ENHANCED ANTIGEN PROCESSING OF HIV SUBUNIT VACCINES

性状（改编自申请人摘要）：α-2-巨球蛋白 (a2M)蛋白质的掺入增强体外抗原呈递， 100-1000-倍，是同样有效的完全弗洛伊德佐剂（CFA） 用于在体内引发抗体。 拟议的长期目标 研究内容包括：（1）评价A2 M作为抗原免疫佐剂的应用 亚单位疫苗的加工，以及（2）确认 使用这种新的公式，作为原理的证明， 有效的体液免疫反应，能够中和 最近证实，HIV-1的白细胞介素-10（IL-10）诱导活性 nef（Nef）。 这些研究可能提供一种安全，有效的方法来提高 各种病毒或细菌蛋白的抗原加工，因此 即使在缺乏免疫系统的情况下， 复制免疫原 用这种新制剂开发的疫苗可 不仅可以预防各种感染的初始感染， 病原体，但也在提高免疫反应，在以前感染 个体 这些目标将通过以下三个具体途径实现： 目的：（1）构建重组Nef的表达载体， 在小鼠、豚鼠和兔白细胞中证实IL-10 Nef诱导;（2）重组Nef掺入小鼠、豚鼠 猪和兔α 2 M的体内免疫原性的测定 与“游离”Nef或配制在其他佐剂中的Nef相比的制剂; （3）Nef免疫动物血清的抗病毒能力测定 在体外，在a2 M中配制以中和Nef的IL-10诱导活性 和/或在体内。 这些具体目标将通过以下方式实现。 通过构建Nef质粒pGEX-Nef获得重组Nef， 通过插入全长Nef基因， 从pUC 19导入谷胱甘肽-S-转移酶（GST）基因融合载体， pGEX-3X。 GST Nef融合蛋白在E.转化大肠杆菌， 将通过谷胱甘肽-琼脂糖凝胶组合纯化pGEX-Nef， 层析，用因子Xa消化以释放GST，并亲和层析， 用偶联至琼脂糖凝胶的高滴度抗Nef兔IgG进行层析。 将使用Deloxi-Gel亲和性去除污染性内毒素 通过凝胶电泳测定最终纯度，质量 分光光度法和氨基酸测序。 IL-10的诱导将是 使用Nef刺激的外周血白细胞测定或引发 腹膜细胞和培养物上清液的ELISA测量或 培养细胞 将使用标准化方法制备每种属的a2 M 制定的程序 研究人员，并将Nef纳入a2 M将完成使用 以前公布的技术。 动物，按每月计划免疫 与Nef，Nef并入a2 M，或Nef与CFA混合，将被流血的 每月一次，通过ELISA和免疫沉淀法测定Nef滴度。 将测试高滴度Nef血清阻断Nef的能力 白细胞培养物中IL-10的诱导作用 sera.

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.