Highly potent, fully recombinant anti-HIV chemokines: reengineering a low-cost microbicide

Abstract

New prevention strategies for use in developing countries are urgently needed to curb the worldwide HIV/AIDS epidemic. The N-terminally modified chemokine PSC-RANTES is a highly potent entry inhibitor against R5-tropic HIV-1 strains, with an inhibitory mechanism involving long-term intracellular sequestration of the HIV coreceptor, CCR5. PSC-RANTES is fully protective when applied topically in a macaque model of vaginal HIV transmission, but it has 2 potential disadvantages related to further development: the requirement for chemical synthesis adds to production costs, and its strong CCR5 agonist activity might induce local inflammation. It would thus be preferable to find a recombinant analogue that retained the high potency of PSC-RANTES but lacked its agonist activity. Using a strategy based on phage display, we set out to discover PSC-RANTES analogs that contain only natural amino acids. We sought molecules that retain the potency and inhibitory mechanism of PSC-RANTES, while trying to reduce CCR5 signaling to as low a level as possible. We identified 3 analogues, all of which exhibit in vitro potency against HIV-1 comparable to that of PSC-RANTES. The first, 6P4-RANTES, resembles PSC-RANTES in that it is a strong agonist that induces prolonged intracellular sequestration of CCR5. The second, 5P12-RANTES, has no detectable G protein-linked signaling activity and does not bring about receptor sequestration. The third, 5P14-RANTES, induces significant levels of CCR5 internalization without detectable G protein-linked signaling activity. These 3 molecules represent promising candidates for further development as topical HIV prevention strategies.

Fig. 1.

Inhibition of HIV-1 infection of primary PBMC cultures by RANTES analogs. Potency (pIC₅₀) of PSC-RANTES and new inhibitors for blocking viral infection of primary PBMC cultures with HIV-1 ADA, and the primary isolate CC1/85. Data are mean pIC₅₀ ± SE for 3 replicate experiments.

Fig. 2.

CCR5 expression after brief exposure to RANTES analogs. Changes in CCR5 staining of activated primary CD4⁺ T cells after a 1-h. pulse with PSC-, 5P12-, 5P14-, or 6P4-RANTES. CCR5 was detected with PA12, a monoclonal Ab directed at the N terminus of CCR5 whose binding is not affected by exposure of the receptor to native or modified RANTES compounds. Error bars show ± SE for 6 replicate experiments with different CD4⁺ T cell donors.

Fig. 3.

Signaling activity of native RANTES and RANTES analogs on CCR5-expressing primary cells. Calcium flux measurements were carried out as described in Methods. Determinations in each of the 2 series shown were in duplicate; error bars indicate minimum and maximum values. Dotted lines indicate limits (min, max) for the negative controls (no chemokine).

Fig. 4.

CCR5-binding affinity of RANTES analogs. Results of competition binding assays using ¹²⁵I MIP-1β/CCL4 on CHO−CCR5 cells. The analogs exhibit similar affinity. Bars indicate mean pIC₅₀ values ± SD, determined in 2–8 independent competition binding assays (see Methods for details).

Fig. 5.

Proposed structure–activity relationship for fully recombinant RANTES analogues. Our data suggest that the tetrapeptide Gln-Gly-Pro-Pro can provide a valid replacement for the PSC-moiety of PSC-RANTES (positions 0–3), provided that it is presented in the context of appropriate combinations of downstream structures at positions 4–9. These structures determine not only anti-HIV potency but G protein-linked signaling activity and capacity to induce intracellular receptor sequestration.