Simultaneous co-infection with swine influenza A and porcine reproductive and respiratory syndrome viruses potentiates adaptive immune responses.

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 CD8ß+ T-cell responses in blood were enhanced. Higher proportions of polyfunctional CD8ß+ 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.

Effect of mucosal adjuvant IL-1ß on heterotypic immunity in a pig influenza model.

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-1ß, 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-1ß 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.

Influence of deoxynivalenol-contaminated feed on the immune response of pigs after PRRSV vaccination and infection.

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.

Immunization with matrix-, nucleoprotein and neuraminidase protects against H3N2 influenza challenge in pH1N1 pre-exposed pigs.

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.

Phenotypic Characterization of a Virulent PRRSV-1 Isolate in a Reproductive Model With and Without Prior Heterologous Modified Live PRRSV-1 Vaccination.

Reproductive disorders induced by porcine reproductive and respiratory syndrome virus (PRRSV) cause high economic losses in the pig industry worldwide. In this study, we aimed to phenotypically characterize a virulent PRRSV-1 subtype 1 isolate (AUT15-33) in a reproductive model. Furthermore, the protective effect of a heterologous modified live virus vaccine (ReproCyc® PRRS EU) was evaluated. In addition, PRRSV AUT15-33 was genotypically compared to other well-characterized isolates. Sixteen gilts were equally divided into four groups: a vaccinated and infected group (V-I), a vaccinated and non-infected group (V-NI), a non-vaccinated and infected group (NV-I), and a non-vaccinated and non-infected (NV-NI) group. After PRRSV infection on gestation day 84, all gilts were clinically examined on a daily basis, and blood samples were taken at five timepoints. Necropsy was performed 3 weeks after infection. The fetal preservation status was assessed, and PRRSV RNA concentrations were measured in the blood and tissue samples from all gilts and fetuses. After infection, all four gilts in the NV-I group were viremic throughout 17 days post-infection (dpi), whereas two gilts in the V-I group were viremic at only one timepoint at 6 dpi. The viral load was significantly higher in gilt serum, tracheobronchial lymph nodes, uterine lymph nodes, maternal endometrium, and fetal placenta of NV-I gilts compared to the V-I ones (p < 0.05). Moreover, the preservation status of the fetuses derived from NV-I gilts was significantly impaired (55.9% of viable fetuses) compared to the other groups (p < 0.001). Upon comparison with other known isolates, the phylogenetic analyses revealed the closest relation to a well-characterized PRRSV-1 subtype 1 field isolate from Belgium. In conclusion, the high virulence of AUT15-33 was phenotypically confirmed in an experimental reproductive model. The vaccination of the gilts showed promising results in reducing viremia, fetal damage, and transplacental transmission of the PRRSV-1 strain characterized in this study.

Porcine Respiratory Coronavirus as a Model for Acute Respiratory Coronavirus Disease.

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.

Simultaneous Infection With Porcine Reproductive and Respiratory Syndrome and Influenza Viruses Abrogates Clinical Protection Induced by Live Attenuated Porcine Reproductive and Respiratory Syndrome Vaccination.

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.

Respiratory and Intramuscular Immunization With ChAdOx2-NPM1-NA Induces Distinct Immune Responses in H1N1pdm09 Pre-Exposed Pigs.

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.

T-Cell Cytokine Response in Salmonella Typhimurium-Vaccinated versus Infected Pigs.

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.

Deoxynivalenol Has the Capacity to Increase Transcription Factor Expression and Cytokine Production in Porcine T Cells.

Deoxynivalenol (DON) is a Fusarium mycotoxin that frequently contaminates the feed of farm animals. Pigs with their monogastric digestive system are in particular sensitive to DON-contaminated feed. At high concentrations, DON causes acute toxic effects, whereas lower concentrations lead to more subtle changes in the metabolism. This applies in particular to the immune system, for which immunosuppressive but also immunostimulatory phenomena have been described. Research in human and rodent cell lines indicates that this may be partially explained by a binding of DON to the ribosome and subsequent influences on cell signaling molecules like mitogen-activated protein kinases. However, a detailed understanding of the influence of DON on functional traits of porcine immune cells is still lacking. In this study, we investigated the influence of DON on transcription factor expression and cytokine production within CD4+, CD8+, and γδ T cells in vitro. At a DON concentration, that already negatively affects proliferation after Concanavalin A stimulation (0.8 µM) an increase of T-bet expression in CD4+ and CD8+ T cells was observed. This increase in T-bet expression coincided with elevated levels of IFN-γ and TNF-α producing T-cell populations. Increases in T-bet expression and cytokine production were found in proliferating and non-proliferating T cells, although increases were more prominent in proliferating cell subsets. Differently, IL-17A production by CD4+ T cells was not influenced by DON. In addition, frequencies of regulatory T cells and their expression of Foxp3 were not affected. In γδ T cells, GATA-3 expression was slightly reduced by DON, whereas T-bet levels were only slightly modulated and hence IFN-γ, TNF-α, or IL-17A production were not affected. Our results show for the single-cell level that DON has the capacity to modulate the expression of transcription factors and related cytokines. In particular, they suggest that for CD4+ and CD8+ T cells, DON can drive T-cell differentiation into a pro-inflammatory type-1 direction, probably depending on the already prevailing cytokine milieu. This could have beneficial or detrimental effects in ongoing immune responses to infection or vaccination.

Magnitude and Kinetics of T Cell and Antibody Responses During H1N1pdm09 Infection in Inbred Babraham Pigs and Outbred Pigs.

We have used the pig, a large natural host animal for influenza with many physiological similarities to humans, to characterize αß, 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.

Vaccination and Infection of Swine With Salmonella Typhimurium Induces a Systemic and Local Multifunctional CD4+ T-Cell Response.

The gram-negative facultative intracellular bacteria Salmonella Typhimurium (STM) often leads to subclinical infections in pigs, but can also cause severe enterocolitis in this species. Due to its high zoonotic potential, the pathogen is likewise dangerous for humans. Vaccination with a live attenuated STM strain (Salmoporc) is regarded as an effective method to control STM infections in affected pig herds. However, information on the cellular immune response of swine against STM is still scarce. In this study, we investigated the T-cell immune response in pigs that were vaccinated twice with Salmoporc followed by a challenge infection with a virulent STM strain. Blood- and organ-derived lymphocytes (spleen, tonsils, jejunal and ileocolic lymph nodes, jejunum, ileum) were stimulated in vitro with heat-inactivated STM. Subsequently, CD4+ T cells present in these cell preparations were analyzed for the production of IFN-γ, TNF-α, and IL-17A by flow cytometry and Boolean gating. Highest frequencies of STM-specific cytokine-producing CD4+ T cells were found in lamina propria lymphocytes of jejunum and ileum. Significant differences of the relative abundance of cytokine-producing phenotypes between control group and vaccinated + infected animals were detected in most organs, but dominated in gut and lymph node-residing CD4+ T cells. IL-17A producing CD4+ T cells dominated in gut and gut-draining lymph nodes, whereas IFN-γ/TNF-α co-producing CD4+ T cells were present in all locations. Additionally, the majority of cytokine-producing CD4+ T cells had a CD8α+CD27- phenotype, indicative of a late effector or effector memory stage of differentiation. In summary, we show that Salmonella-specific multifunctional CD4+ T cells exist in vaccinated and infected pigs, dominate in the gut and most likely contribute to protective immunity against STM in the pig.

Cytokine production and phenotype of Histomonas meleagridis-specific T cells in the chicken.

The protozoan parasite Histomonas meleagridis is the causative agent of the re-emerging disease histomonosis of chickens and turkeys. Due to the parasite's extracellular occurrence, a type-2 differentiation of H. meleagridis-specific T cells has been hypothesized. In contrast, a recent study suggested that IFN-γ mRNA+ cells are involved in protection against histomonosis. However, the phenotype and cytokine production profile of H. meleagridis-specific T cells still awaits elucidation. In this work, clonal cultures of a virulent monoxenic strain of H. meleagridis were used for infecting chickens to detect IFN-γ protein and IL-13 mRNA by intracellular cytokine staining and PrimeFlow™ RNA Assays, respectively, in CD4+ and CD8ß+ T cells. Infection was confirmed by characteristic pathological changes in the cecum corresponding with H. meleagridis detection by immunohistochemistry and H. meleagridis-specific antibodies in serum. In splenocytes stimulated either with H. meleagridis antigen or PMA/ionomycin, IFN-γ-producing CD4+ T cells from infected chickens increased in comparison to cells from non-infected birds 2 weeks and 5 weeks post-infection. Additionally, an increase of IFN-γ-producing CD4-CD8ß- cells upon H. meleagridis antigen and PMA/ionomycin stimulation was detected. Contrariwise, frequencies of IL-13 mRNA-expressing cells were low even after PMA/ionomycin stimulation and mainly had a CD4-CD8ß- phenotype. No clear increase of IL-13+ cells related to H. meleagridis infection could be found. In summary, these data suggest that H. meleagridis infection induces a type-1 differentiation of CD4+ T cells but also of non-CD4+ cells. This phenotype could include γδ T cells, which will be addressed in future studies.

Deoxynivalenol Affects Proliferation and Expression of Activation-Related Molecules in Major Porcine T-Cell Subsets.

The Fusarium mycotoxin deoxynivalenol (DON) contaminates animal feed worldwide. In vivo, DON modifies the cellular protein synthesis, thereby also affecting the immune system. However, the functional consequences of this are still ill-defined. In this study, peripheral blood mononuclear cells from healthy pigs were incubated with different DON concentrations in the presence of Concanavalin A (ConA), a plant-derived polyclonal T-cell stimulant. T-cell subsets were investigated for proliferation and expression of CD8α, CD27, and CD28, which are involved in activation and costimulation of porcine T cells. A clear decrease in proliferation of all ConA-stimulated major T-cell subsets (CD4+, CD8+, and γδ T cells) was observed in DON concentrations higher than 0.4 µM. This applied in particular to naïve CD4+ and CD8+ T cells. From 0.8 µM onwards, DON induced a reduction of CD8α (CD4+) and CD27 expression (CD4+ and CD8+ T cells). CD28 expression was diminished in CD4+ and CD8+ T cells at a concentration of 1.6 µM DON. None of these effects were observed with the DON-derivative deepoxy-deoxynivalenol (DOM-1) at 16 µM. These results indicate that DON reduces T-cell proliferation and the expression of molecules involved in T-cell activation, providing a molecular basis for some of the described immunosuppressive effects of DON.

Bovine Peripheral Blood Mononuclear Cells Are More Sensitive to Deoxynivalenol Than Those Derived from Poultry and Swine.

Deoxynivalenol (DON) is one of the most prevalent mycotoxins, contaminating cereals and cereal-derived products. Its derivative deepoxy-deoxynivalenol (DOM-1) is produced by certain bacteria, which either occur naturally or are supplemented in feed additive. DON-induced impairments in protein synthesis are particularly problematic for highly proliferating immune cells. This study provides the first comparison of the effects of DON and DOM-1 on the concanavalin A-induced proliferation of porcine, chicken, and bovine peripheral blood mononuclear cells (PBMCs). Therefore, isolated PBMCs were treated with DON (0.01-3.37 µM) and DOM-1 (1.39-357 µM) separately, and proliferation was measured using a bromodeoxyuridine (BrdU) assay. Although pigs are considered highly sensitive to DON, the present study revealed a substantially higher sensitivity of bovine (IC50 = 0.314 µM) PBMCs compared to chicken (IC50 = 0.691 µM) and porcine (IC50 = 0.693 µM) PBMCs. Analyses on the proliferation of bovine T-cell subsets showed that all major subsets, namely, CD4⁺, CD8ß⁺, and γδ T cells, were affected to a similar extent. In contrast, DOM-1 did not affect bovine PBMCs, but reduced the proliferation of chicken and porcine PBMCs at the highest tested concentration (357 µM). Results confirm the necessity of feed additives containing DON-to-DOM-1-transforming bacteria and highlights species-specific differences in the DON sensitivity of immune cells.