BCG) is used worldwide, tuberculosis (TB) is still a global concern due to the poor efficacy of BCG. Novel vaccine candidates are therefore urgently required. In this study, two attenuated recombinant  Listeria  strains, LMΔ -msv  and LIΔ -msv  were constructed by deletion of  actA  and  plcB  and expression of a fusion protein consisting of T cell epitopes from four  Mycobacterium tuberculosis  ( Mtb ) antigens ( Rv2460c, Rv2660c, Rv3875 , and  Rv3804c ). The safety and immunogenicity of the two recombinant strains were evaluated in C57BL/6J mice. After intravenous immunization individually, both recombinant strains entered liver and spleen but eventually were eliminated from these organs after several days. Simultaneously, they induced antigen-specific cell-mediated immunity, indicating that the recombinant  Listeria  strains were immunogenic and safe  in vivo . LMΔ -msv  immunization induced stronger cellular immune responses than LIΔ -msv  immunization, and when boosted with LIΔ -msv , antigen-specific IFN-γ CD8 +  T cell responses were notably magnified. Furthermore, we evaluated the protection conferred by the vaccine candidates against mycobacterial infection via challenging the mice with 1 × 10 7  CFU of BCG. Especially, we tested the feasibility of application of them as heterologous BCG supplement vaccine by immunization of mice with BCG firstly, and boosted with LMΔ -msv  and LIΔ -msv  sequentially before challenging. Combination immune strategy (LMΔ -msv  prime-LIΔ -msv  boost) conferred comparable protection efficacy as BCG alone. More importantly, BCG-vaccinated mice acquired stronger resistance to Mycobacterial challenge when boosted with LMΔ -msv  and LIΔ -msv  sequentially. Our results inferred that heterologous immunization with  Listeria -based recombinant strains boosted BCG-primed protection against pulmonary mycobacterial infection.

Heterologous Boosting With Listeria-Based Recombinant Strains in BCG-Primed Mice Improved Protection Against Pulmonary Mycobacterial Infection