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Journal of Virology, February 2001, p. 1339-1347, Vol. 75, No. 3
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.3.1339-1347.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Rapid Definition of Five Novel HLA-A*3002-Restricted Human
Immunodeficiency Virus-Specific Cytotoxic T-Lymphocyte Epitopes by
Elispot and Intracellular Cytokine Staining Assays
Philip J. R.
Goulder,1,2,*
, Marylyn
M.
Addo,1
Marcus A.
Altfeld,1
Eric S.
Rosenberg,1
Yanhua
Tang,1
Ugene
Govender,3
Nolwandle
Mngqundaniso,3
Ken
Annamalai,3
Thorsten U.
Vogel,4
Mike
Hammond,5
Michael
Bunce,6
Hoosen M.
Coovadia,3 and
Bruce D.
Walker1
Partners AIDS Research Center, Massachusetts
General Hospital and Harvard Medical School, Charlestown, Massachusetts
021291; Division of Infectious Diseases,
The Children's Hospital, Boston, Massachusetts
021152; Department of Paediatrics,
University of Natal, Durban,3 and Natal
Blood Transfusion Service, Pinetown,5 South
Africa; Wisconsin Regional Primate Center, Madison, Wisconsin
537154; and Oxford Transplant
Centre, Churchill Hospital, Oxford OX3 7LJ, United
Kingdom6
Received 31 July 2000/Accepted 15 November 2000
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ABSTRACT |
Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes
(CTL) play a major role in control of viral replication. To understand
the contribution of this antiviral response, an initial step is to
fully define the specific epitopes targeted by CTL. These studies
focused on CTL responses restricted by HLA-A*3002, one of the HLA-A
molecules most prominent in African populations. To avoid the
time-consuming effort and expense involved in culturing CTL prior to
defining epitopes and restricting alleles, we developed a method
combining Elispot assays with intracellular gamma interferon staining
of peripheral blood mononuclear cells to first map the optimal epitopes
targeted and then define the HLA restriction of novel epitopes. In two
A*3002-positive subjects whose CTL responses were characterized in
detail, the strongest response in both cases was to an epitope in p17
Gag, RSLYNTVATLY (residues 76 to 86). Using this method, CTL epitopes
for which there were no motif predictions were optimized and the HLA
restriction was established within 48 to 72 h of receipt of blood.
This simple and convenient approach should prove useful especially in
the characterization of CTL responses specific to HIV and other
viruses, particularly in localities where performing cytotoxicity
assays would be problematic.
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INTRODUCTION |
Human immunodeficiency virus
(HIV)-specific cytotoxic T lymphocytes (CTL) play a major role in
controlling virus replication (1, 8, 9, 16, 21, 24).
Development of vaccines designed to generate protective anti-HIV immune
responses requires a fundamental knowledge of the CTL epitopes that are
targeted by infected persons in populations most severely affected by
the epidemic. So far, however, relatively little is known about the HIV-specific CTL epitopes that are presented by HLA class I molecules prevalent in sub-Saharan African populations, despite the estimate that
75% of HIV infection worldwide has occurred in this region. (UNAIDS website
[http://www.unaids.org/epidemic_update/report/Epi_report.htm]). This study therefore focused on one allele prominent in southern Africa, HLA-A*3002 (7, 15). HLA-A30 is in several
countries the commonest HLA-A allele. For example, over 50% of
Zimbabweans express HLA-A30 (14). In African Zulu,
the phenotypic frequency of A30 is 44%, split approximately equally
between A*3001 and A*3002 (13; M. Hammond, unpublished
data). In contrast, in Caucasoids, A30 is uncommon (phenotypic
frequency, 5%). No virus-specific A30-restricted epitopes have been
optimally defined to date.
Initially one subject with A*3002 was studied in detail; five
novel A*3002-restricted CTL epitopes were defined using a
recently described method exploiting the sensitivity of the
Elispot assay that allows CTL epitopes to be rapidly defined
using peripheral blood mononuclear cells (PBMC), and the results were
confirmed by the traditional approach via cytotoxicity assays
(2). The HLA restriction, however, could not be defined by
Elispot assays using PBMC incubated with a panel of peptide-pulsed
Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines (BCL)
because the background level became too high, possibly due to
EBV-specific responses. The requirement to generate CTL clones or
peptide-specific lines in order to define new epitopes is technically
demanding and costly, which limits the number of laboratories that can
be equipped to define new epitopes. To circumvent this need for
culturing cells, a method for defining the HLA restriction of CTL
responses by intracellular cytokine staining (ICS) assays was
developed. Thus, using the Elispot assay for rapid epitope optimization
and the ICS assay for rapid HLA restriction, novel CTL epitopes can now
be defined from PBMC within 48 to 72 h of phlebotomy. In addition, this approach enables HIV-specific CTL responses to be characterized in
laboratories that are not specialized in tissue culture techniques.
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MATERIALS AND METHODS |
Subjects studied.
Subject 199 (HLA A*0201/*3002
B*4402/51 Cw2/5) is a Caucasian whose precise date of infection is
unknown but who had had documented HIV infection for more than 6 years
at the time of study. He is antiretroviral therapy naive. His viral
load and CD4 count at the time of study were 3,700 HIV type 1 (HIV-1)
RNA copies/ml of plasma and 811 cells/µl, respectively. Subject 6007 (HLA A*3002/
B53/*5801 Cw4/7) is an African-Caribbean who had
been treated with highly active antiretroviral therapy for 1 year.
Viral load at time of CTL analysis was below the level of detection
(<50 copies of plasma HIV-1 RNA/ml), and the CD4+ T-cell
count was 645 cells/µl.
HLA typing and subtyping.
HLA typing and subtyping were
performed by sequence-specific primer PCR (6, 17).
Peptides.
Lymphocytes were tested for recognition of a panel
of 290 overlapping peptides, 12 to 20 amino acids in length, spanning
p17 Gag, p24 Gag, Nef, reverse transcriptase (RT), gp120, gp41, Rev, and Tat clade B SF2 sequence (35 Gag peptides provided by the NIBSC
Centralized Facility for AIDS Reagents, supported by EU Program EVA and
the United Kingdom Medical Research Council; the remainder synthesized
either commercially [Research Genetics, Huntsville, Ala.] or at the
Massachusetts General Hospital Peptide Synthesis Core). Peptides in
each case overlapped by at least 10 amino acids.
Elispot assays.
Fresh (PBMC) were separated from whole blood
by Ficoll-Hypaque (Sigma, St. Louis, Mo.) density gradient
centrifugation and placed in 96-well polyvinylidene plates (Millipore,
Bedford, Mass.) which had been precoated with (0.5 µg/ml; anti-gamma
interferon (IFN-
) monoclonal antibody (MAb) 1-DIK (Mabtech,
Stockholm, Sweden). The peptides were added in a volume of 20 µl, and
then PBMC were added at between 15,000 and 80,000 cells per well in a
volume of 180 µl. The end concentration of the peptides was 10 µM.
The plates were incubated overnight at 37°C and 5% CO2
and then washed with phosphate-buffered saline before addition of the
second, biotinylated anti-IFN- MAb, 7-B6-1 biotin (Mabtech), at 0.5 µg/ml and incubation at room temperature for 100 min. Following
washing, streptavidin-conjugated alkaline phosphatase (Mabtech) was
added at room temperature for 40 min. Individual cytokine-producing cells were detected as dark spots after a 20-min reaction with 5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium using an
alkaline phosphatase-conjugate substrate (Bio-Rad, Richmond, Calif.).
The number of specific T cells was calculated by subtracting the
negative control values. Background was <40/million PBMC (2 spots/well
at 50,000 PBMC/well).
Generation of peptide-specific CTL lines.
Peptide-specific
CTL lines were generated as previously described (19).
Briefly, PBMC were incubated together with 200 µM peptide for 1 h and
then resuspended in R10 medium (RPMI 1640 [Sigma], 10% fetal calf
serum [Sigma], 10 mM HEPES buffer [Sigma]) with interleukin-7
(IL-7; R&D systems) added at 40 ng/ml. Following 1 week in culture at
37°C and 5% CO2 the medium was replaced twice weekly
with R10 medium containing recombinant IL-2 (50-U/ml; kindly provided
by M. Gately, Hoffmann-La Roche, Nutley, N.J.) instead of IL-7.
Chromium release assays were performed following 2 to 4 weeks of culture.
Intracellular IFN- staining.
ICS assays were performed as
described elsewhere (12, 22). Briefly, 0.2 × 106 to 1.0 × 106 PBMC were incubated with
4 µM peptide and anti-CD28 and anti-CD49d MAbs (each at 1 µg/ml;
Becton Dickinson) at 37°C and 5% CO2 for 1 h before the
addition of brefeldin A (10 µg/ml; Sigma). Following a further 6-h
incubation at 37°C and 5% CO2, the cells were placed at
4°C overnight. PBMC were then washed and stained with surface antibodies, anti-CD8, and anti-CD3 (Becton Dickinson) at 4°C for 20 min. Following washing, the PBMC were fixed and permeabilized (Caltag,
Burlingame, Calif.), and anti-IFN- MAb (Becton Dickinson) was added.
Cells were then washed and analyzed. For assays using HLA-matched or
mismatched BCL, BCL that were pulsed with 10 µM peptide for 1 h were
washed thrice prior to incubation with PBMC or effectors at
105 BCL and 5 × 105 effectors in 1 ml of
R10 medium. The anti-CD28 and anti-CD49d MAbs were then added, and the
assay was carried out exactly as described above.
Generation of precursor frequency assays.
Precursor
frequency assays were set up as previously described (26),
using seven dilutions of PBMC from 8,000 down to 50 cells per well in
24 replicate wells. In brief, PBMC in 96-well plates were cultured with
irradiated allogeneic feeder PBMC at 50,000 cells/well in a final
volume per well of 200 µl of R10 medium with antibiotics (2 mM
L-glutamine, 50 U of penicillin-streptomycin/ml). The
anti-CD3 MAb 12F6 was added at 10 µg/ml. On day 5 and once weekly
thereafter, the medium was replaced with R10 medium containing recombinant IL-2 (50 U/ml). Wells were screened for specific
recognition of HLA-matched, peptide-pulsed, 51Cr (New
England Nuclear, North Billerica, Mass.)-labelled EBV-transformed BCL
target cells after 17 days in culture.
Bulk cultured lymphocyte assay.
As previously described
(20), phytohemagglutinin-activated lymphoblasts were
washed thrice and added to autologous PBMC in a ratio of 1:4 and
incubated in R10 medium at 1.5 × 106/ml in 50-ml
flasks (Costar). Cells were cultured in the same way as described above
for the precursor frequency assays but with medium replacement with R10
medium containing recombinant IL-2 (50 U/ml) on day 7 and thereafter
twice weekly. Expansions into additional 50-ml flasks were made as
necessary to maintain cell numbers at 1 × 106 to
2 × 106/ml. The chromium release assay was performed
on day 17.
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RESULTS |
Definition of a strong A*3002-restricted response to epitope
RSLYNTVATLY in p17 Gag.
The approach adopted initially to screen
for HIV-specific CTL responses is illustrated in Fig.
1, with 290 overlapping peptides spanning
eight HIV-1 proteins used in the Elispot assay. As shown for subject
199, and as also observed in subject 6007 (not shown), strong responses
were observed within the 15-mer peptide ELRSLYNTVATLYCV (p17 Gag
residues 74 to 88). As previously described (2),
optimization of CTL epitopes can be achieved with much greater rapidity
by Elispot assays using PBMC, and the same optimal epitope RY11 was defined by this method (Fig. 2A). This
was confirmed as the optimal epitope by generation of a CTL line
specific for the longer 15-mer peptide
ELRSLYNTVAT LYCV, followed by cytotoxicity assays using serial truncated peptides (Fig. 2B). This RY11-specific response was
determined as HLA-A*3002-restricted in standard chromium release assays using the peptide-specific CTL line (Fig. 2C), and the failure
of A*3001-positive targets to present this epitope to A*3002-positive effectors (Fig. 2D and data not shown) demonstrated the need for accurate subtyping of A30-positive subjects.
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FIG. 1.
Screening of PBMC from donor 199 for recognition of
overlapping peptides spanning eight HIV-1 proteins as shown.
Frequencies of responses >50 IFN- spot-forming cells (sfc) per
million PBMC to individual peptides are indicated.
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FIG. 2.
(A) Recognition in an Elispot assay using PBMC from
donor 199 of peptides differing by one residue at the N and C termini
of the optimal epitope peptide RSLYNTVATLY (RY11). (B) Recognition in a
chromium release assay of truncations of the p17 Gag peptide
ELRSLYNTVATLYCV by a peptide-specific CTL line from donor 199. Target
BCL were from donor EBV-522 (HLA-A*3002/0 B*14/Cw8/). (C)
HLA-A*3002 restriction of the RY11-specific response using the same
peptide-specific CTL line as in panel A and HLA-matched targets as
shown. (D) HLA-A*3001-positive targets do not present the RY11
peptide to A*3002-positive effectors. A*3002-positive BCL: EBV-522
(as in panels A and C) (diamonds); 009-BMC (A*0201/*3002 B14/27
Cw1/8) (squares); 016-TCH (A*3001/ B42/ Cw17/) (circles). Open
symbols, BCL pulsed with no peptide; closed symbols; targets pulsed
with 10 µM peptide.
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Definition of HLA restriction by ICS.
Previous studies had
shown that although the optimal epitope sequence can be determined very
rapidly and conveniently via Elispot assays using PBMC
(2), this approach did not allow the HLA restriction of
the response to be defined, since a high background of spot-forming
cells was evident in Elispot plates following incubation of PBMC with
HLA-matched BCL even when the BCL were not pulsed with any peptide. To
circumvent this problem, an approach to defining HLA restriction via
intracellular IFN- staining assays was adopted. Incubation of the
RY11 peptide-specific effector cells with BCL matched through various
of the individual HLA-A, -B, and -C alleles expressed by donor 199 enabled the HLA restriction of the response to be determined
unequivocally as HLA-A*3002 (Fig. 3).
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FIG. 3.
HLA restriction of the A*3002-RY11 response by
intracellular IFN- staining assay using peptide-specific CTL as
effectors. The BCL targets and CTL effectors used were the same as for
Fig. 2C. (A) IFN- staining following incubation of effectors with
BCL that had not been pulsed with RY11 peptide; (B) IFN- staining
following incubation of effectors with BCL that had been pulsed with 10 µM RY11 peptide and washed thrice. Percentage of total lymphocytes
gated is indicated for each plot.
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To determine whether this approach could also define the HLA
restriction of a CTL response without the labor-intensive requirement
for a peptide-specific CTL line, we repeated the assay using PBMC
and
the same panel of HLA-matched and mismatched BCL that had
been employed
previously. The same result was obtained by this
method, as illustrated
for the same A*3002-restricted RY11-specific
response in donor 6007 (Fig.
4A and B). A comparison of the
methods
of defining HLA restriction using peptide-specific CTL lines or
clones in standard chromium release assays and using PBMC from
a
separate subject (described in reference
2) in
intracellular
IFN-
staining assays is shown also for a
B60-restricted Nef-specific
response (Fig.
4C and D). These data show
that this method of
defining the HLA restriction of a CTL response by
intracellular
IFN-
staining assay is equivalent to the standard
method using
CTL clones or peptide-specific CTL lines in terms of
achieving
the same result. However, this flow-based method represents a
great saving in terms of the speed at which the result can be
achieved.
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FIG. 4.
HLA restriction of A*3002- and B60-restricted CTL
responses by intracellular IFN- staining assay using effectors
within PBMC. (A and B) HLA-A*3002 restriction of the RY11 response
described above but using PBMC from donor 6007 (A*3002/ B53/*5801
Cw4/7) and A*3002-matched BCL from donor 009-BMC (A*0201/*3002
B14/27 Cw1/8). HLA-mismatched BCL were from donor 027-BMC (A32/34
B51/71 Cw8/16). (C and D) Comparison of HLA restriction of a
B60-restricted Nef-specific response using a CTL clone from donor KM
(A3/ B14/60 Cw3/8) in a chromium release assay (C) and using PBMC
from donor KM in an intracellular IFN- staining assay with the same
BCL targets (D).
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Definition of four further novel HLA-A*3002-restricted CTL
epitopes.
From the initial screening Elispot assays (Fig. 1),
several other responses had been detected in PBMC from donor 199 using the sets of overlapping peptides, particularly in RT and gp41. None
were in regions containing described epitopes for the class I alleles
that this individual expressed (5). Optimization of one of
the gp41-specific responses was determined by both chromium release
assays using a peptide-specific line and Elispot assays using PBMC
(Fig. 5A and B); optimization of three
additional epitopes was determined by the latter approach alone (Fig.
5C to E). In each case, the restriction for these responses was again
HLA-A*3002 (Fig. 6 and data not shown),
as determined in chromium release assays using peptide-specific lines.
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FIG. 5.
Optimization of four additional novel
A*3002-restricted CTL epitopes. (A and B) Optimization of the
gp41-specific epitope IVNRVRQGY (IY9) by chromium release assay using a
peptide-specific line (A; BCL targets from donor EBV-522: A*3002/
B14/ Cw8/) and in an Elispot assay using PBMC from donor 199 (B).
(C to E) Optimization of A*3002-restricted epitopes KYCWNLLQY
(KY9-gp41; C), KLNWASQIY (KY9-RT-35; D), and KQNPDIVIY (KY9-RT-53; E)
by Elispot assay using PBMC incubated with peptide. The HLA restriction
was confirmed in each case using peptide-specific CTL lines in standard
chromium release assays (not shown). sfc, spleen-forming cells.
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FIG. 6.
Comparison of the hierarchy of the A*3002-restricted
responses as determined by Elispot assays with the hierarchy determined
from assays of cytolytic activity. (A) Frequencies of four of the
A*3002-restricted epitopes as shown in Elispot assays and in
precursor frequency assays (PFAs). (B) Hierarchy of responses toward
the same four epitope peptides in a chromium release assay using bulk
cultured lymphocytes as effectors (E) and EBV-522 A*3002-matched
(A*3002/ B14/Cw8/) BCL as targets (T).
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Cytolytic function of A*3002-restricted responses.
In assays
using antigen-specific CD8+ T cells from HIV-infected
subjects, we have observed a strong correlation between IFN- production and cytolytic function (12). However, recent
data have described HIV-specific CD8+ T cells that can
produce antiviral cytokines but are impaired in cytolytic function
(3). We therefore compared cytolytic activities toward
these newly described A*3002-restricted epitopes to determine whether
the hierarchy of response observed from measurements of IFN-
production following peptide stimulation in Elispot or intracellular
cytokine assays was equivalent to the hierarchy observed from two
separate measurements of cytolytic function (Fig. 6). Comparisons of
the CTL precursor frequencies in limiting dilution assays of responses
toward four of the newly described epitopes with the Elispot assays
supported other studies showing that the Elispot assay is more
sensitive (12, 25), but the hierarchy of the responses was
the same in the different assays. Similarly, the hierarchy was the same
in cytotoxicity assays using bulk cultured lymphocytes as in Elispot
assays. These data, albeit from study of a single subject showing
successful control of viremia, are consistent with our previous studies
(12) that show a strong correlation between levels of
IFN--producing cells in response to HIV epitope peptides and
cytotolytic function.
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DISCUSSION |
These data show that strong HIV-specific responses toward
HLA-A*3002-restricted epitopes can be generated in infected persons. Five novel HLA-A*3002-restricted epitopes are defined, of which the
strongest in two subjects described was the epitope RSLYNTVATLY (RY11)
within p17 Gag. All of the peptides have a tyrosine at the C terminus,
indicating that this is an important anchor residue for F pocket
binding in the A*3002 binding groove. This epitope RY11 lies in a
region of clustered epitopes that includes the dominant
HLA-A*0201-restricted epitope SLYNTVATL (5, 11). Characterization of A30-restricted CTL responses is important since A30
is so prevalent, found in up to 50% of populations such as those in
southern Africa that are worst afflicted by the global HIV
epidemic (UNAIDS website
[http://unaids.org/epidemic_update/report/Epi_report.htm]).
The second principal result of the data described is that HLA
restriction can be defined by use of PBMC incubated with peptide-pulsed BCL as antigen-presenting cells in ICS assays. This is a valuable advance in approach to characterizing CTL responses, since the combination of the Elispot assay as previously described
(2) and the ICS assay described here would enable novel
responses to be defined precisely within 2 to 3 days of receipt of the
blood sample. This increases the speed with which novel epitopes can be
defined and obviates the need for labor-intensive culture of peptide-specific CTL lines or CTL clones followed by chromium release
assays. These factors together restrict CTL work to specialized laboratories. To make the dramatic progress that is urgently needed for
a detailed understanding of the anti-HIV responses made by infected
persons in developing countries, these assays ideally should be carried
out on-site where access to the patients is least problematic. The
radical simplifications to the process of defining CTL epitopes that
are described here and recently (2) now enable the
characterization of HIV-specific CTL responses to be undertaken in
laboratories worldwide.
In common with many of the HLA class I molecules that are prevalent in
areas most affected by the HIV epidemic, no HIV-specific HLA-A30-restricted CTL epitopes had previously been described. In
recent studies of infected African Zulu and Xhosa in Durban, South
Africa, and of African-Americans in Boston, Mass., it is noteworthy
that strong responses to the RY11 epitope described here and detectable
responses to the gp41- and RT-specific epitopes were observed in
A*3002-positive subjects (reference 11 and data not
shown). However, despite detailed characterization of five
A*3001-positive subjects using methods similar to those described here, no A*3001-restricted CTL epitopes have been defined to date (reference 11 and data not shown). The reasons for the
infrequency of HIV-specific epitopes presented by prevalent HLA class I
molecules such as HLA-A1 (5) and A*3001 remain unknown.
A recently undertaken comparison of the peptide binding motifs of
A*3001 and A*3002 reveals distinctive differences between the
peptides bound by these two subtypes (18). As previously described for HLA-A2 subtypes (4), these binding
differences correspond to functional differences, in keeping with the
data above showing that A*3001-positive targets pulsed with the
A*3002-defined epitopes were not recognized by A*3002-positive
effectors. These data underline the value of accurate HLA subtyping in
order to define novel CTL responses.
A feature of the A*3002-restricted epitopes described above that is
somewhat unusual but that is consistent with the described motif for
A*3002 is the variability of residues that can be accommodated in
position 2 (P2), normally a primary anchor position. To illustrate this
point, the five HLA-A*3002 epitope peptides defined above are aligned
in Table 1 with the A*3002-binding peptides that were sequenced
(18). In contrast, there appears to be a greater restriction in the residues that occupy P1 than P2 for the
A*3002-binding peptides aligned in Table
1, with seven of nine peptides having either Arg or Lys in this position. However, pool sequencing of peptides eluted from A*3002 did not identify a preference for positively charged residue at P1 (18). The significance of
the variability of residues that can be accommodated at P2 is in the approach that can successfully be adopted to defining further A*3002-restricted CTL epitopes. The method of reverse immunogenetics (13), which first predicts the sequence of peptides on the
basis of the the peptide-binding motif, then tests candidate peptides fitting the motif for adequacy of binding, and finally tests the binding peptides for CTL recognition, is feasible only if the motif is
relatively restricted. As can be seen from the first five
A*3002-restricted epitopes described here (Table 1), each peptide
carries a different residue at P2. This suggests that an approach based
on overlapping peptides as was used here is likely to be more
successful for fully characterizing A*3002-restricted virus-specific
CTL responses.
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TABLE 1.
Comparison of five newly defined A*3002-restricted CTL
epitopes with the A*3002 peptide-binding motif and the sequences of
four eluted A*3002-binding peptides
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One important aspect of CTL epitope characterization that has not been
addressed in these studies is the relevance to vaccine design of CTL
epitopes that are detectable in chronically infected patients using the
approach described above. The use of overlapping peptides whose
sequence is based on a published consensus sequence is clearly limited
by the fact that this sequence may not correspond to the autologous
virus-specific responses being assayed. Previously this limitation was
believed to be minor, since it was argued that conserved epitopes
within which viral escape mutations would not occur early in infection
would represent the most effective CTL responses that should be induced
by vaccines (9, 20). However, recent data from the simian
immunodeficiency virus macaque model (1) make the opposing
argument, that epitopes in which escape occurs earliest may indeed
represent the very responses that should be incorporated into vaccine
design. If the epitopes that are most effective in containing virus are
those that lie in variable regions, then the entire virus in each
HIV-infected subject needs to be sequenced before initiation of CTL
studies. This would clearly represent a huge investment of time and
funding and thus would not be feasible for all patients investigated; however, studies should be undertaken in selected patients to determine
the most effective CTL responses that should be induced by a future HIV
vaccine. In the meantime, this critical issue is perhaps most likely to
be resolved by future challenge experiments carried out in macaques
previously given vaccines to induce different CTL specificities in
different groups of animals.
In conclusion, strong HIV-specific HLA-A*3002-restricted CTL
responses in two infected subjects are described. These are the first
A30-restricted CTL epitopes to be fully defined. These are of
importance since A30 is one of the most prevalent HLA class I molecules
expressed in populations such as those in southern Africa that have
been most seriously affected by the global HIV epidemic. Alignment of
the novel epitopes described illustrates a high degree of variability
in the amino acid residues that can reside at P2 within
A*3002-binding epitope peptides. This finding is of significance,
suggesting that an approach to defining further A*3002-restricted
epitopes specific to HIV-1 or indeed other viruses using reverse
immunogenetics will be relatively unsuccessful. Finally, a rapid method
of defining the HLA restriction of CTL responses using PBMC and
intracellular IFN- staining assays is described. This will be of
substantial value in reducing the time necessary to fully define novel
CTL epitopes.
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ACKNOWLEDGMENTS |
This work was supported by grants to P.J.R.G. from the Elizabeth
Glaser Pediatric AIDS Foundation, the United Kingdom Medical Research
Foundation (G108/274), and the National Institutes of Health (NIH)
(AI46995); to M.M.A. from the German Research Foundation; to M.A.A.
from the German Academic Exchange Foundation; to E.S.R. from the Doris
Duke Charitable Foundation and NIH (AI 01541); and to B.D.W. from the
NIH (AI28568 and AI30914) and Doris Duke Charitable Foundation.
P.J.R.G. is an Elizabeth Glaser Scientist of the Elizabeth Glaser
Pediatric AIDS Foundation. B.D.W. is a Doris Duke Distinguished
Clinical Science Professor.
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FOOTNOTES |
*
Corresponding author. Present address: Department
of Paediatrics, Nuffield Department of Medicine, Level 7, John
Radcliffe Hospital, Oxford OX3 9DU, United Kingdom. Phone:
44-1865-221335. Fax: 44-1865-220993. E-mail:
philip.goulder{at}ndm.ox.ac.uk.
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Journal of Virology, February 2001, p. 1339-1347, Vol. 75, No. 3
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.3.1339-1347.2001
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Abstract
Introduction
