Influenza virus NS vectors expressing the mycobacterium tuberculosis ESAT-6 protein induce CD4+ Th1 immune response and protect animals against tuberculosis challenge

Abstract

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 Calmette-Guérin (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. tuberculosis-specific 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.

FIG. 1.

Expression of the ESAT-6 antigen in Vero cells infected with the recombinant influenza virus vectors. (a) Diagram of the recombinant NS1 protein expressed by influenza A vectors. (b) Western blot of lysates of Vero cells infected at an MOI of 2 with recombinant Flu/ESAT-6 viruses of different subtypes (H1N1, H2N2, and H3N2), using monoclonal antibodies to ESAT-6. (c) Expression of the ESAT-6 protein in the nuclei of infected cells, shown for the Flu/ESAT-6 (H1N1) vector by immunofluorescence using polyclonal serum to ESAT-6.

FIG. 2.

ELISPOT assay results. C57BL/6 mice were immunized i.n. at a 3-week interval with Flu/ESAT-6 vectors of different subtypes and combinations as indicated. Ten days after the second immunization, splenocytes were stimulated by adding the ESAT-6 protein (5 μg/ml) or the ESAT-6_1-20 peptide (5 μg/ml). The number of IFN-γ-secreting cells per 10⁶ splenocytes is shown. H1, H2, and H3 indicate the subtypes of Flu/ESAT-6 viral vectors. Tri indicates the mixture of all three Flu/ESAT-6 vector subtypes, and wt indicates the PR8 wild-type virus. Error bars indicate standard errors of the means.

FIG. 3.

Development of lung pathology in mice 40 days after challenge with M. bovis. Lungs of nonvaccinated mice show marked foci of an existing tuberculosis infection (C) and development of necrosis and decreased lung aeration (A). Mice vaccinated with Flu/ESAT-6 vectors show lesser signs of lung pathology (D) and single and small infiltrative foci and aerated lungs (B). The images show representative views of the lungs from vaccinated and control groups of mice. In the control group of mice, the most pronounced pathological changes (large necrosis foci) are shown. Magnification for panels A and B, ×280.

FIG. 4.

T-cell responses of (a) spleens and (b) MLNs. At 40 days after Mycobacterium challenge, mice were sacrificed and the amounts of IFN-γ in the supernatants from cultures of lymphocytes from spleens and MLNs stimulated with ConA or ESAT-6 were examined by ELISA. Groups 2 to 5 were challenged with 10⁶ CFU of virulent M. bovis. Group 1, naïve mice; group 2, nonvaccinated challenged mice; group 3, mice immunized with wt PR8; group 4, BCG-vaccinated mice; group 5, mice vaccinated twice with Flu/ESAT-6 vectors (H1N1-H3N2). Error bars indicate standard errors of the means.

FIG. 5.

Mean weight gain of guinea pigs. Two groups of animals were immunized twice with a trivalent Flu/ESAT-6 vaccine i.n. or s.c., respectively. One group of guinea pigs was immunized with BCG s.c. once at the time of the first immunization. Naïve guinea pigs were used as the control group. Six weeks after the first immunization, the animals were challenged s.c. with M. tuberculosis H37Rv and weighed weekly. Each time point represents the mean weight gain for four guinea pigs. Error bars indicate standard errors of the means.

FIG. 6.

Bacterial load in guinea pigs, showing the protective efficacy in guinea pigs of Flu/ESAT-6 and BCG vaccine nine weeks after s.c. challenge with M. tuberculosis H37Rv (10⁶ CFU). Bacterial loads in lungs and spleens were determined. Data are presented as log₁₀ CFU/organ. Dashes indicate the geometric means of the bacterial loads in the organs of the animals. In all vaccinated groups, the decrease in CFU titers was significant (P < 0.05) based on the Mann-Whitney test.