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International audience; 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™ adjuvantsdeveloped 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.

International audience; PB1-F2 is a virulence factor of influenza A virus known to increase viral pathogenicity in mammalian hosts. PB1-F2 is an intrinsically disordered protein displaying a propensity to form amyloid-like fibers. However, the correlation between PB1-F2 structures and the resulting inflammatory response is unknown. Here, we used synchrotron-coupled Fourier transform-IR and deep UV microscopies to determine the presence of PB1-F2 fibers in influenza A virus–infected mice. In order to study the correlation between PB1-F2 structure and the inflammatory response, transgenic mice expressing luciferase under the control of an NF-κB promotor, allowing in vivo monitoring of inflammation, were intranasally instilled with monomeric, fibrillated, or truncated forms of recombinant PB1-F2. Our intravital NF-κB imaging, supported by cytokine quantification, clearly shows the proinflammatory effect of PB1-F2 fibers compared with N-terminal region of PB1-F2 unable to fibrillate. It is noteworthy that instillation of monomeric PB1-F2 of H5N1 virus induced a stronger inflammatory response when compared with prefibrillated PB1-F2 of H1N1 virus, suggesting mechanisms of virulence depending on PB1-F2 sequence. Finally, using whole-body plethysmography to measure volume changes in the lungs, we quantified the effects of the different forms of PB1-F2 on respiratory parameters. Thus, we conclude that PB1-F2–induced inflammation and respiratory distress are tightly correlated with sequence polymorphism and oligomerization status of the protein.

International audience; Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in young children. Currently, there is no RSV vaccine or universally accessible antiviral treatment available. Addressing the urgent need for new antiviral agents, we have investigated the capacity of a non-coding single-stranded oligonucleotide (ssON) to inhibit RSV infection. By utilizing a GFP-expressing RSV, we demonstrate that the ssON significantly reduced the proportion of RSV infected A549 cells (lung epithelial cells). Furthermore, we show that ssON’s antiviral activity was length dependent and that both RNA and DNA of this class of oligonucleotides have antiviral activity. We reveal that ssON inhibited RSV infection by competing with the virus for binding to the cellular receptor nucleolin in vitro . Additionally, using a recombinant RSV that expresses luciferase we show that ssON effectively blocked RSV infection in mice. Treatment with ssON in vivo resulted in the upregulation of RSV-induced interferon stimulated genes (ISGs) such as Stat1 , Stat2 , Cxcl10 , and Ccl2. This study highlights the possibility of using oligonucleotides as therapeutic agents against RSV infection. We demonstrate that the mechanism of action of ssON is the inhibition of viral entry in vitro , likely through the binding of the receptor, nucleolin and that ssON treatment against RSV infection in vivo additionally results in the upregulation of ISGs.

Summary: Secondary bacterial infections often complicate viral respiratory infections. We hypothesize that perturbation of the gut microbiota during influenza A virus (IAV) infection might favor respiratory bacterial superinfection. Sublethal infection with influenza transiently alters the composition and fermentative activity of the gut microbiota in mice. These changes are attributed in part to reduced food consumption. Fecal transfer experiments demonstrate that the IAV-conditioned microbiota compromises lung defenses against pneumococcal infection. In mechanistic terms, reduced production of the predominant short-chain fatty acid (SCFA) acetate affects the bactericidal activity of alveolar macrophages. Following treatment with acetate, mice colonized with the IAV-conditioned microbiota display reduced bacterial loads. In the context of influenza infection, acetate supplementation reduces, in a free fatty acid receptor 2 (FFAR2)-dependent manner, local and systemic bacterial loads. This translates into reduced lung pathology and improved survival rates of double-infected mice. Lastly, pharmacological activation of the SCFA receptor FFAR2 during influenza reduces bacterial superinfection. : Sencio et al. provide insights into the mechanisms that underlie bacterial superinfection post-influenza. The authors demonstrate that influenza infection remotely alters the production of short-chain fatty acids (SCFAs) by the gut microbiota. Supplementation with acetate or pharmacological activation of the SCFA receptor FFAR2 reduces susceptibility to secondary bacterial infection. Keywords: influenza A virus, bacterial superinfection, gut microbiota, microbial dysbiosis, food restriction, short-chain fatty acid, acetate, free fatty acid receptor 2, macrophages

National audience

SESSION 3 « Dialogues agresseurs-Hôtes, adaptations réciproques»; Détournement d'une voie métabolique par le virus influenza chez le poulet. Journées d'Animation Scientifique du Département Santé Animale

National audience

Each year, the infection of infants with RSV is the cause of a large number of hospitalizations and deathsworldwide. The current standard of care, Synagis, is used as a preventive medicine for premature children only, and there is no drug available to treat the patients who are suffering from this infection. Targeting the polymerase replication complex is a strategy that has been used successfully to develop drugs against other viruses such as HIV, HCV and HBV. In this project, we wish to inhibit the replication complex by disrupting the N0/P interaction. N0 is the nucleoprotein species that is bound as a complex to the phosphoprotein prior to nucleic acid encapsidation. Recently, the N-terminal region (1-29) of the RSV P protein has been identified as sufficient to bind to N0, and the importance of each P residues has been assessed through alanine scanning mutagenesis experiments. Additionally, NMR studies have provided evidence that P (13-25) has a propensity to fold into an alpha helix in solution. We used these data to design stabilized alpha-helical peptides, with the aim to screen for dominant negative inhibitors of the RSV N0/P interaction. The peptideswere stabilized as macrocyclic peptides using the stapled peptide technology, because this technique can improve their potency, proteolytic stability and cellular permeability. The putative P alpha-helix was plotted onto an alpha-helical wheel, and the amino-acids located on the non-interacting site of the helix were selected for modification with the non-natural amino-acids required for stapling. We will present the results of this stapled peptide scan by reporting i) the conformational analysis by circular dichroism, ii) the biological activity in a novel N0-P fluorescence polarization assay, a viral replication assay and a cytotoxicity assay and iii) the in vivo proof of concept by intranasal administration of a hit peptide, HEVS 124, to BALB/c mice inoculated with a Luc-encoding RSV.

International audience

International audience; Influenza A virus (IAV) is the etiological agent of a highly contagious acute respiratory disease that causes epidemics and considerable mortality annually. Recently, we demonstrated, using an in vitro approach, that the pattern recognition Toll-like receptor (TLR)3 plays a key role in the immune response of lung epithelial cells to IAV. In view of these data and the fact that the functional role of TLR3 in vivo is still debated, we designed an investigation to better understand the role of TLR3 in the mechanisms of IAV pathogenesis and host immune response using an experimental murine model. The time-course of several dynamic parameters, including animal survival, respiratory suffering, viral clearance, leukocyte recruitment into the airspaces and secretion of critical inflammatory mediators, was compared in infected wild-type and TLR3−/− mice. First, we found that the pulmonary expression of TLR3 is constitutive and markedly upregulated following influenza infection in control mice. Notably, when compared to wild-type mice, infected TLR3−/− animals displayed significantly reduced inflammatory mediators, including RANTES (regulated upon activation, normal T cell expressed and secreted), interleukin-6, and interleukin-12p40/p70 as well as a lower number of CD8+ T lymphocytes in the bronchoalveolar airspace. More important, despite a higher viral production in the lungs, mice deficient in TLR3 had an unexpected survival advantage. Hence, to our knowledge, our findings show for the first time that TLR3-IAV interaction critically contributes to the debilitating effects of a detrimental host inflammatory response.

Biomolecular condensates have emerged as an important subcellular organizing principle1. Replication of many viruses, including human respiratory syncytial virus (RSV), occurs in virus-induced compartments called inclusion bodies (IBs) or viroplasm2,3. IBs of negative-strand RNA viruses were recently shown to be biomolecular condensates that form through phase separation4,5. Here we report that the steroidal alkaloid cyclopamine and its chemical analogue A3E inhibit RSV replication by disorganizing and hardening IB condensates. The actions of cyclopamine and A3E were blocked by a point mutation in the RSV transcription factor M2-1. IB disorganization occurred within minutes, which suggests that these molecules directly act on the liquid properties of the IBs. A3E and cyclopamine inhibit RSV in the lungs of infected mice and are condensate-targeting drug-like small molecules that have in vivo activity. Our data show that condensate-hardening drugs may enable the pharmacological modulation of not only many previously undruggable targets in viral replication but also transcription factors at cancer-driving super-enhancers6. Cyclopamine and its chemical analogue A3E inhibit replication of human respiratory syncytial virus (RSV) by hardening the liquid–liquid phase-separated inclusion bodies, resulting in the inhibition of virus replication in the lungs of RSV-infected mice.

Airway inflammation plays a major role in the pathogenesis of influenza viruses and can lead to a fatal outcome. One of the challenging objectives in the field of influenza research is the identification of the molecular bases associated to the immunopathological disorders developed during infection. While its precise function in the virus cycle is still unclear, the viral protein PB1-F2 is proposed to exert a deleterious activity within the infected host. Using an engineered recombinant virus unable to express PB1-F2 and its wild-type homolog, we analyzed and compared the pathogenicity and host response developed by the two viruses in a mouse model. We confirmed that the deletion of PB1-F2 renders the virus less virulent. The global transcriptomic analyses of the infected lungs revealed a potent impact of PB1-F2 on the response developed by the host. Thus, after two days post-infection, PB1-F2 invalidation severely decreased the number of genes activated by the host. PB1-F2 expression induced an increase in the number and level of expression of activated genes linked to cell death, inflammatory response and neutrophil chemotaxis. When generating interactive gene networks specific to PB1-F2, we identified IFN-γ as a central regulator of PB1-F2-regulated genes. The enhanced cell death of airway-recruited leukocytes was evidenced using an apoptosis assay, confirming the pro-apoptotic properties of PB1-F2. Using a NF-kB luciferase adenoviral vector, we were able to quantify in vivo the implication of NF-kB in the inflammation mediated by the influenza virus infection; we found that PB1-F2 expression intensifies the NF-kB activity. Finally, we quantified the neutrophil recruitment within the airways, and showed that this type of leukocyte is more abundant during the infection of the wild-type virus. Collectively, these data demonstrate that PB1-F2 strongly influences the early host response during IAV infection and provides new insights into the mechanisms by which PB1-F2 mediates virulence., Author Summary Influenza A viruses may cause severe respiratory disease. PB1-F2, a viral protein identified in 2001 is suspected to play a role in influenza-related pneumonia. In order to understand the impact of PB1-F2 in the pathogenesis underlying Influenza A virus infection, we engineered a mutant virus unable to express PB1-F2. By the use of high-throughput gene expression assays, we compared the host responses of the wild-type-infected and the PB1-F2 mutant-infected mice. We identified that PB1-F2 expression enhances the immune cell death and inflammatory responses of mice. The inflammatory response mediated by the PB1-F2 expression leads to a massive recruitment of leukocytes within the air spaces, a feature that characterizes the influenza-mediated immunopathology. Our results suggest that PB1-F2 is a virulence factor implicated in the deregulation of the inflammatory response observed in acute influenza virus pneumonia. These data underlie the complexities of virus-host interactions and help us understand by which mechanisms Influenza viruses mediate severe respiratory diseases.

Loïc Guillot: Supported by the Delegation General pour l’Armement Ronan Le Goffic: Supported by “Vaincre la Mucoviscidose” (Paris, France); International audience; Influenza A is a highly contagious single-stranded RNA virus that infects both the upper and lower respiratory tracts of humans. The host innate immune Tolllike receptor (TLR) 3 was shown previously in cells of myeloid origin to recognize the viral replicative, intermediate double-stranded RNA (dsRNA). Thus, dsRNA may be critical for the outcome of the infection. Here we first compared the activation triggered by either influenza A virus or dsRNA in pulmonary epithelial cells. We established that TLR3 is constitutively expressed in human alveolar and bronchial epithelial cells, and we describe its intracellular localization. Expression of TLR3 was positively regulated by the influenza A virus and by dsRNA but not by other inflammatory mediators, including bacterial lipopolysaccharide, the cytokines tumor necrosis factor-␣ and interleukin (IL)-1␤, and the protein kinase C activator phorbol 12-myristate 13-acetate. We also demonstrated that TLR3 contributes directly to the immune response of respiratory epithelial cells to influenza A virus and dsRNA, and we propose a molecular mechanism by which these stimuli induce epithelial cell activation. This model involves mitogen-activated protein kinases, phosphatidylinositol 3-kinase/ Akt signaling, and the TLR3-associated adaptor molecule TRIF but not MyD88-dependent activation of the transcription factors NF-B or interferon regulatory factor/interferon-sensitive response-element pathways. Ultimately, this signal transduction elicits an epithelial response that includes the secretion of the cytokines IL-8, IL-6, RANTES (regulated on activation normal T cell expressed and secreted), and interferon-␤ and the up-regulation of the major adhesion molecule ICAM-1.

International audience; Phtf1 is a gene evolutionarily conserved from Drosophila to human that is abundantly expressed in testis. In adult rat, transcripts were abundant in germinal meiotic and postmeiotic cells. Phtf1-specific antibodies revealed weak activity in a juxtanuclear region of early pachytene spermatocytes. Labeling progressively extended to the entire cytoplasm of step 2-3 spermatids, became intense from step 4, and persisted until the end of spermiogenesis, when it was eliminated in the residual bodies. Phtf1 displayed the properties of an integral membrane protein. In transfected cells and haploid cells of rat seminiferous epithelium, it colocalized with ER markers (calnexin and calmegin, respectively). By using both ER and Golgi markers (TGN-38, p58), we were able to show that, in pachytene spermatocytes and in Golgi phase spermatids, phtf1 labeled a region neighboring the cis-Golgi that probably corresponded to the peripheral Golgi region. Phtf1 staining was not related to ␤-COP, AP1, or AP2 aptamers, indicating that it was not transported between Golgi saccules or between the Golgi complex and plasma membrane. However, aptamer labeling showed that chlatrin vesicles could be engaged in a new traffic route, raising the possibility of a meiotic proacrosomal vesicle origin. Colocalization between phtf1 and calmegin decreased during the acrosomal phase. During the maturation phase, phtf1 was able to identify different ER domains, as described previously for the peripheral Golgi region. Phtf1 provides a potential new marker for Golgi modifications as well as for many of the obscure transformations undergone by the endoplasmic reticulum. It could help to elucidate the morphogenic events connected with the transformation of spermatogenic cells.