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Analysis of the affinity of influenza A virus protein epitopes for swine MHC I by a modified in vitro refolding method indicated cross-reactivity between swine and human MHC I specificities

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Abstract

In vitro refolding assays can be used to investigate the affinity and stability of the binding of epitope peptides to major histocompatibility complex (MHC) class I molecules, which are key factors in the presentation of peptides to cytotoxic T lymphocytes (CTLs). The recognition of peptide epitopes by CTLs is crucial for protection against influenza A virus (IAV) infection. The peptide-binding motif of the swine SLA-3*hs0202 molecule has been previously reported and partly overlaps with the binding motif of the most abundant human MHC allele, HLA-A*0201. In this study, we screened all the protein sequences of the swine-origin epidemic IAV strain A/Beijing/01/2009 (H1N1), and a total of 73 9-mer epitope peptides were predicted to fit the consensus motif of the swine SLA-3*hs0202 or HLA-A*0201 molecule. Then, 14 peptides were selected, and their affinities to SLA-3*hs0202 were tested by a modified in vitro refolding assay. Our results show that ten epitopes could tolerate gel filtration, indicating that these epitopes formed stable or partly stable complexes with SLA-3*hs0202. Eight out of the ten epitopes have been previously reported as HLA-A2-restricted epitopes, which implied cross-reactivity between swine and human MHC I specificities. Furthermore, the modified mini-system refolding method could be applied for the screening of peptides because the refolding efficiency remained almost unchanged with the positive peptide (HA-KMN9) subjected to size-exclusion chromatography and Resource Q anion-exchange chromatography. The results presented here provide new insight into the development of epitope-based vaccines to control IAV and increase our understanding of swine molecular immunology.

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Funding

This study was funded by the State Key Program of the National Natural Science Foundation of China (Grant No. 31702232), by the Zhoukou Normal University for doctoral researchers on scientific research and innovation (Grant No. ZKNUC2016029), and by the Youth Teacher Scientific Research and Innovation Program of Zhoukou Normal University (Grant No. ZKNUB2201710).

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Author notes

  1. Shuhua Fan and Yongli Wang contributed equally to this work.

Authors and Affiliations

  1. College of Life Science and Agronomy, Zhoukou Normal University, Zhoukou, 466001, People’s Republic of China

    Shuhua Fan, Yongli Wang, Xian Wang, Li Huang, Yunxia Zhang, Xiaomeng Liu & Wenshuai Zhu

  2. Institute of Neuroscience and Translational Medicine, Zhoukou Normal University, Zhoukou, People’s Republic of China

    Shuhua Fan, Li Huang, Yunxia Zhang, Xiaomeng Liu & Wenshuai Zhu

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  1. Shuhua Fan
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  2. Yongli Wang
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  4. Li Huang
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Correspondence to Shuhua Fan.

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Online Resource 1

Potential 9-mer epitope peptides selected from the IAV genome by an in silico prediction method based on the HLA-A*0201 and SLA-3*hs0202 motifs. IAV type, 2009 pandemic H1N1 (A/Beijing/01/2009). Estimated binding affinity to SLA-3*hs0202 and HLA-A*0201 was calculated using the website http://www.cbs.dtu.dk/services/NetMHCpan-2.8/ (Hoof et al. 2009). A total of 73 nonapeptides were predicted from IAV proteins based on the SLA-3*hs0202 and HLA-A*0201 binding motifs using the NetMHCpan 2.8 server, and the estimated binding affinities to SLA I are represented as IC50 values. The peptides were selected with theoretical IC50 values ranging from 3 to 29047.6 nM, and IC50 values are inversely proportional to binding affinities. (DOCX 37 kb)

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Fan, S., Wang, Y., Wang, X. et al. Analysis of the affinity of influenza A virus protein epitopes for swine MHC I by a modified in vitro refolding method indicated cross-reactivity between swine and human MHC I specificities. Immunogenetics 70, 671–680 (2018). https://doi.org/10.1007/s00251-018-1070-6

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