Screening antivirals with a mCherry-expressing recombinant bovine respiratory syncytial virus: a proof of concept using cyclopamine.

Bovine respiratory syncytial virus (BRSV) is a pathogenic pneumovirus and a major cause of acute respiratory infections in calves. Although different vaccines are available against BRSV, their efficiency remains limited, and no efficient and large-scale treatment exists. Here, we developed a new reverse genetics system for BRSV expressing the red fluorescent protein mCherry, based on a field strain isolated from a sick calf in Sweden. Although this recombinant fluorescent virus replicated slightly less efficiently compared to the wild type virus, both viruses were shown to be sensitive to the natural steroidal alkaloid cyclopamine, which was previously shown to inhibit human RSV replication. Our data thus point to the potential of this recombinant fluorescent BRSV as a powerful tool in preclinical drug discovery to enable high throughput compound screening.

Depletion of TAX1BP1 Amplifies Innate Immune Responses during Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is the main cause of acute respiratory infections in young children and also has a major impact on the elderly and immunocompromised people. In the absence of a vaccine or efficient treatment, a better understanding of RSV interactions with the host antiviral response during infection is needed. Previous studies revealed that cytoplasmic inclusion bodies (IBs), where viral replication and transcription occur, could play a major role in the control of innate immunity during infection by recruiting cellular proteins involved in the host antiviral response. We recently showed that the morphogenesis of IBs relies on a liquid-liquid-phase separation mechanism depending on the interaction between viral nucleoprotein (N) and phosphoprotein (P). These scaffold proteins are expected to play a central role in the recruitment of cellular proteins to IBs. Here, we performed a yeast two-hybrid screen using RSV N protein as bait and identified the cellular protein TAX1BP1 as a potential partner of this viral protein. This interaction was validated by pulldown and immunoprecipitation assays. We showed that TAX1BP1 suppression has only a limited impact on RSV infection in cell cultures. However, RSV replication is decreased in TAX1BP1-deficient (TAX1BP1 knockout [TAX1BP1KO]) mice, whereas the production of inflammatory and antiviral cytokines is enhanced. In vitro infection of wild-type or TAX1BP1KO alveolar macrophages confirmed that the innate immune response to RSV infection is enhanced in the absence of TAX1BP1. Altogether, our results suggest that RSV could hijack TAX1BP1 to restrain the host immune response during infection. IMPORTANCE Respiratory syncytial virus (RSV), which is the leading cause of lower respiratory tract illness in infants, remains a medical problem in the absence of a vaccine or efficient treatment. This virus is also recognized as a main pathogen in the elderly and immunocompromised people, and the occurrence of coinfections (with other respiratory viruses and bacteria) amplifies the risks of developing respiratory distress. In this context, a better understanding of the pathogenesis associated with viral respiratory infections, which depends on both viral replication and the host immune response, is needed. The present study reveals that the cellular protein TAX1BP1, which interacts with the RSV nucleoprotein N, participates in the control of the innate immune response during RSV infection, suggesting that the N-TAX1BP1 interaction represents a new target for the development of antivirals.

The microbiota plays a critical role in the reactivity of lung immune components to innate ligands.

The gut microbiota contributes to shaping efficient and safe immune defenses in the gut. However, little is known about the role of the gut and/or lung microbiota in the education of pulmonary innate immune responses. Here, we tested whether the endogenous microbiota in general can modulate the reactivity of pulmonary tissue to pathogen stimuli by comparing the response of specific-pathogen-free (SPF) and germ-free (GF) mice. Thus, we observed earlier and greater inflammation in the pulmonary compartment of GF mice than that of SPF mice after intranasal instillation to lipopolysaccharide (LPS), a component of Gram-negative bacteria. Toll-like receptor 4 (TLR4) was more abundantly expressed in the lungs of GF mice than those of SPF mice at steady state, which could predispose the innate immunity of GF mice to strongly react to the environmental stimuli. Lung explants were stimulated with different TLR agonists or infected with the human airways pathogen, respiratory syncytial virus (RSV), resulting in greater inflammation under almost all conditions for the GF explants. Finally, alveolar macrophages (AM) from GF mice presented a higher innate immune response upon RSV infection than those of SPF mice. Overall, these data suggest that the presence of microbiota in SPF mice induced a process of innate immune tolerance in the lungs by a mechanism which remains to be elucidated. Our study represents a step forward to establishing the link between the microbiota and the immune reactivity of the lungs.

Turning off NADPH oxidase-2 by impeding p67phox activation in infected mouse macrophages reduced viral entry and inflammation.

BACKGROUND: Targeting cells of the host immune system is a promising approach to fight against Influenza A virus (IAV) infection. Macrophage cells use the NADPH oxidase-2 (NOX2) enzymatic complex as a first line of defense against pathogens by generating superoxide ions O2- and releasing H2O2. Herein, we investigated whether targeting membrane -embedded NOX2 decreased IAV entry via raft domains and reduced inflammation in infected macrophages. METHODS: Confocal microscopy and western blots monitored levels of the viral nucleoprotein NP and p67phox, NOX2 activator subunit, Elisa assays quantified TNF-α levels in LPS or IAV-activated mouse or porcine alveolar macrophages pretreated with a fluorescent NOX inhibitor, called nanoshutter NS1. RESULTS: IAV infection in macrophages promoted p67phox translocation to the membrane, rafts clustering and activation of the NOX2 complex at early times. Disrupting rafts reduced intracellular viral NP. NS1 markedly reduced raft clustering and viral entry by binding to the C-terminal of NOX2 also characterized in vitro. NS1 decrease of TNF-α release depended on the cell type. CONCLUSION: NOX2 participated in IAV entry and raft-mediated endocytosis. NOX2 inhibition by NS1 reduced viral entry. NS1 competition with p67phox for NOX2 binding shown by in silico models and cell-free assays was in agreement with NS1 inhibiting p67phox translocation to membrane-embedded NOX2 in mouse and porcine macrophages. GENERAL SIGNIFICANCE: We introduce NS1 as a compound targeting NOX2, a critical enzyme controlling viral levels and inflammation in macrophages and discuss the therapeutic relevance of targeting the C-terminal of NADPH oxidases by probes like NS1 in viral infections.

Pig skin includes dendritic cell subsets transcriptomically related to human CD1a and CD14 dendritic cells presenting different migrating behaviors and T cell activation capacities.

Swine skin is one of the best structural models for human skin, widely used to probe drug transcutaneous passage and to test new skin vaccination devices. However, little is known about its composition in immune cells, and among them dendritic cells (DC), that are essential in the initiation of the immune response. After a first seminal work describing four different DC subpopulations in pig skin, we hereafter deepen the characterization of these cells, showing the similarities between swine DC subsets and their human counterparts. Using comparative transcriptomic study, classical phenotyping as well as in vivo and in vitro functional studies, we show that swine CD163(pos) dermal DC (DDC) are transcriptomically similar to the human CD14(pos) DDC. CD163(pos) DDC are recruited in inflamed skin, they migrate in inflamed lymph but they are not attracted toward CCL21, and they modestly activate allogeneic CD8 T cells. We also show that CD163(low) DDC are transcriptomically similar to the human CD1a(pos) DDC. CD163(low) DDC migrate toward CCL21, they activate allogeneic CD8 and CD4 T cells and, like their potential human lung counterpart, they skew CD4 T cells toward a Th17 profile. We thus conclude that swine skin is a relevant model for human skin vaccination.

Structure-based discovery of the novel antiviral properties of naproxen against the nucleoprotein of influenza A virus.

The nucleoprotein (NP) binds the viral RNA genome and associates with the polymerase in a ribonucleoprotein complex (RNP) required for transcription and replication of influenza A virus. NP has no cellular counterpart, and the NP sequence is highly conserved, which led to considering NP a hot target in the search for antivirals. We report here that monomeric nucleoprotein can be inhibited by a small molecule binding in its RNA binding groove, resulting in a novel antiviral against influenza A virus. We identified naproxen, an anti-inflammatory drug that targeted the nucleoprotein to inhibit NP-RNA association required for NP function, by virtual screening. Further docking and molecular dynamics (MD) simulations identified in the RNA groove two NP-naproxen complexes of similar levels of interaction energy. The predicted naproxen binding sites were tested using the Y148A, R152A, R355A, and R361A proteins carrying single-point mutations. Surface plasmon resonance, fluorescence, and other in vitro experiments supported the notion that naproxen binds at a site identified by MD simulations and showed that naproxen competed with RNA binding to wild-type (WT) NP and protected active monomers of the nucleoprotein against proteolytic cleavage. Naproxen protected Madin-Darby canine kidney (MDCK) cells against viral challenges with the H1N1 and H3N2 viral strains and was much more effective than other cyclooxygenase inhibitors in decreasing viral titers of MDCK cells. In a mouse model of intranasal infection, naproxen treatment decreased the viral titers in mice lungs. In conclusion, naproxen is a promising lead compound for novel antivirals against influenza A virus that targets the nucleoprotein in its RNA binding groove.

An Isolate of Streptococcus mitis Displayed In Vitro Antimicrobial Activity and Deleterious Effect in a Preclinical Model of Lung Infection.

Microbiota studies have dramatically increased over these last two decades, and the repertoire of microorganisms with potential health benefits has been considerably enlarged. The development of next generation probiotics from new bacterial candidates is a long-term strategy that may be more efficient and rapid with discriminative in vitro tests. Streptococcus strains have received attention regarding their antimicrobial potential against pathogens of the upper and, more recently, the lower respiratory tracts. Pathogenic bacterial strains, such as non-typable Haemophilus influenzae (NTHi), Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus), are commonly associated with acute and chronic respiratory diseases, and it could be interesting to fight against pathogens with probiotics. In this study, we show that a Streptococcus mitis (S. mitis) EM-371 strain, isolated from the buccal cavity of a human newborn and previously selected for promising anti-inflammatory effects, displayed in vitro antimicrobial activity against NTHi, P. aeruginosa or S. aureus. However, the anti-pathogenic in vitro activity was not sufficient to predict an efficient protective effect in a preclinical model. Two weeks of treatment with S. mitis EM-371 did not protect against, and even exacerbated, NTHi lung infection.

Influenza A virus protein PB1-F2 exacerbates IFN-beta expression of human respiratory epithelial cells.

The PB1-F2 protein of the influenza A virus (IAV) contributes to viral pathogenesis by a mechanism that is not well understood. PB1-F2 was shown to modulate apoptosis and to be targeted by the CD8(+) T cell response. In this study, we examined the downstream effects of PB1-F2 protein during IAV infection by measuring expression of the cellular genes in response to infection with wild-type WSN/33 and PB1-F2 knockout viruses in human lung epithelial cells. Wild-type virus infection resulted in a significant induction of genes involved in innate immunity. Knocking out the PB1-F2 gene strongly decreased the magnitude of expression of cellular genes implicated in antiviral response and MHC class I Ag presentation, suggesting that PB1-F2 exacerbates innate immune response. Biological network analysis revealed the IFN pathway as a link between PB1-F2 and deregulated genes. Using quantitative RT-PCR and IFN-ß gene reporter assay, we determined that PB1-F2 mediates an upregulation of IFN-ß expression that is dependent on NF-κB but not on AP-1 and IFN regulatory factor-3 transcription factors. Recombinant viruses knocked out for the PB1-F2 and/or the nonstructural viral protein 1 (the viral antagonist of the IFN response) genes provide further evidence that PB1-F2 increases IFN-ß expression and that nonstructural viral protein 1 strongly antagonizes the effect of PB1-F2 on the innate response. Finally, we compared the effect of PB1-F2 variants taken from several IAV strains on IFN-ß expression and found that PB1-F2-mediated IFN-ß induction is significantly influenced by its amino acid sequence, demonstrating its importance in the host cell response triggered by IAV infection.

PB1-F2 influenza A virus protein adopts a beta-sheet conformation and forms amyloid fibers in membrane environments.

The influenza A virus PB1-F2 protein, encoded by an alternative reading frame in the PB1 polymerase gene, displays a high sequence polymorphism and is reported to contribute to viral pathogenesis in a sequence-specific manner. To gain insights into the functions of PB1-F2, the molecular structure of several PB1-F2 variants produced in Escherichia coli was investigated in different environments. Circular dichroism spectroscopy shows that all variants have a random coil secondary structure in aqueous solution. When incubated in trifluoroethanol polar solvent, all PB1-F2 variants adopt an alpha-helix-rich structure, whereas incubated in acetonitrile, a solvent of medium polarity mimicking the membrane environment, they display beta-sheet secondary structures. Incubated with asolectin liposomes and SDS micelles, PB1-F2 variants also acquire a beta-sheet structure. Dynamic light scattering revealed that the presence of beta-sheets is correlated with an oligomerization/aggregation of PB1-F2. Electron microscopy showed that PB1-F2 forms amorphous aggregates in acetonitrile. In contrast, at low concentrations of SDS, PB1-F2 variants exhibited various abilities to form fibers that were evidenced as amyloid fibers in a thioflavin T assay. Using a recombinant virus and its PB1-F2 knock-out mutant, we show that PB1-F2 also forms amyloid structures in infected cells. Functional membrane permeabilization assays revealed that the PB1-F2 variants can perforate membranes at nanomolar concentrations but with activities found to be sequence-dependent and not obviously correlated with their differential ability to form amyloid fibers. All of these observations suggest that PB1-F2 could be involved in physiological processes through different pathways, permeabilization of cellular membranes, and amyloid fiber formation.

Control of IFN-I responses by the aminopeptidase IRAP in neonatal C57BL/6 alveolar macrophages during RSV infection.

Respiratory Syncytial Virus (RSV) is the major cause of lower respiratory tract infection in infants, in whom, the sensing of RSV by innate immune receptors and its regulation are still poorly described. However, the severe bronchiolitis following RSV infection in neonates has been associated with a defect in type I interferons (IFN-I) production, a cytokine produced mainly by alveolar macrophages (AMs) upon RSV infection in adults. In the present study, neonatal C57BL/6 AMs mobilized very weakly the IFN-I pathway upon RSV infection in vitro and failed to restrain virus replication. However, IFN-I productions by neonatal AMs were substantially increased by the deletion of Insulin-Responsive AminoPeptidase (IRAP), a protein previously involved in the regulation of IFN-I production by dendritic cells. Moreover, neonatal IRAPKO AMs showed a higher expression of IFN-stimulated genes than their wild-type C57BL/6 counterpart. Interestingly, depletion of IRAP did not affect adult AM responses. Finally, we demonstrated that newborn IRAPKO mice infected with RSV had more IFN-I in their lungs and eliminated the virus more efficiently than WT neonates. Taken together, early-life susceptibility to RSV infection may be related to an original age-dependent suppressive function of IRAP on the IFN-I driven-antiviral responses in neonatal AMs.

The Composition of Circulating Leukocytes Varies With Age and Melanoma Onset in the MeLiM Pig Biomedical Model.

Immunological research in pigs benefits from many improvements with a direct impact on the veterinary control of pig husbandry and on biomedical models. We compiled the available knowledge to develop gating strategies to monitor simultaneously all blood immune cell types by multicolor flow cytometry in Melanoblastoma-bearing Libechov Minipigs (MeLiM). The MeLiM pig spontaneously develops cutaneous melanomas that regress few months later. We monitored lymphoid and myeloid cell subsets in 3 to 21 weeks old pigs. Interestingly, neutrophils, type III monocytes (CD163+ CD14+ MHC II-) and CD4- CD8α- T cells are less abundant in oldest animals in contrast to eosinophils, type II monocytes (CD163- CD14low MHC II+), B cells, γδ T cells, CD4+ CD8α+ and CD4- CD8α+ T cells. Melanoma occurrence led to changes in the blood cell composition. Higher proportions of NK cells, CD4+ and CD4+ CD8α+ T cells, and CD21- B cells among B cells are found in young melanoma-bearing piglets, consistent with the immune-mediated spontaneous regression in the MeLiM model.

Distinctive Cellular and Metabolic Reprogramming in Porcine Lung Mononuclear Phagocytes Infected With Type 1 PRRSV Strains.

Porcine reproductive and respiratory syndrome (PRRS) has an extensive impact on pig production. The causative virus (PRRSV) is divided into two species, PRRSV-1 (European origin) and PRRSV-2 (North American origin). Within PRRSV-1, PRRSV-1.3 strains, such as Lena, are more pathogenic than PRRSV-1.1 strains, such as Flanders 13 (FL13). To date, the molecular interactions of PRRSV with primary lung mononuclear phagocyte (MNP) subtypes, including conventional dendritic cells types 1 (cDC1) and 2 (cDC2), monocyte-derived DCs (moDC), and pulmonary intravascular macrophages (PIM), have not been thoroughly investigated. Here, we analyze the transcriptome profiles of in vivo FL13-infected parenchymal MNP subpopulations and of in vitro FL13- and Lena-infected parenchymal MNP. The cell-specific expression profiles of in vivo sorted cells correlated with their murine counterparts (AM, cDC1, cDC2, moDC) with the exception of PIM. Both in vivo and in vitro, FL13 infection altered the expression of a low number of host genes, and in vitro infection with Lena confirmed the higher ability of this strain to modulate host response. Machine learning (ML) and gene set enrichment analysis (GSEA) unraveled additional relevant genes and pathways modulated by FL13 infection that were not identified by conventional analyses. GSEA increased the cellular pathways enriched in the FL13 data set, but ML allowed a more complete comprehension of functional profiles during FL13 in vitro infection. Data indicates that cellular reprogramming differs upon Lena and FL13 infection and that the latter might keep antiviral and inflammatory macrophage/DC functions silent. Although the slow replication kinetics of FL13 likely contribute to differences in cellular gene expression, the data suggest distinct mechanisms of interaction of the two viruses with the innate immune system during early infection.

Macrophage-B Cell Interactions in the Inverted Porcine Lymph Node and Their Response to Porcine Reproductive and Respiratory Syndrome Virus.

Swine lymph nodes (LN) present an inverted structure compared to mouse and human, with the afferent lymph diffusing from the center to the periphery. This structure, also observed in close and distant species such as dolphins, hippopotamus, rhinoceros, and elephants, is poorly described, nor are the LN macrophage populations and their relationship with B cell follicles. B cell maturation occurs mainly in LN B cell follicles with the help of LN macrophage populations endowed with different antigen delivery capacities. We identified three macrophage populations that we localized in the inverted LN spatial organization. This allowed us to ascribe porcine LN MΦ to their murine counterparts: subcapsular sinus MΦ, medullary cord MΦ and medullary sinus MΦ. We identified the different intra and extrafollicular stages of LN B cells maturation and explored the interaction of MΦ, drained antigen and follicular B cells. The porcine reproductive and respiratory syndrome virus (PRRSV) is a major porcine pathogen that infects tissue macrophages (MΦ). PRRSV is persistent in the secondary lymphoid tissues and induces a delay in neutralizing antibodies appearance. We observed PRRSV interaction with two LN MΦ populations, of which one interacts closely with centroblasts. We observed BCL6 up-regulation in centroblast upon PRRSV infection, leading to new hypothesis on PRRSV inhibition of B cell maturation. This seminal study of porcine LN will permit fruitful comparison with murine and human LN for a better understanding of normal and inverted LN development and functioning.

A DNA-Modified Live Vaccine Prime-Boost Strategy Broadens the T-Cell Response and Enhances the Antibody Response against the Porcine Reproductive and Respiratory Syndrome Virus.

The Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) induces reproductive disorders in sows and respiratory illnesses in growing pigs and is considered as one of the main pathogenic agents responsible for economic losses in the porcine industry worldwide. Modified live PRRSV vaccines (MLVs) are very effective vaccine types against homologous strains but they present only partial protection against heterologous viral variants. With the goal to induce broad and cross-protective immunity, we generated DNA vaccines encoding B and T antigens derived from a European subtype 1 strain that include T-cell epitope sequences known to be conserved across strains. These antigens were expressed either in a native form or in the form of vaccibodies targeted to the endocytic receptor XCR1 and CD11c expressed by different types of antigen-presenting cells (APCs). When delivered in skin with cationic nanoparticles and surface electroporation, multiple DNA vaccinations as a stand-alone regimen induced substantial antibody and T-cell responses, which were not promoted by targeting antigens to APCs. Interestingly, a DNA-MLV prime-boost strategy strongly enhanced the antibody response and broadened the T-cell responses over the one induced by MLV or DNA-only. The anti-nucleoprotein antibody response induced by the DNA-MLV prime-boost was clearly promoted by targeting the antigen to CD11c and XCR1, indicating a benefit of APC-targeting on the B-cell response. In conclusion, a DNA-MLV prime-boost strategy, by enhancing the potency and breadth of MLV vaccines, stands as a promising vaccine strategy to improve the control of PRRSV in infected herds.

Electroporation of a nanoparticle-associated DNA vaccine induces higher inflammation and immunity compared to its delivery with microneedle patches in pigs.

DNA vaccination is an attractive technology, based on its well-established manufacturing process, safety profile, adaptability to rapidly combat pandemic pathogens, and stability at ambient temperature; however an optimal delivery method of DNA remains to be determined. As pigs are a relevant model for humans, we comparatively evaluated the efficiency of vaccine DNA delivery in vivo to pigs using dissolvable microneedle patches, intradermal inoculation with needle (ID), surface electroporation (EP), with DNA associated or not to cationic poly-lactic-co-glycolic acid nanoparticles (NPs). We used a luciferase encoding plasmid (pLuc) as a reporter and vaccine plasmids encoding antigens from the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a clinically-significant swine arterivirus. Patches were successful at inducing luciferase expression in skin although at lower level than EP. EP induced the cutaneaous recruitment of granulocytes, of MHC2posCD172Apos myeloid cells and type 1 conventional dendritic cells, in association with local production of IL-1ß, IL-8 and IL-17; these local responses were more limited with ID and undetectable with patches. The addition of NP to EP especially promoted the recruitment of the MHC2posCD172Apos CD163int and CD163neg myeloid subsets. Notably we obtained the strongest and broadest IFNγ T-cell response against a panel of PRRSV antigens with DNA + NPs delivered by EP, whereas patches and ID were ineffective. The anti-PRRSV IgG responses were the highest with EP administration independently of NPs, mild with ID, and undetectable with patches. These results contrast with the immunogenicity and efficacy previously induced in mice with patches. This study concludes that successful DNA vaccine administration in skin can be achieved in pigs with electroporation and patches, but only the former induces local inflammation, humoral and cellular immunity, with the highest potency when NPs were used. This finding shows the importance of evaluating the delivery and immunogenicity of DNA vaccines beyond the mouse model in a preclinical model relevant to human such as pig and reveals that EP with DNA combined to NP induces strong immunogenicity.

Prolactin promotes the secretion of active cathepsin D at the basal side of rat mammary acini.

Cathepsin D (CD), a lysosomal aspartic protease present in mammary tissue and milk in various molecular forms, is also found in the incubation medium of mammary acini in molecular forms that are proteolytically active on prolactin at a physiological pH. Because prolactin controls the vesicular traffic in mammary cells, we studied, in vivo and in vitro, its effects on the polarized transport and secretion of various forms of CD in the rat mammary gland. CD accumulated in vesicles not involved in endocytosis in the basal region of cells. Prolactin increased this accumulation and the release of endosomal active single-chain CD at the basal side of acini. The CD-mediated proteolysis of prolactin, leading to the antiangiogenic 16-kDa form, at a physiological pH, was observed only in conditioned medium but not milk. These data support the novel concept that an active molecular form of CD, secreted at the basal side of the mammary epithelium, participates in processing blood-borne prolactin outside the cell, this polarized secretion being controlled by prolactin itself.

Porcine Reproductive and Respiratory Syndrome Virus Type 1.3 Lena Triggers Conventional Dendritic Cells 1 Activation and T Helper 1 Immune Response Without Infecting Dendritic Cells.

Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is an arterivirus responsible for highly contagious infection and huge economic losses in pig industry. Two species, PRRSV-1 and PRRSV-2 are distinguished, PRRSV-1 being more prevalent in Europe. PRRSV-1 can further be divided in subtypes. PRRSV-1.3 such as Lena are more pathogenic than PRRSV-1.1 such as Lelystad or Flanders13. PRRSV-1.3 viruses trigger a higher Th1 response than PRRSV-1.1, although the role of the cellular immune response in PRRSV clearance remains ill defined. The pathogenicity as well as the T cell response inductions may be differentially impacted according to the capacity of the virus strain to infect and/or activate DCs. However, the interactions of PRRSV with in vivo-differentiated-DC subtypes such as conventional DC1 (cDC1), cDC2, and monocyte-derived DCs (moDC) have not been thoroughly investigated. Here, DC subpopulations from Lena in vivo infected pigs were analyzed for viral genome detection. This experiment demonstrates that cDC1, cDC2, and moDC are not infected in vivo by Lena. Analysis of DC cytokines production revealed that cDC1 are clearly activated in vivo by Lena. In vitro comparison of 3 Europeans strains revealed no infection of the cDC1 and cDC2 and no or little infection of moDC with Lena, whereas the two PRRSV-1.1 strains infect none of the 3 DC subtypes. In vitro investigation of T helper polarization and cytokines production demonstrate that Lena induces a higher Th1 polarization and IFNγ secretion than FL13 and LV. Altogether, this work suggests an activation of cDC1 by Lena associated with a Th1 immune response polarization.

Porcine Alveolar Macrophage-like cells are pro-inflammatory Pulmonary Intravascular Macrophages that produce large titers of Porcine Reproductive and Respiratory Syndrome Virus.

Lung inflammation is frequently involved in respiratory conditions and it is strongly controlled by mononuclear phagocytes (MNP). We previously studied porcine lung MNP and described a new population of cells presenting all the features of alveolar macrophages (AM) except for their parenchymal location, that we named AM-like cells. Herein we showed that AM-like cells are macrophages phagocytosing blood-borne particles, in agreement with a pulmonary intravascular macrophages (PIM) identity. PIM have been described microscopically long time ago in species from the Laurasiatheria superorder such as bovine, swine, cats or cetaceans. We observed that PIM were more inflammatory than AM upon infection with the porcine reproductive and respiratory syndrome virus (PRRSV), a major swine pathogen. Moreover, whereas PRRSV was thought to mainly target AM, we observed that PIM were a major producer of virus. The PIM infection was more correlated with viremia in vivo than AM infection. Finally like AM, PIM-expressed genes were characteristic of an embryonic monocyte-derived macrophage population, whose turnover is independent of bone marrow-derived hematopoietic precursors. This last observation raised the interesting possibility that AM and PIM originate from the same lung precursor.

A Rift Valley fever virus Gn ectodomain-based DNA vaccine induces a partial protection not improved by APC targeting.

Rift Valley fever virus, a phlebovirus endemic in Africa, causes serious diseases in ruminants and humans. Due to the high probability of new outbreaks and spread to other continents where competent vectors are present, vaccine development is an urgent priority as no licensed vaccines are available outside areas of endemicity. In this study, we evaluated in sheep the protective immunity induced by DNA vaccines encoding the extracellular portion of the Gn antigen which was either or not targeted to antigen-presenting cells. The DNA encoding untargeted antigen was the most potent at inducing IgG responses, although not neutralizing, and conferred a significant clinical and virological protection upon infectious challenge, superior to DNA vaccines encoding the targeted antigen. A statistical analysis of the challenge parameters supported that the anti-eGn IgG, rather than the T-cell response, was instrumental in protection. Altogether, this work shows that a DNA vaccine encoding the extracellular portion of the Gn antigen confers substantial-although incomplete-protective immunity in sheep, a natural host with high preclinical relevance, and provides some insights into key immune correlates useful for further vaccine improvements against the Rift Valley fever virus.

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.