Rosa D, Campagnoli S, Moretto C, Guenzi E, Cousens L, Chin M, Dong C, Weiner A J, Lau J Y, Choo Q L, Chien D, Pileri P, Houghton M, Abrignani S
Chiron-Biocine, Immunobiology Research Institute of Siena (IRIS), Italy.
Proc Natl Acad Sci U S A. 1996 Mar 5;93(5):1759-63. doi: 10.1073/pnas.93.5.1759.
Hepatitis C virus (HCV) is a major cause of chronic hepatitis. The virus does not replicate efficiently in cell cultures, and it is therefore difficult to assess infection-neutralizing antibodies and to evaluate protective immunity in vitro. To study the binding of the HCV envelope to cell-surface receptors, we developed an assay to assess specific binding of recombinant envelope proteins to human cells and neutralization thereof. HCV recombinant envelope proteins expressed in various systems were incubated with human cells, and binding was assessed by flow cytometry using anti-envelope antibodies. Envelope glycoprotein 2 (E2) expressed in mammalian cells, but not in yeast or insect cells, binds human cells with high affinity (Kd approximately 10(-8) M). We then assessed antibodies able to neutralize E2 binding in the sera of both vaccinated and carrier chimpanzees, as well as in the sera of humans infected with various HCV genotypes. Vaccination with recombinant envelope proteins expressed in mammalian cells elicited high titers of neutralizing antibodies that correlated with protection from HCV challenge. HCV infection does not elicit neutralizing antibodies in most chimpanzees and humans, although low titers of neutralizing antibodies were detectable in a minority of infections. The ability to neutralize binding of E2 derived from the HCV-1 genotype was equally distributed among sera from patients infected with HCV genotypes 1, 2, and 3, demonstrating that binding of E2 is partly independent of E2 hypervariable regions. However, a mouse monoclonal antibody raised against the E2 hypervariable region 1 can partially neutralize binding of E2, indicating that at least two neutralizing epitopes, one of which is hypervariable, should exist on the E2 protein. The neutralization-of-binding assay described will be useful to study protective immunity to HCV infection and for vaccine development.
丙型肝炎病毒（HCV）是慢性肝炎的主要病因。该病毒在细胞培养中不能有效复制，因此难以评估感染中和抗体以及在体外评估保护性免疫。为了研究HCV包膜与细胞表面受体的结合，我们开发了一种检测方法来评估重组包膜蛋白与人类细胞的特异性结合及其中和作用。将在各种系统中表达的HCV重组包膜蛋白与人细胞一起孵育，并使用抗包膜抗体通过流式细胞术评估结合情况。在哺乳动物细胞而非酵母或昆虫细胞中表达的包膜糖蛋白2（E2）以高亲和力（解离常数约为10^(-8) M）与人细胞结合。然后，我们评估了能够中和接种疫苗的黑猩猩和携带病毒的黑猩猩血清以及感染各种HCV基因型的人类血清中E2结合的抗体。用在哺乳动物细胞中表达的重组包膜蛋白进行疫苗接种可引发高滴度的中和抗体，这些抗体与免受HCV攻击的保护作用相关。尽管在少数感染中可检测到低滴度的中和抗体，但HCV感染在大多数黑猩猩和人类中不会引发中和抗体。中和源自HCV-1基因型的E2结合的能力在感染HCV基因型1、2和3的患者血清中均匀分布，这表明E2的结合部分独立于E2高变区。然而，一种针对E2高变区1产生的小鼠单克隆抗体可以部分中和E2的结合，这表明E2蛋白上应该至少存在两个中和表位，其中一个是高变的。所描述的结合中和检测方法将有助于研究针对HCV感染的保护性免疫以及疫苗开发。
