BACTERIAL VECTOR SYSTEMS FOR ANTI-SIV MUCOSAL IMMUNITY

这项研究的长期目标是制定一项免疫策略， 这将产生保护性粘膜免疫，防止感染 无论是母亲或后代时，攻击与SIV阴道。我们计划 开发减毒细菌载体（沙门氏菌或BCG）， 代表SlVmac 239包膜肽414至434的DNA片段插入物 和IL-5诱导肽特异性粘膜免疫。DNA片段 编码氨基酸将通过PCR扩增产生， 寡核苷酸引物，其被设计为允许它们直接插入到 vector.将通过SDS-聚丙烯酰胺凝胶电泳分析克隆载体的全细胞裂解物。 使用针对肽414的抗体进行PAGE和蛋白质印迹， 434和IL-5，以确保克隆的载体表达两种分子。 细菌载体将被整合到可生物降解的 在口服后提供疫苗控释的微粒 管理和提高能力的载体采取 进入派尔集合淋巴结初始微球制备 将掺入缀合至钥匙孔的SIV包膜肽414至434 血蓝蛋白（KLH），并用于口服免疫恒河猴。的 抗肽和抗KLH应答将用于访问 这些微粒诱导粘膜反应的有效性。的 将产生的恒河猴伊加抗体用于建立标准曲线 用于我们的ELISA测定，并作为免疫原产生抗IgA亚类 抗体的 我们将探讨一些参数，旨在制定一个最佳的 产生保护性粘膜抗SIV免疫的免疫方案 反应 考虑的参数将包括：1）载体剂量 给药途径;和3）抗原频率 局血清样本，支气管灌洗，子宫冲洗液，和 粘膜刮片将用于评估体液和细胞 针对SIV肽414至434的应答。 采集的样本将 通过ELISA测定肽414至434特异性抗体，病毒 中和抗体滴度，粘膜组织学分析 刮片和T细胞介导的肽414至434特异性ADCC和CTL 应答产生令人满意的中和抗体水平的动物 共同定位于粘膜组织中的病毒将在阴道内用活的 SIVmac 239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.