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*
Partners AIDS Research Center and Infectious Disease Division, and
Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02129;
AIDS Research Center, First Affiliated Hospital China Medical University, Shenyang, China;
Nuffield Department of Medicine, John Radcliffe Hospital, Oxford, United Kingdom;
¶ Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115; and
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Fenway Community Health Center, Boston, MA 02116
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production was measured by ELISPOT assay and flow-based
intracellular cytokine quantification. HLA class I restriction and
cytotoxic activity were confirmed after isolation of peptide-specific
CD8+ T cell lines. CD8+ T cell responses
against Vpr, Vpu, and Vif were found in 45%, 2%, and 33% of
HIV-1-infected individuals, respectively. Multiple CTL epitopes were
identified in functionally important regions of HIV-1 Vpr and Vif.
Moreover, in infected individuals in whom the breadth of HIV-1-specific
responses was assessed comprehensively, Vpr and p17 were the most
preferentially targeted proteins per unit length by CD8+ T
cells. These data indicate that despite the small size of these
proteins Vif and Vpr are frequently targeted by CTL in natural HIV-1
infection and contribute importantly to the total HIV-1-specific
CD8+ T cell responses. These findings will be important in
evaluating the specificity and breadth of immune responses during acute
and chronic infection, and in the design and testing of candidate HIV
vaccines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To better understand immune control of viremia and for the design of potential HIV-1 vaccines, those regions of HIV-1 that induce strong virus-specific immune responses should be identified. To date, the analysis of HIV-1-specific immunity has largely focused on assessment of immune responses directed against the structural HIV-1 proteins Gag, Pol, and Env, as well as the accessory protein Nef (7). Recent data suggest that CTL responses directed against the early expressed regulatory proteins Tat and Rev also play a central role in the HIV-1-specific immune response (5, 8, 9). However, very little is known about the role of cellular immune responses directed against the other accessory proteins.
The relatively small accessory HIV-1 proteins Vpu, Vif, and Vpr have important functions in viral disassembly, nuclear transport of the preintegration complex (PIC),4 viral assembly, and down-regulation of CD4 on the cell surface (10, 11, 12, 13, 14). Thus these accessory proteins appear to be essential for viral replication. However, the extent to which these proteins are targeted by CTL in natural infection remains to be defined, but their cytoplasmic production within infected cells suggests that they should be HLA class I presented and candidate targets for CTL-mediated lysis of infected cells.
In this study, we performed a detailed characterization of CTL
responses to Vpu, Vif, and Vpr, using recently developed techniques
including IFN- ELISPOT and intracellular flow cytometric analyses,
as well as T cell cloning. Our data provide evidence that the Vpr and
Vif proteins represent important targets of cellular host defenses,
whereas Vpu is infrequently recognized. In addition, the Vpr protein is
one of the most frequently targeted HIV-1 proteins by CTL relative to
the length of the protein. This report also provides a characterization
of discrete CTL epitopes within these proteins, identified using
overlapping peptides spanning the entire HIV-1 Vpr, Vpu, and Vif
sequence.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sixty HIV-1-infected and 10 HIV-1-negative individuals were studied at the Massachusetts General Hospital. HIV-1-infected individuals included 45 subjects who were treated with highly active antiretroviral therapy within 180 days of HIV-1 infection, nine individuals with chronic treated HIV-1 infection, and six individuals with long-term nonprogressive HIV-1 infection, defined as viremia below 1000 HIV-1 RNA copies per milliliter for >6 years in the absence of any antiretroviral treatment. At the time of the CTL analysis, subjects on highly active antiretroviral therapy had been effectively treated for at least 6 mo and all had viral loads below 50 copies RNA/ml. The study was approved by the Massachusetts General Hospital Institutional Review Board, and all individuals gave informed consent for participation in the study.
HLA typing
HLA class I molecular typing was performed at the Massachusetts General Hospital Tissue Typing Laboratory using sequence-specific primer-PCR (15).
Synthetic HIV-1 peptides
Peptides were synthesized on an automated peptide synthesizer (MBS 396; Advanced Chemtech, Louisville, KY) using F-moc chemistry. Seventeen overlapping peptides spanning the HIV-1 SF2 B clade Vpr sequence, 14 overlapping peptides spanning the HIV-1 SF2 B clade Vpu sequence, and 36 overlapping peptides spanning the HIV-1 SF2 B clade Vif sequence (12–18 mers with 10 aa overlap) were generated. In addition, peptides corresponding to described optimal HIV-1 CTL epitopes (7) and a panel of 321 overlapping peptides (15–20 mers) spanning the entire p15 Gag, p17 Gag, p24 Gag, gp41 Env, gp120 Env, reverse transcriptase (RT), Rev, Tat, and Nef sequence (16), were used.
Cell lines and media
EBV-transformed B lymphoblastoid cell lines were established and maintained in R20 medium (RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 2 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, 10 mM HEPES, and 20% heat-inactivated FCS (Sigma)) as previously described (17). For culture of CTL clones, medium containing 10% FCS (R10) supplemented with 50 U/ml rIL-2 (provided by Dr. M Gately, Hoffmann-LaRoche, Nutley, NJ) was used.
Generation of peptide-specific CD8+ T cell lines
CD8+ T cells were nonspecifically expanded
from PBMC over 10 days, using a bispecific CD3/CD4 Ab. Peptide-specific
CD8+ T cell lines were subsequently isolated
using an IFN- catching assay (18, 19, 20), according to the
manufacture’s instructions. Briefly, after expansion 10–20 x
106 CD8+ T cells were
incubated on 24-well plates with 20 µM peptide and 1 µg/ml each of
the mAbs anti-CD28 and anti-CD49d (BD Biosciences, Mountain
View, CA) at 37°C, 5% CO2, for 6–8 h. Cells
were subsequently labeled with a bispecific CD45/IFN- catching Ab
and incubated for 45 min at 37°C, 5% CO2.
After several washes, the IFN--producing cells were stained with a
second IFN--PE detection Ab and separated by anti-PE mAb labeled
with magnetic beads (MACS; Miltenyi Biotech, Hamburg, Germany). The
isolated cells were then expanded for 10 days using autologous
irradiated feeders, as described previously (21).
Generation of CTL clones
CTL clones were isolated by limiting dilution as previously described (21, 22), using the anti-CD3-specific mAb 12F6 as stimulus for T cell proliferation. Developing clones were screened for HIV-1-specific CTL activity by 51chromium-release assay (17) against autologous B cell lines (BCLs) pulsed with the peptides recognized in the ELISPOT assays or infected with recombinant vaccinia virus (rVV) expressing either HIV-1 Vpr or Vif (provided by G. P. Mazzara, Therion Biologics, Cambridge, MA). HIV-1-specific clones were maintained by stimulation every 14–21 days with an anti-CD3 mAb and irradiated allogeneic PBMC. HLA-restriction of CTL epitopes was determined using a panel of target cells matched through only one of the HLA-A, HLA-B, or HLA-C class I alleles expressed by the effector cells (21).
ELISPOT assay
PBMC were plated on 96-well polyvinylidene difluoride-backed
plates (MAIP S45; Millipore, Bedford, MA) that had been previously
coated with 100 µl of an anti-IFN- mAb 1-D1k (0.5 µg/ml;
Mabtech, Stockholm, Sweden) overnight at 4°C. Peptides were added
directly to the wells at a final concentration of 1 x
10-5 molar. Cells were added to the wells at
50,000–100,000 cells/well. The plates were incubated at 37°C, 5%
CO2 overnight (14–16 h) and then processed as
described (23, 24). IFN--producing cells were counted
by direct visualization and are expressed as spot-forming cells (SFCs)
per 106 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 <20 SFC per 106 input cells.
Responses 
40 SFC per 106 input cells above
background were considered positive. The positive control consisted of
incubation of 100,000 PBMC with PHA. CD8+ T cell
dependence of all responses to synthetic peptides was confirmed by loss
of IFN- production after CD8+ T cell depletion
using magnetic beads (MACS; Miltenyi Biotech), according to the
manufacturer’s protocol. Fine mapping of epitopes by ELISPOT assay was
performed as described (23), using peptide truncations.
Briefly, 100,000 PBMC/well were incubated with concentrations from
10-4 to 10-11 M peptide
overnight on the ELISPOT plate. All assays were run in duplicate. The
optimal peptide was defined as the peptide that induced 50% maximal
specific IFN- production of T cells at the lowest peptide
concentration (23).
Flow cytometric detection of Ag-induced intracellular IFN-
Intracellular cytokine staining assays were performed as
described elsewhere with minor modifications (25, 26).
Briefly, 0.5–1.0 million PBMC were incubated on 24-well plates with 2
µM peptide and 1 µg/ml each of the mAbs anti-CD28 and
anti-CD49d (BD Biosciences) at 37°C, 5%
CO2, for 1 h, before the addition of 10
µg/ml brefeldin A (Sigma). Following another 5-h incubation at
37°C, 5% CO2, the cells were placed at
4°C overnight. PBMC were then washed with PBS/1% BSA and stained
with surface Abs, anti-CD8 and anti-CD4 (BD Biosciences) at
4°C for 20 min. Following three more washes, the PBMC were fixed and
permeabilized using Caltag Fixation/Permeabilization Kit (Caltag
Laboratories, Burlingame, CA) and anti-IFN- mAb (BD Biosciences)
was added. Cells were then washed and analyzed on a FACSort
Flowcytometer (BD Immunocytometry Systems, San Jose, CA) using PerCP,
allophycocyanin, and FITC as fluorescent parameters. Control
conditions were established by the use of autologous PBMC, which had
not been stimulated with peptide, but otherwise had been treated
identically. Cell population boundaries were established by exclusion
of 99.97% of control lymphocytes. For the determination of HLA class I
restriction of responses by flow cytometry, assays using HLA-matched or
mismatched BCLs were run as described (27). Briefly, BCLs
that were pulsed with 10 µM peptide for 1 h were washed five
times before incubation with effectors (105 BCL
and 5 x 105 effectors) in 1 ml of R10. The
mAbs anti-CD28 and anti-CD49d were then added, and the assay
was run exactly as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether the Vif, Vpr, and/or Vpu proteins are
targeted by the CTL response in HIV-1 infection, we performed a pilot
study in individual AC-06 in whom we had previously demonstrated
responses to a total of nine CTL epitopes in p17, p24, RT, gp41, and
Nef, and defined the optimal epitopes recognized within those gene
products (16, 28). PBMC from subject AC-06 were analyzed
by ELISPOT assay with a set of 388 overlapping peptides spanning the
expressed HIV-1 clade B sequence. This allowed us to assess the
relative contribution of Vpr-, Vpu-, and Vif-specific
CD8+ T cell responses to the overall CD8-mediated
immune response. Fig. 1
shows the
comprehensive characterization of HIV-1-specific CTL responses in this
person using the entire panel of overlapping peptides including 17
peptides spanning Vpr, 14 peptides spanning Vpu, and 36 peptides
spanning Vif, as well as peptides spanning p15, p17, p24, RT, gp41,
gp120, Nef, Rev, and Tat. CD8+ T cell dependence
of all responses was confirmed by CD8/CD4 depletion studies (data not
shown). In addition to the nine previously demonstrated CTL responses,
we detected at least two responses to Vif and one to Vpr (Fig. 1). Of
all of the responses detected, the response to an epitope contained in
the Vif peptide THPRVSSEVHIPLG was the third highest in magnitude (1180
SFC/106 PBMC). These data indicate that multiple
accessory proteins can be targeted simultaneously by the CTL response
in a single individual, and failure to assess these responses would
lead to an underestimation of total CTL responses.
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The above data indicate that Vif and Vpr were targeted by CD8
cells in this individual, but do not indicate the number of epitopes
contained within each of the proteins. For example, the p17 Gag peptide
WEKIRLRPGGKKKYK actually contains two discrete A3-restricted epitopes,
KIRLRPGGK and RLRPGGKKK (7), and responses to two peptides
in Vif that share a 10-aa overlap could represent targeting of an
epitope shared by both peptides, or two discrete epitopes. To determine
the contribution of epitopes within the accessory proteins to the
overall breadth of the CTL response in this person, we next determined
the optimal epitopes within these longer peptides, using newly adapted
techniques (23, 27). This is exemplified for the two CTL
epitopes within HIV-1 Vif (Figs. 2 and 3). The optimal sequences of these novel
CTL epitopes were determined by ELISPOT assay, using PBMC and serial
dilutions of truncated peptides (Fig. 2, A and
B). The peptide that induced 50% of maximal specific
IFN- production at the lowest peptide concentration was defined as
the optimal CTL epitope (Fig. 2A: RIRTWKSLVK (RK10) for
Vif-3; Fig. 2B: HPRVSSEVHI (HI10) for Vif-10). The HLA
restriction of these CTL epitopes was subsequently determined by flow
cytometric quantification of peptide-specific IFN- production using
PBMC and partially HLA class I-matched APCs (Fig. 3). The Vif-RK10
peptide induced IFN- production of CD8+ T
cells only when presented by autologous APC or APC expressing HLA-A3,
demonstrating that this epitope was restricted by HLA-A3. This
peptide also conforms precisely to the predicted motif for
HLA-A3-presented peptides, with a nonpolar residue in position 2 and a
lysine at the C-terminal of the peptide (29, 30). In
contrast, the Vif-HI10 peptide was restricted by HLA-B7 (Fig. 3), and
conformed to the predicted motif for this allele (30). The
sequence of the optimal CTL epitope as well as the HLA class I
restriction was subsequently reconfirmed using a standard
51Cr release assayfollowing the
isolation of peptide-specific CTL clones by limiting dilution (data not
shown). In all, three novel CTL epitopes in two accessory HIV-1
proteins were identified by the use of overlapping peptides in this
single patient (Table I). Of the total of
12 epitopes targeted by individual AC-06, 3 (25%) were located in the
accessory proteins Vif and Vpr.
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quantification by flow cytometry (Fig. 4). Interassay variation, defined as the
SD divided by the mean of the responses between assays run multiple
times in parallel, was <12%, as determined in previous experiments to
evaluate this technique (data not shown). Of the
CD8+ T cells, 15.6% were specific for the tested
HIV-1 CTL epitopes. The immunodominant response in this subject was
directed against the HLA-B7-restricted CTL epitope GPGHKARVL (GL9) in
p24 Gag. A total of 5.6% of CD8+ T cells were
directed against this epitope, contributing 32% to the total
HIV-1-specific CD8 responses. CD8+ T cell
responses directed against the accessory proteins Vif and Vpr
contributed importantly to the total responses (17% of total
HIV-1-specific CD8 responses) in this subject. Therefore, these data
indicate that the breadth and magnitude of the CTL responses in this
person would have been underestimated by 25 and 17%, respectively, had
responses directed against the accessory HIV-1 proteins Vif, Vpr, and
Vpu not been assessed.
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The data described above indicate that CD8+
T cell responses can target epitopes within the accessory HIV-1
proteins Vif, Vpr, and Vpu. To characterize CD8+
T cell responses directed against these accessory proteins in more
detail, 60 HIV-1-infected individuals at different stages of infection
(45 individuals with treated acute HIV-1 infection, 9 individuals with
treated chronic infection, and 6 individuals with long-term
nonprogressive infection) were screened for CD8+
T cell responses against these proteins by using overlapping peptides
in an IFN- ELISPOT assay. PBMC from 27/60 (45%) HIV-1-positive
study subjects recognized at least one overlapping Vpr peptide (Table II), 20/60 (33%) individuals had
responses against one or more Vif peptides (Table III), and only one individual had
responses against the Vpu peptides (individual CMW with long-term
nonprogressive HIV-1 infection had confirmed responses against the
Vpu-6 peptides at 170 SFC/106 PBMC). Magnitude of
responses against the Vpr peptides ranged from 40 to 1400
SFC/106 PBMC (median 245), with Vpr-12 being the most
frequently targeted. Responses against the Vif peptides range from 40
to 1100 SFC/106 PBMC (median 215), with Vif-6 being the
most frequently targeted. CD4 depletion assays and flow-based
analysis of peptide-specific intracellular IFN- production
determined that all responses against the accessory proteins were
CD8+ T cell mediated (data not shown).
Individuals with long-term nonprogressive and treated chronic HIV-1
infection targeted the Vpr more frequently than individuals with
treated acute infection (p = 0.03, two-tailed
Fischer’s exact test), but not Vif (p = 0.8).
This observation for HIV-1 Vpr is in line with our previous observation
that individuals treated during acute HIV-1 infection have a narrowly
directed CTL response, compared with individuals treated later in the
course of infection (16). Responses against the tested
Vif, Vpr, and Vpu peptides were <20 SFC/106 PBMC (range
0–20 SFC/106 PBMC, median 0 SFC/106 PBMC) in
the 10 HIV-1-negative individuals used as controls.
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We next studied how frequently the accessory proteins Vif, Vpr,
and Vpu were recognized compared with other HIV-1 proteins. Therefore,
a subset of 29 HIV-1-infected individuals (4 with long-term
nonprogressive infection, 17 with acute treated infection and 8
with chronic treated infection) were screened with a panel of 388
overlapping peptides spanning the entire Gag, gp41, RT, Rev, Tat, Vpr,
Vpu, Vif, and Nef sequence, as described above for subject AC-06 (Fig. 1
), and frequencies of recognition were compared (Table IV). Furthermore, the frequency of
recognition was adjusted for the length of the proteins by dividing
frequency by the number of amino acids per protein. Adjusted for its
length, the HIV-1 Vpr and p17 Gag proteins were the most frequently
targeted HIV-1 proteins in natural HIV-1 infection, whereas HIV-1 Vif
was targeted as frequently as HIV-1 RT, gp41, or Tat. To exclude
a potential bias toward an overrepresentation of responses directed
against HIV-1 Vpr and p17 Gag due to the high frequency of individuals
with HLA-A2 in the study cohort (40%) and the presence of epitopes
restricted by this allele in both proteins (SLYNTVATL in p17 and
AIIRLLQQL in Vpr; Refs. 31, 32, 33), we repeated the analysis
after the exclusion of individuals expressing HLA-A2. HIV-1 Vpr
and p17 Gag remained the most frequently targeted proteins within
HIV-1, with length-adapted scores of 0.44 and 0.61. Comparing the
frequency of recognition of different HIV-1 proteins among
individuals with acute treated, chronic treated, and long-term
nonprogressive HIV-1 infection, individuals with long-term
nonprogressive infection and individuals with chronic treated infection
had CD8+ T cell responses directed against more
epitopes and more HIV-1 proteins than individuals with acute treated
infection, as described previously for the structural HIV-1 proteins
(16), but these differences did not reach statistical
significance (Fisher’s exact test, p > 0.1). Taken
together, these data extend the studies in individual AC-06 to show
that the accessory proteins are frequently targeted by
CD8+ T cells in HIV-1 infection.
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Despite over 140 optimal CTL epitopes defined to date, no Vif and
Vpu epitopes have been mapped (34) and only two
HLA-A2-restricted epitopes within Vpr, predicted by the HLA-A2
peptide-binding motif, have been described previously
(31). In addition to the three novel HLA-A3- and
B7-restricted CTL epitopes described above in subject AC-06, we
characterized the optimal CTL epitopes restricted by different HLA
class I molecules for peptides recognized frequently in the studied
individuals and determined the percentage of individuals with the
corresponding HLA type recognizing the epitope (Table V). For all novel CTL epitopes, the
optimal peptide sequence was defined using serial dilutions of
truncated peptides as described above and the HLA class I restriction
was determined by using peptides presented by autologous and partially
HLA-matched cell lines. Optimal CTL epitopes were defined by ELISPOT
and flow-based assays using PBMC in most cases and reconfirmed after
isolation of peptide-specific cytotoxic CD8+ T
cell lines in a standard 51Cr release assay (data
not shown). For the HLA-B*1801- and HLA-B*5701-restricted CTL responses
in Vif and the HLA-B*5701-restricted CTL response in Vpr, the frequency
of peptide-specific CD8+ T cells in the
peripheral blood was not sufficiently high to determine the optimal
epitope sequence and the HLA restriction of the response directly using
PBMC. For the characterization of these epitopes,
CD8+ T cells were nonspecifically expanded using
a CD4/CD3-bispecific Ab, and peptide-specific
CD8+ T cell lines were subsequently isolated
using a newly adapted IFN- catching assay that allows for the
isolation of peptide-activated CD8+ T cells using
magnetic beads. This is shown for the CD8+ T
cells specific for the Vif-20 peptide in individual AC-04 (Fig. 5). Using fresh PBMC, 0.3% of
CD8+ T cells targeted this peptide (Fig. 5A). After a 10-day expansion of CD8+
T cells using a bispecific CD3/CD4 Ab and IL-2, the peptide-specific
CD8 population was expanded to 1.3% (Fig. 5B). The
frequency of these Vif-20-specific CD8+ T cells
was further increased to 67% after isolation of peptide-specific cells
using the IFN- catching assay (Fig. 5C).
CD8+ T cell lines isolated this way showed strong
(>80%) peptide-specific cytotoxic activity at different E:T ratios in
a standard 51Cr release assay (Fig. 5D) and were used to determine the HLA class I
restriction and minimal sequence of these novel epitopes by standard
cytotoxicity assays (data not shown). These peptide-specific lines were
also used in a cytotoxicity assay with autologous B lymphoblastoid
cells infected with rVV expressing Vpr and Vif to demonstrate that the
novel epitopes defined were effectively processed intracellularly, as
shown for the HLA-B*1801-restricted CTL response in Vif (Fig. 5D).
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The most frequently recognized CTL epitopes were the
HLA-A*0201-restricted epitope AIIRILQQL (AL9) and the
HLA-B*0702-restricted epitope FPRIWLHGL (FL9) in Vpr. AL9 was targeted
by one-third (8/24) of the individuals expressing the HLA-A2 allele.
FL9 was recognized by 4/8 individuals expressing the HLA-B7, as well as
by the two individuals in this cohort that expressed HLA-B*8101, an HLA
class I allele common in the African population, indicating a high
degree of cross-presentation and -recognition between these closely
related HLA class I alleles. Also, two novel epitopes restricted by
HLA-B*5701, an allele associated with long-term nonprogressive HIV-1
infection, were identified and frequently recognized in individuals
expressing the corresponding allele (Table V). These studies not only
characterize multiple epitopes within these accessory proteins, but
show that the CD8+ T cell responses detected are
associated not only with IFN- production but also with
cytolysis. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In these studies we used a comprehensive set spanning expressed HIV-1 proteins to characterize the relative role of each protein as a target for CD8+ T cell responses. Interestingly, HIV-1 Vpr was the most frequently targeted HIV-1 protein, together with p17 Gag, when adapted to the amino acid length of the protein. It can be speculated that several factors may contribute to the high recognition of Vpr and p17 by virus-specific CTL. First, the high frequency of HLA-A2 in our Caucasian study population may have contributed to an overrepresentation of Vpr- and p17 (Gag)-specific responses due to immunodominant HLA-A2-restricted CTL epitopes within p17 and Vpr. However, a re-evaluation of our data after the exclusion of individuals expressing HLA-A2 from the analysis showed that the frequent recognition of HIV-1 Vpr was independent from the overrepresentation of this allele in the study cohort. Another factor influencing the recognition of viral proteins by CTL may be the amount of expression of these proteins during viral infection, with proteins expressed at high frequencies being more frequently targeted by CTL. Indeed, HIV-1 Gag and HIV-1 Vpr are expressed at higher levels in infected cells compared with HIV-1 Pol and also at higher levels compared with HIV-1 Vif (37, 38). However, a potential relationship between frequency of recognition by CTL and expression levels of the corresponding protein remain to be determined. Finally, the degree of sequence conservation within a particular HIV-1 protein may influence its recognition by CTL, as these proteins are less likely to escape from CTL-mediated immune pressure, leading to the potential accumulation of responses directed against these more conserved proteins. HIV-1 p17 and Vpr are reported to be relatively highly conserved, but are not more conserved than RT, which was much less frequently targeted (39). Additional studies, including analysis of the autologous viral sequence, are needed to better understand the relation between frequencies of CTL recognition and virus variability. Taken together, these data suggest that responses directed against the accessory HIV-1 proteins are frequently detected in natural HIV-1 infection and may contribute importantly to the total virus-specific CD8+ T cell responses.
Several novel techniques adapted during the last year have allowed a
more rapid characterization of novel CTL epitopes, using PBMC with no
prior in vitro expansion. These newly adapted techniques include the
fine-mapping of novel CTL epitopes using serial dilutions of truncated
peptides in an ELISPOT assay (23) and the determination of
HLA restriction of these novel epitopes using APCs only sharing one HLA
class I allele with the corresponding donor in a flow-based restriction
assay (27). However, these techniques require a frequency
of CD8+ T cells specific for the studied epitope
of at least 0.3%, in particular for the flow-based HLA restriction
assay, to provide significant and reliable results. In this study, we
describe an additional technique that allows for the rapid generation
of peptide-specific CD8+ T cell lines, including
lines specific for epitopes recognized by CD8+ T
cells at low frequencies. The nonspecific stimulation of
CD8+ T cells with a bispecific CD3/CD4 Ab allows
for the expansion of CD8+ T cells to levels that
enable the sorting of peptide-specific CD8+ T
cells in an IFN- catching assay using magnetic beads. These
peptide-specific CD8+ T cells can be further
expanded after restimulation with autologous feeder cells and exhibit
strong peptide-specific cytotoxic activity in a standard
51chromium release assay. This newly adapted
assay will further facilitate the identification of novel CTL epitopes
and allow for the rapid generation of epitope-specific T cell lines
that can subsequently be used for functional assays.
The accessory HIV-1 proteins Vpr, Vpu, and Vif are essential for viral
replication and have important roles in the early phases of the viral
life cycle (10, 11, 12, 13, 14). HIV-1 Vpr is implicated in the
nuclear translocation of the PIC after infection (40, 41, 42, 43)
and the arrest of the cell cycle in the G2 phase
(44, 45). HIV-1 Vif may play an important role in
viral disassembly after infection, the transport of incoming viral PIC
to the nucleus, and HIV-1 provirus formation (46, 47, 48, 49, 50).
Different structural regions within these proteins have been linked
with their function. The Vpr protein contains four structural regions:
the N-terminal region, the arginine-rich C-terminal region, and one
central domain containing two putative 
helices (H1 and H2)
(41, 51, 52, 53). It has been shown that the C-terminal basic
region appears to be critical for the cell cycle arrest
(52, 53, 54) and that the two helices are involved in the
nuclear localization of the protein as well as the incorporation of Vpr
into virus particle (41, 51, 52, 53, 54, 55). The association between
function and structure is less well established for HIV-1 Vif
(56, 57, 58, 59, 60).
Several of the newly defined CTL epitopes map to important functional
domains within the Vpr protein. The most frequently recognized novel
CTL epitope AIIRILQQL, restricted by HLA-A*0201, is located within the
helix (H2) of Vpr. This region within Vpr is highly
conserved (61), and single point mutations, involving
either the isoleucine residue in position 60 that represents the
P2 anchor residue for the HLA-*0201-restricted epitope or the
C-terminal anchor residue (position 67) of the novel epitope, have been
recently shown to result in a loss of the perinuclear localization of
the wild-type protein (51), indicating the important
function of this region. It has been suggested that CTL responses
directed against functionally important regions within the virus could
be more effective, as CTL-induced viral escape mutations within these
domains are less likely to occur or may lead to reduced viral
competence (62). In particular, mutations within Vpr and
Vif have been observed in individuals with long-term nonprogressive
HIV-1 infection and were associated with replication-deficient viruses
in human and macaque infection (63, 64, 65). Longitudinal
analysis will be required to evaluate whether the virus is less likely
to escape from CTL targeting epitopes located within these accessory
proteins and whether these responses have any impact on disease
progression.
In conclusion, this study demonstrates that the accessory HIV-1 proteins Vpr and Vif are frequently targeted by virus-specific CD8+ T cells, whereas Vpu is not. Adapted to the length of the proteins, Vpr was preferentially targeted by HIV-1-specific CD8+ T cells compared with other viral proteins. Despite the small size of these proteins, multiple novel CTL epitopes within Vpr and Vif were defined, several of them located within functionally important sites of these proteins. These novel CTL epitopes will allow for a more detailed study of CTL responses directed against HIV-1 and may represent potential candidates for future HIV-1 vaccines.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Bruce D. Walker, Massachusetts General Hospital-East, CNY 5212, 149 13th Street, Charlestown, MA 02129. E-mail address: bwalker{at}helix.mgh.harvard.edu
3 For the HIV Study Collaboration: Nesli Basgoz,* Gregory K. Robbins,* Ben Davis,* Paul E. Sax,¶ Steve Boswell,|| and Dan S. Cohen||.
4 Abbreviations used in this paper: PIC, preintegration complex; RT, reverse transcriptase; rVV, recombinant vaccinia virus; SFC, spot-forming cell; BCL, B cell line.
Received for publication March 23, 2001. Accepted for publication June 18, 2001.
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References |
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helix 2 of human immunodeficiency virus type 1 Vpr contains a determinant which is responsible for the nuclear translocation of proviral DNA in growth-arrested cells. J. Virol. 72:4104.-helical domains of human immunodeficiency virus type 1 Vpr mediate nuclear localization by at least two mechanisms. J. Virol. 74:7179.This article has been cited by other articles:
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  H. Streeck, M. Lichterfeld, G. Alter, A. Meier, N. Teigen, B. Yassine-Diab, H. K. Sidhu, S. Little, A. Kelleher, J.-P. Routy, et al. 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 J. Virol., July 15, 2007; 81(14): 7725 - 7731. [Abstract] [Full Text] [PDF]
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 R. Draenert, T. M. Allen, Y. Liu, T. Wrin, C. Chappey, C. L. Verrill, G. Sirera, R. L. Eldridge, M. P. Lahaie, L. Ruiz, et al. Constraints on HIV-1 evolution and immunodominance revealed in monozygotic adult twins infected with the same virus J. Exp. Med., March 20, 2006; 203(3): 529 - 539. [Abstract] [Full Text] [PDF]
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Z. Wang, B. Metcalf, R. M. Ribeiro, H. McClure, and A. Kaur Th-1-Type Cytotoxic CD8+ T-Lymphocyte Responses to Simian Immunodeficiency Virus (SIV) Are a Consistent Feature of Natural SIV Infection in Sooty Mangabeys J. Virol., March 15, 2006; 80(6): 2771 - 2783. [Abstract] [Full Text] [PDF]
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T. M. Allen, M. Altfeld, S. C. Geer, E. T. Kalife, C. Moore, K. M. O'Sullivan, I. DeSouza, M. E. Feeney, R. L. Eldridge, E. L. Maier, et al. Selective Escape from CD8+ T-Cell Responses Represents a Major Driving Force of Human Immunodeficiency Virus Type 1 (HIV-1) Sequence Diversity and Reveals Constraints on HIV-1 Evolution J. Virol., November 1, 2005; 79(21): 13239 - 13249. [Abstract] [Full Text] [PDF]
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M. Lichterfeld, D. E. Kaufmann, X. G. Yu, S. K. Mui, M. M. Addo, M. N. Johnston, D. Cohen, G. K. Robbins, E. Pae, G. Alter, et al. Loss of HIV-1-specific CD8+ T Cell Proliferation after Acute HIV-1 Infection and Restoration by Vaccine-induced HIV-1-specific CD4+ T Cells J. Exp. Med., September 20, 2004; 200(6): 701 - 712. [Abstract] [Full Text] [PDF]
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 M. Lichterfeld, X. G. Yu, M. T. Waring, S. K. Mui, M. N. Johnston, D. Cohen, M. M. Addo, J. Zaunders, G. Alter, E. Pae, et al. HIV-1-specific cytotoxicity is preferentially mediated by a subset of CD8+ T cells producing both interferon-{gamma} and tumor necrosis factor-{alpha} Blood, July 15, 2004; 104(2): 487 - 494. [Abstract] [Full Text] [PDF]
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T. M. Allen, M. Altfeld, X. G. Yu, K. M. O'Sullivan, M. Lichterfeld, S. Le Gall, M. John, B. R. Mothe, P. K. Lee, E. T. Kalife, et al. Selection, Transmission, and Reversion of an Antigen-Processing Cytotoxic T-Lymphocyte Escape Mutation in Human Immunodeficiency Virus Type 1 Infection J. Virol., July 1, 2004; 78(13): 7069 - 7078. [Abstract] [Full Text] [PDF]
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S. Cardinaud, A. Moris, M. Fevrier, P.-S. Rohrlich, L. Weiss, P. Langlade-Demoyen, F. A. Lemonnier, O. Schwartz, and A. Habel Identification of Cryptic MHC I-restricted Epitopes Encoded by HIV-1 Alternative Reading Frames J. Exp. Med., April 19, 2004; 199(8): 1053 - 1063. [Abstract] [Full Text] [PDF]
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R. Draenert, C. L. Verrill, Y. Tang, T. M. Allen, A. G. Wurcel, M. Boczanowski, A. Lechner, A. Y. Kim, T. Suscovich, N. V. Brown, et al. Persistent Recognition of Autologous Virus by High-Avidity CD8 T Cells in Chronic, Progressive Human Immunodeficiency Virus Type 1 Infection J. Virol., January 15, 2004; 78(2): 630 - 641. [Abstract] [Full Text] [PDF]
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 A. Ali, S. Pillai, H. Ng, R. Lubong, D. D. Richman, B. D. Jamieson, Y. Ding, M. J. McElrath, J. C. Guatelli, and O. O. Yang Broadly Increased Sensitivity to Cytotoxic T Lymphocytes Resulting from Nef Epitope Escape Mutations J. Immunol., October 15, 2003; 171(8): 3999 - 4005. [Abstract] [Full Text] [PDF]
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 S. Corbet, H. V. Nielsen, L. Vinner, S. Lauemoller, D. Therrien, S. Tang, G. Kronborg, L. Mathiesen, P. Chaplin, S. Brunak, et al. Optimization and immune recognition of multiple novel conserved HLA-A2, human immunodeficiency virus type 1-specific CTL epitopes J. Gen. Virol., September 1, 2003; 84(9): 2409 - 2421. [Abstract] [Full Text] [PDF]
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M. Altfeld, M. M. Addo, R. Shankarappa, P. K. Lee, T. M. Allen, X. G. Yu, A. Rathod, J. Harlow, K. O'Sullivan, M. N. Johnston, et al. Enhanced Detection of Human Immunodeficiency Virus Type 1-Specific T-Cell Responses to Highly Variable Regions by Using Peptides Based on Autologous Virus Sequences J. Virol., July 1, 2003; 77(13): 7330 - 7340. [Abstract] [Full Text] [PDF]
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D. Meyer-Olson, K. W. Brady, J. T. Blackard, T. M. Allen, S. Islam, N. H. Shoukry, K. Hartman, C. M. Walker, and S. A. Kalams Analysis of the TCR {beta} Variable Gene Repertoire in Chimpanzees: Identification of Functional Homologs to Human Pseudogenes J. Immunol., April 15, 2003; 170(8): 4161 - 4169. [Abstract] [Full Text] [PDF]
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M. M. Addo, X. G. Yu, A. Rathod, D. Cohen, R. L. Eldridge, D. Strick, M. N. Johnston, C. Corcoran, A. G. Wurcel, C. A. Fitzpatrick, et al. Comprehensive Epitope Analysis of Human Immunodeficiency Virus Type 1 (HIV-1)-Specific T-Cell Responses Directed against the Entire Expressed HIV-1 Genome Demonstrate Broadly Directed Responses, but No Correlation to Viral Load J. Virol., February 1, 2003; 77(3): 2081 - 2092. [Abstract] [Full Text] [PDF]
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V. Novitsky, H. Cao, N. Rybak, P. Gilbert, M. F. McLane, S. Gaolekwe, T. Peter, I. Thior, T. Ndung'u, R. Marlink, et al. Magnitude and Frequency of Cytotoxic T-Lymphocyte Responses: Identification of Immunodominant Regions of Human Immunodeficiency Virus Type 1 Subtype C J. Virol., September 11, 2002; 76(20): 10155 - 10168. [Abstract] [Full Text] [PDF]
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X. G. Yu, M. M. Addo, E. S. Rosenberg, W. R. Rodriguez, P. K. Lee, C. A. Fitzpatrick, M. N. Johnston, D. Strick, P. J. R. Goulder, B. D. Walker, et al. Consistent Patterns in the Development and Immunodominance of Human Immunodeficiency Virus Type 1 (HIV-1)-Specific CD8+ T-Cell Responses following Acute HIV-1 Infection J. Virol., July 29, 2002; 76(17): 8690 - 8701. [Abstract] [Full Text] [PDF]
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K. Yusim, C. Kesmir, B. Gaschen, M. M. Addo, M. Altfeld, S. Brunak, A. Chigaev, V. Detours, and B. T. Korber Clustering Patterns of Cytotoxic T-Lymphocyte Epitopes in Human Immunodeficiency Virus Type 1 (HIV-1) Proteins Reveal Imprints of Immune Evasion on HIV-1 Global Variation J. Virol., July 29, 2002; 76(17): 8757 - 8768. [Abstract] [Full Text] [PDF]
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G. M. Lauer, K. Ouchi, R. T. Chung, T. N. Nguyen, C. L. Day, D. R. Purkis, M. Reiser, A. Y. Kim, M. Lucas, P. Klenerman, et al. Comprehensive Analysis of CD8+-T-Cell Responses against Hepatitis C Virus Reveals Multiple Unpredicted Specificities J. Virol., May 13, 2002; 76(12): 6104 - 6113. [Abstract] [Full Text] [PDF]
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