Rafique Shazia, Idrees Muhammad, Ali Amjad, Iqbal Muhammad
Centre of Applied Molecular Biology, Ministry of Science & Technology Govt. of Punjab, 87-West Canal Bank Road, Thokar Niaz Baig, Lahore, Pakistan,
Mol Biol Rep. 2014 Jun;41(6):3945-50. doi: 10.1007/s11033-014-3262-y. Epub 2014 Feb 25.
Characterization of antibodies targeting the attachment and entry of the viral particles into host cells is important for studding antibody mediated neutralization. Antibodies against the envelope glycoproteins (EGP) have neutralizing capacity and can prevent HCV infections. System based on HCV pseudo typed-particles (HCVpp) stably expressing EGP can be used for screening of HCV anti envelope neutralizing antibodies in the serum of patients with acute and chronic HCV infections. The aim of the current study was to check HCVpp as a useful tool for the detection of anti-HCV envelope antibodies in the serum of HCV infected patients and to test the binding potential of these antiviral molecules to EGP of HCV 3a. Previously developed HCVpp harboring unmodified glycoproteins from local isolates in 293T cell line were used in this study. HCVpp were pre incubated with different concentrations of anti E1 antibody and different E2 antibodies to check antiviral activity. Further we used serum samples with low/medium (≤800,000 IU/mL), and high (>800,000 IU/mL) viral titer from chronic HCV male and female patients. Infection was done in Huh-7 cells for 1 h at 37 oC. Infectivity was checked through Luciferase assay. Considerable decrease in HCVpp infectivity with anti-envelope antibodies was observed in dose dependent manner. Maximum inhibition was seen when 5 µg/ml of monoclonal anti E1 antibody used. Further increase in concentration exhibited no decrease in infectivity which suggests that other factors are also involved in causing infection. Various well characterized E2-specific monoclonal antibodies (mAbs) have been screened for their capability to reduce infection in Huh-7 cells. Three of the four mAbs specific for the E2 had no effect on the infectivity of HCVpp. Confirmation sensitive antibody H53 showed maximum inhibition of infectivity. HCV ELISA positive samples from both male and female patients were used to neutralize the HCVpp. The neutralizing antibody response was observed in both males and females patients and do not assemble the rapidly evolving HCV envelope glycoproteins. That is why in spite the presence of neutralizing antibodies in the blood they fail to resolve infections. Moreover E1 antibodies insignificantly (>0.05) inhibit HCVpp infectivity while E2 antibodies significantly (<0.05) inhibit HCVpp infection. Based on the results of this study it is concluded that anti-envelope antibodies particularly the anti-E2 could be extremely valuable for characterizing the humoral immune response to HCV and for evaluating the potential for developing passive and active immunization for hepatitis C along with interferon therapy.
鉴定靶向病毒颗粒附着和进入宿主细胞的抗体对于研究抗体介导的中和作用很重要。针对包膜糖蛋白（EGP）的抗体具有中和能力，可预防丙型肝炎病毒（HCV）感染。基于稳定表达EGP的HCV假型颗粒（HCVpp）的系统可用于筛选急性和慢性HCV感染患者血清中的HCV抗包膜中和抗体。本研究的目的是检验HCVpp作为检测HCV感染患者血清中抗HCV包膜抗体的有用工具，并测试这些抗病毒分子与HCV 3a的EGP的结合潜力。本研究使用了先前在293T细胞系中构建的携带来自本地分离株的未修饰糖蛋白的HCVpp。将HCVpp与不同浓度的抗E1抗体和不同的E2抗体预孵育，以检测抗病毒活性。此外，我们使用了来自慢性HCV男性和女性患者的低/中（≤800,000 IU/mL）和高（>800,000 IU/mL）病毒滴度的血清样本。在37℃下于Huh-7细胞中进行感染1小时。通过荧光素酶测定法检查感染性。观察到抗包膜抗体使HCVpp感染性呈剂量依赖性显著降低。当使用5μg/ml的单克隆抗E1抗体时观察到最大抑制作用。浓度进一步增加时感染性未见降低，这表明其他因素也参与了感染过程。已筛选了各种特征明确的E2特异性单克隆抗体（mAb），以检测其降低Huh-7细胞感染的能力。四种E2特异性mAb中的三种对HCVpp的感染性没有影响。确认敏感抗体H53显示出最大的感染性抑制作用。使用来自男性和女性患者的HCV ELISA阳性样本中和HCVpp。在男性和女性患者中均观察到中和抗体反应，并且不能聚集快速进化的HCV包膜糖蛋白。这就是为什么尽管血液中存在中和抗体，但它们仍无法消除感染。此外，E1抗体对HCVpp感染性的抑制作用不显著（>0.05），而E2抗体对HCVpp感染有显著抑制作用（<0.05）。基于本研究结果得出结论，抗包膜抗体尤其是抗E2抗体对于表征针对HCV的体液免疫反应以及评估与干扰素疗法一起开发丙型肝炎被动和主动免疫的潜力可能极具价值。
