Induction of T cell responses by chimeric bacterial proteins expressing several copies of a viral T cell epitope

A viral T cell epitope was genetically inserted within the periplasmic MalE protein of Escherichia coli in two different permissive insertion sites and resulting hybrid proteins were used to study the in vitro and in vivo immunogenicity of the foreign T cell epitope. Purified hybrid MalE proteins containing the T cell epitope 120-132 (PreS:T) from PreS2 region of hepatitis B virus HBsAg inserted alone or with its adjacent B cell epitope (132-145) were able to induce strong peptide-specific T cell responses in mice. In vitro stimulation of primed lymph node cells or specific T cell hybridomas by the hybrid proteins required processing of the inserted T cell epitope and was inhibited by antigen-presenting cells fixation. The inserted T cell epitope was presented in vitro, in association with appropriate major histocompatibility complex molecules, as efficiently as free synthetic peptide. The in vitro immunogenicity of MalE hybrid proteins was increased by inserting four tandemly repeated copies of PreS:T, either at site 133 or 303. These results were confirmed in vivo by comparing the proliferative responses of lymph node cells from DBA/1 mice primed with MalE hybrid proteins containing one or four copies of PreS:T. Thus, the use of MalE hybrid proteins expressing multiple copies of a given foreign T cell epitope allows the induction of peptide-specific T cell response with a lower dose of priming antigen.     

Characterizations of four monoclonal antibodies against M2 protein ectodomain of influenza A virus

M2 protein of influenza A virus has been implicated as a target for vaccines with broad cross-strain coverage. Studies in small animal models have shown that antibody responses induced by 23-mer M2 peptide vaccines can provide protection against influenza A virus challenge. To study antiviral mechanisms of Merck M2-OMPC conjugate vaccine, we generated and characterized four M2 peptide-specific monoclonal antibodies (mAbs). Here we demonstrated that the protection by our M2 mAbs is independent of NK-mediated effector functions in mice. The protective mAbs preferentially bind to M2 multimers composed of two or more M2 peptides in parallel orientation. Our findings indicate that the protective M2 Ab prefer to bind to epitopes located within the N-terminal 10 amino acids of the M2 peptide, and the epitopes are likely formed by two M2 peptides in parallel orientation. The implications of these results in antiviral mechanisms of immune responses induced by M2 vaccines are discussed.     

Degeneracy of T cell receptor recognition of an influenza virus hemagglutinin epitope restricted by HLA-DQ and -DR class II molecules

The T cell receptor (TcR) recognizes antigens in the form of short peptide fragments bound to major histocompatibility (MHC) molecules. TcR have an immunoglobulin (Ig)-like structure and, in an analogous manner to antigen recognition by Ig, the third complementarity determining regions (CDR3) of the TcR are believed to provide the primary contact with the peptide lying in the MHC groove. CDR1 and CDR2 are thought to contact the presenting MHC molecule. We have analyzed seven human CD4⁺ T cell clones that recognize a conserved peptide epitope (residues 255–270) within the influenza virus hemagglutinin (H3) HA1 subunit. Two T cell clones recognized the peptide in the context of HLA-DRB1*1001 and HLA-DQB1* 0602/DQA1*0102, respectively, and shared V_α, V_β and J_β gene segments. Only the junctional regions encoding the CDR3 regions of the two TcR chains were different. This suggests that the CDR3 regions of these TcR interact with the MHC class II molecule. Six of the T cell clones were restricted by the HLA-DRB1*1001. Two of these T cell clones expressed Vβ9.1 and three expressed Vβ13 gene segments; the remaining clone expressed Vβ7.2, a close homologue of V_β9.1. A diverse selection of V_α and J gene segments contributed to the junctional heterogeneity of the TcR, indicating a diversity of sequence combinations recognizing the epitope. Nevertheless, five out of six T cell clones bore a motif in the V_α CDR3 loop consisting of adjacent acidic and polar amino acid residues, eight residues from the carboxyl end of each CDR3.     

Definition of three minimal T helper cell epitopes of rubella virus E1 glycoprotein

To characterize T cell-recognized epitopes on rubella virus (RV) E1 glycoprotein, IL-2-dependent RV-specific T cell lines were established from 14 rubella-seropositive healthy donors. The responses of these lines were studied by using a panel of 94 partially overlapping synthetic peptides of 15 amino acids (aa) length covering the known nucleotide sequence of RVE1 glycoprotein. Two to seven peptide-defined epitopes were recognized by the T cell lines, but a large interindividual variation was found. T cell reactivity was most often localized to the regions between aa 276 and 290, aa 381 and 395 and aa 410 and 420. Analysis of overlapping, truncated peptides revealed three minimal T helper cell epitopes VIGSQARK, KFVTAALLN and RVIDPAAQ in aa positions 280–287, 385–393 and 412–419, respectively.     

流感病毒膜蛋白M2e应用研究进展

流行性感冒是一种传染性较高的急性呼吸道疾病,极大地威胁了人类健康.由于流感病毒易发生变异,致目前的防治措施不能及时有效地应对,使防治效果严重降低.流感病毒膜蛋白M2e(extracellular domain of the M2 protein)高度保守,是流感病毒的通用性作用靶点,基于M2e的免疫预防措施可提供广谱的保护作用.M2e疫苗及M2e抗体已成功在动物模型中发挥抗流感作用,临床应用则报道较少,因而研究M2e对于流感病毒的防治具有重要意义.     

CD8 T cell responses to influenza virus infection in aged mice

Influenza is one of the most common infectious diseases afflicting humans, particularly the elderly. The murine model has been widely employed for investigation of immunity to influenza virus infection. In this paper, we review the recent advances in understanding the diminished CD8 T cell immune response to influenza virus infection in aged mice. Possible mechanisms of impaired CD8 T cell responses with aging are addressed, including: (1) the role of dendritic cells (DCs); (2) the effect of age-associated changes in the T cell repertoire; and (3) the interactions with CD4 T cells, including T regulatory (Treg) cells and CD4 T helper cells. The aged murine model of the CD8 T cell response to influenza virus is helping to elucidate the mechanisms of immunosenescence which can lead to therapeutic improvements in the primary CD8 T cell response to new infections, as well as the development of new strategies for immunization to prevent influenza in the elderly.     

Expression of the influenza M2 protein in three different eukaryotic cell lines

Current influenza virus vaccines provide protection in part by antibodies induced to the two surface glycoproteins, the hemagglutinin and the neuraminidase. As a result of the continuous antigenic drift of these glycoproteins, a frequent update of the composition of influenza vaccines is required. The search for more conserved viral epitopes which would induce protective immunity against seasonal influenza viruses and eventually also to novel pandemic influenza viruses has a long history. The ectodomain of the Influenza A Virus M2 Protein has been identified as a possible candidate immunization against influenza. The present study describes the expression of cloned M2 gene in MDCK, HeLa, and COS-7 cells, i.e., in three established eukaryotic cell lines. The expression efficiency was demonstrated by immunofluorescent staining of transfected cells by ELISA, by SDS-PAGE-, and by Western blot-analysis. High level of expression was observed in COS-7 cells. Expression in HeLa and MDCK cells was less efficient. The plasmids constructed in this study may, after modifications, be used for the production of a DNA vaccine. Alternatively the expression product could be refined and used as a purified antigen for the vaccine. Thus, the M2 recombinant protein provides an ideal product for further antigenic, biochemical, structural and functional characterization of the protein and for evaluating its potential for immunodiagnosis and in vaccine studies.     

Rescue of influenza virus expressing GFP from the NS1 reading frame

In this study, several influenza NS1 mutants were examined for their growth ability in interferon (IFN)-deficient Vero cells treated with human interferon alpha (IFN-alpha). Mutants with an intact RNA binding domain showed similar growth properties as the wild-type virus, whereas viruses carrying an impaired RNA binding domain were dramatically attenuated. Relying on the ability of the first half of the NS1 protein to antagonize the IFN action, we established a rescue system for the NS gene based on the transfection of one plasmid expressing recombinant NS vRNA and subsequent coinfection with an IFN sensitive helper virus followed by adding of human IFN-alpha as a selection drug. Using this method, a recombinant influenza A virus expressing green fluorescence protein (GFP) from the NS1 reading frame was rescued. To ensure the posttranslational cleavage of GFP from the N-terminal 125 amino acids (aa) of NS1 protein, a peptide sequence comprising a caspase recognition site (CRS) was inserted upstream the GFP protein. Although a rather long sequence of 275 aa was inserted into the NS1 reading frame, the rescued recombinant vector appeared to be genetically stable while passaging in Vero cells and was able to replicate in PKR knockout mice.     

Microheterogeneity in the recognition of a HLA-DR2-restricted T cell epitope from a meningococcal outer membrane protein

The trimolecular interaction of T cell receptor (TcR), antigen and major histocompatibility complex (MHC) class II was analyzed using a panel of HLA-DR2-restricted T cell clones recognizing the 49-61 region of a meningococcal class I outer membrane protein (OMP). The clones, all CD3⁺CD4⁺CD8^?TcRct/β⁺, were selected by restimulation with the synthetic peptide OMP(49-61), which contains an immunodominant T helper determinant. Using a series of peptides that were sequentially truncated from the N or C terminus, four different epitope fine-specificity patterns were identified. Furthermore, each clone was found to exhibit a distinct recognition pattern for a panel of 20 single-residue substitution analogues of the minimal epitope OMP(50-58). Most substitutions that were not tolerated in the nonamer were allowed when the analogues were prepared departing from the native peptide OMP(49-61). Obviously, the residues outside the minimal epitope contribute to stabilization of the trimolecular complex. These findings suggest that defining the minimal size of T cell determinants may be of limited value. By performing proliferation competition assays putative MHC and TcR contact residues were identified in the peptide. Most likely, He 51 and Phe 54 act as MHC-anchoring residues, whereas Asp 53 represents a critical TcR contact residue for all of the clones. MHC anchoring may be provided by other residues as well, since He 51 and Phe 54 can be substituted by conservative residues [as OMP(50-58) and OMP(49-61) analogues] and with Ala [as OMP(49-61) analogues only]. Some evidence was found for interaction of particular side chains at other positions with TcR molecules, but this contribution was not equally important for all clones. Apparently, the clonotypic TcR can see a single epitope in different ways in the context of the same MHC restriction element. Since most clones use different V_α and V_β genes (which encompass the putative MHC-binding regions first and second complementarity-determining regions, CDR1 and CDR2) different modes of interaction with the HLA-DR2 molecule indeed are likely to occur.     

Protective immunity against H5N1 influenza virus by a single dose vaccination with virus-like particles

We generated influenza virus-like particles (VLPs) containing the wild type (WT) H5 hemagglutinin (HA) from A/Viet Nam/1203/04 virus or a mutant H5 HA with a deletion of the multibasic cleavage motif. VLPs containing mutant H5 HA were found to be as immunogenic as VLPs containing WT HA. A single intramuscular vaccination with either type of H5 VLPs provided complete protection against lethal challenge. In contrast, the recombinant H5 HA vaccine was less immunogenic and vaccination even with a 5 fold higher dose did not induce protective immunity. VLP vaccines were superior to the recombinant HA in inducing T helper type 1 immune responses, hemagglutination inhibition titers, and antibody secreting cells, which significantly contribute to inducing protective immunity after a single dose vaccination. This study provides insights into the potential mechanisms of improved immunogenicity by H5 VLP vaccines as an approach to improve the protective efficacy against potential pandemic viruses.     

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。     

Definition of an immunodominant T cell epitope contained in the envelope gp41 sequence of HIV-1.

The majority of the immunodominant amino acid sequences of HIV-1 that have been characterized to date are coded for by hypervariable gene sequences. These variable sequences are however interspersed with sequences that are highly conserved between HIV strains. Immunogenic viral products with amino acid sequences that vary minimally between strains, and that consistently elicit both humoral and cellular immune responses, may be ideal for inclusion in a subunit vaccine. We studied HIV-seronegative and HIV-infected persons, classified as asymptomatic (AS), ARC or AIDS. Initially, we assessed the cellular immune status of each subject from results of T cell phenotype analyses, assays for serum levels of surrogate markers of disease progression, and responses to mitogens and recall antigen. In addition, we tested whether three short synthetic peptides derived from the conserved sequences of the envelope gp120 (aa 262-284) and gp41 (aa 579-601), and core p17 (aa 106-125) regions of the HTLV-IIIB isolate, could elicit B cell as well as T cell responses in HIV-infected subjects. Only the gp41-derived sequence was immunogenic at both B and T cell levels. To further characterize the gp41 epitope, we used a series of overlapping synthetic peptides derived from a conserved region of the envelope gp41 (aa 572-613). We thus identified an immunodominant 12-mer peptide sequence, gp41(8)(aa 593-604), which consistently elicited both T cell blastogenic and B cell (antibody) responses in AS HIV-seropositive individuals but not in ARC and AIDS patients. Linear regression analysis showed that in AS persons there was a strong positive correlation (P less than 0.0005) between the absolute CD8+ T cell numbers and the magnitude of blastogenic responses to the gp41(8)(aa 593-604). Furthermore, those AS subjects with T cells that proliferated in response to this gp41 analogue also had significantly greater serum levels of antibody to the same short peptide sequence than symptomatic ARC and AIDS patients. These results suggest that cellular responses to the immunodominant and highly conserved envelope sequences of HIV-1, associated with increased CD8+ T cells, may be important in the pathogenesis of HIV disease.     

The polybasic region is not essential for membrane binding of the matrix protein M1 of influenza virus

The matrix protein M1, the organizer of assembly of influenza virus, interacts with other virus components and with cellular membranes. It has been proposed that M1 binding to lipids is mediated by its polybasic region, but this could hitherto not been investigated in vivo since M1 accumulates in the nucleus of transfected cells. We have equipped M1 with nuclear export signals and showed that the constructs are bound to cellular membranes. Exchange of the complete polybasic region and of further hydrophobic amino acids in its vicinity did not prevent association of M1 with membranes. We therefore suppose that M1 probably interacts with membranes via multiple binding sites.     

流感病毒RNA聚合酶蛋白PB1在小鼠中可诱导亚型间交叉免疫保护

目的探索流感病毒RNA聚合酶PB2和PB1亚基作为实验性流感疫苗候选抗原的可能性.方法以复制型质粒（pSCA）为载体分别构建表达甲1型和甲3型流感PB2和PB1的复制型DNA疫苗,免疫小鼠后分别用甲1型流感病毒（A/PR/8/34）进行鼻腔攻击,观察针对不同亚型流感病毒的复制型DNA疫苗的免疫保护效果.结果本实验所构建的复制型DNA疫苗在真核细胞中均可表达外源基因;本实验采用的复制型质粒载体（pSCA）与传统质粒载体（pcDNA3）在诱导小鼠产生抗体方面无差异,并且都诱导了偏向TH1类的免疫反应;表达甲1型和甲3型流感PB1基因的复制型DNA疫苗均可保护小鼠抵御甲1型流感病毒（A/PR/8/34）的攻击.结论表达甲型流感病毒PB1的复制型DNA疫苗能保护小鼠抵御同型和异型流感病毒的攻击,本实验为流感疫苗研究提供新的候选抗原.     

Immunoinformatics approach for a novel multi-epitope subunit vaccine design against various subtypes of Influenza A virus

Vaccination is the best strategy for the control and prevention of contagious diseases caused by Influenza A viruses. Extraordinary genetic variability and continual evolvability are responsible for the virus having survival and adaptation to host cell immune response, thus rendering the current influenza vaccines with suboptimal effectiveness. Therefore, in the present study, using a novel immunoinformatics approach, we have designed a universal influenza subunit vaccine based on the highly conserved epitopic sequences of rapidly evolving (HA), a moderately evolving (NP) and slow evolving (M1) proteins of the virus. The vaccine design includes 2 peptide adjuvants, 26 CTL epitopes, 9 HTL epitopes, and 7 linear BCL epitopes to induce innate, cellular, and humoral immune responses against Influenza A viruses. We also analyzed the physicochemical properties of the designed construct to validate its thermodynamic stability, hydrophilicity, PI, antigenicity, and allergenicity. Furthermore, we predicted a highly stable tertiary model of the designed subunit vaccine, wherein additional disulfide bonds were incorporated to enhance its stability. The molecular docking and molecular dynamics simulations of the refined vaccine model with TLR3, TLR7, TLR8, MHC-I and MHC-II showed stable vaccine and receptors complexes, thus confirming the immunogenicity of the designed vaccine. Collectively, these findings suggest that our multi-epitope vaccine construct may confer protection against various strains of influenza A virus subtypes, which could prevent the need for annual reformulation of vaccine and alleviate disease burden.     

甲型流感病毒NS1抗原捕获ELISA的建立

目的 建立基于单克隆抗体的甲型流感病毒非结构蛋白1(NS1)抗原检测的酶联免疫吸附(ELISA)法.方法 用甲型流感病毒NS1特异性单克隆抗体,通过抗体的优化组合,建立双抗体夹心抗原捕获ELISA,检测不同来源的流感病毒及副流感病毒.结果 对多种抗体组合进行反复筛选,最终确定了特异性检测到甲型流感病毒的NS1蛋白,而与乙型流感病毒和副流感病毒不发生交叉反应的最佳抗体组合.该方法 检测重组H5N1-NS1[A/HongKong/486/97(H5N1)-NS1和A/Vietnam/1194/04(H5N1)-NS1]蛋白的灵敏度最低检测值分别为15.6 ng/ml和240 pg/ml.结论 成功建立了甲型流感病毒NS1抗原捕获ELISA,为建立甲型流感病毒感染早期诊断新方法 奠定基础.     

Analysis of antibody response to the measles virus using synthetic peptides of the fusion protein Evidence of non-random pairing of T and B cell epitopes

The measles virus induces a life-long immune response associated with antibodies specific for the fusion protein. To map the linear immunodominant recognition sites of the fusion (F) protein of the measles virus, we have reacted a complete set of 108 overlapping pentadecapeptides with purified IgG obtained from donor sera with elevated anti-measles titers. The antibodies recognized about 20% of the peptides and generated a characteristic binding pattern, defining about 6 or 7 distinctive regions (31–75; 111–145; 151–165; 191–215; 271–320; 421–440; 481–530) which include the major hydrophobic segment (111–145) of the intersubunit region and the C-terminal Cys-cluster region. The binding sites were located in close proximity of the few experimentally defined T cell epitopes. This pairing of T and B cell epitopes was corroborated by computer-assisted T cell prediction. The significance of a non-random association of T and B cell epitopes for processing and presentation is discussed. It is speculated that in long-term immunity against measles (F protein), B cells of the same sIg specificity play an important role both as antigen presenting cells and as antibody producing cells. In contrast to human sera from late convalescent donors, mouse and rabbit MV antisera with high neutralizing titers as well as neutralizing MV-F specific monoclonal antibodies did not react with the peptides.