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Abstract There is an urgent need for influenza vaccines providing broader protection that may decrease the need for annual immunization of the human population. We investigated the efficacy of heterologous prime boost immunization with chimpanzee adenovirus (ChAdOx2) and modified vaccinia Ankara (MVA) vectored vaccines, expressing conserved influenza virus nucleoprotein (NP), matrix protein 1 (M1) and neuraminidase (NA) in H1N1pdm09 pre-exposed pigs. We compared the efficacy of intra-nasal, aerosol and intra-muscular vaccine delivery against H3N2 influenza challenge. Aerosol prime boost immunization induced strong local lung T cell and antibody responses and abrogated viral shedding and lung pathology following H3N2 challenge. In contrast, intramuscular immunization induced powerful systemic responses and weak local lung responses but also abolished lung pathology and reduced viral shedding. These results provide valuable insights into the development of a broadly protective influenza vaccine in a highly relevant large animal model and will inform future vaccine and clinical trial design.

Porcine respiratory disease is multifactorial and most commonly involves pathogen co-infections. Major contributors include swine influenza A (swIAV) and porcine reproductive and respiratory syndrome (PRRSV) viruses. Experimental co-infection studies with these two viruses have shown that clinical outcomes can be exacerbated, but how innate and adaptive immune responses contribute to pathogenesis and pathogen control has not been thoroughly evaluated. We investigated immune responses following experimental simultaneous co-infection of pigs with swIAV H3N2 and PRRSV-2. Our results indicated that clinical disease was not significantly exacerbated, and swIAV H3N2 viral load was reduced in the lung of the co-infected animals. PRRSV-2/swIAV H3N2 co-infection did not impair the development of virus-specific adaptive immune responses. swIAV H3N2-specific IgG serum titers and PRRSV-2-specific CD8β+ T-cell responses in blood were enhanced. Higher proportions of polyfunctional CD8β+ T-cell subset in both blood and lung washes were found in PRRSV-2/swIAV H3N2 co-infected animals compared to the single-infected groups. Our findings provide evidence that systemic and local host immune responses are not negatively affected by simultaneous swIAV H3N2/PRRSV-2 co-infection, raising questions as to the mechanisms involved in disease modulation.

Nairobi sheep disease (NSD), caused by the viral agent NSD virus (NSDV), is a haemorrhagic fever disease affecting and inducing high mortality in sheep and goat populations. NSDV belongs to the genus Orthonairovirus of the Nairoviridae family from the order Bunyavirales. Other viruses circulating in livestock such as Crimean–Congo haemorrhagic fever virus (CCHFV) and Dugbe virus (DUGV) are members of the same genus and are reported to share antigenic features. There are very few available materials to study NSDV infection both in vitro and in vivo. In the present work, we characterised two monoclonal antibodies generated in mice that recognise NSDV specifically but not CCHFV or DUGV, along with a potential use to define virus-infected cells, using flow cytometry. We believe this tool can be useful for research, but also NSDV diagnostics, especially through immunological staining.

Porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) causes huge economic losses to the European pig industry. PRRSV-1 is divided into 3 subtypes and exhibits considerable antigenic heterogeneity. Due to its high mutation rate, PRRSV-1 is constantly evolving, and highly virulent, particularly subtype 3 strains, are continually emerging. The mechanism(s) underlying PRRSV-1 virulence have not been fully elucidated. In vivo studies have implicated replication kinetics, cell tropism and an enhanced pro-inflammatory cytokine response as potential contributing factors. However, few strains have been directly compared and differences in in vivo study design have hindered comparison, thus limiting our understanding of PRRSV-1 virulence. To address this knowledge gap, we sought to develop a reverse genetics and ex vivo model system, to attempt to identify correlates of PRRSV-1 virulence and attenuation in vitro. Herein we describe the use of primary porcine bone marrow-derived macrophages (BMDM) to investigate the growth kinetics and induced cytokine profiles of the highly virulent SU1-Bel strain, the low virulence 215-06 strain and the attenuated Olot/91 strain. We show that infection of BMDM with virulent PRRSV-1 strains induced higher expression of IL-6 and IL-8 and lower expression of TNF-α when compared with the attenuated strain. In addition, BMDM infected with SU1-Bel secreted significantly more IFN-α than those infected with PRRSV-1 strains of lower virulence. Interestingly, despite inducing less IFN-α than SU1-Bel, Olot/91 induced much higher levels of expression of several interferon-stimulated genes (ISGs), suggesting that Olot/91 may be less able to counteract type I IFN signaling which may contribute to its attenuated phenotype.

There is a critical need to develop superior influenza vaccines that provide broader protection. Influenza vaccines are traditionally tested in naive animals, although humans are exposed to influenza in the first years of their lives, but the impact of prior influenza exposure on vaccine immune responses has not been well studied. Pigs are an important natural host for influenza, are a source of pandemic viruses, and are an excellent model for human influenza. Here, we investigated the immunogenicity of the ChAdOx2 viral vectored vaccine, expressing influenza nucleoprotein, matrix protein 1, and neuraminidase in H1N1pdm09 pre-exposed pigs. We evaluated the importance of the route of administration by comparing intranasal, aerosol, and intramuscular immunizations. Aerosol delivery boosted the local lung T-cell and antibody responses, while intramuscular immunization boosted peripheral blood immunity. These results will inform how best to deliver vaccines in order to harness optimal protective immunity.

The porcine respiratory disease complex (PRDC) is responsible for significant economic losses in the pig industry worldwide. Porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza virus are major viral contributors to PRDC. Vaccines are cost-effective measures for controlling PRRS, however, their efficacy in the context of co-infections has been poorly investigated. In this study, we aimed to determine the effect of PRRSV-2 and swine influenza H3N2 virus co-infection on the efficacy of PRRSV modified live virus (MLV) vaccination, which is widely used in the field. Following simultaneous challenge with contemporary PRRSV-2 and H3N2 field isolates, we found that the protective effect of PRRS MLV vaccination on clinical disease and pathology was abrogated, although viral load was unaffected and antibody responses were enhanced. In contrast, co-infection in non-immunized animals reduced PRRSV-2 viremia and H3N2 virus load in the upper respiratory tract and potentiated T cell responses against both PRRSV-2 and H3N2 in the lung. Further analysis suggested that an upregulation of inhibitory cytokines gene expression in the lungs of vaccinated pigs may have influenced responses to H3N2 and PRRSV-2. These findings provide important insights into the effect of viral co-infections on PRRS vaccine efficacy that may help identify more effective vaccination strategies against PRDC in the field.

Mucosal-associated invariant T (MAIT) cells are a population of innate-like T cells that utilize a semi-invariant T cell receptor (TCR) α chain and are restricted by the highly conserved antigen presenting molecule MR1. MR1 presents microbial riboflavin biosynthesis derived metabolites produced by bacteria and fungi. Consistent with their ability to sense ligands derived from bacterial sources, MAIT cells have been associated with the immune response to a variety of bacterial infections, such as Mycobacterium spp., Salmonella spp. and Escherichia coli. To date, MAIT cells have been studied in humans, non-human primates and mice. However, they have only been putatively identified in cattle by PCR based methods; no phenotypic or functional analyses have been performed. Here, we identified a MAIT cell population in cattle utilizing MR1 tetramers and high-throughput TCR sequencing. Phenotypic analysis of cattle MAIT cells revealed features highly analogous to those of MAIT cells in humans and mice, including expression of an orthologous TRAV1-TRAJ33 TCR α chain, an effector memory phenotype irrespective of tissue localization, and expression of the transcription factors PLZF and EOMES. We determined the frequency of MAIT cells in peripheral blood and multiple tissues, finding that cattle MAIT cells are enriched in mucosal tissues as well as in the mesenteric lymph node. Cattle MAIT cells were responsive to stimulation by 5-OP-RU and riboflavin biosynthesis competent bacteria in vitro. Furthermore, MAIT cells in milk increased in frequency in cows with mastitis. Following challenge with virulent Mycobacterium bovis, a causative agent of bovine tuberculosis and a zoonosis, peripheral blood MAIT cells expressed higher levels of perforin. Thus, MAIT cells are implicated in the immune response to two major bacterial infections in cattle. These data suggest that MAIT cells are functionally highly conserved and that cattle are an excellent large animal model to study the role of MAIT cells in important zoonotic infections.

Host-microbiota interactions are important in shaping immune responses that have the potential to influence the outcome of pathogen infection. However, most studies have focused on the gut microbiota and its possible association with disease outcome, while the role of the nasal microbiota and respiratory pathogen infection has been less well studied. Here we examined changes in the composition of the nasal microbiota of pigs following experimental infection with porcine reproductive and respiratory syndrome virus 2 (PRRSV-2), swine influenza A H3N2 virus (H3N2) or both viruses. DNA extracted from nasal swabs were subjected to 16S rRNA sequencing to study the composition of the nasal microbiota. Bacterial richness fluctuated in all groups, with a slight reduction in pigs singly infected with PRRSV-2 and H3N2 during the first 5 days of infection compared to uninfected controls. In contrast, nasal bacterial richness remained relatively stable after PRRSV-2/H3N2 co-infection. PRRSV-2 and H3N2, alone or in combination differentially altered the abundance and distribution of bacterial families. Single and co-infection with PRRSV-2 or H3N2 was associated with the expansion of the Neisseriaceae family. A positive correlation between H3N2 viral load and the relative abundance of the Neisseriaceae was observed. However, further mechanistic studies are required to understand the significance of the changes in specific bacterial families following these viral infections.

Abstract 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.

The Rift Valley fever virus (RVFV) is responsible for a serious mosquito-borne viral disease in humans and ruminants. The development of a new and safer vaccine is urgently needed due to the risk of introduction of this arbovirus into RVFV-free continents. We recently showed that a DNA vaccine encoding eGn, the ectodomain of the RVFV Gn glycoprotein, conferred a substantial protection in the sheep natural host and that the anti-eGn IgG levels correlated to protection. Addressing eGn to DEC205 reduced the protective efficacy while decreasing the antibody and increasing the IFNγ T cell responses in sheep. In order to get further insight into the involved mechanisms, we evaluated our eGn-encoding DNA vaccine strategy in the reference mouse species. A DNA vaccine encoding eGn induced full clinical protection in mice and the passive transfer of immune serum was protective. This further supports that antibodies, although non-neutralizing in vitro, are instrumental in the protection against RVFV. Addressing eGn to DEC205 was also detrimental to protection in mice, and in this species, both the antibody and the IFNγ T cell responses were strongly decreased. Conversely when using a plasmid encoding a different antigen, i.e., mCherry, DEC205 targeting promoted the antibody response. Altogether our results show that the outcome of targeting antigens to DEC205 depends on the species and on the fused antigen and is not favorable in the case of eGn. In addition, we bring evidences that eGn in itself is a pertinent antigen to be included in a DNA vaccine and that next developments should aim at promoting the anti-eGn antibody response.