AIDS RESEARCH AND HUMAN RETROVIRUSESVolume 14, Number 5, 1998Mary Ann Liebert, Inc.

Administration of Recombinant Human Interleukin 12 to

Chronically SIVmac-Infected Rhesus Monkeys

NORIAKI WATANABE,1 JOSEPH P. SYPEK,2 STUART MITTLER,1 KEITH A. REIMANN,1PEDRO FLORES-VILLANUEVA,1 GERALD VOSS,1 CAROL I. LORD,1 and NORMAN L. LETVIN1

ABSTRACT

With the demonstration that interleukin 12 can enhance natural killer (NK) cell activity and drive CD4+ lym-phocytes toward T helper type 1 (Thl) responses, there is a strong rationale for exploring the use of this cy-tokine as an immunomodulatory therapy in HIV-1-infected individuals. To assess its potential safety andeffects on both immune and virologie aspects of HI V-1 infection, recombinant human IL-12 (rhIL-12) was as-

sessed in rhesus monkeys chronically infected with the simian immunodeficiency virus of macaques (SIVmac).The activity of rhIL-12 on rhesus monkey lymphocytes was confirmed with the demonstration that periph-eral blood lymphocyte lysis of the NK-sensitive cell line Colo was enhanced by this recombinant cytokine. Fur-ther, rhIL-12 was shown to induce interferon-y production by rhesus monkey lymphocytes in vitro. Then, inseparate studies, two treatment regimens of rhIL-12 were assessed in SIVmac-infected monkeys: a low-dose

regimen (0.1 fig/kg, daily for 4 weeks) and a high-dose regimen (2.5 /ug/kg, every 3-4 days, for 3 weeks). BothrhIL-12 treatment regimens were well tolerated by these virus-infected animals. The high-dose regimen ofrhIL-12 induced transient decreases in circulating lymphocytes in the SIVmac-infected monkeys. Furthermore,no changes in lymphocyte-associated SIVmac DNA or SIVmac plasma RNA levels were seen in the treated mon-

keys. These studies indicate that short-term treatment with rhIL-12 is well tolerated and causes no measur-

able changes in virus load in chronically SIV,na,-infected rhesus monkeys.

INTRODUCTION

With the accumulating evidence that effector T cell re-

sponses play a central role in containing the spread ofHIV-1 in infected individuals,1-6 attention is being focused on

the possibility that cytokine therapies that augment these cel-lular responses might prove useful in the treatment of AIDS.The cytokine interleukin 12 (IL-12) is particularly attractive as

a potential AIDS therapeutic. Initially characterized as a me-

diator that can directly enhance natural killer (NK) cell and cy-tolytic T lymphocyte (CTL) activity,7-11 this cytokine may be

capable of augmenting the specific immune response respon-sible for limiting viral replication in HIV-1-infected individu-als. Moreover, it has been shown that IL-12 can inhibit the

generation of helper T type 2 (Th2) responses (the T cell re-

sponses that enhance antibody production) and drive CD4+ in-

flammatory or helper T type 1 (Thl) responses (the T cell re-

sponses that augment cell-mediated immunity).12,13 Provoca-tive data generated in some laboratories suggest that a declinein the clinical status of HIV-1-infected individuals may be as-

sociated with a shift in the helper T cell response from a Thlto a Th2 response.14-16 If this is indeed the case, there is a

strong rationale for employing a therapeutic cytokine in HIV-1-infected individuals that might reverse or minimize thatCD4+ T cell shift.

The SIVmac-infected rhesus monkey provides a powerfulmodel system for exploring AIDS pathogenesis and novel AIDS

therapeutic strategies.17,18 This simian lentivirus has substan-tial sequence homology with HIV, infects CD4+ lymphocytesand macrophages, and induces an AIDS-like illness inmacaques. To explore the safety and potential utility of IL-12in the therapy of AIDS, we assessed the virologie and im-munologie consequences of recombinant human IL-12 (rhTL-12) administration in SIVmac-infected rhesus monkeys.

'Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215.2Preclinical Biology, Genetics Institute, Andover, Massachusetts 01810.

393

394 WATANABE ET AL.

MATERIALS AND METHODS

Animals and cytokine administration

The rhesus monkeys (Macaca mulatto) used in this studywere maintained in accordance with the guidelines of the Com-mittee on Animals for the Harvard Medical School and theGuide for the Care and Use of Laboratory Animals [Depart-ment of Health and Human Services Publication No. (NIH) 85-23, revised 1985]. All monkeys were infected by intravenousinoculation with uncloned simian immunodeficiency virus of

macaques (SIVmac), strain 251, which was propagated in hu-man peripheral blood mononuclear cells (PBMCs). Monkeyswere anesthetized with ketamine-HCl for all procedures.

Monkeys were administered rhIL-12 in separate studies bytwo regimens. In a low-dose treatment regimen, animals re-

ceived daily, subcutaneous injections of 0.1 pg/kg for 4 weeks.In a high-dose treatment regimen, animals received subcuta-neous injections of 2.5 /¿.g/kg, every 3-4 days, for 3 weeks.

Natural killer cell assays

Either Colo (NK lysis sensitive) or Daudi (NK lysis resis-tant) target cells were 51Cr-labeled and then incubated for 5 hrwith density gradient-isolated rhesus monkey peripheral blood

lymphocytes (PBLs) at effector-to-target ratios of 100:1, 50:1,25:1, and 12.5:1. rhIL-12 was added at concentrations rangingfrom 0.1 to 100 U/ml. Spontaneous release varied from 10 to

20%. Percent lysis was calculated as [(experimental release-

spontaneous release)/(100% release—

spontaneous release)] X

100.

rhIL-12 induction of IFN-y production by monkeyPBLs

Rhesus monkey PBLs were isolated by gradient centrifuga-tion. PBLs (5 X 105) were cultured for 24 hr in 48-well plates(Costar, Cambridge, MA) in 1 ml (final volume) of completeRPMI medium supplemented with 25 mM HEPES, 200 mM

glutamine (Cellgro; Mediatech, Hemdon, VA), 10% fetal calfserum (FCS; Bio-Wittaker, Walkersville, MD), and

penicillin-streptomycin (5000 U/ml; GIBCO, Grand Island, NY).Concanavalin A (ConA; Sigma, St. Louis, MO) was added at a

3 pg/ml final concentration. rhIL-12 was added at concentrations

ranging from 0.1 to 100 U/ml. Supernatants were harvested af-ter 24 hr of culture, centrifuged for 10 min at 1500 rpm, andstored at -70°C. rhIL-12-induced interferon y (IFN-y) produc-tion was measured using a monkey IFN-y ELISA kit (Biosource,Camarillo, CA) according to the protocol provided with the kit.IFN-y concentrations were expressed as picograms per milliliter.

Immunophenotyping of monkey PBLs

Immunophenotyping was performed as previously de-scribed,19 using the following monoclonal antibodies: CD14,MY4-FITC (fluorescein isothiocyanate) (Coulter, Miami, FL);CD2, T11-RD1 (Coulter); CD4, OKT4-PE (phycoerythrin)(Ortho Diagnostics Systems, Raritan, NJ); CD8, T8-PE (Dako,Carpenteria, CA); CD16, Leu lla-FTTC (Becton-Dickinson,San Jose, CA); CD3, FN18-FITC (Biosource); CD20,Bl-FITC (Coulter). In the high-dose experiment, a CD3-spe-cific antibody (FN18-FITC; Biosource) was used instead of

anti-CD2. Aliquots of 50 pi of EDTA-anticoagulated bloodwere mixed with 100 pi of phosphate-buffered saline(PBS)-0.1% bovine serum albumin (BSA) and monoclonal an-

tibodies at the recommended concentrations and incubated for30 min at room temperature. Cells were washed once with 1ml of PBS. Erythrocytes were lysed using a commercial lysingreagent (whole blood lysing kit; Coulter) according to manu-

facturer recommendations. After two additional washes, cellswere resuspended in 0.25-0.50 ml of PBS-1% formalin and re-

frigerated until analyzed.Samples were analyzed routinely on an EPICS-XL (Coulter)

flow cytometer. The lymphocyte acquisition gate was set on

forward and log 90° light scatter parameters and verified to in-clude predominantly lymphocytes using CD2, CD 14, and CD20fluorescence. For each sample, a minimum of 5000 cells withinthe lymphocyte acquisition gate was analyzed.

Automated cell counts on EDTA-anticoagulated rhesus

monkey blood samples were performed using a Series 9000

hematology series cell counter (Serono Baker Diagnostics, Al-lentown, PA) in which the cell size discriminators were opti-mized for this species. Leukocyte differential counts were per-formed manually by counting 100 white blood cells on a

Wright's-stained blood smear.

Quantitation of SIV^c provirus and RNA

A semiquantitative polymerase chain reaction (PCR) was

performed on DNA extracted from density gradient-isolatedPBLs of SIVmac-infected monkeys, using a method similar to

that previously described.20 Two hundred nanograms of PBLDNA was amplified by PCR through 30 cycles of 1 min at 50°Cand 1 min at 72°C, using primers to produce a 200-bp productfrom the SIVmac gaglpol region. After 30 cycles, an 8-min ex-

tension cycle was performed at 72°C. Each tube contained 10pi of 10X PCR buffer, 16 pi of dNTPs, 20 pi of primer, 0.5

pi of Taq polymerase, and 33.5 pi of water. These reagentswere prepared as master mix solutions and aliquotted into eachtube. Sample tubes were run with rhesus monkey CD4-specificprimers that produced a 333-bp product to control for DNA

loading. DNA (200 ng) was added in a volume of 20 pi. ThePCR products were electrophoresed through a 2% agarose gel,transferred to a nylon membrane, and hybridized with an in-ternal probe that was radiolabeled with 32P. Quantitation was

performed with a gel scan program by comparison with a

standard generated from a gaglpol fragment of known concen-

tration.Quantitative assays for the measurement of SIV RNA were

performed by Chiron Diagnostics (Emeryville, CA), using a

branched DNA signal amplification assay similar to the Quan-tiplex HIV-RNA branched DNA assay.21 In the SIV assay, tar-

get probes were designed to hybridize with the pol region ofthe SIVmac group of strains, including SIVmac251. The assayresults were quantified by comparison with purified and quan-titated in v/iro-transcribed SIV pol RNA.

RESULTS

Before initiating in vivo studies of rhIL-12 in SIVmac-infectedrhesus monkeys, we assessed the in vitro responsiveness of rhe-

IL-12-TREATED SIVmac-INFECTED MONKEYS

sus monkey PBLs to this recombinant cytokine. PBLs of twonaive rhesus monkeys (196 and 275) were assessed for lysis oftwo tumor cell lines in vitro: the NK-sensitive cell line Coloand the NK-resistant cell line Daudi. As shown in Fig. 1, an

augmentation of NK killing of Colo cells was seen followingaddition of increasing concentrations of rhIL-12. This cytokinedid not augment PBL killing of the Daudi cells (data not shown).

The ability of rhIL-12 to induce IFN-y production by rhesusmonkey PBLs was also evaluated (Fig. 2). PBLs from two nor-

mal monkeys (161 and 254) were stimulated for 24 hr withConA in the presence of increasing concentrations of rhIL-12,and IFN-y was measured in the culture supernatants. rhIL-12induced IFN-y production by the monkey lymphocytes in a con-

centration-dependent fashion. Thus, rhIL-12 had functional ac-

tivity in vitro in rhesus monkey PBLs. These observations, as

well as the demonstration that rhIL-12 can protect macaquesfrom infection with Plasmodium yoelii,22 suggested that the rhe-

control 100 10 1 0.1

IL-12 (U/ml)

IL-121 U/ml

IL-120.1 U/ml

FIG. 2. rhIL-12 induces rhesus monkey PBL production ofIFN-y in vitro. ConA-stimulated PBLs from two normal mon-

keys (161 and 254) were cultured for 24 hr with increasing con-

centrations Of rhIL-12 as noted and culture supernatants were

assayed for IFN-y by ELISA. Bars of different shades repre-sent data from cultures established with PBLs of different mon-

keys.

fr- io

control IL-12100 U/ml

IL-121 U/ml

IL-120.1 U/ml

E:T ratio1100:1 «50:1 25:1 012.5:1

FIG. 1. rhIL-12 augments rhesus monkey PBL killing of theNK-sensitive target cell line Colo. 51Cr-labeled target cells were

incubated for 5 hr with PBLs in the noted concentrations of re-

combinant human IL-12 at E:T ratios of 100:1, 50:1, 25:1, and12.5:1. Spontaneous release varied from 10 to 20%. No cy-tokine-induced change in PBL lysis of Daudi cells was seen

(data not shown). Each panel represents results from an indi-vidual monkey.

sus monkey could be a valid model for assessing the effects ofrhIL-12 on the clinical course of AIDS.

We then assessed the consequences of rhIL-12 treatment ofSIVmac-infected monkeys. Four infected monkeys received sub-cutaneous inoculations of rhIL-12 at a dose of 0.1 /ug/kg dailyfor 4 weeks; four infected monkeys received vehicle alone ac-

cording to the same schedule. This dosage of rhIL-12 was se-

lected on the basis of observations made in repeated dose stud-ies in healthy monkeys.23,24 The animals were assessed

prospectively for changes in white blood cell count, PBL

phenotype, and SIVmac DNA load in PBLs.There was no apparent systemic or hématologie toxicity of

the course of treatment with rhIL-12. Hématologie parametersnever changed during the period of treatment with this doselevel of rhIL-12 (data not shown). Immunologie parameters inthe rhIL-12-treated rhesus monkeys demonstrated no signifi-cant change from pretreatment baseline values or in compari-son with monkeys treated with vehicle alone. No change in rel-ative representation of CD2, CD4, CD8, CD 16, or CD20 cellswas seen following treatment with rhIL-12 (data not shown).

SIVmac proviral load in the PBLs of the rhIL-12-treated an-

imals showed no consistent changes over the course of the ex-

periment (Fig. 3B). The four monkeys treated with rhIL-12 hada pretreatment mean proviral copy number of 61 in 200 ng ofDNA. At the end of the study (the last week of rhIL-12

396 WATANABE ET AL.

Vehicle treated

1000

rhlL-12 treated

-20 -10 0 10 20 30 40 50 60

s 1000

-20 -10 0 10 20 30 40 50 60

D

100

10

mum

-10 0 10 20 30 40

Days

FIG. 3. SIVmac provirus levels do not change in PBLs of infected rhesus monkeys following treatment with rhIL-12. Semi-quantitative PCR to detect SIV provirus was performed on DNA extracted from PBLs of SIVmac-infected monkeys. Quantitationwas by comparison with a standard generated from a gaglpol fragment of known concentration. Each line represents data froman individual animal. In (A) and (B) the 4-week period of daily treatment with 0.1 pg of rhIL-12 per kilogram is bracketed; in(C) and (D) downward pointing arrows indicate days of treatment with rhIL-12 (2.5 /xg/kg). (A) Monkeys treated daily for 4weeks with vehicle alone; (B) monkeys treated daily for 4 weeks with rhIL-12; (C) monkeys treated every 3 to 4 days with ve-

hicle alone; (D) monkeys treated every 3 to 4 days with rhIL-12.

administration) the mean proviral copy number was 68 in 200 ngof DNA. The control animals had a mean proviral copy numberof 91 in 200 ng of DNA before commencement of the experi-ment and 80 in 200 ng of DNA at the end of the study (Fig. 3A).

With no evidence of rhIL-12-related effects in these SIVmac-infected monkeys, a further study was initiated in infected mon-

keys, using a higher dosage of rhIL-12. Three infected mon-

keys received subcutaneous inoculations of rhIL-12 at a doseof 2.5 /Ag/kg on days 1, 4, 8, 11, 15, and 18; three uninfected

monkeys received the same dosage of rhIL-12 according to thesame schedule; and three infected monkeys received vehiclealone, according to the same schedule. The animals were as-

sessed prospectively for changes in white blood cell count, he-moglobin, hematocrit, PBL phenotype, and both SIVmac DNAand RNA load.

There was no apparent systemic toxicity during the course

of treatment with rhIL-12 in infected or uninfected monkeys.Body weights of the infected monkeys never changed duringthe period of treatment with rhIL-12 (data not shown). Fur-thermore, there was no consistent decline in blood hemoglobin,hematocrit, or platelet counts in the SIVmac-infected monkeysfollowing rhIL-12 treatment. Circulating total lymphocyte pop-ulations were transiently decreased during the time of treatment.

These decreases were seen in absolute numbers of circulatingCD20+, CD3+, CD3+CD4+, CD3+CD8+, CD3-CD8+, andCD8+CD16+ cells (Fig. 4). However, these treatment-associ-ated changes in SIVmac-infected monkeys were similar to the

changes seen in uninfected monkeys.

Analysis of viral DNA load in the SIVmac-infected animalstreated with rhIL-12 and those treated with placebo showed no

consistent changes over the course of the experiment (Fig. 3Cand D). While the provirus copy number per 200 ng of DNA var-

ied from animal to animal and there was some variability be-tween sampling points in individual animals, no consistent treat-

ment-associated trend was seen. No significant change in SIVmacRNA was detected in the plasma of the monkeys following high-dose rhIL-12 treatment (Table 1). Plasma SIVmac RNA was es-

sentially unchanged through the period of rhBL-12 treatment.

rhIL-12 treatment of all of these animals but for one in theSIVmac-infected group (320-91) elicited anti-rhIL-12 antibodyresponses, detected as early as 4 days following the last ad-ministration of recombinant cytokine (data not shown). In threeof the six rhIL-12-treated monkeys, successive administrationsof rhIL-12 resulted in lower concentrations of circulating cy-tokine. Thus, the elicited rhIL-12-specific antibodies may haveneutralized the administered cytokines in these monkeys.

DISCUSSION

The present studies indicate that systemic administration ofrhIL-12 is well tolerated in an AIDS virus-infected nonhuman

primate species. In view of the interest in assessing rhIL-12 as

an immune modulatory therapeutic in HIV-infected humans,this observation provides reassurance that the recombinant cy-tokine should be well tolerated in individuals with AIDS.

IL-12-TREATED SIVmac-INFECTED MONKEYS 397

-20 -10

Days0 10 20

[ Lymphocytes MCD3 *CD20

FIG. 4. Administration of rhIL-12 (2.5 /xg/kg) to SIVmac-infected rhesus monkeys causes a transient lymphopenia during theperiod of treatment. Immunophenotyping of PBLs was performed using a whole-blood lysis technique: (•) total lymphocytes;(D) CD3+ T cells; (*) CD20+ B cells. (A) Mean absolute counts from uninfected monkeys treated with rhIL-12; (B) mean ab-solute counts from SrVmac-infected monkeys treated with the control vehicle; (C) mean absolute counts from SIVmac-infectedmonkeys treated with rhIL-12. Arrows indicate treatment days.

If an IL-12-mediated shift in a Th2- to a Thl-biased immuneresponse will prove beneficial in an AIDS virus-infected indi-vidual, one might have predicted a therapeutic response in thecytokine-treated SIVmac-infected monkeys. That such a re-

sponse was not seen could be explained by the fact that the re-

peated administration of rhIL-12 at pharmacologie doses to non-

human primates in the present studies elicited an anti-cytokineantibody response that may have neutralized the immunologieactivity of the cytokine. If a prolonged period of elevated, bi-ologically active IL-12 is needed to generate a Th2 —»Thl shift,such a shift might not have occurred in this setting. The ab-sence of a beneficial therapeutic effect in these rhIL-12-treated,SIVmac-infected monkeys may also indicate that the biologic

events involved in the generation and maintenance of an an-

tiviral cell-mediated immune response in a higher primate maybe more complex than is implied by the simple Thl/Th2 para-digm.

In vitro studies have suggested that HIV-1 replication in hu-man PBLs can actually be increased by the addition of IL-12in vitro.25 The fact that even a transient increase in viral loadwas not seen in the rhIL-12-treated, SIVmac-infected monkeyssuggests this may not prove to be true in vivo. Thus, trials ofrhIL-12 as an immune modulator in HIV-1-infected humansshould be safe to pursue. Therapeutic trials of rhIL-12 in HIV-1-infected individuals will be needed to assess the utility of this

cytokine as an immune modulator in AIDS.

398 WATANABE ET AL.

Table 1. Plasma SIVmac RNA Levelsfollowing Initiation of Treatment of

Rhesus Monkeys with High-Dose rhIL-12

Plasma SIVnac RNA(kEq/mP)

Days after initiation of treatment*

Group Monkey —7 8 22

Placebo treated 172-93 894 74 506315-91 636 331 339371-92 13,000 26,000 33,000

rhIL-12 treated 209-93 510 295 400312-93 142 278 274320-91 2,100 860 670

"Viral load in plasma was quantified by a branched DNA(bDNA) signal amplification assay.

bSpecimens were obtained at the indicated times after initi-ation of the high-dose regimen (2.5 ¿tg/kg, every 3-4 days, for21 days) of rhIL-12.

ACKNOWLEDGMENTS

The authors thank Anna Weinces and Zareen Kapadia for

analyzing specimens, and Debra Ayles for preparation of the

manuscript. We also thank Page Bouchard, Michael DeBruin,Robert Schaub, and Victor van Cleave for their suggestions andcomments in reviewing this manuscript. This work was sup-ported by NIH Grants AI-20729 and RR-00168 and by fundsfrom the Genetics Institute.

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Norman L. LetvinDivision of Viral Pathogenesis

Department of MedicineBeth Israel Deaconess Medical Center

RE-113330 Brookline Ave.

Boston, Massachusetts 02215

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6. Eduardo O'Neill, Vanda Bostik, David C. Montefiori, Edmundo Kraiselburd, François Villinger. 2003. IL-12/GM-CSF Coadministration in an SIV DNA Prime/Protein Boost Protocol Enhances Gag-Specific T Cells But NotVirus-Specific Neutralizing Antibodies in Rhesus Macaques. AIDS Research and Human Retroviruses 19:10, 883-890.[Abstract] [Full Text PDF] [Full Text PDF with Links]

7. M.B. Gardner, P.A. Luciw, M.P. CarlosSimian Retroviruses 195-262. [CrossRef]8. P LENA. 2002. Co-immunization of rhesus macaques with plasmid vectors expressing IFN-$gamma;, GM-CSF, and

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