Methodologies that permit us to monitor the uptake, processing and presentation of viral proteins by antigen presenting cells (APC) in vitro, will assist in the development of strategies that maximize antigen presentation for the induction of vigorous and sustained MHC class I and class II immune responses in vivo. This in turn will support our efforts in the development of effective anti-HIV vaccine strategies. We propose to evaluate the potential of an alternative method of antigen delivery to APC for the induction of strong immune responses in vivo. Specifically, a novel formulation of liposomes, called sterically stabilized liposomes (?SL?), will be used to deliver simian immunodeficiency virus (SIV) proteins to various APC and these antigen loaded APC will then be assessed for their capacity to induce both MHC class I and class II responses in vitro and in vivo. Unlike ?conventional? liposomes, animal and human studies have demonstrated that ?SL? evade rapid elimination from t he liver and spleen and accumulate at body surfaces such as the skin) and lymph nodes. We propose that at these areas, ?SL? and the antigens encapsulated in them, will be taken up by resident APC, such as dendritic cells (DCs), macrophages and possibly B cells and thereby induce strong immune responses to the encapsulated antigens. By modifying the lipid composition of ?SL? (without altering their bio-distribution) we will generate pH-dependent and pH-independent ?SL? formulations that will allow us to preferentially deliver antigens to the cytoplasm or intracellular vacuoles respectively, for the preferential induction of class I and/or class II MHC-restricted T cell responses. First, using established techniques, we will monitor the uptake of these two ?SL? types by macaque blood-derived DCs and macrophages at various stages of maturation and their loading with SIV antigens, such as gag and envelope proteins. In parallel, we will assess the potential of APC loaded with ?SL?-enca psulated SIV gag and/or envelope proteins, to stimulate SIV-specific T cell responses (T cell proliferation and cytolytic activities) in vitro, using T cells from SIV infected or immunized macaques. The magnitude and type of the responses induced will be correlated with the type of ?SL? used to deliver the antigens, the antigen itself, the type of APC, and the quantity and cellular localization of the delivered antigens. Finally, we will evaluate the ability of DCs carry ?SL?-encapsulated SIV antigens, to generate immune responses in vivo. DCs will be isolated from naive animals, treated with ?SL?-containing SIV antigens and re-infused into the same animals, or ?SL?-containing SIV antigens will be administered to the animals directly. The generation of anti-SIV responses by these two methods will be monitored and compared. FUNDING NIH (R21 AI42670) PUBLICATIONS None
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