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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.

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; Development of protein vaccine to prevent congenital infection is a major public health priority. Our goal is the design of mucosal synthetic pathogen inducing protective immune responses against congenital toxoplasmosis. Mice were immunized intranasally, establishing pregnancy and challenging orally. Placental immune response, congenital infection, pup growth, parasitic load rates were studied. Pups born to vaccinated infected dams had significantly fewer brain cysts, no intraocular inflammation and normal growth. Protection was associated with a placental cellular Th1 response downregulated by IL-6 and correlated with persistence of vaccine for few hours in the nose before being totally eliminated. Our vaccine conferred high protection against congenital toxoplasmosis. These results provide support for future studies of other congenital vaccine.

Session : Giardia-Cryptosporidium; International audience

National audience; Regulatory T cells are involved in the maintenance of tolerance in the body and are responsible for persistence of some parasitic infections in mice like leishmaniosis and malaria. In our experiments we strived to explore the role of Treg cells in the persistence of Toxoplasma gondii infection. Outbred Swiss OFI mice were depleted of Treg cells by use of anti-CD25 monoclonal antibody during acute toxoplasmosis. No difference of mortality and brain parasitic load was observed in infected mice from depleted or non-depleted groups. In addition to CD25+ Treg cells, we observed another potential target of anti-CD25 mAb in mice acutely infected with Toxoplasma gondii. This potential target is CD25+ effector cells. A singificantly higher production ofiL-12 was also observed in mice injected with anti-CD25 mAb as compared to the control group injected with isotype control antibody. The simultaneous depletion of CD25+ Treg and CD25+ Teffector cells prohibits concluding the role of Treg cells during acute toxoplasmosis and led us to fmd another strategy to know the function of Treg cells. Swiss OFl mice were infected with type-! Toxoplasma gondii RH tachyzoites (wild type) and attenuated type-I Toxoplasma gondii Micl-3KO tachyzoites, intraperitoneally. The high expansion of CD4+CD25+Foxp3+ Treg at the local site of infection followed by expansion of CD4+CD25+ Teffector cells resulted into control of parasitemia and survival of mice infected with attenuated Micl-3KO parasites. On the other hand, the mice infected with wildtype RH parasites, died because of uncontrolled immune response and high parasitemia.

International audience

International audience