A Mycobacterium tuberculosis-specific subunit vaccine that provides synergistic immunity upon co-administration with Bacillus Calmette-Guérin.

Given the encouraging clinical results of both candidate subunit vaccines and revaccination with Bacillus Calmette-Guérin (BCG) against tuberculosis (TB), there is support for combining BCG and subunit vaccination for increased efficacy. BCG and Mycobacterium tuberculosis (Mtb) share ~98% of their genome and current subunit vaccines are almost exclusively designed as BCG boosters. The goal of this study is to design a TB subunit vaccine composed of antigens not shared with BCG and explore the advantages of this design in a BCG + subunit co-administration vaccine strategy. Eight protective antigens are selected to create an Mtb-specific subunit vaccine, named H107. Whereas traditional vaccines containing BCG-shared antigens exhibit in vivo cross-reactivity to BCG, H107 shows no cross-reactivity and does not inhibit BCG colonization. Instead, co-administering H107 with BCG leads to increased adaptive responses against both H107 and BCG. Importantly, rather than expanding BCG-primed T cells, H107 broadens the overall vaccine repertoire with new T cell clones and introduces 'adjuvant-imprinted' qualities including Th17 responses and less-differentiated Th1 cells. Collectively, these features of H107 are associated with a substantial increase in long-term protection.

In Vivo Antigen Expression Regulates CD4 T Cell Differentiation and Vaccine Efficacy against Mycobacterium tuberculosis Infection.

New vaccines are urgently needed against Mycobacterium tuberculosis (Mtb), which kills more than 1.4 million people each year. CD4 T cell differentiation is a key determinant of protective immunity against Mtb, but it is not fully understood how host-pathogen interactions shape individual antigen-specific T cell populations and their protective capacity. Here, we investigated the immunodominant Mtb antigen, MPT70, which is upregulated in response to gamma interferon (IFN-γ) or nutrient/oxygen deprivation of in vitro-infected macrophages. Using a murine aerosol infection model, we compared the in vivo expression kinetics of MPT70 to a constitutively expressed antigen, ESAT-6, and analyzed their corresponding CD4 T cell phenotype and vaccine protection. For wild-type Mtb, we found that in vivo expression of MPT70 was delayed compared to ESAT-6. This delayed expression was associated with induction of less differentiated MPT70-specific CD4 T cells but, compared to ESAT-6, also reduced protection after vaccination. In contrast, infection with an MPT70-overexpressing Mtb strain promoted highly differentiated KLRG1+CX3CR1+ CD4 T cells with limited lung-homing capacity. Importantly, this differentiated phenotype could be prevented by vaccination, and against the overexpressing strain, vaccination with MPT70 conferred protection similar to vaccination with ESAT-6. Together, our data indicate that high in vivo antigen expression drives T cells toward terminal differentiation and that targeted vaccination with adjuvanted protein can counteract this phenomenon by maintaining T cells in a protective less differentiated state. These observations shed new light on host-pathogen interactions and provide guidance on how future Mtb vaccines can be designed to tip the immune balance in favor of the host.IMPORTANCE Tuberculosis, caused by Mtb, constitutes a global health crisis of massive proportions and the impact of the current coronavirus disease 2019 (COVID-19) pandemic is expected to cause a rise in tuberculosis-related deaths. Improved vaccines are therefore needed more than ever, but a lack of knowledge on protective immunity hampers their development. The present study shows that constitutively expressed antigens with high availability drive highly differentiated CD4 T cells with diminished protective capacity, which could be a survival strategy by Mtb to evade T cell immunity against key antigens. We demonstrate that immunization with such antigens can counteract this phenomenon by maintaining antigen-specific T cells in a state of low differentiation. Future vaccine strategies should therefore explore combinations of multiple highly expressed antigens and we suggest that T cell differentiation could be used as a readily measurable parameter to identify these in both preclinical and clinical studies.

In vivo antigen expression regulates CD4 T cell differentiation and vaccine efficacy against Mycobacterium tuberculosis infection.

New vaccines are urgently needed against Mycobacterium tuberculosis (Mtb), which kills more than 1.4 million people each year. CD4 T cell differentiation is a key determinant of protective immunity against Mtb, but it is not fully understood how host-pathogen interactions shape individual antigen-specific T cell populations and their protective capacity. Here, we investigated the immunodominant Mtb antigen, MPT70, which is upregulated in response to IFN-γ or nutrient/oxygen deprivation of in vitro infected macrophages. Using a murine aerosol infection model, we compared the in vivo expression kinetics of MPT70 to a constitutively expressed antigen, ESAT-6, and analysed their corresponding CD4 T cell phenotype and vaccine-protection. For wild-type Mtb, we found that in vivo expression of MPT70 was delayed compared to ESAT-6. This delayed expression was associated with induction of less differentiated MPT70-specific CD4 T cells but, compared to ESAT-6, also reduced protection after vaccination. In contrast, infection with an MPT70-overexpressing Mtb strain promoted highly differentiated KLRG1+CX3CR1+ CD4 T cells with limited lung-homing capacity. Importantly, this differentiated phenotype could be prevented by vaccination and, against the overexpressing strain, vaccination with MPT70 conferred similar protection as ESAT-6. Together our data indicate that high in vivo antigen expression drives T cells towards terminal differentiation and that targeted vaccination with adjuvanted protein can counteract this phenomenon by maintaining T cells in a protective less-differentiated state. These observations shed new light on host-pathogen interactions and provide guidance on how future Mtb vaccines can be designed to tip the immune-balance in favor of the host.

Rescuing ESAT-6 Specific CD4 T Cells From Terminal Differentiation Is Critical for Long-Term Control of Murine Mtb Infection.

In most cases, Mycobacterium tuberculosis (Mtb) causes life-long chronic infections, which poses unique challenges for the immune system. Most of the current tuberculosis (TB) subunit vaccines incorporate immunodominant antigens and at this point, it is poorly understood how the CD4 T cell subsets recognizing these antigens are affected during long-term infection. Very little is known about the requirements for sustainable vaccine protection against TB. To explore this, we screened 62 human-recognized Mtb antigens during chronic murine Mtb infection and identified the four most immunodominant antigens in this setting (MPT70, Rv3020c, and Rv3019c and ESAT-6). Combined into a subunit vaccine, this fusion protein induced robust protection both in a standard short-term model and in a long-term infection model where immunity from BCG waned. Importantly, replacement of ESAT-6 with another ESAT-6-family antigen, Rv1198, led to similar short-term protection but a complete loss of bacterial control during chronic infection. This observation was further underscored, as the ESAT-6 containing vaccine mediated sustainable protection in a model of post-exposure vaccination, where the ESAT-6-replacement vaccine did not. An individual comparison of the CD4 T cell responses during Mtb infection revealed that ESAT-6-specific T cells were more terminally differentiated than the other immunodominant antigens and immunization with the ESAT-6 containing vaccine led to substantially greater reduction in the overall T cell differentiation status. Our data therefore associates long-term bacterial control with the ability of a vaccine to rescue infection-driven CD4T cell differentiation and future TB antigen discovery programs should focus on identifying antigens with the highest accompanying T cell differentiation, like ESAT-6. This also highlights the importance of long-term readouts in both preclinical and clinical studies with TB vaccines.

Cyclooxygenase inhibitors impair CD4 T cell immunity and exacerbate Mycobacterium tuberculosis infection in aerosol-challenged mice.

Tuberculosis, caused by infection with Mycobacterium tuberculosis (Mtb), kills over 1.6 million people each year despite availability of antibiotics. The increase in drug resistant Mtb strains is a major public health emergency and host-directed therapy as adjunct to antibiotic treatment has gained increased interest. Cyclooxygenase inhibitors (COXi) are frequently used drugs to alleviate tuberculosis related symptoms. Mouse studies of acute intravenous Mtb infection have suggested a potential benefit of COXi for host-directed therapy. Here we show that COXi treatment (ibuprofen and celecoxib) is detrimental to Mtb control in different mouse models of respiratory infection. This effect links to impairments of the Type-1 helper (Th1) T-cell response as CD4 T-cells in COXi-treated animals have significantly decreased Th1 differentiation, reduced IFNγ expression and decreased protective capacity upon adoptive transfer. If confirmed in clinical trials, these findings could have major impact on global health and question the use of COXi for host-directed therapy.

An attenuated Mycobacterium tuberculosis clinical strain with a defect in ESX-1 secretion induces minimal host immune responses and pathology.

Although Mycobacterium tuberculosis (M.tb) DK9897 is an attenuated strain, it was isolated from a patient with extrapulmonary tuberculosis and vaccination with a subunit vaccine (H56) induced poor protection against it. Both attenuation and lack of protection are because M.tb DK9897 cannot secrete the EsxA virulence factor nor induce a host response against it. Genome sequencing identified a frameshift mutation in the eccCa1 gene. Since the encoded EccCa1 protein provides energy for ESX-1 secretion, it suggested a defect in the ESX-1 type VII secretion system. Genetic complementation with a plasmid carrying the M.tb H37Rv sequence of eccCa1-eccCb1-pe35 re-established EsxA secretion, host specific EsxA T-cell responses, and increased strain virulence. The ESX-1 secretion defect prevents several virulence factors from being functional during infection and therefore attenuates M.tb. It precludes specific T-cell responses against strong antigens and we found very little in vivo cytokine production, gross pathology or granuloma formation in lungs from M.tb DK9897 infected animals. This coincides with M.tb DK9897 being unable to disrupt the phagosome membrane and make contact to the cytosol.