The objective of this proposal is the development of vaccination strategies which result in the production of neutralizing antibodies in mucosal secretions as well as cytotoxic T-lymphocyte (CTL) responses at mucosal sites. In this project, we will develop novel DNA vectors and delivery approaches to effectively prime the mucosal immune response, as well as particulate protein antigens which should be effective for boosting the resulting levels of mucosal immunity. We will construct DNA vectors which produce non-infectious VLPs which contain SIV Gag and HIV Env proteins (SHIV-89.6P VLPs). In addition, we will develop SHIV virus- like particles as protein antigens for use in boosting immune responses to DNA vaccines or poliovirus replicons. We will determine antigens for use in boosting immune responses to DNA vaccines or poliovirus replicons. We will determine humoral and cellular immune responses induced by SHIV-89.6P DNA vaccine and VLPs in preliminary studies in mice, in order to define the optimum protocol for subsequent use in rhesus macaques. The first specific aim is to develop more effective approaches for delivery of DNA expression vectors to mucosal surface. We have recently observed enhanced systemic and mucosal immune responses to DNA-encoded antigens after intranasal or oral delivery of DNA vectors when administered in a liposome preparation or when co-administered with a bioadhesive polymer. These approaches will be further developed by use of combinations of such polymers together with liposomes or other agents with known potential for enhancing DNA vaccine efficacy, such as CpG liposomes or other agents with known potential for enhancing DNA vaccine efficacy, such as CpG oligonucleotides. We will also determine the effects of co-administration of DNA plasmids encoding selected cytokines to mucosal surfaces. The second specific aim is to evaluate the use of virus-like particles (VLPs) for boosting mucosal immune response to SHIV DNA vaccine and poliovirus replicons (Project 2). During the current project, we have developed conditions for the production of VLPs using baculovirus and vaccinia expression systems. For the proposed studies, we will use constructs expressing the env gene of SHIV 89.6P, along with the SIVmac239 gag gene which is present in this SHIV construct. We will evaluate the potential of VLPs as a component of a mucosal vaccination strategy, in collaboration with projects 2-4. We will initially compare the systemic and mucosal immune responses to alternative types of immunogens in mice, and those approaches which are found to be most effective will be selected for subsequent studies in primates in projects 3 and 4.
