An Immunoglobulin Fusion Protein Based on the gp120-CD4 Receptor Complex Potently Inhibits Human Immunodeficiency Virus Type 1in Vitro

Abstract

Fusion proteins containing immunoglobulin Fc domains attached to bioactive moieties have been developed as therapeutic agents against several diseases. Here, we describe the development and characteristics of a novel fusion protein (FLSC R/T-IgG1) that targets CCR5, the major coreceptor for HIV-1 during primary infection. FLSC R/T-IgG1 was expressed from a synthetic gene that linked a single chain gp120-CD4 complex containing an R5 gp120 sequence with the hinge-CH2-CH3 portion of human immunoglobulin gamma subtype 1. Purified FLSC R/T-IgG1 exhibited a molecular mass of 189 kDa under reducing conditions, which matched the expected size of one polypeptide chain. Chemically crosslinked or untreated FLSC R/T-IgG1 exhibited a mass of a 360-kDa polypeptide under reducing and nonreducing conditions, which indicated that the molecule adopts a disulfide-linked bivalent structure. The chimeric molecule bound specifically to CCR5-expressing cells and to peptides derived from the CCR5 N-terminus. Such binding was more efficient than what was obtained with a monomeric single chain gp120-CD4 complex. FLSC R/T-IgG1 binding to CCR5 was blocked by preincubation of coreceptor-expressing cells with CCR5 ligands and by antibody to the coreceptor binding domain of gp120. Conversely, FLSC R/T-IgG1 blocked the binding of chemokine to CCR5. However, FLSC R/T-IgG1 did not trigger intracellular Ca2+ mobilization in peripheral blood mononuclear cells. FLSC R/T-IgG1 potently neutralized primary R5 HIV-1 in both a PBMC-based assay and cell line-based assays but did not affect the replication of X4 viruses. These findings suggest that FLSC R/T-IgG1 might be used as a possible therapeutic agent against HIV.