An Immunoglobulin Fusion Protein Based on the gp120-CD4 Receptor Complex Potently Inhibits Human Immunodeficiency Virus Type 1 in 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–C_H2–C_H3 portion of human immunoglobulin γ 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.