Porcine circovirus type 2 (PCV2) is one of the economically most important pathogens for swine production worldwide. Vaccination is a powerful tool to control porcine circovirus diseases (PCVD). However, it is not fully understood how PCV2 vaccination interacts with the porcine immune system. Especially knowledge on the cellular immune response against PCV2 is sparse. In this study we analysed antigen-specific T cell responses against PCV2 in a controlled vaccination and infection experiment. We focused on the ability of CD4+ T cells to produce cytokines using multicolour flow cytometry (FCM). Vaccination with a PCV2 subunit vaccine (Ingelvac CircoFLEX®) induced PCV2-specific antibodies only in five out of 12 animals. Conversely, vaccine-antigen specific CD4+ T cells which simultaneously produced IFN-γ and TNF-α and had a phenotype of central and effector memory T cells were detected in all vaccinated piglets. After challenge, seroconversion occurred earlier in vaccinated and infected pigs compared to the non-vaccinated, infected group. Vaccinated pigs were fully protected against viremia after subsequent challenge. Therefore, our data suggests that the induction of IFN-γ/TNF-α co-producing T cells by PCV2 vaccination may serve as a potential correlate of protection for this type of vaccine.

Although swine are natural hosts for influenza A viruses, the porcine T-cell response to swine influenza A virus (FLUAVsw) infection has been poorly characterized so far. We have studied Ki-67 expression and FLUAVsw-specific production of IFN-γ, TNF-α and IL-2 in CD4+ and CD8β+ T cells isolated from piglets that had been intratracheally infected with a H1N2 FLUAVsw isolate. IFN-γ+TNF-α+IL-2+ multifunctional CD4+ T cells were present in the blood of all infected animals at one or two weeks after primary infection and their frequency increased in four out of six animals after homologous secondary infection. These cells produced higher amounts of IFN-γ, TNF-α and IL-2 than did CD4+ T cells that only produced a single cytokine. The vast majority of cytokine-producing CD4+ T cells expressed CD8α, a marker associated with activation and memory formation in porcine CD4+ T cells. Analysis of CD27 expression suggested that FLUAVsw-specific CD4+ T cells included both central memory and effector memory populations. Three out of six animals showed a strong increase of Ki-67+perforin+ CD8β+ T cells in blood one week post infection. Blood-derived FLUAVsw-specific CD8β+ T cells could be identified after an in vitro expansion phase and were multifunctional in terms of CD107a expression and co-production of IFN-γ and TNF-α. These data show that multifunctional T cells are generated in response to FLUAVsw infection of pigs, supporting the idea that T cells contribute to the efficient control of infection.

Porcine contagious pleuropneumonia caused by Actinobacillus pleuropneumoniae (APP) remains one of the major causes of poor growth performance and respiratory disease in pig herds. While the role of antibodies against APP has been intensely studied, the porcine T cell response remains poorly characterized. To address this, pigs were intranasally infected with APP serotype 2 and euthanized during the acute phase [6–10 days post-infection (dpi)] or the chronic phase of APP infection (27–31 dpi). Lymphocytes isolated from blood, tonsils, lung tissue and tracheobronchial lymph nodes were analyzed by intracellular cytokine staining (ICS) for IL-17A, IL-10 and TNF-α production after in vitro stimulation with crude capsular extract (CCE) of the APP inoculation strain. This was combined with cell surface staining for the expression of CD4, CD8α and TCR-γδ. Clinical records, microbiological investigations and pathological findings confirmed the induction of a subclinical APP infection. ICS-assays revealed the presence of APP-CCE specific CD4+CD8αdim IL-17A-producing T cells in blood and lung tissue in most infected animals during the acute and chronic phase of infection and a minor fraction of these cells co-produced TNF-α. APP-CCE specific IL-17A-producing γδ T cells could not be found and APP-CCE specific IL-10-producing CD4+ T cells were present in various organs but only in a few infected animals. The frequency of identified putative Th17 cells (CD4+CD8αdimIL-17A+) in lung and blood correlated positively with lung lesion scores and APP-specific antibody titers during the chronic phase. These results suggest a potential role of Th17 cells in the immune pathogenesis of APP infection.

The re-emerging disease histomonosis is caused by the protozoan parasite Histomonas meleagridis that affects chickens and turkeys. Previously, protection by vaccination with in vitro attenuated H. meleagridis has been demonstrated and an involvement of T cells, potentially by IFN-γ production, was hypothesized. However, comparative studies between chickens and turkeys on H. meleagridis-specific T cells were not conducted yet. This work investigated IFN-γ production within CD4+, CD8α+ and TCRγδ+ (chicken) or CD3ε+CD4-CD8α- (turkey) T cells of spleen and liver from vaccinated and/or infected birds using clonal cultures of a monoxenic H. meleagridis strain. In infected chickens, re-stimulated splenocytes showed a significant increase of IFN-γ+CD4+ T cells. Contrariwise, significant increments of IFN-γ-producing cells within all major T-cell subsets of the spleen and liver were found for vaccinated/infected turkeys. This indicates that the vaccine in turkeys causes more intense systemic immune responses whereas in chickens protection might be mainly driven by local immunity.

The protozoan parasiteHistomonas meleagridisis the causative agent of the re-emerging disease histomonosis of chickens and turkeys. Due to the parasite’s extracellular occurrence, a type-2 differentiation ofH. 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 ofH. meleagridis-specific T cells still awaits elucidation. In this work, clonal cultures of a virulent monoxenic strain ofH. meleagridiswere 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 CD8β⁺T cells. Infection was confirmed by characteristic pathological changes in the cecum corresponding withH. meleagridisdetection by immunohistochemistry andH. meleagridis-specific antibodies in serum. In splenocytes stimulated either withH. meleagridisantigen 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⁻CD8β⁻cells uponH. meleagridisantigen 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⁻CD8β⁻phenotype. No clear increase of IL-13⁺cells related toH. meleagridisinfection could be found. In summary, these data suggest thatH. meleagridisinfection 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.

Chlamydia trachomatis (Ct) infections are the most common sexually transmitted infection (STI). Despite effective antibiotics for Ct, undetected infections or delayed treatment can lead to infertility, ectopic pregnancies, and chronic pelvic pain. Besides humans, chlamydia poses similar health challenges in animals such as C. suis (Cs) in pigs. Based on the similarities between humans and pigs, as well as their chlamydia species, we use pigs as a large biomedical animal model for chlamydia research. In this study, we used the pig model to develop a vaccine candidate against Ct. The vaccine candidate consists of TriAdj-adjuvanted chlamydial-protease-like activity factor (CPAF) protein. We tested two weekly administration options—twice intranasal (IN) followed by twice intramuscular (IM) and twice IM followed by twice IN. We assessed the humoral immune response in both serum using CPAF-specific IgG (including antibody avidity determination) and also in cervical and rectal swabs using CPAF-specific IgG and IgA ELISAs. The systemic T-cell response was analyzed following in vitro CPAF restimulation via IFN-γ and IL-17 ELISpots, as well as intracellular cytokine staining flow cytometry. Our data demonstrate that while the IN/IM vaccination mainly led to non-significant systemic immune responses, the vaccine candidate is highly immunogenic if administered IM/IN. This vaccination strategy induced high serum anti-CPAF IgG levels with strong avidity, as well as high IgA and IgG levels in vaginal and rectal swabs and in uterine horn flushes. In addition, this vaccination strategy prompted a pronounced cellular immune response. Besides inducing IL-17 production, the vaccine candidate induced a strong IFN-γ response with CD4 T cells. In IM/IN-vaccinated pigs, these cells also significantly downregulated their CCR7 expression, a sign of differentiation into peripheral-tissue-homing effector/memory cells. Conclusively, this study demonstrates the strong immunogenicity of the IM/IN-administered TriAdj-adjuvanted Ct CPAF vaccine candidate. Future studies will test the vaccine efficacy of this promising Ct vaccine candidate. In addition, this project demonstrates the suitability of the Cs pre-exposed outbred pig model for Ct vaccine development. Thereby, we aim to open the bottleneck of large animal models to facilitate the progression of Ct vaccine candidates into clinical trials.