A simian immunodeficiency virus envelope V3 cytotoxic T-lymphocyte epitope in rhesus monkeys and its restricting major histocompatibility complex class I molecule Mamu-A*02.

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A

Simian

Immunodeficiency Virus Envelope V3 Cytotoxic

T-Lymphocyte

Epitope in Rhesus

Monkeys

and

Its

Restricting

Major

Histocompatibility Complex

Class I

Molecule

Mamu-A*02

NORIAKI

WATANABE,'

STEPHEN N.

McADAM,2

JONATHANE.

BOYSON,2

MARIAN S. PIEKARCZYK,2

YASUHIRO YASUTOMI,1 DAVID I.WATKINS,2 ANDNORMAN L.

LETVIN1*

HarvardMedicalSchool, Beth IsraelHospital, Boston, Massachusetts 02215,1and WisconsinRegional PrimateResearch Center and Departmentof Pathology, University ofWisconsin

MedicalSchool, Madison, Wisconsin 537152

Received 25 January 1994/Accepted 18July1994

The use ofthe simianimmunodeficiency virus(SIV) macaquemodel forassessinghumanimmunodeficiency

virusvaccine strategieswill befacilitatedbythecharacterizationofpredominantSIV

cytotoxic

T-lymphocyte

(CTL) epitopes and their restricting majorhistocompatibilitycomplex (MHC) class I molecules inmacaque species.We now define a rhesusmonkey SIVmacCTLepitopein the thirdhypervariable regionof theenvelope glycoprotein of the virus. Thisepitope,YNLTMKCR,contains the first twoamino acidsofacysteine-cysteine loop which is the

SIVmac

analog ofthe human immunodeficiency virus type 1 V3 loop. We also employed one-dimensional isoelectricfocusingtocharacterize the MHC class I molecule of the rhesusmonkeythatbinds this

SIVmaC

envelopepeptide fragment.Cloningandsequencingthe cDNAencodingthis rhesusmonkeyMHC class I moleculedemonstratesthat it is anewly described HLA-Ahomolog, Mamu-A*02.This viral CTLepitope

and itsrestricting MHCclass I molecule willfacilitatethe useofthe

SIVmac

rhesusmonkeymodel for studies ofenvelope-basedvaccinestrategiesand forexploring AIDSimmunopathogenesis.

Aneffective human immunodeficiency virus (HIV) vaccine should be capable of stimulating HIV-specific cytotoxic T

lymphocytes (CTL) (20, 34). Since HIV is spread at least in partthrough exposuretocell-associatedvirus,vaccine-elicited neutralizing antibody responses alone maynotprevent infec-tion with this virus. Considerable experimental evidence has implicated virus-specific CTL in the control of HIV infection.

Inanimals and inhumans, the early containment of AIDS virus replication appears to coincide with the development of

virus-specificCTL responses (27,28, 44). Moreover, the replication ofHIV inCD4+ lymphocytescanbe inhibitedby CD8+ cells withphenotypic characteristics of CTL (33, 35). It is, therefore, crucial that the evaluation ofHIVvaccine strategies includes assessing the elicitation of virus-specific CTL.

A number of HIV vaccine strategies currently being

ex-plored employ the envelope glycoprotein of the virus as the soleimmunogen(4).Theseapproaches include the use of live

vectors carrying the HIV env gene and recombinant HIV

envelope glycoprotein formulated with adjuvant. A model system in which to explore such envelope-based AIDS virus vaccines for the ability to elicit envelope-specific CTL would be extremely valuable.

The simianimmunodeficiency virus (SIV) macaque model has provenuseful in assessing vaccine strategies for the prevention ofHIVinfection (19). Our previous definition of a

predomi-nant

SIVmac

Gag CTL epitope and its restricting major histo-compatibility complex (MHC) class I allele Mamu-A*01 has facilitated the use of this model system for exploring vaccine approaches to the induction of AIDS virus-specific CTL re-sponses (23, 39). A detailed understanding of an

SIVmac

*Corresponding author.Mailing address:

Harvard

Medical School,

BethIsraelHospital, 330 Brookline Ave., Boston, MA 02215. Phone: (617)735-2766. Fax:(617)278-8210.

envelope epitopewould be of considerable value in devising strategiesforevaluatingtheefficacyof variousenvelope-based

vaccines. Moreover, the definition of further SIV CTL epitopes beyond thesingle definedGagpeptideand its restrict-ing MHC class I allele would greatly expand the ability to performAIDS immunopathogenesis studies with this

nonhu-manprimatemodel. Wenowdefinean

SIVmac

envelopeCTL

epitopeand itsrestrictingMHC classImolecule in the rhesus

monkey. Interestingly,thisepitopeincludesaportionofthe V3

loop analog in the third hypervariable region of the

SIVmac

envelope.

MATERUILSANDMETHODS

Animals.Heparinized bloodwasobtainedfrom rhesus

mon-key 381 after infection with

SIVmac

isolate 251. This animal

wasmaintained inaccordance with guidelines of the

Commit-tee onAnimalsforthe Harvard Medical School and the Guide

forthe Care and Useof Laboratory Animals (33a).

Generation of CTL effector cells. Peripheralblood lympho-cytes (PBL) isolated by Ficoll-diatrizoate density gradient

centrifugationwerecultured for 2 to 3 days at 1 x 106cells per mlwith 5

[Lg

ofconcanavalinAper ml in RPMI 1640(GIBCO, GrandIsland, N.Y.) supplemented with L-glutamine (2 mM),

penicillin (50 U/ml), streptomycin (40 ,ug/ml), and 10% fetal calfserum (ICN Biomedicals, Costa Mesa, Calif.). This me-diumwasthen supplemented with human recombinant inter-leukin-2 at a concentration of 20 U/ml, and cultures were maintained another 3 days. For studies to assess the fine

specificityof the CTL epitope recognition, PBL cultures were established initially with either a pool of 4

SIVmac

Env

synthetic peptides, eachat a5-,ug/ml concentration, orwith 5 ,ugofasingle peptide alone with no concanavalinAadded.

Isolation ofCD8+ lymphocytes. CD8+ cells were isolated by incubating the cultured cells with CD8 monoclonal

anti-6690

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41

V)

.' 3

U)

2

0

af)

41_U1)

a-0 5 10 15 20 25 30 35 40 45 50 >C

SlVmac Env peptide number

FIG. 1. SIVmacenvelope peptide recognition byPBLeffector cells from rhesus monkey 381. B-LCL were pulsed with a panel of 54

peptides representing sequential overlapping fragments of the entire

SIVmaCenvelope protein and assessedastargetpopulations ina5'Cr

releaseassaywith effector PBL from rhesus monkey381. Asacontrol

target, autologous B-LCLwereinfected witharecombinant vaccinia

virus-SIVmac Envconstruct (V-Env). An EST ratio of 80:1 was

em-ployed in this study.

body (7Pt3F9; 1:500 dilution of ascites; S. Schlossman,

Dana-Farber Cancer Institute, Boston, Mass.) and thensubjectedto

immunomagnetic bead separation (Dynal, Oslo, Norway). After furtherculture, the magnetic beadswereremoved from

thepurified CD8+ cells by magnet. The live cellswere then

isolated by Ficoll-diatrizoate density centrifugation and

as-sessedfor effector function.

Cytotoxicity assay. Target cellswere autologous or

alloge-neic B-lymphoblastoid cell lines(B-LCL), generated through herpesvirus papio immortalization of PBL,ortransfected ClR

cells (23). These cells were incubated at 37°C in a 5% CO2

atmosphere with S ,ug of peptide per ml or recombinant

vaccinia virus expressing SIVmac env or the irrelevant equine

herpesvirus gHgene(40) and 0.5 ,uCi of Na2CrO4perml for

16 h and then washed twice. Cultured PBL were used as

effector cells. 51Cr-labeledtarget cellswereincubated for 5 h

with effector cells at EST ratios of 80:1, 40:1, 20:1, and 10:1. Spontaneous release varied from 10to 20%. Specific release

was calculated as [(experimental release - spontaneous

re-lease)/(100% release - spontaneousrelease)] x 100.

SIVmaC

envelope peptides. Overlapping25-amino-acid pep-tidesnumbered 1through54weresynthesized bythe method

ofHoughtonetal.(10), usingtheSIVmacenvelopesequenceof Franchini et al. (5). Each consecutive peptide overlaps the preceding oneby 8 amino acids.

Radioimmunoprecipitation. Aliquots (5 x 106 cells) from

the B-LCL were cultured in methionine-free medium for 30

min. [35S]methionine (200 ,Ci; ICNRadiochemicals, Irvine, Calif.; specific activity, 1,000 Ci/mmol)wasthen addedtoeach culture. At the end ofa5-h incubationperiod, the cellswere

pelleted and frozenat -70°C. Cellswerelysed for 30minon

ice in 500,lIoflysisbuffer(20mMTris-HCl[pH 8.0],0.15M

NaCl, 1 mM MgCl2, 0.1 mM phenylmethylsulfonyl fluoride, 5% TritonX-114). The Triton X-114 used in the lysis buffer had been precondensed three times by adding cold distilled

H20, heatingat37°Cfor 5min, separatingintodetergentand

aqueousphases by centrifugation at300 Xgatroom

temper-ature, anddiscarding theaqueous phase. Following lysis,the

sampleswere separatedintodetergentandaqueousphasesas

described above, the aqueous phase was discarded, and the lower detergent phase was brought up to 1 ml with NET-N

A

u) 30

~20 -|

>0

O

4U)

10-none 17 18 19

Peptide number 20

B

V

30-

20-U)

10

none 18 19 cont

Peptide number

FIG. 2. Peptides 18 and 19arerecognized by CD8+ SIVmac

enve-lope-specific effector cells from rhesus monkey 381. (A) PBL of monkey 381werestimulated in vitro withSIVmacEnv peptides 17, 18,

19, and 20 and assessed for lysis of B-LCL pulsed with each individual peptide. (B)PBLofmonkey 381werestimulated in vitro withSIVmac Env 18, positively selected for CD8+ cells, and assessed for lysis of B-LCL pulsed with SIVmac Env peptides 18 and 19 and a control (cont.) peptide. 51Cr releasewasassessed in both studies at anE/T

ratio of 10:1.

(150mMNaCl, 5 mM EDTA, 50 mM Tris-HCl [pH 8.0], 0.5%

NonidetP-40;Sigma). Sampleswereprecleared bytwo

succes-siveincubationson30,ul (50%

[vol/voll)

of protein

A-Sepha-rose CL-4B beads(Sigma) previously saturated with an

irrel-evant ascites and then precleared on protein A-Sepharose

beads alone. Allpreclearing stepswerecarried outfor 1 h at

4°Cwith continuousshaking. Forimmunoprecipitation,50 ,ul

ofprotein-A Sepharosebeadswasincubated for 4 h with5 ,ul

ofW6/32ascites and washed twice with NET-N. Immunopre-cipitation was performed overnight, and the beads were

washed once with NET-N. Each immunoprecipitate was

treated with 20

[lI

of neuraminidase (10 U/ml; type VIII; Sigma)in0.1 M sodiumacetatebuffer(pH 5.5)-i mMCaCl2

on ashaking platform at37°C for 3 h and then washed three times with NET-N.

1-D IEF. One-dimensional isoelectric focusing (1-D IEF)

wascarriedoutaspreviouslydescribed(37). Briefly,a0.75-mm

polyacrylamide gel was made with 5.4% ampholines (LKB,

Gaithersburg, Md.) comprising4%pH5to7,1%pH3.5to10,

and0.4%pH7to9.Immunoprecipitateswereresuspendedin IEFsamplebuffer(9.5Murea,4%[vol/vol] ampholine [pH3.5 to 10],5%2-mercaptoethanol, 2% Nonidet P-40,0.01%

bro-mophenol blue), and the gelwas run on a DNAsequencing

apparatus (SQU-2000; American Bionetics, Hayward, Calif.)

foratotal of 13 h, with thevoltage risingfrom 400to 1,500V ataconstantpowerof 10 W. Afterelectrophoresis,thegelwas

treated with IEF fixative (0.13 M sulfosalicylic acid, 0.7 M trichloroacetic acid, 30% methanol), fixed in 10% methanol and 7.5% acetic acid, treated with En3Hance (New England Nuclear, Boston, Mass.) for 60 min, washed for 15 min in distilled water, dried, and visualized by autoradiographywith XAR-5 film(Eastman Kodak, Rochester, N.Y.).

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TABLE 1. TheSIVmacenvelope CTL epitope (residues 306to313) iswell conserved among macaque andmangabeySIV isolatesa

Isolate Sequenceb Species

SIVMM251 YNLTMKCR Macaque(rhesus)

SIVMM32H --- Macaque (rhesus)

SIVMM142 --- Macaque (rhesus)

SIVMM239 --- Macaque (rhesus)

SIVMNE --- Macaque (pigtailed)

SIVMMlA1 ----I--- Macaque(rhesus)

SIVSTM ---s-- Macaque(stump tailed)

SIVSMMH4 ---R-- Sooty mangabey

SIVSMMPBJ ---R-- Sooty mangabey

SIVAGMTYO ---SVT-- African greenmonkey

HIV2ROD ---SLH-K Human

SIVMNDGB1 -G-KIE-H Mandrill

SIVSYK LR-QII-E Sykes' monkey

HIVlHXB2R TSVEIN-T Human

HIVMN ESVZIN-T Human

HIVSF2 ESVAIN-T Human

a Sequences (withaccession numbers shown inparentheses) wereobtained from the Human RetrovirusandAIDS databaseattheLos Alamos National Laboratory: SIVMM251 (M19499), SIVMM32A (D01065), SIVMM142

(M16403), SIVMM239(M33252),SIVMNE(M32742), SIVMMlAl(M76764),

SIVSTM(M83293),SIVSMMH4(X14307),SIVSMMPBJ(M31325), SIVAG-MTYO(X07805), HIV2ROD (M15390), SIVMNDGB1 (M27270), SIVSYK

L06042), HIVlHXB2R(K03455),HIVMN(M17449),and HIVSF2(K02007).

bDashes indicateidentitywith the SIVMM251 sequence.

RNA extraction, cDNA synthesis, and PCR. mRNA was

extracted from PBL of monkey 458 by using oligo(dT)25

Dynabeads (Dynal, Great Neck, N.Y.) accordingto the

man-ufacturer's instructions. PBL(3 x 106)werewashed with cold

phosphate-buffered saline andresuspended in 100

RI

oflysis

buffer(10mMTris [pH 7.5], 140 mM NaCl, 5 mM KCl, 1% NonidetP-40,3 ,u of RNasin[Promega, Madison, Wis.]).The

lysates were centrifuged, and the supernatants were trans-ferredto 1.2 mg of washedoligo(dT)25 beads. Aftera 4-min

incubation at room temperature, samples were magnetically

isolatedandwashed twice. Beadswereresuspendedin20 pIof

elution buffer (2 mM EDTA, pH 7.5) containing 1 pul of

RNasin,and thesuspensionwasheatedto65°Cfor 2minand

placed inthe magnet,after which the eluatecontaining poly(A)

RNA was withdrawn to a new tube on ice. cDNA synthesis

wasperformedunder thefollowingconditions. RNasin(3 pI),

0.5 mM (each) deoxynucleoside triphosphates (dNTP), 100 mMdithiothreitol, 30 pmol of 3' primer (3' PRH3; 5'-GCAA

GCTFIAGTCCCACACAAGGCAGCTG-3'), and 12U of

Su-perscript M-MLV (Gibco/BRL, Gaithersburg, Md.) were

30 05

._.

- 20 Li

10

o

303-1 303-2 303-3

transfected C 1 R targets

FIG. 3. Effector cells from rhesus monkey 381 lyse SIVmac

env-expressingClR cells transfected with the MHC class I cDNA Mamu-A*02 (303-3). Peptide 18-stimulated PBL from rhesus monkey 381

were assessed for the ability to lyseSIVmac env-expressing ClR cells transfected with threedifferent rhesus monkey MHC class I cDNAs. The

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assaywasdoneatEST ratios of 80:1 (R), 40:1 (0), 20:1 (C]), and 10:1 (X).

FIG. 4. ClR cellstransfected with Mamu-A*02(303-3)expressan

MHCclass I molecule that hasanisoelectricpointthat is identical to that ofan MHC class I molecule expressed by a B-LCL of rhesus monkey 458.

present.Sampleswereincubated for 1 hat37°Cand heatedto

94°Cfor 3 min.Ten microliters ofsampleswasemployedina PCRmixture that contained 50 mMKCl,10 mMTris-HCl,1.5 mMMgCl2, 0.01%gelatin,and 0.25 mM(each)dNTP. Primers

wereaddedasfollows: 60pmolof the 5'primerLPXI(5'-GC

CTCGAGATGACGGTCATGGCTCCCCGAACC-3') and 30

pmolof 3'primer (3'PRH3). Sampleswereheatedto95°Cfor 2 minand thencooledto 37°Cfor 5min, after which 5 U of

Taq polymerasewas added. Cycleconditions usedwere94°C

for 1 min20s;37°Cfor3min20s; and72°Cfor 10min. Subcloning, sequencing, and transfection. After amplifica-tion,thePCRproductwasligatedintopKG5,andthe first 200 bases encoding the leader peptide and part of the alpha-1 domain of plasmids with full-length inserts were sequenced

(29). Three distinct clones were isolated from the rhesus

monkey cDNA, and at least three copies of each of these

cDNAsweresequenced.ClRcellsweretransfected with 20 ,ug

oflinearizedplasmid by electroporation (0.2kV, 960 ,uF) and

selected in mediumcontaining 1.8mgof G418 (GIBCO,Life

Technologies, Inc.)per ,ul. Expressed MHC classI molecules wereprecipitated withW6/32andfocused by1-D IEF.

RESULTS

Definition ofSIVmacenvelope CTL epitopeinvirus-infected rhesus monkey. Since CD8+ CTL recognize fragments of endogenously synthesized viral protein expressed in

associa-tion with MHC classImoleculesonthe surface of autologous

infectedcells,theprecise regionsofaviralproteinrecognized

byaspecific CTL population canbe determinedby assessing

CTLrecognition oftarget cellspulsedwith syntheticpeptide fragments representing regions of viral protein (30, 32). PBL

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LeaderPeptide Alpha 1

C CTCCTCCTG GTG CTC TCA GGG GCC CTGGCC CTG ACT CAG ACCCGGGCA GGC TCT CACTCC ATG AGG TATTTCTAC ACC TCC L L L V L S G A L A L T Q T R A G S H S M R Y F Y T S ATG TCC CGG CCCGGC CGC TGG GAG CCC CGCTTC ATC GCCGTG GGC TAC GTG GAC GACACG CAGTTC GTG CGGTTCGAC AGC

M S R P G R W E P R F I A V G Y V D D T Q F V R F D S GAC GCC GCG AGC CAGAGGAGGAGCCG CGG GCG CCG TGGGTGGAG CAG GAGGGTCCAGAG TATTCG GACCGGGAG ACACGG

D A A S Q R H E P R A P W V E Q E G P E Y W D R E T R

Alpha 2 AACATGAAG GCCGAG ACA CAG AAT GCC CCAGTGAACCTG CGG AAC CTG CGC GGC TACTAC AAC CAG AGC GAG GCC GGG TCT N M K A E T Q N A P V N L R N L R G Y Y N Q S E A G S CAC ACC ATC CAGAGG AT TAC GGC TGCGACCTGGGGCCG GACGGG CGC CTC CTC CGCGGG TAT CAC CAGTCC GCC TAC GAC

H T I Q R M Y G C D L G P D G R L L R G Y H Q S A Y D GGCAAGGATTAC ATC GCCCTG AAC GAG GACCT CGCTCCTGG ACC GCC GCG GACAM GCG GCT CAGAAC ACC CAG CGCAAG G K D Y I A L N E D L R S W T A A D M A A Q N T Q R K TGGGAG GCG GCGGGT GAG GCG GAG CAG CAC AGA ACC TAC CTG GAG GGC GAGTGCCTGGAG TGGCTC CGC AGA TAC CTGGAG

W E A A G E A E Q H R T Y L E G E C L E W L R R Y L E

Alpha3

AACGGGAAG GAGACG CTGCAG CGCGCG GAC CCCCCC AAG ACA CATGTGACC CAC CACCCC GTC TCT GACCAAGAG GCC ACC N G K E T L Q R A D P P K T H V T H H P V S D Q E A T CTGAGGTGCTGG GCC CTG GGC TTCTAC CCT GCG GAG ATC ACACTGACC TGG CAG CGGGATGGAGAG GACCAAACT CAG GAC

L R C W A L G F Y P A E I T L T W Q R D G E D Q T Q D ACG GAG CTCGTGGAG ACC AGG CCTGCAGGG GACGGAACCTTCCAG AAGTGGGCG GCTGTG GTG GTGCCTTCTGGAAAGGAG

T E L V E T R P A G D G T F Q K W A A V V V P S G K E

Tranamembran.

CAG AGATAC ACC TGT CATGT CAG CAT GAG GGTCTG CGTGAG CCCCTC ACCCTG AGATGG GAG CCG TCTTCCCAG TCCACC Q R Y T C H V Q H E C L R E P L T L R W E P S S Q S T ATCCTC ATCGTGGGC ATCATT'GCTGGCCTl GTTCTCCTT'GGAATT' GIG GTC ATT GGA GCTGIG ATT GCTGCTG1GATATOG

I L I V G I I A G L V L L G I V V I G A V I A A V I W Cytoplasmic Dosainj

AGG AGG AAG AGCTCAGATAGA AMA GGA GGG AGC TAC TCT CAG GCT GCA AGCAGTGAC AGT GCC CAG GGC TCT GATGIG TCT R R K S S D R K G G S Y S Q A A S S D S A Q G S D V S CTC ACGGCTTGT AAAGO MTA

L T A C K V

FIG. 5. Nucleotide and predicted amino acid sequences of Mamu-A*02. The predicted amino acid sequence is shown below the nucleotide sequence for clone303-3.

fromSIVmac-infectedmonkey 381 were assessed for the ability

to lyse B-LCL pulsed withapanel of25-amino-acid peptides representing sequential, overlapping fragments of the entire

SIVmac

Envprotein. The B-LCLemployedastargetsincluded both autologous and allogeneic cells. The effector cells from

monkey 381recognized the adjacent Env peptidesp18andp19

pulsed ontoboth autologous B-LCL and one of the allogeneic B-LCL, cell line 458 (Fig. 1 anddata not shown). p37-pulsed targets were also lysed by these effector cells, although less

efficiently (Fig. 1).

Toconfirm the fine specificityof the Envpeptide 18 and19 recognition, PBLofmonkey381 werestimulated in vitro with the four adjacent, overlapping

SIVmac

Env peptides, p17 to p20, andassessed for theability tolyse458 B-LCLpulsed with each of these individualpeptides (Fig. 2A). Significant lysisof B-LCL pulsed with either p18 or p19 was observed.

p18-stimulated PBLofmonkey 381, positively selected forCD8+

cells by using immunomagnetic beads, lysed B-LCL pulsed

withp18 orp19 (Fig. 2B). Therefore, effectorcellrecognition

of these peptidesis mediated by CD8+ cells. These observa-tions suggested that the

SIVmac

Env epitope lies in the

overlapping 8 amino acids shared

by peptides

p18

and

p19,

YNLTMKCR. This sequence includes a portion of the third hypervariableregionof

SIVmac

Env.Itis well conserved among macaqueandmangabeySIVisolates (Table 1).

Cloningandsequencingof therhesusmonkeycDNA

encod-ing the MHC class I molecule that presents

SIV.c

Env

peptide 18 toCD8+ CTL. To characterize further the interac-tion leading to CD8+ CTL recognition of

SIVmac

Envin the infected monkey381, the MHC class I molecule that binds p18 and p19 and presents these peptides to CD8+ CTL was

characterized. Three distinct MHC classIcDNAswerecloned from the 458B-LCL, the allogeneic rhesus monkey B-LCL that binds these peptides and presents them to the CTL of monkey 381. These cDNAs were transfected into ClR cells. p18-stimulated PBL from

SIVmac-infected

rhesusmonkey381were

then assessed for theabilitytolyse each of thetransfected ClR cells lines following infection with the vacciniavirus-SIVmac

envelope. The effector cellslysed the cell line transfected with the 303-3 clone butnotthose transfected with the other rhesus monkey MHC class IcDNAs (Fig. 3).

The moleculeencoded by this cDNAwascharacterized by

1-DIEF (Fig. 4),and theMHCclass IcDNA wassequenced (Fig. 5 and6).Comparisonof the nucleotide sequence ofthis cDNA to otherprimateMHC class Isequencesrevealed that the 303-3 cDNA was most closelyrelated to alleles of theA

locus.Accordingly, this allelewasrenamedMamu-A*02,

con-sistent with the 1990 nomenclature proposal of Klein et al. (15).ThesimilarityofMamu-A*02tootherA-locus alleleswas especially evident in the exons

encoding

the transmembrane andcytoplasmic domains. Alleles of theB locus are 9 nucle-otides shorter than alleles of the A locus, and many alleles of

Mamu-A*02 EPSSQSTILIVGIIAGLVLLGIVVIGAVIAAVIWRRKSS DRKGGSYSQAASSDSAQGSDVSLTACKV*

Mamu-A*01 --F---PM---A--T---V---M--- ---*

Mamu-B*01 ---P----V---AV-AV--T---V---M--- GG---*

L

Patr-C*01 G----P--P----A---AV-AVA-L---V-V-MC--- GG----C---N---E--I---A*

FIG. 6. Mamu-A*02 isanHLA-Ahomolog.The Mamu-A*02 sequence iscomparedtoseveralprimate HLA-A,-B,and-C

homologs,

including Mamu-A*01 (23), Mamu-B*01 (ourunpublished observations), and Patr-C*01 (16). The sequencesshown representthe transmembrane and cytoplasmicdomains of nonhumanprimateMHC class I molecules. Dashesindicateidentitywith Mamu-A*02.

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TABLE 2. SIVmacEnvp18-pulsedClR cellstransfectedwith Mamu-A*02 arelysed by CTL fromrhesus monkey381

NoSpecific releasec

Targetcellsa E/Tratiob

V-env V-cont p18 p196

458 B-LCL 80:1 37 10 21 4

40:1 29 7 18 2

20:1 21 7 13 1

10:1 15 5 8 0

ClR transfected with 80:1 37 7 20 7

Mamu-A*02 40:1 28 4 12 3

20:1 22 3 7 3

10:1 14 2 5 1

aTarget cells were B-LCL thatexpressedthe MHC class Imolecule

Mamu-A*02(458B-LCL)orClR cells transfected withMamu-A*02.

bEffector cells generatedfrom PBL of theSIVmac-infectedrhesusmonkey381 were stimulatedwithconcanavalin A andexpandedininterleukin-2-containing

medium.

c Target cells were infectedwith vacciniavirus-SIVmacEnv(V-env)orvaccinia

viruscarryingthe controlequineherpesvirus gHgene(V-cont)or werepulsed

with theSIVmacenvelopep18 peptideor acontrolHIV-1 reversetranscriptase peptide (IAKEPVHGV)(p196).

the C locus have a 3-nucleotide insertion in exon 5. As

expected,thepredictedaminoacid sequence of the

transmem-brane andcytoplasmicdomains of Mamu-A*02 is also similar

tothepredictedamino acid sequences of allelesof theAlocus

(Fig. 6).

Finally, we demonstrated that Mamu-A*02 is therestricting

MHCclass Imolecule for thisdefined

SIVmac

Envepitope in the rhesus monkey. Concanavalin A-stimulated PBL from monkey 381wereassessed inparallelfor theabilitytolyse458 B-LCL andthe Mamu-A*02-transfected ClR cell line follow-ingp18pulsing(Table 2). Equivalentp18-specific lysisof these target cells was observed. Thus, the 8-amino-acid peptide

fragment spanning residues 306to313 of

SIVmac

Env canbe bound by the rhesus monkey MHC class I molecule

Mamu-A*02 and presented toCD8+ CTL.

DISCUSSION

The immune response to HIV type 1 (HIV-1) gpl20 is of central importance in containing anAIDS virus infection (3, 6). Moreover, accumulating data are pointing to the impor-tance of the cell-mediated immune response in blocking the spread of the AIDS virus(28, 44).Nevertheless, little is known about the cell-mediated immune response to the AIDS virus

envelope glycoprotein. Surprisingly few HIV-1 gpl20 CTL

epitopes have been confirmed employing cloned effector T

lymphocytes(13, 14, 15a). This might be attributable, at least in part, to technical difficulties that arise as a result of extreme sequence variation in large portions of the HIV-1 envelope that occurs through mutation (7). Because of envelope se-quence variation among HIV isolates, it is difficult to match virus-elicited CTL responses with synthetic peptides employed

todefine CTL recognition epitopes. Our ability to study CTL

epitoperecognition in rhesus monkeys by employing an AIDS virus with a known envelope sequence and synthetic peptides generatedto match that sequence provides a powerful model forcircumventing thistechnical problem.

The CTL epitope suggested by the results of this study is composed of amino acid residues 306 to 313, which includes the first two amino acids of the V3-loop analog in the third hypervariable region of the

SIVmac

envelope glycoprotein. The

early, type-specificHIV-1-neutralizing activity in the serum of

infected humans appears to be mediated by antibodies that

recognizetheloopstructureinthe third

hypervariable

domain of the HIVenvelope.For this reason, tremendous interest has focused upon this region of the envelope glycoprotein as a

target forvaccine-mediatedprotective immunity. Thisregion

of HIV-1 also has been implicatedin the cell tropismof the virus

(1, 25).

Accumulating data, however,

suggest that the

analogous region of

SIVmac

is not the principal neutralizing

domainof

SIVmae (11).

Moreover,this

region

of the

envelope

does not appear to dictate the cell

tropism

of

SIVmac

(24).

Interestingly, recent studies suggest that this

portion

of the

SIVmac

envelope does

play

an

important

role in

determining

the pathogenicity of the virus in macaques (9). Our demon-stration of a predominant

SIVmac

Env CTL

epitope

in this

regionof theenvelope

glycoprotein

inMamu-A*02+

monkeys

should further facilitate theuseof the

SIVmac

rhesus

monkey

model for studies of

envelope-dependent

issues of AIDS

immunopathogenesis.

Many of the MHC class I loci are conserved in macaques and

humans,

making

SIV infection of rhesus

monkeys

a

relevant model for

investigating

the cell-mediated immune responsetothe AIDS virus in humans

(38).

Mamu-A*02 is the second example ofanA-locus allele

cloned,

sequenced,

and

expressed from Old World

primates.

A-locus alleles have

previouslybeenclonedand

sequenced

from

chimpanzees

(16,

21),

gorillas

(17,

36),

orangutans,

gibbons

(2),

and rhesus

monkeys

(23).

The transmembrane and

cytoplasmic

domains

arewell conserved among A-locus alleles in these nonhuman

primate

species (Fig.

6) (38).

HLA-B-like lociarealso present in rhesus

monkeys

(data

not

shown),

and alleles of HLA-E-like

(data

not

shown)

and HLA-F-like

(26)

loci from macaques have also beencloned. Evidence for

homologs

of HLA-C and

HLA-G,

however,

has been difficultto obtain

by

PCR

ampli-fication of both

genomic

DNAand cDNAderived from RNA from a

variety

of tissues.

Only in-depth

analysis

ofa

genomic

library will determine whether these two loci are indeed present intherhesus

monkey.

The definition of this CTLepitopeand its

restricting

MHC class I molecule will

significantly

facilitatefurther use of the

SIVmac

model in AIDS research. We have

previously

employed

the single defined Gag CTL epitope in an

SIVmac-infected

monkey to explore live-vector- and

peptide-based

vaccine

strategies

for

inducing

AIDS

virus-specific

CTL

(22, 31, 42,

43).

Wehave also madeuseof thisdefined

epitope

in

exploring

the

immunopathogenesis

of AIDS virus infections

(27,

41).

Thedefinition ofan

SIVmac

envelope

epitope

inMamu-A*02+ rhesus

monkeys

will facilitate the use of the

SIVmac

rhesus

monkey model for studies ofenvelope-based vaccine strate-gies.

ACKNOWLEDGMENTS

WethankShelleyKotlikoff forpreparingthemanuscript.

This work was supported by NIH grants AI-20729, AI-35351, AI-32426, CA-50139, DK-43351, RR-00167,and RR-00168.

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