USING C3D TO INCREASE THE IMMUNOGENICITY OF HIV1 ENV

描述(改编自申请人的摘要)：一个中心问题 HIV-1疫苗的开发一直是为了识别免疫原决定因素 能够提高高滴度、持久、中和的抗体 主要分离株的包膜糖蛋白(Env)。HIV-1环境病毒已经 被证明是一种弱免疫原，能产生低滴度的抗体，这种抗体对 亲和力成熟。在这项提案中，DNA和蛋白质免疫原 将用于测试补体的C3d成分作为分子佐剂 对于环境。在正常免疫反应中，C3d与抗原的结合 促进抗原特异性抗原的启动和成熟 抗体。为了测试C3d对环境的潜在佐剂作用，DNA构建 将产生的编码Env融合到C3d。将使用这些构造 用于DNA免疫和生产用于蛋白质免疫的蛋白质。这个 研究将按照三个具体目标进行。(L)接种疫苗 编码分泌型单体(Gpl20)和寡聚体(Gpl40)形式的质粒 初级环境病毒(89.6)，并使用这些相同形式的环境病毒在羧基上融合 将建造一个、两个或三个C3d串联拷贝的终点站。(2) 将表达质粒构建物以(A)确定 各种包膜蛋白的表达和分泌，(B)检测各种包膜蛋白 用于维持天然/功能构象，以及(C)纯化 精选的env用于蛋白质免疫。(3)环境构造将是 用DNA免疫对小鼠的免疫原性进行比较。为了更多 有利的构建，DNA免疫和蛋白质增强，以及蛋白质 将进行免疫接种。升高的抗体将被滴定用于 环境特异性免疫球蛋白水平和中和活性 原代和T细胞系适应的HIV-1分离株。我们的目标是 确定最有利于提高效价的Env-C3d融合 艾滋病病毒中和抗体-L。整个研究的假设是 C3d融合将通过以下两种方式提高Env的免疫原性 提高启动抗环境抗体应答的效率，并通过 增强抗Env抗体亲和成熟的能力。

DESCRIPTION (Adapted from applicant's abstract): A central problem for the development of HIV-1 vaccines has been to identify immunogen determinant capable of raising high titer, long lasting, neutralizing antibody against 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 and protein immunogens will be used to test the C3d component of complement as a molecular adjuvant for Env. In normal immune responses the attachment of C3d to an antigen enhances both the initiation and the maturation of antigen-specific antibody. To test a potential adjuvant role of C3d for Env, DNA constructs will be produced encoding Env fused to C3d. These constructs will be used for DNA immunizations and to produce protein for protein immunizations. The study will be executed according to three specific aims. (l) Vaccinate with plasmids encoding secreted monomeric (gpl20) and oligomeric (gpl40) forms of a primary Env (89.6) and using these same forms of Env fused at the carboxyl terminus to one, two or three tandem copies of C3d will be constructed. (2) Plasmid constructs will be expressed to (a) determine the levels of expression and secretion of the various Envs, (b) to test the various Envs for the maintenance of native/functional conformations, and (c) to purify selected Envs for protein immunizations. (3) Env constructs will be compared for immunogenicity in mice, using DNA immunizations. For the more favorable constructs, DNA immunizations plus protein boosts, and protein immunizations will be conducted. Raised antibody will be titered for the level of Env-specific IgG and for neutralizing activity for a panel of primary as well as T-cell line adapted HIV-1 isolates. Our goal is to identify the most favorable Env-C3d fusion for raising high titer neutralizing antibody for HIV-l. The hypothesis throughout the study is that the C3d fusions will increase the immunogenicity of Env both by increasing the efficiency of the initiation of anti-Env Ab responses and by increasing the ability of anti-Env Ab to undergo affinity maturation.