Recent advances in epicutaneous immunotherapy and potential applications in food allergy.

Given the potent immunological properties of the skin, epicutaneous immunotherapy (EPIT) emerges as a promising treatment approach for inducing immune tolerance, particularly for food allergies. Targeting the highly immunocompetent, non-vascularized epidermis allows for the application of microgram amounts of allergen while significantly reducing the risk of allergen passage into the bloodstream, thus limiting systemic allergen exposure and distribution. This makes EPIT highly suitable for the treatment of potentially life-threatening allergies such as food allergies. Multiple approaches to EPIT are currently under investigation for the treatment of food allergy, and these include the use of allergen-coated microneedles, application of allergen on the skin pretreated by tape stripping, abrasion or laser-mediated microperforation, or the application of allergen on the intact skin using an occlusive epicutaneous system. To date, the most clinically advanced approach to EPIT is the Viaskin technology platform. Viaskin is an occlusive epicutaneous system (patch) containing dried native allergen extracts, without adjuvants, which relies on frequent application for the progressive passage of small amounts of allergen to the epidermis through occlusion of the intact skin. Numerous preclinical studies of Viaskin have demonstrated that this particular approach to EPIT can induce potent and long-lasting T-regulatory cells with broad homing capabilities, which can exert their suppressive effects in multiple organs and ameliorate immune responses from different routes of allergen exposure. Clinical trials of the Viaskin patch have studied the efficacy and safety for the treatment of life-threatening allergies in younger patients, at an age when allergic diseases start to occur. Moreover, this treatment approach is designed to provide a non-invasive therapy with no restrictions on daily activities. Taken together, the preclinical and clinical data on the use of EPIT support the continued investigation of this therapeutic approach to provide improved treatment options for patients with allergic disorders in the near future.

Immunogenicity and Protective Potential of Mucosal Vaccine Formulations Based on Conserved Epitopes of Influenza A Viruses Fused to an Innovative Ring Nanoplatform in Mice and Chickens.

Current inactivated vaccines against influenza A viruses (IAV) mainly induce immune responses against highly variable epitopes across strains and are mostly delivered parenterally, limiting the development of an effective mucosal immunity. In this study, we evaluated the potential of intranasal formulations incorporating conserved IAV epitopes, namely the long alpha helix (LAH) of the stalk domain of hemagglutinin and three tandem repeats of the ectodomain of the matrix protein 2 (3M2e), as universal mucosal anti-IAV vaccines in mice and chickens. The IAV epitopes were grafted to nanorings, a novel platform technology for mucosal vaccination formed by the nucleoprotein (N) of the respiratory syncytial virus, in fusion or not with the C-terminal end of the P97 protein (P97c), a recently identified Toll-like receptor 5 agonist. Fusion of LAH to nanorings boosted the generation of LAH-specific systemic and local antibody responses as well as cellular immunity in mice, whereas the carrier effect of nanorings was less pronounced towards 3M2e. Mice vaccinated with chimeric nanorings bearing IAV epitopes in fusion with P97c presented modest LAH- or M2e-specific IgG titers in serum and were unable to generate a mucosal humoral response. In contrast, N-3M2e or N-LAH nanorings admixed with Montanide™ gel (MG) triggered strong specific humoral responses, composed of serum type 1/type 2 IgG and mucosal IgG and IgA, as well as cellular responses dominated by type 1/type 17 cytokine profiles. All mice vaccinated with the [N-3M2e + N-LAH + MG] formulation survived an H1N1 challenge and the combination of both N-3M2e and N-LAH nanorings with MG enhanced the clinical and/or virological protective potential of the preparation in comparison to individual nanorings. Chickens vaccinated parenterally or mucosally with N-LAH and N-3M2e nanorings admixed with Montanide™ adjuvants developed a specific systemic humoral response, which nonetheless failed to confer protection against heterosubtypic challenge with a highly pathogenic H5N8 strain. Thus, while the combination of N-LAH and N-3M2e nanorings with Montanide™ adjuvants shows promise as a universal mucosal anti-IAV vaccine in the mouse model, further experiments have to be conducted to extend its efficacy to poultry.

Epicutaneous immunization using synthetic virus-like particles efficiently boosts protective immunity to respiratory syncytial virus.

Despite the substantial health and economic burden caused by RSV-associated illness, no vaccine is available. The sole licensed treatment (palivizumab), composed of a monoclonal neutralizing antibody, blocks the fusion of the virus to the host cell but does not prevent infection. The development of a safe and efficacious RSV vaccine is therefore a priority, but also a considerable challenge, and new innovative strategies are warranted. Most of the adult population encounter regular RSV infections and can elicit a robust neutralizing antibody response, but unfortunately it wanes over time and reinfections during subsequent seasons are common. One approach to protect the mother and young infant from RSV infection is to administer a vaccine capable of boosting preexisting RSV immunity during pregnancy, which would provide protection to the fetus through passive transfer of maternal antibodies, thus preventing primary RSV infection in newborns during their first months of life. Here, we describe the preclinical evaluation of an epicutaneous RSV vaccine booster that combines epicutaneous patches as a delivery platform and a Synthetic Virus-Like Particles (SVLP)-based vaccine displaying multiple RSV F-protein site II (FsII, palivizumab epitope) mimetic as antigen (V-306). We demonstrated in mice that epicutaneous immunization with V-306 efficiently boosts preexisting immunity induced by the homologous V-306 administered subcutaneously. This boosting was characterized by a significant increase in F- and FsII-specific antibodies capable of competing with palivizumab for its target antigen and neutralize RSV. More importantly, epicutaneous booster immunization with V-306 significantly decreased lung viral replication in experimental mice after intranasal RSV challenge, without inducing enhanced RSV disease. In conclusion, an epicutaneous booster vaccine based on V-306 is safe and efficacious in enhancing RSV preexisting immunity in mice. This needle-free vaccine candidate would be potentially suited as a booster vaccine for vulnerable populations such as young infants via pregnant women, and the elderly.

Pre-Existing Humoral Immunity Enhances Epicutaneously-Administered Allergen Capture by Skin DC and Their Migration to Local Lymph Nodes.

Due to its richness in antigen presenting cells, e.g., dendritic cells (DC), the skin has been identified as a promising route for immunotherapy and vaccination. Several years ago, a skin delivery system was developed based on epicutaneous patches allowing the administration of antigen through intact skin. Using mouse models, we have shown that epicutaneous allergen application leads to a rapid uptake and transport of allergen-positive cells to skin-draining lymph nodes (LN). This occurred primarily in animals previously sensitized to the same allergen. In that context, we sought to better understand the role of the specific preexisting immunity in allergen capture by skin DC and their subsequent migration to LN. Specifically, we investigated the role of humoral immunity induced by sensitization and the involvement of IgG Fc receptors (FcγR). Epicutaneous patches containing fluorescently-labeled ovalbumin (OVA) were applied to naïve mice that had previously received either sera or purified IgG isolated from OVA-sensitized mice. To investigate the involvement of FcγR, animals received 2.4G2 (anti-FcγRII/RIII) blocking antibody, 24 hours before patch application. Mice that received sera or purified IgG originating from OVA-sensitized mice showed an increase in the quantity of OVA-positive DC in skin and LN. Moreover, the blockade of FcγR reduced the number of OVA-positive DC in LN to a level similar to that observed in naïve animals. Overall, these results demonstrate that preexisting specific-IgG antibodies are involved in allergen capture by skin DC following EPIT through the involvement of antigen-specific IgG-FcγR.

Epicutaneous immunotherapy protects cashew-sensitized mice from anaphylaxis.

BACKGROUND: The prevalence of tree nut allergy has increased worldwide, and cashew has become one of the most common food allergens. More critically, cashew allergy is frequently associated with severe anaphylaxis. Despite the high medical need, no approved treatment is available and strict avoidance and preparedness for prompt treatment of allergic reactions are considered dual standard of care. In the meantime, Phase III study results suggest investigational epicutaneous immunotherapy (EPIT) may be a relevant and safe treatment for peanut allergy and may improve the quality of life for many peanut allergic children. OBJECTIVE: We aimed to evaluate the capacity of EPIT to provide protection against cashew-induced anaphylaxis in a relevant mouse model. METHODS: The efficacy of EPIT was evaluated by applying patches containing cashew allergens to cashew-sensitized mice. As negative control, sham mice received patches containing excipient. Following treatment, mice were challenged orally to cashew and anaphylactic symptoms, as well as plasmatic levels of mast-cell proteases (mMCP)-1/7, were quantified. RESULTS: Of 16 weeks of EPIT significantly protects against anaphylaxis by promoting a faster recovery of challenged mice. This protection was characterized by a significant reduction of temperature drop and clinical symptoms, 60 minutes after challenge. This was associated with a decrease in mast-cell reactivity as attested by the reduction of mMCP-1/7 in plasma, suggesting that EPIT specifically decrease IgE-mediated anaphylaxis. CONCLUSION: We demonstrate that EPIT markedly reduced IgE-mediated allergic reactions in a mouse model of cashew allergy, which suggests that EPIT may be a relevant approach to treating cashew allergy.

Epidermal micro-perforation potentiates the efficacy of epicutaneous vaccination.

The skin is an immune organ comprised of a large network of antigen-presenting cells such as dendritic cells, making it an attractive target for the development of new vaccines and immunotherapies. Recently, we developed a new innovative and non-invasive vaccination method without adjuvant based on epicutaneous vaccine patches on which antigen forms a dry deposit. Here we describe in mice a method for potentiating the efficacy of our epicutaneous vaccination approach using a minimally invasive and epidermis-limited skin preparation based on laser-induced micro-perforation. Our results showed that epidermal micro-perforation increased trans-epidermal water loss, resulting in an enhancement of antigen solubilization from the surface of the patch, and increased the quantity of antigen delivered to the epidermis. Importantly, this was not associated with an increase in systemic passage of the antigen. Skin micro-perforation slightly activated keratinocytes without inducing an excessive level of local inflammation. Moreover, epidermal micro-perforation improved antigen capture by epidermal dendritic cells and specifically increased the level of Langerhans cells activation. Finally, we observed that epidermal micro-perforation significantly increased the level of the specific antibody response induced by our epicutaneous Pertussis vaccine candidate containing non-adsorbed recombinant Pertussis Toxin and reduced the amount of antigen dose required. Overall, these data confirm the benefit of a minimal and controlled epidermal preparation for improving the effectiveness of an epicutaneous patch-based vaccine, without adversely affecting the safety of the method.

The anti-influenza M2e antibody response is promoted by XCR1 targeting in pig skin.

XCR1 is selectively expressed on a conventional dendritic cell subset, the cDC1 subset, through phylogenetically distant species. The outcome of antigen-targeting to XCR1 may therefore be similar across species, permitting the translation of results from experimental models to human and veterinary applications. Here we evaluated in pigs the immunogenicity of bivalent protein structures made of XCL1 fused to the external portion of the influenza virus M2 proton pump, which is conserved through strains and a candidate for universal influenza vaccines. Pigs represent a relevant target of such universal vaccines as pigs can be infected by swine, human and avian strains. We found that cDC1 were the only cell type labeled by XCR1-targeted mCherry upon intradermal injection in pig skin. XCR1-targeted M2e induced higher IgG responses in seronegative and seropositive pigs as compared to non-targeted M2e. The IgG response was less significantly enhanced by CpG than by XCR1 targeting, and CpG did not further increase the response elicited by XCR1 targeting. Monophosphoryl lipid A with neutral liposomes did not have significant effect. Thus altogether M2e-targeting to XCR1 shows promises for a trans-species universal influenza vaccine strategy, possibly avoiding the use of classical adjuvants.

Host Response Comparison of H1N1- and H5N1-Infected Mice Identifies Two Potential Death Mechanisms.

Highly pathogenic influenza A viruses (IAV) infections represent a serious threat to humans due to their considerable morbidity and mortality capacities. A good understanding of the molecular mechanisms responsible for the acute lung injury observed during this kind of infection is essential to design adapted therapies. In the current study, using an unbiased transcriptomic approach, we compared the host-responses of mice infected with two different subtypes of IAV: H1N1 vs. H5N1. The host-response comparison demonstrated a clear difference between the transcriptomic profiles of H1N1- and H5N1-infected mice despite identical survival kinetics and similar viral replications. The ontological analysis of the two transcriptomes showed two probable causes of death: induction of an immunopathological state of the lung for the H1N1 strain vs. development of respiratory dysfunction in the case of the H5N1 IAV. Finally, a clear signature responsible for lung edema was specifically associated with the H5N1 infection. We propose a potential mechanism of edema development based on predictive bioinformatics tools.

Respiratory Syncytial Virus Infects Regulatory B Cells in Human Neonates via Chemokine Receptor CX3CR1 and Promotes Lung Disease Severity.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infections in infants and is characterized by pulmonary infiltration of B cells in fatal cases. We analyzed the B cell compartment in human newborns and identified a population of neonatal regulatory B lymphocytes (nBreg cells) that produced interleukin 10 (IL-10) in response to RSV infection. The polyreactive B cell receptor of nBreg cells interacted with RSV protein F and induced upregulation of chemokine receptor CX3CR1. CX3CR1 interacted with RSV glycoprotein G, leading to nBreg cell infection and IL-10 production that dampened T helper 1 (Th1) cytokine production. In the respiratory tract of neonates with severe RSV-induced acute bronchiolitis, RSV-infected nBreg cell frequencies correlated with increased viral load and decreased blood memory Th1 cell frequencies. Thus, the frequency of nBreg cells is predictive of the severity of acute bronchiolitis disease and nBreg cell activity may constitute an early-life host response that favors microbial pathogenesis.

RSV N-nanorings fused to palivizumab-targeted neutralizing epitope as a nanoparticle RSV vaccine.

Respiratory syncytial virus (RSV) is the leading cause of acute respiratory infections in children, yet no vaccine is available. The sole licensed preventive treatment against RSV is composed of a monoclonal neutralizing antibody (palivizumab), which targets a conformational epitope located on the fusion protein (F). Palivizumab reduces the burden of bronchiolitis but does not prevent infection. Thus, the development of RSV vaccines remains a priority. We previously evaluated nanorings formed by RSV nucleoprotein (N) as an RSV vaccine, as well as an immunostimulatory carrier for heterologous antigens. Here, we linked the palivizumab-targeted epitope (called FsII) to N, to generate N-FsII-nanorings. Intranasal N-FsII immunization elicited anti-F antibodies in mice that were non-neutralizing in vitro. Nevertheless, RSV-challenged animals were better protected against virus replication than mice immunized with N-nanorings, especially in the upper airways. In conclusion, an N-FsII-focused vaccine is an attractive candidate combining N-specific cellular immunity and F-specific antibodies for protection.

Non-invasive epicutaneous vaccine against Respiratory Syncytial Virus: Preclinical proof of concept.

To put a Respiratory Syncytial Virus (RSV) vaccine onto the market, new vaccination strategies combining scientific and technical innovations need to be explored. Such a vaccine would also need to be adapted to the vaccination of young children that are the principal victims of acute RSV infection. In the present project, we describe the development and the preclinical evaluation of an original epicutaneous RSV vaccine that combines two technologies: Viaskin® epicutaneous patches as a delivery platform and RSV N-nanorings (N) as a subunit antigen. Such a needle-free vaccine may have a better acceptability for the vaccination of sensible population such as infants since it does not require any skin preparation. Moreover, this self-applicative vaccine would overcome some issues associated to injectable vaccines such as the requirement of sterile medical devices, the need of skilled health-care professionals and the necessity of stringent store conditions. Here, we demonstrate that Viaskin® patches loaded with a formulation containing N-nanorings (Viaskin®-N) are highly immunogenic in mice and promotes a Th1/Th17 oriented immune response. More importantly, Viaskin®-N epicutaneous vaccine confers a high level of protection against viral replication upon RSV challenge in mice, without exacerbating clinical symptoms. In swine, which provides the best experimental model for the transcutaneous passage of drug/antigen in human skin, we have shown that GFP fluorescent N-nanorings, delivered epicutaneously with Viaskin® patches, are taken up by epidermal Langerhans cells. We have also demonstrated that Viaskin®-N induced a significant RSV N-specific T-cell response in pig. In conclusion, Viaskin®-N epicutaneous vaccine seems efficient to protect against RSV infection in animal model.

Addition of N-glycosylation sites on the globular head of the H5 hemagglutinin induces the escape of highly pathogenic avian influenza A H5N1 viruses from vaccine-induced immunity.

Highly pathogenic avian influenza A H5N1 viruses remain endemic in poultry in several countries and still constitute a pandemic threat. Since the early 20th century, we experienced four influenza A pandemics. H3N2 and H1N1pdm09 viruses that respectively emerged during 1968 and 2009 pandemics are still responsible for seasonal epidemics. These viruses evolve regularly by substitutions in antigenic sites of the hemagglutinin (HA), which prevent neutralization by antibodies directed against previous strains (antigenic drift). For seasonal H3N2 viruses, an addition of N-glycosylation sites (glycosites) on H3 contributed to this drift. Here, we questioned whether additional glycosites on H5 could induce an escape of H5N1 virus from neutralization, as it was observed for seasonal H3N2 viruses. Seven H5N1 mutants were produced by adding glycosites on H5. The most glycosylated virus escaped from neutralizing antibodies, in vitro and in vivo. Furthermore, a single additional glycosite was responsible for this escape.

Visualizing the replication of respiratory syncytial virus in cells and in living mice.

Respiratory syncytial virus (RSV) is the most important cause of severe lower-respiratory tract disease in calves and young children, yet no human vaccine nor efficient curative treatments are available. Here we describe a recombinant human RSV reverse genetics system in which the red fluorescent protein (mCherry) or the firefly luciferase (Luc) genes are inserted into the RSV genome. Expression of mCherry and Luc are correlated with infection rate, allowing the monitoring of RSV multiplication in cell culture. Replication of the Luc-encoding virus in living mice can be visualized by bioluminescent imaging, bioluminescence being detected in the snout and lungs of infected mice after nasal inoculation. We propose that these recombinant viruses are convenient and valuable tools for screening of compounds active against RSV, and can be used as an extremely sensitive readout for studying effects of antiviral therapeutics in living mice.

A novel subnucleocapsid nanoplatform for mucosal vaccination against influenza virus that targets the ectodomain of matrix protein 2.

In this study, subnucleocapsid nanorings formed by the recombinant nucleoprotein (N) of the respiratory syncytial virus were evaluated as a platform to anchor heterologous antigens. The ectodomain of the influenza virus A matrix protein 2 (M2e) is highly conserved and elicits protective antibodies when it is linked to an immunogenic carrier, making it a promising target to develop universal influenza vaccines. In this context, one or three M2e copies were genetically linked to the C terminus of N to produce N-M2e and N-3M2e chimeric recombinant nanorings. Mice were immunized intranasally with N-M2e or N-3M2e or with M2e or 3M2e control peptides. N-3M2e-vaccinated mice showed the strongest mucosal and systemic antibody responses. These mice presented a reduced viral load and minor weight loss, and all survived upon challenge with influenza virus A/PR8/34 (H1N1) (PR8). We compared the intranasal route to the subcutaneous route of N-3M2e immunization. Only the intranasal route induced a strong local IgA response and led to the protection of mice upon challenge. Finally, we demonstrated that the induction of anti-M2e antibodies by N-3M2e is not impaired by preexisting anti-N immunity. Overall, these results show that the N nanoring is a potent carrier for mucosal delivery of vaccinal antigens.

Kinetic characterization of PB1-F2-mediated immunopathology during highly pathogenic avian H5N1 influenza virus infection.

The PB1-F2 protein encoded by influenza A viruses can contribute to virulence, a feature that is dependent of its sequence polymorphism. Whereas PB1-F2 from some H1N1 viruses were shown to exacerbate the inflammatory response within the airways, the contribution of PB1-F2 to highly pathogenic avian influenza virus (HPAIV) virulence in mammals remains poorly described. Using a H5N1 HPAIV strain isolated from duck and its PB1-F2 knocked-out mutant, we characterized the dynamics of PB1-F2-associated host response in a murine model of lethal pneumonia. The mean time of death was 10 days for the two viruses, allowing us to perform global transcriptomic analyses and detailed histological investigations of the infected lungs at multiple time points. At day 2 post-infection (pi), while no histopathological lesion was observed, PB1-F2 expression resulted in a significant inhibition of cellular pathways involved in macrophage activation and in a transcriptomic signature suggesting that it promotes damage to the epithelial barrier. At day 4 pi, the gene profile associated with PB1-F2 expression revealed dysfunctions in NK cells activity. At day 8 pi, PB1-F2 expression was strongly associated with increased transcription of genes encoding chemokines and cytokines implicated in the recruitment of granulocytes, as well as expression of a number of genes encoding enzymes expressed by neutrophils. These transcriptomic data were fully supported by the histopathological analysis of the mice lungs which evidenced more severe inflammatory lesions and enhanced recruitment of neutrophils in the context of PB1-F2 expression, and thus provided a functional corroboration to the insight obtained in this work. In summary, our study shows that PB1-F2 of H5N1 HPAIV markedly influences the expression of the host transcriptome in a different way than its H1N1 counterparts: H5N1 PB1-F2 first delays the initial immune response but increases the pulmonary inflammatory response during the late stages of infection.