HIV-1 entry into cells is mediated by interactions between the envelope (Env) gp120 and gp41 proteins with CD4 and chemokine receptors via an intermediate called the viral fusion complex (vFC). Here, mAbs were used to find the dynamic changes in expression of antigenic epitopes during vFC formation. A CD4-specific mAb (R275) and anti-vFC mAbs, designated F12-1, F13-6 and F18-4 that recognize the epitopes only appeared by the co-culture of env-transfected 293FT and CD4-transfected 293 cells, were developed by immunizing ganp-gene transgenic mice with an vFC-like structure formed by the same co-culture. The epitopes recognized by the mAbs appeared at different time points during vFC formation: F18-4 appeared first, followed by F13-6, and finally F12-1. The anti-vFC mAbs had little effect on vFC formation or virus neutralization; however, interestingly F12-1 and F18-4 increased exposure of the OKT4-epitope on the domain 3 in the extracellular region of CD4. R275, which recognizes the epitope closely associated with the OKT4-determinant on the domain 3, showed the marked inhibition of vFC formation and viral neutralization activity. The Ab binding to the epitopes appeared during viral membrane fusion might reinforce the appearance of the target epitopes for effective neutralization activity. (C) 2011 Elsevier GmbH. All rights reserved.