A five-antigen Esx-5a fusion delivered as a prime-boost regimen protects against M.tb challenge

The development of tuberculosis (TB) vaccines has been hindered by the complex nature of Mycobacterium tuberculosis (M.tb) and the absence of clearly defined immune markers of protection. While Bacillus Calmette-Guerin (BCG) is currently the only licensed TB vaccine, its effectiveness diminishes in adulthood. In our previous research, we identified that boosting BCG with an intranasally administered chimpanzee adenovirus expressing the PPE15 antigen of M.tb (ChAdOx1.PPE15) improved its protection. To enhance the vaccine’s efficacy, we combined PPE15 with the other three members of the Esx-5a secretion system and Ag85A into a multi-antigen construct (5Ag). Leveraging the mucosal administration safety of ChAdOx1, we targeted the site of M.tb infection to induce localized mucosal responses, while employing modified vaccinia virus (MVA) to boost systemic immune responses. The combination of these antigens resulted in enhanced BCG protection in both the lungs and spleens of vaccinated mice. These findings provide support for advancing ChAdOx1.5Ag and MVA.5Ag to the next stages of vaccine development

A five-antigen Esx-5a fusion delivered as a prime-boost regimen protects against M.tb challenge

The development of tuberculosis (TB) vaccines has been hindered by the complex nature of Mycobacterium tuberculosis (M.tb) and the absence of clearly defined immune markers of protection. While Bacillus Calmette-Guerin (BCG) is currently the only licensed TB vaccine, its effectiveness diminishes in adulthood. In our previous research, we identified that boosting BCG with an intranasally administered chimpanzee adenovirus expressing the PPE15 antigen of M.tb (ChAdOx1.PPE15) improved its protection. To enhance the vaccine’s efficacy, we combined PPE15 with the other three members of the Esx-5a secretion system and Ag85A into a multi-antigen construct (5Ag). Leveraging the mucosal administration safety of ChAdOx1, we targeted the site of M.tb infection to induce localized mucosal responses, while employing modified vaccinia virus (MVA) to boost systemic immune responses. The combination of these antigens resulted in enhanced BCG protection in both the lungs and spleens of vaccinated mice. These findings provide support for advancing ChAdOx1.5Ag and MVA.5Ag to the next stages of vaccine development

Assessment of the reproducibility and inter-site transferability of the murine direct splenocyte mycobacterial growth inhibition assay (MGIA)

Tuberculosis (TB) vaccine candidates must be tested for safety and efficacy using preclinical challenge models prior to advancement to human trials, because of the lack of a validated immune correlate or biomarker of protection. New, unbiased tools are urgently needed to expedite the selection of vaccine candidates at an early stage of development and reduce the number of animals experimentally infected with virulent Mycobacterium tuberculosis (M.tb). In recent years, there has been a concerted effort to develop standardised functional ex vivo mycobacterial growth inhibition assays (MGIAs) as a potential surrogate read-out of vaccine efficacy. We have previously described a direct MGIA for use with mouse splenocytes. In the current study, we set out to systematically compare co-culture conditions for the murine direct splenocyte MGIA with respect to both intra-assay repeatability and inter-site reproducibility. Common sample sets were shared between laboratory sites and reproducibility and sensitivity to detect a BCG-vaccine induced response were assessed. Co-culturing 5×106 splenocytes in 48-well plates resulted in improved reproducibility and superior sensitivity to detect a vaccine response compared with standing or rotating sealed 2ml screw-cap tubes. As the difference between naïve and BCG vaccinated mice was not consistently detected across both sample sets at both sites, we sought to further improve assay sensitivity by altering the multiplicity of infection (MOI). Cell viability at the end of the co-culture period was improved when splenocyte input number was reduced, with the highest viability for the condition of 3×106 splenocytes in 48-well plates. This cell input was also associated with the greatest sensitivity to detect a BCG vaccine-mediated MGIA response using an M.tb inoculum. Based on our findings, we recommend optimal co-culture conditions in a move towards aligning direct MGIA protocols and generating a cross-species consensus for early evaluation of TB vaccine candidates and biomarker studies

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 to 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

Evaluation of the immunogenicity of a promising vaccine regimen to identify immune correlates of protection

A vaccine against tuberculosis (TB), a disease resulting after 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 is variable throughout the world. A promising vaccination regimen composed of BCG prime, followed by vaccination with a chimpanzee adenoviral vector (ChAdOx1) and a modified vaccinia Ankara virus (MVA), both expressing Ag85A, was identified in our laboratory. This regimen significantly improved BCG efficacy in animal studies. Antibody, effector and memory T cell responses induced by BCG-ChAdOx1.85A-MVA85A (B-C-M), were evaluated, to identify immune correlates of protection. This regimen induced Ag85A-specific IgG and IgA responses as well as cytokine responses in CD4+ and CD8+ T cells, both in the systemic and pulmonary system. To discriminate lymphocytes in the vasculature from lymphocytes in the lung parenchyma, a fluorochrome-conjugated antibody was injected intravascularly before harvesting the lungs. Lung parenchymal Ag85A-specific memory CD4+ T cells that were CXCR3+ KLRG1-, significantly increased in B-C-M immunised mice compared to BCG alone at 4, 8 and 20 weeks post vaccination but the cell number decreased at the latter time point. These memory cells associated with protection against M.tb challenge, which was observed at 4 and 8 weeks but not when mice were challenged 20 weeks post vaccination. These putative T cells may have an important role against M.tb infection and could be a target for designing a protective vaccine. In parallel, six novel M.tb antigens were selected and expressed individually in a ChAdOx1 vector. The immunogenicity and protective efficacy of these vaccines were evaluated. Three antigens were not immunogenic when expressed in viral vectors and DNA. The immunogenic antigens induced cytokine production but failed to confer protection when delivered alone or with B-C-M regimen.</p

Evaluation of the immunogenicity of a promising vaccine regimen to identify immune correlates of protection

A vaccine against tuberculosis (TB), a disease resulting after infection with Mycobacterium tuberculosis (M.tb), is urgently needed to prevent more than a million deaths per year. Bacillus CalmetteâGuérin (BCG) is the only available vaccine against TB but its efficacy is variable throughout the world. A promising vaccination regimen composed of BCG prime, followed by vaccination with a chimpanzee adenoviral vector (ChAdOx1) and a modified vaccinia Ankara virus (MVA), both expressing Ag85A, was identified in our laboratory. This regimen significantly improved BCG efficacy in animal studies. Antibody, effector and memory T cell responses induced by BCG-ChAdOx1.85A-MVA85A (B-C-M), were evaluated, to identify immune correlates of protection. This regimen induced Ag85A-specific IgG and IgA responses as well as cytokine responses in CD4+ and CD8+ T cells, both in the systemic and pulmonary system. To discriminate lymphocytes in the vasculature from lymphocytes in the lung parenchyma, a fluorochrome-conjugated antibody was injected intravascularly before harvesting the lungs. Lung parenchymal Ag85A-specific memory CD4+ T cells that were CXCR3+ KLRG1-, significantly increased in B-C-M immunised mice compared to BCG alone at 4, 8 and 20 weeks post vaccination but the cell number decreased at the latter time point. These memory cells associated with protection against M.tb challenge, which was observed at 4 and 8 weeks but not when mice were challenged 20 weeks post vaccination. These putative T cells may have an important role against M.tb infection and could be a target for designing a protective vaccine. In parallel, six novel M.tb antigens were selected and expressed individually in a ChAdOx1 vector. The immunogenicity and protective efficacy of these vaccines were evaluated. Three antigens were not immunogenic when expressed in viral vectors and DNA. The immunogenic antigens induced cytokine production but failed to confer protection when delivered alone or with B-C-M regimen.</p

Regulatory T Cells Decreased during Recovery from Mild COVID-19

Depending on the intensity and duration of SARS-CoV-2 infection, the host immune response plays a significant role in immunological protection. Here, we studied the regulatory T-cell (Treg) response in relation to kinetic change and cytokine production in patients with mild COVID-19. Nineteen SARS-CoV-2-positive patients were recruited, and blood was collected at four time points, i.e., seven days after admission, after discharge, and one and three months after recovery. CD3+CD4+CD25+CD127low was marked as the Treg population, with IL-10 and TGF-&beta; used to study cytokine-producing Tregs. IFN-&gamma;-producing CD8+ T cells were observed for an effector response. The Treg percentage in patients with mild COVID-19 increased during hospitalization compared to during the recovery period. Peripheral blood mononuclear cells (PBMCs) were quantified, and the T-cell response was characterized by re-stimulation with S1 and N peptides. IL-10 and TGF-&beta; were produced by CD25+CD127low T cells during the active infection phase, especially with N peptide stimulation. Compared to N peptide stimulation, S1 peptide stimulation provided superior IFN-&gamma;-secreting CD8+ T-cell responses. Our results suggest that while IFN-&gamma;+CD8+ T cells confer antiviral immunity, cytokine-producing Tregs may have a substantial role in regulating inflammatory responses in mild SARS-CoV-2 infection. Novel vaccine development may also consider enhancing T-cell repertoires

Image_2_A five-antigen Esx-5a fusion delivered as a prime-boost regimen protects against M.tb challenge.tiff

The development of tuberculosis (TB) vaccines has been hindered by the complex nature of Mycobacterium tuberculosis (M.tb) and the absence of clearly defined immune markers of protection. While Bacillus Calmette-Guerin (BCG) is currently the only licensed TB vaccine, its effectiveness diminishes in adulthood. In our previous research, we identified that boosting BCG with an intranasally administered chimpanzee adenovirus expressing the PPE15 antigen of M.tb (ChAdOx1.PPE15) improved its protection. To enhance the vaccine’s efficacy, we combined PPE15 with the other three members of the Esx-5a secretion system and Ag85A into a multi-antigen construct (5Ag). Leveraging the mucosal administration safety of ChAdOx1, we targeted the site of M.tb infection to induce localized mucosal responses, while employing modified vaccinia virus (MVA) to boost systemic immune responses. The combination of these antigens resulted in enhanced BCG protection in both the lungs and spleens of vaccinated mice. These findings provide support for advancing ChAdOx1.5Ag and MVA.5Ag to the next stages of vaccine development.</p

Heterologous COVID-19 Vaccination and Booster with mRNA Vaccine Provide Enhanced Immune Response in Patients with Cirrhosis: A Prospective Observational Study

This study aimed to evaluate the antibody and cellular responses to different coronavirus 2019 (COVID-19) vaccination regimens in patients with cirrhosis and to assess the antibody response after a vaccine booster. We conducted a prospective observational study of 89 patients with cirrhosis and 41 healthy volunteers who received two COVID-19 vaccine doses. Next, we prospectively evaluated 24 patients with cirrhosis who received a booster COVID-19 vaccine dose. In both studies, blood samples were collected before and 4 weeks after vaccination, and anti-spike receptor-binding domain protein IgG levels, T-cell phenotypes, and effector functions were assessed. The heterologous vaccine regimen (CoronaVac [SV]/AstraZeneca [AZ]) produced a better antibody response and CD4+IFNg+ T cell response compared to homogeneous vaccine regimens. The antibody response after the second dose of the vaccine was similar in patients with cirrhosis and healthy volunteers. Patients who received a booster dose of the mRNA vaccine had significantly increased antibody titers compared to those who received the AZ vaccine. In patients with cirrhosis, heterologous vaccination with SV/AZ resulted in a better immune response than the AZ/AZ and SV/SV regimens. Moreover, a booster dose of the mRNA vaccine led to a greater increase in antibody titers compared to the AZ vaccine