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. 2019 Jul 2;10(4):e01255-19.
doi: 10.1128/mBio.01255-19.

Differences in the Binding Affinity of an HIV-1 V2 Apex-Specific Antibody for the SIVsmm/mac Envelope Glycoprotein Uncouple Antibody-Dependent Cellular Cytotoxicity from Neutralization

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Differences in the Binding Affinity of an HIV-1 V2 Apex-Specific Antibody for the SIVsmm/mac Envelope Glycoprotein Uncouple Antibody-Dependent Cellular Cytotoxicity from Neutralization

Benjamin von Bredow et al. mBio. .

Abstract

As a consequence of their independent evolutionary origins in apes and Old World monkeys, human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency viruses of the SIVsmm/mac lineage express phylogenetically and antigenically distinct envelope glycoproteins. Thus, HIV-1 Env-specific antibodies do not typically cross-react with the Env proteins of SIVsmm/mac isolates. Here we show that PGT145, a broadly neutralizing antibody to a quaternary epitope at the V2 apex of HIV-1 Env, directs the lysis of SIVsmm/mac-infected cells by antibody-dependent cellular cytotoxicity (ADCC) but does not neutralize SIVsmm/mac infectivity. Amino acid substitutions in the V2 loop of SIVmac239 corresponding to the epitope for PGT145 in HIV-1 Env modulate sensitivity to this antibody. Whereas a substitution in a conserved N-linked glycosylation site (N171Q) eliminates sensitivity to ADCC, a lysine-to-serine substitution in this region (K180S) increases ADCC and renders the virus susceptible to neutralization. These differences in function correlate with an increase in the affinity of PGT145 binding to Env on the surface of virus-infected cells and to soluble Env trimers. To our knowledge, this represents the first instance of an HIV-1 Env-specific antibody that cross-reacts with SIVsmm/mac Env and illustrates how differences in antibody binding affinity for Env can differentiate sensitivity to ADCC from neutralization.IMPORTANCE Here we show that PGT145, a potent broadly neutralizing antibody to HIV-1, directs the lysis of SIV-infected cells by antibody-dependent cellular cytotoxicity but does not neutralize SIV infectivity. This represents the first instance of cross-reactivity of an HIV-1 Env-specific antibody with SIVsmm/mac Env and reveals that antibody binding affinity can differentiate sensitivity to ADCC from neutralization.

Keywords: ADCC; antibody function; human immunodeficiency virus; neutralizing antibodies; simian immunodeficiency virus.

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Figures

FIG 1
FIG 1
PGT145 mediates ADCC against HIV-, SHIV-, and SIV-infected cells. CEM.NKR-CCR5-sLTR-Luc cells infected with the indicated strains of HIV-1, SHIV, and SIV (A) and SIV alone (B) were incubated with an NK cell line (KHYG-1 cells) expressing human CD16 in the presence of the indicated concentrations of PGT145. ADCC activity was measured as the dose-dependent loss of luciferase activity in % RLU, compared to control wells containing no antibody and NK cells with either infected (maximal) or uninfected (background) target cells. The dotted line represents half-maximal lysis of infected cells, and the error bars represent the standard deviation of the mean from triplicate wells.
FIG 2
FIG 2
PGT145 stains cells infected with diverse lentiviral isolates. (A) Overlay plots show PGT145 (blue) versus DEN3 (red) staining of CEM.NKR-CCR5-sLTR-Luc cells infected with the indicated viruses. (B) Area-under-the-curve (AUC) values for ADCC responses were compared to the geometric mean fluorescence intensity (gMFI) of PGT145 staining on the surface of virus-infected cells by Pearson correlation. Virus-infected cells were identified by gating on the Gag+ CD4low population, and PGT145 staining was detected with PE-conjugated anti-human IgG F(ab′)2.
FIG 3
FIG 3
PGT145 binds to SIV-infected primary rhesus macaque CD4+ T cells and is recognized by rhesus macaque CD16. (A) Overlay plots show PGT145 (blue) versus DEN3 (red) staining of activated rhesus macaque CD4+ T cells infected with wild-type SIVmac239 or SIVmac239 Y721G. Virus-infected cells were identified by gating on the Gag+ CD4low population, and PGT145 staining was detected with PE-conjugated anti-human IgG F(ab′)2. (B and C) ADCC responses were measured using an NK cell line expressing either human or rhesus macaque CD16 (B) or unstimulated PBMCs from two different macaques (C) by incubating SIV-infected CEM.NKR-CCR5-sLTR-Luc cells at a 10:1 effector-to-target-cell ratio in the presence of the indicated concentrations of PGT145. The dotted line indicates half-maximal killing, and the error bars represent standard deviation of the mean from triplicate wells.
FIG 4
FIG 4
PGT145 neutralizes HIV-1 and SHIV but not SIV. The indicated viruses were incubated with serial dilutions of PGT145 for 1 h at 37°C before addition to TZM-bl cells (A) or C8166-SEAP cells (B). After 3 days, neutralization was measured as a decrease in luciferase (A) or secreted alkaline phosphatase (SEAP) (B) activity (RLU) compared to mock-infected or virus-infected control wells without antibody. The dotted line indicates 50% neutralization.
FIG 5
FIG 5
PGT145 binds to the V2 apex of SIVmac239 Env. (A) Amino acid substitutions were introduced in the V2 region of SIVmac239 Env. The amino acid sequence of HIV-1HXB2 is shown for reference, and the positions indicated in the legend correspond to SIVmac239 Env. Dots indicate identity, and dashes indicate gaps. (B) CEM.NKR-CCR5-sLTR-Luc cells were infected with SIVmac239 carrying the indicated Env mutations and incubated with an NK cell line expressing human CD16 in the presence of serial dilutions of PGT145. ADCC activity was measured as the dose-dependent loss of luciferase activity in % RLU. The dotted line represents half-maximal lysis of infected cells, and the error bars represent the standard deviation of the mean from triplicate wells. (C) Area-under-the-curve (AUC) values for ADCC were calculated and compared by one-way ANOVA with a Holm-Sidak adjustment for multiple comparisons. (D) Wild-type SIVmac239 and SIVmac239 K180S were incubated with serial dilutions of PGT145 for an hour before infecting C8166-SEAP cells. Neutralization was determined by calculating the loss of secreted alkaline phosphatase (SEAP) activity. The dotted line indicates 50% neutralization, and the error bars represent the standard deviation of the mean from triplicate wells. (E to G) CEM.NKR-CCR5-sLTR-Luc cells were infected with SIVmac239 V2 variants and stained with either PGT145 (E) or plasma from an SIVmac239-infected rhesus macaque (G). Fluorescence intensities are shown for viable infected (Gag+ CD4low) cells. PGT145 staining (AUC) was compared to ADCC (AUC) by Pearson correlation (F).
FIG 6
FIG 6
PGT145 binding to SIV Env trimers with V2 apex substitutions. The binding kinetics of PGT145 (A), CD4-IgG2 (B), VRC01 (C), and DEN3 (D) to soluble SIVmac239 SOSIP.664 Env trimers with the indicated substitutions in the V2 region was assessed by biolayer interferometry. Monoclonal antibodies immobilized on anti-human IgG-Fc-coated biosensors were dipped into trimer solutions (500 nM). The MAb-trimer interaction is represented as binding curves showing the rates of association (180 to 300 s) and dissociation (300 to 540 s). The dissociation constants (Kd) (E) and dissociation rates (Koff) (F) are plotted for PGT145 binding to each of the Env trimers.

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