Functional deletion of the CCR5 receptor by intracellular immunization produces cells that are refractory to CCR5-dependent HIV-1 infection and cell fusion

Abstract

Studies of naturally occurring polymorphisms of the CCR5 gene have shown that deletion of the functional receptor or reduced expression of the gene can have beneficial effects in preventing HIV-1 infection or delaying disease. Because these polymorphisms are found in otherwise healthy people, strategies that aim to prevent or limit expression of CCR5 should be beneficial in the treatment of HIV-1 disease. To test this approach we have developed a CCR5-specific single-chain antibody that was expressed intracellularly and retained in the endoplasmic reticulum. This CCR5-intrabody efficiently blocked surface expression of human and rhesus CCR5 and thus prevented cellular interactions with CCR5-dependent HIV-1 and simian immunodeficiency virus envelope glycoprotein. Intrabody-expressing cells were shown to be highly refractory to challenge with R5 HIV-1 viruses or infected cells. These results suggest that gene therapy approaches that deliver this intracellular antibody could be of benefit to infected individuals. Because the antibody reacts with a conserved primate epitope on CCR5 this strategy can be tested in nonhuman lentivirus models of HIV-1 disease.

T-cell protection and enrichment through lentiviral CCR5 intrabody gene delivery

“…16 If cellular modifications of chemokine receptors by gene delivery are to be effective for abrogating HIV-1 entry in primary CD4 + T cells, CCR5 intrabodies must function effectively when expressed from the gene delivery vehicle of choice at low vector copy number. To determine whether CCR5 intrabodies can provide protection to leukocytes, lentiviral vectors were developed that expressed the CCR5 intrabody gene, CAD-R5, and a control vector, CAD, which did not include the CCR5 intrabody gene ( Figure 1a).…”

“…The CCR5 intrabody is specific for the N-terminal extracellular domain of CCR5 and engineered with the Lys-Asp-Glu-Leu (KDEL) endoplasmic reticulum retention signal. 16,25 We determined that expression of the CCR5 intrabody from the CAD-R5 vector resulted in almost complete disruption of CCR5 cell surface expression and protected both T-cell lines and primary CD4 + T cells from robust infection with free R5-tropic viruses. Furthermore, intrabody-expressing CD4 + T cells, obtained from non-obese diabetic/severely combined immunodeficient (NOD/SCID)-human thymus/liver (hu thy/liv) mice transplanted with CAD-R5-transduced CD34 + cells, were protected from high-titer R5-tropic HIV-1 challenge in tissue culture.…”

“…[13][14][15][16] However, these viral entry disruption strategies have shown varying levels of protection when the viral challenge dose is increased, thus providing the caution that therapeutic efficacy may be breached by high viral loads. [17][18][19] Recent studies have indicated that HIV-1 may be more effectively transmitted during antigen presentation between CD4 + T cells and HIV-1 exposed or infected dendritic cells (DCs), termed an infectious immunological synapse, thus providing a means for increased local viral levels.…”

Protecting from R5-tropic HIV: individual and combined effectiveness of a hammerhead ribozyme and a single-chain Fv antibody that targets CCR5

“…Several antibodies have been reported to bind CCR5 and inhibit R5-tropic HIV. 20,21,29 The CTC8 monoclonal antibody was reported to block both chemokine binding and HIV coreceptor activity effectively. 40,41 Delivery of the ERlocalized CCR5 ligand RANTES has been reported to protect cells from R5-tropic HIV.…”

“…[15][16][17][18][19][20][21] Intracellular expression of CCR5-binding chemokines to which intracellular targeting sequences have been added, in an attempt to bind and sequester CCR5, has shown some promise in limiting viral entry. 22 However, poor transduction efficiencies, waning transgene expression, immune responses directed against transduced cells, and inability to readminister vectors following initial treatment have all limited the effectiveness of such anti-HIV genetic therapies.…”