Journal of Immunological Methods 308 (2006) 53 67 www.elsevier.

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Research paper

A simplified method for the rapid fluorometric assessment of antibody-dependent cell-mediated cytotoxicity
V. Rau l Go mez-Roma n a, Ruth H. Florese a, L. Jean Patterson a, Bo Peng a, David Venzon b, Kris Aldrich a, Marjorie Robert-Guroff a,*
a b

Vaccine Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 41 Medlars Drive, Room D804, Bethesda, MD 20892-5065, United States Biostatistics and Data Management Section, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, United States Received 3 June 2004; received in revised form 27 July 2005; accepted 15 September 2005 Available online 28 November 2005

Abstract We demonstrate that the FATAL cytolysis assay can be adapted into a rapid and fluorometric antibody-dependent cellular cytotoxicity assay (RFADCC). The RFADCC relies on double-staining target cells with a membrane dye (PKH-26) and a viability dye (CFSE) prior to the addition of antibody and effector cells. We used the RFADCC to assess dose-dependent and envelopespecific anti-human immunodeficiency virus (HIV) ADCC responses mediated by monoclonal antibody-2G12 and human sera. Using the assay, we also detected early anti-simian immunodeficiency virus (SIV) ADCC responses in rhesus macaques infected with pathogenic SIVmac251. Importantly, the RFADCC was further useful in monitoring anti-HIV and anti-SIV ADCC responses elicited by immunizing chimpanzees and rhesus macaques with replicating adenovirus-based AIDS vaccine candidates. In comparison to the standard chromium release assay, the RFADCC provides a higher cell killing readout and is advantageous in allowing use of viably frozen as well as fresh effector cells, thus facilitating assay standardization. The RFADCC is therefore a simple, reliable, and highly sensitive method that can be applied to assess the ADCC activity of monoclonal antibodies as well as ADCC responses elicited by HIV or SIV infection or by AIDS vaccine candidates. D 2005 Elsevier B.V. All rights reserved.
Keywords: ADCC; FATAL; HIV vaccine; Flow cytometry; PKH-26; CFSE

1. Introduction Antibodies can eliminate virus-infected cells via antibody-dependent cellular cytotoxicity (ADCC), a mechanism requiring antibody, antigen-coated target cells and Fcg-receptor-bearing effectors such as NK cells, gy T cells, neutrophils and macrophages. An in vivo protective role of ADCC has been well established in experimental mouse models of lethal infection with herpes simplex virus (Kohl, 1991). Serum ADCC responses against several other viruses, including influenza virus (Hashimoto et al., 1983) and the human and

Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; CRA, chromium release assay; RFADCC, rapid and fluorometric antibody-dependent cellular cytotoxicity assay; HIV, human immunodeficiency virus; SIV, simian immunodeficiency virus; Ad5hr, adenovirus type 5 host range mutant; PBMC, peripheral blood mononuclear cells; CFSE, 5-(and 6-)-carboxyfluorescein diacetate succinimidyl ester. * Corresponding author. Tel.: +1 301 496 2114; fax: +1 301 496 8394. E-mail address: guroffm@mail.nih.gov (M. Robert-Guroff). 0022-1759/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2005.09.018

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simian immunodeficiency viruses (HIV and SIV), have also been described and may be relevant to the control of these viral infections in vivo (Ahmad and Menezes, 1996; Brenner et al., 1991; Tyler et al., 1989). In HIVinfected humans and in SIV-infected rhesus macaques, for example, ADCC responses have been associated with a better clinical outcome and lower viral loads (Ahmad et al., 2001; Forthal et al., 2001; Banks et al., 2002). Recognizing the importance of ADCC responses in host defense, the World Health Organization has acknowledged elicitation of ADCC as one of the goals of vaccination against viral diseases in man (WHO, 1983). Consistent with this recommendation, there is a need for a convenient and reliable method to screen for ADCC responses in assessing the immunogenicity of viral vaccine candidates during clinical and preclinical trials. Traditionally, ADCC responses against viral pathogens have been assessed using cytolytic assays in which target cells are labeled with radioactive chromium isotopes (51Cr) prior to the addition of antibody and effector cells. Radioactive labeling of target cells in these standard chromium-release assays, or CRAs, however, may often have disadvantages including difficulty with labeling certain cell types, low assay sensitivity and high spontaneous chromium release resulting in high background values (Slezak and Horan, 1989; Volgmann et al., 1989; Kruger-Krasagakes et al., 1992; Oumouna et al., 2001). In addition, complying with the increasing regulations on use and disposal of radioisotopes, together with dependence on a radioactive material delivery schedule, can make CRAs rather cumbersome and unappealing for screening ADCC responses during large vaccine trials, particularly in developing countries. An inexpensive and non-radiometric ADCC assay would be more appropriate for this purpose. Several flow cytometry-based alternatives have been designed to circumvent the problems associated with radioactive labeling of target cells in cytotoxicity assays (Slezak and Horan, 1989; Volgmann et al., 1989; Kruger-Krasagakes et al., 1992; Oumouna et al., 2001; Sheehy et al., 2001; Wilkinson et al., 2001). Among these, the FATAL assay, originally designed to test T cell responses (Sheehy et al., 2001), relies on doublelabeling target cells with a lipid membrane associated dye (PKH-26) and a viability dye (CFSE, or 5-(and-6-)carboxyfluorescein diacetate succinimidyl ester). In the FATAL assay, effectors remain unlabeled and the killing of targets is calculated from the fraction of membrane-labeled cells that lose the viability dye during cytolysis (i.e. CFSE-negative cells within the PKH-26

positive gate). Aside from obviating the use of radioisotopes, the main advantage of the FATAL assay over the standard CRA is reduced background values resulting in higher sensitivity and detection of low levels of cytotoxic T lymphocyte (CTL)-mediated lysis. Here we show that dual labeling with PKH-26 and CFSE can be applied to effectively assess ADCC. Using 2G12, a monoclonal antibody (mab) with known ADCC activity against HIV (Trkola et al., 1996), we first illustrate the ease and applicability of rapid and fluorometric antibody-dependent cellular cytotoxicity (RFADCC) in assessing antibody-mediated killing of HIV envelope-coated target cells. We then show that the RFADCC can be applied to assess antiHIV ADCC activity mediated by HIV-positive patient sera and anti-SIV ADCC responses during acute infection of rhesus monkeys with pathogenic SIV. Both antiHIV and anti-SIV ADCC responses were also detected during the course of immunization with adenovirusbased AIDS vaccine candidates. The RFADCC is shown to be simple, reliable, and more sensitive than the standard chromium release assay (CRA). Further we show that the RFADCC is a useful procedure for assessing the ADCC titer of mabs and for measuring the induction of ADCC activity by viral infection or candidate AIDS vaccines during clinical and preclinical trials. 2. Materials and methods 2.1. Antibodies, sera and animals HIV-1 gp120 monoclonal antibody 2G12 was obtained through the AIDS Research and Reference Program, NIAID, NIH. Human IgG1 (Ancell Immunology Research Products, Bayport, MN, USA), obtained from human myeloma plasma, was used as an isotype control for the mab2G12 ADDC experiments. Two samples of patient V HIV-positive human sera, designated as WG393 and WG395 and obtained 20 months apart, were negative for neutralizing antibodies against HIV, positive for reactivity against HIV-1 envelope by ELISA, and positive against HIV-1 gp41 and gp120 by Western blot (Robert-Guroff et al., 1987). Patient W0881 HIV-positive human serum possessed HIV neutralizing activity as well as anti-envelope binding antibodies (MRG, unpublished data). A preparation of HIV immune globulin (HIVIG) with maximal ADCC activity at a dilution of 1/2000 was generously provided by Dr. Kent Weinhold, Duke University. Pooled normal human serum, obtained from clotted whole blood of HIV-negative human males and normal human IgG

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(SIGMA, St. Louis, MO, USA), were used as negative ADCC controls. For experiments involving SIV-infected monkeys, we used mock-immunized control rhesus macaques (Macacca mulatta ) from a retrospective vaccine study (Patterson et al., 2004). The macaques were infected intrarectally with ten macaque-infectious doses of pathogenic SIVmac251. All macaques became infected and highly viremic. Sera were obtained from clotted blood on days 0 and 28 post-infection. For experiments involving vaccine-elicited ADCC activity, retrospective sera from sixteen rhesus macaques were obtained before (day 0) and after (day 98) two immunizations with 5 108 pfu of replicationcompetent adenovirus type 5 host range mutant (Ad5hr)-SIVenv/rev recombinant (Patterson et al., 2003, 2004). Retrospective sera obtained on days 0 and 98 from eight control macaques mock-immunized with the Ad5hr-empty vector were used as negative controls. ADCC was also assessed following vaccination with replication-competent Ad-HIV recombinant vaccines. Sera from two chimpanzees (Pan troglodytes ) were obtained before and 2 weeks after two intranasal immunizations, the first (day 0) with 1 108 pfu Ad5-HIVMNenv/rev recombinant and the second (day 91) with 1 108 pfu Ad7-HIVMNenv/rev. (Peng et al., 2005). All sera from humans and nonhuman primates were stored at 70 8C. Upon thawing, all sera were diluted tenfold in R-10, passed through a 0.2-Am pore syringe filter, heat-inactivated for 1 h at 56 8C, stored at 4 8C and used within a month at the indicated dilutions. 2.2. Effector cells Human peripheral blood mononuclear cells (PBMCs) were used as effector cells. The PBMCs were isolated by Ficoll-Hypaque density centrifugation of buffy coats or leukopheresis preparations obtained from healthy, HIV-seronegative volunteerdonors (Department of Transfusion Medicine, Clinical Center, NIH) and stored viably frozen until use. In some cases, freshly obtained effector cells were used. The human effector cells, including the leukopheresis samples and the buffy coats, a discardable by-product of another type of donation, were irreversibly anonymized before distribution and are exempt from Institutional Review Board review as determined by the Office of Human Subjects Research, CC, NIH. For the anti-SIVADCC experiments, we avoided using monkey PBMCs as effector cells due to their strong NK activity and their capacity to spontaneously kill SIV-infected human tar-

get cells in the absence of antibody (Vowels et al., 1990; Go mez-Roma n et al., 2005). 2.3. Preparation of target cells Target cells varied according to each experiment. To test for HIV or SIV envelope-specific ADCC activity, target cells were prepared by incubating 5 106 CEMNKr (NK-resistant) cells (AIDS Research and Reference Reagent Program, NIAID, NIH) with 15 Ag native, monomeric gp120 derived from HIVIIIB, HIVBal or SIVmac251 (Advanced Bioscience Laboratories, Inc., (ABL), Kensington, MD) for 1 h at room temperature in a total volume of 300 Al of R-10, followed by washing twice in ice-cold R-10. Control targets were non-coated CEM-NKr cells. Saturation (N 97%) of both HIVIIIB- and HIVBal-gp120-coated targets was verified by staining targets with human mab2G12 (1 Ag/ml), followed by staining with a 1/100 dilution of fluorescein-conjugated goat anti-human IgG (Biosource, Camarillo, CA). To test for ADCC activity against SIV-infected cells, chronically SIVmac251-infected H9 T cell targets were prepared by infecting H9 cells with cell-free SIVmac251 (ABL) and culturing in R-10 with twiceweekly passage and addition of fresh, uninfected H9 cells once per week. The cells were monitored weekly for SIV expression using indirect immunofluorescent assays on fixed cells with a mouse anti-SIVmac251 p27 monoclonal antibody (#13-113-100; Advanced Biotechnologies, Inc. (ABI), Columbia, MD) and on live cells using a mouse anti-SIV gp120 monoclonal antibody (#13-115-100; ABI). The secondary antibody was a fluorescein-conjugated goat anti-mouse F(ab) V2 (Biosource, Camarillo, CA). Both assays established that equivalent numbers of SIV Gag- and Env-expressing cells were present in chronically SIVmac251-infected H9 cultures. The cells were routinely monitored for SIV Gag expression and used in the RFADCC assay within 24 h of confirming an infected cell frequency of 90% or higher. 2.4. Rapid fluorometric ADCC assay (RFADCC) Based on methods adapted from the FATAL assay (Sheehy et al., 2001), one million target cells were double-stained with 2.5 10 6 M PKH-26 (Sigma, St. Louis, MO, USA) and 2.5 10 6M CFSE (5-(and 6-)-carboxyfluorescein diacetate, succinimidyl ester; Molecular Probes, Eugene, OR, USA). After staining, viral-infected targets were washed and used directly, while gp120-coated targets were prepared as described

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above. The final target cell preparations were resuspended in R-10 at a cell density of 1 105 cells/ml and 50 Al (5000 cells) were dispensed into each well of a microtiter plate. 100 Al of diluted sera or mabs were added to duplicate wells and the resulting target cells + antibody mixture was incubated for 15 min at room temperature in order to allow the antibody to bind to the antigens on the surface of the target cells. 50 Al of effector cells were then added to each well at E /T ratios of 50:1 or 10:1, as indicated. Plates were centrifuged for 3 min at 400g to promote cell-to-cell interactions and were then incubated for 4 h at 37 8C to allow ADCC to occur. Cells were washed once with PBS, fixed in 3.7% paraformaldehydePBS (v/v) and stored at 4 8C overnight. 2.5. Flow cytometry acquisition and analysis Ten thousand non-gated events were acquired within 24 h of the ADCC assay using a FACSCalibur instrument (Becton Dickinson, San Jose, CA, USA). Flow cytometry data was acquired using CellQuest Software, setting FL1 as the CFSE emission channel (530/25 nm filter) and FL2 as the PKH-26 emission channel (585/ 40 nm filter). Analysis and density plots were generated with WinMDI Version 2.8nJoseph Trotter. Percent killing was obtained by back-gating on the PKH-26high population of targets and is reported here as the percentage of membrane-labeled target cells having lost the viability dye, i.e. % CFSEnegative within PKH26high. Non-stained and single-stained targets were included in every experiment to compensate for singlestained CFSE and PKH-26 emissions. 2.6. Assessment of ADCC by CRA A 51Cr release assay (CRA) was carried out using one million CEM-NKr cells labeled with 200 ACi of 51 Cr (Na2 51CrO4 Perkin Elmer Life and Analytical Sciences, Boston, MA). Similar results were obtained in either a 5- or 6-h CRA when cells were labeled for 2 h at 37 8C, washed and coated as above with HIVIIIB gp120 for the RFADCC assay, or labeled for 90 min at 37 8C with simultaneous coating with gp120 as previously described (Lyerly et al., 1987). The cells were washed twice with cold R-10 before use in the assay. PBMCs from healthy HIV negative donors were used as effector cells. Dilutions of test and control sera or monoclonal antibodies (100 Al) were mixed with 5000 target cells and allowed to react for 30 min at room temperature. The effectors were subsequently added at a 50:1 E /T ratio, the mixtures were incubated for

5 h at 37 8C, and supernatants were harvested. The maximum release control was obtained by lysis of target cells with culture medium containing 1% Triton-X 100 and the spontaneous release control was obtained by reacting the target cells alone with R-10. All samples were run in duplicate and the control samples were run in triplicate. Percent cell lysis was calculated by the following formula: (experimental release spontaneous spontaneous release)/(maximum release spontaneous release) 100. 2.7. Statistics The Wilcoxon rank sum test was used to assess differences in ADCC activity between immunized animals and mock-immunized controls. The comparison between gp120-coated and uncoated cells was made as the fixed effect in a mixed model analysis of variance. The Wilcoxon signed rank test was used to assess differences before and after infection or immunizations. Mean percent ADCC killing F standard deviations (mean F S.D.) are reported. 3. Results 3.1. Assessment of anti-HIV ADCC activity by RFADCC The mechanism by which ADCC mediates killing of targets is illustrated in Fig. 1a. Initially, we used mab2G12, a humanized mab with well-documented ADCC activity against the HIV envelope (Trkola et al., 1996), to establish the RFADCC assay. We first illustrate the components of the method in the absence of antibody (Fig. 1be). Human PBMCs used as effectors were not labeled and were thus visualized in the lower left quadrant of the flow cytometry density plot (Fig. 1b). HIVIIIB gp120-coated CEM-NKr cells used as targets were double-labeled with PKH-26 and CFSE and were visualized in the upper right quadrant (Fig. 1c). Thus, when effectors and targets were mixed at a 50:1 ratio in the absence of antibody (Fig. 1d), both cell populations were easily distinguished from each other at the time of data acquisition. By back-gating on the PKHhigh population at the time of flow analysis, (Fig. 1e), it was evident that the majority of the targets were viable and only a small percentage had spontaneously lost the viability dye, becoming CFSEnegative. Using this RFADCC methodology, we added mab2G12 into the assay to assess its ADCC activity. A 4-h incubation of effectors and targets in the presence of 20 ng of mab2G12 resulted in 60.6% killing, shown

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antibody antigen

granules Fc receptors (CD16, CD32, CD64)

(a)

EFFECTOR CELL

TARGET CELL

(b)
Effectors (E)

(c)

(d)

(e)
high

5.7 %

94.3 %

PKH-26 CFSE
Fig. 1. Components of the ADCC response. (a) Upon recognition of antibodies bound to antigens on the surface of the virus-infected cell, crosslinking of the Fcg-receptors triggers the release of granules from effector cells, followed by killing. In the RFADCC assay, effectors remain unlabeled (b) and targets are dual-stained (c), so that both cell populations are distinguishable when mixed (d). By gating on the PKHhigh population (e), percent killing and viability of targets can be calculated. E /T = 50:1.

as a percentage of the PKH-26-labeled targets having lost the CFSE viability dye (Fig. 2a,c). A background killing of 7.1% was observed in the presence of 20 ng of an IgG1 isotype control (Fig. 2b,d). Dose-dependent ADCC activity was next demonstrated using the RFADCC and serial dilutions of mab2G12 (Fig. 3). While human donors can exhibit different effector cell activity and background nonspecific lysis of target cells, once good donors are identified, their killing of targets recognized by specific antibodies is very reproducible in the RFADCC assay. This is illustrated in Fig. 3 by the similar results obtained in two experiments using effector PBMCs from two different human donors. We defined a cutoff value for positive ADCC activity as greater than

the mean percent killing with the negative control plus three standard deviations. For this experiment, the mean background ADCC activity F the standard deviation over the same antibody concentration range using an IgG isotype control was 12.7 F 2.04% killing for Donor B3 and 13.05 F 0.31% killing for Donor B4. As shown, ADCC activity above the defined cutoff values of 18.8% and 14.0% for donors B3 and B4, respectively, were observed at mab2G12 concentrations of 1 to 10 ng/ml or higher. In comparison, an ADCC endpoint of 100 ng/ml was previously obtained for a titration of mab2G12 killing of similar gp120coated targets using a CRA (Trkola et al., 1996), suggesting that the sensitivity of the RFADCC is significantly higher.

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(a)

mab 2G12

(b) IgG1 control

non-gated

(c)
60.6

(d)
7.1

PKH-26 high gated PKH-26

CFSE
Fig. 2. HIV-envelope specific ADCC. CD4+CEM-NKr cells were coated with HIVIIIB gp120. A 4-h incubation in the presence of 20 ng of 2G12 (a,c) results in significant killing. Background killing is observed by incubating targets with 20 ng of an IgG1 isotype control (b,d). Numbers in the upper left quadrant of the lower panels indicate the percent killing calculated after PKHhigh gating. E /T = 50:1.

Dose-dependent ADCC activity was also observed for an HIV-positive human serum (Fig. 4) and was consistently higher than the background ADCC activity of pooled normal human sera used as a negative control. For this experiment, the mean background killing over the six serial dilutions of normal human sera was 14.4 F 3.54%. As previously defined, the cutoff value was therefore 25.02% for positive anti-HIV ADCC activity. Significant ADCC above this cutoff was observed for HIV-positive human serum V at dilutions ranging from 1/10 to 1/10,000 (from 47.1% to 40.8% killing). A prozone effect was also observed, with ADCC activity at a 1/10 dilution slightly lower than that observed at 1/100 and 1/1000 dilutions. The prozone effect is in agreement with previous reports describing anti-HIV ADCC activity in human sera using a CRA (Weinhold, 1990). 3.2. Comparison of RFADCC and CRA To pursue the indication from the 2G12 titration (Fig. 3) that the RFADCC is more sensitive than the standard CRA, the ADCC activity of 2G12 was evaluated using both methods. Identical target and effector cells were used at the same E /T ratio. Using the CRA, we were never able to obtain greater than 6% net lysis of HIVIIIB gp120-coated target cells (data not shown). Although low, this level of cytolysis is in line with previously published results in which no greater than 10% net lysis of similarly coated CEM-NKr target cells

was obtained over a range of 2G12 concentrations from 0.3 to 20 Ag/ml (Trkola et al., 1996). The slightly lower net percent lysis obtained here may be due to glycosylation differences in the HIVIIIB gp120 preparations used to coat the target cells. The 2G12 antibody recognizes epitopes located within carbohydrate residues on the surface of the envelope protein (Trkola et al., 1996). The previous results were obtained using baculovirusexpressed gp120, while the gp120 used here was purified from the supernatant of an HIVIIIB-infected human cell line (Kalyanaraman et al., 1990). In contrast to the results with the CRA, net percent cell lysis using the RFADCC method was much greater, peaking at 30% (data not shown). An ADCC prozone effect was evident, since ADCC activity at the highest 2G12 concentrations was somewhat lower than the peak activity level observed at 1 Ag/ml. We conducted further comparative evaluations of ADCC activity using patient V serum, reasoning that a polyclonal human serum, in comparison to the monoclonal 2G12 antibody, would be less susceptible to possible carbohydrate differences of the gp120 preparation used to coat target cells. As shown in Fig. 5a and b, greater sensitivity of the RFADCC method was confirmed. Patient V serum exhibited peak ADCC activity of 30% net lysis at a dilution of 1/1000 by RFADCC, while the highest net lysis by CRA was 12% (mean of two experiments) at the same serum dilution. Nevertheless, similar endpoint titers of 1/100,000 were achieved by both methods. No ADCC activity was observed with mock-coated target cells (data not shown). We further evaluated another HIV-positive human serum, W0881, with high titer anti-envelope binding and neutralizing activity, and a preparation of HIVIG using the same viably frozen effector cells (designated A) in both assays. Serum W0881 was evaluated using CEM-NKr target cells coated with HIVIIIB gp120, and the HIVIG using CEM-NKr coated with HIVBal gp120. Here, ADCC titers were higher using the RFADCC method in both cases, with a greater than 100-fold difference in titer seen for the W0881 serum (Fig. 5c,d) and a 16-fold difference for the HIVIG (Fig. 5e,f). Higher percent lysis values were also consistently observed using the RFADCC method compared to the CRA. In the literature, the standard CRA is conducted using freshly prepared effector cells; in these studies, we routinely used viably frozen effector cells. To evaluate the effect of cell freezing on the RFADCC and CRA methods, we obtained another sample of fresh effector cells (designated G) and conducted a comparative evaluation using HIVIG and CEM-NKr target

final concentration of mab 2G12 (ng/ml)

V.R. Go mez-Roma n et al. / Journal of Immunological Methods 308 (2006) 5367

Donor B3

Donor B4

PKH-26

59.7

50.3

32.2

20.8

14.9

10.8

mean ADCC activity

CFSE

(% CFSE-within PKH-26high gate)

Fig. 3. Reproducibility and dose-dependence of the RFADCC assay. CD4+CEM-NKr cells were coated with HIVIIIB gp120 and incubated with serial dilutions of mab-2G12. Effectors from two different donors mediated similar dose-dependent ADCC. Numbers in the upper left quadrants indicate the percent ADCC killing for each experiment. Numbers below the lower panels indicate the average of both experiments. E /T = 50:1.

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fold serum dilution 10 100 1,000 10,000 100,000 1,000,000 Patient V HIV+ Serum

Normal Human Serum PKH-26 CFSE

Fig. 4. Sensitivity and specificity of the RFADCC assay using human sera. CD4+CEM-NKr cells were coated with HIVIIIB gp120 and incubated with serial tenfold dilutions of HIV antibody positive serum from patient V (WG393) or pooled normal human control serum. E /T = 50:1.

(a) RFADCC
50 40
50 40 30

(c) RFADCC
60
P a tient serum W0881 Normal human serum

(e) RFADCC
50 40
HIVIG Control IgG

V.R. Go mez-Roma n et al. / Journal of Immunological Methods 308 (2006) 5367

ADCC (%CSFE high Within PKHhigh

30

Titer: 100,000
20 10 0 0.1 1 10 100 1,000
20 10
P a tient V serum Normal human serum

30

Titer: >10,000,000

20 10 0

Titer: 200,000

0.1

10

100

1,000 10,000

0.02

0.2

20

200

2,000

(b) CRA
16 14 12
P a tient V serum Normal human serum

(d) CRA
30 25 20 15
Patient serum W 0881 Normal human serum

(f) CRA
16 14 12 10 8 6
HIVIG Control IgG

% Lysis

10 8 6 4 2 0

Titer: 12,500

Titer: 100,000

10

Titer: 100,000
5 0

4 2 0

0.1

10

100

1,000

0.1

10

100

1,000 10,000

0.02

0.1

0.5

2.5

12.5

62.5

Reciprocal dilution X 10-3

Fig. 5. Comparative sensitivity of CRA and RFADCC in determining ADCC activity mediated by HIV antibody positive patient sera and HIVIG. ADCC activity of patient V (sample WG395) and patient W0881 sera were evaluated using CEM-NKr cells coated with HIVIIIB gp120. HIVIG was evaluated using CEM-NKr cells coated with HIVBal gp120. Donor effector cells (designated A) differed from those used in Fig. 4. The cutoffs for positive activity and determination of endpoint titer for the RFADCC method were 16.7%, 5.9% and 16.0% for panels a, c, and e, respectively, and for the CRA were 2.9%, 7.8%, and 7.5% for panels b, d, and f, respectively. E /T = 50:1.

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cells coated with HIVBal gp120. Using fresh effector cells, both the RFADCC and CRA methods resulted in the expected peak ADCC activity at a dilution of 1/ 2000 (see Materials and methods) and the same endpoint titer (1/20,000; Fig. 6a,b). However, when the same viably frozen effector cells (designated G) were used, peak ADCC activity measured by RFADCC was at the same level as with fresh cells (Fig. 6c), whereas in the CRA method peak ADCC activity was not distinguishable from the negative control (Fig. 6d). Again note the reproducibility of the RFADCC in evaluating the HIVIG preparation using two different viably frozen effectors (Figs. 5e and 6c). Similar endpoint titers were obtained. The A effector cells (Fig. 5e) gave higher peak cell lysis compared to the G effector cells (Fig. 6c) suggesting better recovery of NK activity upon cell thawing. As a result, ADCC activity was still measurable by CRA using the A effectors (Fig. 5f) but not using the G effectors (Fig. 6d).
Fresh Cells

3.3. Anti-SIV ADCC in the sera of SIV-infected macaques In establishing a method to reliably and easily measure ADCC, our main goal was to apply it to the characterization of humoral immune responses in clinical and preclinical AIDS vaccine trials. Antibodies with ADCC activity are among the first to be detectable in sera shortly after HIV or SIV infection, often preceding the appearance of neutralizing antibodies (Sawyer et al., 1991; von Gegerfelt et al., 1994; Ohkawa et al., 1995). We first addressed whether the RFADCC would detect any ADCC responses at all in rhesus macaque sera which were collected 28 days after a rectal inoculation with pathogenic SIVmac251. Fig. 7 shows that potent dose-dependent anti-SIV gp120 ADCC responses were observed in sera of all SIV-infected rhesus monkeys. Mean SIV-gp120specific ADCC activity at 1/10 serum dilution was
Frozen Cells

(a) RFADCC
35 30 25 35

(c) RFADCC
HIVIG Control IgG
30 25 20

HIVIG Control IgG

ADCC (%CFSEhigh within PKHhigh)

20

Titer: 20,000
15 10 5 0 0.02 0.2 2 20 200 2,000 15 10 5 0 0.02

Titer: 200,000

0.20

20

200

2,000

(b) CRA
14 12 10 14

(d) CRA
HIVIG Control IgG
12 10 8 6

HIVIG Control IgG

Titer: <20

% Lysis

8 6 4 2 0 0 .02 0.2 2 20 200 2,000

Titer: 20,000

4 2 0 0.02 0.2 2 20 200 2,000

Reciprocal dilution X 10-3

Fig. 6. Comparative evaluation of RFADCC and CRA using fresh and viably frozen effector cells. ADCC activity of HIVIG was evaluated using CEM-NKr cells coated with HIVBal gp120. Freshly prepared donor effector cells (designated G) or viably frozen effector G cells obtained on the same day were used. The cutoffs for positive activity and determination of endpoint titer for RFADCC were 19.2% and 10.9% for panels a and c, respectively, and for the CRA were 3.7% and 10.7% for panels b and d, respectively.

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60 50

63

ADCC

40

Rh8 Rh12 Rh13 Rh27 Rh44 Rh46

(% CFSEneg within PKHhigh) 30

20 10 0 10 100 1,000 10,000 100,000

Fold serum dilution

Fig. 7. Use of RFADCC to monitor anti-SIV ADCC responses in SIVinfected rhesus macaques. Rhesus macaques were inoculated intrarectally with ten macaque-infectious doses of pathogenic SIVmac251. On day 28 post-infection, dose-dependent serum ADCC responses were detected against gp120-coated CEM-NKr cells (solid symbols) but not against non-coated CEM-NKr cells (open symbols). Symbols represent the average ADCC value obtained from duplicate experiments. Bars denote the standard deviation from the mean. E /T = 50:1.

49.8 F 4.4% killing compared to 15.4 F 4.5% background activity against non-coated CEM-NKr targets (open symbols, P b 0.0001). Using the defined cutoff value of 18.68% killing, sera of all the SIV-infected macaques were positive over a range of dilutions from 1/10 to 1/10,000. All but two of the sera (Rh44 and Rh46) were positive at a dilution of 1/100,000 (Fig. 7). Note further, the absence of a prozone effect in these early-infection sera indicates that initial screening for

ADCC activity can be reliably carried out at 1/10 serum dilutions. We corroborated that the observed anti-SIV ADCC activity was indeed induced by SIVmac251 infection, as we observed no significant ADCC activity before rectal SIV inoculation when using gp120-coated CEM-NKr target cells (Table 1, day 0 vs. day 28; P b 0.05). Further, to measure this activity in an infected cell context, we performed an additional experiment in which SIVmac251-infected H9 T cells were used as targets. Table 1 also shows that SIV-infected cells were susceptible targets of in vitro ADCC using sera from day 28 post-SIV infection (33.4 F 4.9% killing). Background ADCC activity before inoculating the animals was substantially lower (7.9 F 1.7% killing, P b 0.01). The extent of killing was higher in the gp120-coated target format than in the SIV-infected cell assay but the former was run at 50:1 effector to target ratios, while the latter was run at ratios of 10:1. Two remaining serum samples were re-assayed using 50:1 E /T ratios and the same effector cells in both assay systems. As shown in the bottom panel of Table 1, very similar results were obtained in both assays indicating that for these sera; the predominant ADCC activity was directed to the SIV envelope and was effectively evaluated by both assays. Taken together, these data indicate that the RFADCC assay is a suitable assay to monitor early ADCC responses arising in rhesus macaques within a month of SIV infection.

Table 1 Anti-SIV ADCC activity in SIVmac251-infected rhesus macaques Animal ID ADCC (% CFSEnegative within PKH-26high) gp120-coated CEM-NKr targets (E /T = 50:1) Day 0 Rh8 Rh12 Rh13 Rh27 Rh34 Rh39 Rh44 Rh46 Mean F S.D. 2.3 3.0 3.2 4.3 5.4 3.4 3.6 5.9 3.9 F 1.2 (E /T = 50:1) Rh8 Rh46 Mean 9.9 15.5 12.7 42.2 43.5 42.9 Day 28 49.6 43.8 49.1 46.6 ND ND 55.5 54.1 49.8 F 4.4* SIVmac251-infected H9 targets (E /T = 10:1) Day 0 10.6 9.0 8.6 7.8 7.4 8.2 7.3 4.5 7.9 F 1.7 (E /T = 50:1) 19.9 14.2 17.1 50.5 39.2 44.9 Day 28 33.1 35.4 41.6 35.0 36.7 28.0 26.1 31.5 33.4 F 4.9**

Sera were tested at a 1/10 dilution. Values are the average of duplicate experiments. *P b 0.05 and **P b 0.01, significant differences between day 0 and day 28 ADCC activity using gp120-coated and SIV-infected targets, respectively. Assays in the upper and lower panels were run with effector cells from different donors.

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V.R. Go mez-Roma n et al. / Journal of Immunological Methods 308 (2006) 5367 Table 3 Anti-HIV ADCC activity in two chimpanzees immunized with replicating Ad-HIVMNenv/rev candidate vaccines ADCC (% CFSEnegative within PKH-26high) HIVIIIB gp120-coated CEM-NKr targets Day 0 Chimp 182D 13.1 F 0.7 Chimp 4X0386 12.0 F 0.9 Day 105 38.0 F 0.6 23.4 F 0.3 HIVBal gp120-coated CEM-NKr targets Day 0 ND ND Day 105 55.2 F 3.1 59.6 F 2.0

3.4. ADCC in non-human primates immunized with AIDS vaccines We next sought to determine whether the RFADCC assay was suitable to screen for serum ADCC responses during AIDS vaccine preclinical trials. For this purpose, we used retrospective sera from non-human primates immunized with replicating Ad-SIV or Ad-HIV vaccines. ADCC activity against SIV-infected targets was elicited after immunizing sixteen rhesus macaques with Ad-SIVenv/rev vaccines (Table 2). Although the overall induction of ADCC activity was low with a mean of 19.2 F 4.68% killing, 15 of the 16 macaques were positive based on the cutoff value of 3 standard deviations above the mean of the mock immunized controls (data not shown). The difference in ADCC activity before and after Ad-SIV immunization was statistically significant (5.2% vs. 19.2% killing, P b 0.0001). In contrast, control macaques immunized with the empty adenovirus vector backbone had a negligible increase in background ADCC activity (5.1% vs. 7.5% killing). The difference in post-immunization ADCC activity at day 98 between the two immunization groups was highly significant (7.5 vs. 19.2% killing; P b 0.0001). Similarly, anti-HIV gp120 ADCC activity was elicited in chimpanzees after immunization with replicating Ad-HIVMNenv/rev vaccine candidates (Table 3). Due to the limited number of chimpanzees available for these immunogenicity studies, we were unable to include empty Ad-vector immunized controls. Nonetheless, it is important to note the breadth of the ADCC activity elicited in the chimpanzees. Positive responses were detected against cells coated with envelope antigen derived from two HIV strains different from the immunizing HIVMN strain, one CXCR4 tropic (HIVIIIB) and one CCR5 tropic (HIVBal). Even though the same amount of purified gp120 was used
Table 2 Anti-SIV ADCC activity in rhesus macaques immunized with replicating Ad-recombinants n ADCC (% CFSEnegative within PKH-26high) Day 0 Ad-vector immunized Ad-SIVenv/rev immunized 8 16 5.1 F 1.4 5.2 F 1.1 Day 98 7.5 F 1.6 19.2 F 4.7*

Sera were tested at a 1/10 dilution. Values are the mean F S.D. from duplicate experiments.

to saturate the target cells (15 Ag gp120/five million CEM-NKr cells), the lysis of HIVBal gp120-coated targets by chimpanzee immune sera was higher than that of the HIVIIIB gp120-coated targets. Experiments using both pre- and post-immunization sera from a larger number of chimpanzees to provide statistical power will be needed to clarify whether this is a reproducible difference in ADCC activity. If so, it may result from the higher amino acid identity between the immunizing HIVMN strain and the HIVBal strain (82.6% homology) compared to the amino acid homology between HIVMN and HIVIIIB (79%). 4. Discussion Here we have shown that the FATAL assay, originally designed to assess CTL responses, can be adapted to measure ADCC against HIV and SIV. As we showed for mab2G12, a convenient feature of the RFADCC is that it is both quantitative and sensitive for ADCC activity in the nanomolar range (Fig. 3). Additional features of this assay are that, unlike the CRA: killing is acquired on a single-cell basis; that the percent killing is recorded directly by back-gating during data analysis; and that there is no need to calculate killing from an artificial bmaximal lysis controlQ which would normally involve the use of detergents that are irrelevant to the effector-mediated lysis. For the purposes of illustrating the low background values observed in our experiments, we have chosen to report the percent killing values obtained directly during data analysis, without subtracting any background killing. A considerable advantage of the RFADCC method is its suitability for use with viably frozen effector cells. This feature allows screening and selection of appropriate donor effector cells that can be stored for later use, enabling assay standardization and validation. Whether using fresh or frozen effectors, the RFADCC consistently provided higher levels of cell killing than CRA. The relatively low percentages of cell killing and

Sera were tested at a 1/10 dilution against SIVmac251-infected H9 targets. n = number of macaques per immunization group. E /T = 10:1. * P b 0.0001, significant difference between immunization groups at day 98 and between day 0 and day 98 ADCC activity in the Ad-SIV immunized group.

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often lower titers we initially obtained by the CRA were unexpected (Fig. 5), but were obtained using frozen effectors. Nevertheless, our results were within the lower range of reported cell killing for patient sera using fresh effector cells and the CRA method (Tyler et al., 1990). Here, when fresh effectors were used, the HIVIG preparation exhibited a similar titer by both the RFADCC and CRA methods (Fig. 6) and the peak lysis exhibited by CRA was comparable to other reported preparations of HIVIG assayed by CRA using fresh effector cells (Cummins et al., 1991). The extent of cell lysis in ADCC assays depends in part on the percentage of NK cells in the effector cell population. In the comparative assays conducted here, the percentage of NK cells (CD3CD16+CD56+) in the effector A and G cells (10.8% and 11.0%, respectively) were at the lower end of reported NK cell percentages for adults, ranging from 6% to 33% (Shahghasempour et al., 2001), similar to the findings of Ohkawa et al. (2001), who showed a similar range in reporting that the proportions of NK cells increase with age. The ability to select and viably freeze effector cells from donors with high proportions of NK cells for use in the RFADCC should greatly facilitate analyses of ADCC activity with high sensitivity and reproducibility. To our knowledge, there is only one other instance in which a flow-based cytolysis assay has been used to assess ADCC responses in vitro (Wilkinson et al., 2001). As the authors showed, however, their method had higher background values, ranging from 21% to 32% killing, resulting in a lower assay sensitivity and detection of ADCC activity only at high antibody concentrations (10 Ag/ml, i.e. micromolar range). Further, a caveat of this previous flow-based assay is that it requires sequential staining steps interrupted by the ADCC assay itself: targets are labeled with PKH-26; antibody and effectors are then added; ADCC is allowed to occur and a TO-PRO-3-DNA binding viability fluorophore is added at the end of the procedure to distinguish dead targets. On the other hand, the RFADCC assay described here has a nanomolar assay sensitivity and uses a dual staining step at the beginning of the assay, allowing for the ability to dispense equally-labeled targets to several wells in a microtiter plate simultaneously, thereby minimizing experimental error and enabling high throughput. Indeed, we saw very little variability in duplicate assays, including assays in which different effectors, pre-screened for low background and high killing ability, were used (Fig. 3). The versatility of the RFADCC is illustrated by the choice of targets. Within a month of SIV infection, sera from rhesus macaques mediated ADCC of both

envelope-coated targets and of SIV-infected cells (Table 1). While the gp120-coated target assay demonstrates that CD4-bound SIV envelope can be a target of ADCC on its own, the SIV-infected target assay embraces the possibility that ADCC is directed toward other envelope and non-envelope SIV antigens expressed on the surface of the infected cell. Nef, for example, has been recently shown to be a target of ADCC, as some Nef antigens are expressed on the surface of HIV-1 infected human cells (Yamada et al., 2004). In this regard, the RFADCC method may prove useful in mapping additional epitopes responsible for mediating ADCC. We established that the RFADCC is suitable for assessing serum ADCC responses elicited by vaccination, here using replicating Ad-HIV and Ad-SIV vaccine candidates (Tables 2 and 3). Future studies will have to clarify whether non-replicating adenoviruses or other viral vaccine vectors elicit levels of ADCC activity similar or greater to those reported here. Although we specifically measured ADCC activity against HIV and SIV antigens, we anticipate that the RFADCC can be applied to assess ADCC activity in the context of other studies. ADCC has been shown to be a relevant effector mechanism in mab cancer immunotherapy (Mellstedt, 2003; Houghton and Scheinberg, 2000). Anti-tumor ADCC activity, for example, is a preferred characteristic of Herceptink, Rituxank and other anti-tumor monoclonal antibody candidates in the pipeline for clinical trials (reviewed by Mellstedt, 2003). ADCC is also relevant in protection against various parasitic and bacterial diseases and other viral infections (Hashimoto et al., 1983; Kohl et al., 2000; Zhou et al., 2000; Moore et al., 2002). The RFADCC assay is expected to provide a simple, reliable method to screen for ADCC activity in multiple applications. In summary, we have demonstrated that the FATAL assay can be adapted into a rapid, quantitative and sensitive method to measure ADCC on a single cell basis. We anticipate that the versatility of the RFADCC assay will facilitate advancement of ADCC studies both inside and outside of the HIV vaccine arena. Acknowledgements We thank Drs. Kent Weinhold and Guido Ferrari for providing the HIVIG preparation and helpful advice with the CRA. The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: 2G12 from Dr. Hermann Katinger; CEM-NKr from Dr. Peter Cresswell. This research was supported by the

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