GPI-anchored single chain Fv - an effective way to capture transiently-exposed neutralization epitopes on HIV-1 envelope spike

Abstract

BackgroundIdentification of broad neutralization epitopes in HIV-1 envelope spikes is paramount for HIV-1 vaccine development. A few broad neutralization epitopes identified so far are present on the surface of native HIV-1 envelope spikes whose recognition by antibodies does not depend on conformational changes of the envelope spikes. However, HIV-1 envelope spikes also contain transiently-exposed neutralization epitopes, which are more difficult to identify.ResultsIn this study, we constructed single chain Fvs (scFvs) derived from seven human monoclonal antibodies and genetically linked them with or without a glycosyl-phosphatidylinositol (GPI) attachment signal. We show that with a GPI attachment signal the scFvs are targeted to lipid rafts of plasma membranes. In addition, we demonstrate that four of the GPI-anchored scFvs, but not their secreted counterparts, neutralize HIV-1 with various degrees of breadth and potency. Among them, GPI-anchored scFv (X5) exhibits extremely potent and broad neutralization activity against multiple clades of HIV-1 strains tested. Moreover, we show that GPI-anchored scFv (4E10) also exhibited more potent neutralization activity than its secretory counterpart. Finally, we demonstrate that expression of GPI-anchored scFv (X5) in the lipid raft of plasma membrane of human CD4+ T cells confers long-term resistance to HIV-1 infection, HIV-1 envelope-mediated cell-cell fusion, and the infection of HIV-1 captured and transferred by human DCs.ConclusionsThus GPI-anchored scFv could be used as a general and effective way to identify antibodies that react with transiently-exposed neutralization epitopes in envelope proteins of HIV-1 and other enveloped viruses. The GPI-anchored scFv (X5), because of its breadth and potency, should have a great potential to be developed into anti-viral agent for HIV-1 prevention and therapy.

Glycosylphosphatidylinositol-Anchored Anti-HIV scFv Efficiently Protects CD4 T Cells from HIV-1 Infection and Deletion in hu-PBL Mice

“…It is likely that after gp120-CD4 binding, the available space is not sufficient to accommodate large molecules. Despite the broad range of neutralization activities of X5, we and others showed that free scFv X5 had a relatively lower level of inhibition of virus entry (37,49). X5 function is great enhanced by the addition of soluble CD4.…”

HIV-1 Virus-Like Particles Produced by Stably Transfected Drosophila S2 Cells: a Desirable Vaccine Component

“…To generate stable S2 clones, a limiting dilution assay was performed as described earlier (54). Briefly, 1.5 ϫ 10 2 S2 transfectants were mixed with 10 6 parental S2 cells (feeder layer cells) in 10 ml of Drosophila Schneider 2 medium supplemented with 10% FBS, 100 U of penicillin/ml, 100 U of streptomycin/ml, and 100 mg of L-glutamine/liter.…”

“…TZM-bl cells were obtained from the NIH AIDS Research and Reference Reagent Program (ARRRP; Germantown, MD) (9,38). The human CD4 ϩ T cell line CEMss-CCR5 was generated as described previously (54). These cells were maintained in complete DMEM.…”

“…HIV-1 and 10A1 pseudotypes were generated and a single-cycle infectivity assay was performed as described previously (32,54).…”

HIV-1 Glycoprotein Immunogenicity

“…Numerous Abs were described for their binding to the CD4-induced site and the most interesting by their breadth neutralizing capacities are the 17b, X5, m18, and m14 which all contains long H3s playing a major role in their mechanism of binding (table 1). The H3s region of X5, m6 and m9 appear to be very flexible and highly potent to neutralize the virus (Wen et al, 2010;Zhu et al, 2006) …”