Background:  Some RANTES analogs are potent inhibitors of R5 HIV-1 infection, but cost of production is likely to limit their use as microbicides if they have to be chemically synthesized> Furthermore,  signaling activity on CCR5 raises potential safety concerns. We have developed novel RANTES analogs that promise low cost recombinant production by fermentation. Molecules with two distinct modes of action have been evaluated for potency, durability of activity, and signaling activity.

Methods:  Phage display libraries of RANTES molecules with diversity engineered into the N-terminal region were screened for interaction with CCR5 presented on live cells. Selected molecules isolated from these libraries were tested for their ability to block infection of peripheral blood mononuclear cells (PBMC) cultures by primary R5 HIV-1 isolates, and to modulate CCR5 expression on activated CD4⁺ human T cells. Signaling activity was measured as induction of Ca²⁺ flux in HeLa-CCR5 cells. The durability of CCR5 modulation and inhibition of infection were determined after removal of the RANTES analog. CCR5 occupancy was measured by differential staining with monoclonal antibodies PA12 (N-terminus specific, binding not impaired by RANTES) and 2D7 (specific for the second extracellular loop, binding blocked by RANTES). The new molecules were compared to the most potent chemically synthesized analog, PSC-RANTES.

Results:  We have found 3 new molecules—6P4-, 5P12-, and 5P14-RANTES—to be at least as potent as PSC-RANTES at preventing R5 HIV-1 infection of PBMC, with IC₅₀ values in the pM range. 6P4-RANTES, like PSC-RANTES, causes prolonged internalization of CCR5 and induced Ca²⁺ flux. By contrast, 5P12- and 5P14-RANTES cause no or modest CCR5 internalization, respectively, and neither induces detectable Ca²⁺ flux. 5P14-RANTES blocks 2D7, but not PA12 staining, for as long as 96 hours following a 1-hour pulse exposure, and 5P12-, 5P14- and 6P4-RANTES blocked HIV-1 infection to a greater extent than PSC-RANTES for at least 24 hours after inhibitor removal. Both 6P4- and 5P12- RANTES were evaluated for protective activity in the rhesus macaque vaginal challenge model.

Conclusions:  Novel, potent CCR5 inhibitors have been developed that promise reduced costs of production. The safety profile of two of them appears to be improved, since we do not detect signaling via CCR5. We are continuing the development of these inhibitors as microbicides.