CTL Escape Mediated by Proteasomal Destruction of an
HIV-1 Cryptic Epitope
Sylvain Cardinaud1*, Gesa Consiglieri2, Romain Bouziat3¤, Alejandra Urrutia1, Ste ´phanie Graff-Dubois1,
Slim Fourati4, Isabelle Malet4, Julien Guergnon1, Ame ´lie Guihot1, Christine Katlama4, Brigitte Autran1,
Peter van Endert5, Franc ¸ois A Lemonnier3¤, Victor Appay1, Olivier Schwartz6, Peter M Kloetzel2, Arnaud
1INSERM, UMR-S945, Universite ´ Pierre et Marie Curie (UPMC), Paris, France, 2Institut fu ¨r Biochemie, Charite ´-Universita ¨tsmedizin, Berlin, Germany, 3Institut Pasteur, Unite ´
Cellulaire Antivirale, Paris, France, 4INSERM, UMR-S943, UPMC, Ho ˆpital Pitie ´-Salpe ˆtrie `re, Paris, France, 5INSERM, U1013, Universite ´ Paris Descartes, Faculte ´ de me ´decine
Rene ´ Descartes, Paris, France, 6Institut Pasteur, Unite ´ Virus et Immunite ´, Paris, France
Cytotoxic CD8+ T cells (CTLs) play a critical role in controlling viral infections. HIV-infected individuals develop CTL responses
against epitopes derived from viral proteins, but also against cryptic epitopes encoded by viral alternative reading frames
(ARF). We studied here the mechanisms of HIV-1 escape from CTLs targeting one such cryptic epitope, Q9VF, encoded by an
HIVgag ARF and presented by HLA-B*07. Using PBMCs of HIV-infected patients, we first cloned and sequenced proviral DNA
encoding for Q9VF. We identified several polymorphisms with a minority of proviruses encoding at position 5 an aspartic
acid (Q9VF/5D) and a majority encoding an asparagine (Q9VF/5N). We compared the prevalence of each variant in PBMCs of
HLA-B*07+ and HLA-B*07- patients. Proviruses encoding Q9VF/5D were significantly less represented in HLA-B*07+ than in
HLA-B*07- patients, suggesting that Q9FV/5D encoding viruses might be under selective pressure in HLA-B*07+ individuals.
We thus analyzed ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N. Around 16% of HLA-B*07+ patients
exhibited CTL responses targeting Q9VF epitopes. The frequency and the magnitude of CTL responses induced with Q9VF/
5D or Q9VF/5N peptides were almost equal indicating a possible cross-reactivity of the same CTLs on the two peptides. We
then dissected the cellular mechanisms involved in the presentation of Q9VF variants. As expected, cells infected with HIV
strains encoding for Q9VF/5D were recognized by Q9VF/5D-specific CTLs. In contrast, Q9VF/5N-encoding strains were
neither recognized by Q9VF/5N- nor by Q9VF/5D-specific CTLs. Using in vitro proteasomal digestions and MS/MS analysis,
we demonstrate that the 5N variation introduces a strong proteasomal cleavage site within the epitope, leading to a
dramatic reduction of Q9VF epitope production. Our results strongly suggest that HIV-1 escapes CTL surveillance by
introducing mutations leading to HIV ARF-epitope destruction by proteasomes.
Citation: Cardinaud S, Consiglieri G, Bouziat R, Urrutia A, Graff-Dubois S, et al. (2011) CTL Escape Mediated by Proteasomal Destruction of an HIV-1 Cryptic
Epitope. PLoS Pathog 7(5): e1002049. doi:10.1371/journal.ppat.1002049
Editor: Jeremy Luban, University of Geneva, Switzerland
Received November 24, 2010; Accepted March 11, 2011; Published May 12, 2011
Copyright: ? 2011 Cardinaud et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Agence Nationale de Recherche sur le SIDA et les hepatitis virales (ANRS), Sidaction, the Deutsche
Forschungsgemeinschaft (DFG), the University Pierre et Maris Curie (UPMC), INSERM, CNRS and the Institut Pasteur. We also thank the NIH AIDS Research and
Reference Reagent Program for providing drugs and compounds. R. Bouziat is a fellow of the ANRS. S. Cardinaud is supported by ANRS and Sidaction. A. Urrutia is
supported by Sidaction. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org (SC); email@example.com (AM)
¤ Current address: INSERM, U986, Universite ´ Paris 5, Ho ˆpital Saint-Vincent-de-Paul, Paris, France
Multiple lines of evidence suggest that CD8+ cytotoxic T
lymphocytes (CTLs) play a critical role in controlling HIV-1
replication. During acute infection, expansion of HIV-specific
CD8+ T cells (HS-CTL), before appearance of neutralizing
antibodies, is associated with decreased viremia  and most likely
determines the viral set point during chronic infection [2,3].
Resistance to disease progression correlates with the detection of
Gag-specific CTLs and with the presence of particular HLA
alleles, such as HLA-B*57 and –B*27 [4,5]. HIV rapidly mutates
to evade virus-specific CD8+ T lymphocyte responses, underlying
the selection pressure exerted by CTLs [2,6,7–11]. In large part
due to its error prone reverse transcriptase activity, HIV possesses
a unique capacity to mutate and evade CTL responses. During
acute and chronic HIV infection, CTL escape mutations have been
well documented [9,12,13]. In most cases, these mutations are intra-
epitopic and affect HLA binding and/or alter TCR interactions
leading to loss of CTL activation or more subtle effects .
However, interference with antigen processing may also lead to a
reduced generation of precursor peptides and consequently peptide/
MHC-I complex formation and T cell activation. This could occur
at any stage of the processing pathway. Mutations in epitope-
flanking regions might affect proteasomal processing or N-terminal
trimming leading to escape from CTL recognition [15–20].
CTLs recognize peptides originating from proteasomal process-
ing of viral proteins or truncated misfolded viral polypeptides, also
called DRiPS (for defective ribosomal products) [21–23]. These
viral polypeptides are classically derived from the fifteen HIV-1
viral proteins encoded by the nine primary open reading frames
PLoS Pathogens | www.plospathogens.org1 May 2011 | Volume 7 | Issue 5 | e1002049
. However CTLs also target peptides translated from
alternative reading frames or ARFs (also called cryptic epitopes).
ARF-derived peptides (ARFPs) result from a differential usage of
the three-letter codon alphabet during protein synthesis. How this
change of reading frame occurs remains elusive but various
mechanisms have been proposed. Ribosomes can initiate transla-
tion at an internal initiation codon (Met or Cys), change reading
frame by shifting, or translate alternatively spliced mRNA.
Nonetheless, ARF polypeptides are processed in cells and thus
constitute an important source of cryptic epitopes for MHC-I
presentation . CTL responses directed against these cryptic
epitopes have been detected in autoimmune disease , in
tumors [27,28] but also in several infectious diseases, including
influenza virus , murine AIDS , SIV  and importantly
HIV infections [32–35].
We previously described six ARFPs presented by HLA-B*0702
overlapping the alternative reading frames of HIV-1 gag, pol or env
genes . CTL responses specific for these ARF-derived peptides
were detected in the blood of HIV+ patients. In addition, HIV-
infected cells were recognized by CTLs specific for the gag-
overlapping ARF epitope (so called Q9VF/5D epitope). Impor-
tantly, we showed that the introduction of a stop codon within gag-
ARF abrogated Q9VF/5D epitope generation and Q9VF/5D–
specific CTL activation . Recent studies further highlighted
the in vivo relevance of ARFP-specific CTL responses [33,34,36].
In two independent cohorts studies, Bansal et al. and Berger et al.
investigated the association between specific HLA alleles and HIV
sequence polymorphisms within ARFs. This ‘‘HLA class I
footprint approach’’ allowed the prediction of numerous ARFPs
within the HIV-1 genome, both from sense and antisense
transcripts. On a restricted number of ARFPs, they also
demonstrated that these cryptic epitopes induced CTL responses
during natural infection that might contribute to viral control in
In the present work, we bring to light a novel mechanism of
CTL escape altering the processing and presentation of the Q9VF
epitope encoded by the gag-overlapping ARF. In PBMCs of HLA-
B*07+ and HLA-B*07- HIV-infected individuals, we first
compared the prevalence of QPRSNTHVF (Q9VF/5N) and
QPRSDTHVF (Q9VF/5D) variants of the gag-ARFP. To this
end, we PCR amplified and sequenced twenty HIV proviral
genomes per individuals. We noticed that the proportion of
proviruses encoding Q9VF/5D was significantly lower in HLA-
B*07+ than in HLA-B*07- patients, suggesting that Q9FV/5D
encoding viruses might be under selective pressure in HLA-B*07+
individuals. In HLA-B*07+ and HLA-B*07- patients, we analyzed
ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N
and we dissected the immunogenicity of Q9VF variants. We
observed that cells infected with HIV-1 strains encoding Q9VF/
5N were neither recognized by Q9VF/5N- nor Q9VF/5D-
specific CTLs. We demonstrate that this single amino acid (AA)
variation is responsible for the lack of CD8+ T cell recognition.
We show that HIV can escape CTL surveillance by introducing
mutations leading to epitope destruction by proteasomes.
Analysis of Q9VF gag proviral sequences and
Q9VF-specific CTL responses in HLA-B*07+ patients
Q9VF was originally predicted from the sequence of the
consensus HIVHxB2 (HIVLAI) isolate . HIVLAI bears an
asparagine (N) to aspartic acid (D) substitution at position 5
(Q9VF/5D) representing less than 5% of HIV-1 clade B strains
retrieved from Genbank. We decided to extend these observations
by sequencing HIV proviral sequences isolated from 10 HLA-
B*07+ and 10 HLA-B*07- patients. HLA-typing, virological and
clinical characteristics of these patients are presented in Table 1.
Both groups were age-matched and did not present any significant
differences in terms of CD4 counts, viral loads or treatments (not
shown). From the PBMCs of each patient, we cloned and
sequenced at least 20 HIV-proviral sequences encompassing the
gag-ARF DNA region (Figure 1A and Supplementary Figure S1).
The isolated HIV sequences encoded either Q9VF/5N (present in
16 out of 20 patients, representing 62% of all isolates), Q9VF/5N
variants (exhibiting within the epitope an additional AA difference
from the consensus sequence, 9 out of 20 patients, 14% of all
isolates) or Q9VF/5D (7 out of 20 patients, 15% of all isolates) and
Q9VF/5D variants (2 out of 20 patients, 1% of all isolates)
(Table 2). Between Q9VF/5N and Q9VF/5N-variants, Q9VF/
5N was the major variant representing 80% of proviral sequences
in this group. Q9VF/5D was the major sequence representing
94% of proviral sequences among Q9VF/5D and Q9VF/5D-
variants. Note that these mutations did not impact the translation
of classical gag ORF (Supplementary Figure S1 and not shown). In
contrast, HIV proviruses harboring a STOP codon prior to Q9VF
(8% of all isolates) that most likely abolishes Q9VF translation
were also identified (Figure 1A). HIV proviral sequences encoding
Q9VF/5N and Q9VF/5N-variants were predominant in both
HLA-B*07+ and HLA-B*07- patients. Q9VF/5D or Q9VF/5D-
variant HIV proviral sequences could be retrieved in two out of
the ten HLA-B*07+ patients and in six out of the ten HLA-B*07-
donors. Taking into consideration the diversity of HIV sequences
per donor with regard to their HLA-B7 status, we observe a
significant lower proportion of Q9VF/5D+ HIV strains in HLA-
B*07+ than in HLA-B*07- donors (p,0.04, mean value 3% vs
29% of proviral sequences in HLA-B*07+ and HLA-B*07-
donors, respectively, Figure 2B). Altogether, these results suggested
that Q9VF/5D-encoding HIV strains might be under negative
selective pressure in HLA-B*07+ donors. We thus analyzed CTL
responses directed against Q9VF/5D and Q9VF/5N epitopes in
PBMCs of patients including the 10 HLA-B*07+ patients used for
the analysis of HIV proviral sequences.
PBMCs from 31 HLA-B*07+ patients were loaded with various
peptides and submitted to IFNc-ELISpot (Figure 1C and not
shown). Incubations with peptides corresponding to well-charac-
terized HLA-B*0702-restricted immunodominant epitopes from
HIV-1 Gag classical ORF (SPRTLNAWV, TPQDLNTML,
YPLASLRSLF) induced a significant IFNc-release, demonstrating
that in the course of natural infection the donors mounted CTL
In addition to the classical open reading frames encoding
for the well characterized HIV proteins, HIV exhibits a vast
number of alternative reading frames that have the
potential to encode proteins or polypeptides. We have
previously shown that such reading frames within gag, pol
and env genes express T cell epitopes. In the present work,
we further characterized the role of T-cell responses
targeting the gag-overlapping reading frame in the
selection of HIV variants in vivo. We demonstrate that
under CD8+ T cell immune pressure, HIV escapes by
introducing mutation that affects T-cell recognition of HIV-
infected cells. We characterized the mechanism of CTL-
escape and demonstrate that HIV manipulates antigen
processing and presentation. Our results highlight the
importance of CTL targeting these alternative reading
frame-encoded antigens in the control of HIV replication.
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responses to HIV-1 antigens. Five out of the 31 HLA-B*07+
donors showed a low but significant activation with Q9VF/5D
and Q9VF/5N peptides (Figure 1C). Note that donors reacted to
both peptides or reacted to none and that the frequencies of CTL
responding to Q9VF/5D and Q9VF/5N peptides were in the
same order of magnitude (from 150 to 300 CTL per million of
PBMCs), suggesting that the reactivity to one or the other peptide
might be due to cross reactivity. We previously demonstrated that
CTL lines raised against Q9VF/5N were indeed cross-reactive on
Q9VF/5D and vice versa ( and Supplementary Figure S2).
Viruses encoding Q9VF/5D were not isolated from PBMCs of
the five Q9VF responders (Figure 1), with the exception of patients
P1 that harbored proviruses encoding a Q9VF/5D variant
(QPRGDTHVF, representing 16% of sequences in this donor).
These data prompt us to study the immunogenicity of the Q9VF/
5N and Q9VF/5D epitope variants.
Q9VF/5D to 5N substitution abrogates CTL recognition of
We asked whether the Q9VF/5N epitope was processed and
presented to HS-CTLs by HIV-infected cells. HLA-B*0702+ cells
were infected with HIVLAI and HIVNL-AD8 strains encoding
Q9VF/5D or Q9VF/5N respectively. Five days post-infection (pi),
50 and 47% of the cells were productively infected by HIVLAIand
HIVNL-AD8respectively (as monitored by intracellular Gag-p24
FACS-staining (not shown)). Infected cells were then co-cultured
with HIV-specific CTL lines and T cell activation measured using
IFNc-ELISpot assays (Figure 2). HLA-transgenic mice offer a
rapid and convenient model to identify human T cell epitopes 
and to generate CTL lines specific for peptides of unknown
immunogenicity in humans, such as Q9VF/5N. For this reason,
Q9VF/5D- and Q9VF/5N-specific CTL lines were generated by
peptide immunization of HLA-B*0702+ transgenic mice and in
vitro restimulations [32,37]. As expected, Q9VF/5D- and Q9VF/
5N- specific CTLs secreted high levels of IFNc in response to
Q9VF/5D and Q9VF/5N peptide loaded cells respectively
(Figure 2A). Note that Q9VF/5D- and Q9VF/5N-specific CTL
lines displayed similar capacity to recognize peptide-loaded cells
(Supplementary Figure S2), suggesting that the Q9VF/5N variant
affects neither MHC nor TCR binding of the peptide. As we
previously reported , HIVLAI-infected cells induced a robust
activation of Q9VF/5D-specific CTLs. Due to their capacity to
cross-react on Q9VF/5D peptide (Supplementary Figure S2 and
), Q9VF/5N-specific CTLs were also stimulated by HIVLAI-
infected cells, thus demonstrating that these CTL lines are fully
competent in recognizing HIV-infected cells. In contrast, Q9VF/
5D- and Q9VF/5N-specific CTLs were not activated upon co-
culture with HIVNL-AD8-infected cells (Figure 2A). This is not due
Table 1. List of patients used in this study.
HLA class I
Patient Age Gender ABC
P1 42M nd ndB*07nd nd nd4914
P2 33M nd nd B*07nd ndnd 6429 17763TC-d4T-NVP5
P338M A*01A*02B*07B*08 C*07 C*0766719
P4 44M A*02 A*03 B*07B*27 C*02 C*07 154622
P5 46F A*02 A*03B*07B*51 C*05C*07414 19
P6 58M nd ndB*07nd nd nd866 2.511482None
P741M ndnd B*07B*18 C*05 C*07 64417
P8 47M A*23A*33B*07B*14C*05 C*07 89216
,20 TDF/FTC-FPV/r 11
P9 50MA*02A*03B*07 B*44 C*07C*0781814124 ABC/3TC-LPV/r-ETR13
P10 48MA*01 A*03 B*07B*08C*07 C*0743424
Patients HLA-B*07 -
P11 28MA*01A*02B*08B*27C*07 C*07613 0.2520293 None
P12 44MA*29 A*31B*44B*67 C*12C*16319 21
P13 53M A*01 A*02 B*14B*51C*05 C*1535123
,20 ddi/3TC-ATV/r 17
P1443MA*01A*68B*14 B*15C*04 C*05358 24
P15 63M A*29A*74B*44 B*56C*01 C*161282 23
P16 36F A*01A*02 B*53B*82 C*03C*06 4406
P1744M A*03 A*03B*27B*35 C*02 C*04529 1620ABC/3TC-DRV/r-TDF 13
P1844MA*03A*11B*14B*27 C*01 C*051461 23
P1936F A*29 A*33B*27 B*39C*03 C*07919 2253 ABC/3TC-LPV/r22
P2023F A*24A*29B*18 B*55 C*03C*12 96 16 38035None
aCopies of HIV-1 RNA per milliliter of plasma at the time of study.
bTreatment at the time of study: d4T, stavudine; ddi, diadanosine; TDF, Tenofovir; FTC, Emtricitabine; ATV, Atazanavir; r, ritonavir; DRV, Darunavir; ETR, Etravirine; LPV,
Lopinavir; RAL, Raltegravir; 3TC, Lamivudine; ABC, Abacavir; EFV, Efavirenz; FPV, Fosamprenavir; NVP, Nevirapine; SQV, Saquinavir; AZT, Zidovudine; MVC, Maraviroc.
ART, antiretroviral therapy; nd, not determined.
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Figure 1. Q9VF/5D-specific CTLs exert a selection pressure on HIV Q9VF gag-overlapping ARF. (A) Analysis of Q9VF proviral sequences in
HIV-infected donors. Using PBMCs, proviral DNA of 20 HIV+ individuals were extracted and the region corresponding to gag-ARF PCR-amplified and
cloned. Twenty clones per donor were sequenced. Results are presented as percentage of provirus encoding for Q9VF/5D and 5D variants exhibiting
within the epitope an additional AA difference from the consensus sequence, Q9VF/5N and 5N variants, and sequence harboring a stop codon prior
the epitope (no epitope). Pies on the right represent percentage of provirus combined for all isolates. Top and bottom panels, results for HLA-B*07+
and HLA-B*07- donors, respectively. (B) Percentage of provirus encoding Q9VF/5D or 5D variants within HLA-B*07+ and HLA-B*07- patients. Each dot
represents percentage within the PBMCs of one donor. In HLA-B*07+ patients, variants with 5D are under-represented (P,0.04). (C) Immunogenicity
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to the incapacity of HIVNL-AD8-infected cells to activate HS-CTLs
since CTL clones specific for an HLA-B*0702-restricted HIV-1
Nef epitope (F10LR), raised as a control in these experiments,
were activated upon co-culture with HIVLAI- and HIVNL-AD8-
To extend these observations to other HIV-1 isolates, HLA-
B*0702+ cells were also infected with HIVMNthat encodes for
Q9VF/5N and used as target cells to activate Q9VF/5D- and
Q9VF/5N-specific CTLs (Supplementary Figure S3). HIVNL-AD8-
and HIVMN-infected cells did not induce Q9VF/5D- nor Q9VF/
5N-specific CTL activation. Overall, these results suggested that
HIV-infected cells did not present the Q9VF/5N peptide.
Epitope flanking regions have a direct impact on antigen
processing and presentation . Thereafter, to exclude the
possibility that HIV sequence variations outside the Q9VF/5N
peptide might be responsible for the lack of presentation, we
introduced in HIVLAIa D to N mutation within the Q9VF epitope
(so called HIVLAI-5D.5N). This mutation did not affect the primary
open reading frame of Gag (Supplementary Figure S1) and did not
alter viral replication in T cell lines or primary CD4+ T cells
(Figure 2B). However, cells infected with HIVLAI-5D.5Ncould not
activate Q9VF/5D- nor Q9VF/5N-specific CTLs (Figure 2C).
Thereafter, this single amino acid substitution was sufficient to
abrogate CTL recognition, thus indicating that this asparagine
alters Q9VF MHC-I presentation. We then sought to dissect the
mechanism responsible for the lack of Q9VF/5N MHC-I
Q9VF/5N binds TAP pumps and HLA-B*0702 molecules
The capacity of antigenic peptides to bind to a given HLA allele
is determined by the so-called anchor residues . Mutating an
anchor residue abrogates peptide HLA-binding and subsequent T
cell activation, a strategy often used by viruses to escape viral-
specific T cell responses. The anchor residues of HLA-B*0702
reside at position 2 and 9 of the peptide-ligands. Thereafter, the D
to N substitution at position 5 was not predicted to influence
Q9VF peptide binding to HLA-B*0702 . However, besides
anchor residues, auxiliary residues might affect peptide binding,
we thus compared the capacity of Q9VF/5D and Q9VF/5N
peptides to bind HLA-B*0702. To this end, T2-HLA-B*0702 cells
were loaded O/N with Q9VF/5D or Q9VF/5N peptides and
binding to HLA-B*0702 molecules at the cell surface monitored
by FACS (Figure 3A, left panel). Q9VF/5D and Q9VF/5N
peptides exhibited similar capacities to bind HLA-B*0702 with a
relative affinity (RA, based on the reference peptide) of 2.6 and 1.5
respectively (Figure 3A, left panel). To further characterize the
impact of the 5D to 5N substitution on peptide-MHC interactions,
we compared the capacity of the peptides to stabilize HLA-B*0702
molecules at the cell surface of T2-HLA-B*0702 (Figure 3A, right
panel). To this end, T2-HLA-B*0702 were cultured O/N at 26uC
to allow surface expression of peptide-receptive MHC molecules,
loaded with a high concentration of peptides, shifted to 37uC and
the stability of HLA-B*0702-peptide complexes monitored by
FACS at various time points. An exponential regression of HLA-
B*0702 mean fluorescence intensity (MFI) vs. time reveals that the
stability (t1/2) of HLA-B*0702 pulsed with an irrelevant peptide
(S9L) is 22 min while binding of Q9VF/5D and Q9VF/5N
peptides prolongs the t1/2 to 211 and 641 min respectively
(Figure 3A, right panel). Thereafter, Q9VF/5D and Q9VF/5N
peptides are very good HLA-B*0702-binders and 5D to 5N
substitution tends to prolong surface expression of HLA-B*0702.
Precursor peptides are transported by the TAP pumps
(transporter associated with antigen processing) from the cytosol
into the endoplasmic reticulum (ER), and then loaded on nascent
MHC-I molecules . N-terminally extended peptide precursors
are also transported and further trimmed in the ER by the
endoplasmic reticulum aminopeptidase ERAAP and bound to
MHC-I molecules [42,43]. We asked whether the absence of
Q9VF/5N peptide presentation by HLA-B*0702 within infected
cells might be the result of inefficient ER-translocation of the
Q9VF/5N epitope and/or Q9VF/5N-peptide precursors by TAP.
Hence, we used a TAP-binding assay  to evaluate the affinities
of Q9VF/5D and Q9VF/5N and their precursors with TAP.
Q9VF/5D and Q9VF/5N exhibited a poor affinity for TAP
(Figure 3B), most likely due to the presence of a proline at position
2 that negatively impacts on TAP-mediated peptide transport
. In contrast, their N-terminally extended peptide precursors
EGF-Q9VF/5D and EGF-Q9VF/5N showed at least a two-log
increased efficiency to compete for TAP with an equal 1/IC50of
0.15. Whatever the precursor, Q9VF/5D and Q9VF/5N
containing peptides did not show differences in their capacity to
bind human TAP molecules.
Overall, these data demonstrated that the D to N substitution
within Q9VF does not impact on TAP transport and HLA
binding. In contrast, the 5N substitution might prolong epitope
presentation on the cell surface.
Q9VF/5D epitope generation is dependent on
The proteasomes, that are the major catalytic enzymes involved
in antigen processing, generate the carboxyl termini of most
MHC-bound peptides [38,45]. We thus asked whether the
generation of Q9VF/5D was dependent on proteasomal process-
ing. To this end, HLA-B*0702+ cells were infected with HIVLAI.
Five days pi, infected cells were incubated with a potent and
selective proteasome inhibitor, epoxomicin , treated with a
citrate-phosphate buffer to remove residual MHC-peptide com-
plexes, washed and cultured with Q9VF/5D-specific CTLs as
previously described. Epoxomicin treatment abolished the capac-
ity of HIVLAI-infected cells to activate Q9VF/5D-specific CTLs,
as measured in IFNc-ELISpot (Figure 3C, left panel). Note that
epoxomycin inhibition affected neither MHC-density (as moni-
tored by FACS, not shown) nor the capacity of treated cells to
present exogenous peptide (at 0.1 mg/ml) (Figure 3C, right panel).
Thereafter, these results demonstrated that the generation of
Q9VF epitope depends on proteasomal processing.
5N introduces an aberrant proteasomal cleavage site
within Q9VF epitope
Proteasomes might also destroy CTL epitopes by generating
aberrant cleavages within the epitope  or in epitope-flanking
regions [19,48]. We thus asked whether aberrant proteasomal
of Q9FV peptide variants. PBMCs of HIV-infected HLA-B*07+ donors were loaded with peptides and T cell activation monitored by IFNc-ELISot. PBMCs
were incubated with HLA-B*07-restricted epitopes: Q9VF/5D, Q9VF/5N, a pool of 3 immunodominant HIV-1 Gag epitopes (SPRTLNAWV, TPQDLNTML,
YPLASLRSLF), a CMV-derived epitope (pp65 TPRVTGGGAM) or an HCV-derived epitope as negative control (GPRLGVRAT). Out of 31 HLA-B*07+
patients 5 reacted to Q9VF/5D and Q9VF/5N. Results for the 5 Q9VF reacting patients (Q9VF CTL +, full symbols) and 5 representative Q9VF non-
reacting patients (Q9VF CTL-, open symbols) are shown. Data are means of triplicates. Dotted line indicates threshold of significant positive
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Table 2. Frequencies of HIV-1 proviruses encoding Q9VF epitope variants in PBMCs of studied patients.
Patients HLA-B*07+ +
Frequency of provirus (%)b
Frequency of provirus (%)b
aNumber of patients in which at least one proviral clone encodes the Q9VF variant epitope/total number of tested patients.
bFrequency of proviral clones encoding Q9VF variant epitope among the twenty clones sequenced per patient.
cAverage frequency of proviruses among the ten studied patients (HLA-B*07+ or HLA-B*07-).
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Figure 2. Q9VF/5D to Q9VF/5N substitution abrogates CTL recognition of HIV-infected cells. (A) T1-B7 cells were infected with HIVLAIand
HIVNL-AD8expressing Q9VF/5D and Q9VF/5N, respectively. Two days p.i., the percentage of HIV-infected cells was monitored by intracellular p24
staining and flow cytometry: 50 and 47% of the cells were infected with HIVLAIand HIVNL-AD8, respectively. In an IFNc-ELISpot assay, infected cells
were then used to activate CTL lines specific for Q9VF/5D, Q9VF/5N or an HLA-B*07-restricted HIV-1 Nef epitope (FPVTPQVPLR, F10LR) used as
control. For each peptide, specific CTL lines were generated in three different HLA-B*0702 transgenic mice and used in two independent
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cleavages might be responsible for the lack of Q9VF/5N
The proteasome is a large multicatalytic protease composed of
standard and inducible subunits that replace the standard subunits
upon exposure to IFNc and form the so-called ‘‘immunoprotea-
somes’’ (IP). IP is found in most cell types after IFNc-exposure, but
is constitutive in APCs and is induced in HIV-infected T cells .
Standard (SP) and IP proteasomes display discrete differences in
their capacity to cleave a given peptide substrate . We
submitted the full-length polypeptides from the gag-overlapping
ARF to IP processing. 27mer peptides encompassing Q9VF/5D
or Q9VF/5N peptides were synthesized and incubated with IP
purified from T2.27 cells . After 1 h incubation, the digestions
were analyzed by mass spectrometry (RP-HPLC SI) and peptide
fragments identified by MS/MS (Figure 4A). IP digestion of
Q9VF/5D encompassing peptide showed the presence of major
proteasomal cleavage sites after amino acids F10, F19, I22 and
R24 representing around 80% of total cleavages. The cleavage at
position F19 generated the C-terminal cut of the N-extended
precursors of Q9VF (M1-F19). After 1 h incubation, when
comparing the IP digestion profiles of Q9VF/5D and Q9VF/
5N encompassing peptides, we noticed the presence of a new
cleavage site within the Q9VF/5N epitope. This cut at position
N15 was the most prevalent among Q9VF/5N representing up to
28% of total IP cleavages. These results demonstrated that the D
to N substitution introduces a major cleavage site within the
Q9VF/5N epitope. Nonetheless the C-terminal cut necessary for
the generation of Nt-extended Q9VF/5N precursors was also
detected following 1 h of proteasomal digestion.
Thereafter, we sought to evaluate the amount of cleavage
products generated during Q9VF/5D and Q9VF/5N digestions.
To this end, we performed kinetics of IP digestion where aliquots
were regularly collected and submitted to mass spectrometry
analysis as before (Figure 4B). To compare the amounts of
cleavage products, we used the MS fragment intensity as a
surrogate marker for quantity since these two parameters correlate
significantly . The variations among the different fragments
generated are presented as the relative intensity of peptides that
exhibit a Q9VF C-terminal cut (epitope or precursors) or peptides
issued from cleavages within the Q9VF epitope (referred to as the
antitopes) (Figure 4B). Kinetics of digestion of peptides encom-
passing either Q9VF/5D or Q9VF/5N were identical: 24%, 59%
and 96% of both substrates was degraded after 30 min, 1 h and
2 h respectively. At latter time points, both 27mers were
undetectable. In the course of Q9VF/5D substrate digestion, the
precursor (M1-F19) was readily produced starting from 30 min
with a peak at 4 h digestion (representing 20% of digested
products). The epitope was detected starting from 1 h digestion
and accumulated reaching 13% of all peptide fragments at time
18 h. At latter time points, Q9VF/5D epitopes and precursors
represented up to 14% of all peptide fragments detected. An
antitope corresponding to a cleavage at position S14 was also
generated but represented less than 2% of detected fragments at
each time point. In contrast, during Q9VF/5N substrate digestion,
the antitopes corresponding to the cleavage at position N15 were
already produced after 30 min of digestion and reached around
77% of all peptides from 4 to 18 h, further demonstrating that
N15 is a major cleavage site within Q9VF/5N. Interestingly,
during Q9VF/5N digestion, the epitope was barely detected even
at latter time points (less than 2% of digested products). The
precursor M1-F19 accumulated from 30 min to 2 h (8% of
digested products) but was undetectable after 4 h, suggesting that
the cleavage at position N15 destroyed this peptide. Overall, the
amounts of Q9VF/5N epitope and precursors produced were
markedly reduced as compared to Q9VF/5D digestion.
Taken together, these results demonstrate that the Q9VF/5D
epitope is efficiently produced by proteasomes and accumulates
with time. In contrast, the D to N substitution introduces a major
cleavage site within the epitope leading to the destruction of the
Q9VF/5N epitope and thus the absence of MHC-I binding and
The three-letter codon alphabet allows protein synthesis in six
possible overlapping reading frames. A vast number of ARFs have
the potential to encode proteins or epitopic peptides (ARFPs).
Using an ‘‘HLA class I footprint’’ approach, Bansal et al and
Berger et al recently predicted the existence of numerous ARFPs
within HIV-1 genome [33,34]. We have previously shown that
ARFP-specific CTLs are induced during natural infection .
These CTL responses might contribute to viral control driving
HIV evolution at the population level. ARFPs can mutate during
the first year of infection, suggesting a possible selection of escapes
variants [33,34]. Such a scenario has been highlighted in the
macaque model of SIV infection . Mamu-B*17+ macaques
generate strong CTL responses against SIV ARF-encoded
epitopes leading to ARF mutation affecting epitope binding to
Mamu-B*17 molecules and subsequent SIV replication rebound
. In the present study, we characterized a novel mechanism of
ARFP-specific CTL escape resulting from HIV epitope destruc-
tion by the proteasomes. We suggest that ARFP-specific CTLs
exert a selection pressure leading to negative selection of targeted
HIV strains. Overall, our work shows that CTL escape mutations
are not limited to epitopes encoded by classical ORF, highlighting
the role of ARFP-specific CTLs in the control of HIV infection.
We previously identified a panel of epitopes encoded by ARFs
within HIV-1 gag, pol and env genes . The gag-overlapping ARF
encoding for the Q9VF epitope presented by HLA-B*0702 drew
our attention due to its polymorphism. In a cross-sectional cohort
study, we report that proviruses encoding the Q9VF/5D epitope
(and 5D variants) are rare and significantly under-represented in
PBMCs of HLA-B*07+ patients, thus suggesting Q9VF/5D-
specific CTLs might exert a negative selection pressure on HIV
strains encoding Q9VF/5D variants. In HIV-1 gag ARF, the virus
might escape CTL immune pressure by introducing a 5D to 5N
substitution or Stop codons but prior the epitope. We thus
analyzed CTL responses directed against Q9VF/5D and Q9VF/
experiments. One representative experiment with one CTL line is shown (mean values of triplicates 6SD). T1-B7 cells loaded with the cognate
peptide were used as positive controls. (B) 5N substitution does not affect HIV replication. T1-B7 cells (left panel) and CD4+ activated T cells (right
panel) were infected (at 100 and 1 ng/ml respectively) with HIVLAIand HIVLAI-5D.5N. HIVLAI-5D.5Nexpressing Q9VF/5N was engineered by PCR
mutagenesis of the HIVLAIstrain. Whatever the viral input (1, 10 or 100 ng/ml), 5N substitution did not alter the replication capacity of HIVLAI-5D.5N.
T1-B7 cell infection (left panel) was monitored using GFP expression (upon trans-activation of LTR-GFP). Data are representative of at least five
independent experiments using various viral inputs. CD4+ T cells infection was monitored using p24-Elisa (right panel) and correspond to the mean
values (6SD) of two infections using activated CD4+ T cells from two donors and are representative of two independent experiments (using various
viral input). NI: not infected. (C) 5N substitution is sufficient to abrogate CTL recognition of HIV-infected cells. As in (A) using T1-B7 cells infected with
HIVLAI, HIVNL-AD8and HIVLAI-5D.5N. Infection rates were around 30% of p24+ cells.
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Figure 3. Q9VF/5N binds TAP pumps and HLA-B*0702 molecules. (A) Q9VF/5N and Q9VF/5D peptides exhibit similar affinities for HLA-
B*0702. (Left panel) Q9VF/5D, Q9VF/5N and their natural EGF Nt-extended precursors were loaded O/N at RT on T2-B7 cells. An HLA-B*07-restricted
CMV-derived reference epitope (pp65 RPHERNGFTV, R10TV) and an HLA-A*02-restricted HIV-1-derived epitope (p17 SLYNTVATL, SL9) were also used
as positive and negative control, respectively. HLA-B*0702 binding was monitored using ME-1 antibody and flow cytometry. Based on the reference
peptide R10TV, a relative affinity (RA) was calculated. Data are representative of three different experiments (mean values of triplicates 6SD). (Right
panel) T2-B7 were cultured O/N at 26uC to increase peptide-receptive cell surface molecules, pulsed with the indicated peptides for 2 h in presence of
b2-microglobulin and BFA to stop delivery of newly synthesized MHC-I molecules. Cells were then shifted to 37uC for 1 h, washed to remove
unbound peptides and incubated at 37uC in presence of BFA (0.5 mg/ml) which is considered as time ‘‘zero’’. At the indicated time points, samples
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5N epitopes in PBMCs of patients. Q9VF/5D and Q9VF/5N
peptides induced CTL responses in 16% of HLA-B*07+
individuals tested. Donors reacted to both peptides or reacted to
none. The frequencies of CTLs responding to Q9VF/5D and
Q9VF/5N peptides were about the same magnitude, suggesting
that the reactivity to one or the other peptide might be due to cross
reactivity. The frequency and magnitude of Q9VF/5D responses
in HLA-B*07+ patients were rather low as compared to
immunodominant HLA-B*07-restricted responses (Figure 1 and
). This might be due to the fact that the patients included in
the study were under retroviral therapy that might affect the
expression of ARF during residual HIV-1 translation (Table 1).
Alternatively in our assays, we are most likely monitoring memory
responses to Q9VF/5D that are usually of low magnitude. This
possibility is supported by the observation from Bansal et al that
ARFP encoding sequences mutate during the first year of infection
. Overall, the low representation of Q9VF/5D encoding HIV
proviral sequences in PBMCs of HLA-B*07+ individuals and the
low frequency and magnitude of CTL responses to Q9VF/5D
strongly supported our initial hypothesis that 5N substitution is an
We dissected the immunogenicity of the Q9VF/5N epitope. We
showed that cells infected with HIV-1 strains encoding Q9VF/5N
(HIVNL-AD8 and HIVMN) were not recognized by Q9VF/5N-
specific CTLs. In contrast, Q9VF/5N- and Q9VF/5D-specific
CTLs were activated by HIV-1 strains encoding Q9VF/5D
(HIVLAI). We demonstrated that the single AA substitution from
5D to 5N in HIVLAI sequence is sufficient and required to
abrogate CTL recognition of HIV-infected cells. Thereafter, the
acquisition of this 5N mutation by HIV might help the virus to
interfere with Q9VF epitope expression or processing and
Viruses can interfere with antigen expression to escape CTL
lysis . Various mechanisms have been proposed for the
biosynthesis of ARF-derived polypeptides. Ribosomes can scan
through conventional initiation codons , initiate translation at
an internal initiation non-AUG-codons (Leu or Cys) [34,52],
change reading frame by shifting , or translate alternatively
spliced mRNA (for review see ). We previously described the
presence of a conserved slippery motif (UUUAAAU) upstream of
gag-ARF start codon that may facilitate ribosomal slippage and
thus Q9VF synthesis . Interestingly, a structured region
(hairpin) in HIV-1 RNA has been identified downstream of this
slippery motif . This highly structured RNA region might
cause ribosomal pausing during gag translation thus facilitating
ribosomal slippery and Q9VF expression. The D to N substitution
within the Q9VF epitope is translated from a codon that is located
in the flexible loop of the RNA hairpin structure . Although it
remains to be formally proven, this D to N substitution most likely
does not impact the RNA structure and hence Q9VF expression.
Viruses also manipulate antigen processing and presentation to
escape CTL responses. Interference with antigen presentation
could arise at any stage in the pathway, including processing by
proteasomes, binding of epitope-precursors to TAP, destruction of
these precursors by peptidases in the ER or cytosol and peptide
binding to the MHC-I molecule. HIV-specific CTL responses
have been shown repeatedly to select for intra-epitope mutations
that affect HLA-binding or TcR recognition. In addition, HIV
escape mutations outside the epitope (extra-epitope mutations) can
interfere with antigen processing by proteasomes [17–19,47,54,55]
or by the ER aminopeptidase ERAAP . To our knowledge,
intra-epitope mutations affecting antigen processing have not been
described thus far. Several studies proposed that intra-epitope
variation might affect processing but did not provide a mechanism
[34,20]. The only evidence that intra-epitope mutations might
affect proteasomal processing of viral antigens comes from mouse
We provide several lines of evidence strongly suggesting that the
D to N substitution within the Q9VF epitope impacts neither TcR
recognition nor MHC binding: i) Q9VF/5N- and Q9VF/5D-
specific CTLs can be generate upon peptide immunization of
HLA-B*07-transgenic mice and cross-react to the alternate
peptide ( and Supplementary Figure S2); and ii) Q9VF/5N
and Q9VF/5D peptides bind HLA-B*0702 (Figure 3A). In
addition, we show that Q9VF/5N and Q9VF/5D peptide and
their precursors (elongated on the N-termini) efficiently bind TAP,
thus demonstrating that the D to N substitution does not affect
peptide translocation into the ER. As previously observed with
peptides bearing a proline at position 2 , the optimal Q9VF/
5N- and Q9VF/5D epitopes had a reduced capacity to bind TAP
as compared to their Nt-extended precursors (Figure 3B),
suggesting that in the ER peptide-trimming is required for proper
HLA-B*0702 binding. The ER aminopeptidase ERAAP provides
peptides for many MHC-I molecules but has been also implicated
in the destruction of CTL epitopes . However, ERAAP cannot
process X-P motifs in peptide sequences . Thereafter, though
it cannot be formally excluded, a role of ERAAP in the destruction
of Q9VF/5N is very unlikely. Overall, these data support the
concept that the intra-epitope D to N substitution interferes with
proteasomal processing. Using in vitro proteasomal digestions, we
demonstrate that the D to N substitution introduces a major
cleavage site within the Q9VF epitope (at position N15). Note that
at 1 h-digestion time point we identify mainly primary cleavage
products since less than 50% of the peptide substrates (the 27mer)
have been digested (Figure 4A). To further highlight the potential
impact of this N15 cleavage site in the generation of the Q9VF
epitope, we performed kinetics of peptide digestion using IP. We
observed that amounts of Q9VF/5N epitope and precursors
produced were markedly reduced as compared to Q9VF/5D.
These results strongly suggest that proteasome cleavages at
were removed to 0uC, stained on ice using ME.1 Ab and analyzed by FACS. Data are mean values of two independent experiments. The capacity of
each peptide to stabilize HLA-B*0702 (t1/2) was compared using exponential regression. T1/2of HLA-B*0702 pulsed with the irrelevant peptide (S9L)
was 22 min while binding of Q9VF/5D and Q9VF/5N peptides prolonged the t1/2to 211 and 641 min respectively. T1/2of CMV (pp65 TPRVTGGGAM,
T10AM) and Gag (p24 TPQDLNTML, T9ML) peptides used as positive were 552 and 124 min respectively. (B) Human TAP transporter binding assay.
Microsomes from insect cells expressing human TAPs were incubated with the labeled reference reporter peptide (RRYNASTEL, R9L) then loaded with
serial dilutions of unlabeled reference peptide or tested peptides with or without EGF Nt-extension. TAP affinities were determined as the
concentrations required to inhibit 50% of reporter peptide binding (IC50) and data are presented as 1/IC50ratios: the highest the ratio, the stronger
the affinity. Results are mean values (6SD) from three independent experiments. (C) Q9VF/5D epitope generation is dependent on proteasomal
processing. T1-B7 cells were infected with HIVLAI(as in Figure 1), monitored for HIV infection by flow cytometry, treated or not (unTx) with
epoxomicin (6 h at 37uC). To remove residual MHC-peptide complexes, cells were then treated with a citrate-phosphate buffer, washed and used as
targets to activate Q9VF/5D-specific CTLs in IFNc-ELISpot assay (8h). Note that epoxomycin inhibition affected neither MHC-density (as monitored by
FACS, not shown) nor the capacity of treated cells to present exogenous peptide (0.1 mg/ml) (right panel). Results are mean values (6SD) of triplicates
and representative of three different Q9VF/5D CTL clones. Mock, non infected cells (left panel) or loaded with the irrelevant HCV peptide (right panel).
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position N15 destroy the Q9VF/5N epitope and precursors
resulting in the lack of MHC-I presentation and CTL activation.
In conclusion, a single amino acid variation within HIV epitope
can result in epitope destruction and absence of HIV-specific CTL
Mutation in HIV-1 genome can be silent or can differentially
impact the fitness of the virus. Due to the redundancy of the codon
alphabet, the 5D to 5N substitution in Q9VF does not impact the
primary gag-ORF and thus viral replication (Figure 2B). Never-
theless, considering the multitude of existing ARFs, some
mutations within ARF encoding sequences most likely affect viral
fitness and these ARF sequences might be unavoidably conserved
throughout HIV-1 isolates. Thereafter, the great diversity of ARF
epitopes produced during HIV infection offers a vast panel of
therapeutic targets to stimulate CTL responses. It is interesting to
note that ARF-specific CD8+ T cells can performed multiple
functions [33,34] and control viral replication in vitro, character-
istics that correlate with slow disease progression . In addition,
CTLs targeting ARF-derived epitopes can be induced upon
vaccination  and tumor infiltrating CTLs specific for ARFPs
have been also identified in various cancers, including melanoma
and breast cancers . Such responses against crytptic epitopes
represent a great potential for future immunotherapeutic strate-
Materials and Methods
HIV-1-infected peripheral blood mononuclear cells (PBMCs)
were obtained from HCV (Hepatitis C virus) negative French
ALT-ANRS-CO15 cohort patients . The 31 HLA-B*07+ and
10 HLA-B*07- individuals were identified using the anti-HLA-
B*07 antibody ME.1. HLA status was further confirmed by
genotyping using PCR  or using the Luminex xMAP
technology . HLA-typing, virological and clinical character-
istics of the ten HLA-B*07+ and ten HLA-B*07- patients included
in the study are presented in Table 1.
Patient samples were collected according to French Ethical
rules. Written informed consent and approval by institutional
review Board at the Pitie ´-Salpe ˆtrie `re Hospital were obtained.
Animals were bred at the Pasteur Institute. The Office of
Laboratory Animal Care at Pasteur Institute reviewed and
approved protocols for compliance with the French and European
regulations on Animal Welfare and with Public Health Service
recommendations (Directive 2010/63/EU).
Human CTL assays
PBMCs were isolated by ficoll-centrifugation, pulsed with
Q9VF peptides (1 mM, 1 h at 37uC), and submitted to IFNc-
ELISpot assays as previously described . The HLA-B*0702-
restricted peptides used were: HCV-derived epitope G9AT
417TPRVTGGGAM426) used as negative and positive control
respectively and a pool of known Gag HIV-1-derived epitopes
121YPLASLRSLF130) as control for HIV reactivity . Respons-
es were considered positive when IFNc production was superior to
PBMCs and at least threefold higher than
background (measured with the HCV peptide).
Mouse CTL recognition of infected T1 cells
Mouse CTL lines were derived from splenocytes of peptide
immunized HLA-B*07ma3transgenic mice. In brief, these mice
express HLA-B*0702 heavy chain with a murine a3 domain and
their H-2Kband H-2Dbclass Ia genes have been inactivated .
Cytolytic activity of splenocyte cultures was first assessed in a51Cr
release assay . Peptide specific CTL lines were stimulated in
vitro (5 mg/mL of peptide) and cultured in RPMI 1640 medium
supplemented with 10% FCS, 0.5 mM 2-b-mercaptoethanol
(Sigma), 100 IU/mL penicillin and 100 mg/mL streptomycin
(Gibco-BRL). Ten days later, 26103, 400 and 80 CTLs in
triplicates were stimulated by 105HIV-1-infected T1-B7 cells and
IFNc release was detected by ELISpot assay. Cross-reactivity of
Q9VF/5D- and Q9VF/5N-specific CTLs was tested in IFNc-
ELISpot and Cr51-release assays  using T1-B7 peptide-loaded
cells. Mouse CTL lines specific for the HLA-B*0702-restricted
HIV-1 Nef-derived epitope F10LR (Nef68FPVTPQVPLR77; )
were used as controls. When stated, HIV-infected T1-B7 cells
were treated with epoxomicin (6 h, 1 mg/ml, Calbiochem). To
remove residual MHC-peptide complexes, epoxomycin-exposed
cells were treated with a citrate-phosphate buffer (pH 3.3)
containing 1% BSA and washed twice, prior co-culture with
CTLs for an additional 8 h.
Virus and infections
HIVLAI 5D.5Nwas generated by a single amino acid mutation
in HIVLAIprovirus. The GAT codon (D) of Gag-ARF (AA in
position 15) was replaced by an AAT codon (N) without affecting
the primary Gag AA coding sequence, using the following primer
(59-GGC TTT CAG CCC AGA AGT AAT ACC CAT GTT
TTC AGC) and Quickchange XL Site-directed Mutagenesis Kit
(Stratagene). HIVLAI, HIVLAI-5D.5N, HIVNL-AD8 and HIVMN
were produced by transfection of 293T cells using routine
procedures . T1 cells (174xCEM, CCR5+ LTR-GFP+) stably
transfected with the HLA-B v T1-B7 cells, ) were infected and
used as antigen-presenting cells. 56106T1-B7 cells were infected
with 500 ng of p24 for 3 h in culture medium containing 10 mM
Hepes and 4 mg/ml DEAE-dextran. 2 to 5 days p.i., infected T1-
B7 cells were used as antigen-presenting cells in IFNc-ELISpot
assay. For the infection kinetics, T1-B7 cells were infected with the
indicated viruses according to the same procedure using 1, 10 or
100 ng/ml of p24. Primary CD4+ T cells were isolated from the
blood of healthy donors using ficoll centrifugation and magnetic
beads (Miltenyi) and activated using PHA (1 mg/ml, PAA) and
Figure 4. 5N introduces an aberrant proteasomal cleavage site within the epitope. (A) 5N introduces a strong cleavage site within Q9VF
epitope. 27mer synthetic peptides encompassing Q9VF/5D or Q9VF/5N were submitted to in vitro immunoproteasome (IP) digestion. Resulting
peptide fragments were analyzed by mass-spectrometry. Proteasome cleavage patterns are presented as C-terminal cleavages to a specific AA
(horizontal axis) of Q9VF/5D (upper panel) and Q9VF/5N (lower panel) substrates. The percentage of C-terminal cuts at each AA is indicated. The most
frequent fragments at 1 h IP digestion are depicted. Data represent one of two independent experiments. (B) The overall production of Q9VF epitope
is drastically reduced by the 5N substitution. Q9VF/5D (upper panel) and Q9VF/5N (lower panel) encompassing peptides were digested by IP from
0 h to 18 h. Resulting peptide fragments were analyzed by MS/MS, as in (A). Proteasome cleavage patterns are presented as the estimated
percentage of peptide fragments corresponding to either the substrate (M1-P27), the epitope Q9VF (Q11-F19), precursors with a C-terminal cut at
F19, peptide fragments with a cleavage within the epitope most likely abolishing epitope production (referred to as ‘‘Antitopes’’), or other fragments,
with the sum of all fragments intensities set as 100%.
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rhIL-2 (50 IU/ml, Chiron) . Seven days post activation, CD4+
PHA blasts were infected with various doses of HIV (from 1 to
100 ng/ml of p24). HIV infection was monitored by FACS
(Becton Dickinson) using intracellular HIV p24 staining (KC57
Ab, Beckman Coulter) or p24-Elisa (PerkinElmer).
Sequencing of the Gag-ARF encoding region from clonal
Total DNA was extracted from PBMCs of HLA-B*07+ and
HLA-B*07- HIV+ patients using QIAampblood DNA minikit
(Qiagen). To analyze the diversity of HIV-1 proviruses in the
PBMCs of patients, a 267-bp fragment encompassing the Gag-ARF
coding sequence was amplified by nested PCRs as followed: 5 min
of initial denaturation at 94uC, 1 min at 94uC, 1 min at 57uC, and
1 min at 72uC for 30 cycles, followed by 7 min at 72uC. The outer
primer pair used was (59- ATC AAG CTT GCA CAG CAA GCA
GCAGCTGAC) and(59- CAGGAACTACTA GTA CCC TTC
AGG AAT TCG G), and the inner primer pair was (59- TAC CCT
ATA GTG CAG AAC ATC CAG GG) and (59- GAT AGA GTG
CAT CCA GTG CAT GCA). Samples were treated separately and
negative controls were systematically included. Purified PCR
products were cloned using a TOPO-TA cloning kit (Invitrogen).
Twenty clones per patient were isolated and gag-ARF inserts from
each clonal DNA plasmid were amplified by PCR using M13
primers and sequenced (Applied Biosystem).
HLA-B*07.02-peptide binding and stabilization assays
The capacity of the peptides to bind HLA-B*0702 was
determined using a classical HLA stabilization assays with the
TAP-deficient cell line T2 HLA-B*0702+ . Briefly, cells were
incubated overnight with 100, 10, 1 and 0.1 mM of peptide in
serum-free medium at room temperature. Cells were then stained
with the anti-HLA-B*07 ME.1 antibody and HLA-B*07 surface
expression analyzed by FACS (Becton Dickinson). The concentra-
tion needed to reach 50% of the maximal fluorescence (as defined
with the R10TV peptide (CMV pp65265RPHERNGFTV274) was
calculated (IC50). The relative affinity (RA) is the IC50ratio of the
tested and R10TV reference peptide (the lower the relative affinity,
the stronger the binding). The HLA-A*02-restricted peptide S9L
(HIV-1 p1777SLYNTVATL85) was used as negative control. To
monitor the capacity of the peptides to stabilize HLA-B*0702, T2-
HLA-B*0702 were cultured O/N at 26uC and pulsed the last 2 h
with peptide (100 mM) in presence of b2-microglubilin (Sigma,
1 mg/ml) and brefeldin-A (BFA, Sigma, 10 mg/ml). Cells were then
shifted to 37uC for 1 h, washed to remove unbound peptides and
incubated at 37uC in presence of BFA (0.5 mg/ml). Samples were
removedto 0uC at the indicatedtime points. Cells werethen stained
at 4uC using the ME.1 antibody and analyzed by FACS. Data
(HLA-B*0702 expression) are expressed as MFI vs. time. The
an exponential regression (one phase decay) using Prism software. A
constrain corresponding to the MFI value obtained for the
irrelevant peptide (S9L) at the latest time point was applied to the
plateaus. T10AM (pp65417TPRVTGGGAM426) and T9ML (p24
48TPQDLNTML56) peptides were used as positive controls.
The capacity of the peptides to bind TAP was measured in a
competitive binding assay as described previously . Briefly,
microsomes were purified from Sf9 insect cells expressing human
TAP1–TAP2 complexes, pulsed with the iodinated reporter
peptide R9L (RRYNASTEL) at 300 nM, and loaded with a
dilution of competitor test peptides (0.1 to 1,000 fold molar excess
relative to radioactive reporter peptide). TAP affinities were
determined as the concentrations required to inhibit 50% of
reporter peptide binding (IC50). Results are expressed as 1/IC50
ratios and are mean values from three independent experiments.
The highest the 1/IC50ratio, the highest the affinity.
In vitro proteasome digestions
Immunoproteasomes were isolated from T2.27mp cells (that
stably express all three immunosubunits) as previously described
. Purified proteasomes were analyzed by SDS-PAGE. The yield
was calculated at 90–95%. The 27mer peptides encompassing
Q9VF/5D or Q9VF/5N were synthesized using standard Fmoc
method on an Applied Biosystems 433A automated synthesizer.
The peptides were purified by HPLC and analyzed by mass
spectrometry. Three nmol of peptides were digested in vitro using
1 mg of proteasomes (for 0.5, 1, 2, 4, 8 and 18 h) in 100 ml of buffer
containing 20 mM Hepes/KOH, pH 7.8, 2 mM magnesium
acetate and 2 mM dithiothreitol. Reactions were stopped by the
addition of trifluoroacetic acid to a final concentration of 0.3%. The
digestions were analyzed, by mass spectrometry (RP-HPLC ESI)
and the products were identified by MS/MS.
A standard two-tailed nonparametric Mann-Whitney U-test
(with P,0.05 considered significant) was used to perform statistical
comparison of HIV-1 proviral sequences frequencies using
statistical analysis Prism software (GraphPad).
Gag-ARF. (A) Nucleotide and corresponding amino acid sequenc-
es of Gag (frame 1) and Gag-ARF (frame 3, bold) are depicted.
Nucleotide numbering is according to HIVHXB2sequence. ATG
start and TGA stop codons of Gag-ARF are in bold and the Q9VF/
5D epitope is underlined. (B) Nucleotide and amino acid
sequences of Gag and Gag-ARF from HIVLAI, HIVNL-AD8,
Amino acid and nucleotide sequences of Gag and
cross-reactivity of Q9VF/5D- and Q9VF/5N-specific CTLs
(generated in HLA-B*0702 transgenic mice) was tested in IFNc-
ELISpot (A) and Cr51-release assays (B) using T1-B7 cells loaded
with a single dose (1 mg/ml) (A) or a titration (B) of Q9VF/5D or
Q9VF/5N peptides. A CMV-derived HLA-B*07-restricted epi-
tope (RPHERNGFTV, R10TV) was used as negative control.
Q9VF/5D- and Q9VF/5N-specific CTLs displayed similar
capacity to recognize cells loaded with their cognate peptides.
CTLs were also equally activated by the alternate peptides. Data
are mean values of triplicates (6SD) and representative of at least
three independent experiments.
Q9VF/5D and Q9VF/5N CTL cross-reactivity. The
by Q9VF-specific CTLs. As in Figure 2A using T1-B7 cells
infected with HIVLAI, HIVNL-AD8or HIVMN(X4-tropic isolate
encoding Q9VF/5N). Infection rates were equivalent (around
30% of p24+ cells). Infected cells were then used in an IFNc-
ELISpot assay to activate Q9VF/5D- and Q9VF/5N-specific
CTLs. For each peptide, specific CTL lines were generated in
three different HLA-B*0702 transgenic mice and used in two
independent experiments. One representative experiment with
one CTL line is shown (mean values of triplicates6SD).
Q9VF/5N encoding HIV strains are not recognized
HIV-1 Escapes CTLs Specific for Cryptic Epitope
PLoS Pathogens | www.plospathogens.org13 May 2011 | Volume 7 | Issue 5 | e1002049
The authors are grateful to F. Guivel, A.Lehmann and K.Textoris-Taube
for technical assistance, D. Duffy for critical reading of the manuscript, L.
Weiss, A. Samri and G. Carcelain for help and for providing reagents. We
thank Zabrina Brumme for help in statistical analysis. We thank the ALT
study group and all ALT patients for participating in the study.
Conceived and designed the experiments: AM SC. Performed the
experiments: SC GC RB AU SGD SF IM JG PvE AM. Analyzed the
data: SC PvE PMK AM. Contributed reagents/materials/analysis tools:
AG CK BA FAL VA OS. Wrote the paper: SC AM.
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PLoS Pathogens | www.plospathogens.org15 May 2011 | Volume 7 | Issue 5 | e1002049