联合检测丙肝病毒核心抗原和抗体在丙型肝炎早期诊断中的价值

目的:通过检测丙型肝炎抗体(HCV-Ab),同时对其进行丙型肝炎病毒核心抗原(HCV-cAg)和丙肝病毒RNA(HCV-RNA)两项指标进行检测,探讨HCV-cAg检测在丙型肝炎早期诊断中的意义。方法:对104例HCV-Ab阳性标本及108例高危人群但HCV-Ab阴性标本(包括血液透析患者55例、医务人员20例和丙型肝炎病人的家庭密切接触者33例)及健康对照组同时进行HCV-cAg和HCV-RNA的检测。结果:104例HCV-Ab阳性组中,HCV-cAg阳性25例,HCV-RNA阳性27例;108例HCV-Ab阴性组中,HCV-cAg阳性2例,HCV-RNA阳性1例;健康对照组全阴性。结论:HCV-cAg和HCV-RNA在丙型肝炎早期诊断上无显著性差异,但HCV-cAg检测在临床的应用前景上具有更大的优势,故可在临床推广HCV-Ab和HCV-cAg联合检测,有条件者可联合检测HCV-Ab、HCV-cAg和HCV-RNA。     

A novel linear neutralizing epitope of hepatitis E virus

Hepatitis E virus (HEV) is a serious public health problem that causes acute hepatitis in humans and is primarily transmitted through fecal and oral routes. The major anti-HEV antibody responses are against conformational epitopes located in a.a. 459–606 of HEV pORF2. All reported neutralization epitopes are present on the dimer domain constructed by this peptide. While looking for a neutralizing monoclonal antibody (MAb)-recognized linear epitope, we found a novel neutralizing linear epitope (L2) located in a.a. 423–437 of pORF2. Moreover, epitope L2 is proved non-immunodominant in the HEV-infection process. Using the hepatitis B virus core protein (HBc) as a carrier to display this novel linear epitope, we show herein that this epitope could induce a neutralizing antibody response against HEV in mice and could protect rhesus monkeys from HEV infection. Collectively, our results showed a novel non-immunodominant linear neutralizing epitope of hepatitis E virus, which provided additional insight of HEV vaccine.     

丙型肝炎病毒疫苗研究进展

近年来,在人体内及黑猩猩试验模型中发现HCV天然免疫与特异性免疫能自发清除病毒,表明发展部分有效的针对不同型的HCV疫苗是可能的,这对慢性丙型肝炎的防治有重大意义。此文综述了HCV感染相关免疫保护机制、疫苗研制情况及今后发展方向。     

Immunity of two novel hepatitis C virus polyepitope vaccines

Hepatitis C virus (HCV) infection remains a serious public health burden around the world. So far there is no effective vaccine against this virus. Neutralizing antibody (NAb) responses to the epitopes within HCV E1 and E2 proteins are related to the resolution of hepatitis C infection. E. coli heat-labile enterotoxin B subunit (LTB) has been described as potent immunity adjuvants. In this study, we constructed recombinant pET vectors: pET-R9-Bp (B cell polyepitopes) expressing 7 epitopes from HCV E1 and E2 proteins including R9 (E2_384-411aa)-Bp (E1_313-327aa-E2_396-424aa-E2_436-447aa-E2_523-540aa-E2_610-627aa-E2_631-648aa) and pET-LTB-R9-Bp expressing LTB adjuvant in combination with R9-Bp. Recombinant proteins R9-Bp and LTB-R9-Bp were expressed successfully in E. coli and purified by the Ni-NTA column. Both R9-Bp and LTB-R9-Bp in BALB/c mice induced robust humoral immune response in the context of intraperitoneal or intramuscular immunization but not oral immunization. Intraperitoneal administration of LTB-R9-Bp induced a higher antibody titer (peak titer: 1:341000) than that of R9-Bp (peak titer: 1:85000) after the second boost (P = 0.0036 or 0.0002). However, comparable antibody peak titers were elicited for both R9-Bp and LTB-R9-Bp in intramuscular immunization albeit with significant difference (P = 0.0032) a week after the second boost. In addition, both R9-Bp and LTB-R9-Bp induced the secretion of cytokines including IFN-γ and IL-4 at similar levels. anti-sera induced by both R9-Bp and LTB-R9-Bp recognized native HCV E1 and E2 proteins. Moreover, these HCV-specific antisera inhibited significantly the entry of HCV (P < 0.0001). Taken together, these findings showed that E. coli-based both R9-Bp and LTB-R9-Bp could become promising HCV vaccines.     

Indications of immune protection from hepatitis C infection

Hepatitis C affects many millions of people worldwide and is at very high prevalence among people who inject drugs. In our study of hepatitis C virus (HCV) in the social networks of injecting drug users (IDUs), five IDUs with injecting careers of 9 years or more were HCV antibody and RNA negative. All injected frequently with HCV RNA-positive IDUs, and two had recently injected with the syringe of an RNA-positive IDU. Our data suggest the existence of immune protection from HCV infection.     

Oral immunization with attenuated Salmonella carrying a co-expression plasmid encoding the core and E2 proteins of hepatitis C virus capable of inducing cellular immune responses and neutralizing antibodies in mice

Hepatitis C virus (HCV) core protein has long been considered an attractive candidate for inclusion in a protective vaccine. However, this protein may hamper the development of systemic immune responses because of its immune suppressive properties. We previously reported that immune responses to HCV core protein could be efficiently induced by attenuated Salmonella carrying the HCV core protein, but not the HCV core DNA vaccine. To optimize the combination of the core protein and envelope protein 2 (E2) into a vaccine formulation to induce cellular immune responses and neutralizing antibodies, we constructed a plasmid containing two expression cassettes. One expression cassette was included to regulate the expression of HCV core protein by an inducible in vivo-activated Salmonella promoter, the other was included to regulate the expression of HCV E2 protein by the cytomegalovirus enhancer/promoter. Oral immunization of BALB/c mice with the attenuated Salmonella strain SL7207 carrying this plasmid efficiently induced HCV core and E2-specific cellular immune responses and antibodies. IgG purified from immunized mice could neutralize the infectivity of HCV pseudoparticles (HCVpp) of both the autologous Con 1 isolate and the heterologous H77 isolate, and cell culture produced HCV (HCVcc) of Con1-JFH1 chimera. These results indicated that this vaccine strategy can effectively deliver core and E2 protein to the immune system and provide a promising approach for the development of prophylactic and therapeutic vaccines against HCV infection.     

江苏省宜兴地区丙型肝炎病毒F蛋白抗体检测

目的检测江苏省宜兴地区丙型肝炎（丙肝）患者血清丙肝病毒（HCV）-F抗体及其分布。方法利用基因重组获得的HCV—F／GST蛋白作为抗原，包被酶联反应板，ELISA间接法检测120例丙肝患者、15例乙肝患者、3例戊肝患者及10份正常人血清HCV—F抗体，结合丙肝患者的临床资料及疾病特征，统计分析HCV—F抗体的分布情况及与HCV感染的关系。结果120例丙肝患者血清HCV—F抗体阳性82例，阳性率68％，而15例乙肝、3例戊肝患者及10份正常人血清标本均未检出阳性；资料分析显示HCV—F抗体阳性率与HCV患者年龄、临床类型之间差异有统计学意义，51岁以上年龄组的HCV—F抗体阳性率是20～50岁组的6．675倍（95％CI：2．407～19．071），并且随着病程进展，阳性率也随之增高（OR=2．749，95％CI：1．470～5．141）。结论江苏省宜兴地区HCV感染者血清中存在HCV—F抗体，并可能与病程相关。     

Antibodies to hepatitis C virus in blood donors

Recently a recombinant polypeptide of hepatitis C virus (HCV) has been developed by the Chiron Corporation in California. This antigen has been used to develop an ELISA test (Ortho Diagnostic Systems) for serum anti-HCV antibodies. Preliminary data have shown that this virus is the major cause of NANB hepatitis in the world. We examined differences in anti-HCV prevalence among subgroups of blood donors (total sera examined 639) classified for past or present exposure to HBV or not, and for ALT levels. The anti-HCV prevalence found in regular blood donors with normal ALT levels and no antibody to HBcAg was 1.2%. No significant difference in the anti-HCV prevalence was found among other subgroups of blood donors except that a higher prevalence (10%) was found in a group with both elevated ALT and HBV markers.These preliminary findings suggest that the policy of blood supply should take into account the advent of HCV antibody test.     

170848例患者丙型肝炎病毒抗体检测结果分析

目的 了解医院就诊患者的丙型肝炎感染情况,为早期诊治丙型肝炎感染提供决策依据.方法 用酶联免疫吸附试验对2008-2011年170 848例住院及门诊患者的血液进行丙型肝炎病毒抗体(抗-HCV)检测.结果 在170 848份血液标本中,共检出抗-HCV阳性4984例,抗-HCV阳性率2.92％;内科61 677份血液标本中,检出抗-HCV阳性2035例,抗-HCV阳性率最高,为3.30％,其次分别为外科、门诊、妇科2545、251和153例,阳性率分别为2.94％、2.07％和1.46％;经统计学分析,不同科室间的阳性率差异有统计学意义(P＜0.05);男性92 068份标本,检出抗-HCV阳性3512例,女性78 780份标本,检出抗-HCV阳性1472例,男、女阳性率分别为3.81％、1.87％,不同性别间的阳性率差异有统计学意义(P＜0.05);感染者年龄段中抗-HCV阳性率最高的是31～40岁,在35 971份血液标本中,检出抗-HCV阳性1428例,阳性率3.97％.结论 加强对公众丙型肝炎防治健康教育工作,及早控制感染源,切断传播途径,防止丙型肝炎病毒的蔓延和传播.     

Hepatitis C virus soluble E2 in combination with QuilA and CpG ODN induces neutralizing antibodies in mice

Several studies have emphasized the importance of an early, highly neutralizing antibody response in the clearance of Hepatitis C virus (HCV) infection. The envelope glycoprotein E2 is a major target for HCV neutralizing antibodies. Here, we compared antibody responses in mice immunized with native soluble E2 (sE2) from the H77 1a isolate coupled with different adjuvants or combinations of adjuvants. Adjuvanting sE2 with Freund's, monophosphoryl lipid A (MPL), cytosine phosphorothioate guanine oligodeoxynucleotide (CpG ODN), or alpha-galactosylceramide (αGalCer) derivatives elicited only moderate antibody responses. In contrast, immunizations with sE2 and QuilA elicited exceptionally high anti-E2 antibody titers. Sera from these mice effectively neutralized HCV pseudoparticles (HCVpp) 1a entry. Moreover, the combination of QuilA and CpG ODN further enhanced neutralizing antibody titers wherein cross-neutralization of HCVpp 4 was observed. We conclude that the combination of QuilA and CpG ODN is a promising adjuvant combination that should be further explored for the development of an HCV subunit vaccine. Our work also emphasizes that the ideal combination of adjuvant and immunogen has to be determined empirically.     

T细胞应答在丙型肝炎病毒持续感染中的作用

HCV感染极易慢性化,已成为困扰全球人类健康的重要公共卫生问题。近年研究发现,T细胞应答可能在HCV感染慢性化中的发挥着重要作用,此文对T细胞应答与HCV持续感染的关系进行综述。     

Antigenicity of hepatitis C virus envelope proteins expressed in Chinese hamster ovary cells

A putative second envelope glycoprotein (E2) of hepatitis C virus (HCV) was constitutively produced in a Chinese hamster ovary cell line stably transformed with a plasmid expressing E2 protein under the control of an exogenous promoter and a signal sequence. E2 protein that lacked part of the C-terminal hydrophobic region was glycosylated with high-mannose type oligosaccharides and retained in the cells. On the other hand, E2 protein lacking the entire C-terminal hydrophobic region was glycosylated with complex type oligosaccharides (complex form) and excreted into the culture medium. Immunoreactivity of the high-mannose and complex forms of E2 proteins against sera from HCV infected patients were analyzed. We found that the antigenicity of the complex form of E2 protein was greater than that of the high-mannose form of E2 protein. This result indicated that the complex form of the E2 protein is superior for use in diagnosing HCV infection.     

Assessing the feasibility of hepatitis C virus vaccine trials: Results from the Hepatitis C Incidence and Transmission Study-community (HITS-c) vaccine preparedness study

Efficacy trials of preventive hepatitis C virus (HCV) vaccine candidates raise challenging scientific and ethical issues. Based on data from the first 3 years of a community-based prospective observational study – the Hepatitis C Incidence and Transmission Study-community (HITS-c) – this paper examines the feasibility of conducting trials of candidate HCV vaccines with people who inject drugs (PWID) in Sydney, Australia. Of the 166 PWID confirmed HCV antibody negative and eligible for enrolment, 156 (94%) completed baseline procedures. Retention was high, with 89% of participants retained at 48 weeks and 76% of participants completing at least 75% of study visits within 2 weeks of schedule. The rate of primary HCV infection was 7.9/100 py (95% CI 4.9, 12.7). Of the 17 incident cases, 16 completed at least one follow-up assessment and 12 (75%) had evidence of chronic viraemia with progression to chronic HCV infection estimated to be 6/100 py. Power calculations suggest a chronic HCV infection rate of at least 12/100 py (primary HCV infection rate 16/100 py) will be required for stand-alone trials of highly efficacious candidates designed to prevent chronic infection. However, elevated primary HCV infection was observed among participants not receiving opioid substitution therapy who reported heroin as the main drug injected (26.9/100 py, 95% CI 14.5, 50.0) and those who reported unstable housing (23.5/100 py, 95% CI 7.6, 72.8), daily or more frequent injecting (22.7/100 py, 95% CI 12.2, 42.2) and receptive syringe sharing (23.6/100 py, 95% CI 9.8, 56.7) in the 6 months prior to baseline. These data suggest that it is possible to recruit and retain at-risk PWID who adhere to study protocols and that modification of eligibility criteria may identify populations with sufficiently high HCV incidence. Results support the feasibility of large multi-centre HCV vaccine trials, including in the Australian setting.     

Sexual transmission of hepatitis C virus infection

The role of sexual transmission in the diffusion of HCV infection, was studied through the seroprevalence of anti-HCV antibodies in the heterosexual habitual partners of 83 anti-HCV positive subjects. The index cases were represented by 10 dialysed subjects, 31 patients with chronic liver disease and 42 healthy carriers. Seroprevalence of anti-HCV positivity reported in partners was 8.43%, with a higher rate in cohabitants of patients with chronic liver disease (16.12% vs 4.76% of carriers); no case was found among partners of dialysed subjects. Laboratory and ultrasonograph signs of chronic hepatitis were reported in 3 cases (3.61%). Control on 70% of the cohabitants' relatives, was negative for HCV infections. These data suggest a possible sexual transmission of HCV infection, even if its prevalence resulted modest, undoubtedly lower than in other disease sexually transmitted.     

IL-2-330基因多态性与慢性乙型肝炎病毒和/或丙型肝炎病毒感染及不同临床转归的关系

目的 研究乙型肝炎病毒(HBV)和/或丙型肝炎病毒(HCV)感染及其不同临床转归者IL-2基因多态性(SNP).方法 对河北省赵县某农村HBV和/或HCV感染者及对照共277人采集空腹静脉血.用ELISA检测抗-HBV、抗-HCV生物标志物,筛出HBV重叠HCV感染79例、单纯HBV感染69例、HCV感染55例和对照74例.用RT-nPCR检测HCVRNA,BeckmanLX-20全自动生化仪检测肝功能丙氨酸氨基转移酶(ALT),RFLP-PCR技术检测IL-2-330 SNP,分析IL-2-T330G SNP与HBV和/或HCV感染、不同临床转归、ALT和HCVRNA表达的关系.结果 (1)不同感染类型即单纯HBV、HCV感染和重叠感染者IL-2-330 TT频率明显高于对照,-330 GG频率明显低于对照(χ2=14.24,P=0.03),OR值(95%CI)分别是7.14(2.13～23.81)、3.46(1.17～10.02)、2.93(1.15～7.46),各感染类型间差异无统计学意义(χ2=2.09,P=0.72);各感染组-330T频率明显升高,-330G频率明显降低(χ2=12.33,P=0.01),OR值(95%CI)分别是2.26(1.39～3.69)、1.82(1.09～3.03)、1.73(1.10～2.73)倍.(2)不同临床转归即轻型、中重型肝炎和肝硬化组IL-2-330 TT频率明显高于对照组、-330 GG频率明显低于对照组(χ2=13.52,P=0.04),OR值(95%CI)分别是3.33(1.75～6.32)、3.31(1.75～6.26)、11.23(3.09～40.76).不同临床转归组-330T频率明显升高,-330 G频率明显降低(χ2=12.32,P=0.01);OR值(95%CI)分别是1.86(1.32～2.63)、1.71(1.27～2.31)、2.77(1.57～4.89)倍.(3)IL-2-330基因型和等位基因频率与HCV的病毒复制无统计关联(χ2=0.83,P=0.66;χ2=0.20,P=0.66),与ALT水平亦无统计关联(χ2=1.10,P=0.58;χ2=0.08,P=0.78).结论 IL-2-330 T/G SNP与HBV和/或HCV感染者慢性化及不同临床转归有一定关联,IL-2-330TT/T能增加HBV和/或HCV感染及其临床转归的危险,-330 GG/G则减低其感染和临床转归风险.     

静脉注射毒品人群中HIV、HBV和HCV感染的现况研究

目的了解静脉注射毒品人群中人类免疫缺陷病毒（HIV）、乙型肝炎病毒（HBV）和丙型肝炎病毒（HCV）的感染情况。方法从四川、湖南、广西和新疆等地静脉注射毒品人群中采集血液样本2025份，应用酶联免疫试剂盒检测抗-HIV、抗-HCV抗体和HBsAg。结果红静脉沣射毒品人群中，抗-HIV、抗-HCV及HBsAg的阳性率分别为14．7％～30．4％、60．7％～85．5％和6．6％～22．4％，其HIV／HBV、HIV/HCV、HCV／HBV和HIV／HCV／HBV合并感染率分别为0％～0．4%、11．6％～27．2％、2．3％～14．3％和1．6％～4．8％。结论静脉注射毒品人群中HIV、HBV和HCV的感染率均高于正常人群，其中HIV与HCV合并感染率最高。     

宁波地区丙型肝炎病毒基因分型检测

目的:通过检测丙型肝炎病毒基因分型,探讨宁波地区HCV感染基因型流行株特征。方法:本文共收集231例HCV感染者的血清,采用基因芯片技术进行HCV基因分型。结果:231例丙型肝炎病毒基因分型1b型162例,占70.12%;2a型25例,占10.82%;1b+2a混合型15例,占6.49%;并有少数的3b3、a6、型和其它混合型,均占<5%。结论:宁波地区HCV感染基因型以lb型为主、其次为2a型。基因芯片法检测丙型肝炎病毒基因分型,具有微量、多位点、准确性好、灵敏度高、特异性强等特点。     

丙型肝炎免疫发病机制的研究进展

丙型肝炎病毒(HCV)是输血后肝炎的主要致病因子,给人类的健康造成了极大的危害。由于HCV基因的高变性,容易逃避机体免疫系统的清除;HCV感染免疫系统细胞,造成机体对HCV感染的免疫应答异常,并引起组织的损伤,导致肝细胞的慢性持续性感染,并可进一步发展成肝硬化,甚至肝细胞肝癌。文中对近年来HCV在免疫致病机制方面的研究进展进行了综述。     

Spreading of hepatitis C virus subtypes 1a and 1b through the central region of Argentina

The recent history of the hepatitis C virus (HCV) subtypes 1a and 1b in the central region of Argentina is hypothesized by phylogeographic reconstruction using coalescent based Bayesian analyses. Direct partial E2 sequences from HCV 1a and 1b infected patients attending different health-care centers of the country were analyzed.The inferred date of the most recent common ancestor (t_MRCA) for HCV-1a was: 1962 (between 1943 and 1977) and for HCV-1b was earlier: 1929 (between 1895 and 1953). Diverse ancestral populations were inferred from both subtypes in Córdoba and in Buenos Aires cities and after that, HCV spread within and between larger cities and to other smaller cities. The analyses suggested that HCV-1b was dispersed first and it is currently in a stationary phase whereas HCV-1a was dispersed latter and it is still in a growth phase.Finally, as it was observed in the developed countries, while the transmission of HCV-1b appears to have been somehow prevented, the HCV-1a may still represent a concern in the public health. Further work should be carried out to address their current transmission rate (and its main transmission route) in the Argentinean population.     

Candidate hepatitis C vaccine trials and people who inject drugs: Challenges and opportunities

People who inject drugs (PWID) are at high risk of HCV. Limited evidence of the effectiveness of prevention interventions and low uptake of treatment in this group highlight the need for increased investment in biomedical interventions, notably safe and efficacious vaccines. While several candidates are currently in development, field trials in PWID present challenges, including ethical issues associated with trial literacy, informed consent and standards of care. Significant biological and social factors and differences between HIV and HCV suggest that HCV warrants targeted vaccine preparedness research to lay the groundwork for successful implementation of future trials.