A novel coronavirus, SARS-CoV-2, has been identified as the causative agent of the current COVID-19 pandemic. Animal models, and in particular non-human primates, are essential to understand the pathogenesis of emerging diseases and to assess the safety and efficacy of novel vaccines and therapeutics. Here, we show that SARS-CoV-2 replicates in the upper and lower respiratory tract and causes pulmonary lesions in both rhesus and cynomolgus macaques. Immune responses against SARS-CoV-2 are also similar in both species and equivalent to those reported in milder infections and convalescent human patients. This finding is reiterated by our transcriptional analysis of respiratory samples revealing the global response to infection. We describe a new method for lung histopathology scoring that will provide a metric to enable clearer decision making for this key endpoint. In contrast to prior publications, in which rhesus are accepted to be the preferred study species, we provide convincing evidence that both macaque species authentically represent mild to moderate forms of COVID-19 observed in the majority of the human population and both species should be used to evaluate the safety and efficacy of interventions against SARS-CoV-2. Importantly, accessing cynomolgus macaques will greatly alleviate the pressures on current rhesus stocks.

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.

b Tuberculosis (TB) is a reemerging disease. The only available vaccine, Mycobacterium bovis BCG, 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-ofconcept data for further evaluation of this route of immunization for use in human clinical trials.

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-Guérin (BCG) induced comparable frequencies of IFN-γ spot forming units (SFU) measured in peripheral blood mononuclear cells (PBMCs) by ELISpot, although the induction of IFN-γ 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.

Nine million cases of tuberculosis (TB) were reported in 2013, with a further 1.5 million deaths attributed to the disease. When delivered as an intradermal (i.d.) injection, the Mycobacterium bovis BCG vaccine provides limited protection, whereas aerosol delivery has been shown to enhance efficacy in experimental models. In this study, we used the rhesus macaque model to characterize the mucosal and systemic immune response induced by aerosol-delivered BCG vaccine. Aerosol delivery of BCG induced both Th1 and Th17 cytokine responses. Polyfunctional CD4 T cells were detected in bronchoalveolar lavage (BAL) fluid and peripheral blood mononuclear cells (PBMCs) 8 weeks following vaccination in a dose-dependent manner. A similar trend was seen in peripheral gamma interferon (IFN-γ) spot-forming units measured by enzyme-linked immunosorbent spot (ELISpot) assay and serum anti-purified protein derivative (PPD) IgG levels. CD8 T cells predominantly expressed cytokines individually, with pronounced tumor necrosis factor alpha (TNF-α) production by BAL fluid cells. T-cell memory phenotype analysis revealed that CD4 and CD8 populations isolated from BAL fluid samples were polarized toward an effector memory phenotype, whereas the frequencies of peripheral central memory T cells increased significantly and remained elevated following aerosol vaccination. Expression patterns of the α4β1 integrin lung homing markers remained consistently high on CD4 and CD8 T cells isolated from BAL fluid and varied on peripheral T cells. This characterization of aerosol BCG vaccination highlights features of the resulting mycobacterium-specific immune response that may contribute to the enhanced protection previously reported in aerosol BCG vaccination studies and will inform future studies involving vaccines delivered to the mucosal surfaces of the lung.