Preparation and application of monoclonal antibodies against hepatitis C virus nonstructural proteins

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Monoclonal antibodies:Principles and applications of immmunodiagnosis and immunotherapy for hepatitis C virus

Hepatitis C virus(HCV) is a major health problem worldwide. Early detection of the infection will help better management of the infected cases. The monoclonal antibodies(m Ab) of mice are predominantly used for the immunodiagnosis of several viral,bacterial,and parasitic antigens. Serological detection of HCV antigens and antibodies provide simple and rapid methods of detection but lack sensitivity specially in the window phase between the infection and antibody development. Human mA b are used in the immunotherapy of several blood malignancies,such as lymphoma and leukemia,as well as for autoimmune diseases. In this review article,we will discuss methods of mouse and human monoclonal antibody production. We will demonstrate the role of mouse mA b in the detection of HCV antigens as rapid and sensitive immunodiagnostic assays for the detection of HCV,which is a major health problem throughout the world,particularly in Egypt. We will discuss the value of HCV-neutralizing antibodies and their roles in the immunotherapy of HCV infections and in HCV vaccine development. We will also discuss the different mechanisms by which the virus escape the effect of neutralizing mA b. Finally,we will discuss available and new trends to produce antibodies,such as egg yolk-based antibodies(Ig Y),production in transgenic plants,and the synthetic antibody mimics approach.

Neutralizing antibodies in hepatitis C virus infection

Hepatitis C virus (HCV) is a major cause of hepatitis world-wide. The majority of infected individuals develop chronic hepatitis which can then progress to liver cirrhosis and hepatocellular carcinoma. Spontaneous viral clearance occurs in about 20%-30% of acutely infected individuals and results in resolution of infection without sequaelae. Both viral and host factors appear to play an important role for resolution of acute infection. A large body of evidence suggests that a strong, multispecific and long-lasting cellular immune response appears to be important for control of viral infection in acute hepatitis C. Due too the lack of convenient neutralization assays, the impact of neutralizing responses for control of viral infection had been less defined. In recent years, the development of robust tissue culture model systems for HCV entry and infection has finally allowed study of antibody-mediated neutralization and to gain further insights into viral targets of host neutralizing responses. In addition, detailed analysis of antibody-mediated neutralization in individual patients as well as cohorts with well defined viral isolates has enabled the study of neutralizing responses in the course of HCV infection and characterization of the impact of neutralizing antibodiesfor control of viral infection. This review will summarize recent progress in the understanding of the molecular mechanisms of antibody-mediated neutralization and its impact for HCV pathogenesis.

Comparative Study on 4 EIA Kits for Screening Antibody to Hepatitis C Virus in Pooled Sera

Four enzyme immunoassay (EIA) test kits, 1 Canadian product and 3 Chinese products,were used in the comparative study. Each pool consisted of 5 sera, and the 5 single sera were tested as controls. The tests were carried out according to the instructions, keeping the same dilution of each serum in single and pool samples. It was found that with the Canadian kit,the positive and negative results of opled sera had no difference from that of the controls (P>0. 10). In the case of Chinese Yali and Kehua kits, the positive results of pooled sera showed no difference from the controls (P >0. 10), but the optical density (OD) of negative opls were increased (P < 0. 01 ), though quite distant from the cut-off values. In the case of Changzheng kit, the OD of opitive opls were significantly lower than those of the controls (P < 0. 05 ), and weak positive samples missed the detection. However this problem could be overcome by blocking the microwells beforehand. Our experiment demonstrate that not all EIA test kits are suitable for screening opls for antithey to hepatitis C virus, and that it is important to assess the sensitivity of the EIA kit to be used for this purpose.

Antibody to El peptide of hepatitis C virus genotype 4 inhibits virus binding and entry to HepG2 cells in vitro

瞄准：对丙肝病毒(HCV ) 的 E1 区域分析抗体的抵销的活动。特定的 polyclonal 抗体与从 HCV 的 E1 区域被导出并且被显示高度在 HCV 之中被保存的合成的肽经由新西兰兔子的免疫被提起出版遗传型。方法：超 HCV E1 抗体与为 HCV RNA 积极的浆液样品在 4 度摄氏在夜里被孵化，与从 615 ～ 3.2 百万 IU/ mL 的病毒的负担。对待的重量的单位一为 90 min 与 HepG2 房间被孵化。由反 E1 抗体病毒的绑定和入口堵住进房间借助于 RT-PCR 和流动血细胞计数被测试。结果：用 FITC 染色的直接免疫结合了分析显示出的流动 cytometric 跟随的 E1 抗体在样品的减少的吝啬的荧光紧张与未经治疗的样品相比与 E1 抗体预先孵化。而且， 13 从 18 积极重量的单位一(72%) 显示出由 RT-PCR 检测了的传染性的完全的抑制。结论：在房子里生产 E1 抗体，块绑定和到在病毒绑定建议这 epitope 的参与的靶细胞的 HCV virion 感染的入口和入口。堵住病毒附件到人的房间的这些抗体的隔离作为治疗学的试剂有用。

Prevalence of HCV Antibody and its Associated Factors: A Study from Sentinel Hospitals in China

Objective The prevalence and related factors of serum anti-HCV in different regions and hospitals have not been studied extensively in China. We used routine screening data to determine the prevalence of HCV antibody in hospital patients, evaluate the epidemic trend of hepatitis C and formulate screening strategies.Methods Patient information and HCV antibody testing results were collected from January 2017 to December 2019 in 77 HCV sentinel hospitals in China. Univariate and multivariate logistic regression was used to determine the characteristics and associations.Results HCV antibody prevalence rates were distinct among patients in different departments, with a range of 0.33%–6.93%. Patients who were admitted to the liver disease-related departments(aOR =10.76;95% CI, 10.27–11.28), Internal Medicine(aOR = 2.87;95% CI, 2.75–3.00), and Department of Surgery(aOR = 1.95;95% CI, 1.87–2.04), were more likely to be tested for HCV antibody positive. HCV antibody prevalence was associated with patients aged 45 years and older(aOR = 2.74;95% CI,2.69–2.80), testing in infetious disease hospitals(aOR = 2.33;95% CI, 2.26–2.40) and secondary hospitals(aOR = 1.72;95% CI, 1.69–1.75). Patients in sentinel hospitals of the Northeast(aOR = 12.75;95% CI,12.40–13.11), the Central(aOR = 1.65;95% CI, 1.61–1.70), and the West(aOR = 1.78;95% CI, 1.73–1.83)China had higher HCV prevalence than those who were in the Eastern coastal area. Conclusion Those who were over 45 years old and saw doctors for liver diseases, and invasive diagnosis and treatment should be referred to HCV antibody testing.

Hepatitis C virus may infect extrahepatic tissues in patients with hepatitis C

AIM To explore the status of extrahepatichepatitis C virus(HCV)infection and replicationin hepatitis C patients,and its potentialimplication in HCV infection and pathogenicity.METHODS By reverse-transcriptase poly-merase chain reaction(RT-PCR),in situhybridization(ISH)and immunohistochemistry,HCV RNA,HCV replicative intermediate(minus-strand of HCV RNA),and HCV antigens weredetected in 38 autopsy extrahepatic tissuespecimens(including 9 kidneys,9 hearts,9pancreas,5 intestines,2 adrenal glands,2spleens,1 lymph node,and 1 gallbladder)from 9hepatitis C patients,respectively;and thestatus of HCV replication in extrahepatic tissueswas studied.RESULTS By RT-PCR,all 9 patients werepositive for HCV RNA in kidney,heart,pancreas,and intestine,but only 6(66.7%)patients were positive for HCV replicativeintermediate.HCV RNA and HCV antigens weredetected in kidney,heart,pancreas,intestine,adrenal gland,lymph node,and gallbladder in 5(55.6%)and 6(66.7%)patients by ISH andimmunohistochemistry,respectively.HCV RNA and HCV antigens were not detected in theseextrahepatic organs in 3(33.3%)patients,although their livers were positive for HCV.HCVreplicative intermediate detected by RT-PCR wasconsistent with HCV RNA and HCV antigensdetected by ISH and immunohistochemistry(Kappa=0.42-0.75).HCV RNA and HCVantigens were detected in myocardial cells,epithelial cells of intestinal gladular,interstitialcells of kidney,epithelial cells of tubules andglomerulus,pancreas acinar cells and epithelialcells of pancreatic duct,epithelial cells ofmucous membrane sinus of gallbladder,cortexand medulla cells in adrenal gland,andmononuclear cells in lymph node.HCV RNA wasalso detected in bile duct epithelial cells,sinusoidal cells,and mononuclear cells in livertissues by ISH.CONCLUSION HCV can infect extrahepatictissues,and many various tissue cells maysupport HCV replication;extrahepatic HCVinfection and replication may be of'concomitantstate'in most of patients with hepatitis C.Theinfected extrahepatic tissues might act as areservoir for HCV,and play a role in both HCVpersistence and reactivation of infection.HCVas an etiologic agent replicating and expressingviral proteins in extrahepatic tissues itselfcontributes to extrahepatic syndromeassociated.HCV infection in a few patients withchronic HCV infection.

Hepatitis C virus micro-elimination:Where do we stand?

Hepatitis C virus (HCV) elimination by 2030, using direct-acting antiviraltreatments, has been promoted by the World Health Organization. Thisachievement is not attainable, however, particularly after the 2020 pandemic ofthe coronavirus disease 2019. Consequently, the more realistic objective ofeliminating HCV from population segments for which targeted strategies ofprevention and treatment are easily attained has been promoted in Europe, as avalid alternative. The underlying idea is that micro-elimination will ultimatelylead to macro-elimination. The micro-elimination strategy may target differentspecific populations and at-risk groups. Different settings, including prisons andhospitals, have also been identified as micro-elimination scenarios. In addition,dedicated micro-elimination strategies have been designed that are tailored at thegeographical level according to HCV epidemiology and individual country’sincome. The main elements of a valid and successful micro-elimination project arereliable epidemiological data and active involvement of all the stakeholders.Community involvement represents another essential component for a successfulprogram.

Spontaneous elimination of hepatitis C virus infection: A retrospective study on demographic, clinical, and serological correlates

AIM: To fi nd correlates to spontaneous clearance of hepatitis C virus (HCV) infection, this study compared individuals with self-limited and chronic infection with regard to clinical, demographic, and serological pa-rameters. METHODS: Sixty-seven anti-HCV positive and repeatedly HCV RNA negative individuals were considered to have resolved HCV infection spontaneously. To determine the viral genotype these patients had been infected with HCV serotyping was performed. For comparison reasons, 62 consecutive patients with chronic hepatitis C were enrolled. Cases and controls were compared stratifi ed for age and sex. RESULTS: Retrospective analysis showed (1) a lower humoral reactivity to HCV in patients with self-limited compared to chronic HCV-infection and (2) that younger age, history of iv drug use, and acute/post-acute hepatitis A or B co-infections, but not viral genotypes, are independent correlates for spontaneous HCV clearance. CONCLUSION: The stronger humoral reactivity to HCV in patients with persistent infections and in those with a history of iv drug use is supposed to be due to continuous or repeated contact(s) to the antigen. Metachronous hepatitis A or hepatitis B infections might favor HCV clearance.

Flow cytometric detection of hepatitis C virus antigens in infected peripheral blood leukocytes: Binding and entry

AIM: We designed two synthetic-core-specific peptides core 1 (C1) and core 2 (C2), and an E1-specific peptide (E1). We produced specific polyclonal antibodies againstthese peptides and used the antibodies for detection of HCV antigens on surface and within infected peripheral blood leukocytes.METHODS: Peripheral blood from a healthy individual who tested negative for HCV RNA was incubated with HCV type 4 infected serum for 1 h and 24 h at 37 ℃. Cells were stained by direct and indirect immunofluorescence and measured by flow cytometry.RESULTS: After 1 h of incubation, antibodies against C1,C2, and E1 detected HCV antigens on the surface of 27%,26% and 73% of monocytes respectively, while 10%, 5% and 9% of lymphocytes were positive with anti-C1, anti-C2 and anti-E1 respectively. Only 1-3% of granulocytes showed positive staining with anti-C1, anti-C2 and anti E1 antibodies. After 24 h of incubation, we found no surface staining with anti-C1, anti-C2 or anti-E1. Direct immunostaining using anti-C2 could not detect intracellular HCV antigens, after 1 h of incubation with the virus, while after 24 h of incubation, 28% of infected cells showed positive staining. Only plus strand RNA was detectable intracellularly as early as 1 h after incubation, and remained detectable throughout 48 h post-infection.Interestingly, minus RNA strand could not be detected after 1 h, but became strongly detectable intracellularly after 24 h post-infection.CONCLUSION: Monocytes and lymphocytes are the preferred target cells for HCV infection in peripheral blood leukocytes. Our specific anti-core and anti-E1 antibodies are valuable reagents for demonstration of HCV cell cycle.Also, HCV is capable of infecting and replicating in peripheral blood mononuclear cells as confirmed by detection of minus strand HCV RNA as well as intracellular staining of core HCV antigen.

Virus-host Interactions during Hepatitis C Virus Entry-Implications for Pathogenesis and Novel Treatment Approaches

Hepatitis C virus (HCV) is a member of the Flaviviridae family and causes acute and chronic hepatitis. Chronic HCV infection may result in severe liver damage including liver cirrhosis and hepatocellular carcinoma. The liver is the primary target organ of HCV, and the hepatocyte is its primary target cell. Attachment of the virus to the cell surface followed by viral entry is the first step in a cascade of interactions between the virus and the target cell that is required for successful entry into the cell and initiation of infection. This step is an important determinant of tissue tropism and pathogenesis; it thus represents a major target for antiviral host cell responses, such as antibody-mediated virus neutralization. Following the development of novel cell culture models for HCV infection our understanding of the HCV entry process and mechanisms of virus neutralization has been markedly advanced. In this review we summarize recent developments in the molecular biology of viral entry and its impact on pathogenesis of HCV infection, development of novel preventive and therapeutic antiviral strategies.

Is there a need for universal double reflex testing of HBsAg-positive individuals for hepatitis D infection?

Hepatitis D virus(HDV)can infect HBsAg-positive individuals,causing rapid fibrosis progression,early decompensation,increased hepatocellular carcinoma risk,and higher mortality than hepatitis B virus(HBV)mono-infection.Most countries lack high-quality HDV prevalence data,and the collection techniques employed often bias published data.In recent meta-analyses,HDV prevalence in HBsAg-positive patients reaches 5%-15%and is even significantly higher in endemic areas.Since HBV vaccination programs were implemented,HDV prevalence has decreased among younger populations.However,owing to immigrant influx,it has increased in some Western countries.The current practice of HDV screening in HBsAg-positive individuals is stepwise,based on physician’s discretion,and limited to at-risk populations and may require numerous visits.Double reflex testing,which includes anti-HDV testing in all HBsAg-positive individuals and then HDV RNA testing for anti-HDV-positive ones,is uncommon.Reflex testing can identify more HDV infection cases and link identified patients to further care and follow-up.Moreover,laboratory-based double reflex screening is less biased than physician-led testing.Therefore,health-care providers should learn about reflex testing,and federal and provincial hepatitis control programs should implement laboratory-based double reflex testing to obtain reliable HDV prevalence estimates.The test’s cost-effectiveness depends on the number of HBV-positive patients screened to identify one HDV-positive patient.Such testing may be viable in areas with low HBsAg but high HDV prevalence.However,its economic impact on areas with low HDV prevalence needs further study.

Immune mechanisms of vaccine induced protection against chronic hepatitis C virus infection in chimpanzees

Hepatitis C virus(HCV) infection is characterized by a high propensity for development of life-long viral persistence. An estimated 170 million people suffer from chronic hepatitis caused by HCV. Currently,there is no approved prophylactic HCV vaccine available.With the near disappearance of the most relevant animal model for HCV,the chimpanzee,we review the progression that has been made regarding prophylactic vaccine development against HCV. We describe the results of the individual vaccine evaluation experiments in chimpanzees,in relation to what has been observed in humans. The results of the different studies indicate that partial protection against infection can be achieved,but a clear correlate of protection has thus far not yet been defined.

Medicinal plants against hepatitis C virus

Hepatitis C virus(HCV)is a global health concern which is responsible for most of the liver diseases.Currently,there is no vaccine available for prevention of HCV infection due to the high degree of strain variation.The current standard of care is a combination of pegylated interferonαwith ribavirin and boceprevir/telaprevir.This treatment was partially effective and had significant side effects.Hence,there is a need to develop new antiviral agents that interfere with different stages of the HCV life cycle.Recent advances in the understanding of both the cellular and molecular mechanisms of HCV replication have provided the basis for novel therapeutic strategies.Several hundred plant species and their phyto-constituents have been isolated for screening against HCV,and some have been shown to have great medicinal value in preventing and/or ameliorating viral diseases in pre-clinical and clinical trials.This review summarizes medicinal plants and their phytochemicals which inhibit different stages of HCV life cycle and discuss their potential use in HCV therapy.

Studies on hepatitis C virus and prevalence of its infection in China

HCV infection is one of the most important health problems in China. In this project starting from 1989, the authors carried out a survey on the prevalence of HCV infection in eastern areas of China, collected large amounts of blood samples from individuals of high risk groups from Shanghai and the Provinces of Jiangsu,Hebei, Shandong and Hunan, constructed a random-primed Chinese HCV cDNA λgt11 library, developed diagnostic systems for the detection of anti-HCV, HCV genomic RNA as well as for HCV genotyping,and investigated the possible relationship between HCV infection and the development of hepatocellular carcinoma (HCC). According to our epidemiological data, about 2%-5% of the population was estimated to be infected by HCV in this country. The high anti-HCV positive rate (4. 1% - 65. 5% ) in blood donors indicated that HCV infection was a principal cause of post-transfusion hepatitis. DNA sequencing data of the clones obtained by immunoscreening or PCR method from the cDNA library showed that HCV genotype Ⅱ was the major strain in China. Some of the antigenic epitopes identified from HCV C and NSS regions-derived clones were chosen for the development of anti-HCV EIA kit. The kit showed good agreement with that from UBI, with the total coincidence of 99. 1 % in 736 specimens,and seemed to be more adaptive in the detection of Chinese hepatitis C patients. It was interesting that HCV RNA was detectable in 33. 3% liver tissue specimens of HCC patients. This rate is much higher than that of anti-HCV (10. 5%) in serum specimens of these patients. By in situ hybridization and peroxidase-antiperoxidase (PAP) detection, 26. 0% and 28. 8% of HCC liver specimens were found positive for HCV RNA and antigens. Our findings demonstrated that HCV infection was also a high risk factor of HCC in China.

Autoantibody profiles in autoimmune hepatitis and chronic hepatitis C identifies similarities in patients with severe disease

AIM To determine how the auto-antibodies(Abs) profiles overlap in chronic hepatitis C infection(CHC) and autoimmune hepatitis(AIH) and correlate to liver disease.METHODS Levels of antinuclear Ab, smooth muscle antibody(SMA) and liver/kidney microsomal-1(LKM-1) Ab and markers of liver damage were determined in the sera of 50 patients with CHC infection, 20 AIH patients and 20 healthy controls using enzyme linked immunosorbent assay and other immune assays. RESULTS We found that AIH patients had more severe liver disease as determined by elevation of total Ig G, alkaline phosphatase, total serum bilirubin and serum transaminases and significantly higher prevalence of the three non-organ-specific autoantibodies(auto-Abs) than CHC patients. Antinuclear Ab, SMA and LKM-1 Ab were also present in 36% of CHC patients and related to disease severity. CHC cases positive for auto-Abs were directly comparable to AIH in respect of most markers of liver damage and total Ig G. These cases had longer disease duration compared with auto-Ab negative cases, but there was no difference in gender, age or viral load. KLM-1+ Ab CHC cases showed best overlap with AIH. CONCLUSION Auto-Ab levels in CHC may be important markers of disease severity and positive cases have a disease similar to AIH. Auto-Abs might have a pathogenic role as indicated by elevated markers of liver damage. Future studies will unravel any novel associations between these two diseases, whether genetic or other.

CHANGES OF ANTIBODIES AGAINST ANTIGENS ENCODED BY DIFFERENT REGIONS OF HCV GENOME IN CHRONIC HEPATITIS C PATIENTS TREATED WITH INTERFERON

Twenty patients with chronic hepatitis c were investigated during the treatment with interferon to explore the changes of antibodies to HCV (anti-RCV). Anti-HCV was tested with recombinant immunoblot assay (RIBA) by using three antigens (C22, C33c, and C100-3) encoded by different regions of HCV genome. The changes of individual anti-HCV and ALT were compared with the change of HCV RNA. The results showed that persistent disappearance of serum HCV RNA was closely related to the changes of anti-C33c (P<0. 01) and anti-C100-3 (P<0. 005), but there was no relation between persistent ALT normality and HCV viremia clearance (P<0. 05). In conclusion, monitoring anti-C33c and anti-C100-3 could indicate the changes or HCV viremia. The normalization of ALT after interferon treatment did not indicate disappearance of HCV viremia.

Anti-CD 20 monoclonal antibodies and associated viral hepatitis in hematological diseases

Over the past decade, the administration of anti-CD20 monoclonal antibodies such as rituximab has demonstrated various degrees of effectiveness and has improved patients' outcomes during the treatment of autoimmune hematological disorders and hematological malignancies. However, the depletion of B-cells, the distribution of T-cell populations, and the reconstruction of host immunity resulting from the use of anti-CD20 monoclonal antibodies potentially lead to severe viral infections, such as hepatitis B virus(HBV), hepatitis C virus(HCV), parvovirus B19, and herpes viruses, in patients who are undergoing immune therapy or immunochemotherapy. Of these infections, HBV- and HCV-related hepatitis are a great concern in endemic areas because of the high morbidity and mortality rates in untreated patients. As a result, prophylaxis against HBV infection is becoming a standard of care in these areas. Parvovirus B19, a widespread pathogen that causes red blood cell aplasia in immunocompromised hosts, also causes hepatitis in healthy individuals. Recently, its association with hepatitis was recognized in a patient treated with rituximab. In addition, adenovirus, varicella-zoster virus hepatitis E virus, and rituximab itself have been linked to the occurrence of hepatitis during or after rituximab treatments. The epidemiologies and pathogeneses of these etiologies remain unknown. Because of the increasing use of anti-CD20 monoclonal antibodies for the treatment of hematological malignancies or autoimmune hematological disorders, it is imperative that physicians understand and balance the risks of hepatotropic virusassociated hepatitis against the benefits of using antiCD20 monoclonal antibodies.

疾病类型和临床指标对CMIA抗HCV抗体检测的影响

目的探讨化学发光微粒子免疫测定法(CMIA)测定抗HCV抗体产生灰区结果的影响因素。方法选取采用CMIA进行抗HCV抗体检测,且结果位于灰区(S/CO值为1.0~5.0)的患者183例(灰区组),包括恶性肿瘤33例、乙型肝炎16例、胆囊炎16例、胆囊结石13例、良性肿瘤13例、肝硬化11例、胆管结石9例、胆管炎9例,其他疾病63例。以抗HCV抗体检测结果为阴性(S/CO值<1.0)的健康体检者33名作为正常对照组。采用逆转录聚合酶链反应(RT-PCR)检测所有患者的HCV RNA。收集所有研究对象的临床资料和实验室指标检测结果,记录相应的酶联免疫吸附试验(ELISA)和电化学发光法抗HCV抗体检测结果。采用多元线性回归分析探讨抗HCV抗体检测结果的影响因素。结果灰区组HCV RNA检测结果均为阴性。CMIA和电化学发光法与RT-PCR的符合率为100.00%,ELISA与RT-PCR的符合率为97.81%。多元线性回归分析结果显示,良性肿瘤(t=2.080,P=0.039)可使CMIA检测抗HCV抗体的S/CO值升高,渗透压升高(t=-2.238,P=0.027)可使CMIA检测抗HCV抗体的S/CO值降低,其他指标对CMIA检测抗HCV抗体无显著影响(P>0.05)。灰区组血清离子渗透压与正常对照组比较,差异无统计学意义(P>0.05)。结论采用CMIA检测有良性肿瘤史患者的样本时S/CO值可能会升高,导致产生灰区结果。