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Journal of General Virology (1998), 79 , 21912201. Printed in Great Britain. . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . .

Characterization of HLA-B57-restricted humanimmunodeciency virus type 1 Gag- and RT-specic cytotoxic Tlymphocyte responses

Miche'

l R. Klein,1 Sjoerd H. van der Burg, 2 Egbert Hovenkamp, 1 Agnes M. Holwerda, 1

Jan Wouter Drijfhout, 2 Cornelis J. M. Melief 2 and Frank Miedema 1, 3

1 Department of Clinical Viro-Immunology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service and Laboratory ofExperimental and Clinical Immunology, University of Amsterdam, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands2 Department of Immunohaematology and Blood Bank, Leiden University Hospital, Leiden, The Netherlands3 Department of Human Retrovirology, Academic Medical Center, Amsterdam, The Netherlands

HLA-B57 has been shown to be strongly associatedwith slow disease progression in human immuno-

deciency virus type 1 (HIV-1)-infected patientsfrom the Amsterdam Cohort. Since HIV-1-specicCTL can control and eliminate virus-infected cells,we sought to characterize the dominant HLA-B57-restricted CTL responses at the epitope level. It wasfound that HLA-B57-restricted CTL responses weretargeted at multiple proteins of HIV-1, with CTLspecic for Gag and RT being the most pronounced.Gag-specic CTL recognized peptides ISPRTLNAW(aa 147155) and STLQEQIGW (aa 241249),which had previously been reported as HLA-B57-restricted.TheRT-specicCTL response in one long-term survivor studied in great detail persisted for

10 years and was dominated by HLA-B57-restricted CTL that recognized the newly denedepitope IVLPEKDSW (RT LAI, aa 244252). This

IntroductionThe clinical course and outcome of human immuno-

deciency virus type 1 (HIV-1) infection are highly variable.The time to AIDS appears to follow a quasi-Gaussiandistribution as a consequence of multiphasic and multifactorialvirushost interactions (Klein & Miedema, 1995; Hayneset al.,1996). In a small proportion of HIV-1-infected individuals,whoseem to represent the extreme of the right-hand tail of thedistribution, an extraordinarily benign disease course beyondthe median time to AIDS is observed. This group of so-called

Author for correspondence: Miche'

l Klein. Present address: MedicalResearch Council Lab., Fajara, PO Box 273, Banjul, The Gambia.

Fax j 220 496513. e-mail petiet ! 2-cool.com

epitope could be recognized in the context of bothHLA-B*5701 and HLA-B*5801. Interestingly, three

epitope variants of IVLPEKDSW were observed,which coincided with the strongest detectable CTLresponse to RT. One variant (T2E7) was not recog-nized by IVLPEKDSW-specic CTL despite the factthat this variant bound to HLA-B*5701 with a similarafnity as the index peptide. Finally, only viruseswhich contained the epitope index sequence wereobtained suggesting efcient virus control by CTL.In conclusion, we report the characterization of dominant HIV-1 Gag- and RT-derived, HLA-B57-restricted CTL epitopes which are associated withlonger time to AIDS. Further characterization of CTL responses restricted by HLA-B57 and other

protective HLA alleles may contribute to thedevelopment of effective AIDS vaccines.

long-term survivors appears heterogeneous and consistslargely of long-term asymptomatic patients; however, in mostcases minor signs of disease progression can be observed.These individuals may harbour peculiar combinations of viruscharacteristics, host genetic determinants, antiviral immuneresponses and environmental co-factors that could favour slowdisease progression. Among the group of long-term survivors,there may also be some individuals who are protected fromprogression to AIDS for life. It seems likely that such a rarephenomenon canonly result from unique features notobservedin other patients progressing to AIDS (Klein & Miedema,1995; Hayneset al., 1996).

It is widely held that HIV-1-specic CTL are among thefavourable determinants for delaying disease progression,since in vitroexperiments have shown that HIV-1-specic CTL

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M. R. Klein and othersM. R. Klein and others

kill infected target cells via MHC class I-restricted recognition(Plata et al., 1987; Walkeret al., 1987) and can suppress virusreplication via secretion of various antiviral cytokines (Walkeret al., 1986; Tsubotaet al., 1989; Cocchiet al., 1995; Buseyneet al., 1996). This view is further underscored byin vivoassociations of emerging HIV-1-specic CTL responses with

the profound reduction of viraemia during the acute phase of infection (Borrowet al., 1994, 1997; Koupet al., 1994; Priceetal., 1997) and with the relatively sustained control of viraemiaduring the asymptomatic phase (Kleinet al., 1995). Interest-ingly, several HLA alleles are consistently associated witheither rapid disease progression (Kaslowet al., 1990; Itescuetal., 1992; Sahmoudet al., 1993; Kleinet al., 1994) or longertime to AIDS (Klein & Miedema, 1995; Goulderet al., 1996;Haynes et al., 1996; Kaslowet al., 1996). This suggests thatthere may be qualitative differences in MHC class I-restrictedHIV-1-specic CTL responses that could variably impact onthe rate of virus replication and hence on time to AIDS.Characterization of HIV-1 CTL responses restricted by theseso-called risk and protective HLA alleles should be one of therst steps taken to reveal the molecular basis of the correlatesof immune protection (Harreret al., 1996; Van der Burget al.,1997).

Since the association between HLA-B57 and long-termsurvival appeared to be the strongest correlate of slow diseaseprogression among patients from the Amsterdam Cohort andindependently conrmed observations in other cohort studies(Haynes et al., 1996; Kaslowet al., 1996), we sought tocharacterize the dominant HLA-B57-restricted CTL responsesto HIV-1 Gag and RT proteins at the epitope level.

MethodsStudy population

Nested case-control study on HLA and long-term survival of HIV-1 in-fection. Within the Amsterdam Cohort Studies on HIV-1 infection andAIDS, a nested case-control study was designed to examine the re-lationship between host genetics and duration of HIV-1 infection.Long-term survivors (cases) were HIV-1-seropositive homosexual men(n l 23) with: (i) at least 9 years of active follow-up; (ii) no clinicalAIDS-dening conditions according to the Centers for Disease Controland Prevention (1993) classications; and (iii) mean CD4+ T cell counts

400 cells\ l. Control men (n l 86) were randomly selected from199 HIV-1-seropositive participants who developed clinical AIDS within8 years after seroconversion or seropositive entry in the Cohort Study.Local Caucasian controls (n l 804) were unrelated healthy male blooddonors (Kleinet al., 1994).

Case report of two patients from the Amsterdam Cohort. PatientH090 is a Caucasian homosexual man with HLA type: A1,*0201;B41,*5701; Cw*0602,*1701; DR13; DR52; DQ1,3 (Kleinet al., 1995).This patient was selected because he displayed the most stable diseasecourse among the 238 HIV-1-seropositive participants recruited be-tween October 1984 and May 1985, and was among the 32 men whoseroconverted for HIV-1 during that same period. Patient H090 enteredthe Cohort Study in November 1984 and was found seropositive for

HIV-1 at the next visit in February 1985. He subsequently attendedthe Municipal Health Service in Amsterdam at three-monthly intervals(at least 50 visits) until October 1997 and so far he has remainedasymptomatic. Standard laboratory measurements were routinely per-formed and were found to be in the normal range: CD4+ T cell counts(meanp SD), 860p 190 cells\ l ; CD8+ T cell counts, 520p 170cells\ l; and CD4+ \ CD8+ T cell ratio, 1n7p 0n37 (Kleinet al., 1995).

HIV-1 RNA was always undetectable in serum samples ( 10$

RNAcopies\ ml). HIV-1 could only be isolated from 3 out of 16 differentblood samples tested with a frequency of 1 tissue culture infectiousdose per 10' CD4+ T cells.

Patient H433 was a Caucasian homosexual man with HLA type:A2,3; B7,*5701; Cw6,7; DR1,3; DR52; DQ1,2. He was found to be HIV-1-seropositive when he entered the Cohort Study in January 1985. After7 years, he developed a generalized cytomegalovirus infection of whichhe died. Since six months before AIDS diagnosis his CD4+ T cell countswere not severely decreased (310 cells\ l), no anti-retroviral therapy wasconsidered at that time. This patient is one of three HLA-B57-positiveparticipants from the Cohort who have developed AIDS so far.

Both long-term survivor H090 and progressor H433 did not carry the32 bp deletion in the C-C chemokine receptor-5 (CCR5) gene (De RodaHusman et al., 1997), and both individuals were infected with non-syncytium-inducing HIV-1 variants only as determined in the MT2 assay(Koot et al., 1993).

In vitro restimulation and expansion of HIV-1-specic CTL.HIV-1-specic CTL in PBMC samples of HIV-1-seropositive individualswere expanded in vitrousing an antigen-specic stimulation protocol aspreviously described (Van Baalenet al., 1993; Kleinet al., 1995). Thismethod results in the generation and expansion of CTL which are biasedtowards recognition of HIV-1LAI -derived sequences, sequences highlysimilar to clade B or highly conserved epitopes among all clades of HIV-1.

T cells were stimulated with autologous EpsteinBarr virus-trans-formed B-lymphoblastoid cell lines (B-LCL) infected at an m.o.i. of 5 witrecombinant vaccinia viruses (rVV) expressing single genes of HIV-1LAI(Myers et al., 1995). Cells were harvested after 2022 h infection at 37mCand 5% CO# , and xed with 1% (w\ v) paraformaldehyde for 15 min atroom temperature. Subsequently, xed cells were incubated with 0n2 Mglycine in PBS for 15 min and washed once with complete medium(RPMI 1640; Life Technologies) containing antibiotics and heat-inactivated, pooled human serum (10%) from 810 healthy HIV-1-seronegative blood donors. Fixed stimulator cells were storedat 4mC andkept for a maximum period of 4 weeks.

HIV-1-specic CTL responses were quantied using standardmethods for limiting dilution analysis (Lefkovits & Waldmann, 1984).Briey, 24 replicate wells of eight serial dilutions of PBMC ranging from20000 to 745 cells per well, were co-cultivated in 96-well round-bottommicrotitre-plateswith 10% xed stimulator cellsand 10% irradiated (30 Gy)autologous PBMC per well (100 l). At days 2 and 9, micro-cultures werefed with complete medium supplemented with recombinant IL-2 (rIL-2;

10 U\ ml) (Proleukin; kindly provided by R. Rombouts, Chiron BeneluxBV, Amsterdam, The Netherlands). The cultures were restimulated at day7 with complete medium supplemented with rIL-2 (10 U\ ml) and 10%

xed stimulator cells. On day 15, wells were split and tested forcytotoxicity (see below).

Cultures from positive wells were further expanded in 96-well round-bottom microtitre-plates by non-specic restimulation as previouslydescribed (Van de Griendet al., 1984). Briey, HIV-1-specic CTL bulkcultures or clones were co-cultivated with a cocktail of (i) 2i 10' per mlirradiated (30 Gy) PBMC of 35 healthy HIV-1-seronegative blooddonors and (ii) 1n5i 10' per ml irradiated (50 Gy) B-LCL (mixture of

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HLA-B57-restricted CTL and slow progression to AIDSHLA-B57-restricted CTL and slow progression to AIDS

equal amounts of allogeneic B-LCL APD, BSM and JY) in completemedium supplemented with rIL-2 (10 U\ ml) and leucoagglutinin (LeucoA, 1 g\ ml; Sigma). When sufficient amounts of cells were obtained,effector cells were tested for cytotoxicity at various effector:target ratios(see below).

Recombinant vaccinia viruses (rVV). Recombinant vaccinia

viruses (rVV) have been constructed from the Copenhagen strain of vaccinia virus. The following rVV expressing single genes of HIV-1LAI(Myers et al., 1995) were used for this study: TG.4163 (RT); TG.1144(Gag) (Rautmannet al., 1989); TG.3183 (Env) (McChesneyet al., 1990);TG.1147 (Nef) (Guy et al., 1987); TG.3196 (Tat); TG.4113 (Rev);TG.1160 (Vif) ; and control rVV 186poly, containing no specic insert.M. P. Kieny (Transge' ne SA, Strasbourg, France) and Y. Rivie' re (InstitutPasteur, Paris, France) kindly provided all the rVV.

Peptides. Peptides were synthesized by solid-phase strategies on anautomated peptide synthesizer (Abimed AMS 422) using Fmoc chem-istry. Peptides were analysed by reverse-phase HPLC; they weredissolved in DMSO at 1050 mg\ ml, aliquotted and stored atk 70 mCuntil use.

51 Cr-release assays. Standard &" Cr-release assays were performedas previously described (Van Baalenet al., 1993; Kleinet al., 1995; Vander Burget al., 1995a). Briey, autologous or (partially) HLA-matched B-LCL were infected at an m.o.i. of 5 with rVV expressing single genes of HIV-1LAI or control rVV 186poly and labelled with 100 Ci Na# &" CrO%(Amersham) for 16 h at 37mC and 5% CO# . After three wash steps, 4000target cells were added to each well. Alternatively,&" Cr-labelled anduninfected B-LCL were pre-incubated for 30 min at room temperaturewith synthetic peptides at a nal concentration of 510 g\ ml or in 10-fold serial dilutions in titration experiments as indicated. After 4 h,supernatants were harvested and radioactivity was measured with a-counter (Cobra-II; Canberra Packard Benelux).&" Cr release was expressedas specic lysis (%) using the following formula: ([experimental releasekspontaneous release]\ [maximum releasek spontaneous release])i 100.Spontaneous &" Cr release was always 15% of the maximum release.

Wells were considered positive when&"

Cr release exceeded 10% speciclysis. This arbitrary threshold was always greater than themeanj (3i SD). CTL precursor (CTLp) frequencies were calculatedusing methods previously described (Strijboschet al., 1987). CTLpfrequencies for specic antigens were computed as differences betweenCTLp frequencies determined on specic versus control targets.

ResultsAssociation of HLA-B57 with long-term AIDS-freesurvival

To address which HLA alleles contribute to delayed diseaseprogression in HIV-1 infection, HLA phenotype frequencies

were analysed in a nested case-control study among homo-sexual men from the Amsterdam Cohort. Long-term survivors(cases) were HIV-1-seropositive participants who remainedAIDS-free for over 9 years (median 10n8 years, range 9n211n1)with normal CD4+ T cell counts (mean 538 cells\ l, range408953 in the ninth year of follow-up). Control subjects wereHIV-1-seropositive participants who developed clinical AIDSwithin 8 years after seroconversion or seroprevalent entry inthe study (median 3n7 years, range 0n87n9). None of the HLAalleles which are frequently present in north European

Table 1. Associations between HLA and long-termsurvival of HIV-1 infection

HLA phenotype frequencies are given.

HLA

Long-term

survivors(n l 23) AIDS cases(n l 86) RR* P

Local

controls(n l 804)

Cw6 50n0 12n5 6n5 0n007 20n0Bw4 78n3 43n8 4n3 0n005 52n0B57 26n1 2n3 12n6 0n0006 5n2B27 13n0 3n5 4n1 8n4B51 21n7 11n8 2n1 10n3B18 8n7 9n3 1n1 7n2

* Relative risks (RR) were odds ratios according to Haldane (1955). RRand corresponding probability (P ) indicate the likelihood of long-termsurvival relative to progression to AIDS in the presence of particularHLA alleles. The Chi-square (# ) test was used to evaluate whether the

calculated RR differed signicantly from unity., Not signicant; indicates that the RR did not differ from unity[taken at the 5% level except where marked ( , P l 0n06)].

Caucasian populations differed signicantly between the totalHIV-1-infected population studied here and the local healthycontrol group (data not shown) (Kleinet al., 1994). The Bw4group of HLA-B alleles was observed more frequently amonglong-term survivors compared to subjects who developedAIDS. This association was mainly due to a signicantlyincreased frequency of HLA-B57 (P l 0n0006) and, to a lesserextent, a slightly increased frequency of HLA-B27 (P l 0n06)(Table 1). In addition, HLA-Cw6 was also over-represented inthe group of long-term survivors as a result of linkagedisequilibrium with HLA-B57. Furthermore, similar associations were observed in a prospective study of 148 cohortparticipants with a known date of HIV-1 seroconversion.KaplanMeier survival analysis showed in this case thatindividuals with HLA-B57 had a relative risk of 0n163 (P l0n07) for developing AIDS compared to individuals withoutHLA-B57 (data not shown). Since the association betweenHLA-B57 and long-term survival appeared to be the strongestcorrelate in the Amsterdam Cohort and independently con-rmed observations made in other cohort studies (Kaslowet al.,1996; Goulderet al., 1996), we sought to further characterize

the HLA-B57-restricted HIV-1-specic CTL responses.Participant H090 was selected for further study since hedisplayed the most stable disease course among the personsrecruited betweenOctober 1984and April 1985. Despite morethan 12 years of documented HIV-1 infection, virus load wasvery low to undetectable and CD4+ T cell counts remainedstable and in the normal range of healthy individuals (Kleinetal., 1995). Patient H433 was also selected for this studybecausehe was one of three HLA-B57-positive patients who haddeveloped AIDS at the start of this study. Patient H433 was

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(a ) (b )

Relative HIV-1

LAIC

TLp frequency

Fig. 1. Distribution of HLA-B57-restricted CTL responses. Using limiting dilution analysis, CTLp frequencies for Gag, RT, Env,Tat, Rev, Vif and Nef were quantied in a blood sample from February 1990 of patient H090 ( a ) and from April 1988 ofpatient H433 ( b ). The contribution of HLA-B57-restricted CTL responses was determined in parallel using HLA-B*5701-matched B-LCL as targets in a split-well 51 Cr-release assay. For each protein, the relative contribution of HLA-B57-restrictedCTL ( ) and the remaining CTL ( ) are expressed as a fraction of the total HIV-1-specic CTLp frequency, which isdetermined by summation of the individual CTLp frequencies for the seven proteins tested.

(a ) (b )

Specific lysis (%) Specific lysis (%)

Fig. 2. Immunodominant HLA-B57-restricted Gag LAI-specic CTL epitopes. Bulk CTL cultures of patient H090 ( a ) and patientH433 ( b ) were specically stimulated for HIV-1 Gag LAI. Cytotoxicity was tested in a standard

51 Cr-release assay withautologous B-LCL pulsed with 5 g/ml of the indicated Gag-derived 9-mer peptides carrying the motif for binding to HLA-B*5701. Effector cells were added to target cells in a ratio of 5:1.

HIV-1-seropositive when he entered the Cohort Study inJanuary 1985 and died of AIDS 7 years later.

HLA-B57-restricted CTL responses against multipleHIV-1 proteins

HIV-1-specic CTL responses against Gag, RT, Env, Tat,Rev, Vif and Nef were quantied using limiting dilution

analysis of a blood sample from long-term survivor H090obtained in February 1990. In addition, the contribution of HLA-B57-restricted CTL responses was determined in parallelwith HLA-B*5701-matched B-LCL as targets in split-well&" Cr-release assays (Fig. 1a). In agreement with our previousobservations, dominant CTL responses were targeted at Gag,RT and Tat (Kleinet al., 1995; Van der Burget al., 1997; VanBaalen et al., 1997). Minor responses were detected against

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(a ) (b )

(c ) (d )

Fig. 3. Characterization of RT LAI-specic CTL from long-term survivor H090. ( a ) HLA-restriction of RT LAI-specic CTL. Effectorcells were obtained from cultures of T cell lines with consistent specicity for HIV-1 RT LAI-expressing target cells. Effector cellswere tested for cytotoxicity on autologous or allogeneic B-LCL matched at one or more HLA class I allele and infected with rVV.

Effector cells were added to target cells in a ratio of 2 n5:1. Specic lysis was determined by subtracting lysis of targets infectedwith rVV 186poly (control) from lysis of those infected with rVV TG.4163 (RT LAI). ( b ) Mapping of epitope specicity of RT LAI-specic CTL. Target cells were prepared by incubating autologous B-LCL with 5 g/ml of the indicated synthetic 10-merpeptides that were derived from the set of overlapping RT peptides. Effector cells were added to target cells in a ratio of 5 :1.( c ) Titration of 9- and 10-mer peptides recognized by RT LAI-specic CTL. Effector cells were tested for their ability to lyseautologous B-LCL infected with rVV TG.4163 (RT LAI) or rVV 186poly (control) or pulsed with varying concentrations of peptidep244/9 IVLPEKDSW ( $ ) or p243 PIVLPEKDSW ( ). Effector cells were added to target cells in a ratio of 5 :1. Horizontaldotted line indicates half-maximum lysis. ( d ) Recognition of RT LAI IVLPEKDSW in the context of HLA-B17 alleles. RTLAIIVLPEKDSW-specic CTL from patient H090 were tested for their ability to recognize autologous HLA-B*5701-positive B-LCL($ ) or HLA-mismatched HLA-B*5801-positive B-LCL ( ) pulsed with various concentrations of peptide p244/9 IVLPEKDSW.Effector cells were added to target cells in a ratio of 2 n5 :1.

Env, Nef, Rev and Vif. The contribution of HLA-B*5701-restricted CTL was 50% in the case of Gag-specic CTL and

85% in the case of RT-specic CTL. HLA-B*5701-restrictedCTL responses against the other proteins tested representedonly minor components of the total HIV-1-specic CTLresponse (Fig. 1a).

Similar experiments were carried out for patient H433 whodeveloped AIDS in about 7 years. For this purpose, a bloodsample from April 1988 was analysed which was obtainedabout 3 years after he entered the Cohort Study. At that time,he was still asymptomatic and both CD4+ T cell numbers andthe CD4+ \ CD8+ T cell ratio were only slightly decreased (450

cells\ l and 0n31, respectively). Dominant CTL responses of progressor H433 comprised CTL against Gag (2165 per 10'

PBMC) followed by the responses to Env and Nef (both 1180per 10' PBMC) and a minor response to RT (157 per 10'

PBMC) (Fig. 1b). Using HLA-matched targets, it was de-termined that 84% of the CTLresponse against Gag was HLA-B*5701-restricted. CTL responses against Env and Nef con-sisted of about 20% of HLA-B57-restricted CTL. The frequency of HLA-B57-restricted RT-specic CTL was 104 per10' PBMC and no CTL responses were detected against Tat,Rev and Vif (Fig. 1b). Since HLA-B57-restricted HIV-1 Gag-and RT-specic CTL constituted dominant responses in the

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long-term survivor with the most stable disease course, wedecided to characterize these further at the epitope level.

Identication of HLA-B57-restricted Gag-specic CTLepitopes

The Gag-specic CTL epitopes were identied with a series

of peptides containing the HLA-B57 peptide-binding motif (Falket al., 1995; Barberet al., 1997). Despite the fact that aphenylalanine has been reported as the C-terminal anchorresidue (Goulderet al., 1996) and that this residue has beenfound in some endogenous peptide sequences eluted fromHLA-B17 alleles (Falket al., 1995; Barberet al., 1997), wedecided to focus on a motif with tryptophan at position 9. Therationale behind this was based on previous observations inour laboratory using functional binding assays (Van der Burget al., 1995b) which showed that the alternative anchor residue(phenylalanine) was less able to promote binding to HLA-B*5701 than tryptophan (not shown).

Gag-specic CTL bulk cultures were expanded from ablood sample of patient H090 from August 1988, and weresubsequently tested against the panel of Gag-derived HLA-B57 motif-bearing peptides. We found that peptide GagLAISTLQEQIGW (aa 241249) was recognized by CTL from thispatient (Fig. 2a). Positive wells from the limiting dilutionexperiments of patient H433 that showed consistent cytolyticactivity for GagLAI were also non-specically expandedinvitro. Bulk cultures were subsequently tested on the panel of 9-mer peptides and strongly recognized peptide STLQEQIGW(aa 241249) and, to lesser extent, peptide ISPRTLNAW (aa147155) (Fig. 2b). Both peptides are highly conserved amongthe published HIV-1 sequences (Myerset al., 1995) and havealso previously been observed as being part of dominant HLA-B57-restricted CTL responses to HIV-1 (Johnsonet al., 1991;Goulder et al., 1996).

Characterization of a novel HLA-B57-restrictedepitope in HIV-1 RT

From a blood sample of subject H090 obtained in October1986, ve oligoclonal cytotoxic T cell lines were obtainedwhich showed consistent specicity for HIV-1LAI RT. TheseCTL were CD8+ and granzyme B-positive (data not shown).Using B-LCL that were partially matched for the HLA type of patient H090, it was shown that HLA-B*5701 was therestriction element for all of these RT-specic CTL (Fig. 3a).

The CTL epitope specicity was determined using a set of 536consecutive 10-mer peptides each with a nine residue overlapand spanning the entire sequence of HIV-1 RTLAI (Van derBurget al., 1997) (Fig. 3b). It was shown that peptides 243245were recognized by all RT-specic CTL from patient H090,with RT peptide p243 (aa 243252; PIVLPEKDSW) being theoptimal. Of note, RT-specic CTL from another HLA-B57-positive long-term survivor previously studied in our lab-oratory also recognized the 10-mer peptides p243 and p244(Van der Burget al., 1997).

Specific lysis (%)

Fig. 4. HLA-B57-restricted RT-specic CTL from progressor H433.Effector cells were derived from bulk CTL cultures specically stimulatedfor GagLAI and added to target cells in a ratio of 5 :1. Target cells wereautologous B-LCL pulsed with 5 g/ml of the indicated RT-derived 9-merpeptides.

Both the 10-mer sequence (p243) and the peptide sequencelacking the N-terminal proline (p244\ 9) were titrated in orderto assess the optimal epitope length (Fig. 3c). The 9-mersequence IVLPEKDSW was recognized at much lower con-centrations than the 10-mer peptide p243 (median value half-max. killing 4n7 nM vs 251 nM, respectively). Therefore, it wasconcluded that HIV-1 RTLAI peptide p244\ 9 (aa 244252;IVLPEKDSW) constituted the optimal epitope sequence.

Positive wells from the limiting dilution experiments of patient H433 which showed consistent cytolytic activity forHIV-1 RTLAI were non-specically expandedin vitro. Bulkcultures were subsequently tested on the panel of RT-derivedHLA-B57 motif-bearing 9-mer peptides. Bulk cultures of RT-specic CTL showed consistent reactivity toward peptidesIVLPEKDSW and RTLAI (aa 375383; ITTESIVIW) (Fig. 4).Of note, two 10-mer peptides containing the latter sequencewere also recognized by another HLA-B57 patient previouslystudied in our laboratory (Van der Burget al., 1997).

According to reported similarities in peptide-binding motifsfor HLA-B17 alleles (Falket al., 1995; Barberet al., 1997), it isto be expected that a number of HLA-B57-restricted CTLepitopes will also be recognized in the context of HLA-B58alleles (Goulder et al., 1996). Similarly to the situationdemonstrated for peptide HIV-1 Gag TSTLQEQIGW(Goulderet al., 1996; Bertolettiet al., 1998), we found here thatHLA-B*5701-restricted RT-specic CTL from patient H090could also recognize peptide RTLAI IVLPEKDSW in thecontext of HLA-B*5801 (Fig. 3d ).

Longitudinal analysis of RT-specic CTL and HIV-1variants

The kinetics of the RT-specic CTL responses of long-termsurvivor H090 were studied at the epitope level (Fig. 5). TheRT-specic CTL response reached peak levels 44 months after

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Fig. 5. Kinetics of RTLAI-specic CTL responses at epitope level. Usinglimiting dilution analysis, the CTLp frequencies for RT were quantied insix blood samples of long-term survivor H090. RT-specic CTLpfrequencies ( ) were determined by subtracting CTLp frequenciesdetermined with targets infected with rVV 186poly (control) from CTLpfrequencies calculated with targets infected with rVV TG.4163 (RT LAI). Inaddition, the contribution of CTL specic for RT LAI (aa 244252)IVLPEKDSW ($ ) was determined in parallel using peptide-pulsed HLA-B*5701-matched B-LCL as targets in a split-well 51 Cr-release assay.Epitope-specic CTLp frequencies were calculated by subtracting CTLpfrequencies determined with targets without peptide from CTLpfrequencies determined with targets pulsed with 10 g/ml RTLAI peptidep244/9 IVLPEKDSW (index). In addition, observed virus variants of thisepitope are boxed and time-points of successful virus isolation areindicated with a down-pointing arrow. Ranges at each point represent thestandard error of the calculated frequencies.

HIV-1 seroconversion (frequencyp standard error is 1132p282 CTLp per 10' PBMC). Thereafter, CTLp frequenciesgradually declined, but clearly remained detectable until 10years after seroconversion (160p 74 per 10' PBMC). Sim-ultaneous testing of RT peptide p244\ 9 revealed that CTLdirected against the HLA-B*5701-restricted epitope IVLPE-KDSW dominated the RT-specic CTL response for manyyears (Fig. 5). Although the CTL response against this epitopeshowed largely the same kinetics as the total response against

the entire RT protein, a slight difference was observed at sometime-points. Separate experiments with HLA-matched targetsindeed showed a minor RT-specic CTL response (Fig. 1a)which appeared to be restricted by HLA-A*0201 (data notshown).

To investigate the relationship between dominant HIV-1-specic CTL responses and selective pressure on the virusquasispecies, we decided to sequence the vicinity of epitopeIVLPEKDSWin replication-competent strains we had obtainedvia biological cloning procedures (Schuitemakeret al., 1991).

Serial dilutions of variant peptides were tested for their abilityto inhibit binding of the uorescein-labelled index peptidep244\ 9 to HLA-B*5701 (Van der Burget al., 1995b). Todetermine whether these peptides could still be recognized byIVLPEKDSW-specic CTL, they were also tested in standard&" Cr-release assays.

Due to the low numbers of infected CD4+

T cells in patientH090, biological variants of HIV-1 could only be obtainedfrom three out of 16 different blood samples tested. Eightclones were obtained from PBMC donated in February 1989,a single clone was from October 1990, and eight clones werefrom August 1994. With respect to the epitope sequence, theisolates from February 1989 consisted of three variants: fourclones contained the index sequence of the epitope, threeclones harboured amino acid substitutions at positions 2 and 7(V2 T2\ D7 E7), and one clone was observed with asingle mutation at position 2 (V2 M2) (Fig. 5). Interestingly,we did not observe any sequence variation in the stretch of 30nucleotides anking either side of the epitope sequence (notshown).

The clone from October 1990, and the eight remainingisolates from August 1994 all contained the index sequenceIVLPEKDSW (Fig. 5). The M2 variant of p244\ 9 bound toHLA-B*5701 with somewhat lower affinity than the indexsequence, but was very well recognized by IVLPEKDSW-specic CTL of patient H090 (not shown). The T2E7 variantwas not recognized by IVLPEKDSW-specic CTL of patientH090 (half-max. killing 10 M), despite the fact that thisvariant bound to HLA-B*5701 with similar affinity (IC&! l6 M) as the index peptide.

In total, seven virus isolates were obtained from patientH433, four from April 1988 and three from October 1989.Sequencing revealed that all biological isolates contained aglutamic acid (E) at position 2 (V2 E2). The latter variantwasclearly recognized by IVLPEKDSW-specic CTLof patientH090, albeit at a lower level than the index peptide p244\ 9(not shown). In addition, previous binding experiments alsoindicated that the E2 variant bound signicantly less stronglyto HLA-B*5701 compared to the index peptide (not shown).

DiscussionTo start unravelling the correlates of immune protection to

AIDS, we have analysed the distribution of HLA class I alleles

in a nested case-control study of long-term survivors andprogressors from the Amsterdam Cohort. HLA-B57 wasstrongly associated with longer time to AIDS and inde-pendently conrmed observations made in other studypopulations (Kleinet al., 1995; Goulderet al., 1996; Haynesetal., 1996; Kaslowet al., 1996). Since MHC class I-restrictedCTL responses to HIV-1 can interfere with virus replication viasecretion of antiviral cytokines (Walkeret al., 1986; Tsubotaetal., 1989; Cocchiet al., 1995; Buseyneet al., 1996) or via cell-mediated cytotoxicity (Plataet al., 1987; Walkeret al., 1987),

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we aimed at the identication of HLA-B57-restricted CTLresponses to HIV-1.

HLA-B57-restricted CTL responses were vigorous andinvolved broad recognition of epitopes from multiple proteinsof HIV-1. Dominant responses appeared to be directed againstHIV-1 Gag and RT, but minor responses to Env, Nef, Tat, Rev,

and Vif were also observed. The major CTL responses directedat Gag and RT were subsequently characterized at the epitopelevel. The Gag-derived epitopes that were recognized by CTLfrom the patients we studied involved two highly conservedsequences, namely ISPRTLNAW (aa 147155) and STLQE-QIGW (aa 241249). Previously, these epitopes have beendescribed by others as being part of dominant HLA-B57-restricted CTL responses to HIV-1 (Johnsonet al., 1991;Goulder et al., 1996).

The major component of the RT-specic CTL response inlong-term survivor H090 was targeted at a newly denedHLA-B57-restricted epitope IVLPEKDSW (aa 244252) andpersisted for more than 10 years. These observations add tothe ndings of Kalamset al. (1994) who studied clonotypes of TcR-V CDR3 sequences of a dominant CTL response to anHLA-B14-restricted epitope in gp41 that persisted for morethan 5 years. However, it remains unclear whether our ndingshere were due to persistent clones or persistent immuno-dominance.

The epitope data obtained here using functional assaysclearly show that peptides with a valine at position 2, maybein conjunction with other auxiliary residues such as a proline atposition 4 (Falket al., 1995; Barberet al., 1997), can clearly bindto HLA-B*5701. So far, this has not been observed in thesequences of endogenous peptides eluted from HLA-B17alleles (Falket al., 1995; Barberet al., 1997). From previousinvitro binding assays in our laboratory, we also concluded thata phenylalanine as C-terminal anchor residue was less efficientthan tryptophan (not shown) (Van der Burget al., 1995b). Sofar, only one CTL epitope has been reported with aphenylalanine as C-terminal anchor residue in contrast to atleast seven with a tryptophan (Johnsonet al., 1991; Goulderetal., 1996; Van der Burget al., 1997).

There is accumulating evidence that HIV-1 escape mutantsemerge in situations where the CTL response is vigorouslydirected at a single epitope (Koeniget al., 1995; Borrowet al.,1997; Goulder et al., 1997; Priceet al., 1997; Mortaraet al.,1998). In situations where CTL responses involve broad

recognition of many epitopes, virus escape mutants may beobserved only temporarily (Haaset al., 1996). Our data mayadd to the latter situation since we noticed three distinctvariants of the IVLPEKDSW sequence at the time when thestrongest CTL response to RT was measured. One variant(T2E7) was not recognized by CTL specic for the indexsequence despite the fact that it did bind to HLA-B*5701 withsimilar affinity. Since a threonine (T) at position 2 is consideredto be one of the primary anchor residues for binding (Falket al.,1995; Barberet al., 1997), the lack of recognition of this variant

is most likely due to the glutamic acid (E) at position 7.Although only limited numbers of virus isolates could beobtained from this individual and sequence variation couldhave accumulated randomly, it appeared that only viruses withthe index sequence persisted. Of interest is the fact thatvariationwasconned to the epitope sequence as no mutations

were observed in 30 neighbouring nucleotides on either side.As similarly demonstrated by Haaset al. (1996), our obser-vations suggest that the TcR escape mutants may have beeneliminated from the virus quasispecies by the vigorous andpolyclonal CTL responses we observed in this patient.

In patient H433, we observed a very marginal HLA-B57-restricted CTL response to RT. In all seven biological isolatesof HIV-1, we observed a mutation in therst anchor residue forbinding to HLA-B57 (i.e. E2). This situation may again pointowards virus escape, but could also suggest that this variantsequence was not sufficient to elicit strong RT-specic CTLresponses. This observation also adds to the growing notion toinclude virus sequencing data in order to fully appreciatefunctional epitope information. The latter has been elegantlydemonstrated by Sipsas et al. (1997) who reported that theactual epitope sequence in autologous viruses may be quitedifferent from the virus index sequence (e.g. HIV-1LAI ) whichis widely used to screen for CTL activity.

What remains controversial is the fact that viruses with theindex sequence do persist in the face of vigorous and broadlydirected CTL responses. This strongly suggests that there maybe virus sanctuaries where HIV-1-specic CTL are less capableof controlling infected cells (Kleinet al., 1998). Furthermore, itshows that mechanisms of immune protection to disease aremore complex than just the recognition of specic epitopesper se(Van der Burget al., 1997). HLA-B57-positive patient H433,who developed AIDS after 7 years, illustrates this since hisCTL recognized four HLA-B57-restricted epitopes. Moreover,these epitopes are shown here, as well as in other studies, to bepart of dominant CTL responses of long-term survivors(Goulder et al., 1996; Van der Burget al., 1997). The obviousdifference in disease course may be explained by taking intoaccount the longevity of these responses as well as the entireclonal composition of dominant and sub-dominant HIV-1-specic CTL. For example, in progressor H433, we did notobserve CTL responses to Tat, Rev and Vif and only a weakresponse against RT was seen. It could well be that CTLresponses to regulatory proteins expressed early during the

virus life-cycle are more efficient in controlling virus load, sincthey are thought to eliminate virus-infected cells before themajor release of virus progeny (Van Baalenet al., 1997).

For future AIDS vaccines, it may be worthwhile to includethe type of immune responses that mimic the dominant andpersistent responses observed in long-term survivors. Withrespect to the HLA restriction elements of HIV-1-specic CTLepitopes, these would ideally have to be alleles common inethnic populations with a high prevalence of HIV-1 infection.Coincidentally,HLA-B17 alleles are quite commonin particular

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sub-Saharan populations living in areas with a high prevalenceof HIV-1 infection (Imanishiet al., 1992). As with the ndingsfor peptide Gag TSTLQEQIGW (Goulderet al., 1996;Bertolettiet al., 1998), we observed here that the HLA-B*5701-restricted epitope RTLAI IVLPEKDSW could also be recog-nized by CTL in the context of HLA-B*5801. This nding

again underscores the striking similarity of the peptide-bindingmotifs of the structurally related HLA-B17 alleles (Falket al.,1995; Barberet al., 1997). Unravelling of the molecular basis of the correlates of immune protection, in particular the role of HIV-1-specic CTL responses restricted by HLA-B57 as wellas other protective HLA class I alleles, will hopefullycontribute to the further development of effective AIDSvaccines.

The authors are greatly indebted to all cohort participants for theircontinuous dedication to the study; to Oscar Pontesilli, Susana Kerkhof-Garde and Neeltje Kootstra for expert technical assistance; to WillemienBenckhuijsen for synthesis of peptides; to Ana-Maria de Roda Husman

for CCR5 genotyping; to Dr Y. Rivie'

re and M. P. Kieny for all therecombinant vaccinia viruses; to Dr R. Rombouts for generouslyproviding Proleukin (rIL-2); to Dr N. M. Lardy and Dr M. Bunce forperforming HLA-typings; and to Professor Andrew McMichael forproviding facilities for carrying out some of the experiments described.

This study was performed as part of the Amsterdam Cohort Studieson HIV-1 infection and AIDS, a collaboration between the MunicipalHealth Service, the Academic Medical Center and the Central Laboratoryof the Netherlands Red Cross Blood Transfusion Service, Amsterdam,The Netherlands.

This study was nancially supported by the Dutch AIDS Fund, theDutch Ministry of Public Health on advice of the Dutch ProgramCommittee of AIDS Research in the context of the National AIDSResearch Stimulation Program (PccAo 94014, 95017) and in part by TheNetherlands Organization for Scientic Research (NWO).

M.R. Klein and S. H. van der Burg contributed equally to this study.

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Received 18 March 1998 ; Accepted 12 May 1998

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