Introduction:CD4+ Th9 cells have been associated with inflammatory and allergic diseases. IL-9/Th9 can function as both positive and negative immune regulators, but their protective effects against Mycobacterium tuberculosis(Mtb) are unknown. We found that Th9 cells were associated with mycobacteria-specific T cell responses primed by latent tuberculosis infection and BCG vaccination. Methods:To study TB-specific Th9 protective effects, we generated Th9 cells from ESAT6-specific TCR transgenic mice and healthy human donors. Results:Both murine and human Th9 cells significantly inhibited intracellular mycobacterial growth. In both in vitro models, IL-9 neutralization strongly reduced Th9 protective effects, and IL-9 treatment alone inhibited intracellular mycobacteria. ESAT-6-specific Th9 and Th1 cells were adoptively transferred into naïve Rag1/2-/- recipients before aerosol Mtb infection. Th9 cells provided robust immunity as protective as Th1 cells, significantly reducing bacteria and pathologic changes post-infection. Differential persistence of Th9 vs. Th1 cell phenotypes was confirmed in vivo, and lung tissue qRT-PCR studies demonstrated the absence of IFN-γ responses in Th9-transferred mice, combined with unique expression of the Th9 specific markers IL-9, IL-10 and PU.1. Discussion:Th9 cells can provide important protection against Mtb infection, and should be targeted with future TB vaccine strategies. Furthermore, Th9 cells appear to utilize a novel protective mechanism independent from Th1-mediated protective responses.

Background: The prevalence of pulmonary nontuberculous mycobacteria (NTM) is increasing in Europe and North America. Most pulmonary NTM cases are caused by Mycobacterium avium complex (MAC). The treatment of pulmonary MAC is suboptimal with failure rates ranging from 30% to 40% and there is a need to develop new vaccines. Methods: We tested the ability of two whole-cell vaccines, DAR-901 (heat-killed M. obuense) and BCG (live-attenuated M. bovis), to induce MAC cross-reactive immunity by first immunizing BALB/c mice and then performing IFN-γ ELISPOT assays after overnight stimulation of splenocytes with live MAC. To study the ability of these vaccines to protect against MAC infection, BALB/c mice were vaccinated with DAR-901 (intradermal) or BCG (subcutaneous or intranasal) and challenged with aerosolized MAC 4 weeks later. A group of mice vaccinated with BCG were also treated with clarithromycin via gavage. Lung colony-forming units (CFU) in immunized mice and unvaccinated controls were quantified 4 weeks after infection. Histopathology was used to quantify lung inflammation and flow cytometry was used to study lung immunity in BCG-vaccinated and unvaccinated mice following MAC infection. To increase the safety profile of mucosal BCG vaccination, we studied BCG with a “kill switch” (tetR BCG) in scnn1b-transgenic mice (i.e., mice prone to cystic fibrosis-type lung diseases). Results: Our results showed that (i) DAR-901 induced cross-reactive immunity to MAC to a similar level as BCG, (ii) DAR-901 and BCG protected against aerosol MAC challenge, (iii) mucosal BCG vaccination, compared to systemic BCG and DAR-901 vaccinations, provided the best protection against MAC challenge, (iv) BCG vaccination did not interfere with anti-MAC activities of clarithromycin, (v) BCG-vaccinated mice had increased inflammation and increased frequencies of activated CD4 and CD8 T cells following MAC infection, and (vi) doxycycline treatment of tetR BCG-vaccinated mice decreased lung BCG CFU without affecting MAC immunity. Conclusions: Both DAR-901 and BCG vaccinations induce MAC cross-reactive immunity and protect against aerosolized MAC challenges. Mucosal BCG vaccination provides the best protection and TetR BCG could enhance the safety of mucosal BCG vaccination.

Mycobacterium tuberculosis (Mtb)-specific γ9δ2 T cells secrete granzyme A (GzmA) protective against intracellular Mtb growth. However, GzmA-enzymatic activity is unnecessary for pathogen inhibition, and the mechanisms of GzmA-mediated protection remain unknown. We show that GzmA homodimerization is essential for opsonization of mycobacteria, altered uptake into human monocytes, and subsequent pathogen clearance within the phagolysosome. Although monomeric and homodimeric GzmA bind mycobacteria, only homodimers also bind cluster of differentiation 14 (CD14) and Toll-like receptor 4 (TLR4). Without access to surface-expressed CD14 and TLR4, GzmA fails to inhibit intracellular Mtb. Upregulation of Rab11FIP1 was associated with inhibitory activity. Furthermore, GzmA colocalized with and was regulated by protein disulfide isomerase AI (PDIA1), which cleaves GzmA homodimers into monomers and prevents Mtb inhibitory activity. These studies identify a previously unrecognized role for homodimeric GzmA structure in opsonization, phagocytosis, and elimination of Mtb in human monocytes, and they highlight PDIA1 as a potential host-directed therapy for prevention and treatment of tuberculosis, a major human disease.

Immune evasion of Mycobacterium tuberculosis (Mtb) facilitates intracellular bacterial growth. The mechanisms of immune evasion, however, are still not fully understood. In this study, we reveal that tristetraprolin (TTP), one of the best characterized RNA‐binding proteins controlling the stability of targeted mRNAs, mediates innate immune evasion of mycobacteria. We found that TTP knockout mice displayed reduced bacterial burden in the early stage after Mtb aerosol challenge. Macrophages deficient in TTP also showed an inhibition in intracellular mycobacterial growth. Live mycobacteria induced TTP protein expression in macrophages, which was blocked by the mTOR inhibitor rapamycin. Rapamycin and AZD8055 specifically blocked 4EBP1 phosphorylation in infected macrophages and suppressed intracellular BCG growth. Rapamycin promoted TTP protein degradation through the ubiquitination pathway, whereas the proteasome inhibitor MG‐132 blocked rapamycin function and thus stabilized TTP protein. TTP induction suppressed the expression of iNOS/TNF‐α/IL‐12/IL‐23, and weakened protective immune responses in macrophages, whereas rapamycin enhanced the bactericidal effects through TTP inhibition. Moreover, blocking TTP binding increased the expression of TNF‐α and iNOS and suppressed intracellular mycobacterial growth. Overall, our study reveals a novel role for RNA‐binding protein TTP in Mtb immune evasion mechanisms and provides a potential target for host‐directed therapy against tuberculosis (TB).

In Europe and North America, the prevalence of pulmonary nontuberculous mycobacteria (NTM) is increasing. Most pulmonary NTM infections are caused by the Mycobacterium avium complex (MAC). Sadly, the treatment of pulmonary MAC is suboptimal with failure rates ranging from 37% to 58%. Therefore, there is a need to develop new therapeutics. Developing new immunotherapies and studying their interaction with standard or new drugs requires reliable assays. Four different assays including CFSE-based flow cytometry, in vitro protection assays, IFN-γ ELISPOT, and murine infection models were optimized using a reference strain of MAC (ATCC 700898) to help with the development of immunotherapies for MAC. Expansion of proliferating and IFN-γ producing human T cells is optimal after 7 days of stimulation with MAC at a multiplicity of infection (MOI) of 0.1, achieving a stimulation index of 26.5 ± 11.6 (mean ± SE). The in vitro protection assay for MAC works best by co-culturing T cells expanded for 7 days with MAC (MOI 1)-infected autologous macrophages. Aerosol MAC infection of mice allows measurement of the effects of the BCG vaccine and clarithromycin. IFN-γ ELISPOT assays with live MAC (MOI 3) stimulation of splenocytes from mice immunized with BCG help identify differences between unimmunized mice and mice immunized with BCG. In conclusion, multiple assays are available for use to identify MAC-specific effector T cells, which will help in the development of new therapeutics or vaccines against pulmonary MAC.

Background A controlled human infection model for assessing tuberculosis (TB) immunity can accelerate new vaccine development. Methods In this phase 1 dose escalation trial, 92 healthy adults received a single intradermal injection of 2 × 106 to 16 × 106 colony-forming units of Bacillus Calmette-Guérin (BCG). The primary endpoints were safety and BCG shedding as measured by quantitative polymerase chain reaction, colony-forming unit plating, and MGIT BACTEC culture. Results Doses up to 8 × 106 were safe, and there was evidence for increased BCG shedding with dose escalation. The MGIT time-to-positivity assay was the most consistent and precise measure of shedding. Power analyses indicated that 10% differences in MGIT time to positivity (area under the curve) could be detected in small cohorts (n = 30). Potential biomarkers of mycobacterial immunity were identified that correlated with shedding. Transcriptomic analysis uncovered dose- and time-dependent effects of BCG challenge and identified a putative transcriptional TB protective signature. Furthermore, we identified immunologic and transcriptomal differences that could represent an immune component underlying the observed higher rate of TB disease incidence in males. Conclusions The safety, reactogenicity, and immunogenicity profiles indicate that this BCG human challenge model is feasible for assessing in vivo TB immunity and could facilitate the vaccine development process. Clinical Trials Registration NCT01868464 (ClinicalTrials.gov).

Zika virus (ZIKV) is a flavivirus primarily transmitted by Aedes species mosquitoes, first discovered in Africa in 1947, that disseminated through Southeast Asia and the Pacific Islands in the 2000s. The first ZIKV infections in the Americas were identified in 2014, and infections exploded through populations in Brazil and other countries in 2015/16. ZIKV infection during pregnancy can cause severe brain and eye defects in offspring, and infection in adults has been associated with higher risks of Guillain-Barré syndrome. We initiated a study to describe the natural history of Zika (the disease) and the immune response to infection, for which some results have been reported. In this paper, we identify ZIKV-specific CD4+ and CD8+ T cell epitopes that induce responses during infection. Two screening approaches were utilized: an untargeted approach with overlapping peptide arrays spanning the entire viral genome, and a targeted approach utilizing peptides predicted to bind human MHC molecules. Immunoinformatic tools were used to identify conserved MHC class I supertype binders and promiscuous class II binding peptide clusters predicted to bind 9 common class II alleles. T cell responses were evaluated in overnight IFN-γ ELISPOT assays. We found that MHC supertype binding predictions outperformed the bulk overlapping peptide approach. Diverse CD4+ T cell responses were observed in most ZIKV-infected participants, while responses to CD8+ T cell epitopes were more limited. Most individuals developed a robust T cell response against epitopes restricted to a single MHC class I supertype and only a single or few CD8+ T cell epitopes overall, suggesting a strong immunodominance phenomenon. Noteworthy is that many epitopes were commonly immunodominant across persons expressing the same class I supertype. Nearly all of the identified epitopes are unique to ZIKV and are not present in Dengue viruses. Collectively, we identified 31 immunogenic peptides restricted by the 6 major class I supertypes and 27 promiscuous class II epitopes. These sequences are highly relevant for design of T cell-targeted ZIKV vaccines and monitoring T cell responses to Zika virus infection and vaccination.

Mycobacterium tuberculosis (Mtb) infection is mainly controlled by protective cell-mediated immunity. TNFα and other mediators, such as nitric oxide (NO), are crucial to the macrophage’s antimicrobial activity. The RNA-binding protein MCPIP1 is a zinc-finger protein with RNase activity that targets the 3’UTR region of cytokine’s mRNA for degradation like IL-6, IL-1β, and IL-12. Here, we aim to investigate the role of MCPIP1 during infection and its contribution to Mtb’s immune evasion. Our observations show that MCPIP1 increases after Mtb infection, correlating with disease progression. Similarly, global MCPIP1 KO (MCPIP1 −/−) mice showed lower lung bacterial burden than WT mice after 30 days. To address wheater the reduced number of bacteria was due to the macrophage’s absent MCPIP1 expression, we generated myeloid-specific MCPIP1 KO mice (M-MCPIP1 −/−). M-MCPIP1 −/−mice showed a similar reduction of bacterial growth as the MCPIP1 −/−mice compared with their WT littermates. Infection of bone marrow-derived macrophages confirmed that myeloid-MCPIP1 deficient cells had improved control of bacterial growth with increased IL-1β, IL-6, IL-12, iNOS, and TNFα. To evidence the mechanism by which M-MCPIP1 −/−macrophages contribute to Mtb growth control, we blocked TNFα and iNOS activity. Results showed that protection in M-MCPIP1 −/−is partly due to these molecule’s high expression, as the blockade of these mediators increased the bacterial burden. Our preliminary data suggest that the induction of MCPIP1 contributes to the bacteria’s immune evasion by targeting critical anti-TB molecules. Further experiments are needed to understand the mechanisms involved and consider MCPIP1 as a target for host-directed therapy for TB. R21AI151936 R21AI171734

Immune evasion of Mycobacterium tuberculosis (Mtb) facilitates intracellular bacterial growth. The mechanisms of immune evasion, however, are still not fully understood. In this study, we reveal that tristetraprolin (TTP), one of the best characterized RNA-binding proteins controlling stability of the target mRNAs, mediates innate immune evasion of mycobacteria. We found that TTP knockout mice displayed reduced bacterial burden in the early stage after Mtb aerosol challenge. Macrophages deficient in TTP also showed an inhibition in mycobacterial growth. Live mycobacteria induced TTP protein expression in macrophages, which was blocked by mTOR inhibitor rapamycin. Rapamycin promoted TTP protein degradation. Proteasome inhibitor MG-132 recovered the shortened TTP protein half-life induced by rapamycin. Ubiquitination mediated TTP protein degradation triggered by rapamycin in infected macrophages. TTP induction suppressed the expression of iNOS/TNF-α/IL-12/IL-23, and weakened the bactericidal effects in infected macrophages, whereas blocking mTOR pathway with rapamycin enhanced the bactericidal effects of infected macrophages through TTP inhibition. Overall, this study reveals a novel role for RNA-binding protein TTP in Mtb immune evasion mechanisms and provides a potential target for host-directed therapy against TB. R21AI171734 & R21AI151936

One fourth of the world population is infected with Tuberculosis (TB). Our lab has identified γ9δ2 T cells that secrete Granzyme A (GzmA) with TB protective effects. In this study, we investigated the mechanism(s) by which human GzmA inhibits the intracellular replication of mycobacteria within infected human primary monocytes. GzmA was added to mycobacteria-infected monocytes for downstream analyses using 2D-DIGE and shotgun proteomics. We generated WT, enzymatically inactive (S195A), and monomeric only (C93S) recombinant GzmA and performed: flow cytometry studies using viable mycobacteria; intracellular inhibition assays neutralizing CD14, TLR4 and TLR2; and GzmA immunoprecipitation experiments. The 2D-DIGE proteomic analyses found the ER-stress response and ATP metabolism pathways as important for GzmA-mediated inhibition. Separately, shotgun proteomics uncovered the upregulation of Rab11FIP1 (important for phagocytosis). Both GzmA-WT and S195A proteins inhibited intracellular mycobacteria, but C93S did not. Neutralization of CD14 and TLR4, but not TLR2, reversed GzmA-inhibitory activity. GzmA-WT, S195A, and C93S all bound mycobacteria. However, GzmA-WT and S195A, but not GzmA-C93S, stably bound to TLR4 and CD14. Collectively, these studies demonstrate key structural, functional, and inter-/intra-molecular features required for GzmA-mediated inhibition of intracellular mycobacteria including interactions between GzmA, mycobacteria, TLR4 and CD14. These interactions result in the ER stress response, altered ATP metabolism, enhanced phagocytosis, and inhibition of mycobacteria. Thus, GzmA’s potential role as opsonin could lead to novel host-directed therapeutics for TB infections. Supported by grants from NIH (F30HL151136-01, R01AI048391-12)

IntroductionTrypanosoma cruzi is the protozoan parasite causing Chagas disease, a Neglected Tropical Disease that affects 8 million people and causes 12,000 deaths per year, primarily because of cardiac pathology. Effective vaccination for T. cruzi remains an elusive goal. The use of a live vaccine vector, especially one that mimics the pathogen target, may be superior to the use of recombinant protein or DNA vaccine formulations. MethodsWe generated recombinant Leishmania major, a related trypanosomatid parasite, as a vaccine vehicle to express the immunogenic T. cruzi trans-sialidase (TS) antigen. The induction of T cell and antibody responses, as well as T. cruzi protective immunity generated by these vaccines were assessed in vivo. ResultsWe demonstrate that mice inoculated with these recombinant TS-expressing L. major parasites mount T cell and antibody responses directed against TS and are protected against future T. cruzi infection. We also show that the partially attenuated dhfr-ts- CC1 L. major strain, previously found to induce protective immunity to virulent L. major infection without causing pathology, can also be engineered to express the TS antigen. This latter recombinant may represent a safe and effective option to explore for ultimate use in humans. DiscussionAltogether, these data indicate that L. major can stably express a T. cruzi antigen and induce T. cruzi-specific protective immunity, warranting further investigation of attenuated Leishmania parasites as vaccine.

Mycobacterium tuberculosis (Mtb), the pathological agent that causes tuberculosis (TB) is the number one infectious killer worldwide with one fourth of the world's population currently infected. Data indicate that γ9δ2 T cells secrete Granzyme A (GzmA) in the extracellular space triggering the infected monocyte to inhibit growth of intracellular mycobacteria. Accordingly, deletion of GZMA from γ9δ2 T cells reverses their inhibitory capacity. Through mechanistic studies, GzmA's action was investigated in monocytes from human PBMCs. The use of recombinant human GzmA expressed in a mammalian system induced inhibition of intracellular mycobacteria to the same degree as previous human native protein findings. Our data indicate that: 1) GzmA is internalized within mycobacteria-infected cells, suggesting that GzmA uptake could prevent infection and 2) that the active site is not required to inhibit intracellular replication. Global proteomic analysis demonstrated that the ER stress response and ATP producing proteins were upregulated after GzmA treatment, and these proteins abundancies were confirmed by examining their expression in an independent set of patient samples. Our data suggest that immunotherapeutic host interventions of these pathways may contribute to better control of the current TB epidemic.

One fourth of the world population is infected with Mycobacterium tuberculosis (Mtb). Currently, BCG strains are the only vaccines available to protect against tuberculosis (TB). Our lab has identified γδ T cells that secrete Granzyme A (GzmA) with TB protective effects. GzmA when released by TB-specific γδ T cells has been shown to inhibit the intracellular replication of the pathogen. To elucidate the mechanism(s) involved in this inhibition, the lab has purified human GzmA. We have proven that Granzyme A’s intact active site is necessary to inhibit the intracellular replication of mycobacteria. The substitution of serine to alanine in the active site rendered the protein unable to control the mycobacterial infection as measured by mycobacterial growth inhibition assays, and the measurement of the pro-inflammatory marker TNF. Conversely, the effects of highly pure and active GzmA on mycobacterial replication inside human macrophages have been investigated using systems biology. A 2D-DIGE proteomic approach using adherent human monocytes has been performed to identify the pathways stimulated by GzmA involved in control of mycobacterial replication. Our analysis indicates that ER-stress responses (HSPA5, HSP90B1, P4HB, PDIA3) and purinergic channel receptor activation (ATP5B/C1/D/H/O) may be important for GzmA-mediated intracellular mycobacterial inhibition. Modulation of key proteins in these pathways are currently being targeted by pharmaceutical intervention and gene alteration to test the effects on GzmA-mediated inhibitory effects. Confirmation that these pathways are involved in pathogen clearance will lead to the development of novel host-directed therapies for control of Mtb infection.

Trypanosoma cruzi is the intracellular parasite of Chagas disease, a chronic condition characterized by cardiac and gastrointestinal morbidity. Protective immunity requires CD4+ T cells, and Th1 cells and gamma interferon (IFN-γ) are important players in host defense. More recently, Th17 cells and interleukin 17A (IL-17A) have been shown to exert protective functions in systemic T. cruzi infection. However, it remains unclear whether Th17 cells and IL-17A protect in the mucosa, the initial site of parasite invasion in many human cases. We found that IL-17RA knockout (KO) mice are highly susceptible to orogastric infection, indicating an important function for this cytokine in mucosal immunity to T. cruzi. To investigate the specific role of Th17 cells for mucosal immunity, we reconstituted RAG1 KO mice with T. cruzi-specific T cell receptor transgenic Th17 cells prior to orogastric T. cruzi challenges. We found that Th17 cells provided protection against gastric mucosal T. cruzi infection, indicated by significantly lower stomach parasite burdens. In vitro macrophage infection assays revealed that protection by Th17 cells is reduced with IL-17A neutralization or reversed by loss of macrophage NADPH oxidase activity. Consistently with this, mice lacking functional NADPH oxidase were not protected by Th17 cell transfer. These data are the first report that Th17 cells protect against mucosal T. cruzi infection and identify a novel protective mechanism involving the induction of NADPH oxidase activity by IL-17A. These studies provide important insights for Chagas vaccine development and, more broadly, increase our understanding of the diverse roles of Th17 cells in host defense.

Trypanosoma cruzi is the intracellular parasite of Chagas disease, a chronic condition characterized by cardiac and gastrointestinal morbidity. Protective immunity requires CD4+ T cells, and Th1 cells and IFN-γ are important players in host defense. More recently, Th17 cells and IL-17 have been shown to exert protective functions in systemic T. cruzi infection. However, it remains unclear whether Th17 cells and IL-17A protect against mucosal infection, which is an important cause of human outbreaks. We found that IL-17RA knock-out (KO) mice are highly susceptible to orogastric infection, indicating an important function for this cytokine in mucosal immunity to T. cruzi . To investigate the specific role of Th17 cells for mucosal immunity, we reconstituted RAG1 KO mice with T. cruzi-specific T cell receptor transgenic Th17 cells prior to orogastric T. cruzi challenges. We found that Th17 cells provided protection against gastric mucosal T. cruzi infection, indicated by significantly lower stomach parasite burdens. In vitro macrophage infection assays revealed that protection by Th17 cells is reversed with IL-17A neutralization or loss of macrophage NADPH oxidase activity. Consistent with this, in vivo , mice lacking functional NADPH oxidase were not protected by Th17 cell transfer. These data are the first report that Th17 cells protect against mucosal T. cruzi infection, and identify a novel protective mechanism involving the induction of NADPH oxidase activity in macrophages by IL-17A. These studies provide important insights for Chagas vaccine development, and more broadly, increase our understanding of the diverse roles of Th17 cells in host defense.

Failure of the most recent tuberculosis (TB) vaccine trial to boost bacillus Calmette-Guérin-mediated anti-TB immunity despite the induction of Th1-specific central memory cell and effector memory cell responses highlights the importance of identifying optimal T cell targets for protective vaccines. In this study, we describe a novel, Mycobacterium tuberculosis-specific IFN-γ+CD4+ T cell population expressing surface markers characteristic of naive-like memory T cells (TNLM), which were induced in both human (CD45RA+CCR7+CD27+CD95-) and murine (CD62L+CD44-Sca-1+CD122-) systems in response to mycobacteria. In bacillus Calmette-Guérin-vaccinated subjects and those with latent TB infection, TNLM were marked by the production of IFN-γ but not TNF-α and identified by the absence of CD95 expression and increased surface expression CCR7, CD27, the activation markers T-bet, CD69, and the survival marker CD74. Increased tetramer-positive TNLM frequencies were noted in the lung and spleen of ESAT-61-20-specific TCR transgenic mice at 2 wk postinfection with M. tuberculosis and progressively decreased at later time points, a pattern not seen with TNF-α+CD4+ T cells expressing naive cell surface markers. Importantly, adoptive transfer of highly purified TNLM alone, from vaccinated ESAT-61-20-specific TCR transgenic mice, conferred equivalent protection against M. tuberculosis infection in the lungs of Rag-/- mice when compared with total memory populations (central and effector memory cells). Thus, TNLM may represent a memory T cell population that, if optimally targeted, may significantly improve future TB vaccine responses.

Tuberculosis (TB) remains a tremendous global health problem, with 1/4 of the world's population infected and causing > 1 million deaths annually. Intradermal Bacillus Calmette-Guérin (BCG) vaccine given during infancy protects against severe forms of acute disease but does not prevent chronic infection or development of pulmonary TB. TB vaccine mucosal targeting potentially could induce mucosal resident immune cells with increased protective capacity against pulmonary infection and disease. Sublingual (SL) administration of vaccines may be an optimal mucosal delivery platform based on the high absorptive capacity of this mucosal surface, the extensive lymphoid tissue, and published preclinical studies demonstrating efficacy of SL vaccination against other pathogens. To this end, we performed preliminary testing of sublingual TB vaccines. Vaccination of mice with SL BCG elicited potent mycobacteria-specific T cell responses which persisted 16 weeks post-immunization. The magnitudes of the T cell responses were similarly induced after sublingual, intranasal, and subcutaneous BCG vaccination. Interestingly, serum mycobacteria-specific antibody responses and systemic recovery of BCG post-vaccination were lower after SL BCG compared with systemic BCG immunization. However, more importantly, SL BCG vaccinated mice were significantly protected against an aerosolized virulent M. tuberculosis challenge (P < 0.0001 compared to unvaccinated mice). Furthermore, this protection was long-lived, persisting for 16 weeks with >1 log CFU reduction compared with naïve challenged mice in both lungs and spleens (P < 0.0001 and P < 0.0028, respectively). These exciting results provide strong support for further studies exploring the mechanisms of protective immunity induced by sublingual BCG vaccination.

Pulmonary non-tuberculous mycobacterial (NTM) infections particularly caused by Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MAB) are becoming major health problems in the U.S. New therapies or vaccines which will help prevent the disease, shorten treatment duration and/or increase treatment success rates are urgently needed. This study was conducted with the objective of testing the hypothesis that Bacillus Calmette Guerin (BCG), a vaccine used for prevention of serious forms of tuberculosis (TB) in children and adolescents in tuberculosis hyperendemic countries, induces cross-protective T cell immunity against Mycobacterium avium (MAV) and MAB. Human TB and NTM cross-protective T cells were quantified using flow cytometric assays. The ability of BCG expanded T cells to inhibit the intracellular growth of MAV and MAB was assessed in co-cultures with infected autologous macrophages. In both BCG-vaccinated and M. tuberculosis (Mtb)-infected mice, NTM cross-reactive immunity was measured using IFN-γ ELISPOT assays. Our results demonstrate the following key findings: (i) peripheral blood mononuclear cells from TB skin test-positive individuals contain MAV and MAB cross-reactive T cells, (ii) both BCG vaccination and Mtb infection of mice induce MAV and MAB cross-reactive splenic cells, (iii) BCG-expanded T cells inhibit intracellular MAV and MAB, (iv) CD4, CD8, and γδ T cells play important roles in inhibition of intracellular MAV and MAB and (v) BCG vaccination of healthy volunteers induces TB and NTM cross-reactive T cells. In conclusion, BCG-vaccination induces NTM cross-reactive immunity, and has the potential for use as a vaccine or immunotherapy to prevent and/or treat pulmonary NTM disease.

Influenza world-wide causes significant morbidity and mortality annually, and more severe pandemics when novel strains evolve to which humans are immunologically nalve. Because of the high viral mutation rate, new vaccines must be generated based on the prevalence of circulating strains every year. New approaches to induce more broadly protective immunity are urgently needed. Previous research has demonstrated that influenza-specific T cells can provide broadly heterotypic protective immunity in both mice and humans, supporting the rationale for developing a T cell-targeted universal influenza vaccine. We used state-of-the art immunoinformatic tools to identify putative pan-HLA-DR and HLA-A2 supertype-restricted T cell epitopes highly conserved among > 50 widely diverse influenza A strains (representing hemagglutinin types 1, 2, 3, 5, 7 and 9). We found influenza peptides that are highly conserved across influenza subtypes that were also predicted to be class I epitopes restricted by HLA-A2. These peptides were found to be immunoreactive in HLA-A2 positive but not HLA-A2 negative individuals. Class II restricted T cell epitopes that were highly conserved across influenza subtypes were identified. Human CD4(+) T cells were reactive with these conserved CD4 epitopes, and epitope expanded T cells were responsive to both H1N1 and H3N2 viruses. Dendritic cell vaccines pulsed with conserved epitopes and DNA vaccines encoding these epitopes were developed and tested in HLA transgenic mice. These vaccines were highly immunogenic, and more importantly, vaccine-induced immunity was protective against both H1N1 and H3N2 influenza challenges. These results demonstrate proof-of-principle that conserved T cell epitopes expressed by widely diverse influenza strains can induce broadly protective, heterotypic influenza immunity, providing strong support for further development of universally relevant multi-epitope T cell-targeting influenza vaccines. (C) 2019 Elsevier Ltd. All rights reserved.

The translationally silent 100S ribosome is a poorly understood form of the dimeric 70S complex that is ubiquitously found in all bacterial phyla. The elimination of the hibernating 100S ribosome leads to translational derepression, ribosome instability, antibiotic sensitivity, and biofilm defects in some bacteria. In Firmicutes, such as the opportunistic pathogen Staphylococcus aureus, a 190-amino acid protein called hibernating-promoting factor (HPF) dimerizes and conjoins two 70S ribosomes through a direct interaction between the HPF homodimer, with each HPF monomer tethered on an individual 70S complex. While the formation of the 100S ribosome in gammaproteobacteria and cyanobacteria is exclusively induced during postexponential growth phase and darkness, respectively, the 100S ribosomes in Firmicutes are constitutively produced from the lag-logarithmic phase through the post-stationary phase. Very little is known about the regulatory pathways that control hpf expression and 100S ribosome abundance. Here, we show that a general stress response (GSR) sigma factor (SigB) and a GTP-sensing transcription factor (CodY) integrate nutrient and thermal signals to regulate hpf synthesis in S. aureus, resulting in an enhanced virulence of the pathogen in a mouse model of septicemic infection. CodY-dependent regulation of hpf is strain specific. An epistasis analysis further demonstrated that CodY functions upstream of the GSR pathway in a condition-dependent manner. The results reveal an important link between S. aureus stress physiology, ribosome metabolism, and infection biology. IMPORTANCE The dimerization of 70S ribosomes (100S complex) plays an important role in translational regulation and infectivity of the major human pathogen Staphylococcus aureus. Although the dimerizing factor HPF has been characterized biochemically, the pathways that regulate 100S ribosome abundance remain elusive. We identified a metaboliteand nutrient-sensing transcription factor, CodY, that serves both as an activator and a repressor of hpf expression in nutrientand temperature-dependent manners. Furthermore, CodY-mediated activation of hpf masks a secondary hpf transcript derived from a general stress response SigB promoter. CodY and SigB regulate a repertoire of virulence genes. The unexpected link between ribosome homeostasis and the two master virulence regulators provides new opportunities for alternative druggable sites.