Structural and Biochemical Analyses of Swine Major Histocompatibility Complex Class I Complexes and Prediction of the Epitope Map of Important Influenza A Virus Strains

Abstract

The lack of a peptide-swine leukocyte antigen class I (pSLA I) complex structure presents difficulties for the study of swine cytotoxic T lymphocyte (CTL) immunity and molecule vaccine development to eliminate important swine viral diseases, such as influenza A virus (IAV). Here, after cloning and comparing 28 SLA I allelic genes from Chinese Heishan pigs, pSLA-3*hs0202 was crystalized and solved. SLA-3*hs0202 binding with s␤2m and a KMNTQFTAV (hemagglutinin [HA]-KMN9) peptide from the 2009 pandemic swine H1N1 strain clearly displayed two distinct conformations with HA-KMN9 peptides in the structures, which are believed to be beneficial to stimulate a broad spectrum of CTL immune responses. Notably, we found that different HA-KMN9 conformations are caused, not only by the flexibility of the side chains of residues in the peptide-binding groove (PBG), but also by the skewing of ␣1 and ␣2 helixes forming the PBG. In addition, alanine scanning and circular-dichroism (CD) spectra confirmed that the B, D, and F pockets play critical biochemical roles in determining the peptide-binding motif of SLA-3*hs0202. Based on biochemical parameters and comparisons to similar pockets in other known major histocompatibility complex class I (MHC-I) structures, the fundamental motif for SLA-3*hs0202 was determined to be X-(M/A/R)-(N/Q/R/F)-X-X-X-X-X-(V/I) by refolding in vitro and multiple mutant peptides. Finally, 28 SLA-3*hs0202-restricted epitope candidates were identified from important IAV strains, and two of them have been found in humans as HLA-A*0201-specific IAV epitopes. Structural and biochemical illumination of pSLA-3*hs0202 can benefit vaccine development to control IAV in swine. IMPORTANCEWe crystalized and solved the first SLA-3 structure, SLA-3*hs0202, and found that it could present the same IAV peptide with two distinct conformations. Unlike previous findings showing that variable peptide conformations are caused only by the flexibility of the side chains in the groove, the skewing of the ␣1 and ␣2 helixes is important in the different peptide conformations in SLA-3*hs0202. We also determined the fundamental motif for SLA-3*hs0202 to be X-(M/A/R)-(N/Q/R/F)-X-X-X-X-X-(V/I) based on a series of structural and biochemical analyses, and 28 SLA-3*hs0202-restricted epitope candidates were identified from important IAV strains. We believe our structure and analyses of pSLA-3*hs0202 can benefit vaccine development to control IAV in swine. Influenza A virus (IAV) has always been a major cause of morbidity and mortality throughout history, and new emerging IAVs have posed an increasing threat to human health in recent years (1-3). IAV consists of eight single-stranded-RNA segments encoding 12 proteins: the nucleoprotein (NP), three polymerase proteins (PA, PB1, and PB2), two matrix proteins (M1 and M2), two nonstructural proteins (NS1 and NS2), two surface glycoproteins, hemagglutinin (HA), neuraminidase (NA), and two newly identified proteins (PB1-F2 and PB1-N40) (4-6). Reassortment of the eight gene segments from...

Illumination of PRRSV Cytotoxic T Lymphocyte Epitopes by the Three-Dimensional Structure and Peptidome of Swine Lymphocyte Antigen Class I (SLA-I)

“…However, in terms of peptide binding, the SLA-1 * 1502 structure not only reflects the common features of SLA-I alleles but also exhibits unique allelic-specific characteristics. Similar to the previously resolved SLA-1 * 0401 and SLA-3 * hs0202, the N-terminus of the SLA-1 * 1502 PBG is open because the amino acid at position 167 of the A pocket is a small Ser (Figure 2A), but in other species such as humans and mice, the amino acid at this position is a large Trp (19,20). The peptide-binding motif of SLA-1 * 1502, like that of SLA-1 * 0401 FIGURE 6 | Peptide predictions from different PRRSV strains according to the binding motifs of the pSLA-1*1502 complex.…”

“…The amino acid compositions of these three pockets in SLA-1 * 1502 are shown in Figures 2C-E. No hydrogen bonds or salt bridges were found in these structures; instead, many VDWs were observed between the three pockets and the NSP9-TMP9 peptide ( Table 3). The D pocket is critical for the peptide selection of SLA-1 * 0401 and SLA-3 * hs0202 because of the charged residue at position 156 (19,20). The non-polar Met 156 causes the D pocket of SLA-1 * 1502 to be hydrophobic, in contrast to the charged D pocket of SLA-1 * 0401 or SLA-3 * hs0202 (Figure 3).…”

“…To determine the peptide-binding motif of SLA-1 * 1502, the peptide NSP9-TMP9 was mutated by alanine scanning (19), and CD spectra were used to test the stability of pSLA-1 * 1502 complexes with these mutant peptides (Figure 5). The in vitro refolding and CD results showed that the binding stabilities of P2-Ala, P3-Ala, and P9-Ala mutant peptides are significantly lower than that of the wild-type NSP9-TMP9 peptide.…”

“…Similar to human MHC (also known as human leukocyte antigen, HLA), SLA-I molecules are a highly polymorphic gene superfamily whose peptide-binding specificities are significantly influenced by highly variable sites (17). Thus, far, more than 100 SLA-I genes have been cloned (IPD; http://www.ebi.ac.uk/ipd/index.html), and two three-dimensional (3D) structures of peptide-SLA-I (p/SLA-I) molecules have been determined, revealing the peptide presentation characteristics of SLA-I molecules in swine (19,20). Thus, the situation is favorable for the design and development of a novel viral CTL vaccine against swine PRRS based on these 3D structures of SLA-I molecules.…”

Safety and immunogenicity of mammalian cell derived and Modified Vaccinia Ankara vectored African swine fever subunit antigens in swine

“…The employed approach was justified by 1) the high degree of structural similarity and identification of cross-species influenza virus cytotoxic T lymphocytes epitopes between HLA and SLA class I molecules (Zhang et al, 2011), 2) phylogenetic analyses that demonstrated strong sequence homology between SLA and HLA class II genes (Smith et al, 2005), and 3) previously reported swine epitope prediction studies (Burgara-Estrella et al, 2013; Díaz et al, 2009;Zimic et al, 2011). Recent advancements associated with swine epitope prediction have been reported (Fan et al, 2016;Gutiérrez et al, 2016Gutiérrez et al, , 2015 and these expanded immunoinformatics knowledge and improved tools may result in better prediction of ASFV SLA epitopes for future analysis.…”