JOURNAL OF VIROLOGY, Feb. 2009, p. 1845–1855
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 4
Protective HLA Class I Alleles That Restrict Acute-Phase CD8?
T-Cell Responses Are Associated with Viral Escape Mutations
Located in Highly Conserved Regions of Human
Immunodeficiency Virus Type 1?‡
Yaoyu E. Wang,1† Bin Li,1† Jonathan M. Carlson,2Hendrik Streeck,1Adrianne D. Gladden,1
Robert Goodman,1Arne Schneidewind,1Karen A. Power,1Ildiko Toth,1Nicole Frahm,1
Galit Alter,1Christian Brander,1¶ Mary Carrington,3Bruce D. Walker,1,4
Marcus Altfeld,1David Heckerman,2and Todd M. Allen1*
Partners AIDS Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts1; Microsoft Research,
Redmond, Washington2; Laboratory of Genomic Diversity, SAIC-Frederick, Inc., National Cancer Institute,
Frederick, Maryland3; and Howard Hughes Medical Institute, Chevy Chase, Maryland4
Received 20 May 2008/Accepted 11 November 2008
The control of human immunodeficiency virus type 1 (HIV-1) associated with particular HLA class I alleles
suggests that some CD8?T-cell responses may be more effective than others at containing HIV-1. Unfortunately,
substantial diversities in the breadth, magnitude, and function of these responses have impaired our ability to
of the virus may be particularly effective, since the development of cytotoxic T-lymphocyte (CTL) escape mutations
in these regions may significantly impair viral replication. To address this hypothesis at the population level, we
I-associated mutations across the genome, reflective of CD8 responses capable of selecting for sequence evolution.
The majority of HLA-associated mutations were found in p24 Gag, Pol, and Nef. Reversion of HLA-associated
escape mutations on viral replication. Although no correlations were observed between the number or location of
HLA-associated mutations and protective HLA alleles, limiting the analysis to mutations selected by acute-phase
immunodominant responses revealed a strong positive correlation between mutations at conserved residues and
protective HLA alleles. These data suggest that control of HIV-1 may be associated with acute-phase CD8 responses
capable of selecting for viral escape mutations in highly conserved regions of the virus, supporting the inclusion of
these regions in the design of an effective vaccine.
Despite substantial advances in antiretroviral therapies, de-
velopment of an effective human immunodeficiency virus type
1 (HIV-1) vaccine remains a critical goal (6, 39, 82). Unfortu-
nately, current vaccine efforts have failed to reduce infection
rates in humans (9, 75) and have only achieved modest de-
creases in viral loads in the simian immunodeficiency virus
(SIV)/SHIV macaque model (21, 44, 81). A majority of these
vaccine approaches have focused on inducing T-cell responses,
utilizing large regions of the virus in an attempt to induce a
broad array of immune responses (6, 34, 44, 81). While it is
well established that CD8?T-cell responses play a critical role
in the containment of HIV-1 (45, 49, 67), supported in part by
the strong association of particular HLA class I alleles with
control of HIV (20, 33, 42, 61), it remains unclear which par-
ticular CD8?T-cell responses are best able to control the virus
and thus should be preferentially targeted by a vaccine. Studies
comparing the magnitude, breadth, and function of CD8?
T-cell responses in subjects exhibiting either enhanced or poor
control of HIV-1 have yielded few clues as to the specific
factors associated with an effective CD8?T-cell response (2,
28, 64, 67). Various differences in the functional capacity of
T-cell responses have been observed in long-term nonprogres-
sors (1, 26, 64), although it is possible that these differences
may be reflective of an intact immune response, as opposed to
having had directly enhanced immune control. As such, efforts
are needed to identify factors or phenotypes associated with
protective CD8?T-cell responses in order to enable vaccines
to induce the most effective responses.
Recent studies have begun to suggest that the specificity of
the CD8?T-cell response, or the targeting of specific regions
of the virus, may be associated with control of HIV-1. Prefer-
ential targeting of Gag, a structurally conserved viral protein
responsible for multiple functions, has been associated with
lower viral loads (25, 43, 56, 60, 77, 85). Furthermore, Kiepiela
et al. (43) recently illustrated in a large cohort of 578 clade
C-infected subjects that Gag-specific responses were associ-
ated with lowered viremia, in contrast to Env-specific re-
sponses, which were associated with higher viremia. These data
* Corresponding author. Mailing address: MGH-East, CNY 6625,
149 13th Street, Charlestown, MA 02129. Phone: (617) 726-7846. Fax:
(617) 724-8586. E-mail: firstname.lastname@example.org.
† Y.E.W. and B.L. contributed equally to this study.
¶ Present address: Laboratori de Retrovirologia, Fundacio ´ irsiCaixa,
Hospital Universitari Germans Trias i Pujol, Ctra del Canyet s/n,
08916 Badalona, Barcelona, Catalonia, Spain.
‡ Supplemental material for this article may be found at http://jvi
?Published ahead of print on 26 November 2008.
are in line with previous observations that many of the major
histocompatibility complex (MHC) class I alleles most strongly
associated with control of HIV-1 and SIV, namely, HLA-B57,
HLA-B27, and Mamu-A*01, restrict immunodominant CD8?
T-cell responses against the Gag protein (8, 10, 24, 63, 68, 83).
However, other alleles associated with slower disease progres-
sion, such as HLA-B51 in humans and Mamu-B08 and B-17 in
the rhesus macaque, do not immunodominantly target Gag,
suggesting that targeting of some other regions of the virus
may also be capable of eliciting control (8, 52–54). In addition,
recent studies investigating the pattern of HIV-1-specific
CD8?T-cell responses during acute infection reveal that only
a small subset of CD8?T-cell responses restricted by any given
HLA allele arise during acute infection and that there exist
clear immunodominance patterns to these responses (8, 77,
85). Since control of HIV-1 is likely to be established or lost
during the first few weeks of infection, these data suggest that
potentially only a few key CD8?T-cell responses may be
needed to adequately establish early control of HIV-1.
One of the major factors limiting the effectiveness of CD8?
T-cell responses is the propensity for HIV-1 to evade these
responses through sequence evolution or viral escape (3, 13,
66). Even single point mutations within a targeted CD8
epitope can effectively abrogate recognition by either the HLA
allele or the T-cell receptor. However, recent studies have
begun to highlight that many sequence polymorphisms will
revert to more common consensus residues upon transmission
of HIV-1 to a new host, including many cytotoxic T-lympho-
cyte (CTL) escape mutations (4, 30, 33, 48, 50). Notably, the
more rapidly reverting mutations have been observed to pref-
erentially occur at conserved residues, indicating that structur-
ally conserved regions of the virus may be particularly refrac-
tory to sequence changes (50). In support of these data, many
CTL escape mutations have now been observed to directly
impair viral replication (15, 23, 55, 74), in particular those
known to either revert or require the presence of secondary
compensatory mutations (15, 23, 73, 74). Taken together, these
data suggest that, whereas CTL escape mutations provide a
benefit to the virus to enable the evasion of host immune
pressures, some of these mutations may come at a substantial
cost to viral replication. These data may also imply that the
association between Gag-specific responses and control of
HIV-1 may be due to the targeting of highly conserved regions
of the virus that are difficult to evade through sequence evo-
The propensity by which HIV-1 escapes CD8?T-cell re-
sponses, and the reproducibility by which mutations arise at
precise residues in targeted CD8 epitopes (3, 48), also enables
the utilization of sequence data to predict which responses may
be most capable of exerting immune selection pressure on the
virus. Studies in HIV-1, SIV, and hepatitis C virus (16, 58, 65,
78) are now rapidly identifying immune-driven CTL escape
mutations across these highly variable pathogens at the popu-
lation level by correlating sequence polymorphisms in these
viruses with the expression of particular HLA alleles. We pro-
vide here an analysis of HLA-associated mutations across the
entire HIV-1 genome using a set of sequences derived from
clade B chronically infected individuals. Through full-length
viral genome coverage, these data provide an unbiased analysis
of the location of these mutations and suggest that the control
of HIV-1 by particular HLA alleles correlates with their ability
to preferentially restrict early CD8?T-cell responses capable
of selecting for viral escape mutations at highly conserved
residues of the virus. These data provide support for the in-
clusion of specific highly conserved regions of HIV-1 into vac-
MATERIALS AND METHODS
Subjects. Ninety-eight chronic untreated HIV-1 subtype B-infected subjects
were enrolled in Boston through the Massachusetts General Hospital, the
Lemuel-Shattuck Hospital, and the Fenway Community Health Center. The
study was approved by the Massachusetts General Hospital Review Board, and
all subjects gave written informed consent.
HLA typing. Molecular HLA typing was performed by the Tissue Typing
Laboratory at the Churchill Hospital in Oxford, the Massachusetts General
Hospital Tissue Typing Laboratory, and the Centre for Clinical Immunology and
Biomedical Statistics at the Royal Perth Hospital and Murdock University in
Viral sequencing. Genomic DNA was extracted from peripheral blood mono-
nuclear cell samples (5 million cells) by using a QIAamp DNA blood minikit
(Qiagen catalog no. 51104). Nested PCR protocols with limiting dilution adapted
from Salminen et al. (71, 72) were used to amplify HIV-1 genomes lacking 5? and
3? long terminal repeat regions using EXL DNA polymerase (Stratagene catalog
no. 600344). The sequences of the primary forward and reverse PCR primers,
respectively, were 5?-AAATCTCTAGCAGTGGCGCCCGAACAG-3? and 5?-T
GAGGGATCTCTAGTTACCAGAGTC-3?, while the nested forward and re-
verse primers were 5?-GCGGAGGCTAGAAGGAGAGAGATGG-3? and 5?-G
CACTCAAGGCAAGCTTTATTGAGGCTTA-3?. The PCR cycling conditions
were as follows: 92°C for 2 min; 10 cycles of 10 s at 92°C, 30 s at 60°C, and 10 min
at 68°C; 20 cycles of 10 s at 92°C, 30 s at 55°C, and 10 min at 68°C; and a final
extension of 10 min at 68°C. Five independent PCR products of each sample
were pooled and purified by using a QIAquick PCR purification kit (Qiagen
catalog no. 28104) and directly population sequenced at the Massachusetts
General Hospital DNA Sequencing Core facility using 70 clade B consensus
sequencing primers as previously described (7).
Phylogenetic analysis of HLA-associated sequence polymorphisms. To iden-
tify associations between HLA alleles and HIV polymorphisms, we used a pre-
viously described phylogenetic correction method (12, 19), with a slight modifi-
cation to account for HLA linkage disequilibrium. Briefly, a maximum-likelihood
tree was generated for each gene. To compute the pairwise correlation between
an HLA allele and an observed amino acid, two models of evolution were
compared by using a likelihood ratio test. In the null model, the amino acid was
allowed to evolve independently down the tree (“independent evolution
model”). In the alternative model, the presence (or absence) of the HLA allele
in a given patient was allowed to influence the final transition at the leaf node
(“conditional evolution model”). To account for HLA linkage disequilibrium, we
used a decision tree built using forward selection. First, for every amino acid at
each codon, the allele with the strongest association was added to the list of
identified associations. Then, individuals expressing this allele were removed
from the data set, and the analysis was repeated. This forward-selection proce-
dure was iterated until no HLA allele yielded an association with an uncorrected
P value of ?0.05, thus tending to eliminate spurious associations due to HLA
linkage disequilibrium. Rare HLA-amino acid pairs were not considered; spe-
cifically, we required that the observed or expected count in each bin of the
two-by-two contingency table was at least three. To account for multiple com-
parisons, P values were converted to q-values (76) by using a permutation test as
previously described (19). Associations with q ? 0.2, corresponding to a 20%
false discovery rate (11), are reported. In the present study, correlations in the
presence of an HLA allele are called “escape associations,” and correlations in
the absence of an HLA allele are called “reversion associations”.
HLA RH and immunodominance. The relative hazards (RHs) for disease
progression for each HLA allele were previously determined (21, 33, 62; data not
shown). Here we used the RH values for progression to AIDS disease definition
of 1987 as a marker of “protective” versus “hazardous” HLA alleles. Based on a
previous study by Altfeld et al. (8), acute-phase immunodominant responses
were defined as any HIV-1-specific CD8?T-cell response that was detected in at
least one HIV-1-infected subject within the first 2 months following presentation
with acute HIV-1 infection. We then ranked the epitopes restricted by each HLA
allele in the order of their frequency of recognition, such that the epitope most
frequently recognized by individuals expressing the corresponding allele received
1846WANG ET AL.J. VIROL.
a ranking of 1, and the second most frequently recognized epitope received a
ranking of 2, and so on.
Conservation and covariation analysis. Conservation scores for each residue
were calculated by using PFAAT (17) for clade B sequence alignments derived
from the LANL HIV Sequence Database (http://www.hiv.lanl.gov), which re-
moved highly similar sequences such as multiple clones from a single isolate and
multiple sequences from a single patient. The conservation score S is defined as
S ? 1 ? H, where H represents the normalized Shannon entropy of a particular
column in the alignment. We applied a previously derived P value and algorithm
(79) for pairwise residue covariation analysis on all clade B sequences, limited to
one sequence per patient, for all HIV coding genes. P values are adjusted by
Bonferroni correction. Sequences were aligned to HXB2, and positions where
either the residue conservation score was ?0.6 or a gap represented the most
abundant variant were removed from the analysis. VisANT was used for network
visualization (37, 38).
Statistical analysis. Mann-Whitney, Spearman correlated coefficient tests, and
Fisher exact tests were conducted using Prism 4.0 (GraphPad, San Diego, CA).
Cook’s distances (D) (22) were calculated using MatLab 7.0 (MathWorks,
Natick, MA). The data points are consider outliers (35) if D ? 4/n, where n is
number of data points.
Predicted CD8 epitopes. Predicted CD8 epitopes were determined based on
scanning the cohort consensus sequence using the epitope prediction algorithm
developed by Heckerman et al. (36).
Nucleotide sequence accession numbers. All sequence data in the present
study were deposited in GenBank under accession numbers FJ469682 to
FJ469772 and DQ886031 to DQ886038 (27).
Identification of HLA-associated sequence polymorphisms
across the HIV-1 genome. Viral escape from CD8?T-cell
responses frequently occurs through evolution of specific res-
idues within or flanking a given CD8 epitope (3, 48). This
phenomenon, typically driven by the selection of mutations at
the most variable residue, enables CD8 escape mutations, or
HLA-associated mutations, to be rapidly identified at the pop-
ulation level (16, 58, 65, 78). In order to identify frequent
HLA-associated mutations across the HIV-1 genome, we se-
quenced the entire coding region (from Gag to Nef; nucleo-
tides 790 to 9414 on HXB2) of viruses derived from 98 chronic
untreated HIV-1 subtype B-infected subjects with an average
sequence length of 8,794 nucleotide bases. A total of 18
HLA-A, 26 HLA-B, and 14 HLA-C alleles were represented in
this cohort (see Table S1 in the supplemental material). HLA-
associated sequence polymorphisms across the viral genome
were then identified by using a phylogenetics-based likelihood-
ratio approach (PhyloD) (12, 19). The PhyloD approach cor-
rects for confounding founder effects by considering phylogeny
as a source of hierarchical structure in a generative model,
which can provide much higher specificity to the assessment of
HLA-associated polymorphisms. The PhyloD approach cap-
tures positive selection and purifying negative selection pro-
cesses within four states: polymorphisms away from a particu-
lar residue (“escape”) or toward a specific nonconsensus
residue (“attraction”) in the presence of a specific HLA allele
and polymorphisms away from nonconsensus residues (“repul-
sion”) or toward a particular residue (“reversion”) in the ab-
sence of a specific HLA. For the current analyses, these four
states were consolidated into either “escaping” (escape or at-
traction in the presence of HLA) or “reverting” (reversion or
repulsion in the absence of HLA).
The PhyloD approach identified a total of 76 HLA-associ-
ated polymorphisms located at 47 distinct amino acid residues
across HIV-1 (see Table S2 in the supplemental material).
These were further broken down into 41 escaping and 35
reverting associations, with 29 positions overlapping between
them. These HLA-associated mutations were then categorized
into four classes based on their location relative to epitopes: (i)
located within previously defined CD8 epitopes (46), (ii) flank-
ing described CD8 epitopes (within 3 amino acid residues), or
within (iii) or flanking (iv) predicted CD8 epitopes. Using
these criteria there were 17, 1, 18, and 5 escaping associations
found in each class, respectively, and 13, 1, 15, and 6 reverting
associations for each class (Table 1). As expected, we observed
HLA-associated escape mutations in common CD8 epitopes
such as B57-TW10, B27-KK10, and A3-RK9, where CTL es-
cape has previously been described in longitudinal analyses
(see Fig. S1 in the supplemental material) (3, 4, 41, 48). More-
over, we also identified HLA-associated reversions in the B57-
TW10 and A3-RK9 epitopes as previously described (48), ver-
ifying the use of the PhyloD approach for identifying both of
these types of associations at the population level. Notably, the
numbers of HLA-associated polymorphism detected are sig-
nificantly different from the frequencies of specific HLA alleles
(P ? 0.0001 [Mann-Whitney test]), illustrating lack of any bias
introduced by high frequency alleles in this analysis. Therefore,
due to the modest size of this cohort (n ? 98), these HLA
associations likely represent some of the strongest associations,
TABLE 1. Distribution of HLA-associated escape and reversing mutations observed across the HIV-1 genome relative to the location of the
CD8?T-cell restricted epitope
No. of epitopes
Total 171 185 131 156
VOL. 83, 2009IDENTIFYING GENOME-WIDE CTL ESCAPE MUTATIONS 1847
reflective of those CD8?T-cell responses capable of exerting
the greatest selective pressure.
HLA-associated polymorphisms are concentrated in Gag,
Pol, and Nef. We first examined whether HLA-associated mu-
tations might be clustering in specific regions of HIV-1, com-
paring the frequency and location of escaping and reverting
HLA-associated polymorphisms across the HIV-1 genome. As
illustrated in Fig. 1, the majority of HLA-associated sequence
changes were located in Gag (n ? 19), Pol (n ? 31), and Nef
(n ? 16), where previous studies have illustrated that HIV-
specific CD8?T-cell responses are most frequently detected
(2, 28). In contrast, very few HLA-associations were detectable
in Env (n ? 3) and the accessory and regulatory proteins.
Furthermore, 32 of 76 of the polymorphisms, both escaping
and reverting mutations, were located within or flanking de-
scribed epitopes in Gag and Nef (Table 1). Therefore, the
HLA-associated mutations reflective of immune-driven viral
evolution support the dominant targeting of the viral proteins
Gag and Nef by CD8?T-cell responses. In contrast, the ma-
jority (24 of 31 [77%]) of the associated polymorphisms occur-
ring in Pol were located within or flanking predicted epitopes
that have not been previously described. In comparing these
predicted epitopes with CD8?T-cell responses detected using
genome-wide overlapping peptides in cells from clade B chron-
ically infected subjects (28), we found that for 10 of 27 (37%)
overlapping peptides containing predicted epitopes, at least
10% of the individuals expressing the specific HLA allele ex-
hibited a response, suggesting that a substantial number of
CD8 epitopes in this protein have not yet been mapped.
HLA-associated mutations arise at both conserved and vari-
able residues across HIV-1. In Fig. 1, the relative proportions
of escaping (n ? 41) and reverting (n ? 35) sequence poly-
morphisms were similar within any given protein, suggesting an
overall balance between positive (escaping) and purifying (re-
verting) evolutionary selective pressures. To more closely ex-
amine the relationship between escaping and reverting HLA-
associated mutations, the location of these associations across
the viral genome was plotted against the conservation score
(1-entropy) of the residue at which they arose. Figure 2 illus-
trates that HLA-associated mutations were equally present at
both highly conserved and more variable residues. Moreover,
in the majority of residues where forward escape mutations
were identified a matching HLA-associated reversion was also
detected (30 of 41 cases [71%]). Our previous study examining
viral evolution in longitudinally monitored subjects illustrated
that reverting mutations preferentially arose within structurally
conserved residues, with mutations in Gag and Pol proteins
reverting much more rapidly than in other proteins (50).
Again, we observed that escaping residues without an associ-
ated reversion were significantly less conserved than escaping
residues with an associated reversion (Fig. 2; mean conserva-
FIG. 1. HLA class I-associated mutations are distributed across
HIV-1. Near-full-length viral genome sequencing was conducted on 98
chronically infected subjects. HLA-associated escape and reverting
mutations were then identified for all positions across the HIV-1 ge-
nome and plotted by their location in HIV-1, illustrating their pre-
dominance in Gag, Pol, and Nef.
FIG. 2. Location and residue conservation of HLA-associated escape and reverting mutations. The location of all HLA-driven escaping (red
circle) and reverting (blue cross) mutations in the HIV-1 proteome were plotted against the conservation scores of those residues at which they
arose. Crosses overlaying circles represent positions where both escape and reversion were observed for the same HLA-associated mutation.
HLA-associated escape and reverting mutations arose at both conserved and variable residues, and the majority of escaping residues were
accompanied by a corresponding reversion.
1848 WANG ET AL.J. VIROL.
tion score of 0.825 versus 0.741, respectively [P ? 0.018]).
Taken together, these data support that many CTL escape
mutations are reverting upon transmission to a new host and
that the inherent conservation of a given residue can strongly
influence the propensity for reversion to occur, likely reflecting
different impacts of mutations on viral fitness.
HLA-associated polymorphisms are predominantly driven
by immunodominant CD8?T-cell responses. Recent studies
have begun to examine the immunodominance patterns of
CD8?T-cell responses, illustrating that only a small subset of
defined CD8?T-cell responses are actually present during
acute infection and therefore may have a disproportional effect
on early control of HIV-1 (8, 77). Since this subset of CD8?
T-cell responses represents the first line of responses that
HIV-1 encounters, we examined whether HLA-associated mu-
tations reflective of viral escape were preferentially occurring
within epitopes targeted by the most frequent acute-phase
responses. We counted the number of the respective HLA-
associated mutations in defined CD8 epitopes targeted during
the early phase of infection. These epitopes were plotted in
ranked order as described in Materials and Methods. Of the 15
described CD8 epitopes recognized during acute-phase infec-
tion that contain HLA-associated escape mutations, close to
half of these (7 of 15 [47%]) were targeted by the most fre-
quently mounted CD8?T-cell response of any given HLA
allele (Fig. 3). These data suggest that the most immunodom-
inant acute-phase CD8?T-cell responses are preferentially
driving viral escape or that, conversely, HIV-1 is actively evad-
ing responses that dominate the acute phase of infection.
These data are in line with previous reports indicating prefer-
ential viral escape from the earliest CD8?T-cell responses in
HIV-1 and SIV infection (40, 62).
Protective HLA alleles restrict acute-phase CD8?T-cell re-
sponses that are associated with viral escape mutations at
highly conserved residues. Numerous studies now illustrate
that targeting of the Gag protein by HIV-specific CD8?T-cell
responses, as measured by a gamma interferon enzyme-linked
immunospot assay, correlates with the control of HIV-1 (25,
56, 60, 77, 85). These data are supported by recent studies
illustrating that certain CTL escape mutations in Gag impair
viral replication (15, 23, 55, 74), suggesting that HIV-1 may be
constrained in its ability to effectively escape from cellular
immune pressures against this highly conserved region of the
virus. To determine whether immune control of HIV-1 may
more broadly correlate with the ability of CD8?T-cell re-
sponses to exhibit immune selection pressure at conserved
residues, we examined the correlation between residue conser-
vation at immune-driven escaping positions with the RH (80)
of each restricting HLA allele for disease progression (20, 32,
61). No correlation was observed when the residue conserva-
tion of all 41 immune-driven escape mutations was compared
to the RH of the mutations’ restricting HLA allele (Fig. 4a,
R ? ?0.1482, P ? 0.3553 [Spearman rank]). This analysis was
the same regardless of whether all associations, or only those
associated with defined CD8 epitopes, were included in the
analysis. However, given the potential for acute-phase CD8?
T-cell responses to disproportionately influence early outcome
of HIV-1 infection, the analysis was then limited only to those
HLA-associated mutations localized within epitopes targeted
FIG. 3. HLA-associated mutations are predominantly selected by
acute-phase immunodominant CD8?T-cell responses. The total num-
ber of CD8 epitopes targeted during the acute phase of HIV-1 infec-
tion (9) were plotted in ranked order of their frequency of recognition
as described in Materials and Methods. Dotted bars represent epitopes
that are immunodominant but do not contain HLA-associated escape
mutations identified in the present study; hatched bars represent those
epitopes containing HLA-associated escape mutations defined in the
present study, illustrating that HLA-associated mutations are predom-
inantly driven by the most immunodominant CD8 responses within
those tested HLA alleles.
FIG. 4. Protective HLA alleles restrict acute-phase CD8?T-cell
responses that select for viral escape mutations at highly conserved
residues. The conservation scores of residues at which HLA-associated
escape mutations arose were plotted against the RH (81) to disease
progression of the HLA allele restricting the response. (a) Plotting of
all HLA-associated escape mutations observed within the HIV ge-
nome revealed no correlation between RH of the HLA allele and the
conservation of the escaping residue. (b) HLA-associated escape mu-
tations observed within CD8 epitopes targeted during acute HIV-1
infection revealed a strong correlation between protective HLA alleles
(low RH) and HLA-associated escape mutations arising at conserved
VOL. 83, 2009 IDENTIFYING GENOME-WIDE CTL ESCAPE MUTATIONS1849
by acute-phase CD8?T-cell responses (8, 77) (see Table S2 in
the supplemental material) to test the hypothesis of whether
escape mutations selected by CD8 responses restricted by pro-
tective alleles are more conserved than those selected by haz-
ardous HLA alleles. Here, we observed a significant inverse
correlation between the RH of the restricting HLA allele and
the conservation of the CD8?T-cell selected escaping residues
(R ? ?0.5650, P ? 0.0282 [Spearman rank test]). In this
analysis, there were two clear potential outliers (T290 in the
B51-TI8 epitope in Pol and Y81 in the B35-VY8 epitope in
Nef) based on Cook’s distance (22) as described in Materials
and Methods. When these outliers were removed from the
analysis, this inverse correlation remained significant and was
further strengthened (R ? ?0.7746, P ? 0.0019 [Spearman
rank test]) (data not shown). Therefore, while Altfeld et al. (8)
have previously shown that epitope sequence heterogenicity
does not influence immunodominance patterns, suggesting
that conserved epitopes are not preferentially targeted during
the acute phase, we examined here residue conservation at the
exact residue where the HLA-associated escape mutation oc-
curs as a measurement of the potential for an escape mutation
to impact viral replication. Taken together, these data suggest
that protective HLA alleles are associated with acute-phase
CD8?T-cell responses that select for viral escape mutations at
more conserved residues, whereas the early responses re-
stricted by hazardous HLA alleles select for mutations at more
variable residues. These results provide broader support for
the observation that targeting of the highly conserved p24
region of Gag by CD8?T-cell responses correlates with con-
trol of HIV-1 (29, 43) and moreover suggest that the capacity
of early responses to select for viral escape mutations may
represent an important component of this phenomenon.
Residue conservation and compensatory mutations influ-
ence the reversion of HLA-associated mutations. We have
previously observed that during acute HIV-1 infection trans-
mitted mutations revert more quickly when arising in highly
conserved residues (50). In this regard, it is notable that of the
11 HLA-associated escape mutations in Fig. 2 that did not
exhibit a matching HLA-associated reversion, 5 were located
at variable residues with entropy values of ?0.75. It is possible
that these mutations may have had only a minimal impact on
viral replication and, therefore, were less apt to revert upon
transmission. It is also possible that compensatory mutations
could have arisen during viral escape which functioned to tem-
per the impact of these escape mutations on viral replication
(15, 23, 31, 74). To examine whether compensatory mutations
might be influencing the nonreverting HLA-associated muta-
tions, we performed a covariation analysis between all pairs of
residues within each individual HIV-1 protein to identify pairs
of mutations having a high probability of occurring in conjunc-
tion with one another. This analysis was conducted on a large
data set of 284 Gag, 118 Pol, and 600 Nef sequences derived
from the Los Alamos National Laboratory (LANL) HIV da-
tabase (www.hiv.lanl.gov). As validation of this approach, we
detected previously described compensatory mutations for the
T242N escape mutation in the B57-TW10 epitope and the
R264K escape mutation in the B27-KK10 epitope (Fig. 5) (15,
74). The analysis identified three of the four known covarying
residues for B57-TW10 (H219Q, M228I, and G248A), where
H219Q and M228I mutations were previously shown to par-
tially restore the viral replication defect of T242N (Fig. 5A). It
also successfully identified both the prerequisite (L268M) and
the compensatory (S173A) mutations for the R264K escape
mutation in B27-KK10 (Fig. 5B). In applying this covariation
analysis to the 98 HIV-1 sequences in our data set, we observed
that six of nine sequences exhibiting the T242N mutation con-
tained one or more of the predicted compensatory residues, in
one case from an HLA-B57-negative subject, suggesting the
stability of some of these mutations upon transmission. Of the
nine sequences containing the R264K mutation, seven con-
tained one or more of the predicted covarying residues.
In extending this analysis to the 11 HLA-associated escape
mutations in Fig. 2 for which no reversions were detected, we
observed strong covarying residues for the three intraepitope
associations at residues Gag E312, Gag G354, and Nef H116
(Fig. 5C to E) (Table 2). Notably, these three HLA-associated
mutations were also among the six most conserved residues
without detectable reversions, a finding suggestive of a possible
contribution of compensatory mutations to the lack of rever-
sions detected at these more conserved residues. Taken to-
gether, these data suggest an important contribution of both
the conservation of escaping residues and covarying mutations,
which potentially compensate for the impact of specific muta-
tions on viral fitness and their propensity to revert upon trans-
Large population-based studies of HLA-associated polymor-
phisms serve to rapidly identify the location and frequency of
CTL escape mutations which function to evade host CD8?
T-cell responses. Although previous population studies of
HLA class I-associated mutations in HIV-1 have demonstrated
immune selection pressures against specific HIV-1 residues
(16, 29, 58, 65, 69), we present here an unbiased assessment of
HLA-mediated sequence polymorphisms across the full sub-
type B HIV genome. Our data reveal that these polymor-
phisms are most frequently observed in Gag, Pol, and Nef,
which are known to be dominantly targeted by CD8?T-cell
responses (2, 28). Escape mutations were also observed to
occur at both highly conserved and variable residues, with
matching reverting HLA-associated mutations also detectable
for most mutations. Notably, escape mutations associated with
acute-phase CD8?T-cell responses restricted by protective
HLA alleles were preferentially located at more conserved
residues than those restricted by hazardous HLA alleles.
Taken together, these data suggest that acute-phase CD8?
T-cell responses capable of selecting for viral escape mutations
within highly conserved regions of the virus may be critical to
the control of HIV-1. These data lend support to recent sug-
gestions that eliciting CTL responses targeting highly con-
served regions of HIV-1 such as Gag may be critical to an
effective HIV-1 vaccine.
Longitudinal studies of CTL escape have observed that
CD8?T-cell responses represent a major driving force of se-
quence evolution in HIV, SIV, and hepatitis C virus (3, 16, 47,
58, 62). Viral escape from CD8?T-cell responses has espe-
cially been described during the acute phases of both HIV-1
and SIV infection, illustrating rapid evasion of specific host
immune responses during the first few weeks or months after
1850WANG ET AL.J. VIROL.
infection (3, 5, 13, 62, 66). Recent studies examining the im-
munodominance of CD8?T-cell responses in HIV-1 infection
are beginning to reveal predictable patterns to these responses,
with some CD8 epitopes preferentially recognized during the
acute phase of infection (8, 77). Moreover, responses restricted
by some HLA alleles appear to dominate over responses re-
stricted by other HLA alleles (8). Such studies have important
implications for vaccine design since control of HIV-1 is pre-
dominantly established during the first few weeks or months
after infection, with steady-state viral set points strongly pre-
dictive of disease progression (57). Therefore, some CD8?
T-cell responses are undoubtedly more critical to this early
control than others. In the present study, not only were HLA-
associated escape mutations preferentially located within the
viral proteins most frequently targeted by CD8?T-cell re-
sponses (Gag, Pol, and Nef) but they were also located within
the most immunodominant CD8 epitopes targeted during
acute infection. These data strongly support that HIV-1 is
preferentially evading the strongest and earliest CD8?T-cell
responses that represent the front line responses.
It has been observed that CTL escape mutations will revert
to consensus residues when transmitted to a new host express-
ing different HLA alleles, since these mutations are no longer
required to evade specific CD8?T-cell responses (4, 30, 33, 48,
50). During the acute phase of infection, such reversions may
account for upward of 60% of mutations and notably arise
most rapidly when located at more conserved residues (50).
These data, and more recent studies directly illustrating the
impact of CTL escape mutations on viral replication, provide
strong support that a majority of adaptive mutations are capa-
ble of impairing the replicative capacity of HIV-1 (15, 23, 50,
73, 74). Our observation that the majority of detected escape
mutations are accompanied by a corresponding reversion pro-
vides further support for these data at a genome-wide level.
Interestingly, however, we observed that a subset of CTL es-
cape mutations were not associated with detectable reversions,
with several of the more conserved residues associated with
compensatory mutations that may have tempered the effects of
the escape mutation on viral replication capacity. These data
support the important role that compensatory mutations play
in the impact of CTL escape mutations on viral fitness (15, 23,
73) and highlight that due to structural constraints some CTL
escape mutations may not be able to acquire the necessary
compensatory mutations and thereby may disproportionately
impact viral replication capacity. It will be important, there-
fore, to more broadly examine the frequency and complexity by
which compensatory mutations can arise, since as in the case of
the B57-TW10 epitope the network of compensatory muta-
tions may be highly complex (15, 74).
The ability to more rapidly generate HIV-1 sequence data
FIG. 5. Identification of covarying residues linked to HLA-associated escape mutations. Covarying residue pairs were determined as described
in Materials and Methods. Residues located within each epitope are present within each circle, with HLA-associated escape residues colored red.
Covarying residues are then connected by lines and located outside the circle. Each residue is labeled by its consensus residue and HXB2 position.
The epitope sequences are noted in the following format:SXXXXE, where S is the starting position, E is the end position, and XXXX is the epitope
sequence. (A and B) Covariation networks for B57-TW10 and B27-KK10 epitopes, respectively. Sequences from subjects in our cohort with T242N
or R264K escape mutations are given. The presence or absence of B57/B58 or B27 allele in these patients is noted. (C to F) Covariation networks
for CD8 epitopes B45-AW11, A11-AK11, and B57-HW9, respectively.
VOL. 83, 2009 IDENTIFYING GENOME-WIDE CTL ESCAPE MUTATIONS1851
and correlate sequence changes with the expression of specific
host HLA alleles has enabled a growing number of studies
identifying HLA-associated mutations in HIV-1 (14, 16, 51, 58,
65). These studies have begun to provide substantial insight
not only into the frequency and complexity of immune-driven
mutations but also into the mechanisms associated with “pro-
tective” HLA alleles. Frater et al. (29) recently illustrated that
HLA alleles associated with slower disease progression were
most often associated with CD8?T-cell responses that drive
viral escape in Gag, Pol, and Nef, supporting that “protective”
HLA alleles may restrict uniquely strong CD8?T-cell re-
sponses. Our study builds upon this work by extending
these analyses to the entire HIV-1 genome, revealing the im-
portance of distinguishing between acute-phase immunodom-
inant responses and chronic responses, and specifically ad-
dressing the issue of viral sequence constraints by taking into
account the entropy of escaping residues. Through these com-
bined analyses we were able to illustrate that early immuno-
dominant CD8 responses restricted by “protective” HLA al-
leles primarily select for viral escape mutations in highly
conserved regions of HIV-1, suggesting that the protection
conferred by specific HLA alleles on HIV-1 disease control
may depend upon the costs to replicative capacities required to
successfully escape from early immunodominant responses.
HIV-1 mutants with diminished replication fitness have been
known to provide clinical benefit in practice. For example,
many clinicians have chosen to continue to use 3TC even after
the 3TC resistance mutation M184V has emerged to take ad-
vantage of the viral replicative defect caused by this mutation
Many of the protective MHC class I alleles, such as HLA-
B27, HLA-B57, and Mamu-A*01, preferentially restrict CD8?
T-cell responses targeting the highly conserved Gag protein
(15, 59, 74, 77). The importance of targeting CD8 epitopes in
Gag not only may be due to the sequence conservation of Gag
but also may be due to the early presentation of CD8 epitopes
in Gag derived from the incoming capsid particle prior to de
novo protein synthesis of other viral proteins (70). It is note-
worthy, however, that other MHC class I alleles such as HLA-
B51, Mamu-B08, and Mamu-B17 associated with the control of
HIV-1 and SIV do not immunodominantly target CD8
epitopes in Gag but rather target other viral proteins such as
Pol, Vif, and Nef (8, 52–54). Indeed, many of the immuno-
dominant CD8 responses restricted by “protective” HLA al-
leles were found to drive viral escape in non-Gag proteins,
although notably still at highly conserved residues. Therefore,
in the development of an HIV-1 vaccine it may be important to
consider other highly conserved regions of HIV-1 outside of
Gag in order to provide ample targets for the diversity of HLA
alleles in the population.
The moderate size of the current data set limited the present
study to HLA-associated mutations restricted by common
HLA alleles, as well as those HLA alleles for which immu-
nodominance patterns are known, since the original study by
Altfeld et al. (8) was not an exhaustive analysis for all known
HLA alleles. Similarly, it is likely that we did not have the
power to detect CTL escape mutations associated with sub-
dominant CD8?T-cell responses. In addition, since our study
does not focus on end-stage individuals, late-arising CTL es-
cape mutations will not be represented in our study, as well as
early escape mutations targeted by acute-phase CD8?re-
sponses. Therefore, while the correlation between the most
immunodominant CD8?responses and likelihood for viral
escape was very strong in our study, this correlation might
diminish as subjects are monitored later into infection. Further
broadening of the data set to a larger cohort will be required to
extend our analyses to examine the relative contribution of
these additional responses to early immune control of HIV-1.
More importantly, the approach of utilizing viral sequence
polymorphisms as a measurement of the impact of particular
CD8?T responses on HIV-1 fitness obviously does not take
into account CD8 epitopes that do not escape. Here, larger
datasets will enable determining whether in fact some epitopes
are completely refractory to escape or escape only late in
infection. Finally, it will be important to extend these analyses
to other clades of HIV-1 to determine whether similar corre-
lates of immune control exist in the setting of different viral
strains and different frequencies of HLA alleles.
In conclusion, these data provide an initial assessment of
HLA-associated sequence polymorphisms across the entire
TABLE 2. Covarying residue positions for HLA-associated escaping residues without detectable reversiona
ProteinHLA Position Optimal epitopeConservation
aMutations are labeled in the form XNNNY, where X is the consensus residue, Y is the mutant residue, and NNN is the HXB2 position. ND, not detected; NA,
bLANL P values were determined by using sequences from the LANL database.
c98Chronic P values were determined by using HIV sequences from our cohort of 98 subjects.
dThat is, the number of patients from our cohort that simultaneously contain both mutations.
1852 WANG ET AL.J. VIROL.
HIV-1 proteome and suggest an important influence of early
immunodominant CD8?T-cell responses not only on viral
evolution but also potentially on the outcome of HIV-1 infec-
tion. More importantly, we show that the HLA alleles that
correlate with disease control are capable of selecting for viral
sequence polymorphisms in highly conserved regions of
HIV-1. Taken together, these findings suggest that vaccines
designed to elicit CD8?T-cell responses may need to focus
responses against highly conserved regions of the virus that
would exact a substantial impact on viral fitness.
This study was supported by the National Institutes of Health grant
R01-AI054178 (T.M.A.) and as part of the Collaboration for AIDS
Vaccine Discovery with support from the Bill & Melinda Gates Foun-
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