Antibody-dependent cellular cytotoxicity, infected cell binding and neutralization by antibodies to the SIV envelope glycoprotein

Abstract

Antibodies specific for diverse epitopes of the simian immunodeficiency virus envelope glycoprotein (SIV Env) have been isolated from rhesus macaques to provide physiologically relevant reagents for investigating antibody-mediated protection in this species as a nonhuman primate model for HIV/AIDS. With increasing interest in the contribution of Fc-mediated effector functions to protective immunity, we selected thirty antibodies representing different classes of SIV Env epitopes for a comparison of antibody-dependent cellular cytotoxicity (ADCC), binding to Env on the surface of infected cells and neutralization of viral infectivity. These activities were measured against cells infected with neutralization-sensitive (SIVmac316 and SIVsmE660-FL14) and neutralization-resistant (SIVmac239 and SIVsmE543-3) viruses representing genetically distinct isolates. Antibodies to the CD4-binding site and CD4-inducible epitopes were identified with especially potent ADCC against all four viruses. ADCC correlated well with antibody binding to virus-infected cells. ADCC also correlated with neutralization. However, several instances of ADCC without detectable neutralization or neutralization without detectable ADCC were observed. The incomplete correspondence between ADCC and neutralization shows that some antibody-Env interactions can uncouple these antiviral activities. Nevertheless, the overall correlation between neutralization and ADCC implies that most antibodies that are capable of binding to Env on the surface of virions to block infectivity are also capable of binding to Env on the surface of virus-infected cells to direct their elimination by ADCC.

Fig 1. Env staining on the surface of infected CEM.NKR-_CCR5-sLTR-Luc cells.

CEM.NKR-_CCR5-sLTR-Luc cells were infected with SIV_mac239, SIV_mac316, SIV_smE543-3, SIV_smE660-FL14 and SHIVAD8-EO. After 3–5 days, the cells were stained with the indicated SIV Env-specific antibodies and with a dengue virus-specific antibody (DEN3) as a negative control, followed by AF647-conjugated anti-human IgG F(ab′)₂. The cells were also stained for surface expression of CD4, intracellular expression of the SIV Gag protein and for viability. The histograms plots depict Env staining in comparison to non-specific staining with DEN3 (shaded) on virus-infected (Gag+ CD4_low) cells. Representative data is shown from two independent experiments.

Fig 2. Comparison of Env staining on SIV-infected CEM.NKR-_CCR5-sLTR-Luc cells and primary rhesus macaque CD4+ T cells.

Antibody binding to the surface of SIV-infected cells was quantified as the ratio of the geometric mean fluorescence intensity (gMFI) of staining with each of the Env-specific antibodies to staining with DEN3 control antibody. The relationship between the gMFI ratios of antibody binding to CEM.NKR-_CCR5-sLTR-Luc cells and to rhesus macaque CD4+ T cells was determined by calculating the Spearman’s rank order correlation.

Fig 3. ADCC activity of SIV Env-specific antibodies.

CEM.NKR-_CCR5-sLTR-Luc cells infected with SIV_mac239, SIV_mac316, SIV_smE543-3, SIV_smE660-FL14 and SHIVAD8-EO were incubated with an NK cell line (KHYG-1 cells) expressing rhesus macaque CD16 at a 10:1 effector-to-target (E:T) ratio in the presence of the indicated concentrations of antibody. The dose-dependent loss of luciferase activity was measured in relative light units (RLU) after an 8-hour incubation. ADCC responses were calculated as the remaining luciferase activity (% RLU) by dividing the difference in RLU between SIV-infected cells in the presence of antibody and uninfected cells without antibody (experimental—background) by the difference in RLU between SIV-infected cells and uninfected cells in the absence antibody (maximal–background) and multiplying by 100. The values indicate the mean and standard deviation (error bars) for triplicate wells at each antibody concentration and the dotted line indicates half-maximal killing of SIV-infected cells. Representative data is shown from two independent experiments.

Fig 4. Comparison of ADCC and antibody binding to SIV-infected cells.

Area above the curve (AAC) values for ADCC were calculated from differences between the theoretical maximal luciferase induction and the reduction in luciferase activity at each antibody concentration. Antibody binding to SIV-infected CEM.NKR-_CCR5-sLTR-Luc cells was quantified by calculating the gMFI ratios of staining with each of the Env-specific antibodies to non-specific staining with the DEN3 control antibody. The dotted lines indicate thresholds for detectable ADCC and antibody binding, which were set at one standard deviation above the absolute value of mean of negative ADCC (AAC) values and one standard deviation above the mean of gMFI ratios of antibody binding less than one. Spearman’s rank order correlation coefficients were calculated for comparisons of ADCC and antibody binding for all antibodies and for those antibodies that were above the thresholds for detectable ADCC and binding.

Fig 5. Neutralization of SIV_mac239, SIV_mac316, SIV_smE543-3 and SIV_smE660-FL14 by Env-specific antibodies.

Dilutions of each virus were prepared and incubated with the indicated antibody concentrations for one hour before addition to TZM-bl cells. Three days post-infection, neutralization was calculated from the luciferase activity (RLU) in lysates from cells inoculated with virus plus antibody relative to cells inoculated with virus without antibody. The error bars indicate standard deviations of the mean for triplicate wells and the dotted line indicates 50% neutralization.

Fig 6. Comparison of ADCC and neutralization of viral infectivity.

Area above the curve (AAC) values for ADCC and neutralization were calculated from the differences between the theoretical maximal luciferase induction and the reduction in luciferase activity at each antibody concentration. The dotted lines indicate thresholds for detectable ADCC and neutralization, which were set at one standard deviation above the absolute value of the mean negative ADCC and neutralization AAC values. Spearman’s rank order correlation coefficients were calculated for comparisons of ADCC and neutralization for all antibodies and for those antibodies that were above the thresholds for detectable responses.

Fig 7. SIV Env binding, ADCC and neutralization by CD4-inducible antibodies.

(A) Env staining on the surface of CEM.NKR-_CCR5-sLTR-Luc cells infected with SIV_mac239, SIV_mac316, SIV_smE543-3, SIV_smE660-FL14 and SHIVAD8-EO was measured in the presence (dotted lines) and absence (solid lines) of soluble CD4 (sCD4) (10 μg/ml) relative to non-specific staining with DEN3 (shaded). Antibody binding to SIV-infected (Gag⁺ CD4_low) cells was detected with an AF647-conjugated anti-human IgG F(ab′)₂. (B) ADCC responses (% RLU) were measured as the dose-dependent loss of luciferase activity from SIV-infected CEM.NKR-_CCR5-sLTR-Luc cells after an 8-hour incubation with a rhesus macaque CD16+ NK cell line at a 10:1 E:T ratio and the indicated concentrations of each antibody in the presence and absence of sCD4 (10 μg/ml). The plotted values represent the mean and standard deviation (error bars) for triplicate wells and the dotted line indicates half-maximal ADCC. (C) Neutralization of SIV_mac239, SIV_mac316, SIV_smE543-3 and SIV_smE660-FL14. Dilutions of SIV were prepared and incubated with and without sCD4 (10 μg/ml) and the indicated concentrations of each antibody for one hour before addition to TZM-bl cells. Three days post-infection, neutralization (% RLU) was calculated from the luciferase activity in cell lysates. The error bars indicate standard deviation of the mean for triplicate wells and the dotted line indicates 50% neutralization.