Identification of an unique CXCR4 epitope whose ligation inhibits infection by both CXCR4 and CCR5 tropic human immunodeficiency type-I viruses

Abstract

Background: Small chemical compounds which target chemokine receptors have been developed against human immunodeficiency virus type 1 (HIV-1) and are under investigation for use as anti-HIV-1 microbicides. In addition, monoclonal antibodies (mAbs) against chemokine receptors have also been shown to have anti-HIV-1 activities. The objective of the present study was to screen a panel of three anti-CXCR4 specific monoclonal antibodies (mAbs) for their ability to block the HIV-1 infection using in vitro activated primary peripheral blood mononuclear cells (PBMCs).

Results: PBMCs from normal donors were pre-activated with anti-CD3 and anti-CD28 mAbs for 1 day, and aliquots were infected with a low dose of CCR5-tropic (R5), CXCR4 tropic (X4) or dual tropic (X4R5) HIV-1 isolates and cultured in the presence of a panel of anti-CXCR4 mAbs. The panel included clones A145 mAb against the N-terminus, A120 mAb against a conformational epitope consisting of extracellular loops (ECL)1 and ECL2, and A80 mAb against ECL3 of CXCR4. Among these mAbs, the A120 mAb showed the most potent inhibition of infection, by not only X4 but surprisingly also R5 and X4R5 HIV-1. The inhibition of R5 HIV-1 was postulated to result from the novel ability of the A120 mAb to induce the levels of the CCR5-binding β-chemokines MIP-1α, MIP-1β and/or RANTES, and the down modulation of CCR5 expression on activated CD4+ T cells. Neutralizing anti-MIP-1α mAb significantly reversed the inhibitory effect of the A120 mAb on R5 HIV-1 infection.

Conclusions: The data described herein have identified a unique epitope of CXCR4 whose ligation not only directly inhibits X4 HIV-1, but also indirectly inhibits R5 HIV-1 infection by inducing higher levels of natural CCR5 ligands.

Figure 1

Inhibition of HIV-1 infection in activated PBMCs by anti-CXCR4 mAbs. (a) PBMCs activated with anti-CD3/CD28 for 1 day were infected with either R5 HIV-1_JR-FL or X4 HIV-1_NL4-3 for 2 hours, washed and then cultured in the presence of 10 μg/ml of the A145, A120, A80 rat IgG mAbs or isotype control rat IgG mAb mixture. After 5 days, virus production in the culture supernatants was determined by p24 ELISA. (b) Activated PBMCs infected with R5 HIV-1_JR-FL, R5 HIV-1_JR-CSF, X4 HIV-1_NL4-3 or X4 HIV-1_IIIB were aliquoted and cultured in the presence of 10 μg/ml of the A120 mAb or isotype control mAb. The p24 levels in the culture supernatants were monitored daily by ELISA. Data shown for both (a) and (b) are representative of 3 independent experiments using PBMCs from different donors.

Figure 2

The A120 mAb-mediated inhibition of HIV-1 infection in activated PBMCs from different donors. Activated PBMCs from 6 different donors were infected with either R5 HIV-1_JR-FL or X4 HIV-1_NL4-3 for 2 hours. After extensive washing, the PBMCs were aliquoted and cultured in the presence of A120 or isotype control IgG at 10 μg/ml. (a) After 3~5 days, virus production was determined by p24 ELISA in the culture supernatants, and values obtained on day 4 are shown as representative. P values were 0.007 and 0.032 for R5 HIV-1 and X4 HIV-1, respectively. (b) The PBMC samples obtained on day 4 after infection were fixed and permeabilized, and then stained with anti-HIV-1 p24 mAb labeled with Alexa Fluor 488 and examined by flow cytometry. The frequencies (percentages) of p24⁺ cells were plotted. P values were 0.026 and 0.031 for R5 HIV-1 and X4 HIV-1, respectively. Representative data from 3 independent experiments are shown.

Figure 3

Dose responses of the A120 mAb-mediated inhibition of R5 and X4 HIV-1 infection in activated PBMCs. Activated PBMCs from the donors were infected with either R5 HIV-1_JR-FL or X4 HIV-1_NL4-3. After washing, the PBMCs were aliquoted and cultured in the presence of graded concentrations of the A120 mAb for 4 days. Virus production in the culture supernatant was determined by p24 ELISA. Representative data from 3 independent experiments using 3 different donors' PBMCs are shown.

Figure 4

The A120 mAb does not affect HIV-1 and HTLV-I production from producer cell lines. The X4 HIV-1_IIIB producer cell line (Molt-4/IIIB) and the HTLV-I producer cell line (MT-2) cells were cultured in the presence of 10 μg/ml of A120 or control mAb for 3 days. The culture supernatants were assayed for HIV-1 p24 and HTLV-I p24 by standard ELISA.

Figure 5

LPS is not involved in the A120 mAb-mediated inhibition of HIV-1 infection. Activated PBMCs infected with R5 HIV-1_JR-FL were cultured in the presence or absence of LPS (0.1 μg/ml) or the A120 mAb with or without anti-CD14 mAb. After 4 days, syncytium formation and virus production in the culture supernatants were determined microscopically (a) and using a p24 ELISA kit (b), respectively.

Figure 6

Reversal of the A120 mAb-mediated inhibition of R5 HIV-1 infection in activated PBMCs with anti-CCR5 ligand-neutralizing mAbs. Activated PBMCs from 6 donors were infected with R5 HIV-1_JR-FL and cultured in the presence of 10 μg/ml A120 mAb or isotype control mAb together or without anti-chemokine mAbs including anti-MIP-1α, anti-MIP-1β or anti-RANTES at 10 μg/ml for 4 days. Virus production in the culture supernatants was determined by p24 ELISA. The p24 levels were plotted as percent of control values obtained in cultures incubated in each anti-β-chemokine mAb for each donor.

Figure 7

The A120 mAb-treatment induces the production of CCR5 binding β-chemokines and the down-modulation of CCR5 expression. PBMCs from 6 donors were activated with anti-CD3/28 mAbs for 1 day, washed, aliquoted and then incubated in the presence of 10 μg/ml A120 mAb or isotype control mAb for an additional day. (a) Changes in the concentrations of MIP-1α, MIP-1β and RANTES in the culture supernatants were assayed by ELISA. (b and c) Cells were analyzed for changes in the cell surface expression of CCR5 and CXCR4 on gated populations of CD4⁺T cells (MFI denotes mean fluorescence intensity). A representative flow cytometry dot blot profile is shown.

Figure 8

The A120 mAb stimulates T cells and monocytes to produce β-chemokines in activated PBMCs. One day-activated PBMCs were depleted of CD19⁺B cells, CD4⁺T cells, CD8⁺T cells and/or CD14⁺monocytes using immunobeads conjugated with appropriate lineage specific mAbs, and then cultured in the presence of A120 mAb or isotype control mAb for one day. Concentrations of MIP-1α, MIP-1β and RANTES in the culture supernatants were assayed by ELISA. Representative data from three independent experiments are shown.