In collaboration with Alain Townsend's laboratory in Oxford, we have developed an extremely simple and very inexpensive serological test based on haemagglutination, which allows the detection of antibodies directed against the RBD domain of the SARS-2 virus. This test, called HAT, can be performed anywhere without any specialized equipment. The performance of this test has so far been documented in serum banks, and has shown a sensitivity greater than 90% and a specificity of 99%. The protocol has now been adapted and optimized so that HAT can be performed anywhere, on capillary blood drawn from the fingertip, and the central aim of this project is to validate this adapted protocol. In parallel, the possibility of using flow cytometry to extend the capabilities of HAT will be explored, in particular for the quantitative evaluation of different classes of serum antibodies reacting against the virus. Our ultimate goal is to be able to make this test available to all laboratories around the world who wish to use it and possibly to health agencies who are interested.

The inopportune stimulation of immune checkpoints (IC) can lead to a temporary silencing of the immune system, which can be deleterious in pathological situations such as infections or cancers. The biological mechanisms controlling the activity of these receptors are still unclear to this day. We have identified a new regulator of IC, which acts as a molecular brake of the inhibitory signals transmitted by those receptors. We propose here to elucidate the mechanisms by which this regulator represses IC and to analyze the consequences of this regulation in models of central nervous system autoimmunity. We will also study the importance of this mechanism in the susceptibility to immunotherapy targeting IC and in the phenomenon of inter-individual variations observed in response to those treatments. This study should shed an original light on the biology of IC whose discovery has recently boomed the field of immunotherapy applied to cancers and autoimmune diseases.

The nature and the strength of the immune response differ between women and men, resulting in sex-based differences in the prevalence, manifestations and outcome of autoimmune and infectious diseases. While women are able to mount more vigorous immune responses to infections, they also suffer more from autoimmunity and subsequent inflammation-induced tissue damage. A growing body of data shows that not only sex hormones but also sex-chromosome associated loci regulate biological pathways common to autoimmune and infectious diseases. This is particularly clear for TLR7, a single-stranded RNA receptor encoded by an X-linked gene. The response initiated by TLR7-mediated sensing of ssRNA by innate immune cells and B cells is an essential line of defense against RNA viruses. However, TLR7 can also respond to endogenous ssRNA containing ligands, potentially leading to autoimmunity or inflammation if not properly controlled. The difference in humoral immunity is one of the best conserved sex differences in immunology. Generally, among adults of reproductive ages, females have greater antibody responses than males, higher basal immunoglobulin (Ig) levels, and higher B cell numbers. Sex-based differences in the immune response to RNA viruses (i.e. influenza virus) are also well documented. Among adults, females produce higher neutralizing antibody titers compared with males following seasonal influenza vaccination. In mice it has been suggested that sex-based differences in expression of Tlr7 in B cells may contribute to greater antibody production in females than males. Gene dosage effects due to escape from X-chromosome inactivation (XCI) could possibly contribute to this sex differences. Indeed, in female mammals, one of the two X chromosomes is randomly inactivated to equalize the dosage of X-linked gene expression between sexes. XCI is established at an early stage of female embryogenesis and results in cellular mosaicism, where about one-half of the cells in a tissue express the maternal X chromosome and the other half the paternal one. However, in certain tissues or individuals, 15-23% of X-linked human genes escape XCI and are thus expressed from both the active (Xa) and inactive (Xi) X chromosomes. Indeed, we recently demonstrated that TLR7 escapes XCI in immune cells from women and Klinefelter syndrome males (Souyris et al., Sci. Immunol. 2018). Strikingly, B cells expressing TLR7 biallelically were more responsive than monoallelic cells at specific checkpoints of B cell differentiation that involve signaling through TLR7. We propose a major role for the XCI-escape of TLR7 in generating plasticity and diversity within the female immune cell compartment, with a likely impact on innate and adaptive immunity against selected pathogens or self-components containing ssRNA. A consortium formed by the group of JC Guéry (CPTP, Toulouse), specialist in the study of sex differences in immunity, J. Chaumeil (Cochin Institute, Paris), specialist in XCI, and in epigenetic & nuclear organization in immune cells, and M. Ducatez (ENVT, Toulouse), specialist in influenza surveillance and pathogenesis will address this hypothesis in this project. We will develop and validate new genetic tools in mice not only to track immune cells with bi-allelic expression of Tlr7 during the course of B cell responses to ssRNA nanoparticules and to inactivated RNA viruses (Influenza, SARS-Cov2); but also, to enforce Tlr7 mono-allelism in order to provide a direct causal link between Tlr7 bi-allelic expression in B cells and the generation of protective humoral immunity to influenza and COVID-19 vaccination in female. We will investigate the molecular mechanism of XCI-escape of Tlr7 and how this can be modulated. Altogether, our project will provide unprecedented knowledges on the nature of the mechanism underlying the female predominance in TLR7-driven B cell immunity, and could open avenues for the development of novel vaccine strategies.

The TANDANSS project proposes an innovative and competitive positioning that could bring about decisive advances to extend the knowledge related to acute health and wellness societal issues associated to chronic skin inflammatory diseases, and in particular psoriasis. Currently, treatment of psoriasis includes topical therapy, systemic therapy and phototherapy. The topical administration of anti-inflammatory drugs and steroidal drugs is the first line of treatment and the most practical medication method for psoriasis patients. However, the conventional topical treatments have low efficiency and are replaced, for the most severe forms of the disease, by systemic administration of synthetic drugs with severe side effects or by aggressive phototherapy. In this regard, biologic immunomodulators are often criticized because they are highly expensive (15000 to 20000 €/y/patient) and because they can cause severe (sometimes lethal) side effects, as shown by the withdrawal of several of them by regulatory agencies after the death of patients under treatment. There is undoubtedly an unmet societal need for the development of alternative, non “biologics-like” new drugs that could provide sustainable remissions and life quality improvements for patients, as an alternative to the current strategies. A promising strategy to overcome these problems includes the development of a suitable drug carrier system to achieve controlled and localized delivery of the anti-inflammatory drugs and to provide penetration enhancement to the drugs deep into the tissue according to the specific therapeutic needs. The challenge of this project is to design nanovectors carrying potent anti-inflammatory compounds to provide deep dermal penetration to the drug. Vesicular systems, and especially fluid and/or deformable vesicles, are of great interest in the dermal delivery field as they offer control on drug bioavailability and skin permeation enhancement, if they can deform or fuse with skin lipids. The TANDANSS project proposes to use catanionic vesicles, i.e., mixtures of cationic and anionic amphiphiles, as an innovative candidate to help the anti-inflammatory drugs to go in the deep tissues taking into account that these catanionic systems have shown to be able to interact and fuse with lipid membranes. It should be emphasized that a proof of concept related to the penetration enhancement potential of such a vesicular system has been patented for cosmetic applications with the Affichem company (Toulouse, France). In addition to classical steroidal and non-steroidal anti-inflammatory drugs, a new generation of dendritic anti-inflammatory drug (in preclinical development to treat Chronic Inflammatory Diseases (CIDs) like Rheumatoid Arthritis and Multiple Sclerosis via systemic administration) will also be tested. This non-formulated drug candidate has actually shown promising topical efficacy in vivo in an Imiquimob-induced mouse model of psoriasis (unpublished results of the consortium). One of the key concern lies in the comparison of the in vitro anti-inflammatory properties of the three formulated drugs with the properties of the non-formulated one. Another aspect concerns the effect of these formulations on skin and the impact of the drug physicochemical properties on the formulations permeation properties. The TANDANSS project will imply the measurement of skin permeation properties thanks to in vitro assays on human skin models and validated ex vivo on human living cell models. Psoriasis being the relevant skin inflammation pathology targeted by the TANDANSS project, key information will also be gathered by studying in vitro the effect of the formulated drugs on immune-competent skin cells, and these data will be completed by in vivo studies on the anti-inflammatory and immunomodulatory properties of the formulations on psoriasis mouse models.

Narcolepsy type 1 (NT1) is a disabling orphan disorder characterized by excessive daytime sleepiness and cataplexy, often associated with hypnagogic hallucinations, sleep paralysis, nighttime sleep disruption and weight gain. NT1 is a chronic disorder, and in the large majority of patients it is a sporadic disorder with the main peak of disease onset at 16 years of age. We showed in 2000 that NT1 is due to the specific loss of hypocretin (orexin) neurons within the hypothalamus. Although the etiology of the disease, the reason why the hypocretin neurons die is still obscure, it is clear that both genetic and environmental factors are important. A main specific genetic susceptibility marker is known since more than 95% of patients carry the human leukocyte antigen DQB1*06:02 allele. Increasing evidence argue for an autoimmune pathogenesis with epidemiological studies underlying a strong association between H1N1 influenza virus vaccination and narcolepsy following the 2009 vaccination campaign. The mechanisms underlying such association remain unclear and may involve either a specific immune response to H1N1 with potential molecular mimicry or a large non-specific stimulation of the immune system with increased brain inflammation/blood-brain permeability, allowing the autoimmune process to reach hypocretin neurons resulting in NT1. Recently we have provided evidence supporting the autoimmune hypothesis. We have generated a mouse model in which an induced and targeted neuro-inflammation was able to kill specifically hypocretin neurons and provoke sleep fragmentation and cataplexy episodes, thus an autoimmune mouse model for NT1. We have also reported an immune signature in NT1, alteration in P2RY11 signaling, and revealed the release of several cytokines and growth factors in the sera of NT1 patients. Now, our goal is to use a variety of complementary approaches to test realistic hypotheses regarding the immune mechanisms that could induce NT1. Our project is based on a translational approach using both human and animal models. Altogether preclinical and clinical research studies within this project have the objectives to decipher the autoimmune mechanisms able to destroy hypocretin-producing neurons and investigate how environmental factors such as infection or vaccination trigger an autoimmune response against CNS neurons. Our approaches include the in-depth study of cases of early-diagnosed NT1, induced or not by H1N1 vaccination, and the use of original animal models to test the different immunological pathways that could lead to NT1. The strong position of the 3 academic partners in the domain of sleep, sleep disorders and neuro-immunology, their real and efficient synergy and the access to the biobank of the French reference national center for narcolepsy in Montpellier are major advantages for the success of the project and will contribute to the scientific valorization of the results. New treatment strategies could emerge from the identification of the precise immunological alteration, especially early after disease onset when the potential immune process targeting hypocretin neurons is not too advanced and could be reverted. Our results may lead to new ways to treat such pathologies by opening up a new field of therapy potentially exploitable in public health politics, and leading to new patentable molecules.