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This article reports that the 120-kb Protein tyrosine phosphatase receptor type C polypeptide (PTPRC) gene co-localizes with marker D1S413 on a 610-kb yeast artificial chromosome (YAC) and therefore considerably refines the previous physical localization of this gene on chromosome 1q31→q32. High molecular weight DNA was prepared in high concentration agarose plugs. The size of the YAC was determined by Pulsed Field Gel Electrophoresis using 1 % PFGE grade agarose with a CHEF DRII system run for 24 h on a 30-70 s pulse ramp, 6 V/cm at 10-15°C in 0.5 x TBE, blotted and then hybridised with PTPRC 5' probe. Linkage analysis has mapped the calcium channel &b.alpha;1 subunit gene, CACNL1A3, to a 5-cM interval at 1q31→q32 shared by D1S413 and has revealed that CACNL1A3 lies telomeric to D1S413.

Cattle express three subclasses of IgG antibody - IgG1, IgG2 and IgG3. Unlike IgG1 and IgG2, IgG3 was described relatively recently and the role of this subclass in immunity is unknown. Using recombinant bovine IgG1, IgG2 and IgG3 monoclonal antibodies (mAbs), we demonstrated that only one of the commercially available anti-bovine IgG mAbs tested was able to recognize IgG3, and no mAb exclusively bound IgG3. Here, we evaluated a small ankyrin repeat protein, called Ankyron™ AZS40101, that was generated to bind to bovine IgG3. Ankyron™ AZS40101 bound specifically to IgG3 with minimal reactivity to IgG1 and IgG2. Ankyron™ AZS40101 was shown to be useful in ELISA assays as either a capture or detection reagent. Utilisation of Ankyron™ AZS40101 alongside IgG1 and IgG2 specific mAbs to detect antigen-specific IgG subclasses in the serum of cattle sequentially vaccinated with heterologous foot-and-mouth disease virus capsid antigens revealed a low-level antigen-specific IgG3 response, in addition to IgG1 and IgG2 responses. Assessment of the total IgG1, IgG2 and IgG3 levels in healthy cattle plasma samples showed that IgG3 was measurable at a mean concentration of 0.19 mg/mL, although this was significantly lower than those of IgG1 (mean 3.28 mg/mL) and IgG2 (mean 3.41 mg/mL). Thus, Ankyron™ AZS40101 is a new reagent that provides utility for measurement of bovine IgG3 responses to infection and vaccination., Competing Interests: Declaration of Competing Interest None, (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Assessing the pharmacokinetics of monoclonal antibodies (mAbs) in relevant animal models is essential for designing improved formulations and developing mAb delivery platforms. We have established the pig, a large natural host animal for influenza with many similarities to humans, as a robust model for testing the therapeutic efficacy of anti-influenza mAbs and evaluating mAb delivery platforms. Here, we compared the pharmacokinetic characteristics of two anti-influenza hemagglutinin mAbs, human 2-12C and porcine pb27, in Göttingen minipigs and Landrace × Large White outbred pigs. Minipigs offer the advantage of a more stable weight, whereas outbred pigs are more readily available but exhibit rapid growth. Outbred pigs and minipigs showed similar pharmacokinetics and a similar porcine pb27 half-life (half-life of 15.7 days for outbred pigs and 16.6 days for minipigs). In contrast, the half-life of human 2-12C was more rapid in two of the minipigs but not in the outbred pigs, correlating with the development of antidrug antibodies in the two minipigs. Our results demonstrate that both outbred pigs and minipigs are appropriate models for pharmacokinetic studies and the evaluation of mAb delivery platforms, potentially bridging the gap between small animals and human trials., Competing Interests: MD was employed by the company Certara. VN and MM were employed by the company Milad Pharmaceutical Consulting LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Paudyal, Moorhouse, Sharma, Dodds, Nguyen, Milad and Tchilian.)

Current influenza vaccines are strain-specific and require frequent updates to combat new strains, making a broadly protective influenza vaccine (BPIV) highly desirable. A promising strategy is to induce T-cell responses against internal proteins conserved across influenza strains. In this study, pH1N1 pre-exposed pigs were immunized by aerosol using viral vectored vaccines (ChAdOx2 and MVA) expressing matrix (M1) and nucleoprotein (NP). Following H3N2 challenge, all immunizations (M1, NP or NPM1) reduced lung pathology, but M1 alone offered the greatest protection. NP or NPM1 immunization induced both T-cell and antibody responses. M1 immunization generated no detectable antibodies but elicited M1-specific T-cell responses, suggesting T cell-mediated protection. Additionally, a single aerosol immunization with the ChAdOx vaccine encoding M1, NP and neuraminidase reduced lung pathology. These findings provide insights into BPIV development using a relevant large natural host, the pig., (© 2024. The Author(s).)

The pig is a natural host for influenza viruses and integrally involved in virus evolution through interspecies transmissions between humans and swine. Swine have many physiological, anatomical, and immunological similarities to humans, and are an excellent model for human influenza. Here, we employed single cell RNA-sequencing (scRNA-seq) and flow cytometry to characterize the major leukocyte subsets in bronchoalveolar lavage (BAL), twenty-one days after H1N1pdm09 infection or respiratory immunization with an adenoviral vector vaccine expressing hemagglutinin and nucleoprotein with or without IL-1β. Mapping scRNA-seq clusters from BAL onto those previously described in peripheral blood facilitated annotation and highlighted differences between tissue resident and circulating immune cells. ScRNA-seq data and functional assays revealed lasting impacts of immune challenge on BAL populations. First, mucosal administration of IL-1β reduced the number of functionally active Treg cells. Second, influenza infection upregulated IFI6 in BAL cells and decreased their susceptibility to virus replication in vitro. Our data provide a reference map of porcine BAL cells and reveal lasting immunological consequences of influenza infection and respiratory immunization in a highly relevant large animal model for respiratory virus infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Muir et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells that recognize small molecule metabolites presented by major histocompatibility complex class I related protein 1 (MR1), via an αβ T cell receptor (TCR). MAIT TCRs feature an essentially invariant TCR α-chain, which is highly conserved between mammals. Similarly, MR1 is the most highly conserved major histocompatibility complex-I-like molecule. This extreme conservation, including the mode of interaction between the MAIT TCR and MR1, has been shown to allow for species-mismatched reactivities unique in T cell biology, thereby allowing the use of selected species-mismatched MR1-antigen (MR1-Ag) tetramers in comparative immunology studies. However, the pattern of cross-reactivity of species-mismatched MR1-Ag tetramers in identifying MAIT cells in diverse species has not been formally assessed. We developed novel cattle and pig MR1-Ag tetramers and utilized these alongside previously developed human, mouse, and pig-tailed macaque MR1-Ag tetramers to characterize cross-species tetramer reactivities. MR1-Ag tetramers from each species identified T cell populations in distantly related species with specificity that was comparable to species-matched MR1-Ag tetramers. However, there were subtle differences in staining characteristics with practical implications for the accurate identification of MAIT cells. Pig MR1 is sufficiently conserved across species that pig MR1-Ag tetramers identified MAIT cells from the other species. However, MAIT cells in pigs were at the limits of phenotypic detection. In the absence of sheep MR1-Ag tetramers, a MAIT cell population in sheep blood was identified phenotypically, utilizing species-mismatched MR1-Ag tetramers. Collectively, our results validate the use and define the limitations of species-mismatched MR1-Ag tetramers in comparative immunology studies., Competing Interests: Conflict of interest J. Y. W. M., L. L., D. P. F., A. J. C., J. M., and S. B. G. E. are inventors on university owned patent rights (patent families WO/2015/149130 and WO/2014/005194) licensed for commercial use to Immudex and for non-profit use to the NIH Tetramer Core Facility. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cattle possess three IgG subclasses. However, the key immune functions, including complement and NK cell activation, and enhancement of phagocytosis, are not fully described for bovine IgG1, 2 and 3. We produced chimeric monoclonal antibodies (mAbs) consisting of a defined variable region linked to the constant regions of bovine IgG1, 2 and 3, and expressed His-tagged soluble recombinant bovine Fc gamma receptors (FcγRs) IA (CD64), IIA (CD32A), III (CD16) and Fcγ2R. Functional assays using bovinized mAbs were developed. IgG1 and IgG3, but not IgG2, activated complement-dependent cytotoxicity. Only IgG1 could activate cattle NK cells to mobilize CD107a after antigen crosslinking, a surrogate assay for antibody-dependent cell cytotoxicity. Both IgG1 and IgG2 could trigger monocyte-derived macrophages to phagocytose fluorescently labelled antigen-expressing target cells. IgG3 induced only weak antibody-dependent cellular phagocytosis (ADCP). By contrast, monocytes only exhibited strong ADCP when triggered by IgG2. IgG1 bound most strongly to recombinant FcγRs IA, IIA and III, with weaker binding by IgG3 and none by IgG2, which bound exclusively to Fcγ2R. Immune complexes containing IgG1, 2 and 3 bound differentially to leukocyte subsets, with IgG2 binding strongly to neutrophils and monocytes and all subclasses binding platelets. Differential expression of the FcγRs on leukocyte subsets was demonstrated by surface staining and/or RT-qPCR of sorted cells, e.g., Fcγ2R mRNA was expressed in monocytes/macrophages, neutrophils, and platelets, potentially explaining their strong interactions with IgG2, and FcγRIII was expressed on NK cells, presumably mediating IgG1-dependent NK cell activation. These data reveal differences in bovine IgG subclass functionality, which do not correspond to those described in humans, mice or pigs, which is relevant to the study of these IgG subclasses in vaccine and therapeutic antibody development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Noble, Paudyal, Schwartz, Mwangi, Munir, Tchilian, Hammond and Graham.)

Monoclonal antibodies (mAbs) can be used to complement immunization for the therapy of influenza virus infection. We have established the pig, a natural large animal host for influenza A, with many physiological, immunological, and anatomical similarities to humans, as an appropriate model for testing mAbs. We have evaluated the protective efficacy of the strongly neutralizing human anti-hemagglutinin mAb, 2-12C in the pig influenza model. Intravenous administration of recombinant 2-12C reduced virus load and lung pathology, however, it did not prevent virus nasal shedding and, consequently, transmission. This may be because the pigs were directly infected intranasally with a high dose of the H1N1pdm09 virus. To address this, we developed a contact challenge model in which the animals were given 2-12C and one day later co-housed with donor pigs previously infected intra-nasally with H1N1pdm09. 2-12C pre-treatment completely prevented infection. We also administered a lower dose of 2-12C by aerosol to the respiratory tract, but this did not prevent shedding in the direct challenge model, although it abolished lung infection. We propose that the direct contact challenge model of pig influenza may be useful for evaluating candidate mAbs and emerging delivery platforms prior to clinical trials., Competing Interests: Author RM is an employee of the company Aerogen Limited. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 McNee, Vanover, Rijal, Paudyal, Lean, MacLoughlin, Núñez, Townsend, Santangelo and Tchilian.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Chrun, Maze, Roper, Vatzia, Paudyal, McNee, Martini, Manjegowda, Freimanis, Silesian, Polo, Clark, Besell, Booth, Carr, Edmans, Nunez, Koonpaew, Wanasen, Graham and Tchilian.)

T cell responses directed against highly conserved viral proteins contribute to the clearance of the influenza virus and confer broadly cross-reactive and protective immune responses against a range of influenza viruses in mice and ferrets. We examined the protective efficacy of mucosal delivery of adenoviral vectors expressing hemagglutinin (HA) and nucleoprotein (NP) from the H1N1 virus against heterologous H3N2 challenge in pigs. We also evaluated the effect of mucosal co-delivery of IL-1β, which significantly increased antibody and T cell responses in inbred Babraham pigs. Another group of outbred pigs was first exposed to pH1N1 as an alternative means of inducing heterosubtypic immunity and were subsequently challenged with H3N2. Although both prior infection and adenoviral vector immunization induced strong T-cell responses against the conserved NP protein, none of the treatment groups demonstrated increased protection against the heterologous H3N2 challenge. Ad-HA/NP+Ad-IL-1β immunization increased lung pathology, although viral load was unchanged. These data indicate that heterotypic immunity may be difficult to achieve in pigs and the immunological mechanisms may differ from those in small animal models. Caution should be applied in extrapolating from a single model to humans., Competing Interests: CT and SiS are employees of SIRION Biotech GmbH, a wholly-owned subsidiary of PerkinElmer Inc.. CT receives shares from the company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Schmidt, Paudyal, Villanueva-Hernández, Mcnee, Vatzia, Carr, Schmidt, Mccarron, Martini, Schroedel, Thirion, Waters, Salguero, Gerner, Tenbusch and Tchilian.)

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., (© 2023. The Author(s).)

The pig is an important agricultural species and powerful biomedical model. We have established the pig, a large natural host animal for influenza with many physiological similarities to humans, as a robust model for testing the therapeutic potential of monoclonal antibodies. Antibodies provide protection through neutralization and recruitment of innate effector functions through the Fc domain. However very little is known about the Fc-mediated functions of porcine IgG subclasses. We have generated 8 subclasses of two porcine monoclonal anti influenza hemagglutinin antibodies. We characterized their ability to activate complement, trigger cytotoxicity and phagocytosis by immune cells and assayed their binding to monocytes, macrophages, and natural killer cells. We show that IgG1, IgG2a, IgG2b, IgG2c and IgG4 bind well to targeted cell types and mediate complement mediated cellular cytotoxicity (CDCC), antibody dependent cellular cytotoxicity (ADCC) and antibody mediated cell phagocytosis (ADCP). IgG5b and IgG5c exhibited weak binding and variable and poor functional activity. Immune complexes of porcine IgG3 did not show any Fc-mediated functions except for binding to monocytes and macrophages and weak binding to NK cells. Interestingly, functionally similar porcine IgG subclasses clustered together in the genome. These novel findings will enhance the utility of the pig model for investigation of therapeutic antibodies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Paudyal, Mwangi, Rijal, Schwartz, Noble, Shaw, Sealy, Bonnet-Di Placido, Graham, Townsend, Hammond and Tchilian.)

In the light of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, we have developed a porcine respiratory coronavirus (PRCV) model for in depth mechanistic evaluation of the pathogenesis, virology and immune responses of this important family of viruses. Pigs are a large animal with similar physiology and immunology to humans and are a natural host for PRCV. Four PRCV strains were investigated and shown to induce different degrees of lung pathology. Importantly, although all four strains replicated equally well in porcine cell lines in vitro and in the upper respiratory tract in vivo , PRCV strains causing more severe lung pathology were also able to replicate in ex vivo tracheal organ cultures as well as in vivo in the trachea and lung. The time course of infection of PRCV 135, which caused the most severe pulmonary pathology, was investigated. Virus was shed from the upper respiratory tract until day 10 post infection, with infection of the respiratory mucosa, as well as olfactory and sustentacular cells, providing an excellent model to study upper respiratory tract disease in addition to the commonly known lower respiratory tract disease from PRCV. Infected animals made antibody and T cell responses that cross reacted with the four PRCV strains and Transmissible Gastroenteritis Virus. The antibody response was reproduced in vitro in organ cultures. Comparison of mechanisms of infection and immune control in pigs infected with PRCVs of differing pathogenicity with human data from SARS-CoV-2 infection and from our in vitro organ cultures, will enable key events in coronavirus infection and disease pathogenesis to be identified., Competing Interests: RM is an employee of company Aerogen. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Keep, Carr, Lean, Fones, Newman, Dowgier, Freimanis, Vatzia, Polo, Everest, Webb, Mcnee, Paudyal, Thakur, Nunez, MacLoughlin, Maier, Hammond, Bailey, Waters, Charleston, Tuthill, Britton, Bickerton and Tchilian.)

For the first time we have defined naïve, central memory, effector memory and differentiated effector porcine CD8 T cells and analyzed their distribution in lymphoid and respiratory tissues after influenza infection or immunization, using peptide-MHC tetramers of three influenza nucleoprotein (NP) epitopes. The hierarchy of response to the three epitopes changes during the response in different tissues. Most NP-specific CD8 T cells in broncho-alveolar lavage (BAL) and lung are tissue resident memory cells (TRM) that express CD69 and downregulate CD45RA and CCR7. NP-specific cells isolated from BAL express genes characteristic of TRM, but gene expression differs at 7, 21 and 63 days post infection. In all tissues the frequency of NP-specific CD8 cells declines over 63 days almost to background levels but is best maintained in BAL. The kinetic of influenza specific memory CD8 T cell in this natural host species differs from that in small animal models., (© 2022. The Author(s).)

We have established the pig, a large natural host animal for influenza, with many physiological similarities to humans, as a robust model for testing the therapeutic potential of monoclonal antibodies (mAbs). In this study we demonstrated that prophylactic intravenous administration of 15 mg/kg of porcine mAb pb18, against the K160-163 site of the hemagglutinin, significantly reduced lung pathology and nasal virus shedding and eliminated virus from the lung of pigs following H1N1pdm09 challenge. When given at 1 mg/kg, pb18 significantly reduced lung pathology and lung and BAL virus loads, but not nasal shedding. Similarly, when pb18 was given in combination with pb27, which recognized the K130 site, at 1 mg/kg each, lung virus load and pathology were reduced, although without an apparent additive or synergistic effect. No evidence for mAb driven virus evolution was detected. These data indicate that intravenous administration of high doses was required to reduce nasal virus shedding, although this was inconsistent and seldom complete. In contrast, the effect on lung pathology and lung virus load is consistent and is also seen at a one log lower dose, strongly indicating that a lower dose might be sufficient to reduce severity of disease, but for prevention of transmission other measures would be needed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Paudyal, McNee, Rijal, Carr, Nunez, McCauley, Daniels, Townsend, Hammond and Tchilian.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chrun, Maze, Vatzia, Martini, Paudyal, Edmans, McNee, Manjegowda, Salguero, Wanasen, Koonpaew, Graham and Tchilian.)

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., Competing Interests: SG is co-founder of Vaccitech and named as an inventor on a patent covering use of ChAdOx2-vectored vaccines. TL is a consultant to Vaccitech. RM is an employee of Aerogen Limited and is a named inventor on inhaled vaccine delivery system patents. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Vatzia, Allen, Manjegowda, Morris, McNee, Martini, Kaliath, Ulaszewska, Boyd, Paudyal, Carr, Chrun, Maze, MacLoughlin, van Diemen, Everett, Lambe, Gilbert and Tchilian.)

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.

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., Competing Interests: SE, JM, LL, and DF are inventors on patents describing MR1 tetramers and MR1–ligand complexes. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Edmans, Connelley, Jayaraman, Vrettou, Vordermeier, Mak, Liu, Fairlie, Maze, Chrun, Klenerman, Eckle, Tchilian and Benedictus.)

Pigs are natural hosts for the same subtypes of influenza A viruses as humans and integrally involved in virus evolution with frequent interspecies transmissions in both directions. The emergence of the 2009 pandemic H1N1 virus illustrates the importance of pigs in evolution of zoonotic strains. Here we generated pig influenza-specific monoclonal antibodies (mAbs) from H1N1pdm09 infected pigs. The mAbs recognized the same two major immunodominant haemagglutinin (HA) epitopes targeted by humans, one of which is not recognized by post-infection ferret antisera that are commonly used to monitor virus evolution. Neutralizing activity of the pig mAbs was comparable to that of potent human anti-HA mAbs. Further, prophylactic administration of a selected porcine mAb to pigs abolished lung viral load and greatly reduced lung pathology but did not eliminate nasal shedding of virus after H1N1pdm09 challenge. Hence mAbs from pigs, which target HA can significantly reduce disease severity. These results, together with the comparable sizes of pigs and humans, indicate that the pig is a valuable model for understanding how best to apply mAbs as therapy in humans and for monitoring antigenic drift of influenza viruses in humans, thereby providing information highly relevant to making influenza vaccine recommendations., Competing Interests: The authors have declared that no competing interests exist.

We have used the pig, a large natural host animal for influenza with many physiological similarities to humans, to characterize αβ, γδ T cell and antibody (Ab) immune responses to the 2009 pandemic H1N1 virus infection. We evaluated the kinetic of virus infection and associated response in inbred Babraham pigs with identical MHC (Swine Leucocyte Antigen) and compared them to commercial outbred animals. High level of nasal virus shedding continued up to days 4 to 5 post infection followed by a steep decline and clearance of virus by day 9. Adaptive T cell and Ab responses were detectable from days 5 to 6 post infection reaching a peak at 9 to 14 days. γδ T cells produced cytokines ex vivo at day 2 post infection, while virus reactive IFNγ producing γδ T cells were detected from day 7 post infection. Analysis of NP tetramer specific and virus specific CD8 and CD4 T cells in blood, lung, lung draining lymph nodes, and broncho-alveolar lavage (BAL) showed clear differences in cytokine production between these tissues. BAL contained the most highly activated CD8, CD4, and γδ T cells producing large amounts of cytokines, which likely contribute to elimination of virus. The weak response in blood did not reflect the powerful local lung immune responses. The immune response in the Babraham pig following H1N1pdm09 influenza infection was comparable to that of outbred animals. The ability to utilize these two swine models together will provide unparalleled power to analyze immune responses to influenza., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Edmans, McNee, Porter, Vatzia, Paudyal, Martini, Gubbins, Francis, Harley, Thomas, Burt, Morgan, Fuller, Sewell, Charleston, Bailey and Tchilian.)

A vaccine providing both powerful Ab and cross-reactive T cell immune responses against influenza viruses would be beneficial for both humans and pigs. In this study, we evaluated i.m., aerosol (Aer), and simultaneous systemic and respiratory immunization (SIM) by both routes in Babraham pigs, using the single cycle candidate influenza vaccine S-FLU. After prime and boost immunization, pigs were challenged with H1N1pdm09 virus. i.m.-immunized pigs generated a high titer of neutralizing Abs but poor T cell responses, whereas Aer induced powerful respiratory tract T cell responses but a low titer of Abs. SIM pigs combined high Ab titers and strong local T cell responses. SIM showed the most complete suppression of virus shedding and the greatest improvement in pathology. We conclude that SIM regimes for immunization against respiratory pathogens warrant further study., (Copyright © 2021 The Authors.)