CD34+ Cell Derived Macrophages Are Protected from HIV-1 Challenge by Intrabody-Mediated Reduction of CCR5.

The three main coreceptors that HIV-1 uses for viral entry are CD4 and the chemokine receptors CXCR4 and/or CCR5. Viral tropism dictates which chemokine receptors are used for viral entry. Naturally occurring CCR5 gene polymorphisms resulting in gene deletion (delta 32) results in loss of receptor expression and provides protection from CCR5 using viruses. There are no known deleterious effects of CCR5 loss in mice or in humans. The focus of our studies is to limit infection of myeloid cells by CCR5 using HIV-1s by decreasing CCR5 cell surface expression. We have successfully accomplished this by using HIV-1 vectors to deliver an intrabody directed against CCR5. The CCR5-specific intrabody gene, containing the endoplasmic KDEL sequence, produces a single chain antibody that sequesters CCR5 in the endoplasmic reticulum. We show that >= 93% of THP-1s, a myelomonocytic cell line, transduced with CCR5 intrabody genes stably expressed intrabodies for 6 months. Of cells expressing intrabody, CCR5 was decreased at least 80% on the cell surface. In macrophages derived from CD34+ cells, CCR5 gene expression was maintained at initial transduction levels throughout the 3-week culture period. Colony forming assays (CFU) were performed to assess the effect of intrabody expression on hematopoietic development. No differences were seen in numbers of CFU and colony types when comparing parental, control vector, and intrabody expressing cells (n = 3). To determine the level of protection afforded by CCR5 intrabody expression, macrophages were exposed to JR-FL, a CCR5 using HIV-1, at 0.3 and 0.51 multiplicity of infection. Cultures were maintained for 15 – 23 days and evaluated for p24, a measure of viral replication, at 3 – 8 day intervals. At the end of culture it was found that macrophages expressing CCR5 intrabody generated 42-, 57-, 52-fold less p24 as compared to parental and control vector containing macrophages. Thus, CCR5 intrabody expression drastically decreased viral load. Therefore, reduction of CCR5 through vector delivery of intrabody genes to CD34+ cells should reduce the likelihood of infection of myeloid cells, their function as viral reservoirs, and has promising therapeutic potential. Currently, studies are underway to evaluate this modality of treatment in humanized NOD/SCID mice.