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Session 3 : La mise en place du protocole : un point clé de sa reproductibilité; National audience; La toxoplasmose est une maladie infectieuse cosmopolite due à un protozoaire intracellulaire, parasite opportuniste, Toxoplasma gondii. La toxoplasmose congénitale représente un problème majeur de santé publique et vétérinaire. Environ 3000 femmes enceintes s'infectent chaque année en France avec, dans 10% des cas, une transmission transplacentaire du parasite au fœtus. En parallèle, elle est l'une des causes majeures d'avortements pour les petits ruminants au sein des élevages français et également responsable depuis des décennies de mortalité pour de nombreuses espèces animales de parcs zoologiques. Du point de vue vétérinaire, la séroprévalence de la toxoplasmose est importante et souvent sous-estimée en France, comme en Europe au sein des élevages de ruminants. Le stress et l'anxiété dus au prélèvement sanguin sont observés chez les éleveurs de petits ruminants, comme pour les vétérinaires soignants les animaux de parcs zoologiques. Actuellement, le suivi immunologique de la toxoplasmose se fait sur sérum suite à une prise de sang dans les élevages lors d'une vague d'avortements Afin de proposer un test de dépistage de la toxoplasmose non invasif et non contraignant (raffinement), nous avons développé un outil de prélèvement (swab) et établi la preuve de concept chez la brebis, hôte naturel du toxoplasme. Ce système de prélèvement a déjà été validé pour l'enfant lors du suivi toxoplasmique. Le prélèvement a consisté en un recueil direct de la salive de 51 brebis Préalpes, infectées expérimentalement, par simple mastication mécanique d'un coton absorbant pendant une minute permettant de mesurer la réponse IgG salivaire et de la corréler avec la réponse IgG sérique. Le coton humidifié par la salive est placé dans un collecteur à l'intérieur d'un tube de centrifugation. Les tubes sont centrifugés pendant 15 min,afin d'essorer le coton sur les parois du tube collecteur percé et ensuite de recueillir environ 1mL de salive dans le fond du tube conique. La séroconversion des brebis a été suivie à sept jours et à deux mois post-infection. Une corrélation significative a été observée entre les cinétiques d'IgG sériques et salivaires (Coefficient de régression = 0,8). De plus, la réponse salivaire montre une très forte spécificité. En conclusion, nous avons développé un outil simple non invasif de prélèvement permettant de diminuer le stress de la brebis au cours du prélèvement. Le suivi salivaire est aussi spécifique et sensible que le suivi sérologique dans le cadre d'une toxoplasmose, tout en préservant le bien-être animal. Cet outil de raffinement permettra donc aux éleveurs d'avoir un meilleur suivi sanitaire pour la toxoplasmose de leur troupeau d'ovins, sans l'intervention du vétérinaire. Ce suivi sanitaire toxoplasmique aura un impact sur l'évaluation des risques de transmission pour l'espèce humaine et donc une amélioration de la prévention efficace de la maladie chez les femmes enceintes.

National audience; Squirrel monkeys, new world primates from South America, are very susceptible to toxoplasmosis which can be completely unnoticed because of non-specific clinical signs but induces necrotic lesions. Many cases of toxoplasmosis epidemics in zoos have been identified around the world, resulting in sudden death after few days. The development of a vaccine against toxoplasmosis is a health priority for these squirrel monkeys. These last years, the BioMAP team in partnership with the Biotech Vaxinano demonstrated the effectiveness of a mucosal subunit vaccine against T. gondii in mouse and sheep models. The vaccine, administrated intranasally, used the total extract of antigens of the parasite (TE) encapsulated in muco-penetrating and muco-adherent nanoparticles consisting of maltodextrin and a lipid core. After 2 immunisations with 3 week apart, this vaccine induces a strong Th1 cellular immune response (Dimier-Poisson et al., 2015; Ducournau et a/., 2017; Ducournau et al., 2020) characterized by an increase of IFN-y specific secretion against TE. The vaccine was used in five French zoo partners by following the principle of the veterinary cascade. About sixty squirrel monkeys have been vaccinated. Animal monitoring was performed, and blood samples were taken by veterinarians to study the humoral and cellular immune response. Our results showed that as vaccination did not induce a humoral response, it is possible to identify naturally infected animals from vaccinated animals. In parallel, we observed a significant increase in the secretion of IFNy specific for T. gondii after administration of the vaccine. Since IFN-y is the key mediator of resistance to T. gondii, these results suggest the development of a protective Th1 cellular immune response for vaccinated squirrel monkeys. In fact, since the introduction of vaccination in partner parks, almost 4 years old, no mortality due to toxoplasmosis has been observed.

National audience; Over the past few years, animal welfare has taken more place in society and particularly in scientific research. It has become necessary to refine the experimental procedures as much as possible in infectiology as in our reference model, toxoplasmosis, in agreement with the 3Rs rule and the different ethical concerns existing since 1959. Thus, the establishment of a treatment using analgesics would provide relief in animals acutely infected with Toxoplasma gondii. However, the use of this analgesic should in no way alter the pathophysiology of the parasite and the immune response of the host, so as not to interfere with the initial scientific study. Currently, little is known about the use of paracetamol in an infectious model. In the present work, we studied the impact of paracetamol in the murine model of toxoplasmosis. To do this, zoonotic, telemetric, behavioral, histological and immune parameters were analyzed to better characterize the consequences of a treatment with paracetamol. Paracetamol administered by force-feeding (30 mg / kg / day) appears to be a good pharmacological tool in a mouse model of toxoplasmosis without modifying the major pathophysiology or the immune responses induced by T. gondii in both acute and chronic phases. These first results in mice validated our initial hypothesis that paracetamol appears to be a pharmacological tool for refining and improving animal welfare. Therefore, our project has highlighted the combination of specific markers to contribute to animal welfare in mice, with a view to transferring it to the target species, the sheep. In the long term, the use of paracetamol could be extended to other infectious models with other target animal species.

International audience; Depuis quelques années, le bien-être animal a pris une place importante dans la société et notamment dans la recherche scientifique. Il est devenu nécessaire de raffiner au maximum les procédures expérimentales en infectiologie comme dans notre modèle de référence, la toxoplasmose, en accord avec la règle des 3Rs et les différentes préoccupations éthiques existant depuis 1959. Ainsi, la mise en place d’un traitement utilisant des analgésiques, comme par exemple le paracétamol, permettrait de soulager les animaux infectés au cours de ce processus infectieux, du à Toxoplasma gondii. Cependant, l’utilisation de cet analgésique ne doit en aucun cas modifier la physiopathologie de l’infection et la réponse immunitaire de l’hôte, afin de ne pas interférer avec l’étude scientifique initiale. Actuellement, peu de données sont disponibles sur l’utilisation du paracétamol dans un modèle infectieux expérimental. Dans le présent travail, nous avons étudié son impact dans le modèle murin CBA/J de toxoplasmose. Pour ce faire, des paramètres zoonotiques, télémétriques, comportementaux, histologiques (lésions et distribution tissulaires) et immunitaires (recrutements cellulaires et cytokines d’intérêt (IL-6, IFN et IL-12) ont été analysés afin de mieux caractériser les conséquences d’un traitement au paracétamol à une dose de référence validée de 30 mg / kg/ j sur 5 jours consécutifs. Le paracétamol administré par gavage (30 mg / kg / jour) semble améliorer le bien-être des souris en phase aiguë de la toxoplasmose, en particulier sur la réduction des signes cliniques, la perte de poids et du maintien d’un comportement normal. . La physiopathologie de l’enkystement du parasite dans le cerveau ainsi que les réponses immunitaires induites par T. gondii ne sont pas impactées. Ces premiers résultats chez la souris sont prometteurs et valident notre hypothèse initiale que le paracétamol semble être un outil pharmacologique permettant d’améliorer le bien-être animal. Sous traitement paracétamolé, la combinaison de marqueurs cliniques, comportementaux, parasitaires et immunitaires a contribué à définir le bien-être animal chez la souris, en vue de la transposer à l’espèce cible de la toxoplasmose, le mouton. A long terme, l’utilisation du paracétamol pourrait être étendue à d’autres modèles infectieux expérimentaux afin de répondre aux attentes éthiques et réglementaires.

National audience

Research on viruses, bacteria and protozoa-based immunotherapy has been on the rise for several years. The antitumoral efficacy of these microorganisms relies on three main mechanisms: Destruction of tumor cells, stimulation of the immune response and reprogramming of the tumor microenvironment. In order to optimize their immunotherapeutic action, these microorganisms can be genetically engineered to enhance their tumor-targeting efficacy or to vectorize immunostimulating molecules and/or antibodies. To this aim, molecular engineering allows the design of new antibody formats optimizing their functions. From whole antibodies to tandem single-chain variable fragments, various antibody formats can be vectorized by microorganisms to target receptors such as immune checkpoints or recruit immune effector cells within the tumor. Such possibilities broaden the arsenal of immunotherapeutic cancer treatment. This review focuses on these innovations and their advantages for immunotherapy.Micro-organismes anti-cancéreux et armement - Le couteau suisse de l’immunothérapie.Depuis plusieurs années, la recherche sur les micro-organismes pour une utilisation à des fins d’immunothérapie antitumorale est en plein essor. L’efficacité antitumorale de ces micro-organismes repose sur trois mécanismes principaux : la destruction des cellules tumorales, la stimulation du système immunitaire et la reprogrammation du microenvironnement tumoral. Afin d’optimiser leur action immunothérapeutique, ces micro-organismes peuvent être génétiquement modifiés pour les rendre capables de vectoriser des molécules immunostimulantes ou des anticorps. Par ingénierie moléculaire, il est désormais possible de diversifier les formats et fonctions de ces anticorps afin d’inhiber les points de contrôle immunitaire ou encore de recruter les cellules immunitaires effectrices au site de la tumeur. Cette Synthèse s’intéresse particulièrement à ces innovations et à leurs avantages en immunothérapie.

Session 2 - Toxoplasmose : quoi de neuf en 2021 ?; National audience; Toxoplasmosis is a cosmopolitan infectious disease caused Toxoplasma gondii. Congenital toxoplasmosis is a major public and veterinary health problem. Approximately 3000 pregnant women become infected each year in France with, in 10% of cases, transplacental transmission of the parasite to the foetus. At the same time, it is one of the major causes of abortions in small ruminants in French farms and has also been responsible for the mortality of many animal species in zoos for decades. From a veterinary point of view, the seroprevalence of toxoplasmosis is important and often underestimated in France, as in Europe, in ruminant farms. In order to propose a non-invasive screening test for toxoplasmosis, we have developed a swab and established the proof of concept in ewes, the natural host of toxoplasma. The sampling consisted of a direct collection of saliva from 51 experimentally infected Prealpes ewes by simple mechanical chewing of an absorbent cotton for one minute to measure the salivary IgG response and to correlate it with the serum IgG response. The swab with saliva is placed in a collector inside a centrifuge tube. The tubes are centrifuged and then collect of saliva in the bottom of the conical tube. Seroconversion of the ewes was monitored at seven days and two months post-infection. A significant correlation was observed between serum and salivary IgG kinetics (R = 0.8). Moreover, the salivary response showed a very high specificity. We have developed a simple non-invasive sampling tool that reduces the stress of the ewe during sampling. Salivary monitoring is as specific and sensitive as serological monitoring in the context of toxoplasmosis, while preserving animal welfare. This refinement tool will allow farmers to have a better sanitary follow-up for toxoplasmosis in their sheep flock. This toxoplasmosis sanitary follow-up will have an impact on the evaluation of the transmission risks for the human species and thus an improvement of the effective prevention of the disease in pregnant women.

International audience

International audience; Background Immunotherapy induces, provides, and/or reactivates anti-tumor immune responses. Some microorganisms also can initiate response that lyzes infected tumor and/or stimulates systemic immunity. Attenuated viruses or bacteria are well studied as oncotherapeutics, but not protozoa except Toxoplasma gondii.1 We assessed the effect on tumors of other protozoa that were naturally non-pathogenic to humans. Thus, we discovered the ability to use Neospora caninum (Nc) in a manner and form that demonstrated a synergistic array of pertinent immunotherapeutic characteristics against solid cancers. Our first Article on Neospora as Onco-immunotherapeutic is currently under revision after review by the JITC. We report on the most recent data notably from Nc engineered to secrete human IL-15 within the tumor.Methods In vitro, the immunostimulatory properties of Nc strains wildtype and engineered to secrete human IL-15 were studied. In vivo experiments of treatment with of Nc tachyzoites2 administered locally (intra and peri tumoral) or remotely (subcutaneous) in a murine thymoma EG7 tumor and in human Merkel cell carcinoma (MCC).Results We demonstrated that the treatment of thymoma EG7 by Nc strongly inhibited tumor development. Analysis of immune responses and interactions between Nc and tumor cells showed that Nc had the ability to lyze infected cancer cells, reactivated immune competence within the Tumor Microenvironment (TME), and activated the systemic immune system by promoting the recruitment of immune cells to the site of tumor. We also established in a NOD/SCID mouse model that Nc was able to induce a strong regression of human MCC. Recently, to further enhance oncotherapeutic effect, we engineered an Nc strain to secrete human IL-15 (cross reactive with mouse cells), associated with alpha subunit of IL-15 receptor, increasing its stability.3 This strain induced proliferation of human PBMCs and their secretion of IFN-γ. In the EG7 model, human IL-15 secreting Nc showed greater protection against tumor development, confirming enhancement of immunotherapy by engineering Nc to deliver/secrete IL-15.Conclusions These results highlight Neospora caninum as a potentially extremely efficient, and non-toxic anti-cancer agent, capable of being engineered to express at its surface or to secrete bio-drugs, like human IL-15 cytokine. Our work has identified the broad clinical possibilities of using N. caninum as an oncolytic protozoan in human medicine capable of vectoring molecular therapy, overcoming TME defenses.

International audience; Background Microorganisms that can be used for their lytic activity against tumor cells as well as inducing or reactivating antitumor immune responses are a relevant part of the available immunotherapy strategies. Viruses, bacteria and even protozoa have been largely explored with success as effective human antitumor agents. To date, only one oncolytic virus-T-VEC-has been approved by the US Food and Drug Administration for use in biological cancer therapy in clinical trials. The goal of our study is to evaluate the potential of a livestock pathogen, the protozoan Neospora caninum, non-pathogenic in humans, as an effective and safe antitumorous agent.Methods/Results We demonstrated that the treatment of murine thymoma EG7 by subcutaneous injection of N. caninum tachyzoites either in or remotely from the tumor strongly inhibits tumor development, and often causes their complete eradication. Analysis of immune responses showed that N. caninum had the ability to 1) lyze infected cancer cells, 2) reactivate the immunosuppressed immune cells and 3) activate the systemic immune system by generating a protective antitumor response dependent on natural killer cells, CD8-T cells and associated with a strong interferon (IFN)-gamma secretion in the tumor microenvironment. Most importantly, we observed a total clearance of the injected agent in the treated animals: N. caninum exhibited strong anticancer effects without persisting in the organism of treated mice. We also established in vitro and an in vivo non-obese diabetic/severe combined immunodeficiency mouse model that N. caninum infected and induced a strong regression of human Merkel cell carcinoma. Finally, we engineered a N. caninum strain to secrete human interleukin (IL)-15, associated with the alpha-subunit of the IL-15 receptor thus strengthening the immuno-stimulatory properties of N. caninum. Indeed, this NC1-IL15hRec strain induced both proliferation of and IFN-gamma secretion by human peripheral blood mononuclear cells, as well as improved efficacy in vivo in the EG7 tumor model.Conclusion These results highlight N. caninum as a potential, extremely effective and non-toxic anticancer agent, capable of being engineered to either express at its surface or to secrete biodrugs. Our work has identified the broad clinical possibilities of using N. caninum as an oncolytic protozoan in human medicine.

International audience; Toxoplasma gondiiis a parasitic protozoan of worldwide distribution, able to infect all warm-blooded animals, but particularly sheep. Primary infection in pregnant sheep leads to millions of abortions and significant economic losses for the livestock industry. Moreover, infected animals constitute the main parasitic reservoir for humans. Therefore, the development of a One-health vaccine seems the best prevention strategy. Following earlier work, a vaccine constituted of total extract ofToxoplasma gondiiproteins (TE) associated with maltodextrin nanoparticles (DGNP) was developed in rodents. In this study we evaluated the ability of this vaccine candidate to protect against latent and congenital toxoplasmosis in sheep. After two immunizations by either intranasal or intradermal route, DGNP/TE vaccine generated specific Th1-cellular immune response, mediated by APC-secretion of IFN-gamma and IL-12. Secretion of IL-10 appeared to regulate this Th1 response for intradermally vaccinated sheep but was absent in intranasally-vaccinated animals. Finally, protection against latent toxoplasmosis and transplacental transmission were explored. Intranasal vaccination led to a marked decrease of brain cysts compared with the non-vaccinated group. This DGNP/TE vaccine administered intranasally conferred a high level of protection against latent toxoplasmosis and its transplacental transmission in sheep, highlighting the potential for development of such a vaccine for studies in other species.