Swine Leukocyte Antigen Class Research Articles

Porcine reproductive and respiratory syndrome virus nsp4-mediated β2M downregulation contributes to SLA-I decrease and virus infection in vivo and in vitro

Porcine reproductive and respiratory syndrome virus (PRRSV) infection inhibits swine leukocyte antigen class I (SLA-I) expression in pigs, resulting in inefficient antigen presentation and subsequent low levels of cellular PRRSV-specific immunity as well as persistent viremia. We previously observed that the non-structural protein 4 (nsp4) of PRRSV contributed to inhibition of the β2-microglobulin (β2M) and SLA-I expression in cells. Here, we constructed a series of nsp4 mutants with different combination of amino acid mutations to attenuate the inhibitory effect of nsp4 on β2M and SLA-I expression. Almost all nsp4 mutants exogenously expressed in cells showed an attenuated effect on inhibition of β2M and SLA-I expression, but the recombinant PRRSV harboring these nsp4 mutants failed to be rescued with exception of the rPRRSV-nsp4-mut10 harboring three amino acid mutations. However, infection of rPRRSV-nsp4-mut10 not only enhanced β2M and SLA-I expression in both cells and pigs but also promoted the DCs to active the CD3+CD8+T lymphocytes more efficiently, as compared with its parental PRRSV (rPRRVS-nsp4-wt). These data suggested that the inhibition of nsp4-mediated β2M downregulation improved β2M/SLA-I expression in pigs.

Rotavirus infection inhibits SLA-I expression on the cell surface by degrading β2 M via ERAD-proteasome pathway

Group A Rotavirus (RVA) is a major cause of diarrhea in infants and piglets. β2-microglobulin (β2 M), encoded by the B2M gene, serves as a crucial subunit of the major histocompatibility complex class I (MHC-I) molecules. β2 M is indispensable for the transport of MHC-I to the cell membrane. MHC-I, also known as swine leukocyte antigen class I (SLA-I) in pigs, presents viral antigens to the cell surface. In this study, RVA infection down-regulated β2 M expression in both porcine intestinal epithelial cells-J2 (IPEC-J2) and MA-104 cells. RVA infection did not down-regulate the mRNA level of the B2M gene, indicating that the down-regulation of β2 M occurred on the protein level. Mechanismly, RVA infection triggered β2 M aggregation in the endoplasmic reticulum (ER) and enhanced the Lys48 (K48)-linked ubiquitination of β2 M, leading to the degradation of β2 M through ERAD-proteasome pathway. Furthermore, we found that RVA infection significantly impeded the level of SLA-I on the surface, and the overexpression of β2 M could recover its expression. In this study, our study demonstrated that RVA infection degrades β2 M via ERAD-proteasome pathway, consequently hampering SLA-I expression on the cell surface. This study would enhance the understanding of the mechanism of how RVA infection induces immune escape.

Comparative analysis of swine leukocyte antigen gene diversity in Göttingen Minipigs.

Worldwide, pigs represent economically important farm animals, also representing a preferred preclinical large animal model for biomedical studies. The need for swine leukocyte antigen (SLA) typing is increasing with the expanded use of pigs in translational research, infection studies, and for veterinary vaccine design. Göttingen Minipigs (GMP) attract increasing attention as valuable model for pharmacological studies and transplantation research. This study represents a first-time assessment of the SLA gene diversity in Göttingen Minipigs in combination with a comparative metadata analysis with commercial pig lines. As Göttingen Minipigs could harbor private as well as potential novel SLA allele combinations, future research projects would benefit from the characterization of their SLA background. In 209 Göttingen Minipigs, SLA class I (SLA-1, SLA-2, SLA-3) and class II (DRB1, DQB1, DQA) genes were characterized by PCR-based low-resolution (Lr) haplotyping. Criteria and nomenclature used for SLA haplotyping were proposed by the ISAG/IUIS-VIC SLA Nomenclature Committee. Haplotypes were assigned based on the comparison with already known breed or farm-specific allele group combinations. In total, 14 SLA class I and five SLA class II haplotypes were identified in the studied cohort, to manifest in 26 SLA class I but only seven SLA class II genotypes. The most common SLA class I haplotypes Lr-24.0 (SLA-1*15XX or Blank-SLA-3*04:04-SLA-2*06:01~02) and Lr-GMP-3.0 (SLA-1*16:02-SLA-3*03:04-SLA-2*17:01) occurred at frequencies of 23.44 and 18.66%, respectively. For SLA class II, the most prevalent haplotypes Lr-0.21 (DRB1*01XX-DQB1*05XX-DQA*04XX) and Lr-0.03 (DRB1*03:02-DQB1*03:01-DQA*01XX) occurred at frequencies of 38.28 and 30.38%. The comparative metadata analysis revealed that Göttingen Minipigs only share six SLA class I and two SLA class II haplotypes with commercial pig lines. More importantly, despite the limited number of SLA class I haplotypes, the high genotype diversity being observed necessitates pre-experimental SLA background assessment of Göttingen Minipigs in regenerative medicine, allo-transplantation, and xenograft research.

T-independent B-cell effect of agents associated with swine grower-finisher diarrhea.

Swine dysentery, spirochetal colitis, and salmonellosis are production-limiting enteric diseases of global importance to the swine industry. Despite decades of efforts, mitigation of these diseases still relies on antibiotic therapy. A common knowledge gap among the 3 agents is the early B-cell response to infection in pigs. Thus, this study aimed to characterize the porcine B-cell response to Brachyspira hyodysenteriae, Brachyspira hampsonii (virulent and avirulent strains), Brachyspira pilosicoli, and Salmonella Typhimurium, the agents of the syndromes mentioned above. Immortalized porcine B-cell line derived from a crossbred pig with lymphoma were co-incubated for 8h with each pathogen, as well as E. coli lipopolysaccharide (LPS) and a sham-inoculum (n = 3/treatment). B-cell viability following treatments was evaluated using trypan blue, and the expression levels of B-cell activation-related genes was profiled using reverse transcription quantitative PCR. Only S. Typhimurium and LPS led to increased B-cell mortality. B. pilosicoli downregulated B-lymphocyte antigen (CD19), spleen associated tyrosine Kinase (syk), tyrosine-protein kinase (lyn), and Tumour Necrosis Factor alpha (TNF-α), and elicited no change in immunoglobulin-associated beta (CD79b) and swine leukocyte antigen class II (SLA-DRA) expression levels, when compared to the sham-inoculated group. In contrast, all other treatments significantly upregulated CD79b and stimulated responses in other B-cell downstream genes. These findings suggest that B. pilosicoli does not elicit an immediate T-independent B-cell response, nor does it trigger antigen-presenting mechanisms. All other agents activated at least one trigger within the T-independent pathways, as well as peptide antigen presenting mechanisms. Future research is warranted to verify these findings in vivo.

Downregulation of Swine Leukocyte Antigen Expression Decreases the Strength of Xenogeneic Immune Responses towards Renal Proximal Tubular Epithelial Cells.

Xenotransplantation reemerged as a promising alternative to conventional transplantation enlarging the available organ pool. However, success of xenotransplantation depends on the design and selection of specific genetic modifications and on the development of robust assays allowing for a precise assessment of tissue-specific immune responses. Nevertheless, cell-based assays are often compromised by low proliferative capacity of primary cells. Proximal tubular epithelial cells (PTECs) play a crucial role in kidney function. Here, we generated immortalized PTECs (imPTECs) by overexpression of simian virus 40 T large antigen. ImPTECs not only showed typical morphology and phenotype, but, in contrast to primary PTECs, they maintained steady cell cycling rates and functionality. Furthermore, swine leukocyte antigen (SLA) class I and class II transcript levels were reduced by up to 85% after transduction with lentiviral vectors encoding for short hairpin RNAs targeting β2-microglobulin and the class II transactivator. This contributed to reducing xenogeneic T-cell cytotoxicity (p < 0.01) and decreasing secretion of pro-inflammatory cytokines such as IL-6 and IFN-γ. This study showed the feasibility of generating highly proliferative PTECs and the development of tissue-specific immunomonitoring assays. Silencing SLA expression on PTECs was demonstrated to be an effective strategy to prevent xenogeneic cellular immune responses and may strongly support graft survival after xenotransplantation.

Co-expression of HLA-E and HLA-G on genetically modified porcine endothelial cells attenuates human NK cell-mediated degranulation.

Natural killer (NK) cells play an important role in immune rejection in solid organ transplantation. To mitigate human NK cell activation in xenotransplantation, introducing inhibitory ligands on xenografts via genetic engineering of pigs may protect the graft from human NK cell-mediated cytotoxicity and ultimately improve xenograft survival. In this study, non-classical HLA class I molecules HLA-E and HLA-G were introduced in an immortalized porcine liver endothelial cell line with disruption of five genes (GGTA1, CMAH, β4galNT2, SLA-I α chain, and β-2 microglobulin) encoding three major carbohydrate xenoantigens (αGal, Neu5Gc, and Sda) and swine leukocyte antigen class I (SLA-I) molecules. Expression of HLA-E and/or HLA-G on pig cells were confirmed by flow cytometry. Endogenous HLA-G molecules as well as exogenous HLA-G VL9 peptide could dramatically enhance HLA-E expression on transfected pig cells. We found that co-expression of HLA-E and HLA-G on porcine cells led to a significant reduction in human NK cell activation compared to the cells expressing HLA-E or HLA-G alone and the parental cell line. NK cell activation was assessed by analysis of CD107a expression in CD3-CD56+ population gated from human peripheral blood mononuclear cells. CD107a is a sensitive marker of NK cell activation and correlates with NK cell degranulation and cytotoxicity. HLA-E and/or HLA-G on pig cells did not show reactivity to human sera IgG and IgM antibodies. This in vitro study demonstrated that co-expression of HLA-E and HLA-G on genetically modified porcine endothelial cells provided a superior inhibition in human xenoreactive NK cells, which may guide further genetic engineering of pigs to prevent human NK cell mediated rejection.

Xenorecognition and costimulation of porcine endothelium-derived extracellular vesicles in initiating human porcine-specific T cell immune responses

Porcine vascular endothelial cells (PECs) form a mechanistic centerpiece of xenograft rejection. Here, we determined that resting PECs release swine leukocyte antigen class I (SLA-I) but not swine leukocyte antigen class-II DR (SLA-DR) expressing extracellular vesicles (EVs) and investigated whether these EVs proficiently initiate xenoreactive T cell responses via direct xenorecognition and costimulation. Human T cells acquired SLA-I+ EVs with or without direct contact to PECs, and these EVs colocalized with T cell receptors. Although interferon gamma-activated PECs released SLA-DR+ EVs, the binding of SLA-DR+ EVs to T cells was sparse. Human T cells demonstrated low levels of proliferation without direct contact to PECs, but marked T cell proliferation was induced following exposure to EVs. EV-induced proliferation proceeded independent of monocytes/macrophages, suggesting that EVs delivered both a T cell receptor signal and costimulation. Costimulation blockade targeting B7, CD40L, or CD11a significantly reduced T cell proliferation to PEC-derived EVs. These findings indicate that endothelial-derived EVs can directly initiate T cell-mediated immune responses, and suggest that inhibiting the release of SLA-I EVs from organ xenografts has the potential to modify the xenograft rejection. We propose a secondary-direct pathway for T cell activation via xenoantigen recognition/costimulation by endothelial-derived EVs.

The Deletion of US3 Gene of Pseudorabies Virus (PRV) ΔgE/TK Strain Induces Increased Immunogenicity in Mice.

Re-emerging pseudorabies (PR) caused by pseudorabies virus (PRV) variant has been prevailing among immunized herds in China since 2011, indicating that commercially available PR vaccine strains couldn’t provide complete protection against novel, epidemic PRV variant. Before this study, a gE/TK-gene-deleted virus (PRV ΔgE/TK) was constructed from PRV QYY2012 variant through homologous recombination and Cre/LoxP system. Here, PRV ΔgE/TK/US3 strain was generated by deleting US3 gene based on PRV ΔgE/TK strain using the same method. The growth characteristics of PRV ΔgE/TK/US3 were analogous to that of PRV ΔgE/TK. Moreover, the deletion of US3 gene could promote apoptosis, upregulate the level of swine leukocyte antigen class I molecule (SLA-I) in vitro, and relieve inflammatory response in inoculated BALB/c mice. Subsequently, the safety and immunogenicity of PRV ΔgE/TK/US3 was evaluated as a vaccine candidate in mice. The results revealed that PRV ΔgE/TK/US3 was safe for mice, and mice vaccinated with PRV ΔgE/TK/US3 could induce a higher level of PRV-specific neutralizing antibodies and cytokines, including IFN-γ, IL-2 and IL-4, also higher level of CD8+ CD69+ Tissue-Resident Memory T cells (TRM). The results show that the deletion of US3 gene of PRV ΔgE/TK strain could induce increased immunogenicity, indicating that the PRV ΔgE/TK/US3 strain is a promising vaccine candidate for preventing and controlling of the epidemic PR in China.

414.6: Porcine ULBP1 Disruption Does Not Significantly Reduce Human NK Cell Activation and Cytotoxicity in an in Vitro Model

Background: Pig-to-human xenotransplantation has the potential to address the critical organ shortage. Genetically-engineered pig organs have significantly prolonged survival in human and nonhuman primates by overcoming hyperacute rejection. Human natural killer (NK) cell-mediated acute xenograft rejection of porcine endothelial cells has been established. Porcine UL-Binding Protein (ULBP1) has been identified as a porcine ligand of human NK cell activating receptor NKG2D; interactions presumably lead to NK cell activation and ultimately porcine cell cytotoxicity. We sought to improve pig-to-human compatibility by eliminating ULBP1 ligand in an immortalized porcine liver-derived endothelial cell line (ipLDEC) with five-gene knockout (5GKO). The 5GKO cell line has mutations in GGTA1/CMAH/β4galNT2/SLA-I α-chain/β2M: three genes encoding enzymes responsible for xenoantigen production and two genes encoding swine leukocyte antigen class I. Methods: CRISPR/Cas9 technology was used to knockout the porcine ULBP1 gene in the 5GKO cell line to create a ULBP1-KO/5GKO cell line. Human peripheral blood mononuclear cells (PBMCs) from four donors were activated with human IL-2 for five days. Human PBMCs were co-cultured with 5GKO, ULBP1-KO/5GKO, and Wild-Type (WT) ipLDECs. Cells were stained with antibodies to CD45, CD3, CD56, and CD107a. Flow cytometry analysis was gated on the CD3-CD56+ NK cell population and monitored for expression of CD107a, a marker of NK cell degranulation. Calcein AM cytotoxicity assay was performed to determine human NK cell-mediated ipLDECs cell death. WT, 5GKO, ULBP1-KO/5GKO, HLA-G+/5GKO (positive control) were incubated in a calcein solution for 30 minutes at a concentration of 106 cells/mL. Stained porcine cells were co-cultured with activated human PBMCs at a 10:1 E:T ratio for 4 hours. Co-culture supernatant was harvested and calcein release was measured at an excitation wavelength of 485 nm and emission wavelength of 530 nm to calculate cytotoxicity percentages. Results: Porcine ULBP1 gene mutation was confirmed by Sanger sequencing (Figure 1a), establishing a porcine ULBP1-KO/5GKO cell line. There is no statistically significant difference in NK cell activation between co-cultures of human PBMCs (n=4) with pULBP1-KO/5GKO and 5GKO ipLDEC using CD107a. (Figure 1b). There is no statistically significant difference in NK-induced cytotoxicity between co-cultures of human PBMCs (n=6) with porcine ULBP1-KO/5GKO and 5GKO cell lines using Calcein AM assays (Figure 1c). As expected, HLA-G+/5GKO cytotoxicity was decreased (p = 0.0168) compared to WT (Figure 1c). Conclusion: We established a porcine ULBP1-KO/5GKO cell line. Our studies demonstrate no statistically significant difference in NK cell activation and NK cell-mediated cytotoxicity between 5GKO and ULBP1-KO/5GKO cell lines. Our studies show that ULBP1 is not a crucial ligand for human NKG2D. Future studies will target different human NK cell activating porcine ligands.

Human IL-17 and TNF-α Additively or Synergistically Regulate the Expression of Proinflammatory Genes, Coagulation-Related Genes, and Tight Junction Genes in Porcine Aortic Endothelial Cells.

Immune rejection is the major limitation for porcine xenograft survival in primate recipients. Proinflammatory cytokines play important roles in immune rejection and have been found to mediate the pathological effects in various clinical and experimental transplantation trials. IL-17 and TNF-α play critical pathological roles in immune disorders, such as psoriasis and rheumatoid arthritis. However, the pathological roles of human IL-17 (hIL-17) and human TNF-α (hTNF-α) in xenotransplantation remain unclear. Here we found that hIL-17 and hTNF-α additively or synergistically regulate the expression of 697 genes in porcine aortic endothelial cells (PAECs). Overall, 415 genes were found to be synergistically regulated, while 282 genes were found to be additively regulated. Among these, 315 genes were upregulated and 382 genes were downregulated in PAECs. Furthermore, we found that hIL-17 and hTNF-α additively or synergistically induced the expression of various proinflammatory cytokines and chemokines (e.g., IL1α, IL6, and CXCL8) and decreased the expression of certain anti-inflammatory genes (e.g., IL10). Moreover, hIL-17 plus hTNF-α increased the expression of IL1R1 and IL6ST, receptors for IL1 and IL6, respectively, and decreased anti-inflammatory gene receptor expression (IL10R). hIL-17 and hTNF-α synergistically or additively induced CXCL8 and CCL2 expression and consequently promoted primary human neutrophil and human leukemia monocytic cell migration, respectively. In addition, hIL-17 and hTNF-α induced pro-coagulation gene (SERPINB2 and F3) expression and decreased anti-coagulation gene (TFPI, THBS1, and THBD) expression. Additionally, hIL-17 and hTNF-α synergistically decreased occludin expression and consequently promoted human antibody-mediated complement-dependent cytotoxicity. Interestingly, hTNF-α increased swine leukocyte antigen (SLA) class I expression; however, hIL-17 decreased TNF-α-mediated SLA-I upregulation. We concluded that hIL-17 and hTNF-α likely promote the inflammatory response, coagulation cascade, and xenoantibody-mediated cell injury. Thus, blockade of hIL-17 and hTNF-α together might be beneficial for xenograft survival in recipients.

Impact of Swine Leukocyte Antigen on renal endothelial cells in pig-to-non-human primate xenotransplantation in vitro

Abstract BACKBROUND The development of a pig lacking the GGTA1, B4GNT2 and CMAH genes (TKO) that produce the three known glycan xenoantigens has provided the decreased background human antibody binding to TKO cells. It can be easily detected for antibody binding to sufficiently low levels of any additional xenoantigens expressed on the pig cells, including swine leukocyte antigen (SLA). The object of this study is to evaluate the role of SLA class I and II in the pig-to-non-human primate (NHP) xenotransplantation in vitro at an organ-specific cell level using renal endothelial cells (REC). METHODS Characteristics of genetically-edited REC and Interferon gamma (IFN-γ)-activated REC were analyzed using flow cytometry. Rhesus IgG/IgM binding to REC with or without SLA class I, or SLA class II expression were examined using flow cytometric crossmatch and complement-dependent cytotoxicity assay (CDC). RESULTS SLA I-deficient REC with GGTA1/B4GNT2/CMAH TKO background had significantly lower Levels of antibody binding (p=0.02 and p=0.0001, respectively for IgG and IgM) using 25% rhesus sera (n=14). SLA class II expression on SLA I-deficient GGTA1/B4GNT2 REC was induced following 3-day treatment of IFN-γ. These cells with SLA class II expression had significantly increased IgM binding by crossmatch (p=0.002), and had significantly high levels of cell lysis by CDC assay (p&amp;lt;0.0001) using 25% rhesus sera (n=11). CONCLUSIONS Gene editing of the SLA class I and II on pig organs might protect against chronic antibody-mediated rejection via reducing antibody binding to REC in pig-to-NHP renal xenotransplantation. Porcine REC cell lines will be a useful reagent for the further study of xenoimmunology. Supported by Partial 5R01AI126322

Production of Triple-Gene (GGTA1, B2M and CIITA)-Modified Donor Pigs for Xenotransplantation.

Activation of human immune T-cells by swine leukocyte antigens class I (SLA-I) and class II (SLA-II) leads to xenograft destruction. Here, we generated the GGTA1, B2M, and CIITA (GBC) triple-gene-modified Diannan miniature pigs, analyzed the transcriptome of GBC-modified peripheral blood mononuclear cells (PBMCs) in the pig's spleen, and investigated their effectiveness in anti-immunological rejection. A total of six cloned piglets were successfully generated using somatic cell nuclear transfer, one of them carrying the heterozygous mutations in triple genes and the other five piglets carrying the homozygous mutations in GGTA1 and CIITA genes, but have the heterozygous mutation in the B2M gene. The autopsy of GBC-modified pigs revealed that a lot of spot bleeding in the kidney, severe suppuration and necrosis in the lungs, enlarged peripulmonary lymph nodes, and adhesion between the lungs and chest wall were found. Phenotyping data showed that the mRNA expressions of triple genes and protein expressions of B2M and CIITA genes were still detectable and comparable with wild-type (WT) pigs in multiple tissues, but α1,3-galactosyltransferase was eliminated, SLA-I was significantly decreased, and four subtypes of SLA-II were absent in GBC-modified pigs. In addition, even in swine umbilical vein endothelial cells (SUVEC) induced by recombinant porcine interferon gamma (IFN-γ), the expression of SLA-I in GBC-modified pig was lower than that in WT pigs. Similarly, the expression of SLA-II DR and DQ also cannot be induced by recombinant porcine IFN-γ. Through RNA sequencing (RNA-seq), 150 differentially expressed genes were identified in the PBMCs of the pig's spleen, and most of them were involved in immune- and infection-relevant pathways that include antigen processing and presentation and viral myocarditis, resulting in the pigs with GBC modification being susceptible to pathogenic microorganism. Furthermore, the numbers of human IgM binding to the fibroblast cells of GBC-modified pigs were obviously reduced. The GBC-modified porcine PBMCs triggered the weaker proliferation of human PBMCs than WT PBMCs. These findings indicated that the absence of the expression of α1,3-galactosyltransferase and SLA-II and the downregulation of SLA-I enhanced the ability of immunological tolerance in pig-to-human xenotransplantation.

Mapping the Key Residues within the Porcine Reproductive and Respiratory Syndrome Virus nsp1α Replicase Protein Required for Degradation of Swine Leukocyte Antigen Class I Molecules.

The nonstructural protein 1α (nsp1α) of the porcine reproductive and respiratory syndrome virus (PRRSV) has been shown to target swine leukocyte antigen class I (SLA-I) for degradation, but the molecular details remain unclear. In this report, we further mapped the critical residues within nsp1α by site-directed mutagenesis. We identified a cluster of residues (i.e., Phe17, Ile81, Phe82, Arg86, Thr88, Gly90, Asn91, Phe94, Arg97, Thr160, and Asn161) necessary for this function. Interestingly, they are all located in a structurally relatively concentrated region. Further analysis by reverse genetics led to the generation of two viable viral mutants, namely, nsp1α-G90A and nsp1α-T160A. Compared to WT, nsp1α-G90A failed to co-localize with either chain of SLA-I within infected cells, whereas nsp1α-T160A exhibited a partial co-localization relationship. Consequently, the mutant nsp1α-G90A exhibited an impaired ability to downregulate SLA-I in infected macrophages as demonstrated by Western blot, indirect immunofluorescence, and flow cytometry analysis. Consistently, the ubiquitination level of SLA-I was significantly reduced in the conditions of both infection and transfection. Together, our results provide further insights into the mechanism underlying PRRSV subversion of host immunity and have important implications in vaccine development.

Comparative Analysis of SLA-1, SLA-2, and DQB1 Genetic Diversity in Locally-Adapted Kenyan Pigs and Their Wild Relatives, Warthogs.

Swine leukocyte antigen (SLA) plays a central role in controlling the immune response by discriminating self and foreign antigens and initiating an immune response. Studies on SLA polymorphism have demonstrated associations between SLA allelic variants, immune response, and disease resistance. The SLA polymorphism is due to host-pathogen co-evolution resulting in improved adaptation to diverse environments making SLA a crucial genomic region for comparative diversity studies. Although locally-adapted African pigs have small body sizes, they possess increased resilience under harsh environmental conditions and robust immune systems with reported tolerance to some diseases, including African swine fever. However, data on the SLA diversity in these pigs are not available. We characterized the SLA of unrelated locally-adapted domestic pigs from Homa Bay, Kenya, alongside exotic pigs and warthogs. We undertook SLA comparative diversity of the functionally expressed SLA class I (SLA-1, SLA-2) and II (DQB1) repertoires in these three suids using the reverse transcription polymerase chain reaction (RT-PCR) sequence-based typing (SBT) method. Our data revealed higher genetic diversity in the locally-adapted pigs and warthogs compared to the exotic pigs. The nucleotide substitution rates were higher in the peptide-binding regions of the SLA-1, SLA-2, and DQB1 loci, indicative of adaptive evolution. We obtained high allele frequencies in the three SLA loci, including some breed-specific private alleles, which could guide breeders to increase their frequency through selection if confirmed to be associated with enhanced resilience. Our study contributes to the growing body of knowledge on genetic diversity in free-ranging animal populations in their natural environment, availing the first DQB1 gene data from locally-adapted Kenyan pigs.

Stillbirth rates and their association with swine leucocyte antigen class II haplotypes in Microminipigs

ObjectiveMicrominipig (MMP) is a miniature pig with an extra small body size for experimental use. In the present study, the occurrence of stillbirths and their genetic association with swine leukocyte antigen (SLA) class II haplotypes were evaluated in a population of MMPs.MethodsThe occurrences of stillbirth and genetic association with SLA class II haplotypes using 483 stillborn and 2,246 live piglets, and their parents were compared among the three groups of newborn piglet litters; an all stillborn (AS) group consisting of only stillborn piglet litters, a partial stillborn (PS) group consisting of stillborn and live piglet litters, and an all alive (AA) group consisting of only live piglet litters.ResultsThe incidence of stillborn piglets was 483/2,729 (17.7%). Distributions of litter sizes, numbers of stillborn piglets in a litter, parities, and gestation periods were distinct among the three groups. The frequencies of low resolution haplotype (Lr)-0.7 or Lr-0.23 were higher in the AS group than in the PS or AA groups. In sires, the frequency of Lr-0.7 associated with the AS group was significantly higher in the AS group than with the AA group. In dams, the frequency of Lr-0.23 was significantly higher in the AS group than in the PS or AA groups, whereas the frequency of Lr-0.7 was not significantly different.ConclusionThe incidence of stillborn piglets in MMPs appears to be higher than those in other pig breeds. Several traits related with stillbirths such as the number of stillborn piglets and parities of the AS group were different from those of the PS and AA groups. Specific SLA class II haplotypes were associated significantly with a high incidence of stillbirths and could be used as genetic markers to adopt breeding strategies to lower the rate of stillbirth in MMPs.

Identification of potential SLA-I-restricted CTL epitopes within the M protein of porcine reproductive and respiratory syndrome virus

CD8+ cytotoxic T lymphocytes (CTLs), are essential for clearance of porcine reproductive and respiratory syndrome virus (PRRSV) infection and regulation of host immune responses. Identification of SLA I-restricted CD8+ CTL epitopes would facilitate PRRSV vaccine development. Here, cells isolated from peripheral blood mononuclear cells (PBMCs) of PRRSV-immunized Large White pigs (JXA1-R strain) were screened for immunodominant PRRSV-2 M protein T cell epitopes via ELISPOT assay. Of nine immunodominant epitopes detected, eight elicited significant IFN-γ secretion responses that varied among individual pigs and according to epitope. To predict which epitopes harbored potential CTL epitopes, swine leukocyte antigen (SLA) class I genes of Large White pigs were cloned and sequenced, yielding fourteen distinct SLA class I gene sequences. Based on ELISPOT and SLA genotyping results, SLA-restricted binding of the fourteen predicted class I proteins to peptides derived from the eight immunodominant epitopes were predicted in-silico. After evaluation of 42 pET-peptide-SLA-I-β2m complexes containing predicted restricted peptides, extracellular SLA class I domains and β2m, ELISA testing of 33 peptide-SLA-I-β2m complexes detected four complexed peptides. These four peptides were evaluated using in vitro complex refolding assays that confirmed that M2−5 and M6−1 peptides each formed complexes with SLA-2*0502 and sβ2m, while M9−1 formed a complex with SLA-2*1201 and sβ2m. ELISPOT results confirmed these three 9-mer potential CTL epitopes efficiently stimulated IFN-γ secretion when presented by SLA class I molecules specified here. This study describes effective CTL epitope identification methods for use in future investigations of swine cellular immunity toward T cell-based vaccine development.

Comparative analysis of swine leukocyte antigen gene diversity in European farmed pigs.

SummaryIn Europe, swine represent economically important farm animals and furthermore have become a preferred preclinical large animal model for biomedical studies, transplantation and regenerative medicine research. The need for typing of the swine leukocyte antigen (SLA) is increasing with the expanded use of pigs as models for human diseases and organ‐transplantation experiments and their use in infection studies and for design of veterinary vaccines. In this study, we characterised the SLA class I (SLA‐1, SLA‐2, SLA‐3) and class II (DRB1, DQB1, DQA) genes of 549 farmed pigs representing nine commercial pig lines by low‐resolution (Lr) SLA haplotyping. In total, 50 class I and 37 class II haplotypes were identified in the studied cohort. The most common SLA class I haplotypes Lr‐04.0 (SLA‐1*04XX‐SLA‐3*04XX(04:04)‐SLA‐2*04XX) and Lr‐32.0 (SLA‐1*07XX‐SLA‐3*04XX(04:04)‐SLA‐2*02XX) occurred at frequencies of 11.02 and 8.20% respectively. For SLA class II, the most prevalent haplotypes Lr‐0.15b (DRB1*04XX(04:05/04:06)‐DQB1*02XX(02:02)‐DQA*02XX) and Lr‐0.12 (DRB1*06XX‐DQB1*07XX‐DQA*01XX) occurred at frequencies of 14.37 and 12.46% respectively. Meanwhile, our laboratory has contributed to several vaccine correlation studies (e.g. Porcine Reproductive and Respiratory Syndrome Virus, Classical Swine Fever Virus, Foot‐and‐Mouth Disease Virus and Swine Influenza A Virus) elucidating the immunodominance in the T‐cell response with antigen specificity dependent on certain SLA‐I and SLA‐II haplotypes. Moreover, these SLA–immune response correlations could facilitate tailored vaccine development, as SLA‐I Lr‐04.0 and Lr‐32.0 as well as SLA‐II Lr‐0.15b and Lr‐0.12 are highly abundant haplotypes in European farmed pigs.

Quantifying the Persistence of Vaccine-Related T Cell Epitopes in Circulating Swine Influenza A Strains from 2013-2017.

When swine flu vaccines and circulating influenza A virus (IAV) strains are poorly matched, vaccine-induced antibodies may not protect from infection. Highly conserved T cell epitopes may, however, have a disease-mitigating effect. The degree of T cell epitope conservation among circulating strains and vaccine strains can vary, which may also explain differences in vaccine efficacy. Here, we evaluate a previously developed conserved T cell epitope-based vaccine and determine the persistence of T cell epitope conservation over time. We used a pair-wise homology score to define the conservation between the vaccine’s swine leukocyte antigen (SLA) class I and II-restricted epitopes and T cell epitopes found in 1272 swine IAV strains sequenced between 2013 and 2017. Twenty-four of the 48 total T cell epitopes included in the epitope-based vaccine were highly conserved and found in >1000 circulating swine IAV strains over the 5-year period. In contrast, commercial swine IAV vaccines developed in 2013 exhibited a declining conservation with the circulating IAV strains over the same 5-year period. Conserved T cell epitope vaccines may be a useful adjunct for commercial swine flu vaccines and to improve protection against influenza when antibodies are not cross-reactive.

Diversity of the Swine Leukocyte Antigen Class I and II in Commercial Pig Populations.

Among swine genetic markers, the highly polymorphic swine leukocyte antigen (SLA) is one of the key determinants, associated with not only immune responses but also reproductive performance and meat quality. The objective of this study was to characterize the SLA class I and II diversities in the commercial pig populations. In this study, a total number of 158 pigs (126 gilts and 32 boars) were randomly selected from different breeding herds of five major pig-producing companies, which covered ~70% of Thai swine production. The results indicate that a moderate level of SLA diversity was maintained in the Thai swine population, despite the performance-oriented breeding scheme. The highly common SLA class I alleles were SLA-1*08:XX, SLA-2*02:XX, and SLA-3*04:XX at a combined frequency of 30.1, 18.4, and 34.5%, respectively, whereas DRB1*04:XX, DQB1*02:XX and DQA*02:XX were the common class II alleles at 22.8, 33.3, and 38.6%, respectively. The haplotype Lr-32.0 (SLA-1*07:XX, SLA-2*02:XX, and SLA-3*04:XX) and Lr-0.23 (DRB1*10:XX, DQB1*06:XX, DQA* 01:XX) was the most common SLA class I and II haplotype, at 15.5 and 14.6%, respectively. Common class I and II haplotypes were also observed, which Lr-22.15 was the most predominant at 11.1%, followed by Lr-32.12 and Lr-4.2 at 10.8 and 7.9%, respectively. To our knowledge, this is the first report of SLA class I and II diversities in the commercial pigs in Southeast Asia. The information of the common SLA allele(s) in the population could facilitate swine genetic improvement and future vaccine design.

Peptidomes and Structures Illustrate Two Distinguishing Mechanisms of Alternating the Peptide Plasticity Caused by Swine MHC Class I Micropolymorphism.

The micropolymorphism of major histocompatibility complex class I (MHC-I) can greatly alter the plasticity of peptide presentation, but elucidating the underlying mechanism remains a challenge. Here we investigated the impact of the micropolymorphism on peptide presentation of swine MHC-I (termed swine leukocyte antigen class I, SLA-I) molecules via immunopeptidomes that were determined by our newly developed random peptide library combined with the mass spectrometry (MS) de novo sequencing method (termed RPLD–MS) and the corresponding crystal structures. The immunopeptidomes of SLA-1*04:01, SLA-1*13:01, and their mutants showed that mutations of residues 156 and 99 could expand and narrow the ranges of peptides presented by SLA-I molecules, respectively. R156A mutation of SLA-1*04:01 altered the charge properties and enlarged the volume size of pocket D, which eliminated the harsh restriction to accommodate the third (P3) anchor residue of the peptide and expanded the peptide binding scope. Compared with 99Tyr of SLA-1*0401, 99Phe of SLA-1*13:01 could not form a conservative hydrogen bond with the backbone of the P3 residues, leading to fewer changes in the pocket properties but a significant decrease in quantitative of immunopeptidomes. This absent force could be compensated by the salt bridge formed by P1-E and 170Arg. These data illustrate two distinguishing manners that show how micropolymorphism alters the peptide-binding plasticity of SLA-I alleles, verifying the sensitivity and accuracy of the RPLD-MS method for determining the peptide binding characteristics of MHC-I in vitro and helping to more accurately predict and identify MHC-I restricted epitopes.