Pauline Maisonnasse

Le porc est un modèle d’étude présentant à la fois une grande importance agronomique et un fort potentiel comme modèle biomédical pour différentes pathologies respiratoires. Pourtant, son système immunitaire pulmonaire est peu connu, limitant l’approfondissement de ces études. Notamment, les cellules dendritiques (DCs) et macrophages (Mθs) tissulaires, qui sont à l’interface entre immunités innée et acquise, n’étaient pas caractérisés. L’objectif de cette thèse était de décrire ces cellules dans le poumon porcin à l’état basal et durant des infections par différentes souches de virus Influenza A (VIA), un pathogène capable d’infecter le porc et l’Homme. Nous avons caractérisé pour la première fois les DCs conventionnelles (cDCs) et dérivées de monocytes (moDCs), ainsi que des macrophages dérivés de monocytes (moMθs) et les macrophages alvéolaires (AMs) porcins. Les cDC1 et cDC2 se sont révélées proches de leurs équivalentes murines et humaines de par leur phénotype et leurs capacités de migration et de présentation de l’antigène in vitro. Les moDCs sont, comme chez la souris, pro-inflammatoires et recrutées lors d’une infection par le VIA in vivo. Nous avons également étudié des cellules tissulaires fortement représentées dans le poumon porcin et dont le phénotype est très similaire à celui des AMs : les cellules AM-like. Ces dernières sont pro-inflammatoires lors d’infections in vitro par un virus H3N2 porcin ou une souche Lena du PRRSV. Nous avons enfin étudié ces différents types cellulaires dans le cadre d’infections par deux souches porcines du VIA, l’une du sous-type H3N2 et l’autre du sous-type H1N2, la première étant plus pathologique en élevage et induisant plus d’inflammation. Il se pourrait que les cellules AM-like aient un rôle important dans cette différence de pathogénicité. En conclusion, nous avons mis en place des moyens pour étudier plus précisément le rôle des DCs et Mθs dans le poumon porcin, et ainsi, de mieux comprendre l’inflammation et la réponse immunitaire pulmonaire. Ce travail ouvre des voies, en santés vétérinaire et biomédicale, qui étaient jusqu’à présent réservées au modèle murin.

Porcine reproductive and respiratory syndrome (PRRS) remains a major concern for the swine industry worldwide due to its significant economic impact and its association with respiratory co-infections. Vaccination is a key strategy for PRRS control; however, the most effective commercial anti-PRRSV vaccines to date—live attenuated vaccines, also named modified live virus (MLV) vaccines—provide only partial protection. This study investigates whether oral supplementation with formulated Ulva-derived seaweed extracts can enhance immune responses to PRRSV-1 MLV vaccination. Two supplementation protocols using commercial seaweed extracts formulated with vitamin A and D (Searup®) were tested: one starting one day post-vaccination for five consecutive days (post-vaccination), and another beginning one day before vaccination and continuing for two days post-vaccination (pre-vaccination). In this exploratory farm-based experimental setting, pigs were divided into six groups: three vaccinated groups (supplemented with seaweed either pre- or post-vaccination or not supplemented), and three non-vaccinated control groups receiving the same supplementation regimes. Serum cytokine levels, PRRSV viral loads, and anti-PRRSV antibody responses were measured to assess immune modulation. Post-vaccination supplementation had no significant effect on vaccine viral load or humoral response, though a modest reduction in viral persistence was noted at 12 days post-vaccination (dpv). In contrast, pre-vaccination supplementation significantly increased vaccine virus levels at 4 dpv (p = 0.048) and enhanced anti-PRRSV IgG levels (p = 0.029). This protocol also induced an early and sustained decrease in pro-inflammatory cytokines, suggesting an immunomodulatory effect of algal extract supplemented with Vitamins A and D. We present here for the first time, in field conditions and in the target species, significant immune modifications upon realistic oral supplementation with Searup®. These findings are consistent with previous studies demonstrating the potential of pre-vaccination seaweed supplementation as a cost-effective, practical strategy to enhance humoral responses to PRRSV vaccination. Further studies are needed to evaluate its impact on neutralizing antibody production, cellular responses and protection against heterologous field PRRSV strains.

L’émergence de maladies infectieuses présentant un risque pour la population humaine requiert des outils et modèles expérimentaux permettant de caractériser ces maladies afin de mieux s’en protéger. Suite à l’apparition du SARS-CoV-2, nous avons mis au point un modèle d’infection chez plusieurs espèces de primate non-humain (babouin olive, macaque rhésus et macaque cynomolgus) afin d’évaluer différents aspects de l’infection. À partir de données recueillies dans notre laboratoire sur plus de 150 primates non-humains, nous avons comparé l’infection SARS-CoV-2 par la souche Wuhan et par plusieurs variants, dont Delta et Omicron. Les analyses montrent que le pic de charge virale a lieu entre 2 et 4 jours après exposition au virus quelle que soit la souche. Cependant, l’intensité du pic et la durée de charge virale détectable sont dépendantes du variant. L’infection induit une diminution transitoire du nombre de lymphocytes ainsi qu’une inflammation avec une élévation d’IL-1RA (antagoniste du récepteur de l’interleukine-1), d’IL-15 (interleukine-15), de CCL-1 (ligand-1 chimiokine motif C-C) et d’haptoglobine dans le plasma. La comparaison des variants montre que le variant Delta entraîne les plus fortes charges virales, ainsi qu’une réponse inflammatoire la plus marquée, tandis que les variants Omicron présentent une virulence atténuée. L’étude des réponses immunitaires a mis en évidence une production d’anticorps spécifiques et neutralisants différente en fonction du variant. Les variants Omicron induisent une réponse humorale plus tardive et moins intense. En revanche, la réponse cellulaire des lymphocytes T montre une forte réactivité croisée, avec une production d’interféron γ (IFN-γ) et d’IL-2 (interleukine-2). Ces travaux confirment la pertinence du macaque cynomolgus comme modèle préclinique pour l’étude du SARS-CoV-2 et l’évaluation de stratégies thérapeutiques et vaccinales. Ce modèle a contribué à l’identification de traitements et vaccins efficaces contre la COVID-19 et continue d’être une référence pour l’étude de l’évolution du virus et des réponses immunitaires associées.

The HIV-1 envelope glycoprotein (Env) trimer is the key target for vaccines aimed at inducing neutralizing antibodies (NAbs) against HIV-1. The clinical candidate immunogen ConM SOSIP.v7 is a stabilized native-like HIV-1 Env trimer based on an artificial consensus sequence of all HIV-1 isolates in group M. In preclinical studies ConM SOSIP.v7 trimers induced strong autologous NAb responses in non-human primates (NHPs). To fine-map these responses, we isolated monoclonal antibodies (mAbs) from six cynomolgus macaques that were immunized three times with ConM SOSIP.v7 protein and boosted twice with the closely related ConSOSL.UFO.664 immunogen. A total of 40 ConM and/or ConS-specific mAbs were isolated, of which 18 were retrieved after the three ConM SOSIP.v7 immunizations and 22 after the two immunizations with ConSOSL.UFO.664. 22 mAbs (55%) neutralized the ConM and/or ConS virus. Cross-neutralization of ConS virus by approximately one-third of the mAbs was seen prior to ConSOSL.UFO.664 immunization, albeit with modest potency. Neutralizing antibodies predominantly targeted the V1 and V2 regions of the immunogens, with an apparent extension towards the V3 region. Thus, the V1V2V3 region is immunodominant in the potent NAb response elicited by two consensus sequence native-like HIV-1 Env immunogens. Immunization with these soluble consensus Env proteins also elicited non-neutralizing mAbs targeting the trimer base. These results inform the use and improvement of consensus-based trimer immunogens in combinatorial vaccine strategies.

The COVID-19 pandemic has exemplified that rigorous evaluation in large animal models is key for translation from promising in vitro results to successful clinical implementation. Among the drugs that have been largely tested in clinical trials but failed so far to bring clear evidence of clinical efficacy is favipiravir, a nucleoside analogue with large spectrum activity against several RNA viruses in vitro and in small animal models. Here, we evaluate the antiviral activity of favipiravir against Zika or SARS-CoV-2 virus in cynomolgus macaques. In both models, high doses of favipiravir are initiated before infection and viral kinetics are evaluated during 7 to 15 days after infection. Favipiravir leads to a statistically significant reduction in plasma Zika viral load compared to untreated animals. However, favipiravir has no effects on SARS-CoV-2 viral kinetics, and 4 treated animals have to be euthanized due to rapid clinical deterioration, suggesting a potential role of favipiravir in disease worsening in SARS-CoV-2 infected animals. To summarize, favipiravir has an antiviral activity against Zika virus but not against SARS-CoV-2 infection in the cynomolgus macaque model. Our results support the clinical evaluation of favipiravir against Zika virus but they advocate against its use against SARS-CoV-2 infection.

The definition of correlates of protection is critical for the development of next-generation SARS-CoV-2 vaccine platforms. Here, we propose a model-based approach for identifying mechanistic correlates of protection based on mathematical modelling of viral dynamics and data mining of immunological markers. The application to three different studies in non-human primates evaluating SARS-CoV-2 vaccines based on CD40-targeting, two-component spike nanoparticle and mRNA 1273 identifies and quantifies two main mechanisms that are a decrease of rate of cell infection and an increase in clearance of infected cells. Inhibition of RBD binding to ACE2 appears to be a robust mechanistic correlate of protection across the three vaccine platforms although not capturing the whole biological vaccine effect. The model shows that RBD/ACE2 binding inhibition represents a strong mechanism of protection which required significant reduction in blocking potency to effectively compromise the control of viral replication.

The well documented association between obesity and the severity of SARS-CoV-2 infection raises the question of whether adipose tissue (AT) is impacted during this infection. Using a model of SARS-CoV-2 infection in cynomolgus macaques, we detected the virus within subcutaneous AT (SCAT) but not in visceral AT (VAT) or epicardial AT on day 7 post-infection. We sought to determine the mechanisms responsible for this selective detection and observed higher levels of angiotensin-converting-enzyme-2 mRNA expression in SCAT than in VAT. Lastly, we evaluated the immunological consequences of SARS-CoV-2 infection on AT: both SCAT and VAT T cells showed a drastic reduction in CD69 expression, a standard marker of resident memory T cell in tissue, that is also involved in the migratory and metabolic properties of T cells. Our results demonstrate that in a model of mild infection, SCAT is selectively infected by SARS-CoV-2 although changes in the immune properties of AT are observed in both SCAT and VAT.

The emergence of SARS-CoV-2 variants that are more resistant to antibody-mediated neutralization pose a new hurdle in combating the COVID-19 pandemic. Although vaccines based on the original Wuhan sequence have been shown to be effective at preventing COVID-19, their efficacy is likely to be decreased against more neutralization-resistant variants-of-concern (VOC), in particular, the Beta variant originating in South Africa. We assessed, in mice, rabbits, and non-human primates, whether a third vaccination with experimental Wuhan-based Spike vaccines could alleviate this problem. Our data show that a third immunization improves neutralizing antibody titers against the variants-of-concern, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2). After three vaccinations, the level of neutralization against Beta was similar to the level of neutralization against the original strain after two vaccinations, suggesting that simply providing a third immunization could nullify the reduced activity of current vaccines against VOC.