ENHANCED ANTIGEN PROCESSING OF HIV SUBUNIT VACCINES

描述(改编自申请者摘要)：α-2-巨球蛋白 (A2M)掺入蛋白质可通过以下途径增强体外抗原提呈 100-1000倍，与完全弗洛伊德佐剂(CFA)同等有效 在体内引发抗体。拟议的长期目标 研究内容如下：(1)评价A2M作为新型抗原佐剂的应用 亚单位疫苗的加工，以及(2)确认其潜在的用途 使用这一新的提法，作为原则的证明，归纳 强大的体液免疫反应能够中和 新近证实的HIV-1的白介素10(IL-10)诱导活性 NEF(Nef)。这些研究可能会提供一种安全、有效的方法来增强 各种病毒或细菌蛋白的抗原处理，因此 即使在没有病毒的情况下也能产生强烈的免疫反应 复制免疫原。使用这种新配方开发的疫苗可以 不仅在预防各种疾病的初始感染方面有用处 病原体，但也在增强先前感染的免疫反应性方面 个人。这些目标将通过三个具体的途径来实现 目的：(1)重组Nef和Nef表达载体的构建及表达 IL-10在小鼠、豚鼠和兔白细胞中的表达 Nef诱导；(2)将重组Nef导入小鼠、豚鼠 猪和兔a2M及其体内免疫原性的测定 与“游离”Nef或在其他佐剂中配制的Nef相比的配方； (3)Nef免疫动物血清能力的测定 A2M中和Nef诱导IL-10活性的体外实验 和/或在体内。这些具体目标将实现如下。 通过构建Nef质粒pGEX-Nef获得重组Nef， 通过插入全长Nef基因编码整个成熟蛋白 将pUC19克隆入谷胱甘肽-S-转移酶基因融合载体， PGEX-3X。GST-Nef融合蛋白在大肠杆菌中的表达 PGEX-Nef将通过谷胱甘肽-琼脂糖的组合进行纯化 层析，Xa因子消化释放GST，亲和 高效价抗Nef兔免疫球蛋白偶联琼脂糖层析法。 利用Deloxi-Gel亲和力去除污染的内毒素 凝胶电泳法测定层析和最终纯度，质量 分光光度法和氨基酸测序法。IL-10的诱导将是 用Nef刺激的外周血白细胞测定或诱导 腹膜细胞及培养上清液的酶联免疫吸附试验或RT-PCR检测 培养的细胞。每个物种的A2M将使用标准化的 由以下机构制定的程序 调查人员，并将Nef并入2M将使用 之前发表的技术。动物，按月计划接种疫苗 有了Nef，Nef合并到2M中，或者Nef与CFA混合，将会出血 月龄和Nef效价采用ELISA法和免疫沉淀法测定。 针对Nef的高滴度血清将被测试其阻断Nef的能力 IL-10在预先暴露于稀释液中的白细胞的诱导 塞拉。

DESCRIPTION (adapted from applicant's abstract): Alpha-2-macroglobulin (a2M) incorporation of proteins enhances in vitro antigen presentation by 100-1000-fold and is equally effective as complete Freud's adjuvant (CFA) for eliciting antibodies in vivo. Long-term objectives of the proposed studies are: (1) to evaluate the use of A2M as a novel adjuvant for antigen processing of subunit vaccines, and (2) to confirm the potential utility of using this novel formulation by showing, as proof of principle, induction of potent humoral immune responses capable of neutralizing the recently-demonstrated, interleukin-10(IL-10)-inducing activity of HIV-1 nef(Nef). These studies may provide a safe, efficient way to enhance antigen processing of a variety of viral or bacterial proteins, thus allowing generation of strong immune responses even in the absence of replicating immunogens. Vaccines developed with this novel formulation may have utility not only in preventing initial infections with various pathogens, but also in boosting immune responsiveness in previously infected individuals. These objectives will be accomplished through three specific aims: (1) generation of vectors for and expression of recombinant Nef and demonstration in murine, guinea pig, and rabbit leukocytes of IL-10 induction by Nef; (2) incorporation of recombinant Nef into murine, guinea pig and rabbit a2M and determination of the in vivo immunogenicity of such formulations compared to "free" Nef or Nef formulated in other adjuvants; and (3) determination of the ability of sera from animals immunized with Nef formulated in a2M to neutralize the IL-10-inducing activity of Nef, in vitro and/or in vivo. These specific aims will be accomplished as follows. Recombinant Nef will be obtained by constructing the Nef plasmid pGEX-Nef, encoding the entire mature protein, by insertion of a full-length Nef gene from pUC19 into a glutathione-S-transferase (GST) gene fusion vector, pGEX-3X. GST-Nef fusion protein, expressed in E. coli transformed with pGEX-Nef, will be purified by a combination of glutathione-Sepharose chromatography, digestion with factor Xa to release the GST, and affinity chromatography with high-titered, anti-Nef rabbit IgG coupled to Sepharose. Contaminating endotoxins will be removed using Deloxi-Gel affinity chromatography and final purity determined by gel electrophoresis, mass spectrophotometry, and amino acid sequencing. Induction of IL-10 will be determined using Nef-stimulated peripheral blood leukocytes or elicited peritoneal cells and ELISA measurements of culture supernatants or RT-PCR of cultured cells. a2M for each species will be prepared using standardized procedures established by the investigators, and incorporation of Nef into a2M will be accomplished using previously published techniques. Animals, immunized on a monthly schedule with Nef, Nef incorporated into a2M, or Nef mixed with CFA, will be bled monthly and titers to Nef determined by ELISA and immunoprecipitation. High-titered sera to Nef will be tested for their ability to block Nef induction of IL-10 in leukocyte cultures pre-exposed to dilutions of the sera.