肿瘤抗原MAGE-A亚家族共同B细胞表位预测及分析

目的预测肿瘤抗原MAGE-A亚家族的B细胞表位,为基于多靶点的肿瘤疫苗设计提供依据。方法基于MAGE亚家族各成员蛋白质序列,采用Kyte-Doolittle的亲水性方案,Emini方案,Karplus方案和Jameson-wolf抗原指数方案,并辅以MAGE蛋白的二级结构柔性区域分析,预测MAGE基因家族的B细胞共同表位。结果共预测出了5条共同表位,且部分B细胞表位高度相似或一致。结论二级结构与B细胞表位相结合的预测方法为一种高效、准确的表位预测方法,可为肿瘤治疗性疫苗的设计提供实验依据。     

H5N1亚型禽流感病毒血凝素Th和B细胞表位预测及抗原性分析

目的:预测H5N1亚型禽流感病毒血凝素Th和B细胞相关抗原表位,并初步分析其抗原性.方法:依据近年H5N1亚型禽流感病毒流行趋势,下载得到相关HA蛋白氨基酸序列.进行生物信息学综合分析预测,获得Th和B细胞相关抗原表位,并比较其保守性和特异性.通过BALB/c小鼠和SPF鸡H5N1亚型禽流感病毒阳性血清,初步鉴定候选表位抗原性.结果:综合多项预测及空间构象模拟结果,我们获得了三条候选Th和B细胞表位,分别为HA141～155、HA206～223、HA302～316.候选表位处于H5N1亚型禽流感HA1 蛋白序列上相对保守的区域内,且与目前流行的H5N1亚型禽流感病毒HA相应区域具有较好的一致性.而不同候选表位在BALB/c小鼠和SPF鸡H5N1亚型禽流感病毒阳性血清反应中显示了不同抗体结合能力,预示了其成为功能表位的可能.结论:所筛选的表位具有成为H5N1亚型禽流感病毒HA Th和B细胞相关抗原表位的可能.本研究为深入揭示流感病毒感染与免疫机制,H5N1亚型禽流感功能表位认知及表位疫苗研究奠定了基础.     

H7N9流感病毒HA、NA蛋白的抗原表位预测及其与HLA-Ⅱ类等位基因的相关性分析

目的:分析H7N9流感病毒的HA、NA蛋白的抗原表位;预测H7N9流感病毒相关的HLA-Ⅱ类等位基因及易感人群。方法:软件分析HA、NA的同源性、B细胞表位、T细胞表位以及与HA、NA结合力强的HLA-Ⅱ类等位基因;并根据该等位基因在亚洲不同地域的基因频率,预测H7N9的易感人群。结果:H7N9流感病毒株之间氨基酸序列相对保守;HA具有10个B细胞表位和15个T细胞表位;NA有12个B细胞表位和9个T细胞表位;HLA等位基因DRB1*0701与HA、NA具有强结合力,在新疆、哈尔滨、山东、辽宁、北京、石家庄、天津等多个中国北方地区人群中的基因频率较高,高于广东、云南、台湾地区、日本和韩国。结论:预测了HA、NA的抗原性表位,为H7N9流感病毒的疫苗研究提供了理论基础;HLA-DRB1*0701与H7N9流感病毒高度相关;H7N9流感病毒在新疆、哈尔滨、山东、辽宁、北京、石家庄、天津地区更易传播。     

人易感H6N1禽流感病毒血凝素蛋白的B细胞抗原表位预测及进化分析北大核心CSCD

目的预测人易感H6N1禽流感病毒血凝素蛋白的B细胞抗原表位,并分析其进化特征。方法从GISAID和Gen Bank两大数据库获得人易感H6N1病毒的血凝素蛋白及近缘序列,采用多款预测软件分别预测了其B细胞线性和构象型抗原表位,并分析了这些表位的保守性、适应性和进化特征。结果综合各种因素共预测出4个线性表位(表位A、B、C和D)和2个构象型表位(表位E和F)。表位C和位点41、157、186、187在进化过程中易发生突变,其余表位较为保守,其中表位D最保守;位点157受到强烈的正选择作用,它可能是H6N1病毒逃避宿主免疫系统攻击的一个关键位点。结论人易感H6N1禽流感病毒血凝素蛋白拥有5个保守的B细胞抗原表位(3个线性、2个构象型)和1个正选择作用位点,将为其疫苗的研制、致病机制的理解和病毒防治提供理论基础。     

人易感H6N1禽流感病毒血凝素蛋白的B细胞抗原表位预测及进化分析

目的预测人易感H6N1禽流感病毒血凝素蛋白的B细胞抗原表位,并分析其进化特征。方法从GISAID和Gen Bank两大数据库获得人易感H6N1病毒的血凝素蛋白及近缘序列,采用多款预测软件分别预测了其B细胞线性和构象型抗原表位,并分析了这些表位的保守性、适应性和进化特征。结果综合各种因素共预测出4个线性表位(表位A、B、C和D)和2个构象型表位(表位E和F)。表位C和位点41、157、186、187在进化过程中易发生突变,其余表位较为保守,其中表位D最保守;位点157受到强烈的正选择作用,它可能是H6N1病毒逃避宿主免疫系统攻击的一个关键位点。结论人易感H6N1禽流感病毒血凝素蛋白拥有5个保守的B细胞抗原表位(3个线性、2个构象型)和1个正选择作用位点,将为其疫苗的研制、致病机制的理解和病毒防治提供理论基础。     

H1N1亚型流感病毒血凝素Th和B细胞表位预测及抗原性分析

目的:应用生物信息学方法预测H1N1亚型流感病毒血凝素Th和B细胞相关抗原表位,并初步分析其抗原性,为研制H1N1亚型流感病毒的表位疫苗奠定基础.方法:依据近年流感病毒流行趋势,从GenBank下载具有代表性的H1N1亚型流感病毒HA蛋白氨基酸序列.进行生物信息学综合分析预测,获得Th和B细胞相关抗原表位,并比较其保守性和特异性.通过Balb/c小鼠H1N1亚型流感病毒阳性血清与表位肽的结合试验,初步鉴定候选表位抗原性.结果:综合多项预测及空间构像模拟结果,我们获得了三条候选Th和B细胞表位,分别为HA_(73～87)、HA_(125～139)、HA_(188～205).候选表位处于H1N1亚型流感HAI蛋白序列上相对保守的区域内,且与目前流行的H1N1亚型流感病毒HA相应区域具有较好的一致性.而不同候选表位在BMB/e小鼠H1N1亚型流感病毒阳性血清反应中显示了不同抗体结合能力,预示了其成为功能表位的可能.结论:所筛选的表位具有成为H1N1亚型流感病毒HA Th和B细胞相关抗原表位的可能.此研究为深入揭示流感病毒感染与免疫机制,H1N1亚型流感功能表位认知及表位疫苗研究奠定了基础.     

E血清型沙眼衣原体主要外膜蛋白B细胞表位的预测

基于E血清型沙眼衣原体(Chlamydia trachomatis,CT)主要外膜蛋白(Major outer membrane protein, MOMP)氨基酸序列,采用Hopp-Woods的亲水性方案、Emini表面可及性方案、Jameson-Wolf 抗原指数方案和Janin可及性方案等,辅以对MOMP蛋白的二级结构中的柔性区域及跨膜区域的分析,预测CT MOMP蛋白的B细胞表位.推测最有可能的B细胞表位位于MOMP蛋白N端第73～81区段、217～225区段、第377～386区段、第261～270区段和第161～175区段内或它们的附近.用多参数预测CT MOMP蛋白的B细胞表位,为进一步研究蛋白特性及表位疫苗研制奠定了基础.     

H1N1流感病毒血凝素B细胞抗原表位的鉴定

目的利用计算机模拟和ELISA阻断实验研究流感病毒血凝素(HA)B细胞抗原表位,建立病原微生物表位检测新方法。方法以2009年H1N1流感病毒裂解疫苗作为免疫原,采用常规杂交瘤融合、筛选技术制备单克隆抗体(mAb),应用ELISA、血凝抑制试验(HI)及Western blot法鉴定获得mAb的特性。以获得的mAb为工具,联合应用ELISA阻断实验和计算机模拟方法预测H1N1流感病毒的B细胞抗原表位。结果获得4株抗H1N1流感病毒HA抗原的mAb,通过ELISA阻断实验将HA的B细胞抗原表位分为两类,通过计算机模拟预测发现4株抗体能与HA上的两类表位相结合。结论计算机模拟和ELISA阻断实验的结果一致,建立了用于预测其他病原微生物表位的新方法。     

结核分枝杆菌α晶体蛋白(Rv2031c)的T、B细胞抗原表位在线分析

目的预测结核分枝杆菌潜伏感染相关蛋白α晶体蛋白(Rv2031c)的抗原表位。方法 BLAST在线分析Rv2031c与人类蛋白的同源性;利用DNAStar软件包中的Protean模块预测其B细胞和T细胞抗原表位;RANKPEP和SYPEPITHI在线预测辅助性T(Th)细胞抗原表位;SYFPEPI、BIMAS和Net CTL方法在线预测细胞毒性T淋巴细胞(CTL)表位。结果 Rv2031c与人类蛋白同源性较低,有8个潜在的B细胞抗原表位,7个候选Th细胞抗原表位和3个候选CTL表位。结论 Rv2031c含有较多潜在的人类B细胞、Th细胞和CTL抗原表位,可作为新的结核病疫苗研发的候选靶蛋白。     

应用免疫信息学技术预测严重急性呼吸综合征冠状病毒2(SARS-CoV-2)的抗原表位

目的运用免疫信息学技术预测严重急性呼吸综合征冠状病毒2(SARS-CoV-2)的B细胞、细胞毒性T淋巴细胞(CTL)和辅助T(Th)细胞的抗原表位。方法从NCBI数据库检索SARS-CoV-2蛋白序列,根据抗原性≥0.5和氨基酸数≥100进行筛选,最终的蛋白序列用于后续的抗原肽的预测。用蛋白质结构预测软件Phyre2进行三维结构的预测、蛋白质模型结构的细化软件GalaxyRefine优化蛋白的三维结构,最后用蛋白质结构同源建模SWISS-MODEL系统对优化后的结构进行准确性评估。蛋白序列用于CTL、Th细胞和线性B细胞抗原肽预测,三维结构用于结构性B细胞抗原预测。免疫表位数据库和分析资源(IEDB)预测SARS-CoV-2的CTL和Th细胞抗原表位,B细胞线性抗原肽预测软件Bepipred Linear Epitope Prediction 2.0和B细胞结构抗原肽预测软件ElliPro-Epitope prediction based upon structural protrusion分别预测B细胞线性和结构抗原肽。结果从NCBI数据库获得了27个SARS-CoV-2的蛋白序列,去掉抗原性0.5和氨基酸数100的蛋白质后,最终选定9个蛋白进行后续抗原肽预测。最终获得了24个CTL、20个Th细胞、12个B细胞线性表位和16个B细胞结构表位。结论获得的抗原表位可用于后续多表位疫苗的设计,相较于只针对单种蛋白靶点的抗原表位而言,多靶点抗原表位具有更强的免疫原性,这些抗原表位对SARS-CoV-2疫苗而言,具有一定的参考价值。     

Genome-wide screening of human T-cell epitopes in influenza A virus reveals a broad spectrum of CD4 T-cell responses to internal proteins, hemagglutinins, and neuraminidases

We performed a genome-wide screening for T-cell epitopes using synthetic peptides that encompass all of the influenza A viral proteins, including subtype variants for hemagglutinin (HA; H1, H3, and H5) and neuraminidase (NA; human and avian N1 and N2) proteins, based on the sequence information of recently circulating strains. We identified a total of 83 peptides, 54 of them novel, to which specific T cells were detectable in interferon-γ (IFN-γ) enzyme-linked immunosorbent spot assays using peripheral blood mononuclear cells from four healthy adult donors. The surface glycoproteins, HA and NA, major components of vaccines, expressed many T-cell epitopes. HA and matrix protein 1 expressed more T-cell epitopes than other viral proteins, most of which were recognized by CD4⁺ T cells. We established several cytotoxic CD4⁺ T-cell lines from these donors. We also analyzed H1 and H3 HA-specific T-cell responses using the peripheral blood mononuclear cells of 30 hospital workers. Fifty-three percent of donors gave a positive response to H3 HA peptides, whereas 17% gave a positive response to H1 HA peptides. Our genome-wide screening is useful in identifying T-cell epitopes and is complementary to the approach based on the predicted binding peptides to well-studied HLA-A, -B, and -DR alleles.     

Contribution of antibody production against neuraminidase to the protection afforded by influenza vaccines

Vaccines are instrumental in controlling the burden of influenza virus infection in humans and animals. Antibodies raised against both major viral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), can contribute to protective immunity. Vaccine-induced HA antibodies have been characterized extensively, and they generally confer protection by blocking the attachment and fusion of a homologous virus onto host cells. Although not as well characterized, some functions of NA antibodies in influenza vaccine-mediated immunity have been recognized for many years. In this review, we summarize the case for NA antibodies in influenza vaccine-mediated immunity. In the absence of well-matched HA antibodies, NA antibodies can provide varying degrees of protection against disease. NA proteins of seasonal influenza vaccines have been shown in some instances to elicit serum antibodies with cross-reactivity to avian-origin and swine-origin influenza strains, in addition to HA drift variants. NA-mediated immunity has been linked to (i) conserved NA epitopes amongst otherwise antigenically distinct strains, partly attributable to the segmented influenza viral genome; (ii) inhibition of NA enzymatic activity; and (iii) the NA content in vaccine formulations. There is a potential to enhance the effectiveness of existing and future influenza vaccines by focusing greater attention on the antigenic characteristics and potency of the NA protein.     

A型H3579亚型流感多表位核酸疫苗小鼠实验免疫研究

目的为了应对流感流行多亚型并存的局面,进行了多表位核酸疫苗设计,并观察其免疫小鼠的细胞和体液免疫应答反应。方法设计并合成了含有H3、H9的HA和H5N1NA中和表位,M、NP基因保守序列CTL表位的多表位基因盒(Epi)。采用pVAX载体对H5HA、H7HA及Epi进行融合表达。经肌肉注射免疫6～8周龄雌性Balb/c小鼠,ELISA法检测小鼠血清中的针对H3579亚型流感抗体。三免后2周,分离脾淋巴细胞,进行T淋巴细胞转化试验和T淋巴细胞亚类数量检测。结果免疫小鼠获得了针对H3579亚型流感的体液和细胞免疫反应。结论完整HA抗原结合多表位的DNA疫苗模式的成功,预示了该DNA疫苗可能可以有效应对当前多种亚型流感并存的局面,并为其他种类流感疫苗的研究奠定了基础。     

Functional balance between haemagglutinin and neuraminidase in influenza virus infections

Influenza A and B viruses carry two surface glycoproteins, the haemagglutinin (HA) and the neuraminidase (NA). Both proteins have been found to recognise the same host cell molecule, sialic acid. HA binds to sialic acid-containing receptors on target cells to initiate virus infection, whereas NA cleaves sialic acids from cellular receptors and extracellular inhibitors to facilitate progeny virus release and to promote the spread of the infection to neighbouring cells. Numerous studies performed recently have revealed that an optimal interplay between these receptor-binding and receptor-destroying activities of the surface glycoproteins is required for efficient virus replication. An existing balance between the antagonistic HA and NA functions of individual viruses can be disturbed by various events, such as reassortment, virus transmission to a new host, or therapeutic inhibition of neuraminidase. The resulting decrease in the viral replicative fitness is usually overcome by restoration of the functional balance due to compensatory mutations in HA, NA or both proteins.     

The immune response of BALB/c mice to influenza hemagglutinin: commonality of the B cell and T cell repertoires and their relevance to antigenic drift

An extensive analysis of the class II (I-Ad)-restricted T cell repertoire for influenza hemagglutinin (HA) of the H3 subtype, elicited by natural infection, has shown that majority of CD4+ memory T cell clones focus on antibody-binding regions of HA, sites B and E, and are sensitive to the residue substitutions that have occurred in these regions during antigenic drift. The proliferative responses of CD4+ clones to synthetic peptides have identified T cell epitopes within site B, HA1 177-199 and HA1 182-199, and site E. HA1 56-76. The recognition specificity of T cell clones for antibody-selected mutant viruses, with single amino acid substitutions within these recognition sites identified residues 63, 189, 193 and 198 as being important for T cell recognition and thus established that BALB/c, CD4+ T cell clones were sensitive to the same substitutions known to abrogate BALB/c antibody recognition of the native HA. Our findings indicate extensive commonality of the B cell and T cell repertoires for HA, which may be relevant to an understanding of the immune pressures for antigenic drift, and, moreover, suggest that the antigen-specific B memory cell may be instrumental in selection of the peripheral T cell repertoire.     

H3N2流感病毒体外细胞传代引起的变异

目的 了解用不同来源MDCK细胞分离H3N2流感病毒时,HA和NA片段氨基酸变异情况.方法 选取H3N2流感病毒核酸阳性的咽拭子标本13份,分别在三种不同来源的MDCK细胞中传5代.对标本中病毒及细胞传代的病毒进行HA和NA基因序列测定,分析病毒传代后HA和NA片段变异情况.结果 与咽拭子中的病毒序列相比,MDCK 5代病毒和MDCK-parent 5代病毒均发生了HA和/或NA的突变.9株MDCK 5代病毒HA发生了P221L/P/H突变;8株病毒发生了NA片段的148位或151位的突变.2株MDCK-parent 5代病毒HA221位为多态性.13株病毒MDCK-parent传代后均发生了NA多态性,大多发生在151位.在MDCK-SIAT传5代后的病毒HA均无突变,3株有NA突变.结论 在对流感病毒进行抗原性分析前对病毒进行细胞传代会发生病毒的适应性突变.H3N2流感病毒在MDCK、MDCK-parent中传代后,会引起HA和/或NA突变;MDCK-SIAT传代,病毒变异明显减少.     

Phenotypic drug susceptibility assay for influenza virus neuraminidase inhibitors

A flow cytometric (fluorescence-activated cell sorter [FACS]) assay was developed for analysis of the drug susceptibilities of wild-type and drug-resistant influenza A and B virus laboratory strains and clinical isolates for the neuraminidase (NA) inhibitors oseltamivir carboxylate, zanamivir, and peramivir. The drug susceptibilities of wild-type influenza viruses and those with mutations in the hemagglutinin (HA) and/or NA genes rendering them resistant to one or more of the NA inhibitors were easily determined with the FACS assay. The drug concentrations that reduced the number of virus-infected cells or the number of PFU by 50% as determined by the FACS assay were similar to those obtained with the more time-consuming and labor-intensive virus yield reduction assay. The NA inhibition (NAI) assay confirmed the resistance patterns demonstrated by the FACS and virus yield assays for drug-resistant influenza viruses with mutations in the NA gene. However, only the FACS and virus yield assays detected NA inhibitor-resistant influenza viruses with mutations in the HA gene but not in the NA gene. The FACS assay is more rapid and less labor-intensive than the virus yield assay and just as quantitative. The FACS assay determines the drug susceptibilities of influenza viruses with mutations in either the HA or NA genes, making the assay more broadly useful than the NAI assay for measuring the in vitro susceptibilities of influenza viruses for NA inhibitors. However, since only viruses with mutations in the NA gene that lead to resistance to the NA inhibitors correlate with clinical resistance, this in vitro assay should not be used in the clinical setting to determine resistance to NA inhibitors. The assay may be useful for determining the in vivo susceptibilities of other compounds effective against influenza A and B viruses.     

Engineering of doubly antigenized immunoglobulins expressing T and B viral epitopes

Background: Concomitant with the advent of molecular biology techniques and the ability of immunoglobulins (Ig) to recognize proteins, carbohydrates, lipopeptides and nucleic acids, vaccinologists have taken advantage to develop a variety of prophylactic and therapeutic vaccine prototypes. Presentation of epitopes to the immune system by Ig molecules as a carrier platform offers several advantages: (i) long exposure of the antigen to antigen processing cells (APCs) by virtue of their long half life, (ii) lack of the immune response to self Ig, focusing the immune response to protective epitopes rather than irrelevant epitopes, (iii) it takes advantage of the properties of Fc fragment of various isotypes like crossing the placenta (IgG) or homing in epithelia (IgA), and (iv) targeting various antigens by virtue of their binding specificity. Objectives: This study was aimed to genetically and enzymatically engineer immunoglobulins (Igs) able to express and to deliver concomitantly immunodominant T and B viral epitopes. Study design: Using a genetic engineering approach we replaced the complementary determining region 3 (CDR3) and complementary determining region 2 (CDR2) of an anti-arsonate 91A3 mAb with the immunodominant HA110–120 T cell epitope and HA150–159 B cell epitope of hemagglutinin (HA) of influenza A/PR8 virus, respectively. The second doubly antigenized Ig (IgHAGalB) was constructed on an Ig in which CDR3 was replaced with HA110–120 T cell epitope while the HA150–159 B cell epitope was enzymatically assembled through an imidic bond on the galactose (Gal) residues of the carbohydrate moiety. Results and conclusions: Both genetically and genetically/enzymatically doubly antigenized Ig constructs (dAIg) were properly folded and they were able to activate peptide-specific T cells and to elicit anti-viral antibody response in mice. This demonstrates that the CDR loops as well as carbohydrate moieties of immunoglobulins represent permissive sites for grafting foreign epitopes without altering the structural integrity of immunoglobulins and the immunogenicity of the viral peptides.     

Phenotypic Drug Susceptibility Assay for Influenza Virus Neuraminidase Inhibitors

A flow cytometric (fluorescence-activated cell sorter [FACS]) assay was developed for analysis of the drug susceptibilities of wild-type and drug-resistant influenza A and B virus laboratory strains and clinical isolates for the neuraminidase (NA) inhibitors oseltamivir carboxylate, zanamivir, and peramivir. The drug susceptibilities of wild-type influenza viruses and those with mutations in the hemagglutinin (HA) and/or NA genes rendering them resistant to one or more of the NA inhibitors were easily determined with the FACS assay. The drug concentrations that reduced the number of virus-infected cells or the number of PFU by 50% as determined by the FACS assay were similar to those obtained with the more time-consuming and labor-intensive virus yield reduction assay. The NA inhibition (NAI) assay confirmed the resistance patterns demonstrated by the FACS and virus yield assays for drug-resistant influenza viruses with mutations in the NA gene. However, only the FACS and virus yield assays detected NA inhibitor-resistant influenza viruses with mutations in the HA gene but not in the NA gene. The FACS assay is more rapid and less labor-intensive than the virus yield assay and just as quantitative. The FACS assay determines the drug susceptibilities of influenza viruses with mutations in either the HA or NA genes, making the assay more broadly useful than the NAI assay for measuring the in vitro susceptibilities of influenza viruses for NA inhibitors. However, since only viruses with mutations in the NA gene that lead to resistance to the NA inhibitors correlate with clinical resistance, this in vitro assay should not be used in the clinical setting to determine resistance to NA inhibitors. The assay may be useful for determining the in vivo susceptibilities of other compounds effective against influenza A and B viruses.