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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.

Vaccines against SARS-CoV-2 are urgently required, but early development of vaccines against SARS-CoV-1 resulted in enhanced disease after vaccination. Careful assessment of this phenomena is warranted for vaccine development against SARS CoV-2. Here we report detailed immune profiling after ChAdOx1 nCoV-19 (AZD1222) and subsequent high dose challenge in two animal models of SARS-CoV-2 mediated disease. We demonstrate in rhesus macaques the lung pathology caused by SARS-CoV-2 mediated pneumonia is reduced by prior vaccination with ChAdOx1 nCoV-19 which induced neutralising antibody responses after a single intramuscular administration. In a second animal model, ferrets, ChAdOx1 nCoV-19 reduced both virus shedding and lung pathology. Antibody titre were boosted by a second dose. Data from these challenge models on the absence of enhanced disease and the detailed immune profiling, support the continued clinical evaluation of ChAdOx1 nCoV-19., Lambe, Spencer, Thomas, Gilbert and colleagues report on the detailed immune profile of rhesus macaques and ferrets vaccinated against SARS-CoV-2 under high dose challenge. Their findings indicate that the ChAdOx1 nCoV-19 (AZD1222) the vaccine induces immune responses and reduces disease symptoms in both models, including SARS-CoV-2 mediated pneumonia and virus shedding.

This study describes the use of cynomolgus macaques of Chinese origin (CCM) to evaluate the efficacy and immunogenicity of the BCG vaccine against high dose aerosol Mycobacterium tuberculosis challenge. Progressive disease developed in three of the unvaccinated animals within 10 weeks of challenge, whereas all six vaccinated animals controlled disease for 26 weeks. Three unvaccinated animals limited disease progression, highlighting the intrinsic ability of this macaque species to control disease in comparison to macaques of other species and genotypes. Low levels of IFNγ were induced by BCG vaccination in CCM suggesting that IFNγ alone does not provide a sufficiently sensitive biomarker of vaccination in this model. An early response after challenge, together with the natural bias towards terminal effector memory T-cell populations and the contribution of monocytes appears to enhance the ability of CCM to naturally control infection. The high dose aerosol challenge model of CCM has value for examination of the host immune system to characterise control of infection which would influence future vaccine design. Although it may not be the preferred platform for the assessment of prophylactic vaccine candidates, the model could be well suited for testing post-exposure vaccination strategies and drug evaluation studies.

Safe and effective vaccines will provide essential medical countermeasures to tackle the COVID-19 pandemic. Here, we evaluate the safety, immunogenicity and efficacy of the intradermal delivery of INO-4800, a synthetic DNA vaccine candidate encoding the SARS-CoV-2 spike protein in the rhesus macaque model. Single and 2 dose vaccination regimens were evaluated. Vaccination induces both binding and neutralizing antibodies, along with IFN-γ-producing T cells against SARS-CoV-2. Upon administration of a high viral dose (5 x 106 pfu) via the intranasal and intratracheal routes we observe significantly reduced virus load in the lung and throat, in the vaccinated animals compared to controls. 2 doses of INO-4800 is associated with more robust vaccine-induced immune responses and improved viral protection. Importantly, histopathological examination of lung tissue provides no indication of vaccine-enhanced disease following SARS-CoV-2 challenge in INO-4800 immunized animals. This vaccine candidate is currently under clinical evaluation as a 2 dose regimen.

This study describes the use of cynomolgus macaques of Chinese origin (CCM) to evaluate the efficacy and immunogenicity of the BCG vaccine against high dose aerosol Mycobacterium tuberculosis challenge. Progressive disease developed in three of the unvaccinated animals within ten weeks of challenge, whereas all six vaccinated animals controlled disease for 26 weeks. Three unvaccinated animals limited disease progression, highlighting the intrinsic ability of this macaque species to control disease in comparison to macaques of other species and genotypes. Low levels of IFNγ were induced by BCG vaccination in CCM suggesting that IFNγ alone does not provide a sufficiently sensitive biomarker of vaccination in this model. An early response after challenge, together with the natural bias towards terminal effector memory T-cell populations and the contribution of monocytes appears to enhance the ability of CCM to naturally control infection. The high dose aerosol challenge model of CCM has value for examination of the host immune system to characterise control of infection which would influence future vaccine design. Although it may not be the preferred platform for the assessment of prophylactic vaccine candidates, the model could be well suited for testing post-exposure vaccination strategies and drug evaluation studies.

A single intradermal vaccination with MTBVAC given to adult rhesus macaques was well tolerated and conferred a significant improvement in outcome following aerosol exposure to M. tuberculosis compared to that provided by a single BCG vaccination. Vaccination with MTBVAC resulted in a significant reduction in M. tuberculosis infection-induced disease pathology measured using in vivo medical imaging, in gross pathology lesion counts and pathology scores recorded at necropsy, the frequency and severity of pulmonary granulomas and the frequency of recovery of viable M. tuberculosis from extrapulmonary tissues following challenge. The immune profiles induced following immunisation with MTBVAC reflect those identified in human clinical trials of MTBVAC. Evaluation of MTBVAC- and TB peptide-pool-specific T-cell cytokine production revealed a predominantly Th1 response from poly- (IFN-γ+TNF-α+IL2+) and multi-(IFN-γ+TNF-α+) functional CD4 T cells, while only low levels of Th22, Th17 and cytokine-producing CD8 T-cell populations were detected together with low-level, but significant, increases in CFP10-specific IFN-γ secreting cells. In this report, we describe concordance between immune profiles measured in clinical trials and a macaque pre-clinical study demonstrating significantly improved outcome after M. tuberculosis challenge as evidence to support the continued development of MTBVAC as an effective prophylactic vaccine for TB vaccination campaigns.

Ten million cases of tuberculosis (TB) were reported in 2018 with a further 1.5 million deaths attributed to the disease. Improved vaccination strategies are urgently required to tackle the ongoing global TB epidemic. In the absence of a validated correlate of protection, highly characterised pre-clinical models are required to assess the protective efficacy of new vaccination strategies. In this study, we demonstrate the application of a rhesus macaque ultra-low dose (ULD) aerosol M. tuberculosis challenge model for the evaluation of TB vaccination strategies by directly comparing the immunogenicity and efficacy of intradermal (ID) and aerosol BCG vaccination delivered using a portable vibrating mesh nebulizer (VMN). Aerosol- and ID-delivered Bacille Calmette-Gu&eacute, rin (BCG) induced comparable frequencies of IFN-&gamma, spot forming units (SFU) measured in peripheral blood mononuclear cells (PBMCs) by ELISpot, although the induction of IFN-&gamma, SFU was significantly delayed following aerosol immunisation. This delayed response was also apparent in an array of secreted pro-inflammatory and chemokine markers, as well as in the frequency of antigen-specific cytokine producing CD4 and CD8 T-cells measured by multi-parameter flow cytometry. Interrogation of antigen-specific memory T-cell phenotypes revealed that vaccination-induced CD4 and CD8 T-cell populations primarily occupied the central memory (TCM) and transitional effector memory (TransEM) phenotype, and that the frequency of CD8 TCM and TransEM populations was significantly higher in aerosol BCG-vaccinated animals in the week prior to M. tuberculosis infection. The total and lung pathology measured following M. tuberculosis challenge was significantly lower in vaccinated animals relative to the unvaccinated control group and pathology measured in extra-pulmonary tissues was significantly reduced in aerosol BCG-vaccinated animals, relative to the ID-immunised group. Similarly, significantly fewer viable M. tuberculosis CFU were recovered from the extra-pulmonary tissues of aerosol BCG-vaccinated macaques relative to unvaccinated animals. In this study, a rhesus macaque ULD M. tuberculosis aerosol challenge model was applied as a refined and sensitive system for the evaluation of TB vaccine efficacy and to confirm that aerosol BCG vaccination delivered by portable VMN can confer a significant level of protection that is equivalent, and by some measures superior, to intradermal BCG vaccination.

This pilot study aimed to determine the utility of a cynomolgus macaque model of coinfection with simian immunodeficiency virus (SIV) for the assessment of vaccines designed to prevent reactivation of TB. Following infection caused by aerosol exposure to an ultralow dose of Mycobacterium tuberculosis (M. tb), data trends indicated that subsequent coinfection with SIVmac32H perturbed control of M. tb infection as evidenced by the increased occurrence of progressive disease in this group, higher levels of pathology and increased frequency of progressive tuberculous granulomas in the lung. BCG vaccination led to improved control of TB-induced disease and lower viral load in comparison to unvaccinated coinfected animals. The M. tb-specific IFNγ response after exposure to M. tb, previously shown to be associated with bacterial burden, was lower in the BCG-vaccinated group than in the unvaccinated groups. Levels of CD4+ and CD8+ T cells decreased in coinfected animals, with counts recovering more quickly in the BCG-vaccinated group. This pilot study provides proof of concept to support the use of the model for evaluation of interventions against reactivated/exacerbated TB caused by human immunodeficiency virus (HIV) infection.

SummaryTuberculosis (TB) remains a global pandemic, in both animals and man, and novel vaccines are urgently required. Heterologous prime-boost of BCG represents a promising strategy for improved TB vaccines, with respiratory delivery the most efficacious to date. Such an approach may be an ideal vaccination strategy against bovine TB (bTB), but respiratory vaccination presents a technical challenge in cattle. Inert bacterial spores represent an attractive vaccine vehicle. Therefore we evaluated whether parenterally administered spores are efficacious when used as a BCG boost in a murine model of immunity against Mycobacterium bovis.Here we report the use of heat-killed, TB10.4 adsorbed, Bacillus subtilis spores delivered via subcutaneous injection to boost immunity primed by BCG. We demonstrate that this approach improves the immunogenicity of BCG. Interestingly, this associated with substantial boosting of IL-17 responses; considered to be important in protective immunity against TB. These data demonstrate that parenteral delivery of spores represents a promising vaccine vehicle for boosting BCG, and identifies potential for optimisation for use as a vaccine for bovine TB.

Recombinant Bacillus subtilis spores expressing a TB antigen, MPT64, were tested for their ability to protect mice against tuberculosis challenge. A chimeric gene consisting of the spore coat gene cotB fused to mpt64 was constructed, and expression of a stable CotB-MPT64 hybrid protein of the spore coat verified. Spores were evaluated as a live vaccine and also formaldehyde inactivated. Mice were given three doses of spores or alternatively used in a prime-boost regimen with BCG. The results showed that inactivated recombinant spores were able to reduce the bacterial burden in the lungs of mice to comparable levels to that of BCG. In the prime-boost regimen, both live and inactivated spores showed a reduction in bacterial load in comparison with BCG. ELISPOT and polyfunctional T-cell analysis were performed to examine cellular responses and showed that antigen-specific secretion of Th1 cytokines was stimulated after immunisation with inactive recombinant spores and BCG. In summary, recombinant spores can elicit Th1 responses, which are important for protection against TB disease.

Intradermal (ID) BCG injection provides incomplete protection against TB in humans and experimental models. Alternative BCG vaccination strategies may improve protection in model species, including rhesus macaques. This study compares the immunogenicity and efficacy of BCG administered by ID and intravenous (IV) injection, or as an intratracheal mucosal boost (ID + IT), against aerosol challenge with Mycobacterium tuberculosis Erdman strain. Disease pathology was significantly reduced, and survival improved, by each BCG vaccination strategy, relative to unvaccinated animals. However, IV induced protection surpassed that achieved by all other routes, providing an opportunity to explore protective immunological mechanisms using antigen-specific IFN-γ ELISpot and polychromatic flow cytometry assays. IFN-γ spot forming units and multifunctional CD4 T-cell frequencies increased significantly following each vaccination regimen and were greatest following IV immunisation. Vaccine-induced multifunctional CD4 T-cells producing IFN-γ and TNF-α were associated with reduced disease pathology following subsequent M.tb challenge; however, high frequencies of this population following M.tb infection correlated with increased pathology. Cytokine producing T-cells primarily occupied the CD4 transitional effector memory phenotype, implicating this population as central to the mycobacterial response, potentially contributing to the stringent control observed in IV vaccinated animals. This study demonstrates the protective efficacy of IV BCG vaccination in rhesus macaques, offering a valuable tool for the interrogation of immunological mechanisms and potential correlates of protection.

Tuberculosis (TB) is a reemerging disease. The only available vaccine,Mycobacterium bovisBCG, is delivered intradermally and confers highly variable efficacy against pulmonary disease. There is an urgent need for improved vaccination strategies. Murine studies suggest that immunizations delivered directly to the respiratory mucosa might be a more effective route of vaccination. This study compared the immunogenicity of a leading candidate tuberculosis (TB) vaccine, modified vaccinia virus Ankara expressing antigen 85A (MVA85A), in rhesus macaques, delivered either as an aerosol or as an intradermal boost immunization 12 weeks after an intradermal BCG prime vaccine. Aerosol vaccination was well tolerated. MVA85A delivered by aerosol or by intradermal injection induced antigen-specific immune responses in the periphery and the lung, with a trend toward the highest response when the compartment and route of delivery were matched. The ability of poxvirus-vectored vaccines delivered by the systemic route to induce responses in the mucosal immune compartment in macaques is in contrast to the independent compartmentalization of mucosal and systemic immune systems described in mice. Unlike intradermal vaccination, aerosol vaccination did not induce a detectable serum anti-vector antibody response. The delivery of vaccines to the lungs might provide an immunization strategy that limits the induction of systemic anti-vector immunity, which would be extremely useful in the development of improved vaccine strategies. This is the first study to show a recombinant MVA-vectored vaccine to be highly immunogenic when delivered by the aerosol route to nonhuman primates. These results provide important safety and proof-of-concept data for further evaluation of this route of immunization for use in human clinical trials.

Tuberculosis is a global health problem. The Mycobacterium bovis Bacille Calmette Guerin (BCG) vaccine has variable efficacy (0-80%) so there is a drive to develop novel vaccines. The cytokine, interferon gamma (IFNγ), is an essential component of the protective response to M. tuberculosis (M. tb) infection and is also produced in response to BCG vaccination. Induction of an IFNγ response is used as a biomarker of successful vaccination in the assessment of new tuberculosis (TB) vaccines. The IFNγ ELISPOT assay provides an important tool for TB research. It is used for both the diagnosis of infection (T.Spot assay), and for the evaluation of the immunogenicity of new TB vaccine candidates in human clinical trials, in the non-human primate (NHP) model of TB infection studies. The ELISPOT assay captures IFNγ produced by peripheral blood mononuclear cells (PBMCs) following specific stimulation, onto a membrane so individual cells can be enumerated and the frequency of responding cells determined. Hence spot forming units (SFU) per 106 cells provide the traditional measure for ELISPOT assays. The discriminatory power of SFU is limited. In some situations, the number of SFU in BCG vaccinated, and unvaccinated, subjects was found to be similar, although the spots were observed to be larger in vaccinated subjects. Spot size potentially provides a measure of the quantity of cytokine produced by individual cells. The AID ELISPOT plate reader software used to determine frequency of spots also has the capability to determine the size of each spot. Consideration of spot size in combination with spot forming units was investigated in our studies of BCG immunogenicity. This additional readout was found to enhance the discriminatory power of the ELISPOT assay, and provide more information on the immune response to BCG vaccination and infection with M.tb.

Needle-free, mucosal immunization is a highly desirable strategy for vaccination against many pathogens, especially those entering through the respiratory mucosa, such as Mycobacterium tuberculosis . Unfortunately, mucosal vaccination against tuberculosis (TB) is impeded by a lack of suitable adjuvants and/or delivery platforms that could induce a protective immune response in humans. Here, we report on a novel biotechnological approach for mucosal vaccination against TB that overcomes some of the current limitations. This is achieved by coating protective TB antigens onto the surface of inert bacterial spores, which are then delivered to the respiratory tract. Our data showed that mice immunized nasally with coated spores developed humoral and cellular immune responses and multifunctional T cells and, most importantly, presented significantly reduced bacterial loads in their lungs and spleens following pathogenic challenge. We conclude that this new vaccine delivery platform merits further development as a mucosal vaccine for TB and possibly also other respiratory pathogens.