PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cytometry A. Author manuscript; available in PMC 2013 June 25.
Published in final edited form as:
PMCID: PMC3692008
NIHMSID: NIHMS305834

High throughput quantitative analysis of HIV-1 and SIV-specific ADCC-mediating antibody responses

Abstract

We have developed a high throughput platform to detect the presence of HIV-1 and SIV-specific ADCC-mediating antibody responses. The assay is based on the hydrolysis of a cell-permeable fluorogenic peptide substrate containing a sequence recognized by the serine protease, Granzyme B (GzB). GzB is delivered into target cells by cytotoxic effector cells as a result of antigen (Ag)-specific Ab-Fcγ receptor interactions. Within the target cells, effector cell-derived GzB hydrolyzes the substrate, generating a fluorescent signal that allows individual target cells that have received a lethal hit to be identified by flow cytometry. Results are reported as the percentage of target cells with GzB activity (%GzB). Freshly isolated or cryopreserved PBMC and/or NK cells can be used as effector cells. CEM.NKR cells expressing the CCR5 co-receptor are used as a target cells following (i) coating with recombinant envelope glycoprotein, (ii) infection with infectious molecular clones expressing the Env antigens of primary and lab adapted viruses, or (iii) chronic infection with a variant of HIV-1/IIIB, termed A1953. In addition, primary CD4+ T cells infected with HIV-1 in vitro can also be used as targets. The assay is highly reproducible with a coefficient of variation of less than 25%. Target and effector cell populations, in the absence of serum/plasma, were used to calculate background (8.6±2.3%). We determined that an initial dilution of 1:50 and 1:100 is required for testing of human and non-human primate samples, respectively. This assay allows for rapid quantification of HIV-1 or SIV-specific ADCC-mediating antibodies that develop in response to vaccination, or in the natural course of infection, thus providing researchers with a new methodology for investigating the role of ADCC-mediating antibodies as correlates of control or prevention of HIV-1 and SIV infection.

Keywords: ADCC, HIV, SIV, NK, Fc gamma receptors, Granzyme B, high throughput

Introduction

The antibody-dependent cellular cytotoxicity (ADCC) response represents one of the effector mechanisms used by the immune system to destroy malignant cells or cells infected with intracellular pathogens. In the context of a viral infection, ADCC contributes to viral clearance via specific recognition and targeted elimination of virus-infected cells through direct cooperation of both innate and acquired immunity (1-3). Specifically, the Fab region of an Ab binds to a specific viral antigen on the surface of infected cells, and the Fc region of the Ab binds to an Fcγ receptor (Fcγ-R) on the surface of effector cells. This interaction results in the release of preformed factors including perforin and granzymes from the effector cell that will ultimately mediate the killing of infected target cells. Other factors such as chemokines and/or cytokines can also be released from the activated effector cells, contributing to mediation of immune responses (4-6). ADCC effector cells express cell-surface Fcγ receptors and include natural killer (NK) cells, monocytes/macrophages, and γδ T cell subsets.

The importance of ADCC in the control of HIV and SIV infection has been reported in several studies (7-9), with the most compelling data demonstrating a direct role after passive transfer of monoclonal Ab (10-12). The presence of high-levels of ADCC-mediating antibodies has also been associated with a delay in disease onset, and with the status of long-term non-progressors (13,14). Additionally, the role that vaccine-induced Ab with Fcγ-R-binding properties may have played in preventing HIV-1 infection in the vaccine recipients enrolled in the RV144 human clinical trial in Thailand (15) is currently under investigation. Taken together, these data point out the importance of studying the presence of HIV-1 ADCC-mediating Ab responses following vaccination with AIDS vaccine candidates to establish correlates of protection.

To date, the measurement of ADCC-mediating Abs by effector cells has been limited by the lack of a quantitative technique that allows for specific and high throughput analysis of target cell killing at the single cell level. We have developed a flow cytometry-based assay that takes advantage of our ability to reproducibly detect the proteolytic activity of Granzyme B after its delivery into target cells, initiated by Ab recognition of viral antigens on the target cell membrane. We have determined that this technique is applicable to cell lines pulsed with HIV-1 and SIV recombinant proteins, chronically or acutely infected with HIV-1 and SIV, and to HIV-1 infected primary CD4+ T cells. We have utilized this assay to evaluate the ability of HIV- and SIV-specific antibodies to mediate ADCC responses during infection and in response to vaccination. We expect that further use of this assay will lead to a greater understanding of the contribution of ADCC to both the natural and vaccine-induced immune responses to HIV-1 and SIV.

Methods

Human and Non-Human Primate Sera

HIV-1 seronegative and seropositive sera and plasma were obtained from patients enrolled in various studies conducted by the Centers for HIV and AIDS Vaccine Immunology. Samples collected from non-human primates were provided by Dr. Mario Roederer (NIH/Vaccine Research Center). The HIV IgG immunoglobulin preparation (HIVIG) (16) was obtained from the NIH AIDS Research and Reagent Program. All human and non-human primate samples were collected in accordance to the local IRB procedures. The humanized monoclonal antibody Synagis® (IgG1k; palivizumab; MedImmune, LLC; Gaithersburg, MD) directed to an epitope in the A antigenic site of the F protein of respiratory syncytial virus was purchased from the manufacturer and used as a control.

Target cells

CEM.NKRCCR5 cell line (17) and activated CD4+ T cells were used as target cells. CEM.NKRCCR5 cells were used either after coating with recombinant gp120 or following infection with HIV-1 infectious molecular clones (IMCs) as described in this study. Activated CD4+ T cells were used as targets after infection with IMCs as described in this study. Activated CD4+ T cells were generated from cryopreserved PBMC as follows. Peripheral Blood Mononuclear Cells (PBMC) obtained from an HIV-seronegative donor were thawed and activated by 72-hour culture at 37°C and 5% CO2 in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 20% FBS (Gemini Bio-Products, West Sacramento, CA) in presence of 30 U/ml rhIL-2 (Peprotech, Rocky Hill, NJ) (R20-IL2), and containing anti-CD3 (clone OKT3, eBioscience, San Diego, CA) and anti-CD28 (clone CD28.2, BD Biosciences, San Jose, CA) at 150 ng/ml. The CD4+ enriched population was obtained by removing CD8+ T cells with magnetic bead separation (Miltenyi Biotec GmbH, Germany) using manual columns. All PBMC samples from the seronegative donors were obtained under informed consent according to appropriate IRB protocols. The chronically infected cell line, termed A1953, was obtained by infection of CEM-NKRCCR5 with the A1953 virus that was derived from the HXB2 isolate selected for the inability to downregulate CD4 from the surface of chronically infected T-cell lines. It was subsequently shown to be defective in Nef expression. The A1953-CEM-NKRCCR5-chronically infected cells (A1953) express high levels of CD4 complexed with envelope glycoprotein on the cell surface (J. Hoxie, unpublished data).

Recombinant gp120 HIV-1 proteins

The recombinant gp120 HIV-1 proteins representing the envelopes of the subtype B BaL (GenBank No. M68893; Immune Technology Corp, New York), the subtype C CAP45.2.00 (GenBank No. DQ435682; Immune Technology Corp, New York), and the subtype E CM243 (GenBank No. AY214109; Protein Sciences, Meiden, CT) HIV-1 isolates were used to coat CEM.NKRCCR5 target cells. The recombinant gp120 representing the SIVE660 isolate was also titrated and used as described for the HIV-1 recombinant proteins (GenBank No. ACQ89608; Immune Technology Corp, New York). For each recombinant gp120, the optimum amount to coat the target cells was determined by competing the binding of the Leu3A antibody (clone SK3; Catalog no. 340133; Final dilution 1:5; BD Bioscience, San Jose, CA, USA) to the CD4 receptor expressed on the surface of the cell line. Briefly, 1×106 cells were incubated with serial two-fold dilutions of gp120 starting at 20μg/ml and incubated for 90 minutes at 4°C in RPMI1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 10mM HEPES (Invitrogen, Carlsbad, CA), 100μg/ml Gentamicin (Invitrogen, Carlsbad, CA), and 1% Penicillin-Streptomycin-Glutamine (Invitrogen, Carlsbad, CA), (R10). The cells were subsequently washed twice and stained with a saturating concentration of the FITC-conjugated anti-Leu3A Ab for 15 minutes. After two washes, the cells were resuspended in 200μl of 1% formaldehyde-PBS fixative. The samples were acquired using a LSR II (BD Bioscience, San Jose, California) within 24 hours. The cytometer optimization and calibration were performed according to published guidelines (18). The first concentration of any recombinant gp120 capable of reducing the mean fluorescence signal by >50% of the control cells (cells not pre-incubated with gp120) was chosen to coat the target cells in the final assay.

HIV-1 Infectious molecular clones (IMC)

HIV-1 reporter viruses used were replication-competent infectious molecular clones (IMC) designed to encode the BaL (subtype B) and CM235 (subtype A/E) env genes in cis within an isogenic backbone that also expresses the Renilla luciferase reporter gene and preserves all viral open reading frames (19). The Env-IMC-LucR viruses used were NL-LucR.T2A-BaL.ecto (IMCBaL) (20) and NL- LucR.T2A-AE.CM235-ecto (IMCCM235) (GenBank No. AF2699954; plasmid provided by Dr.Jerome Kim, US Military HIV Research Program). Reporter virus stocks were generated by transfection of 293T cells with proviral IMC plasmid DNA and tittered on TZM-bl cells for quality control

Infection of CEM.NKRCCR5 cell line with HIV-1 reporter IMC

Env-IMC-LucR reporter virus stocks were titrated on CEM.NKRCCR5 in order to determine the input that achieved optimal viral gene expression within 72 hours post-infection, as measured by detection of Renilla Luciferase activity (19) and intra-cellular p24 expression. We subsequently infected 2×106 cells with IMCBAL and IMCCM235 by incubation with the appropriate TCID50/cell dose of IMC for 0.5 hour at 37°C and 5% CO2 in the presence of DEAE-Dextran (7.5 μg/ml). The cells were subsequently resuspended at 0.5×106/ml and cultured for three days in complete medium containing 7.5μg/ml DEAE-Dextran. On assay day, the infection was monitored by measuring the frequency of cells expressing intracellular p24. The assays performed using the IMC-infected target cells were considered reliable if the percentage of viable p24+ target cells on assay day was ≥20%. The data are presented after normalizing to the % of target cells positive for intracellular p24.

Infection of primary CD4+ T cells with HIV-1 reporter IMC

The activated CD4+-enriched T cells were obtained as described in the target cell section. The CD4+ T cell blasts were infected with 12 TCID50/cell (determined in TZM-bl cells) of the HIV-1 IMCBaL by spinocluation (21,22) at 1200 x g for 2 hours. Cells spinoculated in the absence of virus (mock-infected) were used as a negative infection control. As described for the CEM.NKRCCR5, the assay was considered reliable if the percentage of viable p24+ target cells on assay day was ≥20%.

Intracellular p24 staining of infected cells

Following 72 hours of infection, cells were washed in PBS, dispensed in 96-well V-bottom plates at 1×105 viable cells/well, and stained with a vital dye (LIVE/DEAD Fixable Aqua Dead Cell Stain, Invitrogen) to exclude non-viable cells from subsequent analyses. The cells were then washed twice with 250μl/well of washing buffer (WB; PBS+1% FBS) and incubated with Cytofix/Cytoperm (BD Bioscience, San Jose, CA) for 20 minutes at 4°C. The cells were then washed twice with 200μl of 1% Cytoperm washing buffer. After the final wash, the anti-p24 Ab (clone KC57-RD1; catalog no. 6604667; final dilution 1:400 to stain infected CEM.NKRCCR5 and 1:100 to stain infected CD4+ T cell; Beckman Coulter) was added, and the plates were incubated for 30 minutes at 4°C. The plates were washed twice with WB, and the cells were resuspended in 200μl 1% formaldehyde-PBS. The samples were acquired within 24 hours using the LSR II flow cytometer. A minimum of 10,000 total singlet events was acquired for each analysis. Gates were set to include singlet and live events. The appropriate compensation beads were used to compensate the spillover signal for the two fluorophores. Data analysis was performed using FlowJo 8.8.4 software (TreeStar Inc., Ashland, OR). The uninfected CEM.NKRCCR5 and the chronically infected A1953 cell lines were used to titer the vital dye and the anti-p24 Ab, and were used as negative and positive controls, respectively, for the described staining procedure.

Effector cell populations

Cryopreserved peripheral blood mononuclear cells (PBMC) were used as the source of the natural killer (NK) cell effectors. The cells were obtained by leukopheresis from a seronegative donor and were processed and cryopreserved within 6 hours of collection from the donor. The cells were subsequently thawed and rested overnight at 2×106 cell/ml in R10 at 37°C and 5% CO2. The following day, the cells were counted for viability and adjusted to the proper concentration to obtain an Effector to Target ratio of 30:1 (E:T = 30:1). In the experiment where infected CD4+-enriched cells were used as targets, the NK cell populations were isolated from the cryopreserved PBMCs by negative selection with magnetic beads (Human NK Cell Isolation Kit; Miltenyi Biotec GmbH, Germany) after overnight rest. The purified NK effector cells were used at E:T ratio of 10:1. For the depletion assay, magnetic bead separation was also used to remove the CD56+ and CD16+ cells from the PBMC preparation according to the protocol suggested by the manufacturer. Cells incubated with APC-conjugated beads were used as a mock control for any potential non-specific effects of the separation columns. The purity of each depleted cell population was verified using the following surface stain panel: Lineage- APC-Cy7 (anti-CD3, -CD14, -CD19; Ab clones SK7, MϕP9, and SJ25C1; catalog no. 557832, 557831, 557791; dilution 1:10, 1:640, 1:10; BD Biosciences), PE Anti-CD16 (clone 3G8; catalog no. 302008; final dilution 1:167; Biolegend, San Diego, CA), PE-Cy7 anti-CD56 (clone B159; catalog no. 557747; final dilution 1:40; BD Biosciences), and viability marker (LIVE/DEAD Fixable Aqua Dead Cell Stain, Invitrogen).

ADCC-GranToxiLux (ADCC-GTL) assay

Antibody Dependent Cellular Cytotoxic activity mediated by the patient plasma/sera and the HIVIG polyclonal IgG preparation was detected according to our modification of the previously described GranToxiLux (GTL) cell-mediated cytotoxicity procedure (23,24). The assay was performed in 96-well plates as follows and outlined in Figure 1A. Infected and uninfected target cells were counted, washed, resuspended in R10 at 1×106 cell/ml, and labeled with a fluorescent target-cell marker (TFL4; OncoImmunin, Inc., Gaithersburg, MD) and a viability marker (NFL1; OncoImmunin, Inc.) for 15 minutes in a 37°C water bath as specified by manufacturer. After two washes using 10 ml of R10, viable cells were counted using a Guava PCA (Millipore, Billerica, MA) and adjusted to reach a final viable effector to viable target ratio of 30:1 or 10:1 when PBMC and NK cells were used as effector cells, respectively. Twenty-five μl of each effector and target cell suspension and 75 μl of GzB substrate (OncoImmunin, Inc.) were dispensed into each well of a 96-well V-bottom plate. After incubation for 5 minutes at room temperature (RT), 25μl of the appropriate antibody or IgG dilutions were added to the target/effector cell suspension and incubated for 15 minutes at RT. The plates were subsequently centrifuged for 1 minute at 300 × g, and incubated for 1 hour at 37°C and 5% CO2. After two washes with WB, cells were resuspended in 225μl of WB, placed at 4°C, and acquired directly from the assay plate with the LSRII (BD Bioscience, San Jose, California) within 6 hours using the High-Throughput-System (HTS, BD Bioscience, San Jose, California). A minimum of 2.5×103 and 5×103 events representing viable gp120-coated and infected target cells, respectively, was acquired for each condition. The signal for each fluorophore was detected using: 1) 640nm/40mW laser and 660/20 filter for TFL4; 405nm/50mW laser and 450/50 filter for NFL1; 3) 488nm/20mW laser and the combination of 505LP with 525/50 filters for the GzB substrate. Because of the spectral properties of the fluorescent molecules utilized in this panel, manual compensation of the signals was not required to analyze the data, as previously reported (24). Data analysis was performed using FlowJo 8.8.4 software. The investigators developed a dedicated analysis template that reflects the gating strategy illustrated in Figure 1B.

Figure 1
Assay overview and gating procedures with results interpretation

Statistical analysis

For each experiment, the appropriate statistical test was performed as reported throughout the manuscript using GraphPad Prism 5 software version 5.0c (GraphPad Software, Inc), and R version 2.12.1 (http://cran.r-project.org) statistical software. Specifically, for each experimental condition, the positivity criterion is determined by controlling the false positive response rate. Based on a set of HIV seronegative samples, the false positive response rate is estimated by the observed fraction of subjects that have a positive response. An exact 95% confidence interval (computed by Clopper-Pearson method (25)) is provided. To characterize the reproducibility of each experimental condition, the percent coefficient of variation (%CV) is reported. For comparison between two experimental conditions (e.g. fresh versus cryopreserved effector cells), a cubic model of % GzB versus log10 (dilution) is fitted under each condition. And the F-test (26), based on an analysis of variance table, is used to test the null hypothesis that the two experimental conditions have the same cubic %GzB vs log10 (dilution) curve against the null hypothesis that the %GzB vs log10 (dilution) curve is different between the two conditions.

Results

Gating strategy

We developed the gating strategy shown in Figure 1B. We used a condition representing the target cells alone to identify our viable target cell population based on their pre-labeling with the cellular fluorophore (TFL4), and we could exclude the non-viable target cell population labeled with the viability dye (NFL1). Out of the viable target cell population, we identified those that contained the hydrolyzed Granzyme B (GzB) substrate and were, therefore, brightly fluorescent (GzB positive). We used this gate to separate the GzB negative from the positive population in subsequent experimental conditions. The results are expressed as %GzB activity, defined as the percentage of cells positive for proteolytically active GzB (i.e. cells recognized by the effectors) out of the total viable target cell population. As shown in Figure 1B, second column, in the presence of effector and target cells alone without plasma/serum, we observed a background of 5.76% GzB activity (average of 8.6% ±2.3%; CV=27% over 26 assays). Upon addition of plasma/serum samples from HIV-1 seronegative (SN) and seropositive (SP) subjects at a representative dilution of 1:1,000, we observed GzB activity of 8.55% and 45.4%, respectively (Figure 1B, third and fourth columns).

Throughout this manuscript, the final results are reported after subtracting the background. The results calculated in this way can be used to determine the maximum %GzB activity (potency) at any given dilution. Moreover, we can calculate the titer of ADCC-mediating Ab present in the plasma/serum of the donor by interpolating the dilution at which the curve intersects the positive cut-off value after excluding the prozone area (Figure 2A). Future studies to be performed in the context of pre-clinical and clinical vaccine trials will allow us to determine whether potency or titer of ADCC responses might better correlate with protection from infection.

Figure 2
Analysis of the effector cell populations

Reproducibility of staining allows using a template-based analysis

The instrument was rigorously optimized and monitored for daily performance according to published QC procedures (18). All reagents were monitored for inter-lot variability. The analysis of results obtained from 287 independent populations acquired in six different sets of experiments revealed a robust separation of the populations of interest as already reported in Figure 1B. We observed consistent median fluorescence intensity (MFI) values for the target cells (TFL4+= 7302±1115) that allowed their segregation from the effector population. The TFL4+/NFL1- viable target cells (MFI of 1028±115) are easily identified from the TFL4+/NFL1+ non-viable target cells (6865±2540). Lastly, as reported in the gating strategy, the Granzyme B positive cells (MFI=5711±1365) were easily identified from the Granzyme B negative cells (MFI=846±183). The separation of these populations was consistent across conditions and target cell types used and allowed setting the gates in the same position for every sample within the same experiment. The investigators could also easily take advantage of these separations to build a template for analysis that can be applied to all experimental platforms.

Positivity criteria

We tested samples collected from 16 HIV-1 seronegative and 3 seropositive subjects to establish our criteria of positivity. We determined that a threshold of 8 %GzB should be used for gp120-coated and A1953 chronically infected target cells after background subtraction. We further qualified these parameters in a separate set of samples obtained from seronegative (n=8) and placebo vaccine recipients (n=70), and determined that the false positive estimate is 1.3 with 95% CI =(0.032%, 6.9%) based on one false positive finding. For the IMC-infected target cells, the results were considered positive if the % GzB activity after background subtraction was >15%, with no false positive results in 28 samples from seronegative donors.

Evaluation of effector cell populations

In our preliminary experiments, we established that 10,000 target cells/well allowed for acquisition of >2,500 events in the gated viable target population. Moreover, we have previously reported that using an Effector-to-Target ratio (E:T) of 30:1 allowed for sensitive detection of ADCC responses in HIV-1 infected subjects, and AIDS vaccine recipients when fresh PBMC samples were used as effector cells (14,27). In this study, we compared the detection of ADCC-mediating Ab when either fresh or cryopreserved effector cells were used. For the HIV-1 seropositive sample, we observed similar levels of maximal %GzB activity (48% vs 44%) and ADCC Ab titer (90,365-1 vs 98,054-1) using both fresh and cryopreserved PBMC, respectively, collected from the same donor (Figure 2A). In addition, cubic curves of %GzB activity versus log10 (dilution) were fitted to fresh and cryopreserved PBMC effector cells respectively. The F-test comparing the cubic model between the two assays did not suggest any significant difference (p=0.478). We did not detect ADCC activity in the HIV-1 seronegative sample (Figure 2A).

We further compared the performance of the cryopreserved PBMC and NK cells obtained from the same donor as effector populations in our assays using both gp120-coated (Figure 2B) and A1953 target cells (Figure 2C). The NK cells were not pre-activated with any cytokines before utilization as effector cells in the assay. Similar results were observed when the two effector population were used at the E:T ratio of 30:1 and 10:1 for PBMC and NK cells respectively.

Lastly, we evaluated separately the contribution of the CD56+ and CD16+ NK populations to the total effector activity of the whole PBMC population. We depleted overnight rested cryopreserved PBMC of the two populations of interest as described in the methods section, and reported the results obtained with gp120-coated (Figure 2D) and A1953 target cells (Figure 2E). Depletion of the CD56+ or CD16+ NK effector cells reduced the ability to detect HIV-1-specific ADCC responses by >66%, consistent with the NK subsets being responsible for the majority of the effector activity detected in our assay. The contribution of the NK population to the results obtained in this assay is also supported by comparing of the level of ADCC activity detectable for the HIVIG IgG preparation when using effector cells obtained from donors with different percentages of NK cells. As shown in the Supplemental Figure 1, there is a significant direct correlation between the level of detectable ADCC reactivity at each dilution and the frequency of NK cells (Spearman correlation coefficient analysis: range 0.9-1.0 and p values always 0.08-0.017).

Target cell platforms

Our results indicated that the gp120-coated CEM.NKRCCR5 and A1953 chronically infected cell lines could be used as universal and reliable target cell platforms to detect ADCC responses. We wanted to determine if the results obtained with these platforms could be recapitulated when IMCBaL-infected CEM.NKRCCR5 or primary CD4+ T cells were used as target cells. The CD4+ T cells were used following three days of PBMC activation and subsequent depletion of the CD8+ T cells. The effector cells used in this experiment were NK cells obtained from the autologous donor and used at an E:T of 10:1 against both the CEM.NKRCCR5 and CD4+ T cell target cells. The ADCC-mediating Ab could be detected when IMCBaL-infected CEM.NKRCCR5 or primary CD4+ T cells were used as target cells (Figure 3); however the maximum %GzB activity was slightly lower when IMCBaL infected CD4+ T cells were used as targets. As expected, no HIV-1-specific ADCC-responses were detected with the RSV-specific IgG Ab Synagis®, regardless of the target cell platform. We have tested in both CEM.NKRCCR5 cells and primary CD4+ T cells additional IMCs representing multiple isolates of HIV-1 currently used in the neutralizing Ab assay and determined that a minimum infection level of 20% of p24+ target cells can be achieved.

Figure 3
Comparison of CEM.NKRCCR5 and CD4+ T cell target cells

Assay Reproducibility

We investigated the reproducibility of data obtained with gp120-coated CEM.NKRCCR5 cells and the chronically infected A1953 cell line. We measured the ADCC activity in a seropositive and a seronegative sample on four consecutive assays against gp120-coated CEM.NKRCCR5 target cells at five different dilution levels (Figure 4A). The percentage coefficient of variation (%CV) for positive results was between 7.6 and 22.9%. We did not detect a positive response using the seronegative samples at any dilution. We further investigated the reproducibility of the results using A1953 chronically infected cell line. The same samples from a seropositive and a seronegative individual were measured in triplicates on two consecutive days at six different dilution levels. We observed a %CV for each positive results ranging between 6.7 and 11.4% (Figure 4B). Once again, no positive responses were detected in the seronegative sample. Using NFL1 we were able to eliminate target cells that were dead before incubation with the effector cells from subsequent analysis; this likely contributes to our ability to obtain these levels of reproducibility.

Figure 4
Assay reproducibility

Sensitivity of the assay and dynamic range of responses

The experiments used to establish the criteria for positive results indicated that a minimum serum/plasma dilution of 1:50 (human samples) and 1:100 (nonhuman primate samples) should be used, because at lower dilutions, the incidence of false positives is dramatically increased (data not reported).

Based on the criteria established for the minimum dilution and the cut-off for positive responses, we analyzed 14 sera from HIV-1 subtype C- and 6 from subtype E-infected individuals and geographically matched control sera. In Figure 5A, we report the data obtained from the subtype C-infected individuals and control tested against the subtype C CAP45.2 gp120-coated target cells. As shown a wide range of responses is detected with average of the maximum %GzB activity of 30.4% ranging from 19.9 and 43.2% at the 1:6,250 dilution. It should be noted that the previously described prozone phenomena, whereby high concentrations of Ab inhibit ADCC (2,28), is apparent, and commonly observed, at low antibody dilutions. The specificity of our assay is supported by the absence of detectable responses in any of the non-infected donors. Similar results were observed when the subtype E CM243 gp120-coated target cells were used to detect responses among the subtype E-infected individuals (Figure 5B). These data demonstrate our ability to specifically detect a dynamic range of results using this assay platform. More importantly, the same specificity and dynamic range of responses could also be observed when the IMCCM235 infected CEM.NKRCCR5 are used as target cells (Figure 5C). However, a lower level of responses above the cut-off was detected in some samples against IMC-infected target cells when compared to that observed with the gp120-coated cells.

Figure 5
Dynamic range of HIV-1 specific responses

The ADCC-GTL assay can also be used to detect ADCC responses in samples collected from immunized and SIV-infected Rhesus macaques. An example of these results is reported in Figure 5D. All of the samples collected from SIVE660-infected animals tested positive for the presence of ADCC-mediating Ab at 24 weeks post-infection against E660-gp120-coated CEM.NKRCCR5 target cells. The peak of the reactivity was detected between 1:1,600 and 1:25,600 dilutions with %GzB activities ranging from 9.5 to 44.4%. We did not detect ADCC-mediating Ab responses above the positive cut-off in any of the 10 samples collected form the non-infected animals.

Discussion

Traditional methods of measuring ADCC (51Cr-release assays, and the modernized equivalent, Rapid Fluorometric ADCC) (reviewed in (29)) have provided semiquantitative analysis, i.e., population as opposed to single cell readouts, with limited capacity for multiple parameter assessment and large-scale measurement of the ADCC-dependent killing of virus-infected target cells. More recent analytic techniques have focused on events that take place in the effector rather than in the target cells (30,31). Additionally, the standard methods used to introduce HIV Ags to target cells, including pulsing with select panels of whole proteins (2,7,32,33) or overlapping HIV peptides (34,35) limit our understanding of the full breadth of ADCC activity that may be mediated by Abs recognizing antigens expressed on the surface of infected cells.

To fulfill the scope of performing a quantitative, sensitive, and reproducible measurement of the level of ADCC-mediating Ab responses, we adapted a previously described procedure to our needs (24). The assay is performed in a 96well/plate format that allows both staining and incubation of the samples without transferring of samples for final acquisition. The assay is based on the detection of GzB activity derived from effectors within individual target cells. In our assay platform, active GzB is identified by its ability to cleave a selective peptide-linked fluorogenic substrate containing a GzB recognition motif. Fluorescence is conferred upon hydrolysis of the substrate, thereby producing a signal that can be used to directly identify individual cells targeted by the cytotoxic effector cells. Enumeration of these cells by high-throughput flow cytometry provides a rapid and quantitative measure of ADCC responses. Importantly, the presence of active GzB within target cells is one of the earliest discernable indications that a cytotoxic hit has been delivered by an effector cell (24). Therefore, the assay can measure the presence of ADCC Ab within 1 hour from the time that effector and target populations are incubated in the presence of plasma/serum samples. The length of incubation time in this assay (1 hour) can thus far compensate for the length of acquisition time for the number of plates that a single operator is able to handle at once (4.5 hour acquisition time for 3 plates). There is currently an ongoing effort to develop reagents that can be used in association with a fixation procedure to allow more flexibility for time of acquisition from assay end.

We decided to use a cut-off value to define positive results and not to use values commonly reported in the neutralizing Ab field such as 50% and 80% maximal activity. The reason for this choice is related to the fact that ADCC titration curves are non-linear, unlike most neutralizing antibody titration curves. Therefore, 50% or 80% maximal titer would fall within a non-linear region of the curve and/or below a positive response. We have tracked the robustness of these cut-off values, and have not observed any instance where adjustment was needed for a given combination of effector cells and target cells. Our experience does not exclude the possibility that results from this assay based on data sets including larger numbers of HIV-1 seronegative or placebo recipients may provide statistical power that will lower these thresholds to increase the sensitivity of the assay.

We have demonstrated that this assay has the requisite sensitivity to detect ADCC responses using cryopreserved samples as source of effector cells. This will allow investigators to rely on large size samples as source of effectors, such as those collected by leukopheresis, that can be cryopreserved in liquid nitrogen and used as needed. The potency and titer of ADCC mediating-Abs in a human or NHP plasma/sera sample can be determined using less than 10μl of sample, and a minimum of 2.1×106 cryopreserved PBMC. Our data indicate that the majority of the cytotoxic activity (over 66%) detected in our assay is related to the presence of NK cells. Importantly, we have also demonstrated that the frequency of NK cells in the effector cell preparation can dramatically influence the results obtained (Supplemental Figure 1). Given the relationship between percentage of NK cells in donor PBMC and ADCC activity, we recommend screening potential PBMC donors for those containing the most (10%-20% of total lymphocytes) NK cells.

One of the critical considerations when detecting ADCC-mediating Ab responses, is which target cells could provide us with useful information to determine immunogenicity and protective quality of antigens detected by the immune system following infection and/or administration of vaccine candidates. We report that this assay can be used to test samples against the traditional gp120-coated target cells, and with the new platforms that include chronically infected, and IMC-infected CEM.NKRCCR5 cell line. Moreover, we also report that HIV-1 infected primary CD4+ T cells are a suitable target population. We found that the sensitivity and dynamic range of the assay differed slightly among these target cell platforms. This is likely due to variations in the overall levels of antigens present on the surface of the target cells, and to the particular antigenic epitopes available (CD4 binding site, CD4 inducible, gp120 monomers/trimers). We are currently testing these target cell platforms with samples collected from different vaccine clinical trials, and panels of HIV-specific monoclonal Abs, to determine the antigen specificity of the detectable Ab responses, and their relevance to the field.

In summary, we report on a sensitive and reproducible ADCC assay amenable to high-throughput analysis of human or NHP samples. This assay provides researchers with a novel method for rapid quantitative analysis of the ADCC-mediating capabilities of Ab generated in response to HIV or SIV infection and vaccination. Further use and development of this assay platform is likely to increase our understanding of the role of ADCC-mediating Abs in HIV or SIV disease progression, and in prevention from infection.

Supplementary Material

Supp Figure S1

Acknowledgments

Grant Sponsor:

This work was made possible with the support from the Collaboration for AIDS Vaccine Discovery (CAVD) as funded by the Bill and Melinda Gates Foundation (grant # 38619) and from 5R01AI050483-09. Additional support came from the Center for HIV/AIDS Vaccine Immunology (A1067854-03) and from the Duke University Center for AIDS Research (CFAR), a NIH funded program (P30 AI 64518).

References

1. Hashimoto G, Wright PF, Karzon DT. Antibody-dependent cell-mediated cytotoxicity against influenza virus-infected cells. J Infect Dis. 1983;148(5):785–94. [PubMed]
2. Lyerly HK, Matthews TJ, Langlois AJ, Bolognesi DP, Weinhold KJ. Human T-cell lymphotropic virus IIIB glycoprotein (gp120) bound to determinants on normal lymphocytes and expressed by infected cells serves as targets for immune attack. Proc. Natl. Acad. Sci. U.S.A. 1987;84:3797–3801. [PubMed]
3. Kohl S, Charlebois ED, Sigouroudinia M, Goldbeck C, Hartog K, Sekulovich RE, Langenberg AG, Burke RL. Limited antibody-dependent cellular cytotoxicity antibody response induced by a herpes simplex virus type 2 subunit vaccine. J Infect Dis. 2000;181(1):335–9. [PubMed]
4. Lanier LL, Le AM, Civin CI, Loken MR, Phillips JH. The relationship of CD16 (Leu-11) and Leu-19 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic T lymphocytes. J Immunol. 1986;136(12):4480–6. [PubMed]
5. Cooper MA, Fehniger TA, Turner SC, Chen KS, Ghaheri BA, Ghayur T, Carson WE, Caligiuri MA. Human natural killer cells: a unique innate immunoregulatory role for the CD56(bright) subset. Blood. 2001;97(10):3146–51. [PubMed]
6. Jacobs R, Hintzen G, Kemper A, Beul K, Kempf S, Behrens G, Sykora KW, Schmidt RE. CD56bright cells differ in their KIR repertoire and cytotoxic features from CD56dim NK cells. Eur J Immunol. 2001;31(10):3121–7. [PubMed]
7. Ljunggren K, Moschese V, Broliden PA, Giaquinto C, Quinti I, Fenyö EM, Wahren B, Rossi P, Jondal M. Antibodies mediating cellular cytotoxicity and neutralization correlate with a better clinical stage in children born to human immunodeficiency virus-infected mothers. J Infect Dis. 1990;161(2):198–202. [PubMed]
8. Nag P, Kim J, Sapiega V, Landay AL, Bremer JW, Mestecky J, Reichelderfer P, Kovacs A, Cohn J, Weiser B. Women with cervicovaginal antibody-dependent cell-mediated cytotoxicity have lower genital HIV-1 RNA loads. J Infect Dis. 2004;190(11):1970–8. [PMC free article] [PubMed]
9. Gómez-Román VR, Patterson LJ, Venzon D, Liewehr D, Aldrich K, Florese R, Robert-Guroff M. Vaccine-elicited antibodies mediate antibody-dependent cellular cytotoxicity correlated with significantly reduced acute viremia in rhesus macaques challenged with SIVmac251. J Immunol. 2005;174(4):2185–9. [PubMed]
10. Hessell AJ, Rakasz EG, Poignard P, Hangartner L, Landucci G, Forthal DN, Koff WC, Watkins DI, Burton DR. Broadly neutralizing human anti-HIV antibody 2G12 is effective in protection against mucosal SHIV challenge even at low serum neutralizing titers. PLoS Pathog. 2009;5(5):e1000433. [PMC free article] [PubMed]
11. Hessell AJ, Hangartner L, Hunter M, Havenith CEG, Beurskens FJ, Bakker JM, Lanigan CMS, Landucci G, Forthal DN, Parren PWHI. Fc receptor but not complement binding is important in antibody protection against HIV. Nature. 2007;449(7158):101–4. [PubMed]
12. Hessell A, Rakasz E, Tehrani D, Huber M, Weisgrau K, Landucci G, Forthal D, Koff W, Poignard P, Watkins D. Broadly Neutralizing Monoclonal Antibodies 2F5 and 4E10, Directed Against the Human Immunodeficiency Virus Type 1 (HIV-1) gp41 Membrane Proximal External Region (MPER), Protect Against SHIVBa-L Mucosal Challenge. J Virol. 2010;84(3):1302–1113. [PMC free article] [PubMed]
13. Baum LL, Cassutt KJ, Knigge K, Khattri R, Margolick J, Rinaldo C, Kleeberger CA, Nishanian P, Henrard DR, Phair J. HIV-1 gp120-specific antibody-dependent cell-mediated cytotoxicity correlates with rate of disease progression. J Immunol. 1996;157(5):2168–73. [PubMed]
14. Lambotte O, Ferrari G, Moog C, Yates NL, Liao H-X, Parks RJ, Hicks CB, Owzar K, Tomaras GD, Montefiori DC. Heterogeneous neutralizing antibody and antibody-dependent cell cytotoxicity responses in HIV-1 elite controllers. AIDS. 2009;23(8):897–906. [PMC free article] [PubMed]
15. Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, Kaewkungwal J, Chiu J, Paris R, Premsri N, Namwat C, de Souza M, Adams E. Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N Engl J Med. 2009;361(23):2209–20. [PubMed]
16. Cummins LM, Weinhold KJ, Matthews TJ, Langlois AJ, Perno CF, Condie RM, Allain JP. Preparation and characterization of an intravenous solution of IgG from human immunodeficiency virus-seropositive donors. Blood. 1991;77(5):1111–7. [PubMed]
17. Trkola A, Matthews J, Gordon C, Ketas T, Moore JP. A cell line-based neutralization assay for primary human immunodeficiency virus type 1 isolates that use either the CCR5 or the CXCR4 coreceptor. J Virol. 1999;73(11):8966–74. [PMC free article] [PubMed]
18. Perfetto SP, Ambrozak D, Nguyen R, Chattopadhyay P, Roederer M. Quality assurance for polychromatic flow cytometry. Nat Protoc. 2006;1(3):1522–30. [PubMed]
19. Edmonds TG, Ding H, Yuan X, Wei Q, Smith KS, Conway JA, Wieczorek L, Brown B, Polonis V. West JT and others. Replication competent molecular clones of HIV-1 expressing Renilla luciferase facilitate the analysis of antibody inhibition in PBMC. Virology. 2010;408(1):1–13. [PMC free article] [PubMed]
20. Adachi A, Gendelman HE, Koenig S, Folks T, Willey R, Rabson A, Martin MA. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol. 1986;59(2):284–91. [PMC free article] [PubMed]
21. O'Doherty U, Swiggard WJ, Malim MH. Human immunodeficiency virus type 1 spinoculation enhances infection through virus binding. J Virol. 2000;74(21):10074–80. [PMC free article] [PubMed]
22. Freel SA, Lamoreaux L, Chattopadhyay PK, Saunders K, Zarkowsky D, Overman RG, Ochsenbauer C, Edmonds TG, Kappes JC, Cunningham CK. Phenotypic and Functional Profile of HIV-inhibitory CD8 T cells Elicited by Natural Infection and Heterologous Prime/boost Vaccination. J Virol. 2010;84(10):4998–5006. [PMC free article] [PubMed]
23. Liu L, Chahroudi A, Silvestri G, Wernett ME, Kaiser WJ, Safrit JT, Komoriya A, Altman JD, Packard BZ, Feinberg MB. Visualization and quantification of T cell-mediated cytotoxicity using cell-permeable fluorogenic caspase substrates. Nat Med. 2002;8(2):185–9. [PubMed]
24. Packard BZ, Telford WG, Komoriya A, Henkart PA. Granzyme B activity in target cells detects attack by cytotoxic lymphocytes. J Immunol. 2007;179(6):3812–20. [PubMed]
25. Clopper C, Pearson ES. The use of confidence of fiducial limits illustrated in the case of bnomial. Biometrika. 1934;26:404–413.
26. Chambers JM. Linear models. In: Chanders JM, Hastie TJ, editors. Statistical Models in S. Chapman & Hall/CRC; New York: 1992. pp. 95–144.
27. Goepfert PA, Tomaras GD, Horton H, Montefiori D, Ferrari G, Deers M, Voss G, Koutsoukos M, Pedneault L, Vandepapeliere P. Durable HIV-1 antibody and T-cell responses elicited by an adjuvanted multi-protein recombinant vaccine in uninfected human volunteers. Vaccine. 2007;25(3):510–8. [PubMed]
28. Tyler DS, Stanley SD, Zolla-Pazner S, Gorny MK, Shadduck PP, Langlois AJ, Matthews TJ, Bolognesi DP, Palker TJ, Weinhold KJ. Identification of sites within gp41 that serve as targets for antibody-dependent cellular cytotoxicity by using human monoclonal antibodies. J. Immunol. 1990;145(10):3276–3282. [PubMed]
29. Chung A, Rollman E, Johansson S, Kent SJ, Stratov I. The utility of ADCC responses in HIV infection. Curr HIV Res. 2008;6(6):515–9. [PubMed]
30. Chung AW, Rollman E, Center RJ, Kent SJ, Stratov I. Rapid degranulation of NK cells following activation by HIV-specific antibodies. J Immunol. 2009;182(2):1202–10. [PubMed]
31. Liu Q, Sun Y, Rihn S, Nolting A, Tsoukas PN, Jost S, Cohen K, Walker B, Alter G. Matrix metalloprotease inhibitors restore impaired NK cell-mediated antibody-dependent cellular cytotoxicity in human immunodeficiency virus type 1 infection. J Virol. 2009;83(17):8705–12. [PMC free article] [PubMed]
32. Broliden K, Von Gegerfelt A, Persson C, Horal P, Svennerholm B, Wahren B, Bjorling E, Vahlne A. Identification of cross-reactive antigenic target regions for HIV type 1-specific antibody-dependent cellular cytotoxicity. AIDS Res Hum Retroviruses. 1996;12(18):1699–702. [PubMed]
33. Baum LL, Cassutt KJ, Knigge K, Khattri R, Margolick J, Rinaldo C, Kleeberger CA, Nishanian P, Henrard DR, Phair J. HIV-1 gp120-specific Antibody-Dependent Cell-Mediated cytotoxicity correlates with rate of disease progression. J. Immunol. 1996;157:2168–2173. [PubMed]
34. Stratov I, Chung A, Kent SJ. Robust NK Cell-Mediated Human Immunodeficiency Virus (HIV)-Specific Antibody-Dependent Responses in HIV-Infected Subjects. J Virol. 2008;82(11):5450–5459. [PMC free article] [PubMed]
35. Tiemessen CT, Shalekoff S, Meddows-Taylor S, Schramm DB, Papathanasopoulos MA, Gray GE, Sherman GG, Coovadia AH, Kuhn L. Cutting Edge: Unusual NK cell responses to HIV-1 peptides are associated with protection against maternal-infant transmission of HIV-1. J Immunol. 2009;182(10):5914–8. [PMC free article] [PubMed]