Novel Approach to Block HIV-1 Infection Complexes with Bifunctional Inhibitors: A Preferential Targeting of CD4-CCR5

Two receptors, CD4 and one of several chemokine receptors, are required for cellular HIV-1 infection, with CCR5 being the main coreceptor for macrophage-tropic strains. We have designed bifunctional fusion proteins, consisting of RANTES/CCL5 and a single-chain Fv Ab fragment against CD4 to simultaneously block CD4 and CCR5. The fusion proteins bind to both receptors, compete with RANTES/CCL5 binding, and induce down-modulation of CCR5 (cid:1) 10 times more efficiently on CD4 (cid:1) compared with CD8 (cid:1) T cells. Moreover, after short incubation and subsequent washout, a significant down-modulation of CCR5 was only seen with the fusion proteins and only on CD4 (cid:1) cells, but not with unmodified RANTES or on CD4 (cid:2) cells, indicating a preferential targeting of CCR5 on CD4 (cid:1) T cells. The fusion proteins block M-tropic HIV infection more efficiently than RANTES/CCL5 and CD4 Abs alone or in combination. To our knowledge this is the first report of simultaneous blockade of an HIV-1 receptor and coreceptor with bifunctional inhibitors. The Journal of Immunology, 2005, 175: 7586–7593. close proximity of CD4 and CCR5 the basis for our present studies. To date, no approaches have been described to simultaneously target the HIV-1 receptor CD4 and a coreceptor (CCR5) with bifunctional constructs. The constructs consist of the chemokine RANTES and a single-chain Fv Ab fragment directed against CD4, fused by a short peptide linker that preserves the full functional activity of both components. We show that the constructs bind to both CD4 and CCR5 and preferentially down-mod-ulate CCR5 from the surface of CD4-positive cells. When cells were exposed for only a short time to RANTES or the fusion proteins, significant down-modulation of CCR5 was only seen on CD4-positive cells with the fusion proteins and not with unmodified RANTES, indicating that the constructs preferentially target the CD4-CCR5 complex. In assays measuring cellular HIV-1 infection of PBMC, inhibition of M-tropic HIV-1 infection was markedly improved by the fusion proteins compared with RANTES and CD4 Abs, alone or in combination. These data indicate that the simultaneous targeting of CD4 and CCR5 might be a novel strategy to suppress cellular HIV-1 infection.

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