Bioinformatics Analysis on B cell Epitopes of Rice Allergen RAG1

[Objective] To predict the secondary structure and B cell epitopes of the rice major allergen RAG1. [Method] The amino acid sequence of rice allergen RAG1 was acquired from Expasy protein database. The secondary structure of RAG1 was predicted by DNAStar Protean software with Gamier-Robson program, Chou-Fasman program and Karplus-Schulz program; the B cell epitopes of RAG1 was predicted with the Kyte Doolittle hydrophilic program, Emini surface accessibility program and Jameson-Wolf antigenic index program. [Result] The predictions on secondary structure and B cell epitopes showed that the regions of 33-44, 119-129, 155-163 were the dominant B cell epitopes. [Conclusion] This study predicted the potential dominant B cell epitopes in rice allergen RAG1 by comprehensive use of multi-methods and multi-parameters, and provided a theoretical basis for further researches on identification, antigen modification and epitope vaccine design of RAG1 B cell epitopes.

The Prediction of B Cell Epitopes for VP73 Protein of African Fever Virus

[Objective] The B cell epitopes for VP73 protein of African swine fever virus was predicted. [ Method] Based on the analysis of the amino acid sequence and the flexible regions of VP73 protein, the B cell epitopes for VP73 protein of African swine fever virus were predicted by method of Kyte-Doolittie, Emini and Jameson-Wolf. [Result] The B cell epitopes were located at or adjacent to the N-terminal No. 11 - 18,26 -48,73 -82,136 - 150,159 - 174,181 - 189,191 - 210,247 - 276,279 - 295,313 - 323 and 382 - 392. [Conclusion] The multi-parameters analytic method was adopted to predict the B cell epitopes for VP73 protein of African swine fever virus, which laid solid foundation for further characterizing the protein of VP73 and researching epitope vaccine.

Prediction of promiscuous T cell epitopes in RNA dependent RNA polymerase of Chikungunya virus

Objective: To explore RNA dependent RNA polymerase of Chikungunya virus(CHIKV) and develop T cell based epitopes with high antigenicity and good binding affinity for the human leukocyte antigen(HLA) classes as targets for epitopes based CHIKV vaccine. Methods: In this study we downloaded 371 non-structural protein 4 protein sequences of CHIKV belonging to different regions of the world from the US National Institute of Allergy and Infectious Diseases(NIAID) virus pathogen resource database. All the sequences were aligned by using CLUSTALW software and a consensus sequence was developed by using Uni Pro U Gene Software version 1.2.1. PropredⅠand Propred software were used to predict HLAⅠ and HLAⅡ binding promiscuous epitopes from the consensus sequence of non-structural protein 4 protein. The predicted epitopes were analyzed to determine their antigenicity through Vaxijen server version 2.0. All the HLAⅠ binding epitopes were scanned to determine their immunogenic potential through the Immune Epitope Database(IEDB). All the predicted epitopes of our study were fed to IEDB database to determine whether they had been tested earlier. Results: Twenty two HLA class Ⅱ epitopes and eight HLA classⅠepitopes were predicted. The promiscuous epitopes WMNMEVKII at position 486–494 and VRRLNAVLL at 331–339 were found to bind with 37 and 36 of the 51 HLA class Ⅱ alleles respectively. Epitope MANRSRYQS at position 58–66 and epitopes YQSRKVENM at positions 64–72 were predicted to bind with 12 and 9 HLAⅠI alleles with antigenicity scores of 0.754 9 and 1.013 0 respectively. Epitope YSPPINVRL was predicted to bind 18 HLAⅠ alleles and its antigenicity score was 1.425 9 and immunogenicity score was 0.173 83. This epitope is very useful in the preparation of a universal vaccine against CHIKV infection. Conclusions: Epitopes reported in this study showed promiscuity, antigenicity as well as good binding affinity for the HLA classes. These epitopes will provide the baseline for development of efficacious vaccine for CHIKV.

Conservation of T cell epitopes between seasonal influenza viruses and the novel influenza A H7N9 virus

A novel avian influenza A(H7N9) virus recently emerged in the Yangtze River delta and caused diseases, often severe, in over 130 people. This H7N9 virus appeared to infect humans with greater ease than previous avian influenza virus subtypes such as H5N1 and H9N2. While there are other potential explanations for this large number of human infections with an avian influenza virus, we investigated whether a lack of conserved T-cell epitopes between endemic H1N1 and H3N2 influenza viruses and the novel H7N9 virus contributes to this observation. Here we demonstrate that a number of T cell epitopes are conserved between endemic H1N1 and H3N2 viruses and H7N9 virus. Most of these conserved epitopes are from viral internal proteins. The extent of conservation between endemic human seasonal influenza and avian influenza H7N9 was comparable to that with the highly pathogenic avian influenza H5N1. Thus, the ease of inter-species transmission of H7N9 viruses(compared with avian H5N1 viruses) cannot be attributed to the lack of conservation of such T cell epitopes. On the contrary, our findings predict significant T-cell based cross-reactions in the human population to the novel H7N9 virus. Our findings also have implications for H7N9 virus vaccine design.

Screening HLA-A-restricted T cell epitopes of SARS-CoV-2 and the induction of CD8^(+)T cell responses in HLA-A transgenic mice

Since severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)-specific T cells have been found to play essential roles in host immune protection and pathology in patients with coronavirus disease 2019(COVID-19),this study focused on the functional validation of T cell epitopes and the development of vaccines that induce specific T cell responses.A total of 120 CD8^(+)T cell epitopes from the E,M,N,S,and RdRp proteins were functionally validated.Among these,110,15,6,14,and 12 epitopes were highly homologous with SARS-CoV,OC43,NL63,HKU1,and 229E,respectively;in addition,four epitopes from the S protein displayed one amino acid that was distinct from the current SARS-CoV-2 variants.Then,31 epitopes restricted by the HLA-A2 molecule were used to generate peptide cocktail vaccines in combination with Poly(I:C),R848 or poly(lactic-co-glycolic acid)nanoparticles,and these vaccines elicited robust and specific CD8^(+)T cell responses in HLA-A2/DR1 transgenic mice as well as wild-type mice.In contrast to previous research,this study established a modified DC-peptide-PBL cell coculture system using healthy donor PBMCs to validate the in silico predicted epitopes,provided an epitope library restricted by nine of the most prevalent HLA-A allotypes covering broad Asian populations,and identified the HLA-A restrictions of these validated epitopes using competitive peptide binding experiments with HMy2.CIR cell lines expressing the indicated HLA-A allotype,which initially confirmed the in vivo feasibility of 9-or 10-mer peptide cocktail vaccines against SARS-CoV-2.These data will facilitate the design and development of vaccines that induce antiviral CD8^(+)T cell responses in COVID-19 patients.

ASFV pp62 Protein Has 9 Dominant B-T Cell Combined Epitopes

[Objective]The paper was to study the basic characteristics of the pp62 protein of the African swine fever virus strain Pig/HLJ/2018,and to provide more basic data for the study of the virus.[Method]We used the ProtParam program to analyze the physical and chemical properties of the pp62 protein.TMHMM-2.0 and SignalP-5.0 were used to analyze protein transmembrane region and signal peptide,and Lasergene 7.0 Protean program was used to study protein antigen index,hydrophilicity,surface accessibility and titration curve.Protein N-glycosylation site and O-glycosylation site came out with NetNGlyc-1.0 and NetOGlyc-4.0 online servers.Then,PSIPRED-4.0,NetSurfP-2.0,and PSRSM were used to analyze protein secondary structure,BepiPred1.0 and IEDB tools were used to analyze protein B-cell epitopes,and NetMHC 4.0 and NetMHCpan tools were used to analyze protein T-cell epitopes.And we used Swiss-Model to analyze the high-level structure of the protein,the EzMol tool to visually analyze B-T cell combined epitopes,and finally,MEGA 7.0 to analyze the genetic evolutionary relationship of the protein.[Result]The pp62 protein of African swine fever viral strain Pig/HLJ/2018 had a molecular weight of 60.5 kDa.It was a hydrophilic acid labile protein,and had no transmembrane region and signal peptide.There were 5 N-glycosylation sites and 4 O-glycosylation sites.Analysis of the secondary structure of the protein showed that the proportions of helix,coil and strands were 45.5%,41.7%and 12.8%,respectively.The study of dominant epitopes revealed that there were 14 dominant B-cell epitopes and 16 dominant T-cell epitopes.And 9 dominant B-T cell combined epitopes located on the surface of the protein molecule were found.The phylogenetic tree constructed with the pp62 protein showed that the evolutionary relationship of Pig/HLJ/2018 strain was the closest to Georgia/2007/1,which belonged to genotype II.[Conclusion]The results will provide basic information for pp62 research.

Prediction of Secondary Structure and B Cell Epitope of GH Protein from Acipenser sinensis

[ Objective] The aim was to predict the secondary structure and B cell epitope of growth hormone （GH） protein from Acipenser sinensis. [Method] With the amino acid sequence of GH protein from A. sinensis as the base, the secondary structure of GH protein from A. sinensis was predicted by the method of Gamier-Robson, Chou-Fasman and Karpius-Schulz, and its cell epitope was predicted by the method of Kyte- Doolittle, Emini and Jameson-Wolf. [Result] The sections of 18 -23, 55 -55, 67 -73, 83 -86,112 -114,151 -157 and 209 -211 in the N terminal of GH protein molecule had softer structure and these sections could sway or fold to produce more complex tertiary structure. The sections of 19 -23, 51 -71,84 -95, 128 -139, 164 -176 and 189 -195 in the N terminal of GH protein could be the epitope of B cell and there were flexible regions in these sections or near these sections of GH protein molecule. So the dominant regions could be in these sections or near these sections. [ Conclusion] The research provided the basis for the preparation of monoctonal antibody of GH protein from A. sinensis and provided the reference for the discussion for the molecular regulation mechanism of A. sinensis.

Prediction on Antigenic Epitope Characteristics of Bt Cry2Ab Protein in Transgenic Crops

[Objective] This study aimed to predict the structural characteristics of Bt Cry2Ab protein in transgenic crops with bioinformatic analysis to provide the theoretical clues for design of antibody Cry2Ab. [Method] The amino acid sequence of Cry2Ab protein was searched from NCBI database. The B cell epitopes were predicted with DNAStar. The binding affinity between Cry2Ab protein and MHC-II molecules was analyzed with NetMHCII 2.2 Server to predict the T cell epitopes. [Result] Prediction result suggested the potential B cell epitope of Cry2Ab locating in the region of 208-215. Analysis of the binding affinity between Cry2Ab and MHC-II molecules suggested the regions of 177-185, 299-307 and 255-263 were the potential T cell epitopes. Human with HLA-DRB10101 alleles and HLA-DRB10701 alleles were more sensitive to Cry2Ab protein. [Conclusion] This study facilitates to understand the structural characteristics of Cry2Ab protein and provides a new clue to improve the assessment method for potential allergenicity of genetically modified food.

A novel recombinant DNA vaccine encodingMycobacterium tuberculosis ESAT-6 and FL protects againstMycobacterium tuberculosis challenge in mice

Mycobacterium tuberculosis 6-kDa early secretory antigenic target （ESAT-6） is a dominant target antigen for cell-mediated immunity in the early phase of tuberculosis. The fms-like tyrosine kinase 3 ligand （FL） that induces potent immune response has been used as an adjuvant in vaccine development. In this study, a new recombinant plasmid （plRES-epitope-peptides-FL） encoding three T cell epitopes of ESAT-6 and FL was constructed, and the immunogenicity of the DNA vaccine was assessed in C57BL/6 mice immunized with the plasmid DNA vaccine. Additionally, a strategy of intramuscular injection with the DNA vaccine （prime） and intranasal administration of the epitope peptides （boost） was employed to induce higher immune reaction of the mice. The results showed that mice vaccinated with the recombinant plasmid DNA vaccine and boosted with the peptides not only increased the levels of Thl cytokines （IFN-γ and IL-12）, the number of IFN-γ＋ T cells and activities of cytotoxic T lymphocytes as well as IgG, but also enhanced protection against Mycobacterium tuberculosis challenge. In conclusion, these data indicate that the novel recombinant plRES-epitope-peptides-FL plasmid is a useful DNA vaccine for pre- venting Mycobacterium tuberculosis infection.

Adeno-associated virus mediated delivery of Tregitope 167 ameliorates experimental colitis

AIM:To explore the anti-inflammatory potential of adeno-associated virus-mediated delivery of Tregitope 167 in an experimental colitis model.METHODS:The trinitrobenzene sulfonate(TNBS) model of induced colitis was used in Balb/c mice.Subsequently after intravenous adeno-associated virusmediated regulatory T-cell epitopes(Tregitope) delivery,acute colitis was initiated by intra-rectal administration of 1.5 mg TNBS in 40% ethanol followed by a second treatment with TNBS(0.75 mg in 20% ethanol) 8 d later.Control groups included mice not treated with TNBS(healthy control group) and mice treated by TNBS only(diseased group).At the time of sacrifice colon weight,the disease activity index and histology damage score were determined.Immunohistochemical staining of the colonic tissues was performed to asses the cellular infiltrate and the presence of transcription factor forkhead Box-P3(Foxp3).Thymus,mesenteric lymph nodes,liver and spleen tissue were collected and the corresponding lymphocyte populations were further assessed by flow cytometry analysis for the expression of CD4+ T cell and regulatory T cell associated markers.RESULTS:The Tregitope 167 treated mice gained an average of 4% over their initial body weight at the time of sacrifice.In contrast,the mice treated with TNBS alone(no Tregitope) developed colitis,and lost 4% of their initial body weight at the time of sacrifice(P < 0.01).The body weight increase that had been observed in the mice pre-treated with Tregitope 167 was substantiated by a lower disease activity index and a decreased colon weight as compared to the diseased control group(P < 0.01 and P < 0.001,respectively).Immunohistochemical staining of the colonic tissues for CD4+ showed that inflammatory cell infiltrates were present in TNBS treated mice with or without administration with tregitope 167 and that these cellular infiltrates consisted mainly of CD4+ cells.For both TNBS treated groups CD4+ T cell infiltrates were observed in the sub-epithelial layer and the lamina propria.CD4+ T cell infiltrates were also present in the muscularis mucosa layer of the diseased control mice,but were absent in the Tregitope 167 treated group.Numerous Foxp3 positive cells were detected in the lamina propria and sub-epithelium of the colon sections from mice treated with Tregitope 167.Furthermore,the Foxp3 and glycoprotein A repetitions predominant markers were significantly increased in the CD4+ T lymphocyte population in the thymus of the mice pre-treated with adeno-associated virus serotype 5(cytomegalovirus promoter-Tregitope 167),as cytomegalovirus promoter compared to lymphocyte populations in the thymus of diseased and the healthy control mice(P < 0.05 and P < 0.001,respectively).CONCLUSION:This study identifies adeno-associated virus-mediated delivery of regulatory T-cell epitope 167 as a novel anti-inflammatory approach with the capacity to decrease intestinal inflammation and induce longterm remission in inflammatory bowel disease.

Sequence Similarity Analysis of Non-Self CTL Epitopes and Mouse Proteins Using Sequence Alignment

This research analyzed amino acid sequence similarity between non-self T cell epitopes recognized by mouse antibodies and mouse proteins. Using sequence alignment,we found that only 8 of 1 108 epitopes are highly similar to mouse protein sequences. The result shows that non-self T cell epitopes are not similar or have little similarity to mouse protein sequences. Furthermore,reviewing the related literature,we also found that the eight epitopes would trigger immune responses in some particular environment,which are ignored by T cells in normal condition. The result suggests that no or low-similarity peptide vaccines can reduce the chance of collateral cross-reactions and enhance the antigen-specific immune response to vaccine.

Identification of cross-reactive CD8^(+)T cell receptors with high functional avidity to a SARS-CoV-2 immunodominant epitope and its natural mutant variants

Despite the growing knowledge of T cell responses in COVID-19 patients,there is a lack of detailed characterizations for T cell-antigen interactions and T cell functions.Here,with a predicted peptide library from SARS-CoV-2 S and N proteins,we found that specific CD8+T cell responses were identified in over 75%of COVID-19 convalescent patients(15/20)and an epitope from the N protein,N361-369(KTFPPTEPK),was the most dominant epitope from our selected peptide library.Importantly,we discovered 2 N361-369-specific T cell receptors(TCRs)with high functional avidity that were independent of the CD8 co-receptor.These TCRs exhibited complementary cross-reactivity to several presently reported N361-369 mutant variants,as to the wild-type epitope.Further,the natural functions of these TCRs in the cytotoxic immunity against SARS-CoV-2 were determined with dendritic cells(DCs)and the lung organoid model.We found that the N361-369 epitope could be normally processed and endogenously presented by these different types of antigen presenting cells,to elicit successful activation and effective cytotoxicity of CD8+T cells ex vivo.Our study evidenced potential mechanisms of cellular immunity to SARS-CoV-2,and illuminated potential ways of viral clearance in COVID-19 patients.These results indicate that utilizing CD8-independent TCRs against SARS-CoV-2-associated antigens may provide functional superiority that is beneficial for the adoptive cell immunotherapies based on natural or genetically engineered T cells.Additionally,this information is highly relevant for the development of the next-generation vaccines with protections against continuously emerged SARS-CoV-2 mutant strains.

Efficient control of chronic LCMV infection by a CD4 T cell epitope-based heterologous prime-boost vaccination in a murine model

CD4^(+)T cells are essential for sustaining CD8^(+)T cell responses during a chronic infection.The adoptive transfer of virus-specific CD4^(+)T cells has been shown to efficiently rescue exhausted CD8^(+)T cells.However,the question of whether endogenous virus-specific CD4^(+)T cell responses can be enhanced by certain vaccination strategies and subsequently reinvigorate exhausted CD8^(+)T cells remains unexplored.In this study,we developed a CD4^(+)T cell epitope-based heterologous prime-boost immunization strategy and examined the efficacy of this strategy using a mouse model of chronic lymphocytic choriomeningitis virus(LCMV)infection.We primed chronically LCMV-infected mice with a Listeria monocytogenes vector that expressed the LCMV glycoprotein-specific I-Ab-restricted CD4^(+)T cell epitope GP61–80(LM-GP61)and subsequently boosted the primed mice with an influenza virus A(PR8 strain)vector that expressed the same CD4^(+)T cell epitope(IAV-GP61).This heterologous prime-boost vaccination strategy elicited strong anti-viral CD4^(+)T cell responses,which further improved both the quantity and quality of the virusspecific CD8^(+)T cells and led to better control of the viral loads.The combination of this strategy and the blockade of the programmed cell death-1(PD-1)inhibitory pathway further enhanced the anti-viral CD8^(+)T cell responses and viral clearance.Thus,a heterologous prime-boost immunization that selectively induces virus-specific CD4^(+)T cell responses in conjunction with blockade of the inhibitory pathway may represent a promising therapeutic approach to treating patients with chronic viral infections.

Yeast display of MHC-II enables rapid identification of peptide ligands from protein antigens(RIPPA)

CD4^(+)T cells orchestrate adaptive immune responses via binding of antigens to their receptors through specific peptide/MHC-II complexes.To study these responses,it is essential to identify protein-derived MHC-II peptide ligands that constitute epitopes for T cell recognition.However,generating cells expressing single MHC-II alleles and isolating these proteins for use in peptide elution or binding studies is time consuming.Here,we express human MHC alleles(HLA-DR4 and HLA-DQ6)as native,noncovalentαβdimers on yeast cells for direct flow cytometry-based screening of peptide ligands from selected antigens.We demonstrate rapid,accurate identification of DQ6 ligands from pre-pro-hypocretin,a narcolepsy-related immunogenic target.We also identify 20 DR4-binding SARS-CoV-2 spike peptides homologous to SARS-CoV-1 epitopes,and one spike peptide overlapping with the reported SARS-CoV-2 epitope recognized by CD4^(+)T cells from unexposed individuals carrying DR4 subtypes.Our method is optimized for immediate application upon the emergence of novel pathogens.

MHC Class Ⅰ Antigen Presentation-Recently Trimmed and Well Presented

Presentation of antigenic peptide to T cells by major histocompatibility complex (MHC) class I molecules is the key to the cellular immune response.Non-self intracellular proteins are processed into short peptides and transported into endoplasmic reticulum (ER) where they are assembled with class I molecules assisted by several chaperone proteins to form trimeric complex.MHC class I complex loaded with optimised peptides travels to the cell surface of antigen presentation cells to be recognised by T cells.The cells presenting non-self peptides are cleared by CD8 positive T cells.In order to ensure that T cells detect an infection or mutation within the target cells the process of peptide loading and class I expression must be carefully regulated.Many of the cellular components involved in antigen processing and class I presentation are known and their various functions are now becoming clearer.Cellular & Molecular Immunology.2004;1(1):22-30.

Computational Analysis of Cysteine Proteases(Clan CA,Family C1) of Leishmania major to Find Potential Epitopic Regions

Leishmania is associated with a broad spectrum of diseases, ranging from simple cutaneous to invasive visceral leishmaniasis. Here, the sequences of ten cysteine proteases of types A, B and C of Leishmania major were obtained from GeneDB database. Prediction of MHC class I epitopes of these cysteine proteases was performed by NetCTL program version 1.2. In addition, by using BcePred server, different structural properties of the proteins were predicted to find out their potential B cell epitopes. According to this computational analysis, nine regions were predicted as B cell epitopes. The results provide useful information for designing peptide-based vaccines.