丙型肝炎病毒多表位抗原在恒河猴中的免疫应答及保护性试验

利用ＨＣＶ抗原多表位来研制ＨＣＶ疫苗是目前的一个新方向。本研　究利用ＨＣＶ的ＨＣＶ的５个保守表位串联,并加入破伤风类毒素上的一个Ｔ细胞激活位点,设计成　一个ＨＣＶ多表位抗原基因ＰＣＸ,在大肠杆菌中表达,用此蛋白免疫恒河猴,诱导猴体产生了较　高的抗体水平,滴度达１∶１０００以上,在免疫后的６０周抗体滴度仍达１∶４０以上。同时,在免　疫后６周用人ＨＣＶ阳性血清攻击猴子,免疫ＰＣＸ的猴子出现一过性ＡＬＴ升高,在攻击后三周内用　ＲＴ－ＰＣＲ检测到猴血清内ＨＣＶ的ＲＮＡ阳性。结果表明,免疫多表位的ＰＣＸ蛋白可以诱导机体产生　高水平的免疫应答。     

丙型肝炎病毒复合高变区1模拟表位蛋白的免疫原性分析

将丙型肝炎病毒高变区1(HVR1)模拟表位融合基因插入原核表达载体pGEX-4T-1,在大肠杆菌BL21(DE3)中进行表达,经亲和层析和凝胶过滤层析获得HCV HVR1模拟表位融合蛋白.用Western blot和ELISA检测融合蛋白与HCV抗体阳性血清的结合情况.皮下注射免疫BALB/c小鼠,用ELISA检测小鼠血清中的抗HCV抗体水平及其与天然HCV高变区1合成肽的交叉反应.结果表明融合蛋白能与HCV抗体阳性血清特异结合,融合蛋白与HCV抗体阳性血清的结合频率为71.6%(25/35).融合蛋白免疫小鼠后能有效诱导免疫应答,其诱生的特异性抗体最高滴度达104(免疫后第8周),且该抗体能同2条天然HCV HVR1合成肽发生交叉反应.本研究提示,HCV复合HVR1模拟表位融合蛋白在丙型肝炎疫苗的研发中可能具有潜在应用价值.     

丙型肝炎病毒复合高变区1模拟表位蛋白的免疫原性分析

将丙型肝炎病毒高变区1(HVR1)模拟表位融合基因插入原核表达载体pGEX-4T-1,在大肠杆菌BL21(DE3)中进行表达,经亲和层析和凝胶过滤层析获得HCVHVR1模拟表位融合蛋白。用Westernblot和ELISA检测融合蛋白与HCV抗体阳性血清的结合情况。皮下注射免疫BALB/c小鼠,用ELISA检测小鼠血清中的抗HCV抗体水平及其与天然HCV高变区1合成肽的交叉反应。结果表明融合蛋白能与HCV抗体阳性血清特异结合,融合蛋白与HCV抗体阳性血清的结合频率为71.6%(25/35)。融合蛋白免疫小鼠后能有效诱导免疫应答,其诱生的特异性抗体最高滴度达104(免疫后第8周),且该抗体能同2条天然HCVHVR1合成肽发生交叉反应。本研究提示,HCV复合HVR1模拟表位融合蛋白在丙型肝炎疫苗的研发中可能具有潜在应用价值。     

HCV多表位抗原基因重组减毒口服活菌苗的研究

把丙型肝炎病毒(hepatitis C virus,HCV)复合多表位抗原基因PCX与β半乳糖苷酶基因(GZ)融合后,构建重组减毒鼠伤寒沙门氏菌口服活菌苗SL3261(pWR/PCX),免疫小鼠及家兔后,于第6周始可检测到低水平的抗GZPCX IgG(1∶200),至3月时最高滴度分别达1∶800及1∶25 600,均显著高于宿主菌SL3261组及空白对照组。在免疫小鼠的肠道灌洗液中可检测到抗GZPCX sIgA\.GZPCX抗原可促进免疫小鼠及家兔淋巴细胞增殖,诱发明显的迟发性超敏反应(DTH)。口服免疫后小鼠体重出现一过性下降,但未见其它明显的毒性作用,安全性较好。本研究从新的角度探讨了HCV复合多表位重组口服活菌苗的可行性,为HCV疫苗的研究提供新的实验依据。     

丙型肝炎病毒高变区1模拟表位的交叉反应性分析

研究丙型肝炎病毒(Hepatitis C virus,HCV)高变区1(Hypervariable region 1,HVR1)抗原表位的交叉反应性,获取高反应性的抗原表位.设计并合成5种HVR1模拟表位基因,构建编码HVR1模拟表位的表达载体,表达并纯化表位蛋白.ELISA法检测表位蛋白与35份HCV抗体阳性血清的交叉反应性.包装HCV假病毒(HCV pseudotype particles,HCVpp),评价表位蛋白免疫BALB/c鼠血清在假病毒感染Huh7.5细胞中的作用.结果表明,表达纯化的5种表位蛋白(P1、P2、P5、P6、P8)均可与HCV抗体阳性血清反应,阳性反应率分别为54.3%(P1)、62.9%(P2)、80%(P5)、68.6%(P6)、54.3%(P8).表位蛋白P6、P8免疫BALB/c鼠血清对HCV假病毒感染Huh7.5细胞具明显的抑制作用.结果提示,选取的HVR1模拟表位在HCV感染免疫与疫苗研制中可能具有潜在的价值.     

人乳头瘤病毒-6 L1蛋白B-细胞优势表位作为多肽疫苗的研究

本研究分析了人乳头瘤病毒-6型L1外壳蛋白之B-细胞优势表位,并拟以此为基础制作表位多肽疫苗.研究中采用Goldkey和PC/Gene软件系统综合分析HPV6之L1蛋白B-细胞优势表位后,Fmoc固相合成表位多肽,通过HPLC纯化和毛细管电泳分析其纯度.与佐剂完全乳化后,免疫小鼠,进行动物水平的免疫效果评价.取免疫小鼠血清,与HPV-6 DNA阳性的尖锐湿疣患者疣体组织上清液结合,以鉴定免疫小鼠所产生抗体的特异性.发现L1蛋白第425-439位和第486-500位具有较高的免疫原性,可明显诱导小鼠血清抗体滴度升高,且该抗体与人尖锐湿疣疣体组织上清液呈阳性反应.说明所选这两个肽段为HPV6之L1蛋白的B-细胞优势表位,但诱导产生的抗体是否具有功能特异性,正在做进一步研究.     

A组轮状病毒多个结构蛋白抗原表位的串联表达及其免疫原性的检测

从北京腹泻婴儿粪便提取的轮状病毒(rotavirus,RV)(T114株)的RNA中,克隆到轮状病毒结构蛋白基因vp4,vp6和vp7的全长cDNA,对它们编码的蛋白质序列和可能的抗原表位肽进行了预测,选择了RV主要抗原蛋白VP7、VP6和VP4的4个抗原表位肽,通过人工合成DNA的方式将这些抗原表位肽基因串联融合成一个阅读框RME(rotavirus multipleepitopes,RME)并构建原核表达载体.大肠杆菌表达的RME在ELISA反应中可被RV多克隆抗体识别,纯化的RME蛋白注射免疫小鼠可诱导特异性免疫应答,产生高滴度的同源氨基酸序列特异抗体和人RV抗体,其中针对RME的IgG抗体滴度达到l∶40 000,针对单个抗原表位EV7、EV6和EV4的IgG抗体滴度达l∶10 000～l∶20 000,针对RV Wa株的IgG抗体滴度较低为l∶2 500,但能特异地中和该病毒对MAC145细胞的侵染.上述结果为新型RV基因工程疫苗的研发提供了论据和基础.     

人乳头瘤病毒-6 L1蛋白B-细胞优势表位作为多肽疫苗的研究

本研究分析了人乳头瘤病毒-6型L1外壳蛋白之B-细胞优势表位,并拟以此为基础制作表位多肽疫苗。研究中采用Goldkey和PC／Gene软件系统综合分析HPV6之L1蛋白B-细胞优势表位后,Fmoc固相合成表位多肽,通过HPLC纯化和毛细管电泳分析其纯度。与佐剂完全乳化后,免疫小鼠,进行动物水平的免疫效果评价。取免疫小鼠血清,与HPV-6 DNA阳性的尖锐湿疣患者疣体组织上清液结合,以鉴定免疫小鼠所产生抗体的特异性。发现L1蛋白第425-439位和第486-500位具有较高的免疫原性,可明显诱导小鼠血清抗体滴度升高,且该抗体与人尖锐湿疣疣体组织上清液呈阳性反应。说明所选这两个肽段为HPV6之L1蛋白的B-细胞优势表位,但诱导产生的抗体是否具有功能特异性,正在做进一步研究。     

大肠杆菌表达的戊型肝炎病毒ORF2多肽对恒河猴的免疫保护研究

在大肠杆菌中表达的一段戊型肝炎病毒（HEV）结构蛋白NE2,纯化后以弗氏佐剂,按0d,10d,30d的方案10μg/针的剂量免疫3只恒河猴,在第2周抗体阳转,第6周时1只滴度达1∶100 000,另2只滴度1∶20 000,此时以106 PCR滴度的HEV病毒粪悬液攻击。对照组3只均出现血清转氨酶（ALT）升高,抗体阳转,粪便持续排毒1月以上;疫苗组无一发病,未检出非疫苗来源的抗体,其中1只始终未检出粪便排毒,另2只仅出现短暂排毒。以一份NE2免疫后猴血清（滴度1∶20 000）与103 PCR滴度的病毒混匀后感染2只恒河猴,结果对照组2只均持续排毒3周以上,抗体阳转,1只ALT明显升高;而抗体中和组2只猴始终未检出粪便排毒,抗NE2抗体缓慢下降,ALT正常。这些结果表明NE2具有良好的免疫原性和免疫保护性,有可能成为有效的戊肝疫苗。     

重组霍乱毒素B亚基与恶性疟原虫多抗原表位融合蛋白对恒河猴的免疫保护作用研究

使用重组霍乱毒素B亚基（CTB）与恶性疟原虫抗原表位融合蛋白（AWTE）免疫恒河猴,研究其免疫应答并观察对食蟹疟原虫攻击的保护作用。结果表明：在0,14,28天分别通过鼻腔和肌肉注射免疫恒河猴,第3次免疫后2周,抗CTB抗体平均滴度可达1∶512（鼻腔免疫）和1∶10000（肌肉免疫）;肌肉免疫后抗疟原虫抗体滴度也显著高于鼻腔免疫组。用125×10⁸个食蟹疟子孢子攻击,对照组5只恒河猴在攻击后10～14d全部感染,其中1只在攻击后21d死亡,另4只重度感染,感染持续30d以上。鼻腔免疫组的5只动物均在攻击20d后出现原虫,其中3只轻度感染,感染持续4d后即恢复,其余2只感染持续36d以上。肌肉注射组3只未受感染,其余2只在攻击后19d后轻度感染,感染4d后即完全恢复。以上结果表明,使用霍乱毒素B亚基为载体蛋白构建的重组疟疾疫苗具有良好的免疫原性,对食蟹疟攻击具有良好的交叉免疫保护作用。     

Identification of HCV genotypes in HCV infected blood donors

HCV infection is a leading cause of chronic liver disease, including cirrhosis of the liver. There are at least six major genotypes and more than 50 subtypes of HCV. The prevalence and distribution of HCV genotypes depend on geographical location. The aim of this study was to identify and compare the HCV genotypes in HCV infected blood donors and patients. In this cross-sectional study, 167 serum samples from 103 blood donors and 64 patients with hepatitis C were investigated for HCV genotypes. HCV genotyping was carried out using type-specific primers from the core region of the viral genome. The highest frequency was for genotype 1a, with 53 and 34 (51.5% versus 53.1%) of subjects in blood donors and patients respectively. Genotype 3a and 1b were the other frequent genotypes with 4 and 16 (3.9% versus 25%) and 39 and 10 (37.9% versus 15.6%) subjects, respectively. There was not any statistical significant association between the place of infection of the patients and genotype. The results of this study indicate that the distribution of genotypes in the two populations was similar. The dominant HCV genotypes between blood donors and patients were 1a, 3a and 1b respectively.     

Inhibition of HCV Replication in HCV Replicon by shRNAs

We show that the vector-derived long dsRNA specifically inhibits the replication of HCV RNA in HCV replicon. We designed a long dsRNA targeted to the full-length HCV IRES/core elements (1-to 377-nt). Our results revealed that the replication of HCV RNA was reduced to near background levels in a sequence-specific manner by the long dsRNAs in the HCV replicon. We also designed four shRNAs against several regions (120- to 139-nt, 260- to 279-nt, 330- to 349-nt, and 340- to 359-nt) of the HCV IRES/Core elements. The two HCV IRES/core-specific shRNAs, 330- to 349-nt and 340- to 359-nt, containing the AUG initiation codon sequence showed stronger HCV inhibitory effects than the other two shRNAs, 120- to 139-nt and 260- to 279-nt.     

HCV entry receptors as potential targets for siRNA-based inhibition of HCV

Hepatitis C virus (HCV) infection is a major worldwide problem causes acute and chronic HCV infection. Current treatment of HCV includes pegylated interferon-α (PEG IFN- α) plus ribavirin (RBV) which has significant side effects depending upon the type of genotype. Currently, there is a need to develop antiviral agents, both from synthetic chemistry and Herbal sources. In the last decade, various novel HCV replication, helicase and entry inhibitors have been synthesized and some of which have been entered in different phases of clinical trials. Successful results have been acquired by executing combinational therapy of compounds with standard regime in different HCV replicons. Even though, diverse groups of compounds have been described as antiviral targets against HCV via Specifically Targeted Antiviral Therapy for hepatitis C (STAT-C) approach (in which compounds are designed to directly block HCV or host proteins concerned in HCV replication), still there is a need to improve the properties of existing antiviral compounds. In this review, we sum up potent antiviral compounds against entry, unwinding and replication of HCV and discussed their activity in combination with standard therapy. Conclusively, further innovative research on chemical compounds will lead to consistent standard therapy with fewer side effects.     

Immune Biomarker Differences and Changes Comparing HCV Mono-Infected,HIV/HCV Co-Infected,and HCV Spontaneously Cleared Patients

Background

Immune biomarkers are implicated in HCV treatment response, fibrosis, and accelerated pathogenesis of comorbidities, though only D-dimer and C-reactive protein have been consistently studied. Few studies have evaluated HIV/HCV co-infection, and little longitudinal data exists describing a broader antiviral cytokine response

Methods

Fifty immune biomarkers were analyzed at baseline(BL) and HCV end of treatment follow-up(FU) time point using the Luminex 50-plex assay in plasma samples from 15 HCV-cleared, 24 HCV mono- and 49 HIV/HCV co-infected patients receiving antiretroviral treatment, who either did or did not receive pegylated-interferon/ribavirin HCV treatment. Biomarker levels were compared among spontaneous clearance patients, mono- and co-infected, untreated and HCV-treated, and sustained virologic responders (SVR) and non-responders (NR) at BL and FU using nonparametric analyses. A Bonferroni correction, adjusting for tests of 50 biomarkers, was used to reduce Type I error

Results

Compared to HCV patients at BL, HIV/HCV patients had 22 significantly higher and 4 significantly lower biomarker levels, following correction for multiple testing. There were no significantly different BL levels when comparing SVR and NR in mono- or co-infected patients; however, FU levels changed considerably in co-infected patients, with seven becoming significantly higher and eight becoming significantly lower in SVR patients. Longitudinally between BL and FU, 13 markers significantly changed in co-infected SVR patients, while none significantly changed in co-infected NR patients. There were also no significant changes in longitudinal analyses of mono-infected patients achieving SVR or mono-infected and co-infected groups deferring treatment

Conclusions

Clear differences exist in pattern and quantity of plasma immune biomarkers among HCV mono-infected, HIV/HCV co-infected, and HCV-cleared patients; and with SVR in co-infected patients treated for HCV. Though >90% of patients were male and co-infected had a larger percentage of African American patients, our findings may have implications for better understanding HCV pathogenesis, treatment outcomes, and future therapeutic targets     

Effect of hepatitis C virus (HCV) NS5B-nucleolin interaction on HCV replication with HCV subgenomic replicon

We previously reported that nucleolin, a representative nucleolar marker, interacts with nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) through two independent regions of NS5B, amino acids 208 to 214 and 500 to 506. We also showed that truncated nucleolin that harbors the NS5B-binding region inhibited the RNA-dependent RNA polymerase activity of NS5B in vitro, suggesting that nucleolin may be involved in HCV replication. To address this question, we focused on NS5B amino acids 208 to 214. We constructed one alanine-substituted clustered mutant (CM) replicon, in which all the amino acids in this region were changed to alanine, as well as seven different point mutant (PM) replicons, each of which harbored an alanine substitution at one of the amino acids in the region. After transfection into Huh7 cells, the CM replicon and the PM replicon containing NS5B W208A could not replicate, whereas the remaining PM replicons were able to replicate. In vivo immunoprecipitation also showed that the W208 residue of NS5B was essential for its interaction with nucleolin, strongly suggesting that this interaction is essential for HCV replication. To gain further insight into the role of nucleolin in HCV replication, we utilized the small interfering RNA (siRNA) technique to investigate the knockdown effect of nucleolin on HCV replication. Cotransfection of replicon RNA and nucleolin siRNA into Huh7 cells moderately inhibited HCV replication, although suppression of nucleolin did not affect cell proliferation. Taken together, our findings strongly suggest that nucleolin is a host component that interacts with HCV NS5B and is indispensable for HCV replication.     

Human Monoclonal Antibody HCV1 Effectively Prevents and Treats HCV Infection in Chimpanzees

Hepatitis C virus (HCV) infection is a leading cause of liver transplantation and there is an urgent need to develop therapies to reduce rates of HCV infection of transplanted livers. Approved therapeutics for HCV are poorly tolerated and are of limited efficacy in this patient population. Human monoclonal antibody HCV1 recognizes a highly-conserved linear epitope of the HCV E2 envelope glycoprotein (amino acids 412–423) and neutralizes a broad range of HCV genotypes. In a chimpanzee model, a single dose of 250 mg/kg HCV1 delivered 30 minutes prior to infusion with genotype 1a H77 HCV provided complete protection from HCV infection, whereas a dose of 50 mg/kg HCV1 did not protect. In addition, an acutely-infected chimpanzee given 250 mg/kg HCV1 42 days following exposure to virus had a rapid reduction in viral load to below the limit of detection before rebounding 14 days later. The emergent virus displayed an E2 mutation (N415K/D) conferring resistance to HCV1 neutralization. Finally, three chronically HCV-infected chimpanzees were treated with a single dose of 40 mg/kg HCV1 and viral load was reduced to below the limit of detection for 21 days in one chimpanzee with rebounding virus displaying a resistance mutation (N417S). The other two chimpanzees had 0.5–1.0 log₁₀ reductions in viral load without evidence of viral resistance to HCV1. In vitro testing using HCV pseudovirus (HCVpp) demonstrated that the sera from the poorly-responding chimpanzees inhibited the ability of HCV1 to neutralize HCVpp. Measurement of antibody responses in the chronically-infected chimpanzees implicated endogenous antibody to E2 and interference with HCV1 neutralization although other factors may also be responsible. These data suggest that human monoclonal antibody HCV1 may be an effective therapeutic for the prevention of graft infection in HCV-infected patients undergoing liver transplantation.