Despite extensive effort on vaccine development, protection against mucosally acquired viruses remains a difficult goal, most vaccines favoring systemic immune responses including T-cell immunity and IgG-based humoral responses. Indeed, to successfully protect against mucosal pathogens, vaccine strategies should combine the use of proper adjuvant and local immunization to trigger efficient and long lasting mucosal immune responses. We recently demonstrated that IL-7, secreted early and transiently after infection of mucosae, acts as a danger signal by inducing local expression of specific panels of chemokines that attracts immune cells at the infection sites. Similarly, locally administered IL-7 at the surface of vaginal mucosa or in the respiratory tract triggers local expression of chemokines and massive immune cell homing into these mucosae. Exploiting this property, VacMucIL7 project proposes that local delivery of IL-7, linked to poly(D, L-lactic acid) nanoparticles (PLA-NP) to favor mucosal barrier penetration, will prepare mucosae to further antigen delivery, improving immune responses to mucosal vaccines. We will establish the proof of concept of the IL-7/PLA-NP efficacy to adjuvant mucosal vaccines in mouse and non-human primate models of lung and vaginal infections (Influenza A and Herpes simplex viruses). In a first work package, we will study in details the immediate consequences of local administration of IL-7 in mice and define the best combinations of IL-7 and antigen, associated with PLA-NP to initiate a mucosal immune response using diphtheria toxoid and ovalbumin as model antigens in both the lungs and the vagina. The efficacy of these combinations will then be confirmed in non-human primates. Once established the best doses, time course and formulations for adequate mucosal vaccine delivery, we will test, both in mice and - non-human primate models, the efficacy of the best vaccine strategy to elicit protective mucosal immunity against respiratory tract infection with Influenza A virus or vaginal challenge with Herpes simplex virus type-2. The expected impacts of VacMuc7 project will be at different levels: - Develop an original approach to adjuvant mucosal vaccines by “preparing” the immunization site before vaccination using mucosal administration of a physiological dose of a natural molecule (IL-7) followed by local antigen delivery. This needle-free strategy will also avoid chemicals, presently disregarded by general population and participating to poor adhesion to vaccination campaigns. - Provide a comprehensive analysis of the IL-7 impact on mucosal immunity, both in lungs and the vagina, strengthening our knowledge concerning this particular arm of the immune system. Whether our approach of mucosal vaccination is successful to protect the animals not only against disease but, more importantly, against infection as a result of a quicker and better clearance at portals of entry of both airborne or sexually transmitted pathogens, it will be possible to adapt this strategy to the development of vaccines against other viral (HIV, Hepatitis…) and bacterial (Chlamydia…) infections that target mucosae and still represent major public health challenge. This project may also allow improving nanoparticles usage in vaccine development and permit elaborating new spray devices aimed at enhancing the efficiency of antigen availability/distribution at mucosal surfaces. VacMucIL7 project combines the complementary skills of 3 research teams, from mucosal immunity to nanomedicine and fully meets the requirements of the Axe 3.10 of the ANR program. Through a pre-clinical approach, we will develop an innovative adjuvant strategy to improve mucosal vaccines. Moreover, the molecular and cellular players implicated in the early phase of mucosal immune reactions will be studied in details not only in mice but also more importantly in non-human primates.

Mucosal tissues are composed by a specialized epithelium covered by a mucus layer, and are the main portal of entry of most of pathogens. The induction of local (mucosal) immune response is highly desirable in order to effectively prevent mucosal infection and transmission, especially against rapidly emerging pathogens. The presence in the small intestine of Peyer’s Patches (PP) as inductive sites of the gut-associated lymphoid tissue (GALT) makes the oral route of administration as a desirable approach to mucosal vaccination. However, the oral vaccine delivery needs to overcome some harsh conditions in the gastrointestinal tract (GIT) such as acid pH, degradation by digestive enzymes, low uptake across mucus layer, and immune tolerance. Lipid-polymer hybrid nanoparticles (NPs) allow the modification of surface properties to overcome the mucosal permeation barriers, enhanced physicochemical stability and drug protection during the transit through the GIT. The objective of SpheOrVac project is to conceive a robust mRNA delivery system able to bypass the GI barrier and to permit a mRNA expression, strong enough to induce a mucosal immune response. Previously, we have developed a lipid-polymer hybrid NPs, named spheroplexes (Sphx), as siRNA delivery system. The oral administration of TNF-a siRNA Sphx to mice with ulcerative colitis induced by DSS indicated a disease regression with a decrease in the level of TNF-a in the colon. Our hypothesis is that by using a combination of dedicated lipids and polymers to form Sphx, we could gather mRNA vectorization, adjuvant properties and muco-penetrating property in a unique delivery system able to induce a robust and long-lasting mucosal and systemic immune response following oral administration. Using an automated microfluidic system, we will synthetize mRNA Sphx made with diverse lipid-polymer compositions and evaluate their correlation with formulation stability and interaction with mucus layer. The immunostimulatory properties of the formulation will be evaluated considering the intrinsic adjuvant property of hybrid nanoparticles and the incorporation of adjuvant molecules in the delivery system to overcome the oral tolerance. To analyze the physiological uptake of fluorescent Sphx formulations, we will use an ex-vivo mice loop model, mimicking in vivo situation, and representing the natural environment of intestinal mucosa of living animal. The biodistribution evaluation will be carried out to select mRNA Sphx formulations able to target the small intestine and able to express mRNA in dendritic cells at PP region. Finally, as a proof of concept, mice immunization will be performed with selected Sphx formulated with N1-methylpseudouridine modified mRNA encoding serodominant secreted effector protein B (SseB), which will be used as immunogenic and protective antigen model against Salmonella enterica infection. If our hypothesis is confirmed, SpherOrVac data could be used as a foundation to future mRNA vaccines capable of preventing not only infection but also the transmission of (re)emergent pathogens.

The goal of the GELIHPARBAL project is to create injectable hydrogels that can be used as an emergency solution for deep and jagged wounds, to decrease bleeding immediate threat while improving muscle repair and functional skin healing. A new avenue of research, based on the development of biomaterials, has appeared in recent years as an alternative to classical tissue reconstruction approaches, such as grafts. Their aim is to provide a scaffold that will activate or guide cellular responses to enhance or trigger tissue regeneration. Nevertheless, if these biomaterials are trying to reproduce specific properties of the extracellular matrix (notably rigidity) to influence cell fate, elasticity has seldom been considered so far. Concomitantly, the possibility to provide haemostatic competences to these biomaterials, through induction of the blood clotting cascade by prothrombotic molecules and through filling and compression of the wound, opens new possibilities to control haemorrhagic risks. The GELIHPARBAL project therefore aims to develop an innovative therapeutic approach for deep wounds based on the use of injectable porous elasto-mimetic hydrogels that, through their intrinsic properties, allow concomitantly (1) the haemostatic filling of deep wounds, while (2) providing a specific guidance of muscle and skin repair. A major innovation will be the injectable formulation of porous elastic hydrogels that can compressively fill and conform accurately to the wound shape; and allow wound healing cells colonization while reinforcing their regenerative properties through mechanic and structural properties. The elasto-mimetic porous hydrogels developed during a previous ANR project (DHERMIC, ANR-11-TECS-016) will serve as technological basis for the development of the injectable formulations. The coordinating laboratory (Laboratory of tissue biology and therapeutic engineering, LBTI) has indeed reached the "biological" proof of concept of these approaches by validating the efficacy of the hydrogels in forming reconstructed skin equivalents and enhancing skin wound healing in vivo. This project therefore falls within the design and creation of innovative, bioinspired biodegradable and bioactive materials. To reach this ambitious goal, the consortium formed by three institutional partners (CNRS and INSERM) possesses the required competences and expertise’s to develop the biomaterials (LBTI), to finely analyse their mechanical, rheological (RMeS) and biological properties (INMG), and to study and evaluate the improvement of tissue healing and the resulting functionality gain (LBTI) The scientific program is divided in 3 scientific work packages (chemistry/biomaterials, mechanobiology and in vivo) and an administrative WP. They have for goal to 1) design elasto-mimetic haemostatic injectable porous hydrogels of large mechanical versatility; 2) Determine the mechanical and structural properties of porous matrices most effective in guiding proliferation and differentiation of muscle stem cells and in inducing muscle regeneration; 3) develop injectable porous hydrogels that can simultaneously promote muscle and skin regeneration. The administrative task is dedicated to the project and intellectual property management.

The aim of vaccination is to generate memory cells endowed with enhanced or novel functional capacities capable of inducing an amplified and faster immune response to subsequent pathogen exposure. As such, immune memory can confer long-term protection of the host against microbial infections. Antibody (Ab) responses to T cell-dependent antigen (Ag) requires cooperation of B cells with T follicular helper cells (Tfh), a T cell subset dedicated to provide help to B cells. Both cell types give rise to memory cells. The B and Tfh memory compartments are heterogeneous and comprise different subsets endowed with distinct functions that are still not fully understood. Vaccine adjuvants have been largely developed without a clear understanding of their mechanism of action but it is accepted that they can be used to achieve not only quantitative but also qualitative alterations of the immune response. We postulate that fitness and quality of the humoral immunity conferred by vaccination rely on the composition of the memory Tfh and B cell compartments and on the efficiency of the dialogue between these two populations. The objective of our present project is fourfold. 1. To explore how different vaccine formulations of the same model Ag shape the anatomy of the memory helper T and B cell compartments. 2. To explore the links between anatomy of the memory compartments and protective immunity thanks to our choice of a bacterial component as our model Ag. We will put emphasis on two aspects of the Ab response: - its cross-reactivity: an ideal vaccine should be able to generate broadly neutralizing Abs susceptible to confer protection against pathogens that display substantial antigenic drift. - its neutralizing capacity, because protective immunity relies on neutralizing Abs. 3. To analyze the influence of vaccine formulation on Ag availability . The fact that a significant proportion of mBCs are located in persistent germinal center (GC)-like structures in the proximity of Ag depots and mTfh suggests that longevity of B and Tfh memory may rely on Ag persistence and low-level Ag-driven stimulations. We postulate that a strong link exists between Ag availability and persistence of B cell memory. 4. To decipher the crosstalk between mTfh and mBC that are known to tightly collaborate during recall responses. Long-term survival and functional competence of these memory subsets is likely to depend on their cognate interactions. We will first determine whether all mTfh subsets are equally able to provide help to mBC during secondary responses. We will then explore the impact of the adjuvant formulation on: i) the location of mBC and mTfh, ii) the B helper function of mTfh. Our present project is of strategic importance in the field of vaccinology because it will help continuing the transition from empiricism to rational vaccine design. We contend that the recently discovered complexity of the mBC and mTfh compartments is an information which has not yet been exploited for vaccinology. We hope the MEMO-SIGN project will contribute to fill this gap. The key innovative elements of this project are: 1. our angle of attack of immune memory that takes into account the likely interdependency between helper T cell memory and B cell memory, two topics that are most often approached separately but are closely interconnected in real life 2. our use of a bacteria-derived vaccine Ag allowing to connect immune memory signature with immune protection. Given the heterogeneity of both memory compartments, it can be envisaged that the quality and efficiency of the recall response relies on the mTfh and mBC subtypes that are recruited to cooperate. Overall, this comprehensive study will give new insights into the field of fundamental immunology that will ultimately be translated to human vaccine design.

Mucosal vaccination mimics natural infection and induces a systemic and a local antibody secretion. Yet, there are still technological barriers to overcome for the development of this route of administration: increase the residence time of the antigen at the mucosal site, protect the antigen from degradation and induce an immune response at distant sites. Herein we propose to develop a natural patch as an innovative carrier for buccal vaccine based on promising preliminary data. Such patch is obtained through the use of biodegradable polysaccharides as building blocks. The patch will contain a Flu antigen (model) and a specific mucosal adjuvant. This biomimetic patch will permit to deliver subunit vaccine components with a spatial and temporal control to optimize antigen uptake at the buccal mucosa/patch interface ensuring efficient immune responses. Patch efficacy will be assessed by sublingual administration on rodent and non-human primate.