BACTERIAL VECTOR SYSTEMS FOR ANTI-SIV MUCOSAL IMMUNITY

本研究的长期目标是制定一种免疫策略

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.