汉滩病毒核衣壳蛋白C-端T细胞表位鉴定

目的　鉴定汉滩病毒核衣壳蛋白 (HTNVNP)C 端T细胞表位 ,为肾综合征出血热(HFRS)发病机理、疫苗研制及抗病毒免疫反应研究奠定基础。方法　采用Ficoll密度梯度离心法分离HFRS恢复期患者外周血单个核细胞 (PBMC)。用IFN γELISPOT实验和T细胞增殖实验 ,测试 7名患者PBMC对 2 3条NPC 端合成多肽的T细胞应答。结果　IFN γELISPOT实验结果表明 ,2名供体(3、4 )可分别检测到对 5 1、70号 2条多肽特异性T细胞应答。在供体 3,70号肽特异性T细胞频率为4 5SFC 10 6 PBMC ;在供体 4 ,5 1号肽特异性T细胞频率为 82SFC 10 6 PBMC。T细胞增殖实验与ELISPOT结果基本一致 ,但 5 3号肽和 6 4号肽还可分别刺激供体 1和供体 4的T细胞增殖 ,而未能诱导IFN γ分泌。结论　 5 1号和 70号多肽可能是NPC 端较强的T细胞表位。     

人类偏肺病毒F蛋白的B细胞表位预测

目的 预测人类偏肺病毒F蛋白的B细胞表位。方法综合分析二级结构、亲水性、表面可及性与抗原性指数，预测F蛋白的抗原表位。结果B细胞表位位于F蛋白N段15-28、88-102、161-189、195-202、206-213、245-257、290-299、302-309、318-334、410-419、515-526区段。结论应用多参数预测F蛋白的B细胞表位，为进一步研究蛋白特征、单克隆抗体制备及表位疫苗研制奠定了基础。     

汉滩病毒核蛋白T细胞表位的鉴定及其特异性T细胞应答规律的初步探讨

目的:鉴定引起肾综合征出血热(HFRS)的汉滩病毒核蛋白(HTNV-NP)的T细胞表位,并探讨其特异性T细胞应答的规律。方法:合成覆盖HTNV-NP全长序列的全套部份重叠15肽,用ELISPOT方法筛选能刺激HFRS患者外周血单个核细胞(PBMC)产生IFN-γ的15肽,并测定其特异性T细胞的频率。结果:47例HFRS患者中,有18例可对HTNV-NP产生特异性T细胞应答,并发现在病程的早期就已开始产生T细胞应答。从11例患者PBMC中,鉴定出了17种HTNV-NP特异的T细胞表位,其中14种T细胞表位为首次报道,T细胞表位主要位于NP一级结构的中部。结论:HFRS病程早期即可产生特异性T细胞应答,T细胞表位主要位于NP序列的中部,T细胞应答可能在汉滩病毒感染过程中起重要的免疫保护作用。     

旋毛虫抗原分子克隆及其T细胞和B细胞表位预测

目的 克隆旋毛虫肌幼虫Mr21 000抗原蛋白基因(Ts21),预测其T细胞和B细胞表位.方法 旋毛虫(河南地理株)感染小鼠后收集肌幼虫,提取肌幼虫总RNA,应用RT-PCR技术获取目的基因Ts21,构建重组质粒pUC18-Ts21并测序,应用生物信息分析软件预测其T细胞和B细胞表位.结果 成功构建克隆载体pUC18-Ts21.旋毛虫Mr21 000抗原的T细胞表位位于第9～23、135～150位氨基酸位点;B细胞表位位于第31～35、53～56、98～104、124～130、133～157、160～172位氨基酸位点.结论 成功克隆了旋毛虫河南地理株肌幼虫Mr21 000抗原的编码基因,T细胞和B细胞表位预测结果表明该抗原蛋白具有较好的抗原性,可作为旋毛虫病免疫诊断和预防的候选抗原.     

类风湿性关节炎患者单个核细胞对合成肽反应性研究

目的：寻找与类风湿性关节性相关的Ｔ细胞表位。方法：用４种合成的短肽；ＤＷ１５肽，ＨＳＰ肽，ＧＲＰ肽，ＣⅡ肽，体外刺激从类风关患者外周血，关节液，滑膜中得到的单个核细胞。结果：关节内（关节液＋滑膜）ＭＮＣ对ＧＲＰ肽的反应与正常对照差异显著。关节内ＭＮＣ对ＰＨＡ刺激的反应显著低于患者及正常人外周血ＭＮＣ。结论：ＧＲＰ肽是部分类风关患者关节内Ｔ细胞的Ｔ细胞表位。     

Induction of T cell response by bluetongue virus core-like particles expressing a T cell epitope of the M1 protein of influenza A virus

A CD4⁺ T cell epitope of the influenza virus matrix protein corresponding to the C terminus (QAYQKRMGVQMQRFK) was inserted into the VP7 gene of bluetongue virus (BTV). The chimeric protein was expressed by a dual recombinant Autographa californica polyhedrosis virus (AcNPV), which encodes the two inner capsid proteins VP3 and VP7 of BTV. When Spodoptera frugiperda cells (Sf9 cells) were infected with this recombinant BTV, core-like particles (CLPs) were formed as demonstrated by electron microscopy. To study the immunogenicity of a foreign epitope deprived of its natural flanking sequences in vitro, purified CLPs expressing the T cell epitope were used to stimulate two different MHC class II-restricted CD4⁺ human T cell clones. One of these T cell clones, ALF 3.7 was specific for the inserted epitope, whereas the other T cell clone ALF 4.4 recognized shorter derivates of the given epitope. CLPs with the inserted epitope were presented as efficiently as purified influenza virus matrix protein to the clone ALF 3.7, whereas clone ALF 4.4 showed no proliferative response. Received: 17 February 1998     

重组卡介苗表达的MalE蛋白T细胞表位的鉴定

目的 鉴定重组卡介苗表达的MalE蛋白T细胞表位。方法 用抗原提呈试验、T细胞增殖试验、ELISPOT试验和表位作图测试重组MalE蛋白的T细胞表位。结果 重组BCG.MalE功能性表达了MalE蛋白的4种H-2^d限制性T细胞表位。结论 在4种表位中，p68-82是重组MalE蛋白的主要T细胞表位，而其余3个为次要表位。     

Identification of human T cell epitopes in the Mycobacterium leprae heat shock protein 70-kD antigen.

In a number of pathogens, heat shock proteins (hsp) stimulate humoral and cellular immune responses despite significant sequence identity with host hsp. The 70-kD hsp of Mycobacterium leprae, which shares 47% identity with human hsp70 at the protein level, elicited a T cell response in most Myco. bovis (bacille Calmette-Guérin (BCG)) vaccinees as well as leprosy and tuberculosis patients and their contacts. In order to locate T cell epitopes, DNA fragments encoding portions of the 70-kD hsp were expressed in the vector pGEX-2T and tested for T cell reactivity in an in vitro proliferative assay. Cultures of peripheral blood mononuclear cells (PBMC) from BCG vaccinees indicated that the C-terminal half of the molecule contained multiple T cell epitopes, as the T cells from a majority of Myco. leprae hsp70-reactive individuals responded to C-344. Lower proportions of patients with paucibacillary leprosy (36%) and tuberculosis patients (16%) responded to C-344. The smaller C-142 fragment which includes the terminal 70 residues unique to Myco. leprae and is the target for the human antibody response elicited a cellular response in few patients and no vaccinees. In order to map T cell epitopes, two series of synthetic peptides encompassing the region 278-502 were prepared. Using overlapping 12mer and 20mer peptides, this region of the molecule was found to contain several potential T cell epitopes. The longer peptides gave a clearer indication of reactive sequences including regions of the molecule which were not identified with the 12mer peptides. Fine mapping of reactive peptide pools using the 12mer peptides identified two T cell epitopes. Although both were located in regions of the molecule shared with Myco. tuberculosis, one appeared to be cross-reactive with the equivalent human sequence, and thus has the potential to initiate autoimmune responses.     

T cell recognition of the posttranslationally cleaved intersubunit region of influenza virus hemagglutinin

The influenza virus hemagglutinin is synthesized as a single polypeptide chain, but upon maturation it will posttranslationally be modified by a host cell related trypsin-like enzyme. The enzymatic cleavage attacks the so-called intersubunit region of the molecule giving rise to covalently linked HA1 and HA2 subunits. An I-E^drestricted T cell epitope was identified in the highly conserved intact intersubunit region of the influenza virus hemagglutinin. T cell recognition of a 25-mer synthetic peptide comprising the intact intersubunit region does not require further processing and the elimination of the intervening Arg residue coupling the fusion peptide to the C-terminal segment of HA1 does not abolish the T cell activating capacity. The fine specificity pattern of a T cell hybridoma similar to that of the polyclonal T cell response demonstrates that a single T cell receptor is able to recognize peptides of different sizes representing not only the uncleaved but also the cleaved form of this hemagglutinin region. Based on specificity studies the epitope was localized to the C-terminal 11 amino acids of the HA1 subunit. The cross-reactivity of peptide-primed T cells with influenza virus infected antigen-presenting cells shows that fragments comprising the identified epitope of the intersubunit region can be generated as a result of natural processing of the hemagglutinin molecule. As antigen-presenting cells are lacking the enzyme which is responsible for the posttranslational modification of newly synthesized hemagglutinin molecules, the role of immature viral proteins in immune recognition is discussed.     

人偏肺病毒黏附蛋白的二级结构及B细胞表位初步预测

目的预测人偏肺病毒G蛋白的二级结构及B细胞表位。方法分别采用SOPMA方法及HMMTOP程序预测G蛋白的二级结构和跨膜区域；综合分析蛋白的柔性结构、亲水性、表面可及性与抗原性指数，预测G蛋白的抗原表位。结果G蛋白的二级结构主要为柔性区域，占62．1％；廿螺旋占22．37％；β-折叠占15．53％；N端第32～51位氨基酸残基为跨膜区。B细胞表位位于G蛋白N端55—77、80-104、111～126、130～167、178—210区段。结论应用多参数预测G蛋白的二级结构与B细胞表位，为进一步研究蛋白特征、单克隆抗体制备及表位疫苗研制奠定了基础。     

Human B-cell epitopes of puumala virus nucleocapsid protein,the major antigen in early serological response

Puumala virus (PUU) is a member of the Hantavi rus genus in the family Bunyaviridae and the etiologic agent of nephropathia epidemica (NE), a form of haemorrhagic fever with renal syndrome (HFRS). In this study we compared the immunofluorescence patterns of NE sera and antibodies raised against recombinant PUU proteins and confirm that the nucleocapsid protein is the major target in the early IgG response of NE patients and provides the molecular basis for simple and rapid differentiation between acute illness and old immunity by granular vs. diffuse fluorescence staining in the indirect immunofluorescence test. The differential kinetics of B-cell responses to PUU nucleocapsid vs. envelope proteins was emphasized further by the end-point titres of IgG antibodies to N, G1 and G2 proteins in NE patients. The granular fluorescence correlated with low IgG avidity in 99.8%. and diffuse fluorescence with high avidity in 100% of 617 NE sera studied. Epitope scanning with overlapping 14-mer peptides covering the whole nucleocapsid protein by a shift of 3 amino acids revealed six major antigenic epitopes recognized by sera from acute-phase NE patients. The epitopes clustered mainly in the hydrophilic regions, and two of them in a highly variable region which could probably serve as an antigen to distinguish serologically between infections of closely related hantaviruses, some apparently apathogenic, some causing lethal infections. The anti-peptide epitope pattern varied between different individuals and a collection of several pinbound peptides was needed to be recognised by most NE sera studied. © 1995 Wiley-Liss, Inc.     

滤泡调节性T细胞在病毒感染与疫苗免疫中的作用及机制研究进展

滤泡调节性T细胞(T follicular regulatory cell, Tfr)是近年来发现的一类CD4 ＋ T细胞亚群,其来源于调节性T细胞(T regulatory cell, Treg),兼具Treg细胞及滤泡辅助性T细胞(T follicular helper cell, Tfh)相似的生物学特...     

日本血吸虫钙蛋白酶肽链文库的制备及其B细胞表位的筛选

目的 筛选钙蛋白酶（calpain)的B细胞表位，探讨其B细胞表位的免疫原性及其所诱导的免疫应答类型。方法 用Genetyx-MAC9.0分析calpain肽连的亲水性，在亲水性区域以9个氨基酸为肽链单位的固相重叠钛链库（每相邻两个肽链之间仅有一个氨基酸不同）被Auto-spot Robot制备，用Dot-ELISA在合成的肽链库上筛选B细胞表位，含有B细胞表位的肽链免疫BALB／c小鼠，ELISA鉴别特异性IgG抗体亚类的产生。结果 由758个氨基酸组成的日本血吸虫calpain蛋白含有2个主要的亲水区，其存在区域分别在氨基酸229－375和555－613；当我们测定合成的肽链文库时，在亲水区内筛选出4个B细胞的表位，分别是氨基酸234－251（YAHLSGGT）；290－297（CLMGCSIH）；338－346（PWGDSHEW）；358－364（AWCDGAPQ）；与对照组鼠相比较免疫鼠血清中IgG1、IgG2a、IgG2b特异性抗体有显著性意义的上升。结论 日本血吸虫calpain是一个具有免疫原性的蛋白，能够激发宿主产生高水平的免疫球蛋白，提示calpain的B细胞表位可以成为日本血吸虫的诊断抗原和日本血吸虫疫苗候选分子。     

Dichotomy between the T and the B cell epitopes of the synthetic polypeptide (T,G)-A–L

Studies with the well-characterized, synthetic, random-multichain polypeptide poly(L Tyr, L Glu)-poly(DL Ala)–poly(L Lys) (T,G)-A–L), led to the discovery of determinant-specific genetic control of the immune response, as well as to other immunological phenomena. Moreover, the tetrapeptide TyrTyrGluGlu built on the same backbone (“T-T-G-G)-A–L”) was found to represent its major B cell epitope. We have recently shown that for interaction with major histocompatibility complex class II molecules and stimulation of T cells, (T, G)-A–L requires proteolytic processing and the resulting T cell epitopes are close to the N termini of the branched polymer's side chains. Thus, we were interested to elucidate the major T cell epitope of (T, G)-A–L, by using the ordered polypeptides (T-T-G-G)-A–L and (T-G-T-G)-A–L, in which only the two internal amino acids of the tetrapeptide attached to the side chains are switched. We established T cell lines to these antigens, and found that the ordered analog (T-T-G-G)-A–L, which was defined as the B cell epitope of (T,G)-A–L, did not represent its T cell epitope, whereas (T-G-T-G)-A–L, to which only a minor anti-(T,G)-A–L Ab response was directed, was found to be its major T cell epitope. In addition, there was no cross-reaction between (T-G-T-G)-A–L and (T-T-G-G)-A–L at the T cell level, similar to the lack of cross-reaction of their antibodies. Analysis of the repertoire of the T cell receptors used by these lines revealed that the (T,G)-A–L and the (T-T-G-G)-A–I specific T cell lines were not restricted in their Vα and Vβ TCR usage, whereas the (T-G-T-G)-A–L-specific line was restricted by both Vα and Vβ T cell receptor gene products. This difference might be due to the thymus-independent characteristics previously described for the latter antigen.     

Induction in vitro of a primary human antiviral cytotoxic T cell response

We report herein the successful priming of human anti-viral cytotoxic T cells (CTL) in vitro using two induction strategies based on the stimulation of peripheral blood mononuclear cells isolated from uninfected donors with synthetic viral peptides. The peptides used contain HLA-A2 binding motifs and have been identified as HLA-A2-restricted CTL epitopes in patients infected by the hepatitis B and C viruses. One approach uses repetitive long-term stimulation and the other uses bulk cultures containing large numbers of naive peripheral blood mononuclear cells. Both approaches successfully induce HLA-A2-restricted CTL specific for several viral epitopes. Some CTL recognize endogenously synthesized antigen on target cells infected with recombinant vaccinia virus expressing the corresponding viral proteins. This simple technique permits easy analysis of the primary human CTL repertoire, and may be exploitable for production of specific CTL effector cells for adoptive immunotherapy and dissection of the cellular and molecular requirements for priming of naive human CTL.     

Identification of epitopes in the nucleocapsid protein of Nipah virus using a linear phage-displayed random peptide library

A random peptide library of heptamers displayed on the surface of M13 bacteriophage was used to identify specific epitopes of antibodies in pooled sera of swine naturally infected by Nipah virus. The selected heptapeptides were aligned with protein sequences of Nipah virus and several putative epitopes were identified within the nucleocapsid protein. A total of 41 of 60 (68%) selected phage clones had inserts resembling a region with the sequence SNRTQGE, located at the C-terminal end (amino acids 503-509) of the nucleocapsid protein. The binding of antibodies in the swine and human antisera to the phage clone was inhibited by a synthetic peptide corresponding to this region. Epitopes identified by phage display are consistent with the predicted antigenic sites for the Nipah virus nucleocapsid protein. The selected phage clone used as a coating antigen discriminated swine and human Nipah virus sera-positive from sera-negative samples exhibiting characteristics, which might be attractive for diagnostic tests.     

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.