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（由申请人提供）：发展的核心问题 HIV-1 疫苗已鉴定出能够提高滴度的免疫原， 针对包膜糖蛋白 (Envs) 的长效中和抗体 初级分离株。 HIV-1 包膜已被证明是一种弱免疫原，会产生低免疫原 亲和力成熟缓慢的滴度抗体。在这个 建议，DNA 免疫原将用于测试可溶形式的 CD4 (sCD4) 融合 Env，以产生针对受保护区域的中和抗体。在 HIV/细胞相互作用，Env 附着在细胞表面的 CD4 上。这 相互作用会导致 Env 的变化，从而暴露出对生命至关重要的神秘区域 Env 与辅助受体结合，随后融合并进入人体细胞。 sCD4/Env 的 DNA 免疫原将允许 Env 区域稳定暴露 用于产生中和抗体（Ab）。此外，为了 增强中和抗体反应，DNA免疫原将被测试 含有与 C3d 融合的 sCD4/Env。 HIV-1 Env 已成为开发 有效的疫苗。我们实验室已经成功使用了C3d组件 补体作为病毒糖蛋白的分子佐剂。正常免疫情况下 反应中，Gd 与抗原的结合增强了反应的起始和 抗原特异性抗体的成熟。测试 sCD4 的潜在作用 和 C3d 为 Env，将产生编码与 sCD4 融合的 Env 的 DNA 构建体 (sCD4/Env) 和 sCD4/Env/C3d 用于免疫接种。该研究将是 根据三个具体目标来执行。 (1) 编码a的疫苗质粒 初级 Env 的分泌单体 (gpl20) 形式和这些相同形式的 Env 在氨基末端与 sCD4 的两种形式之一（2 个结构域或 4 个结构域）融合 结构域）和/或在 C3d 的三个串联拷贝的羧基末端将是 建造的。各种质粒的表达和分泌水平 结构将被确定。 (2) 将比较 Env 构造 兔子的免疫原性，使用DNA免疫。升高的抗体将是 测定 Env 特异性 IgG 的水平和中和活性 主要 HIV-1 分离株组。 (3) 密码子优化的Env基因将用于 这些相同的构建体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 提高高滴度中和抗体。贯穿整个研究的假设 是 sCD4 融合会增加中和抗体反应和 C3d 融合将通过提高效率来增强 Env 的免疫原性 抗 Env Ab 反应的启动并通过增加 抗-Env Ab 经历亲和力成熟。