Background:  Results from cell lines co-expressing CD4 and CCR5 have suggested that CD4 in primary T cells is present at saturating levels whereas CCR5 is present at rate-limiting levels for efficient infection by R5 strains. However, the significance of these observations in donor T cells is not known. Similarly, although the relative antiviral activity of the fusion inhibitor enfuvirtide (T-20) against R5 HIV-1 has been shown to be dependent on CCR5 density levels on cell lines, the role of CCR5 density on the antiviral activity of T-20 in donor T cells is unknown. In the present study, we have evaluated the effects of CCR5 and CD4 density levels on primary CD4 T cells on the replication efficiency and T-20 susceptibility of R5 HIV-1. In addition, we have evaluated the effects of Rapamycin (RAPA), an immunomodulatory drug previously shown to reduce CCR5 expression, on the antiviral activity of T-20.

Methods:  Densities of CD4 and CCR5 were determined by Quantitative FACS Analysis. Infectivity assays were done using donor peripheral blood mononuclear cells (PBMC). Associations between receptor density levels, virus replication levels and T-20 sensitivity (IC₅₀) were tested using the non-parametric Spearman’s rank correlation test. The effect of RAPA on T-20 antiviral activity was assessed on a cell-cell fusion assay and in infectivity assays.

Results:  Replication of R5 HIV-1 varied as much as 12-fold among donors and replication was positively correlated with CCR5 density levels on donor CD4 T cells (r = 0.55, p = 0.011), but not with CD4 density levels. In addition, sensitivity of R5 HIV-1 to T-20 varied ~100-fold among donors and CCR5 density levels were positively correlated with decreased susceptibility of R5 HIV-1 to T-20 (r = 0.84, p = 0.00004). Moreover, inhibition of CCR5 expression by non-toxic concentrations of RAPA resulted in potentiation of the antiviral activity of T-20 against R5 HIV-1 in a fusion assay and in infectivity assays.

Conclusions:  These in vitro results using primary cells indicate that CCR5 density levels on CD4 T cells determine the replicative efficiency and T-20 susceptibility of R5 strains of HIV-1. Furthermore, they demonstrate that reduction of CCR5 levels by the drug RAPA results in enhancement of T-20 antiviral activity against R5 HIV-1. These results suggest that therapeutic and preventive approaches to alter CCR5 density may have clinical consequences and warrant further investigation.