Genomic characterization of equine influenza A subtype H3N8 viruses by long read sequencing and functional analyses of the PB1-F2 virulence factor of A/equine/Paris/1/2018.

Equine influenza virus (EIV) remains a threat to horses, despite the availability of vaccines. Strategies to monitor the virus and prevent potential vaccine failure revolve around serological assays, RT-qPCR amplification, and sequencing the viral hemagglutinin (HA) and neuraminidase (NA) genes. These approaches overlook the contribution of other viral proteins in driving virulence. This study assesses the potential of long-read nanopore sequencing for fast and precise sequencing of circulating equine influenza viruses. Therefore, two French Florida Clade 1 strains, including the one circulating in winter 2018-2019 exhibiting more pronounced pathogenicity than usual, as well as the two currently OIE-recommended vaccine strains, were sequenced. Our results demonstrated the reliability of this sequencing method in generating accurate sequences. Sequence analysis of HA revealed a subtle antigenic drift in the French EIV strains, with specific substitutions, such as T163I in A/equine/Paris/1/2018 and the N188T mutation in post-2015 strains; both substitutions were in antigenic site B. Antigenic site E exhibited modifications in post-2018 strains, with the N63D substitution. Segment 2 sequencing also revealed that the A/equine/Paris/1/2018 strain encodes a longer variant of the PB1-F2 protein when compared to other Florida clade 1 strains (90 amino acids long versus 81 amino acids long). Further biological and biochemistry assays demonstrated that this PB1-F2 variant has enhanced abilities to abolish the mitochondrial membrane potential ΔΨm and permeabilize synthetic membranes. Altogether, our results highlight the interest in rapidly characterizing the complete genome of circulating strains with next-generation sequencing technologies to adapt vaccines and identify specific virulence markers of EIV.

Non-Steroidal Estrogens Inhibit Influenza Virus by Interacting with Hemagglutinin and Preventing Viral Fusion.

Influenza virus is one of the main causes of respiratory infections worldwide. Despite the availability of seasonal vaccines and antivirals, influenza virus infections cause an important health and economic burden. Therefore, the need to identify alternative antiviral strategies persists. In this study, we identified non-steroidal estrogens as potent inhibitors of influenza virus due to their interaction with the hemagglutinin protein, preventing viral entry. This activity is maintained in vitro, ex vivo, and in vivo. Therefore, we found a new domain to target on the hemagglutinin and a class of compounds that could be further optimized for influenza treatment.

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.

Corrigendum: A Universal Influenza Vaccine Can Lead to Disease Exacerbation or Viral Control Depending on Delivery Strategies.

[This corrects the article on p. 641 in vol. 7, PMID: 28082980.].

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.

Expanding the tools for identifying mononuclear phagocyte subsets in swine: Reagents to porcine CD11c and XCR1.

Pig is a domestic species of major importance in the agro-economy and in biomedical research. Mononuclear phagocytes (MNP) are organized in subsets with specialized roles in the orchestration of the immune response and new tools are awaited to improve MNP subset identification in the pig. We cloned pig CD11c cDNA and generated a monoclonal antibody to pig CD11c which showed a pattern of expression by blood and skin MNP subsets similar to humans. We also developed a porcine XCL1-mCherry dimer which specifically reacted with the XCR1-expressing dendritic cell subset of the type 1 lineage in blood and skin. These original reagents will allow the efficient identification of pig MNP subsets to study their role in physiological and pathological processes and also to target these cells in novel intervention and vaccine strategies for veterinary applications and preclinical evaluations.

A Universal Influenza Vaccine Can Lead to Disease Exacerbation or Viral Control Depending on Delivery Strategies.

The development of influenza A virus (IAV) vaccines, which elicits cross-strain immunity against seasonal and pandemic viruses is a major public health goal. As pigs are susceptible to human, avian, and swine-adapted IAV, they would be key targets of so called universal IAV vaccines, for reducing both the zoonotic risk and the economic burden in the swine industry. They also are relevant preclinical models. However, vaccination with conserved IAV antigens (AGs) in pigs was reported to elicit disease exacerbation. In this study, we assessed whether delivery strategies, i.e., dendritic cell (DC) targeting by the intradermal (ID) or intramuscular (IM) routes, impact on the outcome of the vaccination with three conserved IAV AGs (M2e, NP, and HA2) in pigs. The AGs were addressed to CD11c by non-covalent binding to biotinylated anti-CD11c monoclonal antibody. The CD11c-targeted AGs given by the ID route exacerbated disease. Conversely, CD11c-targeted NP injected by the IM route promoted T cell response compared to non-targeted NP. Furthermore, the conserved IAV AGs injected by the IM route, independently of DC targeting, induced both a reduction of viral shedding and a broader IgG response as compared to the ID route. Our findings highlight in a relevant animal species that the route of vaccine delivery impacts on the protection induced by conserved IAV AGs and on vaccine adverse effects. Finally, our results indicate that HA2 stands as the most promising conserved IAV AG for universal vaccine development.