Specificity Characterization of SLA Class I Molecules Binding to Swine-Origin Viral Cytotoxic T Lymphocyte Epitope Peptides in Vitro

Abstract

Swine leukocyte antigen (SLA) class I molecules play a crucial role in generating specific cellular immune responses against viruses and other intracellular pathogens. They mainly bind and present antigens of intracellular origin to circulating MHC I-restricted cytotoxic T lymphocytes (CTLs). Binding of an appropriate epitope to an SLA class I molecule is the single most selective event in antigen presentation and the first step in the killing of infected cells by CD8+ CTLs. Moreover, the antigen epitopes are strictly restricted to specific SLA molecules. In this study, we constructed SLA class I complexes in vitro comprising viral epitope peptides, the extracellular region of the SLA-1 molecules, and β2-microglobulin (β2m) using splicing overlap extension polymerase chain reaction (SOE-PCR). The protein complexes were induced and expressed in an Escherichia coli prokaryotic expression system and subsequently purified and refolded. Specific binding of seven SLA-1 proteins to one classical swine fever virus (CSFV) and four porcine reproductive and respiratory syndrome virus (PRRSV) epitope peptides was detected by enzyme-linked immunosorbent assay (ELISA)-based method. The SLA-1∗13:01, SLA-1∗11:10, and SLA-1∗11:01:02 proteins were able to bind specifically to different CTL epitopes of CSFV and PRRSV and the MHC restrictions of the five epitopes were identified. The fixed combination of Asn151Val152 residues was identified as the potentially key amino acid residues influencing the binding of viral several CTL epitope peptides to SLA-1∗13:01 and SLA-1∗04:01:01 proteins. The more flexible pocket E in the SLA-1∗13:01 protein might have fewer steric limitations and therefore be able to accommodate more residues of viral CTL epitope peptides, and may thus play a critical biochemical role in determining the peptide-binding motif of SLA-1∗13:01. Characterization of the binding specificity of peptides to SLA class I molecules provides an important basis for epitope studies of infectious diseases in swine, and for the rational development of novel porcine vaccines, as well as for detailed studies of CTL responses in pigs used as animal models.

Functional analysis of prv-miR-LLT11a encoded by pseudorabies virus

“…The current study demonstrated that SLA-1 was a potential target gene of prv-miR-LLT11a by using RNAhybrid software based on the miRNAs and target gene's secondary structures; in addition, a dual luciferase assay method was utilized for verification of results. In previous study, SLA class 1 molecular (SLA-1) genes have been shown to have pivotal roles in specific cellular immune responses against viral infections [32]. Furthermore, it was reported that SLA-1-mediated antigen presentation pathways can be antagonized by PRV to evade the immune responses of the hosts [33].…”

Positive selection pressure on E2 protein of classical swine fever virus drives variations in virulence, pathogenesis and antigenicity: Implication for epidemiological surveillance in endemic areas

“…To identify the T‐cell epitopes for E2 protein of CSFV with higher accuracy, we established the following strategy: (a) we mapped the promising epitopes using IEDB 2.19 methodology (Recommended) of IEDB Analysis Resource (Lundegaard et al, ; Nielsen et al, ) (http://tools.iedb.org/mhci/) for pig species, restricted to the Swine Leukocyte Antigen (SLA) alleles: SLA‐1*0101, SLA‐1*1101, SLA‐1*1301 SLA‐2*0101, SLA‐3*0101, SLA‐3*0602. The restriction for SLA haplotypes was based on two criteria: the alleles present in the swine breeds Large White and Landrace (Sorensen et al, ), both present in Cuban pig population (Rios et al, ), and the SLA alleles identified which specifically bind different CTL epitopes of CSFV (Gao et al, ). The cut‐off score was set to IEDB ≤1 to guarantee maximum epitope‐like properties (Table S2; Lundegaard et al, ; Nielsen et al, ), for the prediction of T‐cell epitopes other software such as SYFPEITHI or BIMAS were not used since in these programs, predictions for alleles from pig are not available; (b) a search for MHC‐I binding was also performed using NetMHC 4.0 Server.…”

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

“…We showed that the arginine at position 156 (156 Arg ) is essential for peptide binding of SLA-1*04:01, and when 156 Arg was mutated to alanine, the peptides bound by the mutant SLA-1*04:01 (R156A) became significantly different from those bound by SLA-1*04:01 (30). Another previous study reported that SLA-1*13:01 is quite similar to SLA-1*04:01 but has different properties in presenting peptides (31). The lack of corresponding cell lines and monoclonal antibodies against SLA-I makes it challenging for us to use the current MS method to comprehensively and accurately determine their differences in peptide binding caused by micropolymorphism.…”