BACTERIAL VECTOR SYSTEMS FOR ANTI-SIV MUCOSAL IMMUNITY

这项研究的长期目标是开发一种免疫策略 这将产生保护性的粘膜免疫并防止感染 经阴道感染SIV的母亲或子女。我们计划 研制含有以下成分的减毒细菌载体(沙门氏菌或卡介苗) 代表SLVmac239包膜多肽414至434的DNA片段插入 和IL-5诱导肽特异性粘膜免疫。DNA片段 编码氨基酸将通过使用 设计的寡核苷酸引物，允许它们直接插入到 向量。将克隆载体的全细胞裂解产物进行SDS-PAGE分析。 使用针对肽414的抗体进行PAGE和Western blotting 434和IL-5，以确保克隆载体同时表达这两种分子。 细菌载体将被整合到可生物降解材料中 口服后可提供疫苗控释的微粒 管理和加强将被携带的病媒的能力 从肠道进入佩耶的补片。微球初始制剂 将结合在锁孔上的SLV包膜多肽414至434 帽状血蓝蛋白(KLH)，并用于口服免疫猕猴。这个 将使用抗肽和抗KLH反应来访问 这些微粒诱导粘膜反应的有效性。这个 将产生的恒河猴IgA抗体用于建立标准曲线 用于我们的ELISA检测，并作为产生抗LGA亚类的免疫原 抗体。 我们将探索一些参数，以开发出最佳的 产生保护性粘膜抗SIV免疫的免疫方案 回应。考虑的参数包括：1)载体的剂量 给药；2)给药途径；3)抗原频率 行政管理。血清样本，支气管灌洗，子宫冲洗，和 黏膜刮片将被用来评估体液和细胞 针对SIV肽414至434的反应。收集的样本将 用酶联免疫吸附试验检测414到434肽的特异性抗体，病毒 粘膜中和抗体滴度、组织学分析 刮片和T细胞介导肽414至434特异性的ADCC和CTL 回应。动物产生令人满意的中和抗体水平 共定位于粘膜组织的LIVE将在阴道内受到挑战 SIVmac239病毒。

The long-range goal of this study is to develop an immunization strategy that will produce protective mucosal immunity and prevent infection of either the mother or offspring when challenged with SIV vaginally. We plan to develop an attenuated bacterial vector (Salmonella or BCG) containing DNA fragment inserts representing SlVmac239 envelope peptide 414 to 434 and lL-5 to induce peptide-specific mucosal immunity. DNA fragments encoding amino acids will be generated by PCR amplification using oligonucleotide primers designed to permit their direct insertion into the vector. Whole cell lysates of the cloned vectors will be analyzed by SDS- PAGE and Western blots using antibodies directed against peptIde 414 to 434 and IL-5 to insure the cloned vectors are expressing both molecules. The bacterial vectors will be incorporated into biodegradable microparticles to provide controlled release of vaccine after oral administration and to enhance the ability of the vectors to be taken up from the intestine into Peyer's patches. Initial microsphere preparations will incorporate the SlV envelope peptide 414 to 434 conjugated to keyhole limpet hemocyanin (KLH), and used to orally immunize rhesus macaques. The anti-peptide and anti-KLH responses will be used to access the effectiveness of these microparticles to induce a mucosal response. The rhesus IgA antibody produced will be used to establish a standard curve for our ELISA assay, and as an immunogen to produce anti-lgA subclass antibodies. We will explore a number of parameters designed to develop an optimal immunization protocol to produce a protective mucosal anti-SIV immune response. Parameters considered will include: 1) dose of vectors administered; 2) route of administration; and 3) frequency of antigen administration. Serum samples, bronchial lavages, uterine washes, and mucosal scrapings will be used to evaluate both humoral and cellular responses directed against SIV peptide 414 to 434. Collected samples will be assayed for peptide 414 to 434-specific antibodies by ELISA, virus neutralization antibody titers, histological analysis of mucosal scrapings, and T cell-mediated peptide 414 to 434-specific ADCC and CTL responses. Animals making satisfactory levels of neutralizing antibodies co-localized in mucosal tissue will be challenged vaginally with live SIVmac239 virus.