CD4 and C3d fusion proteins-antibodies to HIV-1 envelope

CD4 和 C3d 融合蛋白 - HIV-1 包膜抗体

• 批准号: 6409076
• 负责人: Ted M Ross
• 金额: $ 20.93万
• 依托单位: EAST CAROLINA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6409076
• 关键词: AIDS vaccines; CD4 molecule; HIV envelope protein; active immunization; antibody specificity; antibody titering; chimeric proteins; drug screening /evaluation; enzyme linked immunosorbent assay; humoral immunity; immunoglobulin G; laboratory rabbit; molecular cloning; neutralizing antibody; plasmids; vaccine development; vector vaccine; western blottings

DESCRIPTION (Provided by applicant): A central problem for the development of HIV-1 vaccines has been to identify immunogens capable of raising high titer, long lasting, neutralizing antibody to the envelope glycoproteins (Envs) of primary isolates. The HIV-1 Env has proven to be a weak immunogen, raising low titer antibodies that are slow to undergo affinity maturation. In this proposal, DNA immunogens will be used to test soluble forms of CD4 (sCD4) fused to Env in order to generate neutralizing antibodies to protected regions. In HIV/cell interactions, Env attaches to CD4 on the cell surface. This interaction leads to changes in Env, which expose cryptic regions important for Env binding to co-receptors and subsequent fusion and entry into human cells. DNA immunogens of sCD4/Env will allow for the stable exposure of Env regions for generation of neutralizing antibodies (Ab). In addition, in order to enhance the neutralizing antibody response, DNA immunogens will be tested containing sCD4/Env fused to C3d. HIV-1 Env has been a target for developing an effective vaccine. Our laboratory has successfully used the C3d component of complement as a molecular adjuvant for viral glycoproteins. In normal immune responses, the attachment of Gd to an antigen enhances both the initiation and the maturation of antigen-specific antibody. To test a potential role of sCD4 and C3d for Env, DNA constructs will be produced encoding Env fused to sCD4 (sCD4/Env) and sCD4/Env/C3d and used for immunizations. The study will be executed according to three specific aims. (1) Vaccine plasmids encoding a secreted monomeric (gpl20) form of primary Envs and these same forms of Env fused at the amino terminus with one of two forms for sCD4 (2 domain or 4 domain) and/or at the carboxyl terminus to three tandem copies of C3d will be constructed. The levels of expression and secretion of the various plasmid constructs will be determined. (2) The Env constructs will be compared for immunogenicity in rabbits, using DNA immunization. Raised antibody will be titered for the level of Env-specific IgG and for neutralizing activity for a panel of primary HIV-1 isolates. (3) Codon-optimized Env genes will be used in these same constructs and efficient expression and immunogenicity will be determined. Our goal is to identify the most favorable sCD4/Env/C3d fusion for raising high titer neutralizing antibody. The hypothesis throughout the study is that the sCD4 fusion will increase the neutralizing Ab response and the C3d fusion will enhance the immunogenicity of Env both by increasing the efficiency of the initiation of anti-Env Ab response and by increasing the ability of anti-Env Ab to undergo affinity maturation.

描述（申请人提供）：开发的核心问题 HIV-1疫苗一直是为了鉴定能够提高高滴度的免疫原子， 持久的，中和对包膜糖蛋白（ENK）的抗体 主要分离株。 HIV-1 Env已被证明是弱的免疫原 滴度抗体的经历缓慢的亲和力成熟。在这个 提案，DNA免疫原用于测试CD4（SCD4）融合的可溶性形式 为了产生对受保护区域的中和抗体的环境。在 HIV/细胞相互作用，ENV附着在细胞表面上的CD4。这 互动会导致Env的变化，这暴露了隐秘区域对 Env与共受体的结合以及随后的融合和进入人类细胞的结合。 SCD4/ENV的DNA免疫原子将允许稳定暴露于Envions 用于中和抗体（AB）。另外，为了 增强中和抗体反应，将测试DNA免疫原 包含与C3D融合的SCD4/ENV。 HIV-1 Env一直是开发 有效的疫苗。我们的实验室已成功使用了 作为病毒糖蛋白的分子佐剂补体。正常免疫 响应，GD对抗原的附着会增强起始和 抗原特异性抗体的成熟。测试SCD4的潜在作用 和C3D用于ENV，将生成DNA构建体，编码与SCD4融合的Env （SCD4/ENV）和SCD4/ENV/C3D，用于免疫。该研究将是 根据三个特定目标执行。 （1）编码A的疫苗质粒 分泌的单体（GPL20）主要Envs和这些相同形式的Env形式 在氨基末端融合，SCD4的两种形式之一（2个域或4个 域）和/或在羧基末端到C3D的三个串联副本将是 构造。各种质粒的表达和分泌水平 将确定构造。 （2）将比较ENV结构 使用DNA免疫，兔子的免疫原性。饲养的抗体将是 用于ENV特异性IgG的水平和中和活性 原代HIV-1分离株的面板。 （3）将使用密码子优化的Env基因 这些相同的结构以及有效的表达和免疫原性将是 决定。我们的目标是确定最有利的SCD4/Env/C3D融合 提高中和抗体的高滴度。整个研究中的假设 是SCD4融合会增加中和AB响应和C3D 融合将通过提高效率来增强ENV的免疫原性 抗ENV AB反应的启动并通过提高 抗ENV AB进行亲和力成熟。