MAVS-deficiency is associated with a reduced T cell response upon secondary RSV infection in mice

Infections with respiratory syncytial virus (RSV) occurs repeatedly throughout life because sustained, protective memory responses fail to develop. Why this occurs is not known. During RSV infection the recognition of the virus via the cytosolic RIG-I like receptors and signaling via the adaptor protein MAVS is crucial for mounting an innate immune response. However, if this signaling pathway is important for T cell responses during primary infection and during re-infection is not fully elucidated. We describe a second peak of pro-inflammatory mediators during the primary immune response to RSV that coincides with the arrival of T cells into the lung. This second peak of cytokines/chemokines is regulated differently than the early peak and is largely independent of signaling via MAVS. This was concurrent with Mavs−/− mice mounting a strong T cell response to primary RSV infection, with robust IFN-γ; and Granzyme B production. However, after RSV re-infection, Mavs−/− mice showed fewer CD4+ and CD8+ short term memory T cells and their capacity to produce IFN-γ; and Granzyme B, was decreased. In sum, cytosolic recognition of RSV is important not only for initiating innate anti-viral responses but also for generating or maintaining efficient, short term T cell memory responses

Using an effective TB vaccination regimen to identify immune responses associated with protection in the murine model

A vaccine against tuberculosis (TB), a disease resulting from infection with Mycobacterium tuberculosis (M.tb), is urgently needed to prevent more than a million deaths per year. Bacillus Calmette–Guérin (BCG) is the only available vaccine against TB but its efficacy varies throughout the world. Subunit vaccine candidates, based on recombinant viral vectors expressing mycobacterial antigens, are one of the strategies being developed to boost BCG-primed host immune responses and efficacy. A promising vaccination regimen composed of intradermal (i.d.) BCG prime, followed by intranasally (i.n.) administered chimpanzee adenoviral vector (ChAdOx1) and i.n. or i.d. modified vaccinia Ankara virus (MVA), both expressing Ag85A, has been previously reported to significantly improve BCG efficacy in mice. Effector and memory immune responses induced by BCG-ChAdOx1.85A-MVA85A (B-C-M), were evaluated to identify immune correlates of protection in mice. This protective regime induced strong Ag85A-specific cytokine responses in CD4+ and CD8+ T cells, both in the systemic and pulmonary compartments. Lung parenchymal CXCR3+ KLRG1- Ag85A-specific memory CD4+ T cells were significantly increased in B-C-M compared to BCG immunised mice at 4, 8 and 20 weeks post vaccination, but the number of these cells decreased at the latter time point. This cell population was associated with the protective efficacy of this regime and may have an important protective role against M.tb infection

Enhanced protection conferred by mucosal BCG vaccination associates with presence of antigen-specific lung tissue-resident PD-1+ KLRG1− CD4+ T cells

BCG, the only vaccine licensed against tuberculosis, demonstrates variable efficacy in humans. Recent preclinical studies highlight the potential for mucosal BCG vaccination to improve protection. Lung tissue-resident memory T cells reside within the parenchyma, potentially playing an important role in protective immunity to tuberculosis. We hypothesised that mucosal BCG vaccination may enhance generation of lung tissue-resident T cells, affording improved protection against Mycobacterium tuberculosis. In a mouse model, mucosal intranasal (IN) BCG vaccination conferred superior protection in the lungs compared to the systemic intradermal (ID) route. Intravascular staining allowed discrimination of lung tissue-resident CD4+ T cells from those in the lung vasculature, revealing that mucosal vaccination resulted in an increased frequency of antigen-specific tissue-resident CD4+ T cells compared to systemic vaccination. Tissue-resident CD4+ T cells induced by mucosal BCG displayed enhanced proliferative capacity compared to lung vascular and splenic CD4+ T cells. Only mucosal BCG induced antigen-specific tissue-resident T cells expressing a PD-1+ KLRG1− cell-surface phenotype. These cells constitute a BCG-induced population which may be responsible for the enhanced protection observed with IN vaccination. We demonstrate that mucosal BCG vaccination significantly improves protection over systemic BCG and this correlates with a novel population of BCG-induced lung tissue-resident CD4+ T cells

Identification and evaluation of novel protective antigens for the development of a candidate TB subunit vaccine

The development of a vaccine against tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (M.tb), is urgently needed. The only currently available vaccine, BCG, has variable efficacy. One approach in the global vaccine development effort is focused on boosting BCG using subunit vaccines. The identification of novel antigens for inclusion in subunit vaccines is a critical step in the TB vaccine development pathway. We selected four novel mycobacterial antigens recognised during the course of human infection. A replication deficient chimpanzee adenovirus (ChAdOx1) was constructed to express each antigen individually and these vectors were evaluated for protective efficacy in murine M.tb challenge experiments. One antigen, PPE15 (Rv1039c), conferred significant and reproducible protection when administered alone and as a boost to BCG vaccination. We identified immunodominant epitopes to define the protective immune responses using tetramers and intravascular staining. Lung parenchymal CD4+ and CD8+ CXCR3+ KLRG1- T cells, previously associated with M.tb protection, were enriched in vaccinated compared to control groups. Further work to evaluate the protective efficacy of PPE15 in more stringent preclinical animal models is now merited, together with the identification of further novel protective antigens using this selection strategy

Identification and evaluation of novel protective antigens for the development of a candidate TB subunit vaccine

The development of a vaccine against tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (M.tb), is urgently needed. The only currently available vaccine, BCG, has variable efficacy. One approach in the global vaccine development effort is focused on boosting BCG using subunit vaccines. The identification of novel antigens for inclusion in subunit vaccines is a critical step in the TB vaccine development pathway. We selected four novel mycobacterial antigens recognised during the course of human infection. A replication deficient chimpanzee adenovirus (ChAdOx1) was constructed to express each antigen individually and these vectors were evaluated for protective efficacy in murine M.tb challenge experiments. One antigen, PPE15 (Rv1039c), conferred significant and reproducible protection when administered alone and as a boost to BCG vaccination. We identified immunodominant epitopes to define the protective immune responses using tetramers and intravascular staining. Lung parenchymal CD4+ and CD8+ CXCR3+ KLRG1- T cells, previously associated with M.tb protection, were enriched in vaccinated compared to control groups. Further work to evaluate the protective efficacy of PPE15 in more stringent preclinical animal models is now merited, together with the identification of further novel protective antigens using this selection strategy

Enhanced protection conferred by mucosal BCG vaccination associates with presence of antigen-specific lung tissue-resident PD-1+ KLRG1− CD4+ T cells

BCG, the only vaccine licensed against tuberculosis, demonstrates variable efficacy in humans. Recent preclinical studies highlight the potential for mucosal BCG vaccination to improve protection. Lung tissue-resident memory T cells reside within the parenchyma, potentially playing an important role in protective immunity to tuberculosis. We hypothesised that mucosal BCG vaccination may enhance generation of lung tissue-resident T cells, affording improved protection against Mycobacterium tuberculosis. In a mouse model, mucosal intranasal (IN) BCG vaccination conferred superior protection in the lungs compared to the systemic intradermal (ID) route. Intravascular staining allowed discrimination of lung tissue-resident CD4+ T cells from those in the lung vasculature, revealing that mucosal vaccination resulted in an increased frequency of antigen-specific tissue-resident CD4+ T cells compared to systemic vaccination. Tissue-resident CD4+ T cells induced by mucosal BCG displayed enhanced proliferative capacity compared to lung vascular and splenic CD4+ T cells. Only mucosal BCG induced antigen-specific tissue-resident T cells expressing a PD-1+ KLRG1− cell-surface phenotype. These cells constitute a BCG-induced population which may be responsible for the enhanced protection observed with IN vaccination. We demonstrate that mucosal BCG vaccination significantly improves protection over systemic BCG and this correlates with a novel population of BCG-induced lung tissue-resident CD4+ T cells

Identification of antigens presented by MHC for vaccines against tuberculosis

Mycobacterium tuberculosis (M.tb) is responsible for more deaths globally than any other pathogen. The only available vaccine, bacillus Calmette-Guérin (BCG), has variable efficacy throughout the world. A more effective vaccine is urgently needed. The immune response against tuberculosis relies, at least in part, on CD4+ T cells. Protective vaccines require the induction of antigen-specific CD4+ T cells via mycobacterial peptides presented by MHC class-II in infected macrophages. In order to identify mycobacterial antigens bound to MHC, we have immunoprecipitated MHC class-I and class-II complexes from THP-1 macrophages infected with BCG, purified MHC class-I and MHC class-II peptides and analysed them by liquid chromatography tandem mass spectrometry. We have successfully identified 94 mycobacterial peptides presented by MHC-II and 43 presented by MHC-I, from 76 and 41 antigens, respectively. These antigens were found to be highly expressed in infected macrophages. Gene ontology analysis suggests most of these antigens are associated with membranes and involved in lipid biosynthesis and transport. The sequences of selected peptides were confirmed by spectral match validation and immunogenicity evaluated by IFN-gamma ELISpot against peripheral blood mononuclear cell from volunteers vaccinated with BCG, M.tb latently infected subjects or patients with tuberculosis disease. Three antigens were expressed in viral vectors, and evaluated as vaccine candidates alone or in combination in a murine aerosol M.tb challenge model. When delivered in combination, the three candidate vaccines conferred significant protection in the lungs and spleen compared with BCG alone, demonstrating proof-of-concept for this unbiased approach to identifying new candidate antigens

Hepcidin deficiency and iron deficiency do not alter tuberculosis susceptibility in a murine M. tb infection model

Tuberculosis (TB), caused by the macrophage-tropic pathogen Mycobacterium tuberculosis (M.tb) is a highly prevalent infectious disease. Since an immune correlate of protection or effective vaccine have yet to be found, continued research into host-pathogen interactions is important. Previous literature reports links between host iron status and disease outcome for many infections, including TB. For some extracellular bacteria, the iron regulatory hormone hepcidin is essential for protection against infection. Here, we investigated hepcidin (encoded by Hamp1) in the context of murine M.tb infection. Female C57BL/6 mice were infected with M.tb Erdman via aerosol. Hepatic expression of iron-responsive genes was measured by qRT-PCR and bacterial burden determined in organ homogenates. We found that hepatic Hamp1 mRNA levels decreased post-infection, and correlated with a marker of BMP/SMAD signalling pathways. Next, we tested the effect of Hamp1 deletion, and low iron diets, on M.tb infection. Hamp1 knockout mice did not have a significantly altered M.tb mycobacterial load in either the lungs or spleen. Up to 10 weeks of dietary iron restriction did not robustly affect disease outcome despite causing iron deficiency anaemia. Taken together, our data indicate that unlike with many other infections, hepcidin is decreased following M.tb infection, and show that hepcidin ablation does not influence M.tb growth in vivo. Furthermore, because even severe iron deficiency did not affect M.tb mycobacterial load, we suggest that the mechanisms M.tb uses to scavenge iron from the host must be extremely efficient, and may therefore represent potential targets for drugs and vaccines