of September 27, 2015.This information is current as Challengemac251against Oral SIV

Protection of Neonatal Rhesus Macaques Cellular Cytotoxicity-Mediating IgG inNonneutralizing Antibody-Dependent Evaluation of Passively Transferred,

Robert-GuroffKalyanaraman, Marta L. Marthas and MarjorieNancy Haigwood, David Venzon, Vaniambadi S.

Mahalanabis,Donald N. Forthal, Gary Landucci, Madhumita Ruth H. Florese, Koen K. A. Van Rompay, Kris Aldrich,

http://www.jimmunol.org/content/177/6/4028doi: 10.4049/jimmunol.177.6.4028

2006; 177:4028-4036; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/177/6/4028.full#ref-list-1

, 22 of which you can access for free at: cites 40 articlesThis article

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2006 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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Evaluation of Passively Transferred, NonneutralizingAntibody-Dependent Cellular Cytotoxicity-Mediating IgG inProtection of Neonatal Rhesus Macaques against OralSIVmac251 Challenge1

Ruth H. Florese,* Koen K. A. Van Rompay,‡ Kris Aldrich,* Donald N. Forthal,§

Gary Landucci,§ Madhumita Mahalanabis,¶ Nancy Haigwood,¶� David Venzon,†

Vaniambadi S. Kalyanaraman,# Marta L. Marthas,‡ and Marjorie Robert-Guroff2*

Previously, Ab-dependent cellular cytotoxicity (ADCC) was significantly correlated with reduced acute viremia upon intrarectalSIVmac251 challenge of immunized rhesus macaques. To directly assess ADCC protective efficacy, six neonatal macaques wereinfused s.c. with immune IgG (220 mg/kg) purified from the immunized animals and positive for ADCC and Ab-dependentcell-mediated viral inhibition (ADCVI) activities. Six neonates received control IgG. The neonates were challenged twice orallywith 105 50% inhibiting tissue culture-infective dose of SIVmac251 2 days post-IgG infusion. At challenge, plasma of neonates thatreceived immune IgG did not neutralize SIVmac251 but had geometric mean ADCC titers of 48,130 and 232,850 against SIVmac251-infected and gp120-coated targets, respectively. Peak ADCVI activity varied from 62 to 81%. ADCC activity declined with the2-wk IgG half-life but was boosted at wk 4, together with de novo ADCC-mediating Abs in controls, by postchallenge viremia.ADCVI activity was similarly induced. No protection, assessed by viral burdens, CD4 counts, and time to euthanasia was observed.

Possible factors contributing to the discrepancy between the previous correlation and lack of protection here include: the high oralchallenge dose compared with the 400-fold lower intrarectal dose; the challenge route with regard to viral dissemination anddistribution of infused IgG; insufficient NK effector activity and/or poor functionality in newborns; insufficient immune IgG; andthe possibility that the previous correlation of ADCC with protection was augmented by cellular immune responses also presentat challenge. Future studies should explore additional challenge routes in juvenile macaques using higher amounts of potent IgGpreparations. The Journal of Immunology, 2006, 177: 4028–4036.

A ntibody-dependent cell-mediated cytotoxicity (ADCC)3

is a powerful immune mechanism that can eliminate vi-rus-infected cells via effector cells armed with viral-spe-

cific Abs. The ADCC process requires three components: targetcells expressing a surface Ag, Ag-specific Abs, mainly of the IgGisotype, and Fc�R-bearing effector cells such as NK cells, ��Tcells, neutrophils, and macrophages. Abs binding to both targetAgs and effector cell Fc�Rs induce target killing by lysis and/orapoptosis.

Abs mediating ADCC are among the first antiviral immune re-sponses to occur following infection. In HIV-infected individuals,Abs with ADCC activity appear early in acute infection, oftenpreceding a neutralizing Ab response (1, 2). In HIV-infected pa-tients, as in SIV-infected rhesus macaques, ADCC responses havebeen associated with a better clinical outcome and/or lower viralloads (3–6). Yet to date, the contribution of ADCC activity toprotection against HIV or SIV infection has not been fullyelucidated.

Ab-dependent cell-mediated virus inhibition (ADCVI) is closelyrelated to ADCC in that it relies on interactions between an in-fected target cell, Ab, and FcR-bearing effector cells. However,rather than target cell lysis, ADCVI measures virus inhibition frominfected target cells and may involve both cytotoxic and noncyto-toxic mechanisms. ADCVI has been associated with the fall inviremia during acute HIV infection and may underlie the protec-tive effect of passively infused, nonneutralizing Ab (7, 8).

Previously, we reported strong protection against intrarectalSIVmac251 challenge following two mucosal immunizations withone or more replication-competent Ad type 5 host range mutant(Ad5hr) expressing SIVenv/rev, SIVgag, and/or SIVnef and boost-ing with SIV gp120 protein or a polypeptide “peptomer” repre-senting the CD4 binding site of the SIV envelope (9). High-titerSIV envelope-specific binding Abs were induced by the vaccineregimen and shown to correlate with reduced acute phase viremia.Notably, these binding Abs did not neutralize primary SIVmac251.Both serum Abs and purified IgG from the immunized rhesus ma-caques mediated ADCC activity that was correlated with reduced

*Vaccine Branch and †Biostatistics and Data Management Section, National CancerInstitute, Bethesda, MD 20892; ‡California National Primate Research Center, Uni-versity of California, Davis, CA 95616; §University of California, Irvine School ofMedicine, Orange, CA 92868; ¶Department of Microbiology, University of Wash-ington, Seattle, WA 98195; �Seattle Biomedical Research Institute, Seattle, WA98109; and #Advanced Bioscience Laboratories, Kensington, MD 20895

Received for publication April 5, 2006. Accepted for publication July 3, 2006.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported in part by the Intramural Research Program of the NationalInstitutes of Health, National Cancer Institute, by National Institutes of Health GrantR01 AI52039 (to D.N.F.), and by a National Center for Research Resources supple-ment to the California National Primate Research Center Base Operating Grant(RR00169).2 Address correspondence and reprint requests to Dr. Marjorie Robert-Guroff, Na-tional Institutes of Health, National Cancer Institute, 41 Medlars Drive, Building 41,Room D804, Bethesda, MD 20892-5065. E-mail address: guroffm@mail.nih.gov3 Abbreviations used in this paper: ADCC, Ab-dependent cellular cytotoxicity; AD-CVI, Ab-dependent cell-mediated virus inhibition; Ad, adenovirus; Ad5hr, Ad type 5host range mutant; TCID50, 50% inhibiting tissue culture-infective dose; RFADCC,rapid fluorometric ADCC.

The Journal of Immunology

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00

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acute viremia (6). These results implied that the Abs possessed afunctional activity different from virus neutralization that contrib-uted to viremia control.

Classically, the role of Abs in protecting against infection ordisease has been studied by passive transfer experiments, wherebyserum, immune globulin, or Ab preparations are infused into a hostbefore or shortly after challenge with an infectious agent. Numer-ous such studies have shown protective efficacy of HIV or SIV Absagainst i.v., vaginal, or oral challenges in nonhuman primates.Both neutralizing mAbs specific for the HIV envelope (10–12) andpolyclonal immune globulin with neutralizing activity against HIVor SIV (13, 14) have been shown to be protective. In contrast, thecontribution of nonneutralizing Abs to protection has been rarelyassessed. Passive transfer of SIV hyperimmune serum lacking theability to neutralize the SIVmac251 challenge virus protected new-born rhesus macaques against oral SIV challenge (15). In vitrostudies recently attributed this protection to ADCVI (7). No pas-sive transfer studies have directly assessed ADCC activity in theabsence of other functional activities in SIV or simian HIVsystems.

To elucidate the role of the previously described vaccine-elic-ited ADCC-mediating Abs in protection against SIVmac251 (6, 9),we purified IgG from a pool of prechallenge macaque sera andplasma and conducted a passive transfer experiment. We also eval-uated the immune IgG transferred and macaque plasma pre- andpost-SIVmac251 challenge for ADCVI activity. As limited IgG wasavailable, we chose the neonatal SIV rhesus macaque model. Oralinoculation of newborn macaques with pathogenic SIV is a usefulmodel of human infant HIV infection (16) and has been used inevaluating both immunologic and therapeutic intervention strate-gies (15, 17, 18). The small size of the newborns allowed us toinclude sufficient animals in the study to provide statistical power.In the present study, we report the challenge outcome and resultsof extensive immunologic analyses pre- and postchallenge.

Materials and MethodsPreparation of IgG fractions

Sera and plasma previously collected from 31 vaccinated rhesus macaquesand stored at �70°C were thawed and combined as pool A. The macaqueshad been immunized intranasally and then intratracheally with an Ad5hrexpressing SIVenv/rev, with or without additional Ad5hr encoding SIVgagand/or SIVnef (9). The animals were subsequently boosted twice i.m. withnative SIV gp120 protein in MPL-SE adjuvant. Samples obtained 2 wkafter the second protein booster immunization (wk 38 postinitial immuni-zation) and at the time of challenge (wk 42) were pooled. The time-of-challenge samples were previously shown to possess high-titered Abs thatmediated ADCC activity (6). Sera and plasma from mock-immunized con-trol macaques from the same study (after monophosphoryl lipid A-stableemulsion immunizations, wk 26, 30, 34, 38, and 42) were also thawed andcombined as pool B. To obtain sufficient control sera and plasma, samplesobtained following the last mock-booster immunization (wk 38 and 42)were added from a previous study in which macaques received two intra-nasal administrations of empty Ad5hr vector and two immunizations withQS-21 adjuvant only (19). The final volume of pool A was 135 ml, whereaspool B contained 109 ml.

IgG was purified from pools A and B by the Immunology Core, Hu-moral Immunity Subcore of the University of Washington Center for AIDSResearch, Seattle Biomedical Research Institute. The purification methodwas described previously (14). Briefly, pools A and B were thawed, heatinactivated at 56°C for 45 min, clarified by centrifugation, and filteredthrough a 0.2-�M mini capsule filter before purification on protein G-Sepharose (Amersham Biosciences) equilibrated with PBS. Pool B waspurified first, and then pool A was purified using the same protein G col-umn. IgG was eluted with 0.5 M acetic acid (pH 3.0) and neutralized with3 M Tris (pH 9.0). IgG containing fractions, identified by OD (OD280),were verified by electrophoresis on reducing SDS-polyacrylamide gels.Peak fractions were pooled, concentrated, dialyzed extensively in PBS, andfiltered through a 0.2-�M filter. The IgG preparations contained �5 en-dotoxin U/ml. Coomassie-stained gels indicated �95% purity for both pool

A and B IgG preparations. Pool A contained 1118 mg of immune IgG (13mg/ml) and pool B contained 739 mg of control IgG (11.2 mg/ml).

Animals and assessment of MHC class

Newborn rhesus macaques (Macaca mulatta) were negative for SIV, HIVtype 2, type D retrovirus, and simian T cell lymphotropic virus type 1. Theywere hand-reared at the California National Primate Research Center andhoused following the guidelines of the American Association for Accred-itation of Laboratory Animal Care. Strict adherence to the “Guide for Careand Use of Laboratory Animals” (20) was observed. When necessary, an-imals were immobilized with 10 mg/kg ketamine hydrochloride (Parke-Davis) injected i.m. Six animals were randomly assigned at birth to theexperimental group and received immune IgG from pool A. Six formed thecontrol group and received control IgG from pool B. MHC typing wasperformed by the Rhesus Macaque MHC Typing Core, University of Wis-consin Hospital and Clinics. Two neonates in the experimental group(36460 and 36495) and one in the control group (36475) were positive forthe Mamu-A*01 allele. Weights of the neonates ranged from 0.45 to 0.61kg with a mean of 0.5 kg for both groups.

IgG administration and SIVmac251 challenge

Within 4 days of birth, neonates were s.c. administered either 220 mg/kgimmune or control IgG while under ketamine anesthesia. Two days later,the animals were bled and orally administered two sequential high-doseoral challenges, 24 h apart, with 105 50% inhibiting tissue culture-infectivedose (TCID50) of SIVmac251 (lot no. 2/02 propagated on rhesus PBMC; titerof 8.6�108 viral RNA copies/ml). Animals were bled on day 7 postchal-lenge and then at weekly or bimonthly intervals for routine monitoring ofblood counts and lymphocyte subsets, viral loads, and Ab responses.Plasma samples were stored at �70°C until use. The macaques were alsomonitored for weight, general health, and clinical signs and symptoms ofdisease progression. Animals were euthanized when necessary according topreviously defined criteria (21).

Postchallenge virologic monitoring and lymphocyte phenotyping

SIV RNA in plasma was quantified by a bDNA signal amplification assay,specific for the SIVmac251 pol gene (22). Lymphocyte phenotypic analysiswas performed using three- and four-color flow cytometry as describedpreviously (22).

Ab assays

The ability of Abs to mediate ADCC activity was assessed using the rapidfluorometric ADCC (RFADCC) assay described elsewhere (23). Briefly,CEM-NKr cells (AIDS Research and Reference Reagent Program, Na-tional Institutes of Allergy and Infectious Diseases) coated with SIVgp120or H9 cells chronically infected with SIVmac251 were used as targets. Thetarget cells were dual labeled with the membrane dye, PKH-26 (Sigma-Aldrich), and CFSE (Molecular Probes), a vital dye that is rapidly lostwhen cell membranes are damaged. Labeled targets were resuspended inRPMI 1640 medium containing 10% FCS (R-10) and allowed to react withheat-inactivated (56°C, 30 min) serially diluted plasma in a 96-well mi-crotiter plate for 30 min at room temperature. Human PBMC used as ef-fector cells were added at a 50:1 E:T ratio. The reaction mixture wasincubated for 4 h at 37°C in 5% CO2, after which the cells were fixed with3.7% paraformaldehyde for flow cytometry. Controls included nonstainedand single-stained target cells. Nongated events (50,000) in duplicate wellswere acquired within 18 h using a FACSCalibur instrument (BD Bio-sciences). Acquisition was done using CellQuest software, and data anal-ysis was performed with WinMDI 2.0. Percent ADCC cell killing is re-ported as the percentage of membrane-labeled target cells having lost theviability dye, i.e., percentage of CFSEnegative within the PKH-26high gate.ADCC titers are defined as the reciprocal dilution or IgG concentration atwhich the percent ADCC killing was greater than the mean percent killingof the negative control plus 3 SDs.

The ADCVI assay was based on methods previously described for mea-sles virus and HIV (8, 24). CEMx174 target cells were infected with theSIVmac251 challenge stock at a multiplicity of infection of 0.01. After ad-sorption for 1 h, cells were washed, incubated in 5% CO2 at 37°C for 48 hin medium, and washed again. Infected target cells (5 � 104) were nextplated in 96-well round-bottom microtiter plates, and various dilutions ofplasma were added along with human PBMC effector cells at an E:T ratioof 10:1. Plasma in the absence of effector cells was also tested. After 7 daysincubation at 37°C in 5% CO2, supernatant fluid was collected and assayedfor p27 by ELISA (Zeptometrix). Virus inhibition due to ADCVI wascalculated as follows: percent inhibition � 100 � (1 � ([p27p]/p27n])),where [p27p] and [p27n] are the concentrations of p27 in supernatant fluid

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from wells containing a source of SIV-positive or -negative Ab, respec-tively. Mean values from experiments using two different donor cells arereported. Titer is expressed as the reciprocal of the plasma dilution at which60% inhibition was observed.

Binding Abs to SIVmac251 gp120 were determined by ELISA (25). Thebinding titer was defined as the reciprocal of the plasma dilution or the IgGconcentration at which the absorbance of the test plasma or IgG was twicethat of a negative control serum diluted 1/200. Binding titers to whole SIVwere determined as described previously (26).

Neutralizing Abs in the IgG fractions and neonate plasma against theSIVmac251 challenge stock were evaluated in sMAGI cells (27) as describedpreviously (28). Positive controls included plasma from a macaque infectedwith SIVmneE11S, known to neutralize SIVmac251. End point titers of 75 and90% are reported.

Statistical analyses

The Wilcoxon rank-sum test was used to compare viral loads and survivaltimes between experimental and control groups and to analyze differencesbetween postchallenge ADCVI activity in experimental and control ma-caques. Spearman rank correlation coefficients were calculated in analyz-ing ADCC titers, percent killing, and survival times.

ResultsCharacterization of passively transferred Ab

We previously reported that sera from vaccinated macaques at thetime of challenge possessed high-titered binding Abs able to me-diate ADCC activity against SIV-infected target cells (6) butlacked neutralizing activity against primary SIVmac251 (9). In thepresent study, we combined sera and plasma obtained at challenge(wk 42 postimmunization) and 2 wk following the last proteinbooster immunization (wk 38) from the same macaques to obtainan experimental pool (pool A) from which immune IgG could bepurified. Sera and plasma from mock-immunized macaques weresimilarly pooled for preparation of control IgG (pool B). Beforepurification, the properties of pools A and B were examined. Se-rum/plasma pool A exhibited high-titered binding Ab to SIVmac251

gp120, whereas pool B was Ab negative (Table I). Similarly, Absin pool A mediated potent ADCC activity against both SIV-in-fected target cells and target cells coated with SIV gp120, whereaspool B was negative with either SIV-infected or gp120-coatedtargets.

Following IgG purification, the pools were reassessed for thesame properties. Immune IgG from pool A exhibited a potent bind-ing Ab titer against SIV gp120 together with ADCC titers againstSIV-infected and gp120-coated target cells in the nanogram range(Table I). In contrast to results with serum/plasma pool A, theimmune IgG of pool A exhibited some loss of ADCC titer againstgp120-coated target cells relative to the ADCC titer against SIV-infected targets, suggesting non-IgG Abs removed from the IgGpool may have possessed some ADCC activity. Pool A immuneIgG was also positive for ADCVI activity, exhibiting �90% in-hibition of SIVmac251 infection at the concentration tested. The

control IgG of pool B lacked binding, ADCC, and ADCVI Abactivity. As expected, based on earlier results showing no neutral-ization of primary SIVmac251 by sera at challenge, 90% neutral-ization of the SIVmac251 challenge stock required a high concen-tration of pool A immune IgG. Pool B was negative.

Prechallenge Ab characterization

Following s.c. administration of immune or control IgG to theneonates, blood samples were obtained 2 days later, and the ma-caques were orally challenged twice, 24 h apart, with SIVmac251.Plasma samples obtained at challenge were evaluated for bindingand functionally active Abs. The passive transfer resulted in theappearance of anti-SIV Abs in the blood of the neonatal macaques2 days later (Table II). Therefore, binding and functionally activeAbs were present at the time of SIVmac251 challenge. As expectedbased on the similar size of the macaques, all six that receivedimmune IgG of pool A exhibited similar binding Ab titers to wholeSIV, as well as to purified native SIV gp120. Similar ADCC titersamong the six neonates were also observed using both gp120-coated (geometric mean titer of 232,850) and SIV-infected targets(geometric mean titer of 48,130). No ADCC activity was observedin plasma of the control neonates. Macaques that received immuneIgG also exhibited ADCVI activity, with peak inhibition rangingfrom 62 to 81%. Peak values were sometimes observed at plasmadilutions of 1/100 rather than the lesser 1/25 dilution due to aprozone effect (29). Plasma from only two animals exhibited�60% ADCVI activity at dilutions � 1/100. Ninety percent ofneutralization titers against the challenge stock of SIVmac251 were�25 for all macaques (data not shown). Plasma samples from ma-caques that received control IgG were negative for binding,ADCC-mediating, and neutralizing Abs and exhibited negligibleinhibition in the ADCVI assay.

Results of oral SIVmac251 challenge

Following the oral SIVmac251 challenges on 2 successive days,both experimental and control macaques became infected, exhib-iting high viral loads (Fig. 1, A and B). No difference was observedbetween groups. High viremia was generally maintained over timeamong the neonates until they had to be euthanized due to pro-gression to AIDS. Rhesus neonates have high CD4� T cell countscompared with adults (21, 30, 31). Thus, this phenotype partlyexplains the high viral burdens because more target cells are avail-able for viral infection. In this regard, CD4 T cell counts declinedsomewhat by wk 2 postchallenge but not to exceedingly low levels(Fig. 1, C and D), which is consistent with previous studies onSIVmac251-infected infant macaques (21, 31). No difference inCD4 count was observed between neonates that received immuneIgG or control IgG.

Table I. Properties of sera/plasma and IgG pools used for passive transfera

Binding to SIVgp120

ADCC: gp120-CoatedTargets

ADCC: SIV-InfectedTargets ADCVI Neutralization

Sera/plasmaPool A 167,500 �10,000,000 98,000 NDb NDPool B �100 �100 �200 ND ND

IgGPool A 0.053 �g/ml 0.033 �g/ml 0.014 �g/ml 95% 3.25 mg/mlPool B �10 �g/ml �10 �g/ml �10 �g/ml 0% Negative

a Results for sera/plasma pools are reported as titers as described in Materials and Methods. Results for IgG pools are reported as the concentrationof IgG required for a positive response. Control IgG of pool B was negative for 90% neutralization at the highest concentration of IgG tested (2.8 mg/ml).ADCVI is reported as percent inhibition for pool A tested at 130 �g/ml and pool B tested at 112 �g/ml.

b ND, Not done.

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We also analyzed length of survival of infant macaques in bothgroups. The majority of infants developed clinical AIDS and hadto be euthanized between 9 and 14 wk, although one Mamu-A*01positive neonate (36460) that received immune IgG survived for24 wk (data not shown). Two other Mamu-A*01-positive ma-caques (36475 and 36495 that received control and immune IgG,respectively) were among the last macaques euthanized at wk 13and 14, respectively. Mamu-A*01-positive macaques control vire-mia more effectively than Mamu-A*01-negative animals (32), andin the present study, the three Mamu-A*01-positive animals hadsignificantly longer survival than the other nine ( p � 0.027 by theexact log-rank test). The viral loads of these three macaques werealso significantly lower than the other nine over wk 4, 6, 8, and9/10 postchallenge ( p � 0.02 for each by the Wilcoxon rank-sumtest). However, after taking the Mamu-A*01 factor into account,there was still no significant difference in time to death or viralburden between the two groups of neonates.

Postchallenge Ab activity

To better understand the similar challenge outcome between thetwo groups of macaques, we evaluated the spectrum of Ab activ-ities postchallenge. Initially, we assayed binding Abs specific forSIVgp120 over the course of infection. The gp120-binding Ab inthe passively transferred immune IgG decayed with a half-life of�2 wk (data not shown). The control macaques failed to developany gp120-specific binding Abs following infection with the ex-ception of macaque 36455 that exhibited a binding titer of 240–270 at wk 8–10 postchallenge.

Examination of ADCC activity in the macaque plasma postchal-lenge revealed an initial drop in ADCC titer as measured usingSIV gp120-coated target cells (Fig. 2, A and B). This paralleled thedecay of the transferred immune IgG. However, at wk 4, theADCC titers were boosted and reached peak titers at 6 wk post-challenge. Similarly, ADCC activity against gp120-coated target

FIGURE 1. Viral loads (A and B)and CD4� lymphocyte levels (C andD) among neonates following oralSIVmac251 challenge. Recipients of im-mune IgG (A and C); control IgG (Band D). An asterisk denotes Mamu-A*01-positive macaques. Values forwk 9, 10, and 11 are combined andplotted as wk 10.

Table II. Ab activities in the plasma of neonatal macaques at the time of challenge, following passive transfer

Neonate

Binding toWhole SIV

(titer)

Binding toSIV gp120

(titer)ADCC: gp120-Coated

Targets (titer)

ADCC:SIV-InfectedTargets (titer)

ADCVIa (percentinhibition)

Immune IgG (pool A) recipients36460 1,600 12,800 250,000 86,000 7736476 1,600 12,800 156,000 39,000 8136492 1,600 12,800 108,000 54,000 8136495 1,600 12,800 310,000 48,000 7736502 1,600 12,800 781,250 27,500 6236507 1,600 12,800 156,250 52,000 65

Control IgG (pool B) recipients36455 Negb �200 �10 �100 236475 Neg �200 �10 �100 2136489 Neg �200 �10 �100 2036494 Neg �200 �10 �100 ND36500 Neg �200 �10 �100 ND36508 Neg �200 �10 �100 ND

a Peak percent inhibition is reported since some macaque plasma exhibited a prozone effect (29) with greater inhibition observed with increasing serum dilution.b Neg, Negative; ND, not done.

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cells appeared at wk 4 in the macaques that received control IgGand peaked at wk 6. These results are consistent with induction ofde novo ADCC activity resulting from SIV infection.

ADCC activity against SIVmac251-infected cells exhibited a sim-ilar pattern (Fig. 2, C and D). In macaques that received immuneIgG, the ADCC titers declined over the first 2 wk and then wereboosted at wk 4. In the control animals, de novo ADCC activityagain appeared at wk 4. When assessed by either gp120-coatedtargets or SIV-infected targets, ADCC titers remained 10- to 100-fold higher in the macaques that received immune IgG comparedwith the controls, reflecting maintenance of ADCC activity in vivofrom the passively transferred IgG in addition to de novo activityresulting from SIV infection. Using SIV-infected targets, peak ti-ters were observed at wk 4 postchallenge, with the experimentalgroup exhibiting a geometric mean ADCC titer of 98,704, and the

controls a geometric mean titer of 7,396. Using gp120-coated tar-gets, peak titers were observed at wk 6 postchallenge, with geo-metric means of 173,564 and 1,339 for the experimental and con-trol groups, respectively. As the immune IgG continued to decayand as disease progression occurred, the ADCC activity observedin plasma samples gradually declined until the time of euthanasia.

We also monitored ADCVI activity in the macaque plasmas at4 wk postchallenge. The results are similar to those for ADCCactivity in showing maintenance of ADCVI activity at wk 4 in theimmune IgG group and induction of de novo ADCVI activity inthe control group (Table III). Peak ADCVI activity remained sig-nificantly higher at wk 4 postchallenge in the macaques that re-ceived immune IgG ( p � 0.0087). Overall, however, ADCVI ti-ters were low, and despite the maintenance of activity, titers werenot boosted in the experimental animals.

FIGURE 2. Titers of ADCC-medi-ating Abs in sequential plasma speci-mens from neonatal macaques usingSIVgp120-coated CEM.NKr cells (Aand B) and H9 cells infected withSIVmac251 (C and D) as targets. Recip-ients of immune IgG (A and C); controlIgG (B and D). Values for wk 9, 10,and 11 are combined as in Fig. 1.

Table III. Evaluation of ADCVI in plasma from experimental and control macaques, pre- and post-oral SIVmac251 challenge

Macaque

Peak Percent Inhibition Titer

At challengePostchallenge

(week 4) At challengePostchallenge

(week 4)

Immune IgG recipients36460 77 98 400 �10036476 81 87 100 �10036492 81 70 100 2536495 77 93 400 2536502 62 96 100 2536507 65 89 100 25

Mean � SEM 73.8 � 3.4 88.8 � 4.1Control IgG recipients

36455 2 64 �25 2536475 21 63 �25 2536489 20 25 �25 �2536494 NDa 88 ND 2536500 ND 55 ND 10036508 ND 61 ND 100

Mean � SEM 14.3 � 6.2 59.3 � 8.3

a ND, Not done.

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In conjunction with induction of de novo ADCC and ADCVIactivity resulting from SIV infection, the macaques also developedneutralizing Abs (Fig. 3). By wk 4 and 6 postchallenge, two of sixmacaques that received control IgG had developed low titer Absable to achieve 90% neutralization of the SIVmac251 challenge vi-rus, whereas in the group that received immune IgG, three of sixdeveloped such Abs. If a 75% end point was used for determiningtiter, five macaques in each group were able to neutralize the pri-mary SIVmac251 stock. Overall, the neutralizing Ab titers observedfollowing infection were similar between the experimental andcontrol macaques, which is consistent with the observed lack ofneutralizing Ab prechallenge in the immune IgG.

Overall, the spectrum of Abs induced or boosted at 4 wk post-challenge as a result of SIV infection did not exert any effect on thechallenge outcomes. Viral loads were not diminished, and CD4counts did not show any consistent increase at wk 4–6.

NK cells in the infant macaques

In view of the ADCC and ADCVI activities mediated by the ma-caque plasma samples in vitro, but the lack of any observableeffect on challenge outcome in vivo, we considered the levels ofpotential ADCC effector cells in the blood of the infant macaquesbefore challenge. ��T cells were not measured; however, bothneutrophils and monocytes were present at adequate levels(8159 � 565 and 960 � 132/�l, respectively). In contrast, theabsolute mean numbers and percentages (Fig. 4) of NK cells inboth macaque groups were low at the time of challenge and sub-sequently increased by 2–4 wk. The percentages observed weresimilar to those reported for healthy rhesus neonates up to 3 wk ofage (30) and for SIV-infected infant macaques (31) but lower thanthe 9–13% reported for juvenile macaques. In older rhesus ma-caques, NK cells peak �2 wk post-SIV infection before returningto prechallenge levels (33), and a similar pattern was observed

here. Unfortunately, blood volumes were too small to investigateNK functionality.

DiscussionAs current vaccine candidates have not induced broadly neutral-izing Abs able to provide “sterilizing immunity” against HIV in-fection, protection that diminishes the initial viral burden to a levellow enough to provide prolonged viremia control, slow diseaseprogression, and avoidance of virus transmission has become thegoal of vaccine design. In this regard, if potent, vaccine-inducedAbs are present before HIV exposure, the ADCC mechanism couldprovide a rapid response since initial expansion of a specific T cellpopulation is not required. The ADCC mechanism targets virus-infected cells, but ADCC-mediating Abs might also prevent infec-tion by rapidly interacting with virus bound to CD4 target cells,leading to cell lysis by ADCC before virus entry. Systemic Abs inunderlying mucosal tissues might contribute to protection frommucosal challenge by blocking expansion of the founder seedstocks of virus-infected cells that lead to systemic infection (34).Neutralizing mAbs and immune IgG shown to mediate postexpo-sure prophylaxis (12, 13) might also protect in part by an ADCCmechanism. Delay of Ab treatment as much as 6 h after initialvirus exposure has blocked subsequent infection (13). While neu-tralizing activity could block cell-to-cell virus transmission, anADCC mechanism could eliminate cells expressing target Ags be-fore release of infectious virions. The ability of potent neutralizingmAbs to mediate ADCC activity has not been explored systemat-ically, although 2G12 and IgG1 b12 both possess such ability (23,35, 36).

In the present study, we addressed the protective efficacy ofADCC activity apart from neutralization, using nonneutralizing Abpreviously shown to mediate ADCC activity that correlated withreduced acute viremia following SIVmac251 intrarectal challenge(6, 9). Initial in vitro characterization of the pooled immune serumrevealed a complexity in the epitope(s) recognized by the ADCC-mediating Abs. ADCC Ab titers against gp120-coated targets were

FIGURE 3. Development of neutralizing Abs against the SIVmac251

challenge virus following oral administration to the neonatal macaques.Reciprocal titers are shown for two end points. �, An equivocal result wasobtained for this macaque: one titer � 25 and one titer of 55. Sufficientplasma was not available for a third repetition. The limit of detection was1:25; reciprocal titers � 25 are plotted as 25.

FIGURE 4. Absolute numbers (A) and percentages of NK cells (B) inthe macaques following oral challenge with SIVmac251. Week 0 is equiv-alent to 2–6 days of age. Values shown are means � SEM.

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higher than Ab binding titers against gp120 (Table I). This sug-gests that when gp120 was bound to CD4 on the target cell, aconformational change exposed alternate epitopes recognized bythe immune sera. This speculation is supported by the observationthat animals that received control IgG developed de novo ADCCactivity against gp120-coated target cells following SIV infection(Fig. 2D) but continued to be negative for gp120 binding Ab (datanot shown). Apparently, similar conformational epitopes appearupon SIV infection and induce Abs able to mediate ADCC killingof gp120-coated targets. It is also possible that ADCC is simplymore sensitive than ELISA, as suggested by Sawyer et al. (1).However, in contrast to our results, this greater sensitivity wasobtained using different Ags for evaluating binding (Dupont HIVELISA) and ADCC activity (vaccinia-HIV gp120-infectedtargets).

Our results show clearly that the immune IgG infused did notprotect newborn rhesus macaques from oral SIV infection asshown by viral burdens, disease progression, and survival timessimilar to those of neonates that received control IgG. Severalfactors may explain the difference between the previously ob-served correlation and the challenge outcome reported here.

First, a high challenge dose was administered to ensure infectionof the control neonates. The previous correlation of ADCC activitywith reduced acute viremia was observed following an intrarectalchallenge with �500 TCID50 of SIVmac251 (9), while in thepresent study, the neonates received two sequential oral dosesof 105 TCID50 SIVmac251 each. Recent studies in neonatal rhe-sus macaques (18) have used repeated lower-dose oral expo-sures of 104 TCID50 each (2 ml of 7 � 107 viral RNA copies/ml), which is still high compared with average levels of SIV inbreast milk during chronic SIV infection (2 � 102–2 � 103 viralRNA copies/ml; Ref. 37) but more relevant to natural infection.However, in the present study, insufficient immune IgG wasavailable for continuous transfer over several days of repeatedlow-dose challenges. Thus, the 400-fold greater dose used incomparison to the previous study may have obscured any pro-tection mediated by the immune IgG.

Second, the route of challenge may have influenced the out-come. Following oral infection of juvenile and neonatal macaques,SIV quickly accesses draining lymph nodes and rapidly spreads totissues throughout the body within 1–2 days (38). In contrast,while some rapid dissemination of SIV to distal sites occurs fol-lowing vaginal exposure, systemic infection is delayed �6 days byestablishment of small “founder” populations of infected cells inthe cervicovaginal mucosa, which then spread SIV throughout thelymphoid tissues (39). Thus, there may be a greater window oftime for immune intervention to control viral replication andspread. Whether rectal SIV transmission and dissemination is sim-ilar to vaginal infection has not been well investigated, nor havevarious challenge routes been directly compared. Nevertheless, itis plausible that the kinetics of oral infection may be more rapiddue to more rapid transfer of virus to the lymphatic system. If so,protection would be easier to achieve following rectal transmis-sion, explaining the positive correlation with ADCC activity in theearlier study and the lack of protection mediated by anti-envelopeAbs observed here.

The distribution of the passively transferred immune IgG in tis-sue/mucosal compartments after s.c. administration is also notknown. ADCC-mediating Ab was present in plasma of the neo-nates at the time of oral challenge. Whether Ab was also present inthe oral mucosa and at a level comparable to that in the rectalmucosa in the earlier study is not known. Rectal secretions in theearlier study were negative for ADCC activity (6). However, con-sistent with the presence of low-titered SIV-specific Ab in saliva

following passive transfer of SIV hyperimmune serum (15), low-titered ADCC activity against gp120-coated and SIVmac251-in-fected target cells (titers of �25 and ranging from �5 to 20, re-spectively) was present in saliva of the experimental macaques 1wk postchallenge. These titers declined over the next 3 wk,whereas saliva samples of the control neonates were consistentlynegative.

Third, the neonatal macaques may have lacked sufficient andfully functional NK effector cells at the time of challenge. NKcells are major effector cells that mediate ADCC and relatedADCVI activities. Monocyte/macrophages also mediate theseactivities and, in older animals, are probably the main effectorcells for ADCVI (8). The contribution of other cell types toADCC and ADCVI effector function in neonatal macaques isnot known. In macaques, as in humans, the proportion of NKcells increases with age. Neonatal macaques have �3.5% NKcells in peripheral blood (31) in contrast to �13% in adults(40). The mean percentage of NK cells in the neonates at chal-lenge was as expected, low (3.5%), and declined following SIVinfection instead of exhibiting the expected age-dependent in-crease (Fig. 4). It is possible that NK cells were insufficient forpotent ADCC activity in vivo. A significant negative correlationamong all 12 neonates between NK cell percentage and viralload was observed at wk 1 postchallenge ( p � 0.01), the firsttime point at which viremia was measured. This correlationdisappeared at later time points but suggests that NK cell num-ber may impact the outcome of viral exposures.

Additionally, human infants are less able to mediate ADCCagainst HIV-infected cells than adults (41). Blood volumes ob-tained from the rhesus neonates were not sufficient for assessmentof NK functionality. However, control of SIV infection by theADCC mechanism postchallenge may have been impaired in theneonatal macaques.

Fourth, too little immune IgG may have been passively trans-ferred. Based on anti-envelope binding titers, plasma Ab concen-trations at challenge were �13-fold lower than that in the originalsera/plasma pool. No benchmarks for protective ADCC levelshave been established. The mean net ADCC killing of SIV-in-fected targets in the previous study in which ADCC activity wascorrelated with reduced acute viremia was 31.1% (6), whereas inthe present study, net ADCC killing mediated by plasma from theexperimental neonates was 12.4%. The 2.5-fold lesser activity mayhave been below the threshold at which protection could be de-tected. ADCVI activity was also low at challenge in comparison toplasma from infected macaques.

Finally, ADCC activity alone may not be adequately protec-tive. In the previous study, although the immunized macaquesdid not possess neutralizing Abs, they exhibited strong cellularimmune responses (9, 42, 43). It is possible that ADCC activity,in conjunction with cellular immunity, acted to partially controlacute infection. Such adaptive cellular immunity was of coursenot present in the neonatal macaques. While confirmation of therole of individual immune parameters in protection is informa-tive, in vivo a single protective correlate may not exist. Rather,the sum total of immune responses may ultimately control in-fectious diseases.

Overall, the passively transferred immune IgG did not protectthe neonatal macaques from oral SIVmac251 challenge in this pilotexperiment. However, the results suggest a design for further stud-ies exploring the potential role in protection of nonneutralizing butADCC-mediating Abs. Passive transfer experiments should beconducted in juvenile or adult animal models, using more potentIgG preparations and exploring additional routes of virus trans-mission that allow lower challenge doses. ADCC remains a

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potentially important mechanism for vaccine development, span-ning innate and adaptive immunity. The extent to which it cancontribute to protective efficacy should be clearly defined.

AcknowledgmentsWe thank William Sutton and members of the Immunology Core, HumoralImmunity Subcore of the University of Washington Center for AIDS Re-search, Seattle Biomedical Research Institute for IgG purification; StephenWhitney for ELISA binding assays; Emily Blackwood, Kimberly Schmidt,and the veterinary staff, Colony Services and Clinical Laboratory of theCalifornia National Primate Research Center for their technical assistance.The following reagent was obtained from the AIDS Research and Refer-ence Reagent Program, National Institute of Allergy and Infectious Dis-eases, National Institutes of Health: CEM-NKr cells from Dr. PeterCresswell.

DisclosuresThe authors have no financial conflict of interest.

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