Potent and broad anti-HIV-1 activity exhibited by a glycosyl-phosphatidylinositol-anchored peptide derived from the CDR H3 of broadly neutralizing antibody PG16

Abstract

PG9 and PG16 are two recently isolated quaternary-specific human monoclonal antibodies that neutralize 70 to 80% of circulating HIV-1 isolates. The crystal structure of PG16 shows that it contains an exceptionally long CDR H3 that forms a unique stable subdomain that towers above the antibody surface to confer fine specificity. To determine whether this unique architecture of CDR H3 itself is sufficient for epitope recognition and neutralization, we cloned CDR H3 subdomains derived from human monoclonal antibodies PG16, PG9, b12, E51, and AVF and genetically linked them to a glycosyl-phosphatidylinositol (GPI) attachment signal. Each fusion gene construct is expressed and targeted to lipid rafts of plasma membranes through a GPI anchor. Moreover, GPI-CDR H3(PG16, PG9, and E51), but not GPI-CDR H3(b12 and AVF), specifically neutralized multiple clades of HIV-1 isolates with a great degree of potency when expressed on the surface of transduced TZM-bl cells. Furthermore, GPI-anchored CDR H3(PG16), but not GPI-anchored CDR H3(AVF), specifically confers resistance to HIV-1 infection when expressed on the surface of transduced human CD4(+) T cells. Finally, the CDR H3 mutations (Y100HF, D100IA, and G7) that were previously shown to compromise the neutralization activity of antibody PG16 also abolished the neutralization activity of GPI-CDR H3(PG16). Thus, we conclude that the CDR H3 subdomain of PG16 neutralizes HIV-1 when targeted to the lipid raft of the plasma membrane of HIV-1-susceptible cells and that GPI-CDR H3 can be an alternative approach for determining whether the CDR H3 of certain antibodies alone can exert epitope recognition and neutralization.

Fig. 1.

Expression of GPI-anchored CDR H3 in transduced TZM-bl cells. (A) Schematic diagram of the lentiviral vectors pRRL-CDR H3/hinge/His tag/DAF. CDR H3s were derived from four human monoclonal antibodies, PG16, b12, E51, and AVF. Hinge, a human IgG3 hinge region; His tag, a six-histidine-residue tag; DAF, the C-terminal 34 amino acid residues of decay-accelerating factor; RSV, Roys sarcoma virus; PGK, human phosphoglycerate kinase gene promoter; RRE, Rev-responsive element; cPPT, central polypurine tract and termination sequence. (B) List of CDR H3 peptide sequences of PG16, b12, E51, and AVF. (C) FACS analysis of cell surface expression of CDR H3/hinge/His tag/DAF in mock- and CDR H3(PG16, b12, E51, and AVF)/hinge/His tag/DAF-transduced TZM-bl cells with or without PI-PLC treatment.

Fig. 2.

Localization of GPI-anchored CDR H3 in transduced TZM-bl cells. Shown are data for confocal analysis of mock- or GPI-CDR H3(PG16 and AVF)-transduced TZM-bl cells. Cells were stained with Alexa 555-conjugated cholera toxin B subunit (CtxB), and cells were stained with a mouse anti-His tag antibody followed by Alexa 488-conjugated goat anti-mouse IgG antibody.

Fig. 3.

Effect of GPI-CDR H3(PG16, PG9, b12, E51, and AVF) on infection of HIV-1 viruses and pseudotypes. (A) Effect of GPI-CDR H3s on efficiency of transduction of HIV-1 and 10A1 pseudotypes into GPI-CDR H3-transduced TZM-bl cells. Shown are percentages of reduction of the relative luciferase activities in TZM-bl cells transduced with GPI-CDR H3(AVF, b12, PG16, and E51) compared with mock-transduced parental TZM-bl cells. Green, ≥50% inhibition; yellow, ≥90% inhibition; red, ≥99% inhibition. The percentage of inhibition was based on the following calculation: (RLA in virus alone in a given transduced cell − RLA in no virus in the same transduced cell)/(RLA in virus alone in the parental cell − RLA in no virus in the parental cell). (B) Effect of GPI-CDR H3s on wild-type HIV-1 and SIVMne027 infection in GPI-CDR H3-transduced TZM-bl cells. w/o, parental TZM-bl cells. (C) FACS analysis of cell surface expression of CDR H3/hinge/His tag/DAF in CDR H3(PG16, PG9, E51, and AVF)/hinge/His tag/DAF-transduced TZM-bl cells. (D) Percentage of the reduction of relative luciferase activity in TZM-bl cells transduced with GPI-CDR H3(AVF, PG9, PG16, and E51) compared with mock-transduced parental TZM-bl cells. Yellow, ≥90% inhibition; red, ≥99% inhibition.

Fig. 4.

Relative susceptibilities to 10A1 and HIV-1 pseudotypes and replication-competent HIV-1 of TZM-bl cells transduced with GPI-CDR H3(PG16) and with GPI-CDR H3 mutants (G7, D100HI, and Y100HF). (A) Amino acid sequence comparison of wild-type CDR H3(PG16) and its mutants. (B) FACS analysis of cell surface expression of TZM-bl cells transduced with GPI-CDR H3(PG16) and with GPI-CDR H3 mutants (G7, D100HI, and Y100HF). (C) Relative susceptibility to pseudotypes and replication-competent HIV-1 of TZM-bl cells transduced with GPI-CDR H3 mutants (G7, D100HI, and Y100HF) compared to cells transduced with GPI-CDR H3(PG16).

Fig. 5.

Effect of GPI-CDR H3(PG16) on anti-HIV-1 activity of transduced human CD4⁺ T cells. (A) GPI-CDR H3(PG16) confers resistance to HIV-1 Bru-3 in human CD4⁺ T cells. (B) GPI-CDR H3(PG16) confers resistance to HIV-1 Bru-Yu2 in human CD4⁺ T cells.

Fig. 6.

EGFP expression in parental CEMss-CCR5 cells and CEMss-CCR5 cells expressing GPI-CDR H3(PG16 and AVF) transduced with a VSV-G-pseudotyped HIV-1 vector. (A) Percent EGFP-positive cells; (B) MFI (mean fluorescence intensity).