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Examining epitope mutagenesis as a strategy to reduce and eliminate human antibody binding to class II swine leukocyte antigens.

ABSTRACT: Xenotransplantation of pig organs into people may help alleviate the critical shortage of donors which faces organ transplantation. Unfortunately, human antibodies vigorously attack pig tissues preventing the clinical application of xenotransplantation. The swine leukocyte antigens (SLA), homologs of human HLA molecules, can be xenoantigens. SLA molecules, encoded by genes in the pig major histocompatibility complex, contribute to protective immune responses in pig. Therefore, simply inactivating them through genome engineering could reduce the ability of the human immune system to surveil transplanted pig organs for infectious disease or the development of neoplasms. A potential solution to this problem is to identify and modify epitopes in SLA proteins to eliminate their contribution to humoral xenoantigenicity while retaining their biosynthetic competence and ability to contribute to protective immunity. We previously showed that class II SLA proteins were recognized as xenoantigens and mutating arginine at position 55 to proline, in an SLA-DQ beta chain, could reduce human antibody binding. Here, we extend these observations by creating several additional point mutants at position 55. Using a panel of monoclonal antibodies specific for class II SLA proteins, we show that these mutants remain biosynthetically competent. Examining antibody binding to these variants shows that point mutagenesis can reduce, eliminate, or increase antibody binding to class II SLA proteins. Individual mutations can have opposite effects on antibody binding when comparing samples from different people. We also performed a preliminary analysis of creating point mutants near to position 55 to demonstrate that manipulating additional residues also affects antibody reactivity.

SUBMITTER: Ladowski JM

PROVIDER: S-EPMC6905188 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Examining epitope mutagenesis as a strategy to reduce and eliminate human antibody binding to class II swine leukocyte antigens.

Ladowski Joseph M JM Martens Gregory R GR Reyes Luz M LM Hauptfeld-Dolejsek Vera V Tector Matthew M Tector Joseph J

Immunogenetics 20190704 7

Xenotransplantation of pig organs into people may help alleviate the critical shortage of donors which faces organ transplantation. Unfortunately, human antibodies vigorously attack pig tissues preventing the clinical application of xenotransplantation. The swine leukocyte antigens (SLA), homologs of human HLA molecules, can be xenoantigens. SLA molecules, encoded by genes in the pig major histocompatibility complex, contribute to protective immune responses in pig. Therefore, simply inactivatin ...[more]

PMID: 31270568

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Project description: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 ?2-microglobulin (?2m) 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.

Dataset Information

Examining epitope mutagenesis as a strategy to reduce and eliminate human antibody binding to class II swine leukocyte antigens.

Publications

Examining epitope mutagenesis as a strategy to reduce and eliminate human antibody binding to class II swine leukocyte antigens.

Similar Datasets

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