Live attenuated vaccines are generally highly efficacious and often superior to inactivated vaccines, yet the underlying mechanisms of this remain largely unclear. Here we identify recognition of microbial viability as a potent stimulus for follicular helper T cell (T cell) differentiation and vaccine responses. Antigen-presenting cells (APCs) distinguished viable bacteria from dead bacteria through Toll-like receptor 8 (TLR8)-dependent detection of bacterial RNA. In contrast to dead bacteria and other TLR ligands, live bacteria, bacterial RNA and synthetic TLR8 agonists induced a specific cytokine profile in human and porcine APCs, thereby promoting T cell differentiation. In domestic pigs, immunization with a live bacterial vaccine induced robust T cell and antibody responses, but immunization with its heat-killed counterpart did not. Finally, a hypermorphic TLR8 polymorphism was associated with protective immunity elicited by vaccination with bacillus Calmette-Guérin (BCG) in a human cohort. We have thus identified TLR8 as an important driver of T cell differentiation and a promising target for T cell-skewing vaccine adjuvants.

Endemically infected European wild boar are considered a major reservoir of African swine fever virus in Europe. While high lethality was observed in the majority of field cases, strains of moderate virulence occurred in the Baltic States. One of these, “Estonia 2014”, led to a higher number of clinically healthy, antibody-positive animals in the hunting bag of North-Eastern Estonia. Experimental characterization showed high virulence in wild boar but moderate virulence in domestic pigs. Putative pathogenic differences between wild boar and domestic pigs are unresolved and comparative pathological studies are limited. We here report on a kinetic experiment in both subspecies. Three animals each were euthanized at 4, 7, and 10 days post infection (dpi). Clinical data confirmed higher virulence in wild boar although macroscopy and viral genome load in blood and tissues were comparable in both subspecies. The percentage of viral antigen positive myeloid cells tested by flow cytometry did not differ significantly in most tissues. Only immunohistochemistry revealed consistently higher viral antigen loads in wild boar tissues in particular 7 dpi, whereas domestic pigs already eliminated the virus. The moderate virulence in domestic pigs could be explained by a more effective viral clearance.

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Since its onset in the Caucasus and Russia in 2007, ASF has spread widely and now affects several European countries. In autumn 2018, the virus also reached China, the world's largest pig producer, and spread subsequently to several Asian countries (Dixon, Sun, & Roberts, 2019). Within Europe, the abundant wild boar population plays an important role in the maintenance of ASFV. Wild boars serve as a reservoir for ASFV and present a risk factor for the

Pigs are important livestock and comprehensive understanding of their immune responses in infections is critical to improve vaccines and therapies. Moreover, similarities between human and swine physiology suggest that pigs are a superior animal model for immunological studies. However, paucity of experimental tools for a systematic analysis of the immune responses in pigs represent a major disadvantage. To evaluate the pig as a biomedical model and additionally expand the knowledge of rare immune cell populations in swine, we established a multicolor flow cytometry analysis platform of surface marker expression and cellular responses for porcine invariant Natural Killer T cells (iNKT). In humans, iNKT cells are among the first line defenders in various tissues, respond to CD1d-restricted antigens and become rapidly activated. Naïve porcine iNKT cells were CD3 + /CD4 − /CD8 + or CD3 + /CD4 − /CD8 − and displayed an effector- or memory-like phenotype (CD25 + /ICOS + /CD5 hi /CD45RA − /CCR7 ± /CD27 + ). Based on their expression of the transcription factors T bet and the iNKT cell-specific promyelocytic leukemia zinc finger protein (PLZF), porcine iNKT cells were differentiated into functional subsets. Analogous to human iNKT cells, in vitro stimulation of porcine leukocytes with the CD1d ligand α-galactosylceramide resulted in rapid iNKT cell proliferation, evidenced by an increase in frequency and Ki-67 expression. Moreover, this approach revealed CD25, CD5, ICOS, and the major histocompatibility complex class II (MHC II) as activation markers on porcine iNKT cells. Activated iNKT cells also expressed interferon-γ, upregulated perforin expression, and displayed degranulation. In steady state, iNKT cell frequency was highest in newborn piglets and decreased with age. Upon infection with two viruses of high relevance to swine and humans, iNKT cells expanded. Animals infected with African swine fever virus displayed an increase of iNKT cell frequency in peripheral blood, regional lymph nodes, and lungs. During Influenza A virus infection, iNKT cell percentage increased in blood, lung lymph nodes, and broncho-alveolar lavage. Our in-depth characterization of porcine iNKT cells contributes to a better understanding of porcine immune responses, thereby facilitating the design of innovative interventions against infectious diseases. Moreover, we provide new evidence that endorses the suitability of the pig as a biomedical model for iNKT cell research.

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Infection with African swine fever virus (ASFV) causes a highly lethal haemorrhagic disease in domestic and Eurasian wild pigs. Thus, it is a major threat to pig populations worldwide and a cause of substantial economic losses. Recently, less virulent ASFV strains emerged naturally, which showed higher experimental virulence in wild boar than in domestic pigs. The reason for this difference in disease progression and outcome is unclear but likely involves different immunological responses. Unfortunately, besides the importance of CD8α+ lymphocytes, little is known about the immune responses against ASFV in suids. Against this background, we used a multicolour flow cytometry platform to investigate the T‐cell responses in wild boar and domestic pigs after infection with the moderately virulent ASFV strain ‘Estonia2014’ in two independent trials. CD4−/CD8α+ and CD4+/CD8α+ αβ T‐cell frequencies increased in both subspecies in various tissues, but CD8α+ γδ T cells differentiated and responded in wild boar only. Proliferation in CD8α+ T cells was found 10 days post infectionem only. Frequencies of T‐bet+ T cells increased in wild boar but not in domestic pigs. Of note, we found a considerable loss of perforin expression in cytotoxic T cells, 5 and 7 dpi. Both subspecies established a regulatory T‐cell response 10 dpi. In domestic pigs, we show increasing levels of ICOS+ and CD8α+ invariant Natural Killer T cells. These disparities in T‐cell responses might explain some of the differences in disease progression in wild boar and domestic pigs and should pave the way for future studies.

Pigs are anatomically, genetically and physiologically comparable to humans and represent a natural host for influenza A virus (IAV) infections. Thus, pigs may represent a relevant biomedical model for human IAV infections. We set out to investigate the systemic as well as the local immune response in pigs upon two subsequent intranasal infections with IAV H1N1pdm09. We detected decreasing numbers of peripheral blood lymphocytes after the first infection. The simultaneous increase in the frequencies of proliferating cells correlated with an increase in infiltrating leukocytes in the lung. Enhanced perforin expression in αβ and γδ T cells in the respiratory tract indicated a cytotoxic T cell response restricted to the route of virus entry such as the nose, the lung and the bronchoalveolar lavage. Simultaneously, increasing frequencies of CD8αα expressing αβ T cells were observed rapidly after the first infection, which may have inhibited uncontrolled inflammation in the respiratory tract. Taking together, the results of this study demonstrate that experimental IAV infection in pigs mimics major characteristics of human seasonal IAV infections.