Study On The Characteristics Of Feline Major Histocompatibility Complex Class ? Molecules Presenting Coronavirus Peptides

Cats can be infected with SARS-coronavirus 2(SARS-CoV-2).As an important companion animal,this makes people worry that cats may act as a host animal to affect the spread of the virus between humans.At the same time,cats infected with SARS-CoV-2 are mostly asymptomatic or mild symptoms and can remove the virus faster than humans,which makes cats can be used as a suitable animal model to study the characteristics of adaptive immunity after infection with coronavirus.The purpose of this study was to explain the immune mechanism of rapid activation of specific T cell immune response and antiviral effect in cats infected with SARS-CoV-2,and whether cats may produce specific cross-reactivity to peptides derived from SARS-CoV-2 and FECV.We renatured FLA-K*00701 heavy chain and beta-2,macroglobulin(?2m)and antigenic peptides from SARS-CoV-2 and FECV in vitro to form FLA complex proteins,and collected protein structure data by crystallization and X-ray diffraction,and analyzed the structural characteristics of feline leukocyte antigen(FLA)class I allele FLA-K*00701 presenting peptides derived from SARS-CoV-2 and FECV at the same time.The trend of the two peptides in the peptide binding groove(PBG)is basically the same,and there may be no difference in the recognition of the two peptides from the two viruses in the CDR3 region of the T cell receptor(TCR).The cross-conserved peptides of SARS-CoV-2 and FECV coronaviruses can stimulate the same specific T cell response in the host.At the same time,we analyzed the structural characteristics of another classic FLA class I allele FLA-E*00301 binding to peptides derived from FECV to form FLA-E*00301/peptide complexes.Through circular dichroism or free peptide elution experiments,We determined that the anchoring sites in the PBG region of these two different genotypes of FLA I molecules,like most mammals,are also in the B and K pockets,and determined their respective anchoring motifs.According to the anchored motifs of FLA I FLA-K*00701 and FLA-E*00301 alleles identified in this study,a large number of peptides consistent with the characteristics of the two FLA I anchoring motifs were predicted from the four structural proteins and ORF1ab polymerase proteins of SARS-CoV-2 and FECV.The renaturation efficiency of the peptides randomly selected according to the two FLA I anchoring motifs was the same as that of the peptides selected according to the affinity score predicted by the website of NetMHCpan.Therefore,the peptides predicted according to the anchoring motifs not only accord with the affinity characteristics of NetMHCpan class I molecules,but also can predict more peptides with strong binding to FLA I molecules on the basis of ensuring accurate prediction compared with the peptides screened according to the affinity score of NetMHCpan.At the same time,we found all the cross-conserved peptides derived from SARS-CoV-2 and FECV,which may be recognized and bound by FLA I molecules and presented to T cells,and established a cross-conserved peptide library of these two viruses.85%of these cross-conserved peptides are distributed on ORF1ab proteins.From this peptide library,we screened out cross-conserved peptides from two viruses that match the characteristics of the anchoring motifs of the two FLA I alleles and can be bound by the corresponding FLA I genes.By studying the distribution of these peptides,it was found that the cross-conserved peptides with different peptide length and different number of amino acids were the most distributed on ORF1ab protein,accounting for more than 90%of all predicted peptides.After showing the positions of these cross-conserved peptides on the same axis,we can directly see that there are three dense regions of conserved epitopes on the ORF1ab protein.The concentrated regions 4911-4986 and 5140-5269 were located on RNA polymerase and 5603-5741 on helicase.In addition,there are also a large number of cross-conserved peptides on 3CL proteases.This provides evidence for the development of universal polypeptide vaccines based on conserved protein epitopes that can achieve cross-protection against multiple coronaviruses.To sum up,the protein structures of feline FLA I presenting SARS-CoV-2 and FECV were analyzed for the first time.The anchoring motifs of the two genotypes were determined.According to the anchoring motifs,a large number of cross-conserved epitopes shared by the two viruses were found,and the dense regions of cross-conserved epitopes were located.It provides a valuable reference for the research and development of universal coronavirus vaccine.