NF-KB在人巨细胞病毒感染人胚肺成纤维细胞中的表达及意义

目的:探讨核转录因子(NF-KB)在人巨细胞病毒(HCMV)感染宿主细胞中的表达及意义.方法:用HCMV AD169株感染人胚肺成纤维细胞(HEL),采用免疫组化和原位杂交方法检测HEL细胞不同时相NF-kB和bcl-2蛋白的表达及NF-KB P 65 mRNA的表达,同时用流式细胞术检测细胞凋亡指数.结果:HCMV感染后48 h细胞核表达NF-KB蛋白阳性数最多,96 h后逐渐减少,120 h无阳性细胞,NF-KBp 65 mRNA表达水平在24h达到最高,bd-2的表达与NF-kB的活性改变一致.HCMV感染在72 h前抑制宿主细胞凋亡,在120 h可诱导宿主细胞凋亡.结论:NF-KB在宿主细胞中的动态表达与HCMV的生活周期及致病有关.     

习惯性流产孕妇巨细胞病毒pp65抗原检测的临床意义

目的:探讨人巨细胞病毒(HCMV)活动性感染与习惯性流产(RPL)的关系。方法:采集习惯性流产孕妇和正常产前体检孕妇外周血,分离外周血单个核细胞(PBMCs)和血浆,分别用免疫荧光法和实时定量PCR检测HCMV pp65抗原和HCMV-DNA,并比较2种方法的一致性。结果:46例RPL患者HCMV pp65抗原有14例阳性,阳性率30.4%,50例正常体检孕妇HCMV pp65抗原有4例阳性,阳性率8%,2组孕妇HCMV活动性感染率有显著性差异(2χ=6.76,P<0.01)。孕妇HC-MV pp65抗原阳性率升高,孕妇流产几率增加(2χ=6.39,P<0.01)。免疫荧光法和实时定量PCR有较好的一致性(93.5%)。结论:习惯性流产孕妇HCMV活动性感染率显著高于正常孕妇,HCMV pp65抗原检测可作为RPL早期诊断指标之一。     

HCMV IE1蛋白对小鼠Raw264．7细胞株TNF-α、IL-1β表达的影响

目的构建人巨细胞病毒(Human cytomegalovirus,HCMV)立即早期蛋白1(IE1)真核表达载体pcDNA3.1(-)-IE1,将其转染小鼠巨噬细胞Raw264.7细胞株,观察IE1蛋白在细胞中的表达及其对小鼠巨噬细胞Raw264.7分泌细胞因子功能的影响。方法双酶切pQE30-IE1,回收目的基因IE1,亚克隆入真核表达载体pcDNA3.1(-),筛选、鉴定阳性克隆得pcDNA3.1(-)-IE1;提取质粒pcDNA3.1(-)-IE1,通过Super FectTM脂转染试剂将质粒pcDNA3.1(-)-IE转染Raw264.7细胞,Western-blot鉴定IE1蛋白的表达,并用酶联免疫吸附试验(ELISA)法检测培养上清液中细胞因子TNF-α、IL-1β的表达水平。结果与结论①成功构建了真核表达载体pcDNA3.1(-)-IE1;②pcD-NA3.1(-)-IE1能在Raw264.7细胞中表达;③IE1的表达可上调巨噬细胞TNF-α、IL-1β的表达水平。     

人巨细胞病毒被膜磷蛋白pp65的检测及其应用

目的:建立一种快速简便诊断肾移植受者人巨细胞病毒(HCMV)活动性感染的方法.方法:运用免疫组织化学的催化信号扩增法检测外周血白细胞中的人巨细胞病毒被膜磷蛋白pp65,巨细胞病毒信使核糖核酸(pp67-mRNA)检.测法作比较.结果:检测105例肾移植受者中,HCMVpp65抗原阳性45例,pp67-mRNA检测阳性40例,pp65抗原阳性细胞数为(70±43)个/2×105个WBC,而有症状的CMV病25例,抗原阳性细胞指数为(85±44)个/2×105个WBC.对照组100名健康人,pp65抗原检测全为阴性.pp65的敏感性、特异性分别是100%、88.9%.结论i该法敏感,简便,可作为肾移植术后HCMV病的早期诊断并可指导抗病毒治疗.     

糖尿病患者活动性人巨细胞病毒感染监测及临床意义

目的 临测糖尿病患者活动性人巨细胞病毒(HCMV)感染,并探讨其临床意义.方法 在HCMV pp65基因内自行设计一对引物,建立RT-PCR检测727例糖尿病患者及230名健康献血者外周血白细胞HCMVpp65 mRNA转录水平,同时应用抗体捕获酶联免疫法检测HCMV pp65-IgM.结果 糖尿病患者HCMV-IgM及mRNA阳性率分别为11.14%和16.64%,与对照组(0.87%、2.17%)相比均差异有统计学意义(P<0.01);比较不同年龄段HCMV感染率,结果发现发病年龄<30岁的患者中HCMV-mRNA阳性率明显高于其他年龄组,从病程来看,病程<1年的糖尿病患者HCMV-mRNA阳性检出率最高.结论 糖尿病患者易发生活动性的HCMV感染,RT-PCR检测HCMV pp65和抗体捕获HCMV pp65特异性IgM可快速有效地确诊HCMV活动性感染的发生.     

人巨细胞病毒活动性感染对早孕绒毛细胞MMP9和TIMP1蛋白活性的影响

目的探究人巨细胞病毒活动性感染对早孕绒毛细胞基质金属蛋白酶9(MMP9)和组织型基质金属蛋白酶抑制剂(TIMP1)活性的影响。方法收集2016年10月-2018年10月在湘潭市妇幼保健院和衡阳市第一人民医院自愿接受流产的健康孕妇的绒毛,人巨细胞病毒感染后,免疫组化检测细胞角蛋白7(CK7)、人波形蛋白(Vim)、HER-2基因(c-erbB-2)的表达以观察绒毛细胞纯度。免疫组化检测pp65抗原用于观察人巨细胞病毒(HCMV)的感染情况。Western Blot、荧光定量PCR(RT PCR)和酶联免疫吸附测定(ELISA)用于检测HCMV感染后早孕绒毛细胞中MMP9和TIMP1的表达水平。结果免疫组化染色显示,分离的原代细胞为高纯度绒毛细胞且HCMV可感染原代早孕绒毛细胞。PCR结果显示,HCMV组MMP9基因表达水平为0.23±0.08,低于对照组(P<0.05),TIMP1基因表达水平为5.49±1.76,高于对照组(P<0.05)。HCMV感染后第4天,HCMV组早孕绒毛细胞胞质中MMP9蛋白表达水平为0.18±0.08,低于对照组(P<0.05),TIMP1蛋白表达水平为0.97±0.16,高于对照组(P<0.05)。此外,与模拟感染细胞相比,感染HCMV的巨噬细胞中MMP-9活性持续降低。感染后第7天HCMV组上清液中MMP9蛋白水平低于对照组(P<0.05);感染后第4天和第7天HCMV组上清液中TIMP1蛋白水平高于对照组(P<0.05)。结论 HCMV感染可显著下调原代早孕绒毛细胞MMP9的基因和蛋白表达,上调TIMP1的基因和蛋白表达。     

金叶败毒制剂对人巨细胞病毒感染宿主细胞周期的调节作用

目的:探讨中药金叶败毒制剂(简称中药)对人巨细胞病毒(HCMV)感染宿主细胞周期的影响。方法:用HCMV AD169感染人胚肺成纤维细胞(HEL),用金叶败毒制剂干预后,采用免疫组织化学法检测HCMV感染及中药干预后宿主细胞P16蛋白的表达,并采用流式细胞技术检测HCMV感染及药物干预后宿主细胞周期。结果:在HCMV感染48h后,金叶败毒制剂能明显抑制宿主细胞P16蛋白的表达。经中药干预后,在病毒感染24h后,宿主细胞处于S期细胞明显减少;病毒感染48h后,宿主细胞处于G2/M期的细胞明显增多;在HCMV感染72h后,宿主细胞均表现为S期细胞明显减少,G2/M期细胞明显增多,而G0/G1期细胞亦明显增多。结论:金叶败毒制剂可能通过抑制HCMV感染细胞P16蛋白的表达,促进感染细胞从S期进展到G2/M期,促进细胞周期进程而发挥抗病毒作用。     

脑胶质瘤人巨细胞病毒感染对TGF-β/Smads信号转导通路的影响

目的探究脑胶质瘤患者人巨细胞病毒(HCMV)感染对转化生长因子β(TGF-β)/Smads信号转导通路的影响。方法选取承德医学院附属医院神经外科脑胶质瘤患者67例,其中WHOⅡ级26例、WHOⅢ～Ⅳ级41例,分别归为低级别瘤组、高级别瘤组,另选取无任何肿瘤性疾病的脑外伤患者行内减压手术切除的新鲜脑组织17例为对照组,采用巢氏聚合酶链反应(Nested PCR)、免疫组织化学法分别检测各组HCMV DNA序列、pp65抗原表达;采用蛋白免疫印迹法、实时荧光定量PCR(qPCR)检测TGF-β/Smads信号通路相关分子(TGF-β1、Smad7)蛋白、mRNA水平,并分析HCMV感染与TGF-β1、Smad7基因表达的相关性。结果高级别瘤组、低级别瘤组HCMV DNA、pp65抗原阳性率均高于对照组,且两组间差异有统计学意义(P0.05);高级别瘤组、低级别瘤组TGF-β1、Smad7蛋白及mRNA水平均高于对照组(P0.05),且两组间差异比较有统计学意义(P0.05);HCMV DNA、pp65抗原阳性组TGF-β1、Smad7蛋白及mRNA相对表达量均高于其对应阴性组,差异比较均具有统计学意义(P0.05)。结论 HCMV感染与脑胶质瘤患者发生、发展密切相关,肿瘤级别越高,HCMV感染越严重,HCMV感染可能通过上调TGF-β1、Smad7基因的表达发挥促癌作用。     

人巨细胞病毒感染人胚肺成纤维细胞对其细胞周期的影响

目的研究人巨细胞病毒（human cytomegalovirus,HCMV）感染人胚肺成纤维细胞（human embryoniclung fibroblast,HELF）后对其细胞周期的影响,探讨HCMV感染的发病机制。方法用HCMV感染同步化的HELF作为感染组,同时设模拟感染组为对照组。用倒置相差显微镜观察两组细胞形态学变化至感染后7d。于感染后12h、24h、48h、72h和96h收获细胞,用免疫细胞化学法检测两组细胞核内HCMVIE172蛋白,流式细胞术检测细胞周期。结果模拟感染组未见细胞形态学改变,亦未出现HCMVIE172蛋白阳性产物。感染组感染后72h时即可见局部细胞变圆,膨胀,胞体及胞核巨大化,7d时可见所有细胞均发生病变;免疫细胞化学检测显示,其各时间点的细胞均在核内出现呈棕黄色颗粒的HCMVIE172蛋白。模拟感染组在感染后12h时有65.7%的细胞处于G1期,24h时有43.8%的细胞处于G1期;感染组在感染后12h时有70.4%的细胞处于G1期,24h时有69.9%的细胞处于G1期;两两比较,差异均有显著性（均P〈0.01）。结论HCMV感染HELF后在细胞核内进行复制、增殖,其影响了HELF周期,使大部分细胞停滞在G期。     

人巨细胞病毒IE1核酸疫苗的免疫效果研究

[目的]用人巨细胞病毒(HCMV)IE1核酸疫苗pcDNA3.1(-)-IE1免疫BALB/c小鼠,初步研究其产生的体液免疫和细胞免疫应答水平。[方法]将pcDNA3.1(-)-IE1通过肌肉注射免疫BALB/c小鼠,通过PCR测定和免疫组化检测其在肌细胞中的表达,细胞因子测定、淋巴细胞转化实验检测免疫效果。[结果]PCR检测到与IE1大小一致的片段,免疫组化结果显示IE1基因在小鼠肌细胞中表达IE1目的蛋白。小鼠脾淋巴细胞经PHA刺激后,实验组IL-4、IL-2、IFN-γ含量以及淋巴细胞转化率显著增高,与对照组比较差异有统计学意义(P﹤0.05)。[结论]pcDNA3.1(-)-IE1核酸疫苗能在BALB/c小鼠肌细胞中稳定存在并能表达HCMV IE1蛋白,pcDNA3.1(-)-IE1核酸疫苗可诱导BALB/c小鼠脾细胞分泌IL-4、IL-2、IFN-r并刺激BALB/c小鼠脾细胞增殖。     

A synthetic human cytomegalovirus pp65-IE1 fusion antigen efficiently induces and expands virus specific T cells

Infection with human cytomegalovirus (HCMV) can cause severe complications in newborns and immunocompromised patients, and a prophylactic or therapeutic vaccine against HCMV is not available. Here, we generated a HCMV vaccine candidate fulfilling the regulatory requirements for GMP-compliant production and clinical testing. A novel synthetic fusion gene consisting of the coding sequences of HCMV pp65 and IE1 having a deleted nuclear localization sequence and STAT2 binding domain was introduced into the genome of the attenuated vaccinia virus strain MVA. This recombinant MVA, MVA-syn65_IE1, allowed for the production of a stable ∼120 kDa syn65_IE1 fusion protein upon tissue culture infection. MVA-syn65_IE1 infected CD40-activated B cells activated and expanded pp65- and IE1-specific T cells derived from HCMV-seropositive donors to at least equal levels as control recombinant MVA expressing single genes for pp65 or IE1. Additionally, we show that MVA-syn65_IE1 induced HCMV pp65- and IE1-epitope specific T cells in HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice. Thus, MVA-syn65_IE1 represents a promising vaccine candidate against HCMV and constitutes a basis for the generation of a multivalent vaccine targeting relevant pathogens in immunocompromised patients.     

Optimized recombinant dense bodies of human cytomegalovirus efficiently prime virus specific lymphocytes and neutralizing antibodies without the addition of adjuvant

Control of human cytomegalovirus (HCMV) infection correlates with the reconstitution of antiviral T lymphocytes in haematopoietic stem cell transplant recipients. A vaccine to foster this reconstitution and to ameliorate the severe consequences of HCMV reactivation is yet unavailable. This work focused on providing a rationale for the amendment of the yields and the antigenic composition of a vaccine, based on subviral dense bodies (DB) of HCMV. Modified DB were generated that contained the HLA-A2 presented IE1 model peptide TMYGGISLL, integrated at different positions in the major DB protein pp65. Insertion at position W175 of pp65 allowed efficient formation of recDB in the cytoplasm of infected cells and resulted in considerable yields of these particles. Even in the absence of adjuvant, these particles proved to be highly immunogenic with respect to CD8 and CD4 T cell and neutralizing antibody responses.     

The next generation recombinant human cytomegalovirus vaccine candidates—Beyond gB

Human cytomegalovirus (HCMV) infects the majority of the global population and persists within the infected host for life; infection of healthy adults rarely leads to severe acute clinical symptoms. In contrast, HCMV is a leading infectious cause of congenital disease and a common cause of complications in transplant recipients. A vaccine to prevent HCMV disease in these populations is a widely recognized medical need. We review recent advances in our understanding of the candidate vaccine antigens and published clinical trial data for the four most recent HCMV vaccine candidates: a gB subunit adjuvanted with MF59, a DNA vaccine expressing gB and pp65, alphavirus replicon particles (VRPs) expressing gB and a pp65–IE1 fusion protein, and a pp65 peptide vaccine. The candidates are safe, although some adverse events were reported for an adjuvanted variant of the pp65 peptide vaccine. The gB/MF59 vaccine elicited strong humoral responses with limited durability. The gB/pp65 DNA vaccine elicited cellular immunity, and the pp65 peptide vaccine elicited modest cellular immunity, but only when formulated with an adjuvant. Only the VRP vaccine expressing gB and pp65–IE1 elicited both humoral and cellular immunity. The gB/MF59 vaccine showed a short-term 50% efficacy at preventing infection of seronegative women and significantly reduced viremia and need for antivirals in solid organ transplant recipients, and the gB/pp65 DNA vaccine showed signs of clinical benefit in hematopoietic stem cell transplant recipients. Importantly, the partial efficacy of the subunit and DNA vaccines is new evidence that both humoral and cellular immunity contribute to controlling HCMV-related disease. These data show the clinical feasibility of a recombinant HCMV vaccine. We discuss areas for potential improvements in the next generation of vaccine candidates.     

Induction of human cytomegalovirus (HCMV)-glycoprotein B (gB)-specific neutralizing antibody and phosphoprotein 65 (pp65)-specific cytotoxic T lymphocyte responses by naked DNA immunization

Plasmids expressing the human cytomegalovirus (HCMV) glycoprotein B (gB) (UL55) or phosphoprotein 65 (pp65) (UL83) were constructed and evaluated for their ability to induce immune responses in mice. The full-length gB as well as a truncated form expressing amino acids 1-680 of gB, and lacking the fragment encoding amino acids 681 907 including the transmembrane domain of gB (gB680) were evaluated. Immunization of mice with plasmids coding for gB or gB680 induced ELISA and neutralizing antibodies, with the highest titres in mice immunized with the gB680 plasmid. Mice immunized with the gB plasmid predominantly produced IgG2a gB-specific antibody, while the gB680 plasmid raised mostly IgG1 anti-gB antibody. Mice immunized with the pp65 plasmid developed pp65-specific cytotoxic T lymphocytes (CTL) and ELISA antibodies. Immunization with a mixture of both gB and pp65 plasmids raised antibodies to both proteins and pp65-specific CTL, indicating a lack of interference between these two plasmids. These results suggest that DNA immunization is a useful approach for vaccination against HCMV disease.     

人巨细胞病毒pp65DNA疫苗诱导小鼠免疫效应的研究

目的探讨人巨细胞病毒pp65 DNA疫苗免疫小鼠产生的细胞免疫应答及相关的细胞因子变化,分析该疫苗是否具高度免疫性。方法将40只健康Balb/c小鼠随机分成A、B两组,每组20只。A组(对照组):注射生理盐水;B组(实验组):注射pp65 DNA疫苗;采用流式细胞仪检测CD3+、CD4+及CD8+;采用双抗体夹心ELISA法检测小鼠血清中的IFN-γ;采用放射免疫法检测小鼠血清中的IL-2。统计学处理用SPSS 10.0统计学软件,组间比较用t检验,P0.05为差异有统计学意义。结果B组小鼠T细胞亚群CD4+、CD8+、CD3+及细胞因子IFN-γ,IL-2水平明显高于A组,差异均有统计学意义(P0.01)。结论本研究表明HCMV pp65 DNA疫苗能诱导Balb/c小鼠产生一定的细胞免疫应答及细胞因子的变化。     

Randomized,double-blind,Phase 1 trial of an alphavirus replicon vaccine for cytomegalovirus in CMV seronegative adult volunteers

Development of a cytomegalovirus (CMV) vaccine is a priority. We evaluated a two component alphavirus replicon particle vaccine expressing CMV gB or a pp65/IE1 fusion protein, previously shown to induce robust antibody and cellular immune responses in mice, in a randomized, double-blind Phase 1 clinical trial in CMV seronegative subjects. Forty subjects received a lower dose (LD) or higher dose (HD) of vaccine or placebo by intramuscular or subcutaneous injection at Weeks 0, 8 and 24. The vaccine was well tolerated, with mild to moderate local reactogenicity, minimal systemic reactogenicity, and no clinically important changes in laboratory parameters. All vaccine recipients developed ex vivo, direct IFN-γ ELISPOT responses to CMV antigens (maximal mean spot-forming cells per 10⁶ PBMC in LD and HD groups of 348 and 504 for pp65, 83 and 113 for IE1, and 138 and 114 for gB), and neutralizing antibodies (maximal geometric mean titer 110 with LD and 218 with HD). Polyfunctional CD4⁺ and CD8⁺ T cell responses were detected by polychromatic flow cytometry. This alphavirus replicon particle vaccine was safe and induced neutralizing antibody and multifunctional T cell responses against three CMV antigens that are important targets for protective immunity.     

DNA vaccines expressing glycoprotein complex II antigens gM and gN elicited neutralizing antibodies against multiple human cytomegalovirus (HCMV) isolates

Human cytomegalovirus (HCMV) glycoprotein complex II (gcII) consists of two glycoproteins, gM and gN. Although gcII specific IgG purified from HCMV positive patient sera can neutralize HCMV, there has been no report describing the generation of virus-neutralizing antibodies by immunization with individual recombinant gM or gN antigens. In the current study, gM and gN antigens were expressed by the mammalian expression vector pJW4303 and used as DNA vaccines to determine the immunogenicity of these proteins. Sera from mice or rabbits immunized with individual or combinations of gM and gN DNA vaccines contained gM and gN specific antibodies as confirmed by ELISA and Western blot analyses. The combined gM and gN antigens induced the strongest antibody responses that recognized both gM and gcII complex while gM DNA vaccine alone could only elicit antibody specific for gM antigen. When given alone, the gN DNA vaccine did not induce detectable gcII specific antibody even though in vitro gN expression was confirmed by the formation of gM/gN complex in FSK cells using a gN-specific monoclonal antibody 14-16A. The neutralizing antibody titer of anti-gM/gN sera (1:128) was higher than that of anti-gM sera (1:32) against the autologous virus, HCMV AD169. Heterologous HCMV strains including Towne and Davis could also be neutralized by the anti-gM/gN antisera. Our data supported the rationale for the use of the HCMV gM/gN protein complex as protective antigens for subunit based HCMV vaccine development. DNA vaccination is an effective approach to express the gM/gN antigen complex in vivo without the need to express and purify these highly insoluble and structurally complicated antigens.