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Infections of neonatal piglets with Cystoisospora suis are responsible for substantial economic losses in pig production. To investigate kinetics of T-cell populations, which are possibly involved in this infection, lymphocytes from blood, spleen, mesenteric lymph nodes and the jejunal mucosa of infected and noninfected piglets were investigated by flow cytometry and immunohistochemistry at five time points during the acute phase of primary infection. Additionally, m RNA expression levels of pattern recognition receptors and immunomodulatory cytokines in the jejunum were investigated. T-cell receptor-γδ+ T cells were found to be increased in the gut mucosa 4 days after infection and were most likely involved in the primary local immune response. Five to eleven days later, cytotoxic T cells peaked in this location, which was preceded by an expansion of this lymphocyte population in the mesenteric lymph nodes. In intestines of infected piglets, m RNA expressions of TLR-2, NOD2 and TNF-α were significantly upregulated, suggesting an involvement in parasite recognition, immune response and possibly also in immunopathology. Taken together, this study identifies cellular and molecular players involved in the early immune responses against C. suis, but their precise role in the pathogenesis and control of this neonatal disease requires further investigation. [ABSTRACT FROM AUTHOR]

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.)

Activation-induced markers (AIMs) are frequently analyzed to identify re-activated human memory T cells. However, in pigs the analysis of AIMs is still not very common. Based on available antibodies, we designed a multi-color flow cytometry panel comprising pig-specific or cross-reactive antibodies against CD25, CD69, CD40L (CD154), and ICOS (CD278) combined with lineage/surface markers against CD3, CD4, and CD8α. In addition, we included an antibody against tumor necrosis factor alpha (TNF-α), to study the correlation of AIM expression with the production of this abundant T cell cytokine. The panel was tested on peripheral blood mononuclear cells (PBMCs) stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin, Staphylococcus enterotoxin B (SEB) or PBMCs from African swine fever virus (ASFV) convalescent pigs, restimulated with homologous virus. PMA/ionomycin resulted in a massive increase of CD25/CD69 co-expressing T cells of which only a subset produced TNF-α, whereas CD40L expression was largely associated with TNF-α production. SEB stimulation triggered substantially less AIM expression than PMA/ionomycin but also here CD25/CD69 expressing T cells were identified which did not produce TNF-α. In addition, CD40L-single positive and CD25 + CD69 + CD40L + TNF-α - T cells were identified. In ASFV restimulated T cells TNF-α production was associated with a substantial proportion of AIM expressing T cells but also here ASFV-reactive CD25 + CD69 + TNF-α - T cells were identified. Within CD8α + CD4 T cells, several CD25/CD40L/CD69/ICOS defined phenotypes expanded significantly after ASFV restimulation. Hence, the combination of AIMs tested will allow the identification of primed T cells beyond the commonly used cytokine panels, improving capabilities to identify the full breadth of antigen-specific T cells in pigs., 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 © 2024 Moorton, Tng, Inoue, Netherton, Gerner and Schmidt.)

Introduction: The mycotoxins deoxynivalenol (DON) and zearalenone (ZEN), produced by Fusarium fungi, are frequently found in the cereal-rich diet of pigs and can modulate the immune system. Some enzymes or bacteria present in the digestive tract can de-epoxydize DON to deepoxy-deoxynivalenol (DOM-1) and biotransform ZEN into hydrolyzed ZEN (HZEN). The effects of these metabolites on immune cells, particularly with respect to the vaccine responses, are poorly documented. The aim of this study was to address the impact of DON and ZEN and their respective derivatives, on proliferation, and antibody production of porcine B cells in vitro ., Methods: Peripheral blood mononuclear cells (PBMCs), isolated from healthy pigs, were stimulated with the Toll-like receptor (TLR) 7/8-agonist Resiquimod (R848) or the TLR/1/2-agonist Pam3Cys-SKKKK in combination with DON [0.1-1.6 µM] or DOM-1 [1.6 µM and 16 µM] and ZEN [2.5-40 µM] or HZEN [40 µM]., Results: A strong decrease in B-cell proliferation was observed at DON concentrations equal to or exceeding 0.8 µM and at ZEN concentrations equal to or exceeding 20 µM. Treatment with 1.6 µM DON or 40 µM ZEN led to almost a complete loss of live CD79α + B cells. Moreover, CD21 expression of proliferating IgG + and IgM + B-cell subsets was decreased at DON concentrations equal to and exceeding 0.4 µM and at ZEN concentrations equal to or exceeding 10 µM. ELISpot assays revealed a decrease of IgG-secreting B cells at concentrations of and exceeding 0.4 µM and at ZEN concentrations equal to and exceeding 10 µM. ELISA assays showed a decrease of IgM, IgG, and IgA secretion at concentrations equal to or exceeding 0.4 µM DON. ZEN reduced IgM secretion at 20-40 µM (both R848 and Pam3Cys-SKKKK), IgG secretion at 40 µM (both R848 and Pam3Cys-SKKKK) and IgA secretion at 20-40 µM., Discussion: Our in vitro experiments show that while DON and ZEN impair immunoglobulin production and B-cell proliferation, this effect is abrogated by HZEN and DOM-1., Competing Interests: EM and AK are employed by dsm-firmenich, which produces animal feed additives. This, however, did not influence the design of the experimental studies or bias the presentation and interpretation of results. 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 Pierron, Kleber, Mayer and Gerner.)

Background: Serological tests are widely used in various medical disciplines for diagnostic and monitoring purposes. Unfortunately, the sensitivity and specificity of test systems are often poor, leaving room for false-positive and false-negative results. However, conventional methods were used to increase specificity and decrease sensitivity and vice versa. Using SARS-CoV-2 serology as an example, we propose here a novel testing strategy: the 'sensitivity improved two-test' or 'SIT²' algorithm., Methods: SIT² involves confirmatory retesting of samples with results falling in a predefined retesting zone of an initial screening test, with adjusted cut-offs to increase sensitivity. We verified and compared the performance of SIT² to single tests and orthogonal testing (OTA) in an Austrian cohort (1117 negative, 64 post-COVID-positive samples) and validated the algorithm in an independent British cohort (976 negatives and 536 positives)., Results: The specificity of SIT² was superior to single tests and non-inferior to OTA. The sensitivity was maintained or even improved using SIT² when compared with single tests or OTA. SIT² allowed correct identification of infected individuals even when a live virus neutralisation assay could not detect antibodies. Compared with single testing or OTA, SIT² significantly reduced total test errors to 0.46% (0.24-0.65) or 1.60% (0.94-2.38) at both 5% or 20% seroprevalence., Conclusion: For SARS-CoV-2 serology, SIT² proved to be the best diagnostic choice at both 5% and 20% seroprevalence in all tested scenarios. It is an easy to apply algorithm and can potentially be helpful for the serology of other infectious diseases., Competing Interests: Competing interests: NP-N received a travel grant from DiaSorin. DWE reports lecture fees from Gilead outside the submitted work. OCB reports grants from GSK, grants from Menarini, grants from Boehringer Ingelheim, grants from Astra, grants from MSD, grants from Pfizer, and grants from Chiesi, outside the submitted work. SH does receive unrestricted research grants (GSK, Boehringer, Menarini, Chiesi, Astra Zeneca, MSD, Novartis, Air Liquide, Vivisol, Pfizer, TEVA) for the Ludwig Boltzmann Institute of COPD and Respiratory Epidemiology, and is on advisory boards for G. SK, Boehringer Ingelheim, Novartis, Menarini, Chiesi, Astra Zeneca, MSD, Roche, Abbvie, Takeda and TEVA for respiratory oncology and COPD. PQ is an advisory board member for Roche Austria and reports personal fees from Takeda outside the submitted work. The Dept. of Laboratory Medicine (Head: OWF) received compensations for advertisement on scientific symposia from Roche, DiaSorin, and Abbott and holds a grant for evaluating an in-vitro diagnostic device from Roche. CJB is a Board Member of Technoclone. HH receives compensations for biobank services from Glock Health Science and Research and BlueSky immunotherapies., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

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.)

This multiplex staining panel was developed to differentiate cattle T cells into conventional (CD4 and CD8) and unconventional (γδ-TCR) subsets as well as their stage of differentiation and activation. The combination of CD45RO and CD62L allows the identification of naïve (T Naïve ), central memory (T CM ), effector memory (T EM ) and terminal effector (T TE ) T cells. Activated cattle T cells (T AV ) can be identified by the cell surface expression of CD25. This panel was developed using cryopreserved cattle peripheral blood mononuclear cells (PBMCs) and tested on fresh as well as stimulated PBMCs. Therefore, this 8-color, 10-parameter flow cytometry panel simultaneously identifies cattle T Naïve , T AV , T CM , T EM , T TE and γδ-TCR cells. This panel will improve our ability to examine T-cell response to pathogens and vaccines in cattle including the potential to identify previously undescribed subpopulations. Furthermore, this panel can be readily optimized for other bovid species as many of these reagents are likely to cross react., (© 2023 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.)

The impact of the Fusarium mycotoxin deoxynivalenol (DON) on the immune response against porcine reproductive and respiratory syndrome virus (PRRSV) vaccination and infection was investigated. Forty-two weaned piglets were separated into seven groups and received three different diets: Low DON (1.09 ppm), High DON (2.81 ppm) or No DON. These three treatments were split further into either vaccinated (Ingelvac PRRSFLEX EU) and challenged with PRRSV 28 days post-vaccination, or only infected at day 28. A seventh group received no DON, no vaccination, and no infection. Two weeks after challenge infection, when pigs were euthanized, the number of IFN-γ producing lymphocytes in the blood of vaccinated animals was lower in pigs on High DON compared to animals on Low DON or No DON. Intracellular cytokine staining showed that vaccinated animals fed with the Low DON diet had higher frequencies of TNF-α/IFN-γ co-producing CD4 + T cells than the other two vaccinated groups, particularly in lung tissue. Vaccinated animals on High DON had similar viral loads in the lung as the non-vaccinated groups, but several animals of the Low DON or No DON group receiving vaccination had reduced titers. In these two groups, there was a negative correlation between lung virus titers and vaccine-specific TNF-α/IFN-γ co-producing CD4 + T cells located either in lung tissue or blood. These results indicate that after PRRSV vaccination and infection, high levels of DON negatively influence immune parameters and clearance of the virus, whereas low DON concentrations have immunomodulatory effects., (© 2023. The Author(s).)

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating viruses for the global swine industry. Infection during late gestation causes reproductive failure but the local immune response in utero remains poorly understood. In this study, an experimental PRRSV-infection model with two different PRRSV-1 field isolates was used to investigate the immune cell phenotypes at the maternal-fetal interface during late gestation. In addition, phenotypic changes induced by a modified live virus (MLV, ReproCyc ® PRRS EU) vaccine were studied. Vaccinated (n = 12) and non-vaccinated pregnant gilts (n = 12) were challenged with either one of the PRRSV-1 field isolates (low vs. high virulent, LV or HV) or sham-inoculated at day 84 of gestation. Twenty-one days post infection all gilts were euthanized and the fetal preservation status for all fetuses per litter was assessed. Leukocytes from the maternal-fetal interface were isolated and PRRSV-induced changes were investigated using ex vivo phenotyping by flow cytometry. PRRSV load in tissue from the maternal endometrium (ME) and fetal placenta (FP) was determined by RT-qPCR. In the ME, a vast increase in CD8β T cells with CD8α pos CD27 dim early effector phenotype was found for fetuses from the non-vaccinated LV and HV-challenged gilts, compared to non-treated and vaccinated-only controls. HV-challenged fetuses also showed significant increases of lymphocytes with effector phenotypes in the FP, including NKp46 pos NK cells, CD8α high γδ T cells, as well as CD8α pos CD27 pos/dim CD4 and CD8 T cells. In vaccinated animals, this common activation of effector phenotypes was more confined and the fetal preservation status significantly improved. Furthermore, a negative correlation between the viral load and CD163 high CD169 pos mononuclear phagocytic cells was observed in the FP of HV-infected animals. These results suggest that the strong expansion of effector lymphocytes in gilts that were only infected causes immune-pathogenesis rather than protection. In contrast, the attenuated MLV seems to dampen this effect, yet presumably induces memory cells that limit reproductive failure. This work provides valuable insights into changes of local immune cell phenotypes following PRRSV vaccination and infection., 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 Stas, Kreutzmann, Stadler, Sassu, Mair, Koch, Knecht, Stadler, Dolezal, Balka, Zaruba, Mötz, Saalmüller, Rümenapf, Gerner and Ladinig.)

Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae affects pig health status and the swine industry worldwide. Despite the extensive number of studies focused on A. pleuropneumoniae infection and vaccine development, a thorough analysis of the A. pleuropneumoniae exoproteome is still missing. Using a complementary approach of quantitative proteomics and immunoproteomics we gained an in-depth insight into the A. pleuropneumoniae serotype 2 exoproteome, which provides the basis for future functional studies. Label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed 593 exoproteins, of which 104 were predicted to be virulence factors. The RTX toxins ApxIIA and ApxIIIA -were found to be the most abundant proteins in the A. pleuropneumoniae serotype 2 exoproteome. Furthermore, the ApxIVA toxin was one of the proteins showing the highest abundance, although ApxIVA is commonly assumed to be expressed exclusively in vivo. Our study revealed several antigens, including proteins with moonlight functions, such as the elongation factor (EF)-Tu, and proteins linked to specific metabolic traits, such as the maltodextrin-binding protein MalE, that warrant future functional characterization and might present potential targets for novel therapeutics and vaccines. Our Ig-classes specific serological proteome analysis (SERPA) approach allowed us to explore the development of the host humoral immune response over the course of the infection. These SERPAs pinpointed proteins that might play a key role in virulence and persistence and showed that the immune response to the different Apx toxins is distinct. For instance, our results indicate that the ApxIIIA toxin has properties of a thymus-independent antigen, which should be studied in more detail., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Progress in the phenotypic characterisation of porcine B cells is ongoing, with recent advances in the identification of B1 cell subsets and plasma cells. However, regulatory B cells, commonly identified by interleukin (IL)-10 production, have not been studied in pigs so far. Here we investigate IL-10 expression in B cell subsets in response to CpG-oligodeoxynucleotides, phorbol 12-myristate 13-acetate and ionomycin stimulation in vitro. Our results reflect similar findings in human and mice. We identify a small subset of IL-10 competent B cells, present within both porcine B1 and B2 cell subsets across blood, spleen, mediastinal lymph nodes and lung tissue, with varied differentiation statuses. The capacity for IL-10 production coincided with CD95 expression, suggesting an activated phenotype of IL-10 competent B cells. These findings support the emerging paradigm that B cell IL-10 production is a function of various B cell subsets influenced by activation history and microenvironmental factors., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

T follicular helper (Tfh) cells provide help to germinal center B cells for affinity maturation, class switch and memory formation. Despite these important functions, this subset has not been studied in detail in pigs due to a lack of species-specific antibodies. We investigated putative Tfh cells from lymphoid tissues and blood of healthy pigs by using cross-reactive antibodies for inducible T-cell costimulator (ICOS) and B-cell lymphoma 6 (Bcl-6). In lymph nodes, we identified a CD4 + T cell population with an ICOS + Bcl-6 + CD8α + phenotype, reminiscent of human and murine germinal center Tfh cells. Within blood-derived CD4 + T cells, sorted ICOS hi CD25 - and ICOS dim CD25 dim cells were able to induce the differentiation of CD21 + IgM +  B cells into Ig-secreting plasmablasts. Compared to naïve CD4 + T cells, these two phenotypes were 3- to 7-fold enriched for cells expressing the Tfh-related transcripts CD28, CD40LG, IL6R and MAF, as identified by single-cell RNA sequencing. These results provide a first characterization of Tfh cells in swine and confirm their ability to provide B-cell help., (Copyright © 2022. Published by Elsevier Ltd.)

The receptor binding domain (RBD) of the SARS-CoV-2 spike (S)-protein is a prime target of virus-neutralizing antibodies present in convalescent sera of COVID-19 patients and thus is considered a key antigen for immunosurveillance studies and vaccine development. Although recombinant expression of RBD has been achieved in several eukaryotic systems, mammalian cells have proven particularly useful. The authors aimed to optimize RBD produced in HEK293-6E cells towards a stable homogeneous preparation and addressed its O-glycosylation as well as the unpaired cysteine residue 538 in the widely used RBD (319-541) sequence. The authors found that an intact O-glycosylation site at T323 is highly relevant for the expression and maintenance of RBD as a monomer. Furthermore, it was shown that deletion or substitution of the unpaired cysteine residue C538 reduces the intrinsic propensity of RBD to form oligomeric aggregates, concomitant with an increased yield of the monomeric form of the protein. Bead-based and enzyme-linked immunosorbent assays utilizing these optimized RBD variants displayed excellent performance with respect to the specific detection of even low levels of SARS-CoV-2 antibodies in convalescent sera. Hence, these RBD variants could be instrumental for the further development of serological SARS-CoV-2 tests and inform the design of RBD-based vaccine candidates., (© 2022 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

Antibody-secreting plasma cells (PCs) have remained largely uncharacterized for years in the field of porcine immunology. For an in-depth study of porcine PCs, we identified cross-reactive antibodies against three key transcription factors: PR domain zinc finger protein-1 (Blimp-1), interferon regulatory factor 4 (IRF4), and paired box 5 (Pax5). A distinct Blimp-1 + IRF4 + cell population was found in cells isolated from blood, spleen, lymph nodes, bone marrow, and lung of healthy pigs. These cells showed a downregulation of Pax5 compared to other B cells. Within Blimp-1 + IRF4 + B cells, IgM-, IgG-, and IgA-expressing cells were identified and immunoglobulin-class distribution was clearly different between the anatomical locations, with IgA + PCs dominating in lung tissue and IgM + PCs dominating in the spleen. Expression patterns of Ki-67, MHC-II, CD9, and CD28 were investigated in the different organs. A high expression of Ki-67 was observed in blood, suggesting a plasmablast stage. Blimp-1 + IRF4 + cells showed an overall lower expression of MHC-II compared to regular B cells, confirming a progressive loss in B-cell differentiation toward the PC stage. CD28 showed slightly elevated expression levels in Blimp-1 + IRF4 + cells in most organs, a phenotype that is also described for PCs in mice and humans. This was not seen for CD9. We further developed a FACS-sorting strategy for live porcine PCs for functional assays. CD3 - CD16 - CD172a - sorted cells with a CD49d high FSC-A high phenotype contained Blimp-1 + IRF4 + cells and were capable of spontaneous IgG production, thus confirming PC identity. These results reveal fundamental phenotypes of porcine PCs and will facilitate the study of this specific B-cell subset in the future., 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 Villanueva-Hernández, Adib Razavi, van Dongen, Stadler, de Luca, Beyersdorf, Saalmüller, Gerner and Mair.)

The activating receptor NKp46 shows a unique expression pattern on porcine leukocytes. We showed already that in swine not all NK cells express NKp46 and that CD3 + NKp46 + lymphocytes form a T-cell subset with unique functional properties. Here we demonstrate the expression of NKp46 on CD4 high CD14 - CD172a + porcine plasmacytoid dendritic cells (pDCs). Multicolor flow cytometry analyses revealed that the vast majority of porcine pDCs (94.2% ± 4) express NKp46 ex vivo and have an increased expression on the single-cell level compared to NK cells. FSC/SSC high CD4 high NKp46 + cells produced high levels of IFN-α after CpG ODN 2216 stimulation, a hallmark of pDC function. Following receptor triggering with plate-bound monoclonal antibodies against NKp46, phosphorylation of signaling molecules downstream of NKp46 was analyzed in pDCs and NK cells. Comparable to NK cells, NKp46 triggering led to an upregulation of the phosphorylated ribosomal protein S6 (pS6) in pDCs, indicating an active signaling pathway of NKp46 in porcine pDCs. Nevertheless, a defined effector function of the NK-associated receptor on porcine pDCs could not be demonstrated yet. NKp46-mediated cytotoxicity, as shown for NK cells, does not seem to occur, as NKp46 + pDCs did not express perforin. Yet, NKp46 triggering seems to contribute to cytokine production in porcine pDCs, as induction of TNF-α was observed in a small pDC subset after NKp46 cross-linking. To our knowledge, this is the first report on NKp46 expression on pDCs in a mammalian species, showing that this receptor contributes to pDC activation and function., 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 Mair, Stadler, Razavi, Saalmüller and Gerner.)

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).)

T cells are an essential component of the adaptive immune system. Over the last 15 years, a constantly growing toolbox with which to study T cell biology in pigs has allowed detailed investigations on these cells in various viral and bacterial infections. This review provides an overview on porcine CD4, CD8, and γδ T cells and the current knowledge on the differentiation of these cells following antigen encounter. Where available, the responses of these cells to viral infections like porcine reproductive and respiratory syndrome virus, classical swine fever virus, swine influenza A virus, and African swine fever virus are outlined. In addition, knowledge on the porcine T cell response to bacterial infections like Actinobacillus pleuropneumoniae and Salmonella Typhimurium is reviewed. For CD4 T cells, the response to the outlined infections is reflected toward the Th1/Th2/Th17/Tfh/Treg paradigm for functional differentiation.

Natural killer (NK) cells have been studied extensively in humans and mice for their vital role in the vertebrate innate immune system. They are known to rapidly eliminate tumors or virus infected cells in an immune response utilizing their lytic properties. The natural cytotoxicity receptors (NCRs) NKp30 (NCR3), NKp44 (NCR2), and NKp46 (NCR1) are important mediators of NK-cell cytotoxicity. NKp44 expression was reported for NK cells in humans as well as in some non-human primates and found exclusively on activated NK cells. Previously, no information was available on NKp44 protein expression and its role in porcine lymphocytes due to the lack of species-specific monoclonal antibodies (mAbs). For this study, porcine-specific anti-NKp44 mAbs were generated and their reactivity was tested on blood and tissue derived NK cells in pigs of different age classes. Interestingly, NKp44 expression was detected ex vivo already on resting NK cells; moreover, the frequency of NKp44 + NK cells was higher than that of NKp46 + NK cells in most animals analyzed. Upon in vitro stimulation with IL-2 or IL-15, the frequency of NKp44 + NK cells, as well as the intensity of NKp44 expression at the single cell level, were increased. Since little is known about swine NK cells, the generation of a mAb (clone 54-1) against NKp44 will greatly aid in elucidating the mechanisms underlying the differentiation, functionality, and activation of porcine NK cells., 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 Mair, Crossman, Wagner, Babasyan, Noronha, Boyd, Zarlenga, Stadler, van Dongen, Gerner, Saalmüller and Lunney.)

Because of its size, anatomical similarities, and now also accessibility to genetic manipulations, pigs are used as animal models for human diseases and immune system development. However, expression and function of CD28, the most important costimulatory receptor expressed by T cells, so far is poorly understood in this species. Using a newly generated mAb (mAb 3D11) with specificity for pig CD28, we detected CD28 on CD8 + and CD4 + αβ T cells. Among γδ T cells, CD28 expression was restricted to a small CD2 + subpopulation of phenotypically naive cells. Functionally, CD28 ligation with mAb 3D11-costimulated porcine T cells, enhanced proliferation and cytokine secretion in vitro. We used a second, likewise newly generated but superagonistic, anti-CD28 mAb (CD28-SA; mAb 4D12) to test the function of CD28 on porcine T cells in a pilot study in vivo. Injection of the CD28-SA into pigs in vivo showed a very similar dose-response relationship as in humans (i.e., 100 µg/kg body weight [BW]) of CD28-SA induced a cytokine release syndrome that was avoided at a dose of 10 µg/kg BW and below. The data further suggest that low-dose (10 µg/kg BW) CD28-SA infusion was sufficient to increase the proportion of Foxp3 + regulatory T cells among CD4 + T cells in vivo. The pig is thus a suitable animal model for testing novel immunotherapeutics. Moreover, data from our pilot study in pigs further suggest that low-dose CD28-SA infusion might allow for selective expansion of CD4 + regulatory T cells in humans., (Copyright © 2021 by The American Association of Immunologists, Inc.)

PRRSV is one of the most important viruses in the global swine industry and is often controlled by the use of modified live virus (MLV) vaccines. This study assessed the impact of a PRRSV-1 MLV vaccine applied to 1-day-old piglets challenged on day 28 of life with a PRRSV-1 field isolate (AUT15-33). Twenty-one piglets were vaccinated within 24 h of birth (T02), whereas 20 piglets were left unvaccinated (T01). Necropsy was performed two weeks post-challenge. Comparing the two groups, T02 piglets showed significantly higher ( p = 0.017) average daily weight gain. In addition, significantly lower ( p < 0.0001) PRRSV RNA loads were measured in serum of T02 piglets at all investigated time points. All T01 piglets were viremic and shed virus in nasal swabs, whereas only 71.4% and 38.1% of the T02 group were viremic or shed virus, respectively. Piglets from T02 had significantly higher numbers ( p < 0.0001) of IFN-γ producing lymphocytes compared to T01. At necropsy, differences in gross and histologic lung lesions were statistically significant ( p = 0.012 and p < 0.0001, respectively) between the two groups. Hence, this MLV vaccine administered to 1-day-old piglets was able to protect piglets against PRRSV infection at weaning.

The receptor binding domain (RBD) of the SARS-CoV-2 spike protein plays a key role in the virus-host cell interaction, and viral infection. The RBD is a major target for neutralizing antibodies, whilst recombinant RBD is commonly used as an antigen in serological assays. Such assays are essential tools to gain control over the pandemic and detect the extent and durability of an immune response in infected or vaccinated populations. Transient expression in plants can contribute to the fast production of viral antigens, which are required by industry in high amounts. Whilst plant-produced RBDs are glycosylated, N -glycan modifications in plants differ from humans. This can give rise to the formation of carbohydrate epitopes that can be recognized by anti-carbohydrate antibodies present in human sera. For the performance of serological tests using plant-produced recombinant viral antigens, such cross-reactive carbohydrate determinants (CCDs) could result in false positives. Here, we transiently expressed an RBD variant in wild-type and glycoengineered Nicotiana benthamiana leaves and characterized the impact of different plant-specific N -glycans on RBD reactivity in serological assays. While the overall performance of the different RBD glycoforms was comparable to each other and to a human cell line produced RBD, there was a higher tendency toward false positive results with sera containing allergy-related CCD-antibodies when an RBD carrying β1,2-xylose and core α1,3-fucose was used. These rare events could be further minimized by pre-incubating sera from allergic individuals with a CCD-inhibitor. Thereby, false positive signals obtained from anti-CCD antibodies, could be reduced by 90%, on average., Competing Interests: JH, MH, KV, and AW were employed by AIT Austrian Institute of Technology GmbH. FA who developed the CCD inhibitor is in a commercial relationship with companies who sell the inhibitor. 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 Schwestka, König-Beihammer, Shin, Vavra, Kienzl, Grünwald-Gruber, Maresch, Klausberger, Laurent, Stadler, Manhart, Huber, Hofner, Vierlinger, Weinhäusel, Swoboda, Binder, Gerner, Grebien, Altmann, Mach, Stöger and Strasser.)

Chlamydia ( C. ) suis can often be isolated from conjunctival swab specimens from pigs with conjunctivitis or keratoconjunctivitis. In the field, it is assumed to be a multifactorial disease triggered by immunosuppressing factors. This is the first experimental study to provoke clinical signs of conjunctivitis in pigs after C. suis primary mono-infection. Five six-week-old male piglets, free of ocular chlamydia shedding and seronegative for Chlamydia, were conjunctivally infected with the C. suis -type strain S45 (1 × 10 9 inclusion forming units), while four piglets served as negative controls. The infection group developed clinical signs of conjunctivitis with a peak in the first week post-infection. Immunohistochemical evaluation revealed the presence of Chlamydia not only in the conjunctival epithelium, but also in the enlarged lacrimal glands, lungs, and intestine. No circulating antibodies could be detected during the whole study period of three weeks, although three different test systems were applied as follows: the complement fixation test, MOMP-based Chlamydiaceae ELISA, and PmpC-based C. suis ELISA. Meanwhile, high numbers of IFN-γ-producing lymphocytes within PBMC were seen after C. suis re-stimulation 14 days post-infection. Hence, these data suggest that entry via the eye may not elicit immunological responses comparable to other routes of chlamydial infections.

Vaccination with the live attenuated vaccine Salmoporc is an effective measure to control Salmonella Typhimurium (STM) in affected swine populations. However, the cellular immune response evoked by the Salmoporc vaccine including differences in vaccinated pigs versus non-vaccinated pigs upon STM infection have not been characterized yet. To investigate this, tissue-derived porcine lymphocytes from different treatment groups (vaccination-only, vaccination and infection, infection-only, untreated controls) were stimulated in vitro with heat-inactivated STM and abundances of IFN-γ, TNF-α and/or IL-17A-producing T-cell subsets were compared across organs and treatment groups. Overall, our results show the induction of a strong CD4 + T-cell response after STM infection, both locally and systemically. Low-level induction of STM-specific cytokine-producing CD4 + T cells, notably for the IFN-γ/TNF-α co-producing phenotype, was detected after vaccination-only. Numerous significant contrasts in cytokine-producing T-cell phenotypes were observed after infection in vaccinated and infected versus infected-only animals. These results suggest that vaccine-induced STM-specific cytokine-producing CD4 + T cells contribute to local immunity in the gut and may limit the spread of STM to lymph nodes and systemic organs. Hence, our study provides insights into the underlying immune mechanisms that account for the efficacy of the Salmoporc vaccine.

In this work, we report on two novel monoclonal antibodies, specific for porcine CD9. CD9 is a tetraspanin that is expressed on a wide variety of cells. We phenotyped porcine immune cell subsets and found that CD9 was expressed on all monocytes as well as a subset of B cells. CD9 was variably expressed on T cells, with CD4 T cells containing the highest frequency of CD9 + cells. CD9 expression positively correlated with the frequency of central memory CD4 T cells in ex vivo PBMC. Therefore, we proceeded to explore CD9 as a marker of T cell function. Here we observed that CD9 was expressed on the vast majority of long-lived influenza A virus-specific effector cells that retained the capacity for cytokine production in response to in vitro recall antigen. Therefore, the new antibodies enable the detection of a cell surface molecule with functional relevance to T cells. Considering the importance of CD9 in membrane remodelling across many cell types, they will also benefit the wider field of swine biomedical research., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Extracellular vesicles produced by different types of cells have recently attracted great attention, not only for their role in physiology and pathology, but also because of the emerging applications in gene therapy, vaccine production and diagnostics. Less well known than their eukaryotic counterpart, also bacteria produce extracellular vesicles, in the case of the Gram-negative E. coli the main species is termed outer membrane vesicles (OMVs). In this study, we show for the first time the functional surface modification of E. coli OMVs with glycosylphosphatidylinositol (GPI)-anchored protein, exploiting a process variably described as molecular painting or protein engineering in eukaryotic membranes, whereby the lipid part of the GPI anchor inserts in cell membranes. By transferring the process to bacterial vesicles, we can generate a hybrid of perfectly eukaryotic proteins (in terms of folding and post-translational modifications) on a prokaryotic platform. We could demonstrate that two different GPI proteins can be displayed on the same OMV. In addition to fluorescent marker proteins, cytokines, growth factors and antigens canb be potentially transferred, generating a versatile modular platform for a novel vaccine strategy.