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Tuberculosis is a major global threat to human health. Since the widely used BCG vaccine is poorly effective in adults, there is a demand for the development of a new type of boost tuberculosis vaccine. We designed a novel intranasal tuberculosis vaccine candidate, TB/FLU-04L, which is based on an attenuated influenza A virus vector encoding two mycobacterium antigens, Ag85A and ESAT-6. As tuberculosis is an airborne disease, the ability to induce mucosal immunity is one of the potential advantages of influenza vectors. Sequences of ESAT-6 and Ag85A antigens were inserted into the NS1 open reading frame of the influenza A virus to replace the deleted carboxyl part of the NS1 protein. The vector expressing chimeric NS1 protein appeared to be genetically stable and replication-deficient in mice and non-human primates. Intranasal immunization of C57BL/6 mice or cynomolgus macaques with the TB/FLU-04L vaccine candidate induced Mtb-specific Th1 immune response. Single TB/FLU-04L immunization in mice showed commensurate levels of protection in comparison to BCG and significantly increased the protective effect of BCG when applied in a “prime-boost” scheme. Our findings show that intranasal immunization with the TB/FLU-04L vaccine, which carries two mycobacterium antigens, is safe, and induces a protective immune response against virulent M. tuberculosis.

Brucellosis is a zoonotic disease caused by Gram-negative bacteria. Most of the brucellosis vaccines in the application are whole-bacteria vaccines. Live-attenuated vaccines are widely used for brucellosis prevention in sheep, goats, pigs, and cattle. Thus, there is also a need for an adjuvanted vaccine for human brucellosis, because the attenuated Brucella vaccines now utilized in animals cause human illness. Here, we developed a live-attenuated Brucella suis strain 2 vaccine (S2) adjuvanted with Ag85a (Ag85a-S2). We found that Ag85a-S2 activated cGAS-STING pathways both in intestinal mucosal cells in vivo and in the BMDM and U937 cell line in vitro. We demonstrated that the cGAS knockout significantly downregulated the abundance of interferon and other cytokines induced by Ag85a-S2. Moreover, Ag85a-S2 triggered a stronger cellular immune response compared to S2 alone. In sum, Ag85a-S2-mediated enhancement of immune responses was at least partially dependent on the cGAS-STING pathway. Our results provide a new candidate for preventing Brucella pathogens from livestock, which might reduce the dosage and potential toxicity compared to S2.

Mycobacterium tuberculosis (Mtb) infection is one of the leading causes of death worldwide. The Modified Vaccinia Ankara (MVA) vaccine vector expressing the mycobacterial antigen 85A (MVA85A) was demonstrated to be safe, although it did not improve BCG efficacy, denoting the need to search for improved tuberculosis vaccines. In this work, we investigated the effect of IL-12 DNA -as an adjuvant- on an Ag85A DNA prime/MVA85A boost vaccination regimen. We evaluated the immune response profile elicited in mice and the protection conferred against intratracheal Mtb H37Rv challenge. We observed that the immunization scheme including DNA-A85A+DNA-IL-12/MVA85A induced a strong IFN-γ production to Ag85A in vitro, with a significant expansion of IFN-γ+CD4+ and IFN-γ+CD8+ anti-Ag85A lymphocytes. Furthermore, we also detected a significant increase in the proportion of specific CD8+CD107+ T cells against Ag85A. Additionally, inclusion of IL-12 DNA in the DNA-A85A/MVA85A vaccine scheme induced a marked augment in anti-Ag85A IgG levels. Interestingly, after 30 days of infection with Mtb H37Rv, DNA-A85A+DNA-IL-12/MVA85A vaccinated mice displayed a significant reduction in lung bacterial burden. Together, our findings suggest that IL-12 DNA might be useful as a molecular adjuvant in an Ag85A DNA/MVA prime-boost vaccine against Mtb infection. Fil: Morelli, María Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: del Medico Zajac, Maria Paula. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Pellegrini, Joaquín Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Amiano, Nicolás Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Tateosian, Nancy Liliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Calamante, Gabriela. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Gherardi, Maria Magdalena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina Fil: García, Verónica Edith. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina

Tuberculosis (TB) is one of the deadliest infectious diseases worldwide and is caused by Mycobacterium tuberculosis (Mtb). An effective vaccine to prevent TB is considered the most cost-effective measure for controlling this disease. Many different vaccine antigen (Ag) candidates, including well-known and newly identified Ags, have been evaluated in clinical and preclinical studies. In this study, we took advantage of a plant system of protein expression using Nicotiana benthamiana to produce N-glycosylated antigen 85A (G-Ag85A), which is one of the most well-characterized vaccine Ag candidates in the field of TB vaccines, and compared its immunogenicity and vaccine efficacy with those of nonglycosylated Ag85A (NG-Ag85A) produced with an Escherichia coli system. Notably, G-Ag85A induced a more robust IFN-γ response than NG-Ag85A, which indicated that G-Ag85A is well recognized by the host immune system during Mtb infection. We subsequently compared the vaccine potential of G-Ag85A and NG-Ag85A by evaluating their immunological features and substantial protection efficacies. Interestingly, G-Ag85A yielded moderately enhanced long-term protective efficacy, as measured in terms of bacterial burden and lung inflammation. Strikingly, G-Ag85A-immunized mice showed a more balanced proportion of multifunctional Th1-biased immune responses with sustained IFN-γ response than did NG-Ag85A-immunized mice. Collectively, plant-derived G-Ag85A could induce protective and balanced Th1 responses and confer long-term protection against a hypervirulent Mtb Beijing strain infection, which indicated that plant-produced G-Ag85A might provide an excellent example for the production of an Mtb subunit vaccine Ag and could be an effective platform for the development of anti-TB vaccines.

Mycobacterium ulcerans (MU) infection causes the disfiguring necrotic skin disease, Buruli ulcer (BU). While vaccination with Mycobacterium bovis BCG provides nominal antigenic cross-reactivity for induction of immunity against experimental MU infection, a mycobacterial species with greater genetic homology to Mycobacterium ulcerans may serve as a richer source of cross-protective immunogens and lack the pathological features of MU-based vaccines. Mycobacterium marinum, a highly homologous genetic relative of MU, could be used to satisfy these criteria and, as such, we have generated a recombinant M. marinum strain expressing the immunodominant, protective MU-Ag85A. The immunogenicity and protection achieved by murine vaccination with this strain are superior to standard BCG vaccination and may serve as a foundation for developing more effective BU vaccines.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is currently the only vaccine available against tuberculosis (TB), however, it has various levels of efficacy for preventing pulmonary TB. In this study, a recombinant BCG (rBCG::Ag85A) strain overexpressing the immunodominant Ag85A antigen was constructed, and its immunogenicity and protective efficacy were evaluated. The results showed that the Ag85A protein was successfully overexpressed in rBCG::Ag85A, the Ag85A peptide–MHC complexes on draining lymph node dendritic cells of C57BL/6 mice infected with rBCG were detectable 4 h post-infection. The C57BL/6 mice infected with this strain had stronger antigen-specific interferon-gamma (IFN-γ) responses and higher antibody titers than those immunized with BCG, and the protective experiments showed that the rBCG::Ag85A can provide higher but not significantly protective efficacy against Mycobacterium tuberculosis (M. tuberculosis) H37Rv infection compared with BCG vaccine. Therefore, our results demonstrated the potential of rBCG::Ag85A as a vaccine candidate against TB.

Toll-like receptor (TLR) triggering is an important step in the induction of T helper (Th) type 1 T cells which are key players in protection against the intracellular pathogen Mycobacterium ( M. ) tuberculosis . Here we report on the construction of a fusion protein consisting of a tuberculosis vaccine candidate mycolyl-transferase antigen 85A (Ag85A, Rv3804c) coupled to the outer membrane lipoprotein I (OprI) from Pseudomonas ( P. ) aeruginosa , a documented TLR2/TLR4 trigger. Subcutaneous boosting with this fusion protein in the absence of adjuvant increased significantly the Ag85A-specific humoral but not cellular immune responses of Ag85A-DNA vaccinated mice. Intranasal priming of C57BL/6 mice with live, attenuated Mycobacterium bovis bacille Calmette-Guerin (BCG) vaccine, followed by intranasal boosting with OprI-Ag85A increased systemic and local antigen-specific interferon (IFN)-γ and interleukin (IL)-2 responses in spleen, draining cervical and mediastinal lymph nodes and particularly in lung tissue, as compared to responses in mice only vaccinated with BCG vaccine. Despite enhanced immune responses, boosting with OprI-Ag85A did not increase protective efficacy against M. tuberculosis of either plasmid DNA or BCG vaccine in this experimental setting.