HLA-A＊02／24限制的HBcAg表位特异性细胞毒T细胞克隆的建立及分析

目的建立HLA-A＊0201／2403限制的HBcAg表位特异性CTL细胞克隆。方法HLA-A＊0201／2403阳性HBV感染者的外周血单个核细胞（PBMC）分别用限制性表位肽HBc18-27和HBc117-125刺激,并以有限稀释法进行克隆化,期间单独使用植物血凝素（PHA）及联合使用表位肽刺激。用免疫荧光、流式细胞术以及乳酸脱氢酶释放实验对所获克隆进行鉴定。结果从HBc18-27活化的细胞中获得29株细胞克隆,其中28株为CD8^＋T克隆,均具有特异性细胞毒活性。从HBc117-125激活的细胞中获得12株CD8＋T克隆,其中9株有明显的细胞毒活性,另3株细胞毒活性则显著低下。克隆化过程中单独使用PHA及联合使用表位肽,克隆形成率分别为15.62％和14.58％。结论分别用表位肽HBc18-27和HBc117-125,能激活HLA-A＊0201／2403阳性HBV感染者的CD8^＋T细胞。建立CD8^＋T克隆过程中,表位肽的使用不能提高克隆形成率。     

血吸虫反向疫苗学理论与技术的研究现状与展望

【提要】 近年发展起来的反向疫苗学,以“序列-结构-功能”思想为依据,以免疫信息学、计算机预测设计以及高通量的各种组学（包括基因组学、 转录本组学、 蛋白质组学等）综合集成技术为核心,可能为血吸虫疫苗候选分子的发现提供一条新途径。本文简要介绍了反向疫苗学常用的理论预测和实验方法;概述了应用反向疫苗学理论与技术筛选和鉴定血吸虫候选疫苗分子（保护性抗原或表位）的进展情况;最后,讨论了通过模拟辐照致弱血吸虫疫苗或人群获得性免疫的效应机制等来发展血吸虫反向疫苗学的可能的研究方向。     

Induction of specific and flavivirus—Cross-reactive CTLs by immunization with a single dengue virus-derived CTL epitope peptide

Specificities of cytotoxic T lymphocyte (CTL) effector cells induced in BALB/c mouse by immunization with the single modified CTL epitope peptide derived from NS3 of dengue virus types 1 and 3, or that of dengue virus types 2 and 4 were examined. The effector cells included CTLs specific for the epitope peptide used for immunization and those cross-reactive to epitope peptides of other flaviviruses. A CTL clone, 2F7, was established from the effector cells. The clone 2F7 was specific for the epitope peptide used for immunization. Recognition by the effector cells was remarkably impaired by amino acid substitutions at positions 3, 5, and 6 of the epitope peptides. These results indicate that immunization with a single CTL epitope peptide of dengue viruses induces serotype-specific CTLs as well as CTLs cross-reactive to the other flaviviruses, and that the a.a. residues at positions 3, 5, and 6 are critical for cross-reaction.     

A族链球菌(GAS)Fba蛋白单克隆抗体对应表位核心氨基酸的确定

目的:对A族链球菌Fba蛋白McAb2所对应的表位进行定位.方法:以初步定位的表位区段为线索合成三段重叠多肽,dot-ELISA检测McAb2与合成肽的亲合力,并以McAb2为靶分子,利用噬菌体随机七肽库进行亲和筛选,用竞争ELISA鉴定阳性克隆.结果:dot-ELISA检测结果表明Fba_(100-112)肽段与McAb2的结合能力最强.经过3轮亲和筛选后,随机挑选20个噬菌体克隆,竞争ELISA对其与McAb2的亲和力做检测,其中12个克隆显示较强的阳性结果.阳性克隆DNA测序,与Fba基因第100位氨基酸至110位氨基酸中ITPDL同源性较高.结论:通过dot-ELISA将A族链球菌Fba蛋白McAb2对应的表位定位于100～112位氨基酸,结果同噬菌体随机肽库筛选的核心氨基酸位置吻合.为进一步研制表位肽疫苗、研究Fba蛋白及其单克隆抗体的生物学功能奠定了基础.     

Functional properties and epitope characteristics of T-cells recognizing natural HIV-1 variants

To understand how broad recognition of HIV-1 variants may be achieved we examined T-cell reactivity in newly infected persons as well as vaccine recipients to a broad spectrum of potential T-cell epitope (PTE) variants containing conservative, semi-conservative and non-conservative amino acid substitutions. Among early infected persons T-cells recognized epitope variants with one substitution at a significantly higher frequency versus those with two (P = 0.0098) and three (P = 0.0125) substitutions. Furthermore T-cells recognized variants containing conservative substitutions at a higher frequency versus those containing semi-conservative (P = 0.0029) and non-conservative (P < 0.0001) substitutions. Similar effects were observed on recognition of variants by vaccine-induced T-cells. Moreover even when variants were recognized, the IFN-γ and granzyme B responses as well as T-cell proliferation were of lower magnitude. Finally, we show that epitope distribution is strongly influenced by both processing preferences and amino acid entropy. We conclude that induction of broad immunity is likely to require immunogen sequences that encompass multiple variants. However, cost-effective design of peptide and sequence based vaccine immunogens that provide maximal coverage of circulating sequences may be achieved through emphasis on virus domains likely to be T-cell targets.     

Peptide mimotopes of rabies virus glycoprotein with immunogenic activity

A random constrained hexapeptide phage display library (Cys-6aa-Cys) was screened with purified neutralizing human anti-rabies virus IgG antibodies (hRABVIgG) to identify peptides that correspond to or mimic natural epitopes on rabies virus glycoprotein (RABVG) and to investigate their immunogenicities in vivo. After four rounds of biopanning, 20 phage clones randomly selected for their specificity to hRABVIgG, effectively blocked the binding of the inactive rabies virus (RABV) to hRABVIgG. The phage clones were sequenced and the deduced amino acid sequences were derived (C-KRDSTW-C; C-KYLWSK-C; C-KYWLSR-C; C-KYWWSK-C; C-KYAWSR-C; C-KYSMSK-C). Alignments to the amino acid sequence of RABVG showed good match with the antigenic site III (at 330–338 aa), indicating that the hRABVIgG antibodies most likely recognize preferentially this antigenic site. The selected mimotopes were able to inhibit the interactions of the hRABVIgG antibodies with RABV in a dose-dependent manner. Subcutaneous administration of phageKRDSTW expressing the RABVG site III mimotope induced an RABVG-specific IgG response in BALB/c mice. The results indicated that peptide mimotopes when displayed on phages, are accessible to the mice immune system to trigger a humoral response and to induce IgG production. The RABVG site III mimotope (C-KRDSTW-C) would provide a new and promising concept for the development of rabies vaccine.     

A novel mouse model for immunogenic evaluation of human HBV vaccines

To prepare a human HBV vaccine, investigators need an animal model that can help them screen and prioritize vaccine candidates. In this study, HBV/HLA-A2.1 double transgenic mice (dTg), confirmed by analysis of genomic integration and by observation of long-term expression of HBV and HLA genes, were generated for the first time. The HBV/HLA-A2.1 dTg not only display tolerance to HBV antigens (Ags), but also have the ability to process and present HLA-A2 restricted antigenic epitopes. The animals vaccinated with HLA-A2 restricted HBc_18–27 or HBs_183–191 epitope peptide vaccine induced effective HLA-A2 restricted peptide-specific cytolytic T lymphocyte responses. This was supported by the evidence of cytotoxicity assay, ELISPOT and tetramer staining analysis. Furthermore, T cell tolerance against HBV Ags in HBV/HLA-A2.1 dTg was broken by the HBc_18–27 or HBs_183–191 peptide vaccine. In conclusion, HBV/HLA-A2.1 dTg was demonstrated to be useful model for in vivo immunogenicity testing of human HBV-based vaccines.     

Therapeutic efficacy of a multi-epitope vaccine against Helicobacter pylori infection in BALB/c mice model

Epitope vaccine is a promising option for therapeutic vaccination against Helicobacter pylori (H. pylori) infection. In this study, we constructed a multi-epitope vaccine with five epitopes and mucosal adjuvant E. coli heat-labile enterotoxin B subunit (LTB) named HUepi-LTB and evaluated its therapeutic effect against H. pylori infection in BALB/c mice model. HUepi-LTB containing three Th epitopes from UreB and two B cell epitopes from UreB and HpaA was constructed and expressed in E. coli. Oral therapeutic immunization with HUepi-LTB significantly decreased H. pylori colonization compared with oral immunization with PBS, and the protection was correlated with antigen-specific CD4⁺ T cells and IgG and mucosal IgA antibody responses. This multi-epitope vaccine may be a promising vaccine candidate that may help to control H. pylori infection.