UC San Diego

Human immunodeficiency virus type 1 (HIV-1) targets CD4+ T -cells, macrophages, and dendritic cells. In addition to the primary CD4 HIV-1 receptor, cell surface levels of the R5- and X4-tropic coreceptors, chemokine receptors CCR5 and CXCR4 respectively, orchestrate the success of HIV-1 entry. Disruption of either the receptor or coreceptor would prevent viral entry and subsequent viral production. HIV-1 derived vector systems successfully delivered an intracellular single chain antibody (intrabody) and short hairpin RNA (shRNA) to disrupt coreceptor cell surface expression and explore viral entry pathogenesis. Delivery of a CCR5 specific intrabody disrupted CCR5 expression and protected primary CD4+ T cells against free viral R5- tropic HIV-1 challenge. In vivo expression of the CCR5 intrabody in NOD/SCID-hu thy/liv thymocytes also protected against an ex vivo R5-tropic infection. Interestingly, due to the relatively higher expression levels of CXCR4, singly expressing a CXCR4 intrabody or short hairpin RNA did not efficiently disrupt CXCR4 levels to provide protection against X4-tropic HIV-1 entry. However, when expressing both intrabody and shRNA from a combination HIV -1 vector, efficient knockdown of CXCR4 on CD4+ T cells was observed. Although a percentage of CXCR4 combination vector CD4+ T cells were CXCR4 positive, the level of CXCR4 available per cell was significantly reduced and was sufficient to protect against X4-tropic HIV-1 entry. In addition to free viral infection, the effect of these vectors during dendritic cell-mediated infection was examined. A small percentage of CD4+ T cells expressing the CCR5 intrabody or CXCR4 combination vector exhibited a selective growth advantage and enriched during a robust dendritic cell-mediated HIV-1 challenge. In order to extend these protection strategies to non-human primate cells, it was necessary to modify the HIV-1 vector capsid to overcome species-specific post-entry restriction barriers. A quadruple capsid mutation (V86P/H87Q/I91V/ M96I) was introduced which abrogated the species-specific block and permitted HIV-1 vector transduction of non-human primate cell lines and human cells. Surprisingly, this mutation did not allow transduction in primary rhesus macaque CD4+ T cells. Optimization of anti-viral HIV-1 vector delivery strategies for future use in the rhesus macaque simian immunodeficiency virus (SIV) model will be beneficial to study HIV-1 pathogenesis and therapy