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HLA-B57 has been shown to be strongly associated epitope could be recognized in the context of both
with slow disease progression in human immuno- HLA-B*5701 and HLA-B*5801. Interestingly, three
deficiency virus type 1 (HIV-1)-infected patients epitope variants of IVLPEKDSW were observed,
from the Amsterdam Cohort. Since HIV-1-specific which coincided with the strongest detectable CTL
CTL can control and eliminate virus-infected cells, response to RT. One variant (T2E7) was not recog-
we sought to characterize the dominant HLA-B57- nized by IVLPEKDSW-specific CTL despite the fact
restricted CTL responses at the epitope level. It was that this variant bound to HLA-B*5701 with a similar
found that HLA-B57-restricted CTL responses were affinity as the index peptide. Finally, only viruses
targeted at multiple proteins of HIV-1, with CTL which contained the epitope index sequence were
specific for Gag and RT being the most pronounced. obtained suggesting efficient virus control by CTL.
Gag-specific CTL recognized peptides ISPRTLNAW In conclusion, we report the characterization of
(aa 147–155) and STLQEQIGW (aa 241–249), dominant HIV-1 Gag- and RT-derived, HLA-B57-
which had previously been reported as HLA-B57- restricted CTL epitopes which are associated with
restricted. The RT-specific CTL response in one long- longer time to AIDS. Further characterization of
term survivor studied in great detail persisted for CTL responses restricted by HLA-B57 and other
" 10 years and was dominated by HLA-B57- protective HLA alleles may contribute to the
restricted CTL that recognized the newly defined development of effective AIDS vaccines.
epitope IVLPEKDSW (RTLAI, aa 244–252). This
kill infected target cells via MHC class I-restricted recognition HIV-1 at the next visit in February 1985. He subsequently attended
(Plata et al., 1987 ; Walker et al., 1987) and can suppress virus the Municipal Health Service in Amsterdam at three-monthly intervals
replication via secretion of various antiviral cytokines (Walker (at least 50 visits) until October 1997 and so far he has remained
asymptomatic. Standard laboratory measurements were routinely per-
et al., 1986 ; Tsubota et al., 1989 ; Cocchi et al., 1995 ; Buseyne formed and were found to be in the normal range : CD4+ T cell counts
et al., 1996). This view is further underscored by in vivo (mean³SD), 860³190 cells}µl ; CD8+ T cell counts, 520³170
associations of emerging HIV-1-specific CTL responses with cells}µl ; and CD4+}CD8+ T cell ratio, 1±7³0±37 (Klein et al., 1995).
the profound reduction of viraemia during the acute phase of HIV-1 RNA was always undetectable in serum samples (! 10$ RNA
infection (Borrow et al., 1994, 1997 ; Koup et al., 1994 ; Price et copies}ml). HIV-1 could only be isolated from 3 out of 16 different
al., 1997) and with the relatively sustained control of viraemia blood samples tested with a frequency of ! 1 tissue culture infectious
during the asymptomatic phase (Klein et al., 1995). Interest- dose per 10' CD4+ T cells.
Patient H433 was a Caucasian homosexual man with HLA type :
ingly, several HLA alleles are consistently associated with A2,3 ; B7,*5701 ; Cw6,7 ; DR1,3 ; DR52 ; DQ1,2. He was found to be HIV-
either rapid disease progression (Kaslow et al., 1990 ; Itescu et 1-seropositive when he entered the Cohort Study in January 1985. After
al., 1992 ; Sahmoud et al., 1993 ; Klein et al., 1994) or longer 7 years, he developed a generalized cytomegalovirus infection of which
time to AIDS (Klein & Miedema, 1995 ; Goulder et al., 1996 ; he died. Since six months before AIDS diagnosis his CD4+ T cell counts
Haynes et al., 1996 ; Kaslow et al., 1996). This suggests that were not severely decreased (310 cells}µl), no anti-retroviral therapy was
there may be qualitative differences in MHC class I-restricted considered at that time. This patient is one of three HLA-B57-positive
HIV-1-specific CTL responses that could variably impact on participants from the Cohort who have developed AIDS so far.
Both long-term survivor H090 and progressor H433 did not carry the
the rate of virus replication and hence on time to AIDS.
32 bp deletion in the C-C chemokine receptor-5 (CCR5) gene (De Roda
Characterization of HIV-1 CTL responses restricted by these Husman et al., 1997), and both individuals were infected with non-
so-called risk and protective HLA alleles should be one of the syncytium-inducing HIV-1 variants only as determined in the MT2 assay
first steps taken to reveal the molecular basis of the correlates (Koot et al., 1993).
of immune protection (Harrer et al., 1996 ; Van der Burg et al., + In vitro restimulation and expansion of HIV-1-specific CTL.
1997). HIV-1-specific CTL in PBMC samples of HIV-1-seropositive individuals
Since the association between HLA-B57 and long-term were expanded in vitro using an antigen-specific stimulation protocol as
survival appeared to be the strongest correlate of slow disease previously described (Van Baalen et al., 1993 ; Klein et al., 1995). This
progression among patients from the Amsterdam Cohort and method results in the generation and expansion of CTL which are biased
independently confirmed observations in other cohort studies towards recognition of HIV-1LAI-derived sequences, sequences highly
(Haynes et al., 1996 ; Kaslow et al., 1996), we sought to similar to clade B or highly conserved epitopes among all clades of
HIV-1.
characterize the dominant HLA-B57-restricted CTL responses T cells were stimulated with autologous Epstein–Barr virus-trans-
to HIV-1 Gag and RT proteins at the epitope level. formed B-lymphoblastoid cell lines (B-LCL) infected at an m.o.i. of 5 with
recombinant vaccinia viruses (rVV) expressing single genes of HIV-1LAI
(Myers et al., 1995). Cells were harvested after 20–22 h infection at 37 °C
and 5 % CO , and fixed with 1 % (w}v) paraformaldehyde for 15 min at
Methods #
room temperature. Subsequently, fixed cells were incubated with 0±2 M
+ Study population glycine in PBS for 15 min and washed once with complete medium
Nested case-control study on HLA and long-term survival of HIV-1 in-
(RPMI 1640 ; Life Technologies) containing antibiotics and heat-
fection. Within the Amsterdam Cohort Studies on HIV-1 infection and
inactivated, pooled human serum (10 %) from 8–10 healthy HIV-1-
AIDS, a nested case-control study was designed to examine the re- seronegative blood donors. Fixed stimulator cells were stored at 4 °C and
lationship between host genetics and duration of HIV-1 infection. kept for a maximum period of 4 weeks.
Long-term survivors (cases) were HIV-1-seropositive homosexual men HIV-1-specific CTL responses were quantified using standard
(n ¯ 23) with : (i) at least 9 years of active follow-up ; (ii) no clinical methods for limiting dilution analysis (Lefkovits & Waldmann, 1984).
AIDS-defining conditions according to the Centers for Disease Control Briefly, 24 replicate wells of eight serial dilutions of PBMC ranging from
and Prevention (1993) classifications ; and (iii) mean CD4+ T cell counts 20 000 to 745 cells per well, were co-cultivated in 96-well round-bottom
" 400 cells}µl. Control men (n ¯ 86) were randomly selected from microtitre-plates with 10% fixed stimulator cells and 10% irradiated (30 Gy)
199 HIV-1-seropositive participants who developed clinical AIDS within autologous PBMC per well (100 µl). At days 2 and 9, micro-cultures were
8 years after seroconversion or seropositive entry in the Cohort Study. fed with complete medium supplemented with recombinant IL-2 (rIL-2 ;
Local Caucasian controls (n ¯ 804) were unrelated healthy male blood 10 U}ml) (Proleukin ; kindly provided by R. Rombouts, Chiron Benelux
donors (Klein et al., 1994). BV, Amsterdam, The Netherlands). The cultures were restimulated at day
7 with complete medium supplemented with rIL-2 (10 U}ml) and 10%
Case report of two patients from the Amsterdam Cohort. Patient fixed stimulator cells. On day 15, wells were split and tested for
H090 is a Caucasian homosexual man with HLA type : A1,*0201 ; cytotoxicity (see below).
B41,*5701 ; Cw*0602,*1701 ; DR13 ; DR52 ; DQ1,3 (Klein et al., 1995). Cultures from positive wells were further expanded in 96-well round-
This patient was selected because he displayed the most stable disease bottom microtitre-plates by non-specific restimulation as previously
course among the 238 HIV-1-seropositive participants recruited be- described (Van de Griend et al., 1984). Briefly, HIV-1-specific CTL bulk
tween October 1984 and May 1985, and was among the 32 men who cultures or clones were co-cultivated with a cocktail of (i) 2¬10' per ml
seroconverted for HIV-1 during that same period. Patient H090 entered irradiated (30 Gy) PBMC of 3–5 healthy HIV-1-seronegative blood
the Cohort Study in November 1984 and was found seropositive for donors and (ii) 1±5¬10' per ml irradiated (50 Gy) B-LCL (mixture of
CBJC
HLA-B57-restricted CTL and slow progression to AIDS
equal amounts of allogeneic B-LCL APD, BSM and JY) in complete Table 1. Associations between HLA and long-term
medium supplemented with rIL-2 (10 U}ml) and leucoagglutinin (Leuco survival of HIV-1 infection
A, 1 µg}ml ; Sigma). When sufficient amounts of cells were obtained,
effector cells were tested for cytotoxicity at various effector : target ratios HLA phenotype frequencies are given.
(see below).
+ Recombinant vaccinia viruses (rVV). Recombinant vaccinia Long-term Local
viruses (rVV) have been constructed from the Copenhagen strain of survivors AIDS cases controls
vaccinia virus. The following rVV expressing single genes of HIV-1LAI HLA (n ¯ 23) (n ¯ 86) RR* P (n ¯ 804)
(Myers et al., 1995) were used for this study : TG.4163 (RT) ; TG.1144
(Gag) (Rautmann et al., 1989) ; TG.3183 (Env) (McChesney et al., 1990) ; Cw6 50±0 12±5 6±5 0±007 20±0
TG.1147 (Nef) (Guy et al., 1987) ; TG.3196 (Tat) ; TG.4113 (Rev) ; Bw4 78±3 43±8 4±3 0±005 52±0
TG.1160 (Vif) ; and control rVV 186poly, containing no specific insert. B57 26±1 2±3 12±6 0±0006 5±2
M. P. Kieny (Transge' ne SA, Strasbourg, France) and Y. Rivie' re (Institut B27 13±0 3±5 4±1 † 8±4
Pasteur, Paris, France) kindly provided all the rVV. B51 21±7 11±8 2±1 10±3
B18 8±7 9±3 1±1 7±2
+ Peptides. Peptides were synthesized by solid-phase strategies on an
automated peptide synthesizer (Abimed AMS 422) using Fmoc chem-
istry. Peptides were analysed by reverse-phase HPLC ; they were * Relative risks (RR) were odds ratios according to Haldane (1955). RR
dissolved in DMSO at 10–50 mg}ml, aliquotted and stored at ®70 °C and corresponding probability (P) indicate the likelihood of long-term
until use. survival relative to progression to AIDS in the presence of particular
HLA alleles. The Chi-square (χ#) test was used to evaluate whether the
+ 51Cr-release assays. Standard &"Cr-release assays were performed
calculated RR differed significantly from unity.
as previously described (Van Baalen et al., 1993 ; Klein et al., 1995 ; Van
, Not significant ; indicates that the RR did not differ from unity
der Burg et al., 1995 a). Briefly, autologous or (partially) HLA-matched B- [taken at the 5 % level except where marked (†, P ¯ 0±06)].
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 µCi Na &"CrO
# %
(Amersham) for 16 h at 37 °C and 5 % CO . After three wash steps, 4 000
# Caucasian populations differed significantly between the total
target cells were added to each well. Alternatively, &"Cr-labelled and
uninfected B-LCL were pre-incubated for 30 min at room temperature HIV-1-infected population studied here and the local healthy
with synthetic peptides at a final concentration of 5–10 µg}ml or in 10- control group (data not shown) (Klein et al., 1994). The Bw4
fold serial dilutions in titration experiments as indicated. After 4 h, group of HLA-B alleles was observed more frequently among
supernatants were harvested and radioactivity was measured with a γ- long-term survivors compared to subjects who developed
counter (Cobra-II ; Canberra Packard Benelux). &"Cr release was expressed AIDS. This association was mainly due to a significantly
as specific lysis (%) using the following formula : ([experimental release® increased frequency of HLA-B57 (P ¯ 0±0006) and, to a lesser
spontaneous release]}[maximum release®spontaneous release])¬100.
Spontaneous &"Cr release was always ! 15 % of the maximum release.
extent, a slightly increased frequency of HLA-B27 (P ¯ 0±06)
Wells were considered positive when &"Cr release exceeded 10 % specific (Table 1). In addition, HLA-Cw6 was also over-represented in
lysis. This arbitrary threshold was always greater than the the group of long-term survivors as a result of linkage
mean(3¬SD). CTL precursor (CTLp) frequencies were calculated disequilibrium with HLA-B57. Furthermore, similar associa-
using methods previously described (Strijbosch et al., 1987). CTLp tions were observed in a prospective study of 148 cohort
frequencies for specific antigens were computed as differences between participants with a known date of HIV-1 seroconversion.
CTLp frequencies determined on specific versus control targets. Kaplan–Meier survival analysis showed in this case that
individuals with HLA-B57 had a relative risk of 0±163 (P ¯
0±07) for developing AIDS compared to individuals without
Results HLA-B57 (data not shown). Since the association between
Association of HLA-B57 with long-term AIDS-free HLA-B57 and long-term survival appeared to be the strongest
survival correlate in the Amsterdam Cohort and independently con-
To address which HLA alleles contribute to delayed disease firmed observations made in other cohort studies (Kaslow et al.,
progression in HIV-1 infection, HLA phenotype frequencies 1996 ; Goulder et al., 1996), we sought to further characterize
were analysed in a nested case-control study among homo- the HLA-B57-restricted HIV-1-specific CTL responses.
sexual men from the Amsterdam Cohort. Long-term survivors Participant H090 was selected for further study since he
(cases) were HIV-1-seropositive participants who remained displayed the most stable disease course among the persons
AIDS-free for over 9 years (median 10±8 years, range 9±2–11±1) recruited between October 1984 and April 1985. Despite more
with normal CD4+ T cell counts (mean 538 cells}µl, range than 12 years of documented HIV-1 infection, virus load was
408–953 in the ninth year of follow-up). Control subjects were very low to undetectable and CD4+ T cell counts remained
HIV-1-seropositive participants who developed clinical AIDS stable and in the normal range of healthy individuals (Klein et
within 8 years after seroconversion or seroprevalent entry in al., 1995). Patient H433 was also selected for this study because
the study (median 3±7 years, range 0±8–7±9). None of the HLA he was one of three HLA-B57-positive patients who had
alleles which are frequently present in north European developed AIDS at the start of this study. Patient H433 was
CBJD
M. R. Klein and others
(a) (b)
Relative HIV-1LAI CTLp 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 quantified in a blood sample from February 1990 of patient H090 (a) and from April 1988 of
patient 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 51Cr-release assay. For each protein, the relative contribution of HLA-B57-restricted
CTL (+) and the remaining CTL (*) are expressed as a fraction of the total HIV-1-specific CTLp frequency, which is
determined by summation of the individual CTLp frequencies for the seven proteins tested.
(a) (b)
HIV-1-seropositive when he entered the Cohort Study in analysis of a blood sample from long-term survivor H090
January 1985 and died of AIDS 7 years later. obtained in February 1990. In addition, the contribution of
HLA-B57-restricted CTL responses was determined in parallel
with HLA-B*5701-matched B-LCL as targets in split-well &"Cr-
HLA-B57-restricted CTL responses against multiple release assays (Fig. 1 a). In agreement with our previous
HIV-1 proteins observations, dominant CTL responses were targeted at Gag,
HIV-1-specific CTL responses against Gag, RT, Env, Tat, RT and Tat (Klein et al., 1995 ; Van der Burg et al., 1997 ; Van
Rev, Vif and Nef were quantified using limiting dilution Baalen et al., 1997). Minor responses were detected against
CBJE
HLA-B57-restricted CTL and slow progression to AIDS
(a) (b)
(c) (d)
Fig. 3. Characterization of RTLAI-specific CTL from long-term survivor H090. (a) HLA-restriction of RTLAI-specific CTL. Effector
cells were obtained from cultures of T cell lines with consistent specificity for HIV-1 RTLAI-expressing target cells. Effector cells
were 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±5 : 1. Specific lysis was determined by subtracting lysis of targets infected
with rVV 186poly (control) from lysis of those infected with rVV TG.4163 (RTLAI). (b) Mapping of epitope specificity of RTLAI-
specific CTL. Target cells were prepared by incubating autologous B-LCL with 5 µg/ml of the indicated synthetic 10-mer
peptides 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 RTLAI-specific CTL. Effector cells were tested for their ability to lyse
autologous B-LCL infected with rVV TG.4163 (RTLAI) or rVV 186poly (control) or pulsed with varying concentrations of peptide
p244/9 IVLPEKDSW (E) or p243 PIVLPEKDSW (+). Effector cells were added to target cells in a ratio of 5 : 1. Horizontal
dotted line indicates half-maximum lysis. (d) Recognition of RTLAI IVLPEKDSW in the context of HLA-B17 alleles. RTLAI
IVLPEKDSW-specific CTL from patient H090 were tested for their ability to recognize autologous HLA-B*5701-positive B-LCL
(E) 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±5 : 1.
Env, Nef, Rev and Vif. The contribution of HLA-B*5701- cells}µl and 0±31, respectively). Dominant CTL responses of
restricted CTL was 50 % in the case of Gag-specific CTL and progressor H433 comprised CTL against Gag (2165 per 10'
85 % in the case of RT-specific CTL. HLA-B*5701-restricted PBMC) followed by the responses to Env and Nef (both 1180
CTL responses against the other proteins tested represented per 10' PBMC) and a minor response to RT (157 per 10'
only minor components of the total HIV-1-specific CTL PBMC) (Fig. 1 b). Using HLA-matched targets, it was de-
response (Fig. 1 a). termined that 84 % of the CTL response against Gag was HLA-
Similar experiments were carried out for patient H433 who B*5701-restricted. CTL responses against Env and Nef con-
developed AIDS in about 7 years. For this purpose, a blood sisted of about 20 % of HLA-B57-restricted CTL. The fre-
sample from April 1988 was analysed which was obtained quency of HLA-B57-restricted RT-specific CTL was 104 per
about 3 years after he entered the Cohort Study. At that time, 10' PBMC and no CTL responses were detected against Tat,
he was still asymptomatic and both CD4+ T cell numbers and Rev and Vif (Fig. 1 b). Since HLA-B57-restricted HIV-1 Gag-
the CD4+}CD8+ T cell ratio were only slightly decreased (450 and RT-specific CTL constituted dominant responses in the
CBJF
M. R. Klein and others
CBJG
HLA-B57-restricted CTL and slow progression to AIDS
CBJH
M. R. Klein and others
we aimed at the identification of HLA-B57-restricted CTL is most likely due to the glutamic acid (E) at position 7.
responses to HIV-1. Although only limited numbers of virus isolates could be
HLA-B57-restricted CTL responses were vigorous and obtained from this individual and sequence variation could
involved broad recognition of epitopes from multiple proteins have accumulated randomly, it appeared that only viruses with
of HIV-1. Dominant responses appeared to be directed against the index sequence persisted. Of interest is the fact that
HIV-1 Gag and RT, but minor responses to Env, Nef, Tat, Rev, variation was confined to the epitope sequence as no mutations
and Vif were also observed. The major CTL responses directed were observed in 30 neighbouring nucleotides on either side.
at Gag and RT were subsequently characterized at the epitope As similarly demonstrated by Haas et al. (1996), our obser-
level. The Gag-derived epitopes that were recognized by CTL vations suggest that the TcR escape mutants may have been
from the patients we studied involved two highly conserved eliminated from the virus quasispecies by the vigorous and
sequences, namely ISPRTLNAW (aa 147–155) and STLQE- polyclonal CTL responses we observed in this patient.
QIGW (aa 241–249). Previously, these epitopes have been In patient H433, we observed a very marginal HLA-B57-
described by others as being part of dominant HLA-B57- restricted CTL response to RT. In all seven biological isolates
restricted CTL responses to HIV-1 (Johnson et al., 1991 ; of HIV-1, we observed a mutation in the first anchor residue for
Goulder et al., 1996). binding to HLA-B57 (i.e. E2). This situation may again point
The major component of the RT-specific CTL response in towards virus escape, but could also suggest that this variant
long-term survivor H090 was targeted at a newly defined sequence was not sufficient to elicit strong RT-specific CTL
HLA-B57-restricted epitope IVLPEKDSW (aa 244–252) and responses. This observation also adds to the growing notion to
persisted for more than 10 years. These observations add to include virus sequencing data in order to fully appreciate
the findings of Kalams et al. (1994) who studied clonotypes of functional epitope information. The latter has been elegantly
TcR-Vβ CDR3 sequences of a dominant CTL response to an demonstrated by Sipsas et al. (1997) who reported that the
HLA-B14-restricted epitope in gp41 that persisted for more actual epitope sequence in autologous viruses may be quite
than 5 years. However, it remains unclear whether our findings different from the virus index sequence (e.g. HIV-1LAI) which
here were due to persistent clones or persistent immuno- is widely used to screen for CTL activity.
dominance. What remains controversial is the fact that viruses with the
The epitope data obtained here using functional assays index sequence do persist in the face of vigorous and broadly
clearly show that peptides with a valine at position 2, maybe directed CTL responses. This strongly suggests that there may
in conjunction with other auxiliary residues such as a proline at be virus sanctuaries where HIV-1-specific CTL are less capable
position 4 (Falk et al., 1995 ; Barber et al., 1997), can clearly bind of controlling infected cells (Klein et al., 1998). Furthermore, it
to HLA-B*5701. So far, this has not been observed in the shows that mechanisms of immune protection to disease are
sequences of endogenous peptides eluted from HLA-B17 more complex than just the recognition of specific epitopes per
alleles (Falk et al., 1995 ; Barber et al., 1997). From previous in se (Van der Burg et al., 1997). HLA-B57-positive patient H433,
vitro binding assays in our laboratory, we also concluded that who developed AIDS after 7 years, illustrates this since his
a phenylalanine as C-terminal anchor residue was less efficient CTL recognized four HLA-B57-restricted epitopes. Moreover,
than tryptophan (not shown) (Van der Burg et al., 1995 b). So these epitopes are shown here, as well as in other studies, to be
far, only one CTL epitope has been reported with a part of dominant CTL responses of long-term survivors
phenylalanine as C-terminal anchor residue in contrast to at (Goulder et al., 1996 ; Van der Burg et al., 1997). The obvious
least seven with a tryptophan (Johnson et al., 1991 ; Goulder et difference in disease course may be explained by taking into
al., 1996 ; Van der Burg et al., 1997). account the longevity of these responses as well as the entire
There is accumulating evidence that HIV-1 escape mutants clonal composition of dominant and sub-dominant HIV-1-
emerge in situations where the CTL response is vigorously specific CTL. For example, in progressor H433, we did not
directed at a single epitope (Koenig et al., 1995 ; Borrow et al., observe CTL responses to Tat, Rev and Vif and only a weak
1997 ; Goulder et al., 1997 ; Price et al., 1997 ; Mortara et al., response against RT was seen. It could well be that CTL
1998). In situations where CTL responses involve broad responses to regulatory proteins expressed early during the
recognition of many epitopes, virus escape mutants may be virus life-cycle are more efficient in controlling virus load, since
observed only temporarily (Haas et al., 1996). Our data may they are thought to eliminate virus-infected cells before the
add to the latter situation since we noticed three distinct major release of virus progeny (Van Baalen et al., 1997).
variants of the IVLPEKDSW sequence at the time when the For future AIDS vaccines, it may be worthwhile to include
strongest CTL response to RT was measured. One variant the type of immune responses that mimic the dominant and
(T2E7) was not recognized by CTL specific for the index persistent responses observed in long-term survivors. With
sequence despite the fact that it did bind to HLA-B*5701 with respect to the HLA restriction elements of HIV-1-specific CTL
similar affinity. Since a threonine (T) at position 2 is considered epitopes, these would ideally have to be alleles common in
to be one of the primary anchor residues for binding (Falk et al., ethnic populations with a high prevalence of HIV-1 infection.
1995 ; Barber et al., 1997), the lack of recognition of this variant Coincidentally, HLA-B17 alleles are quite common in particular
CBJI
HLA-B57-restricted CTL and slow progression to AIDS
sub-Saharan populations living in areas with a high prevalence cytotoxic T lymphocyte clone : inhibition of HIV replication by nonlytic
of HIV-1 infection (Imanishi et al., 1992). As with the findings mechanisms and lysis of HIV-infected CD4 cells. Virology 225,
248–253.
for peptide Gag TSTLQEQIGW (Goulder et al., 1996 ;
Bertoletti et al., 1998), we observed here that the HLA-B*5701- Centers for Disease Control and Prevention (1993). 1993 revised
classification system for HIV infection and expanded surveillance case
restricted epitope RTLAI IVLPEKDSW could also be recog- definition for AIDS among adolescents and adults. Mortality and
nized by CTL in the context of HLA-B*5801. This finding Morbidity Weekly Report 41, 1–19.
again underscores the striking similarity of the peptide-binding Cocchi, F., DeVico, A. L., Garzino-Demo, A., Arya, S. K., Gallo, R. C. &
motifs of the structurally related HLA-B17 alleles (Falk et al., Lusso, P. (1995). Identification of RANTES, MIP-1α, and MIP-1β as the
1995 ; Barber et al., 1997). Unravelling of the molecular basis of major HIV-suppressive factors produced by CD8 T cells. Science 270,
the correlates of immune protection, in particular the role of 1811–1815.
HIV-1-specific CTL responses restricted by HLA-B57 as well De Roda Husman, A. M., Koot, M., Cornelissen, M., Keet, I. P. M.,
as other ‘ protective ’ HLA class I alleles, will hopefully Brouwer, M., Broersen, S. M., Bakker, M., Roos, M. T. L., Prins, M., De
Wolf, F., Coutinho, R. A., Miedema, F., Goudsmit, J. & Schuitemaker, H.
contribute to the further development of effective AIDS
(1997). Association between CCR5 genotype and the clinical course of
vaccines. HIV-1 infection. Annals of Internal Medicine 127, 882–890.
Falk, K., Ro$ tzschke, O., Takiguchi, M., Gnau, V., Stevanovic, S., Jung,
The authors are greatly indebted to all cohort participants for their G. & Rammensee, H. G. (1995). Peptide motifs of HLA-B58, B60, and
continuous dedication to the study ; to Oscar Pontesilli, Susana Kerkhof- B62 molecules. Immunogenetics 41, 165–168.
Garde and Neeltje Kootstra for expert technical assistance ; to Willemien Goulder, P. J. R., Bunce, M., Krausa, P., McIntyre, K., Crowley, S.,
Benckhuijsen for synthesis of peptides ; to Ana-Maria de Roda Husman Morgan, B., Edwards, A., Giangrande, P., Phillips, R. E. & McMichael,
for CCR5 genotyping ; to Dr Y. Rivie' re and M. P. Kieny for all the A. J. (1996). Novel, cross-restricted, conserved, and immunodominant
recombinant vaccinia viruses ; to Dr R. Rombouts for generously cytotoxic T lymphocyte epitopes in slow progressors in HIV type-1
providing Proleukin (rIL-2) ; to Dr N. M. Lardy and Dr M. Bunce for infection. AIDS Research and Human Retroviruses 12, 1691–1698.
performing HLA-typings ; and to Professor Andrew McMichael for Goulder, P. J. R., Phillips, R. E., Colbert, R. A., McAdam, S., Ogg, G.,
providing facilities for carrying out some of the experiments described. Nowak, M. A., Giangrande, P., Luzzi, G., Morgan, B., Edwards, A.,
This study was performed as part of the Amsterdam Cohort Studies McMichael, A. J. & Rowland-Jones, S. (1997). Late escape from an
on HIV-1 infection and AIDS, a collaboration between the Municipal immunodominant cytotoxic T-lymphocyte response associated with
Health Service, the Academic Medical Center and the Central Laboratory progression to AIDS. Nature Medicine 3, 212–217.
of the Netherlands Red Cross Blood Transfusion Service, Amsterdam,
Guy, B., Kieny, M. P., Rivie' re, Y., LePeuch, C., Dott, K., Girard, M.,
The Netherlands.
Montagnier, L. & Lecocq, J. P. (1987). HIV F}3« orf encodes a
This study was financially supported by the Dutch AIDS Fund, the
phosphorylated GTP-binding protein resembling an oncogene product.
Dutch Ministry of Public Health on advice of the Dutch Program
Nature 330, 266–269.
Committee of AIDS Research in the context of the National AIDS
Research Stimulation Program (PccAo 94014, 95017) and in part by The Haas, G., Plikat, U., Debre! , P., Lucchiari, M., Katlama, C., Dudoit, Y.,
Netherlands Organization for Scientific Research (NWO). Bonduelle, O., Bauer, M., Ihlenfeldt, H. G., Jung, G., Maier, B.,
Meyerhans, A. & Autran, B. (1996). Dynamics of viral variants in HIV-
M. R. Klein and S. H. van der Burg contributed equally to this study.
1 Nef and specific cytotoxic T lymphocytes in vivo. Journal of Immunology
157, 4212–4221.
Haldane, J. B. S. (1955). The estimation and significance of the
References logarithm of a ratio of frequencies. Annals of Human Genetics 20,
Barber, L. D., Percival, L., Arnett, K. L., Gumperz, J. E., Chen, L. & 309–311.
Parham, P. (1997). Polymorphisms in the α1 helix of the HLA-B heavy Harrer, T., Harrer, E., Kalams, S. A., Elbeik, T., Staprans, S. I., Feinberg,
chain can have an overriding influence on peptide-binding specificity. M. B., Cao, Y., Ho, D. D., Yilma, T., Caliendo, A. M., Johnson, R. P.,
Journal of Immunology 158, 1660. Buchbinder, S. P. & Walker, B. D. (1996). Strong cytotoxic T cell and
Bertoletti, A., Cham, F., McAdam, S., Rostron, T., Rowland-Jones, S., weak neutralizing antibody responses in a subset of persons with stable
Sabally, S., Corrah, T., Ariyoshi, K. & Whittle, H. (1998). Cytotoxic T non-progressing HIV type-1 infection. AIDS Research and Human
cells from human immunodeficiency virus type 2-infected patients Retroviruses 12, 585–592.
frequently cross-react with different human immunodeficiency virus type Haynes, B. F., Pantaleo, G. & Fauci, A. S. (1996). Toward an
1 clades. Journal of Virology 72, 2439–2448. understanding of the correlates of protective immunity to HIV infection.
Borrow, P., Lewicki, H., Hahn, B. H., Shaw, G. M. & Oldstone, M. B. A. Science 271, 324–328.
(1994). Virus-specific CD8 cytotoxic T-lymphocyte activity associa- Imanishi, T., Akaza, T., Kimura, A., Tokunaga, K. & Gojobori, T. (1992).
ted with control of viremia in primary human immunodeficiency virus Allele and haplotype frequencies for HLA and complement loci in various
type 1 infection. Journal of Virology 68, 6103–6110. ethnic groups. In HLA 1991, Proceedings of the Eleventh International
Borrow, P., Lewicki, H., Wei, X., Horwitz, M. S., Peffer, N., Meyers, H., Histocompatibility Workshop and Conference held in Yokohama, pp.
Nelson, J. A., Gairin, J. E., Hahn, B. H., Oldstone, M. B. A. & Shaw, 1065–1220. Edited by K. Tsuji, M. Aizawa & T. Sasazuki. Oxford :
G. M. (1997). Antiviral pressure exerted by HIV-1 specific cytotoxic T Oxford University Press.
lymphocytes (CTLs) during primary infection demonstrated by rapid Itescu, S., Mathur, W. U., Skovron, M. L., Brancato, L. J., Marmor, M.,
selection of CTL escape virus. Nature Medicine 3, 205–211. Zeleniuch, J. A. & Winchester, R. (1992). HLA-B35 is associated with
Buseyne, F., Fevrier, M., Garcia, S., Gougeon, M. L. & Rivie' re, Y. accelerated progression to AIDS. Journal of Acquired Immune Deficiency
(1996). Dual function of a human immunodeficiency virus (HIV)-specific Syndromes 5, 37–45.
CBJJ
M. R. Klein and others
Johnson, R. P., Trocha, A., Yang, L., Mazzara, G. P., Panicali, D. L., McChesney, M., Tanneau, F., Regnault, A., Sansonetti, P., Montagnier,
Buchanan, T. M. & Walker, B. D. (1991). HIV-1 gag-specific cytotoxic L., Kieny, M. P. & Rivie' re, Y. (1990). Detection of primary cytotoxic T
T lymphocytes recognize multiple highly conserved epitopes : fine lymphocytes specific for the envelope glycoprotein of HIV-1 by deletion
specificity of the gag-specific response defined by using unstimulated of the env amino-terminal signal sequence. European Journal of Immunology
peripheral blood mononuclear cells and cloned effector cells. Journal of 20, 215–220.
Immunology 147, 1512–1521. Mortara, L., Letourneur, F., Gras-Masse, H., Venet, A., Guillet, J. G. &
Kalams, S. A., Johnson, R. P., Trocha, A. K., Dynan, M. J., Ngo, H. S., Bourgault-Villada, I. (1998). Selection of virus variants and emergence
D’Aquila, R. T., Kurnick, J. T. & Walker, B. D. (1994). Longitudinal of virus escape mutants after immunization with an epitope vaccine.
analysis of T cell receptor (TCR) gene usage by human immunodeficiency Journal of Virology 72, 1403–1410.
virus 1 envelope-specific cytotoxic T lymphocyte clones reveals a limited Myers, G., Hahn, B. H., Henderson, L. E., Korber, B. T. M., Jeang, K. T.,
TCR repertoire. Journal of Experimental Medicine 179, 1261–1271. McCutchan, F. E. & Pavlakis, G. N. (1995). Human Retroviruses and
Kaslow, R. A., Duquesnoy, R., Van Raden, M., Kingsley, L., Marrari, M., AIDS 1995 : A Compilation and Analysis of Nucleic Acid and Amino Acid
Friedman, H., Su, S., Saah, A. J., Detels, R., Phair, J. P. & Rinaldo, C. Sequences. Los Alamos, NM : Los Alamos National Laboratory.
(1990). A1, Cw7, B8, DR3 HLA antigen combination associated with Plata, F., Autran, B., Martins, L. P., Wain-Hobson, S., Raphael, M.,
rapid decline of T-helper lymphocytes in HIV-1 infection. Lancet 335, Mayaud, C., Denis, M., Guillon, J. M. & Debre, P. (1987). AIDS virus-
927–930. specific cytotoxic T lymphocytes in lung disorders. Nature 328, 348–351.
Kaslow, R. A., Carrington, M., Apple, R., Park, L., Munoz, A., Saah, Price, D. A., Goulder, P. J., Klenerman, P., Sewell, A. K., Easterbrook,
A. J., Goedert, J. J., Winkler, C., O’Brien, S. J., Rinaldo, C., Detels, R., P. J., Troop, M., Bangham, C. R. & Phillips, R. E. (1997). Positive
Blattner, W., Phair, J. P., Erlich, H. & Mann, D. L. (1996). Influence of selection of HIV-1 cytotoxic T lymphocyte escape variants during
combinations of human major histocompatibility complex genes in the primary infection. Proceedings of the National Academy of Sciences, USA 94,
course of HIV-1 infection. Nature Medicine 2, 405–411. 1890–1895.
Keet, I. P. M., Klein, M. R., Just, J. & Kaslow, R. A. (1996). The role of Rautmann, G., Kieny, M. P., Brandely, R., Dott, K., Girard, M.,
host genetics in the natural history of HIV-1 infection : the needles in the Montagnier, L. & Lecocq, J. P. (1989). HIV-1 core proteins expressed
haystack. AIDS 10 (Suppl. A), S59–S67. from recombinant vaccinia viruses. AIDS Research and Human Retroviruses
Klein, M. R. & Miedema, F. (1995). Long-term survivors of HIV-1 5, 147–157.
infection. Trends in Microbiology 3, 386–391. Sahmoud, T., Laurian, Y., Gazengel, C., Sultan, Y., Gautreau, C. &
Klein, M. R., Keet, I. P. M., D’Amaro, J., Bende, R. J., Hekman, A., Costagliola, D. (1993). Progression to AIDS in French haemophiliacs :
Mesman, B., Koot, M., de Waal, L. P., Coutinho, R. A. & Miedema, F. association with HLA-B35. AIDS 7, 497–500.
(1994). Associations between HLA frequencies and pathogenic features Schuitemaker, H., Kootstra, N. A., De Goede, R. E. Y., De Wolf, F.,
of human immunodeficiency virus type 1 infection in seroconverters from Miedema, F. & Tersmette, M. (1991). Monocytotropic human immuno-
the Amsterdam Cohort of homosexual men. Journal of Infectious Diseases deficiency virus 1 (HIV-1) variants detectable in all stages of HIV
169, 1244–1249. infection lack T-cell line tropism and syncytium-inducing ability in
Klein, M. R., Van Baalen, C. A., Holwerda, A. M., Kerkhof-Garde, S. R., primary T-cell culture. Journal of Virology 65, 356–363.
Bende, R. J., Keet, I. P. M., Eeftinck Schattenkerk, J. K. M., Osterhaus, Sipsas, N. V., Kalams, S. A., Trocha, A., He, S., Blattner, W. A., Walker,
A. D. M. E., Schuitemaker, H. & Miedema, F. (1995). Kinetics of Gag- B. D. & Johnson, R. P. (1997). Identification of type-specific cytotoxic
specific CTL responses during the clinical course of HIV-1 infection : a T lymphocyte responses to homologous viral proteins in laboratory
longitudinal analysis of rapid progressors and long-term asymptomatics. workers accidentally infected with HIV-1. Journal of Clinical Investigation
Journal of Experimental Medicine 181, 1365–1372. 99, 752–762.
Klein, M. R., Van der Burg, S. H., Pontesilli, O. & Miedema F. (1998). Strijbosch, L. W. G., Buurman, W. A., Does, R. J. M. M., Zinken, P. H. &
Cytotoxic T lymphocytes in HIV-1 infection, a killing paradox? Groenewegen, G. (1987). Limiting dilution assays. Experimental design
Immunology Today (in press). and statistical analysis. Journal of Immunological Methods 97, 133–140.
Koenig, S., Conley, A. J., Brewah, Y. A., Jones, G. M., Leath, S., Boots, Tsubota, H., Lord, C. I., Watkins, D. I., Morimoto, C. & Letvin, N. L.
L. J., Davey, V., Pantaleo, G., Demarest, J. F., Carter, C., Wannebo, C., (1989). A cytotoxic T lymphocyte inhibits acquired immunodeficiency
Yannelli, J. R., Rosenberg, S. A. & Clifford Lane, H. (1995). Transfer of syndrome virus replication in peripheral blood lymphocytes. Journal of
HIV-1 specific cytotoxic T lymphocytes to an AIDS patient leads to Experimental Medicine 169, 1421–1434.
selection for mutant HIV variants and subsequent disease progression. Van Baalen, C. A., Klein, M. R., Geretti, A. M., Keet, I. P. M., Miedema,
Nature Medicine 1, 330–336. F., Van Els, C. A. C. M. & Osterhaus, A. D. M. E. (1993). Selective in
Koot, M., Keet, I. P. M., Vos, A. H. V., De Goede, R. E. Y., Roos, M. T. L., vitro expansion of HLA class I-restricted HIV-1 gag specific CD8 T
Coutinho, R. A., Miedema, F., Schellekens, P. T. A. & Tersmette, M. cells from seropositive individuals : identification of CTL epitopes and
(1993). Prognostic value of human immunodeficiency virus type 1 precursor frequencies. AIDS 7, 781–786.
biological phenotype for rate of CD4+ cell depletion and progression to Van Baalen, C. A., Pontesilli, O., Huisman, R. C., Geretti, A. M., Klein,
AIDS. Annals of Internal Medicine 118, 681–688. M. R., De Wolf, F., Miedema, F., Gruters, R. A. & Osterhaus, A. D.
Koup, R. A., Safrit, J. T., Cao, Y., Andrews, C. A., McLeod, G., (1997). Human immunodeficiency virus type 1 Rev- and Tat-specific
Borkowsky, W., Farthing, C. & Ho, D. D. (1994). Temporal associations cytotoxic T lymphocyte frequencies inversely correlate with rapid
of cellular immune responses with the initial control of viremia in primary progression to AIDS. Journal of General Virology 78, 1913–1918.
human immunodeficiency virus type 1 syndrome. Journal of Virology 68, Van de Griend, R. J., Van Krimpen, B. A., Bol, S. J. L., Thompson, A. &
4650–4655. Bolhuis, R. L. H. (1984). Rapid expansion of human cytotoxic T cell
Lefkovits, I. & Waldmann, H. (1984). Limiting dilution analysis of the clones : growth promotion by a heat-labile serum component and by
cells of immune system. I. The clonal basis of the immune response. various types of feeder cells. Journal of Immunological Methods 66,
Immunology Today 5, 265–268. 285–298.
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HLA-B57-restricted CTL and slow progression to AIDS
Van der Burg, S. H., Klein, M. R., Van de Velde, C. J. H., Kast, W. M., HIV-1 reverse transcriptase-specific cytotoxic T lymphocytes targeted at
Miedema, F. & Melief, C. J. M. (1995 a). Induction of primary human epitopes under structural or functional constraints do not protect against
CTL response against a novel conserved epitope in a functional sequence progression to AIDS. Journal of Immunology 159, 3648–3654.
of HIV-1 reverse-transcriptase. AIDS 9, 121–127. Walker, C. M., Moody, D. J., Stites, D. P. & Levy, J. A. (1986). CD8
Van der Burg, S. H., Ras, E., Drijfhout, J. W., Benckhuijsen, W. E., lymphocytes can control HIV infection in vitro by suppressing virus
Bremers, A. J. A., Melief, C. J. M. & Kast, W. M. (1995 b). An HLA class replication. Science 234, 1563–1566.
I peptide-binding assay based on competition for binding to class I Walker, B. D., Chakrabarti, S., Moss, B., Paradis, T. J., Flynn, T., Durno,
molecules on intact human B cells : identification of conserved HIV-1 A. G., Blumberg, R. S., Kaplan, J. C., Hirsch, M. S. & Schooley, R. T.
polymerase peptides binding to HLA-A*0301. Human Immunology 44, (1987). HIV-1 specific cytotoxic T lymphocytes in seropositive
189–198. individuals. Nature 328, 345–348.
Van der Burg, S. H., Klein, M. R., Pontesilli, O., Holwerda, A. M.,
Drijfhout, J. W., Kast, W. M., Miedema, F. & Melief, C. J. M. (1997). Received 18 March 1998 ; Accepted 12 May 1998
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