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

描述(由申请人提供)：发展的一个中心问题 HIV-1疫苗一直是为了识别能够提高高滴度的免疫原， 针对猪瘟病毒包膜糖蛋白(Env)的持久中和抗体 初级分离株。HIV-1 env已被证明是一种弱免疫原，其免疫力较低 亲和力成熟较慢的滴度抗体。在这 提议，DNA免疫原将用于检测融合的可溶性形式的CD4(SCD4) 以产生针对受保护区域的中和抗体。在……里面 HIV/细胞相互作用时，Env附着在细胞表面的CD4上。这 相互作用导致环境发生变化，从而暴露出对 Env与辅助受体结合，随后融合并进入人类细胞。 SCD4/env的DNA免疫原将允许env区域的稳定暴露 用于产生中和抗体(Ab)。此外，为了 增强中和抗体反应，将检测DNA免疫原 含有与C3d融合的sCD4/env。HIV-1环境病毒一直是开发一种 有效的疫苗。我们实验室已经成功地使用了C3d组件 补体作为病毒糖蛋白的分子佐剂。在正常免疫状态下 在反应中，Gd与抗原的结合既增强了起始，也增强了 抗原特异性抗体的成熟。测试sCD4的潜在作用 对于Env和C3d，将产生编码Env与sCD4融合的DNA构建体 (sCD4/Env)和sCD4/Env/C3d，用于免疫。这项研究将是 根据三个具体目标执行。(1)编码a基因的疫苗质粒 分泌单体(Gpl20)形式的原生环境和这些相同形式的环境 在氨基末端与sCD4(2个结构域或4个)的两种形式之一融合 结构域)和/或在C3d的三个串联拷贝的羧基末端 建造的。不同性质粒的表达和分泌水平 构造将被确定。(2)将对环境构造进行比较 用DNA免疫兔的免疫原性。提高的抗体将是 滴度为环境特异性免疫球蛋白水平和中和活性 HIV-1主要分离株小组。(3)密码子优化的env基因将用于 这些相同的结构和有效的表达和免疫原性将是 下定决心。我们的目标是确定最有利的sCD4/env/C3d融合 提高高效价中和抗体。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 通过增强抗-Env抗体的免疫应答能力 抗Env抗体亲和力成熟。

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.