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Two proof of concept clinical trials with TB vaccines demonstrate that new approaches can prevent sustained TB infection in adolescents (BCG revaccination) and TB disease in adults (M72/ASO1E) (Nemes et al., 2018; Tait et al., 2019) [1,2]. Both approaches are in late stage development and provide motivation and rationale to invest into a global TB vaccine pipeline. This pipeline needs to be diverse to address TB-specific challenges including variation in target populations, uncertainties in animal model predictivity and lack of immune correlates of protection. It requires that individual vaccine candidates must be advanced rationally and that the global pipeline must be managed in the most nimble and resource-efficient way, especially in the current constrained funding environment. The TB Vaccine Development Pathway is a webtool which has been developed as an offer to the field to provide a source of information and guidance covering vaccine development from discovery to implementation. It is underpinned by generic and TB vaccine-specific guidelines, regulatory frameworks and best practice, and was compiled by a multi-disciplinary team of scientific and technical experts with the input of the TB vaccine community. The Pathway is a unique tool to guide and accelerate the development of TB vaccine candidates and may be useful for other vaccine development fields.

Summary BCG vaccination can strengthen protection against pathogens through the induction of epigenetic and metabolic reprogramming of innate immune cells, a process called trained immunity. We and others recently demonstrated that mucosal or intravenous BCG better protects rhesus macaques from Mycobacterium tuberculosis infection and TB disease than standard intradermal vaccination, correlating with local adaptive immune signatures. In line with prior mouse data, here, we show in rhesus macaques that intravenous BCG enhances innate cytokine production associated with changes in H3K27 acetylation typical of trained immunity. Alternative delivery of BCG does not alter the cytokine production of unfractionated bronchial lavage cells. However, mucosal but not intradermal vaccination, either with BCG or the M. tuberculosis-derived candidate MTBVAC, enhances innate cytokine production by blood- and bone marrow-derived monocytes associated with metabolic rewiring, typical of trained immunity. These results provide support to strategies for improving TB vaccination and, more broadly, modulating innate immunity via mucosal surfaces., Graphical Abstract, Highlights Nonhuman primates recapitulate trained immunity upon live attenuated TB vaccination Intravenous BCG induces changes in H3K27 acetylation and enhances cytokine production Mucosal BCG improves induction of trained immunity of monocytes over intradermal BCG The M. tuberculosis-derived candidate vaccine MTBVAC appears equally potent as BCG, Vierboom et al. demonstrate the induction of trained immunity in blood and bone marrow monocytes after vaccination with live attenuated TB vaccines in nonhuman primates. Mucosal respiratory delivery of BCG or MTBVAC induces trained immunity more efficiently compared to standard intradermal vaccination.

Summary Background Infants are a key target population for new tuberculosis vaccines. We assessed the safety and immunogenicity of the live-attenuated Mycobacterium tuberculosis vaccine candidate MTBVAC in adults and infants in a region where transmission of tuberculosis is very high. Methods We did a randomised, double-blind, BCG-controlled, dose-escalation trial at the South African Tuberculosis Vaccine Initiative site near Cape Town, South Africa. Healthy adult community volunteers who were aged 18–50 years, had received BCG vaccination as infants, were HIV negative, had negative interferon-γ release assay (IGRA) results, and had no personal history of tuberculosis or current household contact with someone with tuberculosis were enrolled in a safety cohort. Infants born to HIV-negative women with no personal history of tuberculosis or current household contact with a person with tuberculosis and who were 96 h old or younger, generally healthy, and had not yet received routine BCG vaccination were enrolled in a separate infant cohort. Eligible adults were randomly assigned (1:1) to receive either BCG Vaccine SSI (5 × 105 colony forming units [CFU] of Danish strain 1331 in 0·1 mL diluent) or MTBVAC (5 × 105 CFU in 0·1 mL) intradermally in the deltoid region of the arm. After favourable review of 28-day reactogenicity and safety data in the adult cohort, infants were randomly assigned (1:3) to receive either BCG Vaccine SSI (2·5 × 105 CFU in 0·05 mL diluent) or MTBVAC in three sequential cohorts of increasing MTBVAC dose (2·5 × 103 CFU, 2·5 × 104 CFU, and 2·5 × 105 CFU in 0·05 mL) intradermally in the deltoid region of the arm. QuantiFERON-TB Gold In-Tube IGRA was done on days 180 and 360. For both randomisations, a pre-prepared block randomisation schedule was used. Participants (and their parents or guardians in the case of infant participants), investigators, and other clinical and laboratory staff were masked to intervention allocation. The primary outcomes, which were all measured in the infant cohort, were solicited and unsolicited local adverse events and serious adverse events until day 360; non-serious systemic adverse events until day 28 and vaccine-specific CD4 and CD8 T-cell responses on days 7, 28, 70, 180, and 360. Secondary outcomes measured in adults were local injection-site and systemic reactions and haematology and biochemistry at study day 7 and 28. Safety analyses and immunogenicity analyses were done in all participants who received a dose of vaccine. This trial is registered with ClinicalTrials.gov, number NCT02729571. Findings Between Sept 29, 2015, and Nov 16, 2015, 62 adults were screened and 18 were enrolled and randomly assigned, nine each to the BCG and MTBVAC groups. Between Feb 12, 2016, and Sept 21, 2016, 36 infants were randomly assigned—eight to the BCG group, nine to the 2·5 × 103 CFU MTBVAC group, nine to the 2·5 × 104 CFU group, and ten to the 2·5 × 105 CFU group. Mild injection-site reactions occurred only in infants in the BCG and the 2·5 × 105 CFU MTBVAC group, with no evidence of local or regional injection-site complications. Systemic adverse events were evenly distributed across BCG and MTBVAC dose groups, and were mostly mild in severity. Eight serious adverse events were reported in seven vaccine recipients (one adult MTBVAC recipient, one infant BCG recipient, one infant in the 2·5 × 103 CFU MTBVAC group, two in the 2·5 × 104 CFU MTBVAC group, and two in the 2·5 × 105 CFU MTBVAC group), including one infant in the 2·5 × 103 CFU MTBVAC group treated for unconfirmed tuberculosis and one in the 2·5 × 105 CFU MTBVAC group treated for unlikely tuberculosis. One infant died as a result of possible viral pneumonia. Vaccination with all MTBVAC doses induced durable antigen-specific T-helper-1 cytokine-expressing CD4 cell responses in infants that peaked 70 days after vaccination and were detectable 360 days after vaccination. For the highest MTBVAC dose (ie, 2·5 × 105 CFU), these responses exceeded responses induced by an equivalent dose of the BCG vaccine up to 360 days after vaccination. Dose-related IGRA conversion was noted in three (38%) of eight infants in the 2·5 × 103 CFU MTBVAC group, six (75%) of eight in the 2·5 × 104 CFU MTBVAC group, and seven (78%) of nine in the 2·5 × 105 CFU MTBVAC group at day 180, compared with none of seven infants in the BCG group. By day 360, IGRA reversion had occurred in all three infants (100%) in the 2·5 × 103 CFU MTBVAC group, four (67%) of the six in the 2·5 × 104 CFU MTBVAC group, and three (43%) of the seven in the 2·5 × 105 CFU MTBVAC group. Interpretation MTBVAC had acceptable reactogenicity, and induced a durable CD4 cell response in infants. The evidence of immunogenicity supports progression of MTBVAC into larger safety and efficacy trials, but also confounds interpretation of tests for M tuberculosis infection, highlighting the need for stringent endpoint definition. Funding Norwegian Agency for Development Cooperation, TuBerculosis Vaccine Initiative, UK Department for International Development, and Biofabri.

High-throughput analyses of RNA and protein expression are increasingly used for better understanding of vaccine-induced immunity and protection against infectious disease. With an increasing number of vaccine candidates in clinical development, it is timely to consider standardisation and harmonisation of sample collection, storage and analysis to ensure results of highest quality from these precious samples. These challenges were discussed by a group of international experts during a workshop organised by TRANSVAC, a European Commission-funded Research Infrastructure project. The main conclusions were: Platforms are rarely standardised for use in preclinical and clinical studies. Coordinated efforts should continue to harmonise the experimental set up of these studies, as well as the establishment of internal standards and controls. This will ensure comparability, efficiency and feasibility of the global analyses performed on preclinical and clinical data sets.

There are nearly ten million new cases and 1.4 million deaths from tuberculosis (TB) each year, and the 90-year old bacille calmette-guérin (BCG) vaccine in widespread use appears to have minimal impact on the worldwide incidence, despite demonstrating reasonable efficacy against complications of infant TB and death. Novel vaccine development has accelerated in the past ten years, with at least 16 candidates entering human trials, and a few vaccines have entered into Phase 2b efficacy studies. However, different vaccines may be needed due to the varying disease states (naïve, latently infected, or active), the ages affected (infants, adolescents and young adults, the elderly), and patient health status (HIV and immunocompromised patients especially). Modeling has shown that mass vaccination of latently infected populations, especially adolescents and young adults, will likely have the largest impact on new infection rates. At present, research and development of TB vaccines is hampered by the lack of validated animal models, the absence of correlates of immunity and a human challenge model, as well as by the size and cost of Proof-of-Concept clinical trials. Nonetheless, ongoing research and clinical studies should remove many of these barriers over the next five years, and lead to an increased understanding of the pathogenicity of Mycobacterium tuberculosis and what may constitute protective immunity during various stages of infection and disease.

Reduction of active disease by preventive therapy has the potential to make an important contribution towards the goal of tuberculosis (TB) elimination. This report summarises discussions amongst a Working Group convened to consider areas of research that will be important in optimising the design and delivery of preventative therapies. The Working Group met in Cape Town on 26th February 2012, following presentation of results from the GC11 Grand Challenges in Global Health project to discover drugs for latent TB. (C) 2012 Elsevier Ltd. All rights reserved.

TB is now the single pathogen that causes the greatest mortality in the world, at over 1.6 million deaths each year. The widely used the 90 year old BCG vaccine appears to have minimal impact on the worldwide incidence despite some efficacy in infants. Novel vaccine development has accelerated in the past 15 years, with 15 candidates entering human trials; two vaccines are now in large-scale efficacy studies. Modeling by three groups has consistently shown that mass vaccination that includes activity in the latently infected population, especially adolescents and young adults, will likely have the largest impact on new disease transmission. At present the field requires better validated animal models, better understanding of a correlate of immunity, new cost-effective approaches to Proof of Concept trials, and increased appreciation by the public health and scientific community for the size of the problem and the need for a vaccine. Such a vaccine is likely to also play a role in the era of increasing antibiotic resistance. Ongoing efforts and studies are working to implement these needs over the next 5 years, which will lead to an understanding that will increase the likelihood of a successful TB vaccine.

This chapter talks about the progress in the study of immune reactions mainly to antigens of Mycobacterium tuberculosis that have been firmly identified and the largely characterized protein structure. An important breakthrough in the characterization of antigens was achieved initially by the production of monoclonal antibodies to mycobacterial antigens and particularly by the development of recombinant DNA systems for efficient expression of mycobacterial genes in Escherichia coli. Heat shock proteins (hsp) have highly conserved amino acid sequences. Their functions as molecular chaperones in the assembly and disassembly of proteins into oligomers during transport through the cell or as proteases that degrade misfolded or foreign proteins are vital for each cell. The significance of lipoproteins for the immune response to mycobacteria has been in focus in view of reports that acylation of proteins enhances their ability to induce delayed-type hypersensitivity (DTH) and that synthetic lipopeptides have the ability to prime cytotoxic T cells. Proteins secreted from viable bacteria could be available for immune recognition at an early stage of infection and may therefore play a special role in protective immune mechanisms. Antibody levels following immunization with soluble extracts of tubercle bacilli in adjuvant demonstrated the codominant control by the H-2 IA locus of the response to individual epitopes. Analysis of the specificity of T-cell responses to the mycobacterial 65-kDa heat shock protein has so far attracted more attention in relation to its potential role in autoimmunity than in respect to tuberculosis.

1. Purpose of TB Vaccine Blueprint . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S6 2. Tuberculosis vaccines: a strategic blueprint for the next decade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S7 2.1. Creativity in research and discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S7 2.1.1. Use out-of-the-box approaches and advanced technologies to describe mechanisms of protective immunity for tuberculosis . . . . . . . S7 2.1.2. Improve the antigenic vaccine repertoire to prevent infection and provide sterilizing immunity . . . . . . . . . . . . . . . . . . . . . . . . . S8 2.1.3. Facilitate translational research, comparative preclinical studies and animal models that mimic human TB disease. . . . . . . . . . . . . . S8 2.2. Correlates of immunity and biomarkers for TB vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S8 2.3. Clinical trials: harmonization & cooperation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S9 2.3.1. Determine TB prevalence and incidence, select trial sites and choose target populations for TB vaccines that result in the greatest reduction in disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S9 2.3.2. Design clinical trials with appropriate endpoints for determining an acceptable efficacy for TB vaccines . . . . . . . . . . . . . . . . . . . . S9 2.3.3. Address regulatory and ethics issues and plan for post-licensure sustainability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S9 2.4. Rational selection of TB vaccine candidates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S10 2.5. Building support through advocacy, communications and resource mobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S10 2.5.1. Expand financing to provide sufficient resources to advance and sustain research on TB vaccines . . . . . . . . . . . . . . . . . . . . . . . . S11 2.5.2. Continue and expand on efforts to raise awareness of the role of new TB vaccines as part of a comprehensive response to the global TB epidemic and build support at all levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S11 2.5.3. Broaden the base of advocates, allies and champions for TB vaccine R&D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S11 3. Implementation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S11 Appendix 1. A summary of the critical activities outlined in the Blueprint . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S12 Appendix 2. The twelve TB vaccines currently in clinical trials (circa 2011) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S13 Contributors/Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S13

In response to recommendations from the Steering Committees responsible for co-ordination of World Health Organization programmes for research on the immunology of leprosy (IMMLEP) and tuberculosis (IMMTUB), a list was prepared summarizing the properties of mycobacterial proteins currently under investigation with respect to their immunological activities. After consultation with more than 40 laboratories world-wide this list was extended to form the compilation shown below and is intended to provide a comprehensive and convenient reference for future studies in this field.

Mycobacteria have the ability to persist within host phagocytes, and their success as intracellular pathogens is thought to be related to the ability to modify their intracellular environment. After entry into phagocytes, mycobacteria-containing phagosomes acquire markers for the endosomal pathway, but do not fuse with lysosomes. The molecular machinery that is involved in the entry and survival of mycobacteria in host cells is poorly characterized. Here we describe the use of organelle electrophoresis to study the uptake of Mycobacterium bovis bacille Calmette Guerin (BCG) into murine macrophages. We demonstrate that live, but not dead, mycobacteria occupy a phagosome that can be physically separated from endosomal/lysosomal compartments. Biochemical analysis of purified mycobacterial phagosomes revealed the absence of endosomal/lysosomal markers LAMP-1 and beta-hexosaminidase. Combining subcellular fractionation with two-dimensional gel electrophoresis, we found that a set of host proteins was present in phagosomes that were absent from endosomal/lysosomal compartments. The residence of mycobacteria in compartments outside the endosomal/lysosomal system may explain their persistence inside host cells and their sequestration from immune recognition. Furthermore, the approach described here may contribute to an improved understanding of the molecular mechanisms that determine the intracellular fate of mycobacteria during infection.

The major goal of the present study was to develop a limiting dilution system for the enumeration of T cells which respond to mycobacterial antigens. Purified T cells from M. tuburculosis-immune mice were restimulated with mycobacterial antigens and accessory cells, and after 4 days expanded with antigen, accessory cells and T cell growth factor. After another 3 days, proliferative responses were determined. Similar cultures performed without antigen served as controls. Limiting dilution analysis revealed that approximately 1/2000 to 1/3000 T cells from M. tuberculosis-immune mice responded to whole M. tuberculosis organisms while T cells from normal mice did not respond. Similar T cell numbers reacted with several mycobacterial strains indicating expression of shared T cell antigens. Using a semi-purified recombinant 64-kDa protein from M. bovis the frequency of T cells generated after immunization with M. tuberculosis which reacted with a single mycobacterial protein could be estimated. We found that approximately 1/5 of the M. tuberculosis-reactive T cells recognized this particular antigen. Immunization with the recombinant 64-kDa protein in an adjuvant containing trehalose dimycolate, monophosphoryl lipid A and mycobacterial cell wall skeleton stimulated an equally high number of M. tuberculosis-reactive T cells (1/2000). These findings demonstrate (a) that a high proportion of tuberculosis-responsive T cells are directed against the 64-kDa protein and (b) that immunization with this antigen in an appropriate adjuvant system is capable of stimulating high numbers of M. tuberculosis-reactive T cells. Limiting dilution analysis with a panel of mycobacterial proteins or peptides may allow their ranking from immunodominant to immunosilent and facilitate identification of antigens or epitopes relevant to protection.

To date, only a limited number of antigens have been described as specific for Mycobacterium leprae, and in many cases, homologues have subsequently been shown to exist in mycobacteria such as M. avium and M. intracellulare. A Leprosy Synthetic Peptide Skin Test Initiative was established by the Steering Committee on the Immunology of Mycobacteria of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, to investigate the potential of synthetic peptides that encode T-cell epitopes as diagnostic tools, which could be used to develop a skin-test reagent specific for leprosy. Such M. leprae-specific peptides should have unique amino acid sequences, or significant sequence-dissimilarity from those in other mycobacteria. Synthetic peptides, 15 amino acids long, were synthesised from 33 genes or open reading frames within the M. leprae genome. Tuberculoid leprosy patients from four leprosy-endemic countries, Brazil, Ethiopia, Nepal and Pakistan, were tested as subjects known to have been infected with M. leprae, and to make good T-cell responses to antigens of M. leprae; UK blood donors were used as non-exposed or non-infected subjects. Peptides inducing potentially specific responses in leprosy patients and not in UK controls, and those inducing cross-reaction responses, present in both leprosy patients and non-exposed, non-infected controls, were identified. A difference from the equivalent M. tuberculosis sequence of five or more amino acid residues did not, by itself, identify peptides that were M. leprae-specific, suggesting that many of these peptides may have homologues in environmental mycobacteria. To date, this approach has identified a number of peptides with greater than 90% specificity and 19-47% sensitivity, which are undergoing further specificity-testing. Such peptides would have great potential as T-cell reagents with which to monitor exposure to M. leprae within communities, formulated either as skin-test reagents, or as antigens for tests in vitro.

Summary To fight tuberculosis, better vaccination strategies are needed. Live attenuated Mycobacterium tuberculosis-derived vaccine, MTBVAC, is a promising candidate in the pipeline, proven to be safe and immunogenic in humans so far. Independent studies have shown that pulmonary mucosal delivery of Bacillus Calmette-Guérin (BCG), the only tuberculosis (TB) vaccine available today, confers superior protection over standard intradermal immunization. Here we demonstrate that mucosal MTBVAC is well tolerated, eliciting polyfunctional T helper type 17 cells, interleukin-10, and immunoglobulins in the airway and yielding a broader antigenic profile than BCG in rhesus macaques. Beyond our previous work, we show that local immunoglobulins, induced by MTBVAC and BCG, bind to M. tuberculosis and enhance pathogen uptake. Furthermore, after pulmonary vaccination, but not M. tuberculosis infection, local T cells expressed high levels of mucosal homing and tissue residency markers. Our data show that pulmonary MTBVAC administration has the potential to enhance its efficacy and justifies further exploration of mucosal vaccination strategies in preclinical efficacy studies., Graphical Abstract, Highlights Pulmonary MTBVAC delivery confers immune signature correlating with TB protection This signature spreads through the lung without a recall response in the skin Vaccine-induced T cells have increased mucosal homing and tissue residency markers Vaccine-induced antibodies enhance phagocytosis of M. tuberculosis, Dijkman et al. show that pulmonary immunization with the M. tuberculosis-derived vaccine candidate MTBVAC confers a local mucosal antigen-specific signature—polyfunctional Th1/Th17 cells exhibiting increased homing and tissue residency marker expression, IL-10, and phagocytosis-promoting immunoglobulins—that has been associated previously with protection from TB infection and disease in rhesus macaques.