Infection with Mycobacterium tuberculosis remains a major cause of morbidity and mortality all over the world. Since the effectiveness of the only available tuberculosis vaccine, Mycobacterium bovis bacillus CalmetteGuérin (BCG), is suboptimal, there is a strong demand to develop new tuberculosis vaccines. As tuberculosis is an airborne disease, the intranasal route of vaccination might be preferable. Live influenza virus vaccines might be considered as potential vectors for mucosal immunization against various viral or bacterial pathogens, including M. tuberculosis. We generated several subtypes of attenuated recombinant influenza A viruses expressing the 6-kDa early secretory antigenic target protein (ESAT-6) of M. tuberculosis from the NS1 reading frame. We were able to demonstrate the potency of influenza virus NS vectors to induce an M. tuberculosisspecific Th1 immune response in mice. Moreover, intranasal immunization of mice and guinea pigs with such vectors induced protection against mycobacterial challenge, similar to that induced by BCG vaccination.Since reverse genetics methods have been developed for negative-strand RNA viruses (30, 33), influenza viruses can be considered as viral vectors for immunization against different pathogens. The influenza A virus contains a segmented genome consisting of eight negative-strand RNA fragments. Among them, the smallest fragment (NS), encoding two proteins (NS1 and Nep), is a suitable target for genetic manipulation. NS1 is the only nonstructural protein of the influenza virus and has been shown to tolerate relatively long insertions of more than 250 amino acids (23). Moreover, as the NS1 protein is produced in large quantities in infected cells, expression of foreign sequences from the NS1 reading frame should result in a strong immune response against the inserted antigen. It was demonstrated that immunization of mice with NS influenza virus vectors could trigger a CD8 ϩ T-cell response, especially when two vectors belonging to different influenza virus subtypes were used for prime-boost immunizations (12, 39). However, influenza virus NS vectors might be less efficient for the induction of insert-specific T-helper and antibody responses due to the intracellular localization of the NS1 protein leading to a less efficient presentation of linked antigen through the major histocompatibility complex class II pathway.In this work, we tested the potential of recombinant influenza virus NS vectors expressing a Mycobacterium tuberculosis antigen to induce T-helper response after intranasal immunization of mice. It is accepted that control over tuberculosis infection depends on the recruitment of antigen-specific T cells, mainly CD4 ϩ cells, to the lungs and the release of cytokines, particularly gamma interferon (IFN-␥), to trigger the bacterium-killing mechanisms in activated macrophages (14). We constructed a recombinant influenza virus NS gene for the expression of the 6-kDa early secretory antigenic target protein (ESAT-6) derived from M. tuberculosis and introduc...

New strategies providing protection against tuberculosis (TB) are still pending. The airborne nature of Mycobacterium tuberculosis (M.tb) infection assumes that the mucosal delivery of the TB vaccine could be a more promising strategy than the systemic route of immunization. We developed a mucosal TB vaccine candidate based on recombinant attenuated influenza vector (Flu/THSP) co-expressing truncated NS1 protein NS1(1–124) and a full-length TB10.4 and HspX proteins of M.tb within an NS1 protein open reading frame. The Flu/THSP vector was safe and stimulated a systemic TB-specific CD4+ and CD8+ T-cell immune response after intranasal immunization in mice. Double intranasal immunization with the Flu/THSP vector induced protection against two virulent M.tb strains equal to the effect of BCG subcutaneous injection in mice. In a guinea pig TB model, one intranasal immunization with Flu/THSP improved protection against M.tb when tested as a vaccine candidate for boosting BCG-primed immunity. Importantly, enhanced protection provided by a heterologous BCG-prime → Flu/THSP vector boost immunization scheme was associated with a significantly reduced lung and spleen bacterial burden (mean decrease of 0.77 lg CFU and 0.72 lg CFU, respectively) and improved lung pathology 8.5 weeks post-infection with virulent M.tb strain H37Rv.