The Post-Acute Phase of SARS-CoV-2 Infection in Two Macaque Species Is Associated with Signs of Ongoing Virus Replication and Pathology in Pulmonary and Extrapulmonary Tissues.

The post-acute phase of SARS-CoV-2 infection was investigated in rhesus (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis). During the acute phase of infection, SARS-CoV-2 was shed via the nose and throat, and viral RNA was occasionally detected in feces. This phase coincided with a transient change in systemic immune activation. Even after the alleged resolution of the infection, computed tomography (CT) and positron emission tomography (PET)-CT revealed pulmonary lesions and activated tracheobronchial lymph nodes in all animals. Post-mortem histological examination of the lung tissue revealed mostly marginal or resolving minimal lesions that were indicative of SARS-CoV-2 infection. Evidence for SARS-CoV-2-induced histopathology was also found in extrapulmonary tissue samples, such as conjunctiva, cervical, and mesenteric lymph nodes. However, 5-6 weeks after SARS-CoV-2 exposure, upon necropsy, viral RNA was still detectable in a wide range of tissue samples in 50% of the macaques and included amongst others the heart, the respiratory tract and surrounding lymph nodes, salivary gland, and conjunctiva. Subgenomic messenger RNA was detected in the lungs and tracheobronchial lymph nodes, indicative of ongoing virus replication during the post-acute phase. These results could be relevant for understanding the long-term consequences of COVID-19 in humans.

Stronger induction of trained immunity by mucosal BCG or MTBVAC vaccination compared to standard intradermal vaccination.

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.

Pulmonary MTBVAC vaccination induces immune signatures previously correlated with prevention of tuberculosis infection.

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.

Recommendations for Standardizing Thorax PET-CT in Non-Human Primates by Recent Experience from Macaque Studies.

Despite the possibilities of routine clinical measures and assays on readily accessible bio-samples, it is not always essential in animals to investigate the dynamics of disease longitudinally. In this regard, minimally invasive imaging methods provide powerful tools in preclinical research. They can contribute to the ethical principle of gathering as much relevant information per animal as possible. Besides, with an obvious parallel to clinical diagnostic practice, such imaging platforms are potent and valuable instruments leading to a more refined use of animals from a welfare perspective. Non-human primates comprise highly relevant species for preclinical research to enhance our understanding of disease mechanisms and/or the development of improved prophylactic or therapeutic regimen for various human diseases. In this paper, we describe parameters that critically affect the quality of integrated positron emission tomography and computed tomography (PET-CT) in non-human primates. Lessons learned are exemplified by results from imaging experimental infectious respiratory disease in macaques; specifically tuberculosis, influenza, and SARS-CoV-2 infection. We focus on the thorax and use of 18F-fluorodeoxyglucose as a PET tracer. Recommendations are provided to guide various stages of PET-CT-supported research in non-human primates, from animal selection, scan preparation, and operation, to processing and analysis of imaging data.

Respiratory Immunization With a Whole Cell Inactivated Vaccine Induces Functional Mucosal Immunoglobulins Against Tuberculosis in Mice and Non-human Primates.

Vaccination through the natural route of infection represents an attractive immunization strategy in vaccinology. In the case of tuberculosis, vaccine delivery by the respiratory route has regained interest in recent years, showing efficacy in different animal models. In this context, respiratory vaccination triggers lung immunological mechanisms which are omitted when vaccines are administered by parenteral route. However, contribution of mucosal antibodies to vaccine- induced protection has been poorly studied. In the present study, we evaluated in mice and non-human primates (NHP) a novel whole cell inactivated vaccine (MTBVAC HK), by mucosal administration. MTBVAC HK given by intranasal route to BCG-primed mice substantially improved the protective efficacy conferred by subcutaneous BCG only. Interestingly, this improved protection was absent in mice lacking polymeric Ig receptor (pIgR), suggesting a crucial role of mucosal secretory immunoglobulins in protective immunity. Our study in NHP confirmed the ability of MTBVAC HK to trigger mucosal immunoglobulins. Importantly, in vitro assays demonstrated the functionality of these immunoglobulins to induce M. tuberculosis opsonization in the presence of human macrophages. Altogether, our results suggest that mucosal immunoglobulins can be induced by vaccination to improve protection against tuberculosis and therefore, they represent a promising target for next generation tuberculosis vaccines.

Evaluation of heterologous prime-boost vaccination strategies using chimpanzee adenovirus and modified vaccinia virus for TB subunit vaccination in rhesus macaques.

Tuberculosis (TB) still is the principal cause of death from infectious disease and improved vaccination strategies are required to reduce the disease burden and break TB transmission. Here, we investigated different routes of administration of vectored subunit vaccines based on chimpanzee-derived adenovirus serotype-3 (ChAd3) for homologous prime-boosting and modified vaccinia virus Ankara (MVA) for heterologous boosting with both vaccine vectors expressing the same antigens from Mycobacterium tuberculosis (Ag85B, ESAT6, Rv2626, Rv1733, RpfD). Prime-boost strategies were evaluated for immunogenicity and protective efficacy in highly susceptible rhesus macaques. A fully parenteral administration regimen was compared to exclusive respiratory mucosal administration, while parenteral ChAd3-5Ag prime-boosting and mucosal MVA-5Ag boosting were applied as a push-and-pull strategy from the periphery to the lung. Immune analyses corroborated compartmentalized responses induced by parenteral versus mucosal vaccination. Despite eliciting TB-specific immune responses, none of the investigational regimes conferred a protective effect by standard readouts of TB compared to non-vaccinated controls, while lack of protection by BCG underpinned the stringency of this non-human primate test modality. Yet, TB manifestation after full parenteral vaccination was significantly less compared to exclusive mucosal vaccination.

Disparate Tuberculosis Disease Development in Macaque Species Is Associated With Innate Immunity.

While tuberculosis continues to afflict mankind, the immunological mechanisms underlying TB disease development are still incompletely understood. Advanced preclinical models for TB research include both rhesus and cynomolgus macaques (Macaca mulatta and Macaca fascicularis, respectively), with rhesus typically being more susceptible to acute progressive TB disease than cynomolgus macaques. To determine which immune mechanisms are responsible for this dissimilar disease development, we profiled a broad range of innate and adaptive responses, both local and peripheral, following experimental pulmonary Mycobacterium tuberculosis (Mtb) infection of both species. While T-cell and antibody responses appeared indistinguishable, we identified anti-inflammatory skewing of peripheral monocytes in rhesus and a more prominent local pro-inflammatory cytokine release profile in cynomolgus macaques associated with divergent TB disease outcome. Importantly, these differences were detectable both before and early after infection. This work shows that inflammatory and innate immune status prior to and at early stages after infection, critically affects outcome of TB infection.

Prevention of tuberculosis infection and disease by local BCG in repeatedly exposed rhesus macaques.

Tuberculosis (TB) remains the deadliest infectious disease1, and the widely used Bacillus Calmette-Guérin (BCG) vaccine fails to curb the epidemic. An improved vaccination strategy could provide a cost-effective intervention to break the transmission cycle and prevent antimicrobial resistance2,3. Limited knowledge of the host responses critically involved in protective immunity hampers the development of improved TB vaccination regimens. Therefore, assessment of new strategies in preclinical models to select the best candidate vaccines before clinical vaccine testing remains indispensable. We have previously established in rhesus macaques (Macaca mulatta) that pulmonary mucosal BCG delivery reduces TB disease where standard intradermal injection fails4,5. Here, we show that pulmonary BCG prevents infection by using a repeated limiting-dose Mycobacterium tuberculosis challenge model and identify polyfunctional T-helper type 17 (TH17) cells, interleukin-10 and immunoglobulin A as correlates of local protective immunity. These findings warrant further research into mucosal immunization strategies and their translation to clinical application to more effectively prevent the spread of TB.

Variable BCG efficacy in rhesus populations: Pulmonary BCG provides protection where standard intra-dermal vaccination fails.

M.bovis BCG vaccination against tuberculosis (TB) notoriously displays variable protective efficacy in different human populations. In non-human primate studies using rhesus macaques, despite efforts to standardise the model, we have also observed variable efficacy of BCG upon subsequent experimental M. tuberculosis challenge. In the present head-to-head study, we establish that the protective efficacy of standard parenteral BCG immunisation varies among different rhesus cohorts. This provides different dynamic ranges for evaluation of investigational vaccines, opportunities for identifying possible correlates of protective immunity and for determining why parenteral BCG immunisation sometimes fails. We also show that pulmonary mucosal BCG vaccination confers reduced local pathology and improves haematological and immunological parameters post-infection in animals that are not responsive to induction of protection by standard intra-dermal BCG. These results have important implications for pulmonary TB vaccination strategies in the future.

MVA.85A boosting of BCG and an attenuated, phoP deficient M. tuberculosis vaccine both show protective efficacy against tuberculosis in rhesus macaques.

BACKGROUND: Continuous high global tuberculosis (TB) mortality rates and variable vaccine efficacy of Mycobacterium bovis Bacille Calmette-Guérin (BCG) motivate the search for better vaccine regimes. Relevant models are required to downselect the most promising vaccines entering clinical efficacy testing and to identify correlates of protection. METHODS AND FINDINGS: Here, we evaluated immunogenicity and protection against Mycobacterium tuberculosis in rhesus monkeys with two novel strategies: BCG boosted by modified vaccinia virus Ankara expressing antigen 85A (MVA.85A), and attenuated M. tuberculosis with a disrupted phoP gene (SO2) as a single-dose vaccine. Both strategies were well tolerated, and immunogenic as evidenced by induction of specific IFNgamma responses. Antigen 85A-specific IFNgamma secretion was specifically increased by MVA.85A boosting. Importantly, both MVA.85A and SO2 treatment significantly reduced pathology and chest X-ray scores upon infectious challenge with M. tuberculosis Erdman strain. MVA.85A and SO2 treatment also showed reduced average lung bacterial counts (1.0 and 1.2 log respectively, compared with 0.4 log for BCG) and significant protective effect by reduction in C-reactive protein levels, body weight loss, and decrease of erythrocyte-associated hematologic parameters (MCV, MCH, Hb, Ht) as markers of inflammatory infection, all relative to non-vaccinated controls. Lymphocyte stimulation revealed Ag85A-induced IFNgamma levels post-infection as the strongest immunocorrelate for protection (spearman's rho: -0.60). CONCLUSIONS: Both the BCG/MVA.85A prime-boost regime and the novel live attenuated, phoP deficient TB vaccine candidate SO2 showed significant protective efficacy by various parameters in rhesus macaques. Considering the phylogenetic relationship between macaque and man and the similarity in manifestations of TB disease, these data support further development of these primary and combination TB vaccine candidates.

Effect of adjuvant on reactogenicity and long-term immunogenicity of the malaria Vaccine ICC-1132 in macaques.

ICC-1132 is a malaria vaccine candidate based on a modified hepatitis B virus core particle (HBc) bearing putative protective epitopes from the circumsporozoite protein (CS) of Plasmodium falciparum. While the epitope carrier itself is immunogenic, its potency can be increased by formulation with adjuvants. As a prelude to Phase I clinical trials, rhesus macaques were immunised twice with GMP grade ICC--1132 in saline or formulated with the adjuvants Alhydrogel (Alhydrogel) or Montanide((R)) ISA 720 (Montanide). Both adjuvant formulations gave significant humoral responses after the first injection, with titres increasing further after the second dose. The Montanide formulation was the most immunogenic, but undesirable reactogenicity in the form of sterile abscesses was associated with higher dosage levels of ICC--1132. These side effects could be avoided with lower antigen load, or by formulation of the second dose in Alhydrogel. Such measures also reduced peak titres and longevity of antibodies against CS, demonstrating the delicate balance between immunogenicity and reactogenicity of new vaccine formulations.

Protection of macaques against Mycobacterium tuberculosis infection by a subunit vaccine based on a fusion protein of antigen 85B and ESAT-6.

Various new tuberculosis (TB) vaccine candidates in combination with new delivery systems, including subunit vaccines, are currently being evaluated by a number of laboratories. One vaccine candidate that has shown promising protective capacity in mice and guinea pigs is a fusion of Ag85B and ESAT-6. In this study, we have investigated the efficacy of this Ag85B-ESAT-6 fusion protein vaccine in a non-human primate model for TB. Vaccination of cynomolgus monkeys with the Ag85B-ESAT-6 fusion protein in two different adjuvant (DDA/MPL, AS02A) resulted in a reduction in bacterial number and/or lung pathology in animals challenged with Mycobacterium tuberculosis. Vaccination prevented an increase in C-reactive protein serum levels, general activation of CD4 and CD8 subsets and boosted development of humoral and cellular immune responses to a spectrum of mycobacterial antigens on exposure to M. tuberculosis infection. We show, in two independent experiments, that vaccination of primates with Ag85B-ESAT-6 induces protective immune responses, suggesting that Ag85B-ESAT-6 is a strong candidate for further clinical evaluation. As far as we are aware this is the first report of protection in primates with a subunit vaccine.

TB diagnosis in non-human primates: comparison of two interferon-gamma assays and the skin test for identification of Mycobacterium tuberculosis infection.

In general non-human primates are highly susceptible to infections with Mycobacterium tuberculosis which therefore presents an explosive health threat to colonies. To screen for M. tuberculosis infections in non-human primates, the skin test is routinely used. However, the reliability of this test in primates is debatable. The aim of this study was to compare relatively easy in vitro diagnostic tests for TB with the skin test for detection of a tuberculosis (TB) infection. Two in vitro assays, a whole blood interferon-gamma (WB IFN-gamma) assay and in vitro stimulation of isolated lymphocytes (PBMC IFN-gamma) were evaluated during both experimental TB infections in macaques as well as during an outbreak of TB in a macaque quarantine facility. The WB IFN-gamma assay was also evaluated on healthy old and new world monkeys. Our results show that both in vitro assays detected TB infection in macaques. All experimentally infected animals showed TB-specific responses in both assays. In contrast, several TB animals were not diagnosed TB positive using the skin test. In addition, during the outbreak in the quarantine facility one animal was not detected using the routinely used skin test, but it showed strong positive responses in the WB assay. In conclusion, the in vitro assays are a valuable tool for screening non-human primates for TB infection, especially because the assays cause relatively less stress for the animals compared to the skin test and give reproducible and reliable results.

Transfected Plasmodium knowlesi produces bioactive host gamma interferon: a new perspective for modulating immune responses to malaria parasites.

Transgenic pathogenic microorganisms expressing host cytokines such as gamma interferon (IFN-gamma) have been shown to manipulate host-pathogen interaction, leading to immunomodulation and enhanced protection. Expression of host cytokines in malaria parasites offers the opportunity to investigate the potential of an immunomodulatory approach by generating immunopotentiated parasites. Using the primate malaria parasite Plasmodium knowlesi, we explored the conditions for expressing host cytokines in malaria parasites. P. knowlesi parasites transfected with DNA constructs for expressing rhesus monkey (Macaca mulatta) IFN-gamma under the control of the heterologous P. berghei apical membrane antigen 1 promoter, produced bioactive IFN-gamma in a developmentally regulated manner. IFN-gamma expression had no marked effect on in vitro parasite development. Bioactivity of the parasite-produced IFN-gamma was shown through inhibition of virus cytopathic effect and confirmed by using M. mulatta peripheral blood cells in vitro. These data indicate for the first time that it is feasible to generate malaria parasites expressing bioactive host immunomodulatory cytokines. Furthermore, cytokine-expressing malaria parasites offer the opportunity to analyze cytokine-mediated modulation of malaria during the blood and liver stages of the infection.