Recognition of a Defined Region within p24 Gag by CD8+ T Cells during Primary Human Immunodeficiency Virus Type 1 Infection in Individuals Expressing Protective HLA Class I Alleles

Article (PDF Available)inJournal of Virology 81(14):7725-31 · July 2007with18 Reads
DOI: 10.1128/JVI.00708-07 · Source: PubMed
Abstract
Human immunodeficiency virus type 1 (HIV-1)-specific immune responses during primary HIV-1 infection appear to play a critical role in determining the ultimate speed of disease progression, but little is known about the specificity of the initial HIV-1-specific CD8+ T-cell responses in individuals expressing protective HLA class I alleles. Here we compared HIV-1-specific T-cell responses between subjects expressing the protective allele HLA-B27 or -B57 and subjects expressing nonprotective HLA alleles using a cohort of over 290 subjects identified during primary HIV-1 infection. CD8+ T cells of individuals expressing HLA-B27 or -B57 targeted a defined region within HIV-1 p24 Gag (amino acids 240 to 272) early in infection, and responses against this region contributed over 35% to the total HIV-1-specific T-cell responses in these individuals. In contrast, this region was rarely recognized in individuals expressing HLA-B35, an HLA allele associated with rapid disease progression, or in subjects expressing neither HLA-B57/B27 nor HLA-B35 (P < 0.0001). The identification of this highly conserved region in p24 Gag targeted in primary infection specifically in individuals expressing HLA class I alleles associated with slower HIV-1 disease progression provides a rationale for vaccine design aimed at inducing responses to this region restricted by other, more common HLA class I alleles.

Figures

JOURNAL OF VIROLOGY, July 2007, p. 7725–7731 Vol. 81, No. 14
0022-538X/07/$08.000 doi:10.1128/JVI.00708-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Recognition of a Defined Region within p24 Gag by CD8
T Cells
during Primary Human Immunodeficiency Virus Type 1 Infection
in Individuals Expressing Protective HLA Class I Alleles
Hendrik Streeck,
1
Mathias Lichterfeld,
1
Galit Alter,
1
Angela Meier,
1
Nickolas Teigen,
1
Bader Yassine-Diab,
2
Harlyn K. Sidhu,
1
Susan Little,
4
Anthony Kelleher,
3
Jean-Pierre Routy,
5
Eric S. Rosenberg,
1
Rafick-Pierre Sekaly,
2
Bruce D. Walker,
1,6
and Marcus Altfeld
1
*
Partners AIDS Research Center, Infectious Disease Unit, Massachusetts General Hospital, and Division of AIDS, Harvard Medical School,
Boston, Massachusetts 02129
1
; Laboratoire d’Immunologie, Centre de Recherche du CHUM, Montreal, Quebec, Canada
2
;
National Centre in HIV Epidemiology and Clinical Research, University of New South Wales, Darlinghurst, Australia
3
;
Department of Medicine, University of California, San Diego, San Diego, California 92093
4
; McGill University, Division of
Hematology and Immunodeficiency Service, Royal Victoria Hospital, Montreal, Quebec, Canada
5
; and
Howard Hughes Medical Institute, Chevy Chase, Maryland 20815
6
Received 3 April 2007/Accepted 2 May 2007
Human immunodeficiency virus type 1 (HIV-1)-specific immune responses during primary HIV-1 infection
appear to play a critical role in determining the ultimate speed of disease progression, but little is known about
the specificity of the initial HIV-1-specific CD8
T-cell responses in individuals expressing protective HLA
class I alleles. Here we compared HIV-1-specific T-cell responses between subjects expressing the protective
allele HLA-B27 or -B57 and subjects expressing nonprotective HLA alleles using a cohort of over 290 subjects
identified during primary HIV-1 infection. CD8
T cells of individuals expressing HLA-B27 or -B57 targeted
a defined region within HIV-1 p24 Gag (amino acids 240 to 272) early in infection, and responses against this
region contributed over 35% to the total HIV-1-specific T-cell responses in these individuals. In contrast, this
region was rarely recognized in individuals expressing HLA-B35, an HLA allele associated with rapid disease
progression, or in subjects expressing neither HLA-B57/B27 nor HLA-B35 (P < 0.0001). The identification of
this highly conserved region in p24 Gag targeted in primary infection specifically in individuals expressing
HLA class I alleles associated with slower HIV-1 disease progression provides a rationale for vaccine design
aimed at inducing responses to this region restricted by other, more common HLA class I alleles.
Acute human immunodeficiency virus type 1 (HIV-1) infec-
tion is marked by high titers of viral load accompanied by fever,
lymphadenopathy, cutaneous rash (18), and a significant loss of
CD4
T cells, in particular in the gut-associated lymphoid
tissue (26, 27). The resolution of these symptoms and the
subsequent decline in viremia are associated with the emer-
gence of HIV-1-specific CD8
T cells (7, 22). The importance
of CD8
T-cell responses in controlling HIV-1 viremia is fur-
ther supported by the successive selection of viral escape vari-
ants within targeted cytotoxic T-lymphocyte (CTL) epitopes
(16). Furthermore, the human leukocyte antigen (HLA) class
I haplotype that restricts virus-specific CD8
T-cell epitopes
has a significant predictive value for the rate of HIV-1 disease
progression as well as the level of HIV-1 replication (9). In
particular, subjects expressing HLA-B27 or -B57 experience a
significantly slower progression to AIDS and constitute up to
50% of the subjects in established long-term nonprogressor
cohorts (12), while subjects expressing HLA-B35 face a more
rapid progression towards AIDS (14, 19). These differences in
HIV-1 disease outcome depending on the HLA class I geno-
type indicate that HIV-1-specific CD8
T-cell responses re-
stricted by different HLA class I molecules are not equally
effective but may differ in their specificity and their ability to
control viral replication (16).
The initial virus-specific CD8
T-cell response in acute
HIV-1 infection is of low magnitude and is narrowly directed
against a limited number of epitopes, but it subsequently
broadens during prolonged antigen stimulation in the chronic
phase of infection (5, 10, 28). However, despite the generally
more broadly directed and vigorous immune response in
chronic infection, control over viral replication wanes during
later stages of infection. These findings further suggest that the
quality and specificity of the initial virus-specific CD8
T-cell
response, rather than the magnitude of the response, may be
associated with the initial control of viral replication (29) and
that responses primed early in infection are crucial for deter-
mining the long-term control over viral replication.
Several cross-sectional studies in chronic HIV-1-infected
individuals have shown that the CD8
T-cell responses in subjects
with low viral load and slow disease progression are preferen-
tially directed against HIV-1 Gag (1, 17, 21, 25). However,
these studies in chronic infection did not allow determination
of whether Gag-specific responses emerged early in infection,
contributing to the initial control of viral replication and the
determination of a viral set point, or simply developed in
chronic infection in the setting of immune control of HIV-1.
We therefore investigated the specificity of the virus-specific
T-cell response in primary infection in individuals expressing
* Corresponding author. Mailing address: Partners AIDS Research
Center, Massachusetts General Hospital, 149 13th Street, Boston, MA
02129. Phone: (617) 724-2461. Fax: (617) 724-8586. E-mail: maltfeld
@partners.org.
Published ahead of print on 9 May 2007.
7725
protective or nonprotective HLA class I alleles and demon-
strate that a specific region within p24 Gag is highly targeted by
HIV-1-specific CD8
T cells in subjects expressing protective
HLA class I molecules but not in subjects expressing nonpro-
tective HLA class I molecules.
MATERIALS AND METHODS
Subjects. Fresh peripheral blood mononuclear cells (PBMCs) from 43 patients
diagnosed with primary HIV-1 infection were screened comprehensively for
HIV-1-specific CD8
T-cell responses against the entire expressed HIV-1 pro
-
teome using a gamma interferon (IFN-) enzyme-linked immunospot (ELISPOT)
assay. In addition, cryopreserved samples from a second large cohort of 293
subjects collected 8 weeks (10 days) after the initial presentation with primary
HIV-1 infection were screened for HIV-1-specific CD8
T-cell responses using
HLA-matched optimal peptides in an IFN- ELISPOT assay (4). Samples were
collected from different sites of the AIEDRP network: Massachusetts General
Hospital, Boston (n 154); Laboratoire d’Immunologie, Centre de Recherche
du CHUM, Montreal, Quebec, Canada (n 85); National Centre in HIV
Epidemiology and Clinical Research, Sydney, Australia (n 50); and Antiviral
Research Center, University of California, San Diego (n 4). More than 95%
of subjects in the cohorts were infected with HIV-1 clade B. The study was
approved by the respective institutional review boards and was conducted ac-
cording to the human experimentation guidelines of the Massachusetts General
Hospital.
HLA tissue typing. High- and intermediate-resolution HLA class I typing was
performed by sequence-specific PCRs according to standard procedures. DNA
was extracted from PBMCs using the Purgene DNA isolation kit for blood
(Gentra Systems, Minneapolis, MN). The HLA class I allele distribution in the
study population generally reflected the distribution in a typical Caucasoid pop-
ulation.
Peptides. Freshly isolated PBMCs of each of the 43 subjects with primary
HIV-1 infection were screened using 410 overlapping peptides (generally 18-
mers), varying from 15 to 20 amino acids (aa) in length, which overlapped by 10
aa and spanned the entire clade B consensus sequence 2001, as described pre-
viously (13). In the additional 293 study subjects, previously described optimal
peptides corresponding to HLA-matched epitopes were tested (4). Peptides were
synthesized commercially (Research Genetics) or at the Massachusetts General
Hospital Peptide Synthesis Core Facility.
ELISPOT assay. PBMCs were plated in 96-well polyvinylidene difluoride-
backed plates (MAIP S45; Milipore) that had been coated previously with 100 l
of an anti-IFN monoclonal antibody, 1-D1k (2 g/ml; Mabtech), overnight at
4°C. Peptides were added directly to the wells at a final concentration of 14
g/ml. Cells were added to the wells at 50,000 to 100,000 cells/well. The plates
were incubated at 37°C in 5% CO
2
overnight (14 to 16 h) and then processed as
described previously (1). IFN--producing cells were counted by direct visual-
ization and are expressed as spot-forming cells (SFCs) per 10
6
cells. The number
of specific IFN--secreting T cells was calculated by subtracting the negative
control value from the established SFC count. The negative controls were always
30 SFCs/10
6
input cells (median, 5; range, 0 to 25 SFCs/10
6
input cells). The
positive control consisted of incubation of 100,000 PBMCs with phytohemagglu-
tinin.
Statistical analysis. Statistical analysis and graphical presentation were done
using SigmaPlot 5.0 (SPSS Inc., Chicago, IL) and GraphPad Prism. Results are
given as the mean standard deviation (SD) or the median and range. Statistical
analysis of significance (P values) was based on a one-way analysis of variance
(ANOVA) test.
RESULTS AND DISCUSSION
Control over viral replication in primary HIV-1 infection has
been linked to the early emergence of HIV-1-specific CD8
T-cell responses leading to a subsequent decline of HIV-1
viremia to a set point (7, 22). To characterize the areas of the
HIV-1 proteome that are initially targeted by these few early
and effective virus-specific responses, we identified 43 subjects
during primary HIV-1 infection. Twenty-seven individuals
were identified prior to complete HIV-1 seroconversion (neg-
ative or indeterminate anti-p24 enzyme-linked immunosorbent
assay and less than three positive bands in the HIV-1 Western
blot) with a median viral load of 750,000 copies/ml (range,
6,220 to 6 10
7
copies/ml) and a median CD4
count of 453
cells/l (range, 191 to 929 cells/l). Sixteen individuals were
identified within 6 months of acute infection with a median
viral load of 28,150 copies/ml (range, 216 to 780,000 copies/ml)
and CD4
count of 480 cells/l (range, 292 to 1,069 cells/l).
We comprehensively screened HIV-1-specific T-cell responses
in these 43 individuals using a panel of 410 overlapping pep-
tides spanning the entire proteome of the HIV-1 clade B 2001
consensus sequence in an IFN- ELISPOT assay (13).
As described previously (5, 10, 28), total HIV-1-specific T-
cell responses in these individuals with primary infection were
of lower magnitude (median of 132 SFCs/10
6
PBMCs; range,
56 to 2,360) and more narrowly directed against a very limited
number of epitopes (median of 9 targeted epitopes; range, 2 to
38) than HIV-1-specific T-cell responses usually detected in
chronic infection (1, 2, 13). The targeted epitopes were located
evenly within the proteins encoded by the three large structural
genes of HIV-1 gag, env, and pol, as well as by the smaller
accessory nef gene (average numbers of recognized epitopes
within the HIV-1 proteins: Gag, 2.9 2.9; Pol, 2.8 3.7; Env,
2.4 2.2; and Nef, 1.7 1.3). Responses directed against each
of these proteins contributed between 14 and 25% to the total
virus-specific T-cell response (Fig. 1A). Taking into account
the amino acid length of each of these four HIV-1 proteins that
represented the major targets of virus-specific T-cell responses
in primary HIV-1 infection (by dividing the magnitude of the
average T-cell response to each protein by the number of
amino acids of the respective protein), Nef was the most im-
munogenic component of HIV-1 (data not shown), as previ-
ously described (24). Taken together, these data demonstrate
that early HIV-1-specific T-cell responses do not generally
favor a certain region of the HIV-1 proteome, but that HIV-1
Nef is predominantly targeted if stratified by the amino acid
length of each protein.
To further investigate whether the recognition of specific
regions within HIV-1 by T cells during primary infection dif-
fered between individuals expressing protective or nonprotec-
tive HLA class I molecules, we separated study subjects into
individuals expressing the two HLA class I alleles most strongly
associated with protection from HIV-1 disease progression
(HLA-B27 and HLA-B57); individuals expressing HLA-B35,
an HLA class I allele that has been associated with more rapid
disease progression; and individuals expressing neither of these
alleles. Of the 43 subjects identified during primary HIV-1
infection, 8 subjects carried the protective HLA class I alleles
HLA-B27 (n 4) and HLA-B57 (n 4), while 6 individuals
expressed HLA-B35 (4 individuals with the subtype HLA-
B3501 and 2 individuals with the subtype HLA-B3502). These
frequencies generally reflect the frequencies of the respective
HLA types in a Caucasoid population (HLA-B27, 4.9%; HLA-
B35, 19%; and HLA-B57, 8.7%) (8).
In the eight individuals expressing HLA-B27 or -B57, the
majority of HIV-1-specific T-cell responses were directed to-
wards Gag early in primary HIV-1 infection (Fig. 1B), and
notably HIV-1 Gag-specific responses contributed significantly
more to the total HIV-1-specific IFN- response in these in-
dividuals (44%; SD, 13.1%) than in subjects expressing non-
protective alleles (21.5%; SD, 19.8%; P 0.01 [Fig. 1B]).
7726 STREECK ET AL. J. VIROL.
Furthermore, Gag-specific T-cell responses contributed only
negligibly to the total virus-specific response in individuals who
expressed HLA-B35 (average of 5.7%; SD, 6.7% [Fig. 1B])
and were significantly lower than those in subjects expressing
HLA-B57/B27 (P 0.001) or expressing neither HLA-B57/
B27 nor HLA-B35 (P 0.01). Instead, HIV-1-specific T-cell
responses in HLA-B35
individuals were preferentially di
-
rected against HIV-1 Env (Fig. 1B). Differences in the recog-
nition of Nef, Pol, or the accessory proteins were not signifi-
cantly different among the three groups.
When we analyzed the contribution of the individual Gag
proteins (p17, p24, and p15 [p2-p7-p1-p6]) to the total HIV-
1-specific T-cell response individually, responses to p24 Gag
contributed significantly more (38%; SD, 17.8%; P 0.001)
to the total response in HLA-B27/57
individuals than in the
individuals expressing nonprotective alleles (13.2% [SD,
15.8] in subjects expressing neither HLA-B27/B57 nor HLA-
B35 and 7.7% [SD, 15.0] in subjects expressing HLA-B35
[Fig. 1C]). Although there was no statistically significant dif-
ference in the magnitude (P 0.18) and breadth (P 0.07) of
the HIV-1-specific T-cell responses between the three groups,
a trend towards lower breadth and magnitude was observed in
FIG. 1. (A) Protein specificity of HIV-1-specific T-cell responses during primary HIV-1 infection. Forty-three subjects with primary HIV-1
infection were screened by IFN- ELISPOT assay using 410 overlapping peptides spanning the entire clade B consensus sequence. The percentage
contributions of HIV-1-specific T cells to the total number of IFN- responses are shown for the different HIV-1 proteins (Gag, Nef, Pol, Env,
Vpr, Vif, Rev, Tat, and Vpu). All three large structural HIV-1 proteins Gag, Env, and Pol, as well as the smaller accessory Nef protein, were
targeted evenly. The contributions to the total number of immune responses against the four most targeted epitopes (Gag, Pol, Nef, and Env) did
not differ significantly (P 0.05). (B) Comparison of protein specificity of T-cell responses between subjects expressing HLA-B27/B57 or HLA-B35
and subjects expressing neither HLA-B27/B57 nor HLA-B35. The contribution to the total HIV-1-specific T-cell response is shown for the different
HIV-1 proteins (Gag, Nef, Pol, Env, Vpr, Vif, Rev, Tat, and Vpu) stratified according to the three HLA groups. Significant differences (P 0.05)
are indicated with asterisks (*). (C) Comparison of protein specificity within the Gag protein of T-cell responses between subjects expressing
HLA-B27/B57 or HLA-B35 and subjects expressing neither HLA-B27/B57 nor HLA-B35. The contribution to the total HIV-1-specific response
is shown for the different proteins encoded within Gag: p17, p24, and p15 (p2-p7-p1-p6) and stratified according to the three HLA groups. In
subjects expressing HLA-B35, no responses against p15 were detected (indicated with X). Significant differences (P 0.05) are indicated with
asterisks.
V
OL. 81, 2007 CD8
T-CELL-TARGETED DEFINED REGION IN HIV-1 p24 Gag 7727
subjects expressing HLA-B27/B57 (median number of recog-
nized epitopes, 4.5; median magnitude, 1,251 SFCs/10
6
cells)
compared to the other two groups (subjects expressing neither
HLA-B27/B57, median number of recognized epitopes, 10;
median magnitude 2,944 SFC/10
6
cells; subjects expressing
HLA-B35, median number of recognized epitopes, 6; median
magnitude, 2,239 SFCs/10
6
cells). These data, derived from
individuals early in primary HIV-1 infection, show that T cells
from subjects expressing HLA-B27 and HLA-B57, two alleles
strongly associated with slower HIV-1 disease progression (9),
preferentially target epitopes within p24 Gag.
To determine whether the identified Gag-specific responses
were indeed CD8
T-cell responses restricted by HLA-B27/
B57, we further investigated the fine specificity of the Gag-
specific T-cell responses on the single-epitope level in these
individuals. Only a very limited number of overlapping pep-
tides (OLPs) within p24 Gag were consistently targeted in the
eight individuals expressing HLA-B27 and HLA-B57, includ-
ing OLP-36 (PVGEIYKRWIILGLNKIV [Gag aa 257 to 274]),
which was targeted by all four HLA-B27
individuals, and
OLP-33 (SDIAGTTSTLQEQIGWM [Gag aa 234 to 250]),
which was targeted by all four HLA-B57
individuals (Fig. 2).
These two OLPs contained two previously described HLA-
B27- and HLA-B57-restricted CD8
T-cell epitopes, B27 (K
RWIILGLNK; KK10) and B57 (TSTLQEQIGW; TW10). Fig-
ure 2 demonstrates the individual OLPs within Gag targeted in
the eight study subjects, as well as the responses to the peptides
corresponding to the optimal described epitopes contained
within these OLPs when tested individually. Strikingly, the
responses against these few immunodominant epitopes con-
tributed 84% (range, 30 to 100%) to the total Gag-specific
CD8
T-cell responses and 36% (range, 14 to 64%) to the total
HIV-1-specific CD8
T-cell responses in these eight B57/B27
subjects. Both epitopes KK10 and TW10 are located between
aa 240 and 272 within the same region of p24 Gag, confined to
the functionally important alpha helices 6 and 7. None of the
6 subjects expressing HLA-B35 and only 3 out of the 29 re-
maining subjects that expressed neither HLA-B57/B27 nor
FIG. 2. Assessment of the fine specificity of Gag-specific responses in subjects expressing HLA-B27/B57. Values show the magnitude of a
response in SFCs/10
6
cells. Each graph represents one subject. The percentages of Gag contribution to the total number of responses for every
subject are as follows: AC-138, 30%; AC-154, 51%; AC-34, 19%; AC-153, 42%; AC-160, 62%; AC-162, 56%; AC-207, 41%; AC-183, 22%. Bars
on the left represent the immune responses detected with overlapping peptides spanning the entire Gag clade B consensus sequence, while bars
on the right represent the respective response to the optimal epitope encoded within the overlapping peptide when tested individually.
7728 STREECK ET AL. J. V
IROL.
HLA-B35 recognized this specific region within p24 Gag. In
these three individuals, responses to this area only contributed
a median of 2.8% (range, 1 to 14%) to the total HIV-1-specific
CD8
T-cell response. None of these three subjects expressed
HLA alleles with a previously reported association with slow
disease progression (Ac-144, HLA-A2, -A2, -B14, -B44, -Cw5,
and -Cw8; Ac-151, HLA-A34, -A24, -B1517, -B49, -Cw07, and
-Cw07; and Ac-158, HLA-A1, -A68, -B7, -B8, -Cw7, and
-Cw7). These data suggest that individuals expressing protec-
tive HLA class I alleles specifically target very few immuno-
dominant epitopes within a very defined region of p24 Gag
during primary HIV-1 infection.
To further evaluate this narrow targeting of a specific region
in HIV-1 p24 Gag by CD8
T-cell responses restricted by
HLA-B27 and -B57 early in HIV-1 infection, we extended the
analysis to a second, larger, multicenter cohort of 293 individ-
uals identified during primary HIV-1 infection. HIV-1-specific
CD8
T-cell responses in these individuals were characterized
using peptides corresponding to optimal CD8
T-cell epitopes
described for the respective HLA class I allotype of the study
subject (4). The assessment of HIV-1-specific CD8
T-cell
responses was performed on frozen PBMC samples collected 8
weeks ( 10 days) following the initial presentation with pri-
mary infection (4). In this large cohort of 293 subjects, we
identified 21 subjects expressing HLA-B57 and 13 subjects
expressing HLA-B27. While 94% of the subjects expressing
HLA-B57/B27 recognized described p24 Gag CD8
T-cell
epitopes, only 64% of non-B27/B57/B35 subjects recognized
p24 Gag epitopes and only 9.8% of HLA-B35-expressing sub-
jects recognized them. Among the 51 individuals expressing
HLA-B35, only 5 recognized the epitope PPIPVGDIY (B-35-
PY9) within p24, with no statistically significant difference be-
tween HLA-B35Px
and HLA-B35Py
individuals in this
small data set (1 HLA-B35Px and 3 HLA-35Py; one subtype
was not available).
In the HLA-B57
individuals, the epitope TW10 in p24 Gag
again represented the immunodominant HIV-1-specific CD8
T-cell response and was targeted in 86% of individuals, fol-
FIG. 3. (A) Frequency of recognition of optimal HIV-1-specific CD8
T-cell epitopes described for the respective HLA class I allele of subjects
expressing HLA-B57. Twenty-one subjects expressing HLA-B57 were screened by optimal HLA-matched epitopes 8 weeks (10 days) postdiag-
nosis. The epitope TSTLQEQIGW (TW10 [p24 Gag]) was recognized in 86% of the subjects, followed by IVLPEKDSW (IW9 [RT]) in 61.9% and
KAFSPEVIPMF (KF11 [p24 Gag]) in 52.4%. (B) Frequency of recognition of optimal HIV-1-specific CD8
T-cell epitopes described for the
respective HLA class I allele of subjects expressing HLA-B27. Thirteen subjects expressing HLA-B27 were screened by optimal HLA-matched
epitopes 8 weeks (10 days) postdiagnosis. The epitope KRWIILGLNK (KK10 [p24 Gag]) was recognized in 92% of the subjects, while the other
HLA-B27-restricted epitopes were less frequently recognized. (C) Contribution of epitope-specific HLA-B57 responses to the total HIV-1-specific
CD8
T-cell response. Twenty-one subjects expressing HLA-B57 were screened for responses against the optimal HIV-1-specific CD8
T-cell
epitopes described for their respective HLA class I alleles. The epitope TW10 (p24 Gag) contributed 31% (33.3%) to the total HIV-1-specific
CD8
T-cell response, while other HLA-B57-restricted responses had a significantly lower contribution to the total HIV-1 specific CD8
T-cell
response calculated by using a one-way ANOVA (P 0.001). (D) Contribution of epitope-specific HLA-B27 responses to the total HIV-1-specific
CD8 T-cell response. Thirteen subjects expressing HLA-B27 were screened for responses against the optimal HIV-1-specific CD8
T-cell epitopes
described for their respective HLA class I alleles. The epitope KK10 (p24 Gag) contributed 39.7% (20.5%) to the total HIV-1-specific CD8
T-cell response, while other HLA-B27-restricted responses had a significantly lower contribution to the total HIV-1-specific CD8
T-cell response,
calculated by using a one-way ANOVA (P 0.001).
V
OL. 81, 2007 CD8
T-CELL-TARGETED DEFINED REGION IN HIV-1 p24 Gag 7729
lowed by IVLPEKDSW in the reverse transcriptase (IW9) in
61.9% and KAFSPEVIPMF in p24 (KF11) in 52.4%, while
other HLA-B57-restricted epitopes were less frequently tar-
geted (Fig. 3A). In HLA-B27
subjects, the epitope KK10
(p24 Gag) was recognized in 92% of subjects, while other
HLA-B27-restricted epitopes were rarely recognized (Fig. 3A).
These data reconfirmed in a second large cohort of 293 study
subjects that the immunodominant epitopes targeted in indi-
viduals expressing the protective HLA class I alleles B27 and
B57 are located consistently within a specific area of p24 Gag
(aa 240 to 272, containing HLA-B57-TW10 and HLA-B27-
KK10).
We next investigated in this cohort of 293 study subjects how
much each targeted HIV-1-specific CD8
T-cell epitope con
-
tributed to the total virus-specific CD8
T-cell response of the
HLA-B57
and HLA-B27
individuals during primary HIV-1
infection. The tested HIV-1-specific CD8
T-cell epitopes de
-
scribed within HIV-1 Gag contributed 44% in subjects with
HLA-B57 and 41% in subjects with HLA-B27 to the total
HIV-1-specific immune response. The HLA-B57-restricted re-
sponse to the epitope TW10 alone contributed already 31%
(SD, 33.3%) to the total virus-specific response in B57
subjects, and the HLA-B27-restricted response to the epitope
KK10 contributed 40% (SD, 20.5%) to the total response in
B27
subjects. Other HLA-B57-restricted responses or re
-
sponses restricted by other HLA class I alleles in the HLA-
B57
individuals (data not shown) had a significantly lower
contribution to the total HIV-1-specific CD8
T-cell response
(Fig. 3B). Similarly, in HLA-B27
subjects, other HLA-B27-
restricted responses outside p24 Gag contributed little to the
total virus-specific CD8
T-cell response (Fig. 3B). In conclu
-
sion, the overall majority of the HIV-1-specific CD8
T-cell
response in subjects expressing HLA-B27 or HLA-B57 was
geared towards p24 Gag and was mainly composed of two
epitopes, KK10 and TW10, located within the same region of
Gag (aa 240 to 272).
Why might CD8
T-cell responses directed against this re
-
gion of p24 Gag be more beneficial than responses directed
against other regions of the virus? The region between aa 240
and aa 272 of p24 Gag is highly conserved (more than 98.3%
[range, 48 to 100%] conservation per amino acid position ac-
cording to the HIV-1 clade B sequences published in the Los
Alamos Database), and previous studies have indicated that
sequence variations within this region may incur a significant
loss of viral fitness (23, 25). It has been shown that under the
selection pressure of the CD8
T-cell response in HLA-B57
individuals, the epitope TW10 mutates after the acute phase of
the infection in 74% of the subjects by a Thr3Asn amino acid
substitution at residue 242 (23, 25). Furthermore, the CTL
escape mutations within the TW10 epitope revert quickly back
to wild type when transmitted to an HLA-B57-negative indi-
vidual (23). This reversion of the escape mutation back to wild
type is most likely due to a fitness cost associated with a loss in
the capability to stabilize helices 6 and 7 within p24 in the
presence of the mutation (25). In contrast, escape mutations
from the HLA-B27-restricted epitope KK10 occur only in later
stages of the infection and after the prior accumulation of
compensatory mutations (20). Furthermore, CTL escape mu-
tations within the KK10 epitope have been associated with
HIV-1 disease progression (6, 11, 15). Overall, these data in-
dicate that individuals expressing HLA-B57 and -B27 are ca-
pable of targeting this vulnerable “Achilles’ heel” of HIV-1
p24 Gag early in acute infection, allowing for the rapid control
of viral replication and the preservation of an effective antiviral
immune response (3). Recent studies also suggest that Gag
epitopes are the earliest presented targets on infected cells,
even prior to the reverse transcription and integration of the
virus, as relative large quantities of Gag protein are contained
in the capsid of the incoming virus, while the intracellular
processing of Env epitopes follows the de novo synthesis of
Env protein (30).
In conclusion, we have demonstrated that the majority of
HIV-1-specific CD8
T-cell responses in individuals express
-
ing the protective HLA class I alleles B27 and B57 are directed
against a highly conserved region within p24 Gag that is rarely
targeted in other individuals during primary HIV-1 infection.
Interestingly however, several potential epitopes restricted by
common HLA class I alleles such as HLA-A2, HLA-A3, HLA-
B7, and HLA-B8, as well as conserved CD4
T-cell epitopes
(31), are contained within this area of p24 Gag studied here
(http://www.hiv.lanl.gov/content/immunology/maps/ctl/p24
.html) but not the immunodominant HLA-A2-restricted p17
Gag epitope SLYNTVATL (A2-SL9). While targeting of these
p24 Gag HIV-1 epitopes by CD8
T cells is rarely observed
early in natural HIV-1 infection (less than 10% of the non
HLA-B27/B57 individuals targeted this region), these epitopes
can be targeted in chronic HIV-1 infection (5, 13). Future
studies will need to evaluate the possibility of overcoming
immunodominance patterns emerging in early natural infec-
tion by prior vaccinations, with the aim of targeting vulnerable
areas of HIV-1 with CD8
T-cell responses restricted by com
-
mon HLA class I alleles (3).
ACKNOWLEDGMENTS
We thank all study subjects for their participation. We thank in
particular Robert Finlayson (Taylor Square Clinic, Darlinghurst, Syd-
ney, Australia), Robert MacFarland (407 Doctors, Darlinghurst, Syd-
ney, Australia), Cassy Workman, (AIDS Treatment Initiative, Darlin-
ghurst, Sydney, Australia), and Mark Bloch (Holdsworth House
General Practice, Darlinghurst, Sydney, Australia) for enrolling study
participants from the respective Australian sites.
This study was supported by the National Institutes of Health (RO1
AI50429; Acute Infection Early Disease Research Network U01
AI052403). In addition, A.K. is supported by a Program grant and
Practitioner fellowship from the Australian National Health and Med-
ical Research Council (NHMRC). The NHMRC is supported by the
Commonwealth Department of Health and Ageing, Australia. The
work of S.L. was supported by National Institutes of Health grant
AI43638. J.-P.R. is a physician-scientist supported by Fonds de
Recherche en Saute´ du Quebec (FRSQ). We thank all the members of
the Montreal HIV Infection Study receiving financial support from
FRSQ.
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VOL. 81, 2007 CD8
T-CELL-TARGETED DEFINED REGION IN HIV-1 p24 Gag 7731
    • "Of note, HLA-B*57:01 appears as somewhat of an outlier, exhibiting higher than expected escape prevalence in early infection given its protective nature. We hypothesize that the reason B*57 can maintain sustained HIV-1 control despite rapid escape in some epitopes (Table 1 and [2,13,16,18,28]) is because the early B*57-restricted CD8+ response often simultaneously targets more than one epitope, notably in p24 Gag [31,414243, where escape is accompanied by fitness costs444546. HLA-associated polymorphisms identified at the population level mark viral sites under strong, reproducible in vivo immune pressure by individual HLA alleles [11]. "
    [Show abstract] [Hide abstract] ABSTRACT: Background The reproducible nature of HIV-1 escape from HLA-restricted CD8+ T-cell responses allows the identification of HLA-associated viral polymorphisms ¿at the population level¿ ¿ that is, via analysis of cross-sectional, linked HLA/HIV-1 genotypes by statistical association. However, elucidating their timing of selection traditionally requires detailed longitudinal studies, which are challenging to undertake on a large scale. We investigate whether the extent and relative timecourse of immune-driven HIV adaptation can be inferred via comparative cross-sectional analysis of independent early and chronic infection cohorts.ResultsSimilarly-powered datasets of linked HLA/HIV-1 genotypes from individuals with early (median¿<¿3 months) and chronic untreated HIV-1 subtype B infection, matched for size (N¿>¿200/dataset), HLA class I and HIV-1 Gag/Pol/Nef diversity, were established. These datasets were first used to define a list of 162 known HLA-associated polymorphisms detectable at the population level in cohorts of the present size and host/viral genetic composition. Of these 162 known HLA-associated polymorphisms, 15% (occurring at 14 Gag, Pol and Nef codons) were already detectable via statistical association in the early infection dataset at p¿¿¿0.01 (q¿<¿0.2) ¿ identifying them as the most consistently rapidly escaping sites in HIV-1. Among these were known rapidly-escaping sites (e.g. B*57-Gag-T242N) and others not previously appreciated to be reproducibly rapidly selected (e.g. A*31:01-associated adaptations at Gag codons 397, 401 and 403). Escape prevalence in early infection correlated strongly with first-year escape rates (Pearson¿s R¿=¿0.68, p¿=¿0.0001), supporting cross-sectional parameters as reliable indicators of longitudinally-derived measures. Comparative analysis of early and chronic datasets revealed that, on average, the prevalence of HLA-associated polymorphisms more than doubles between these two infection stages in persons harboring the relevant HLA (p¿<¿0.0001, consistent with frequent and reproducible escape), but remains relatively stable in persons lacking the HLA (p¿=¿0.15, consistent with slow reversion). Published HLA-specific Hazard Ratios for progression to AIDS correlated positively with average escape prevalence in early infection (Pearson¿s R¿=¿0.53, p¿=¿0.028), consistent with high early within-host HIV-1 adaptation (via rapid escape and/or frequent polymorphism transmission) as a correlate of progression.Conclusion Cross-sectional host/viral genotype datasets represent an underutilized resource to identify reproducible early pathways of HIV-1 adaptation and identify correlates of protective immunity.
    Full-text · Article · Aug 2014
    • "Because of this adaptive process, epitopes presented by common HLA variants are generated in low amounts, whereas epitopes presented by rare HLA variants are made in abundance. Taken together with the reported weak and infrequent CTL responses associated with common HLA variants, and the strong and frequent CTL responses associated with rare HLA variants in these Gag regions (Goulder and Walker, 2012; Streeck et al., 2007 ), our results support previous findings linking epitope-abundance to the magnitude and frequency of epitope-specific CTL responses (Lazaro et al., 2009; Ranasinghe et al., 2011; Schmidt et al., 2012; Tenzer et al., 2009 ). Our data provide clear evidence that all epitope-presenting HLA class I variants exert selective pressures on subtypespecific positions in these key HIV Gag regions, resulting in population-level immune evasion in both HIV B and HIV C infection. "
    [Show abstract] [Hide abstract] ABSTRACT: The recent HIV-1 vaccine failures highlight the need to better understand virus-host interactions. One key question is why CD8(+) T cell responses to two HIV-Gag regions are uniquely associated with delayed disease progression only in patients expressing a few rare HLA class I variants when these regions encode epitopes presented by ∼30 more common HLA variants. By combining epitope processing and computational analyses of the two HIV subtypes responsible for ∼60% of worldwide infections, we identified a hitherto unrecognized adaptation to the antigen-processing machinery through substitutions at subtype-specific motifs. Multiple HLA variants presenting epitopes situated next to a given subtype-specific motif drive selection at this subtype-specific position, and epitope abundances correlate inversely with the HLA frequency distribution in affected populations. This adaptation reflects the sum of intrapatient adaptations, is predictable, facilitates viral subtype diversification, and increases global HIV diversity. Because low epitope abundance is associated with infrequent and weak T cell responses, this most likely results in both population-level immune evasion and inadequate responses in most people vaccinated with natural HIV-1 sequence constructs. Our results suggest that artificial sequence modifications at subtype-specific positions in vitro could refocus and reverse the poor immunogenicity of HIV proteins.
    Full-text · Article · Apr 2014
    • "The large diversity of viable viral strains makes it difficult for the adaptive immune system to mount natural responses that effectively control the virus (Autran et al., 2008; Goulder and Watkins, 2004). Immune responses or therapeutic agents that target regions of the viral proteome where mutations lead to a large cost in replicative fitness can be very effective for viral control or aborting the infection (Dahirel et al., 2011; Goulder and Watkins, 2004; Streeck et al., 2007). Systematic means to derive the viral fitness landscape permits the identification of such regions. "
    [Show abstract] [Hide abstract] ABSTRACT: A prophylactic or therapeutic vaccine offers the best hope to curb the HIV-AIDS epidemic gripping sub-Saharan Africa, but it remains elusive. A major challenge is the extreme viral sequence variability among strains. Systematic means to guide immunogen design for highly variable pathogens like HIV are not available. Using computational models, we have developed an approach to translate available viral sequence data into quantitative landscapes of viral fitness as a function of the amino acid sequences of its constituent proteins. Predictions emerging from our computationally defined landscapes for the proteins of HIV-1 clade B Gag were positively tested against new in vitro fitness measurements and were consistent with previously defined in vitro measurements and clinical observations. These landscapes chart the peaks and valleys of viral fitness as protein sequences change and inform the design of immunogens and therapies that can target regions of the virus most vulnerable to selection pressure.
    Full-text · Article · Mar 2013
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