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The tuberculosis vaccine, Bacille Calmette-Guerin (BCG), also affords protection against non-tuberculous diseases attributable to heterologous immune mechanisms such as trained innate immunity, activation of non-conventional T-cells, and cross-reactive adaptive immunity. Aerosol vaccine delivery can target immune responses toward the primary site of infection for a respiratory pathogen. Therefore, we hypothesised that aerosol delivery of BCG would enhance cross-protective action against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and be a deployable intervention against coronavirus disease 2019 (COVID-19). Immune parameters were monitored in vaccinated and unvaccinated rhesus macaques for 28 days following aerosol BCG vaccination. High-dose SARS-CoV-2 challenge was applied by intranasal and intrabronchial instillation and animals culled 6–8 days later for assessment of viral, disease, and immunological parameters. Mycobacteria-specific cell-mediated immune responses were detected following aerosol BCG vaccination, but SARS-CoV-2-specific cellular- and antibody-mediated immunity was only measured following challenge. Early secretion of cytokine and chemokine markers associated with the innate cellular and adaptive antiviral immune response was detected following SARS-CoV-2 challenge in vaccinated animals, at concentrations that exceeded titres measured in unvaccinated macaques. Classical CD14+ monocytes and Vδ2 γδ T-cells quantified by whole-blood immunophenotyping increased rapidly in vaccinated animals following SARS-CoV-2 challenge, indicating a priming of innate immune cells and non-conventional T-cell populations. However, viral RNA quantified in nasal and pharyngeal swabs, bronchoalveolar lavage (BAL), and tissue samples collected at necropsy was equivalent in vaccinated and unvaccinated animals, and in-life CT imaging and histopathology scoring applied to pulmonary tissue sections indicated that the disease induced by SARS-CoV-2 challenge was comparable between vaccinated and unvaccinated groups. Hence, aerosol BCG vaccination did not induce, or enhance the induction of, SARS-CoV-2 cross-reactive adaptive cellular or humoral immunity, although an influence of BCG vaccination on the subsequent immune response to SARS-CoV-2 challenge was apparent in immune signatures indicative of trained innate immune mechanisms and primed unconventional T-cell populations. Nevertheless, aerosol BCG vaccination did not enhance the initial clearance of virus, nor reduce the occurrence of early disease pathology after high dose SARS-CoV-2 challenge. However, the heterologous immune mechanisms primed by BCG vaccination could contribute to the moderation of COVID-19 disease severity in more susceptible species following natural infection.

Mycobacterium tuberculosis (Mtb) is the leading cause of death from infection worldwide 1 . The only available vaccine, BCG (Bacillus Calmette–Guérin), is given intradermally and has variable efficacy against pulmonary tuberculosis, the major cause of mortality and disease transmission 1 , 2 . Here we show that intravenous administration of BCG profoundly alters the protective outcome of Mtb challenge in non-human primates ( Macaca mulatta ). Compared with intradermal or aerosol delivery, intravenous immunization induced substantially more antigen-responsive CD4 and CD8 T cell responses in blood, spleen, bronchoalveolar lavage and lung lymph nodes. Moreover, intravenous immunization induced a high frequency of antigen-responsive T cells across all lung parenchymal tissues. Six months after BCG vaccination, macaques were challenged with virulent Mtb. Notably, nine out of ten macaques that received intravenous BCG vaccination were highly protected, with six macaques showing no detectable levels of infection, as determined by positron emission tomography–computed tomography imaging, mycobacterial growth, pathology and granuloma formation. The finding that intravenous BCG prevents or substantially limits Mtb infection in highly susceptible rhesus macaques has important implications for vaccine delivery and clinical development, and provides a model for defining immune correlates and mechanisms of vaccine-elicited protection against tuberculosis. The delivery route and dose of the BCG vaccine profoundly alters the protective outcome after Mycobacterium tuberculosis challenge in non-human primates.

Intradermal MVA85A, a candidate vaccine against tuberculosis, induces high amounts of Ag85A-specific CD4 T cells in adults who have already received the BCG vaccine, but aerosol delivery of this vaccine might offer immunological and logistical advantages. We did a phase 1 double-blind trial to compare the safety and immunogenicity of aerosol-administered and intradermally administered MVA85A In this phase 1, double-blind, proof-of-concept trial, 24 eligible BCG-vaccinated healthy UK adults were randomly allocated (1:1) by sequentially numbered, sealed, opaque envelopes into two groups: aerosol MVA85A and intradermal saline placebo or intradermal MVA85A and aerosol saline placebo. Participants, the bronchoscopist, and immunologists were masked to treatment assignment. The primary outcome was safety, assessed by the frequency and severity of vaccine-related local and systemic adverse events. The secondary outcome was immunogenicity assessed with laboratory markers of cell-mediated immunity in blood and bronchoalveolar lavage samples. Safety and immunogenicity were assessed for 24 weeks after vaccination. Immunogenicity to both insert Ag85A and vector modified vaccinia virus Ankara (MVA) was assessed by ex-vivo interferon-γ ELISpot and serum ELISAs. Since all participants were randomised and vaccinated according to protocol, our analyses were per protocol. This trial is registered with ClinicalTrials.gov, number NCT01497769. Both administration routes were well tolerated and immunogenic. Respiratory adverse events were rare and mild. Intradermal MVA85A was associated with expected mild local injection-site reactions. Systemic adverse events did not differ significantly between the two groups. Three participants in each group had no vaccine-related systemic adverse events; fatigue (11/24 [46%]) and headache (10/24 [42%]) were the most frequently reported symptoms. Ag85A-specific systemic responses were similar across groups. Ag85A-specific CD4 T cells were detected in bronchoalveolar lavage cells from both groups and responses were higher in the aerosol group than in the intradermal group. MVA-specific cellular responses were detected in both groups, whereas serum antibodies to MVA were only detectable after intradermal administration of the vaccine. Further clinical trials assessing the aerosol route of vaccine delivery are merited for tuberculosis and other respiratory pathogens. The Wellcome Trust and Oxford Radcliffe Hospitals Biomedical Research Centre.

Mycobacterium tuberculosis remains a global health threat despite the world-wise use of the vaccine Bacille Calmette Guerin (BCG). Introducing a new vaccine could help to combat this pandemic, and efforts are underway to develop novel candidates, including by modifying parental BCG. Here, we use the genetically and phenotypically heterogenous Diversity Outbred mice to test the efficacy of the novel vaccine candidate BCGΔBCG1419c. BCGΔBCG1419c contains a mutation in BCG1419c, a phosphodiesterase linked with biofilm formation. BCGΔBCG1419c was previously shown to be more attenuated than the parental BCG strain in inbred mice and to provide protection against aerogenic M. tuberculosis challenge in inbred mice and guinea pigs. In the current study, we find that protection afforded by BCGΔBCG1419c in outbred DO mice is at least equivalent to, if not better than, that provided by parental BCG.

Tuberculosis (TB) is the leading cause of death worldwide from a single infectious agent. Bacillus Calmette-Guérin (BCG), the only licensed vaccine, provides limited protection. Controlled human infection models (CHIMs) are useful in accelerating vaccine development for pathogens with no correlates of protection; however, the need for prolonged treatment makes an unethical challenge agent. Aerosolised BCG provides a potential safe surrogate of infection. A CHIM in BCG-vaccinated as well as BCG-naïve individuals would allow identification of novel BCG-booster vaccine candidates and facilitate CHIM studies in populations with high TB endemicity. The purpose of this study was to evaluate the safety and utility of an aerosol BCG CHIM in historically BCG-vaccinated volunteers. There were 12 healthy, historically BCG-vaccinated UK adults sequentially enrolled into dose-escalating groups. The first three received 1 × 10 CFU aerosol BCG Danish 1331 via a nebuliser. After safety review, subsequent groups received doses of 1 × 10 CFU, 1 × 10 CFU, or 1 × 10 CFU. Safety was monitored through self-reported adverse events (AEs), laboratory tests, and lung function testing. Immunology blood samples were taken pre-infection and at multiple timepoints post-infection. A bronchoalveolar lavage (BAL) taken 14 days post-infection was analysed for presence of live BCG. No serious AEs occurred during the study. Solicited systemic and respiratory AEs were frequent in all groups, but generally short-lived and mild in severity. There was a trend for more reported AEs in the highest-dose group. No live BCG was detected in BAL from any volunteers. Aerosol BCG induced potent systemic cellular immune responses in the highest-dose group 7 days post-infection. Aerosol BCG infection up to a dose of 1 × 10 CFU was well-tolerated in historically BCG-vaccinated healthy, UK adults. No live BCG was detected in the BAL fluid 14 days post-infection despite potent systemic responses, suggesting early clearance. Further work is needed to expand the number of volunteers receiving BCG via the aerosol route to refine and establish utility of this aerosol BCG CHIM. https://clinicaltrials.gov/, identifier NCT04777721.

A new and more effective tuberculosis (TB) vaccine is urgently needed, but development is hampered by the lack of validated immune correlates of protection. Bacillus Calmette Guérin (BCG) vaccination by the aerosol (AE) and intravenous (IV) routes has been shown to confer superior levels of protection from challenge with Mycobacterium tuberculosis (M.tb) in non-human primates (NHP) compared with standard intradermal (ID) administration. This finding offers a valuable opportunity to investigate which aspects of immunity are associated with improved control of M.tb and may represent biomarkers or correlates of protection. As TB vaccine research to date has focused largely on cellular immunity, we aimed to better characterize the poorly-understood serum antibody response to BCG administered by different routes of vaccination in NHP. We demonstrate superior M.tb-specific IgG, IgA, and IgM titers in serum following IV BCG vaccination compared to the ID or AE routes. We also observe improved capacity of IgG induced by IV BCG to opsonize the surface of mycobacteria, and report for the first time that M.tb-specific IgG from IV BCG vaccinated animals is of higher avidity compared with IgG from ID or AE BCG vaccinated animals. Notably, we identified a significant correlation between IgG avidity and measures of protection from aerosol M.tb challenge. Our findings highlight a potential role for antibodies as markers and/or mediators of the superior vaccine-induced protection IV BCG confers against TB and suggest that quality, as well as quantity, of antibodies should be considered when developing and evaluating TB vaccine candidates. •Superior serum antibody titers following IV vs. ID or aerosol BCG vaccination.•IgG induced by IV BCG has improved mycobacterial opsonization vs. ID or aerosol BCG.•Higher avidity M.tb-specific IgG following IV vs. ID or aerosol BCG vaccination.•Association between IgG avidity and protection from aerosol M.tb challenge.

Alveolar macrophages (AMs) play a critical role during Mycobacterium tuberculosis (Mtb) infection as the first cells in the lung to encounter bacteria. We previously showed that AMs initially respond to Mtb in vivo by mounting a cell-protective, rather than pro-inflammatory response. However, the plasticity of the initial AM response was unknown. Here, we characterize how previous exposure to Mycobacterium , either through subcutaneous vaccination with Mycobacterium bovis (scBCG) or through a contained Mtb infection (coMtb) that mimics aspects of concomitant immunity, impacts the initial response by AMs. We find that both scBCG and coMtb accelerate early innate cell activation and recruitment and generate a stronger pro-inflammatory response to Mtb in vivo by AMs. Within the lung environment, AMs from scBCG vaccinated mice mount a robust interferon-associated response, while AMs from coMtb mice produce a broader inflammatory response that is not dominated by Interferon Stimulated Genes. Using scRNAseq, we identify changes to the frequency and phenotype of airway-resident macrophages following Mycobacterium exposure, with enrichment for both interferon-associated and pro-inflammatory populations of AMs. In contrast, minimal changes were found for airway-resident T cells and dendritic cells after exposures. Ex vivo stimulation of AMs with Pam3Cys, LPS and Mtb reveal that scBCG and coMtb exposures generate stronger interferon-associated responses to LPS and Mtb that are cell-intrinsic changes. However, AM profiles that were unique to each exposure modality following Mtb infection in vivo are dependent on the lung environment and do not emerge following ex vivo stimulation. Overall, our studies reveal significant and durable remodeling of AMs following exposure to Mycobacterium , with evidence for both AM-intrinsic changes and contributions from the altered lung microenvironments. Comparisons between the scBCG and coMtb models highlight the plasticity of AMs in the airway and opportunities to target their function through vaccination or host-directed therapies.

Despite widespread immunization with Bacille-Calmette-Guérin (BCG), the only currently licensed tuberculosis (TB) vaccine, TB remains a leading cause of mortality globally. There are many TB vaccine candidates in the developmental pipeline, but the lack of a robust animal model to assess vaccine efficacy has hindered our ability to prioritize candidates for human clinical trials. Here we use a murine ultra-low dose (ULD) Mycobacterium tuberculosis (Mtb) challenge model to assess protection conferred by BCG vaccination. We show that BCG confers a reduction in lung bacterial burdens that is more durable than that observed after conventional dose challenge, curbs Mtb dissemination to the contralateral lung, and, in a small percentage of mice, prevents detectable infection. These findings are consistent with the ability of human BCG vaccination to mediate protection, particularly against disseminated disease, in specific human populations and clinical settings. Overall, our findings demonstrate that the ultra-low dose Mtb infection model can measure distinct parameters of immune protection that cannot be assessed in conventional dose murine infection models and could provide an improved platform for TB vaccine testing.

The current tuberculosis (TB) vaccine, Mycobacterium bovis Bacillus Calmette-Guérin (BCG), provides insufficient protection against pulmonary TB. Previously, we generated a listeriolysin-expressing recombinant BCG strain, which to date has successfully completed phase I and phase IIa clinical trials. In an attempt to further improve efficacy, we deleted the antiapoptotic virulence gene nuoG , encoding NADH dehydrogenase 1 subunit G, from BCG Δ ureC :: hly . In vitro , deletion of nuoG unexpectedly led to strongly increased recruitment of the autophagosome marker LC3 to the engulfed vaccine, suggesting that nuoG also affects xenophagic pathways. In mice, BCG Δ ureC :: hly Δ nuoG vaccination was safer than BCG and improved protection over that of parental BCG Δ ureC :: hly , significantly reducing TB load in murine lungs, ameliorating pulmonary pathology, and enhancing immune responses. Transcriptome analysis of draining lymph nodes after vaccination with either BCG Δ ureC :: hly or BCG Δ ureC :: hly Δ nuoG demonstrated earlier and stronger induction of immune responses than that with BCG SSI and suggested upregulation of inflammasome activation and interferon-induced GTPases. In summary, BCG Δ ureC :: hly Δ nuoG is a promising next-generation TB vaccine candidate with excellent efficacy and safety. IMPORTANCE Autophagy and apoptosis are fundamental processes allowing cells to degrade their components or kill themselves, respectively. The immune system has adopted these mechanisms to eliminate intracellular pathogens. Residing in host cells, the causative agent of tuberculosis, Mycobacterium tuberculosis , has evolved strategies to set cellular programs of autophagy and apoptosis “on hold.” The mycobacterial gene nuoG was found to prevent host cell apoptosis. We have deleted nuoG in the live vaccine candidate BCG Δ ureC :: hly , which is in phase II clinical development, to leave cellular apoptosis “on go” upon immunization. In preclinical models, this strategy boosted immunity and improved protection from M. tuberculosis infection. Unexpectedly, we obtained compelling evidence that mycobacterial nuoG facilitates inhibition of autophagic pathways, suggesting a new role for this gene in the host-pathogen interplay in tuberculosis. Autophagy and apoptosis are fundamental processes allowing cells to degrade their components or kill themselves, respectively. The immune system has adopted these mechanisms to eliminate intracellular pathogens. Residing in host cells, the causative agent of TB, Mycobacterium tuberculosis , has evolved strategies to set cellular programs of autophagy and apoptosis “on hold.” The mycobacterial gene nuoG was found to prevent host cell apoptosis. We have deleted nuoG in the live vaccine candidate BCG Δ ureC :: hly , which is in phase II clinical development, to leave cellular apoptosis “on go” upon immunization. In preclinical models, this strategy boosted immunity and improved protection from M. tuberculosis infection. Unexpectedly, we obtained compelling evidence that mycobacterial nuoG facilitates inhibition of autophagic pathways, suggesting a new role for this gene in the host-pathogen interplay in TB.

The currently licensed anti-tuberculosis (TB) vaccine, Mycobacterium bovis BCG, provides limited protection against pulmonary TB in adolescents and adults, the main cause of TB transmission and mortality. To obtain an improved BCG-based vaccine candidate with increased immune signalling but still low virulence, we have previously generated a recombinant BCG strain named BCG::ESX-1 Mmar , which is heterologously expressing ESX-1 functions of Mycobacterium marinum and thereby modulates the host innate immune responses via phagosomal rupture-associated induction of type I interferon production and enhanced inflammasome activity, leading to superior protection against TB disease in murine infection models. As protection may also vary with the route of vaccination, here, we have explored aerosol vaccination relative to subcutaneous vaccination, using BCG Pasteur and BCG::ESX-1 Mmar . We found that mice vaccinated via the aerosol route with BCG Pasteur or BCG::ESX-1 Mmar both yielded higher frequencies of CD4 + and CD8 + Th1 activated effectors and T effector memory cells in the lungs compared to subcutaneously immunised mice, whereas comparable polyfunctional Th1 (IL-2, TNF-α and IFN-γ) cytokine-producing subsets were observed in the spleens of all vaccinated mice. Significantly higher IL-17A responses without severe lung pathology were seen in the lungs of aerosol-vaccinated mice associated to local and transient inflammatory cytokine responses and immune cell infiltrations. In contrast to the subcutaneous route, aerosol vaccination elicited high amounts of humoral IgG and IgA responses in the bronchoalveolar lavage fluid and induced a substantial number of lung CD4 + and CD8 + T cells expressing CD69 + CD103 + tissue-residency markers. These effects led to significant improved protection against M. tuberculosis and reduced lung pathology in aerosol-vaccinated mice compared to subcutaneously vaccinated mice. Moreover, BCG::ESX-1 Mmar vaccine induced enhanced T-cell immunity and superior protection compared to parental BCG Pasteur for both vaccination routes and thereby represents an interesting candidate for developing improved vaccination strategies against TB.