Marked Epitope- and Allele-Specific Differences in Rates of Mutation in Human Immunodeficiency Type 1 (HIV-1) Gag, Pol, and Nef Cytotoxic T-Lymphocyte Epitopes in Acute/Early HIV-1 Infection

Article (PDF Available)inJournal of Virology 82(18):9216-27 · August 2008with44 Reads
DOI: 10.1128/JVI.01041-08 · Source: PubMed
During acute human immunodeficiency virus type 1 (HIV-1) infection, early host cellular immune responses drive viral evolution. The rates and extent of these mutations, however, remain incompletely characterized. In a cohort of 98 individuals newly infected with HIV-1 subtype B, we longitudinally characterized the rates and extent of HLA-mediated escape and reversion in Gag, Pol, and Nef using a rational definition of HLA-attributable mutation based on the analysis of a large independent subtype B data set. We demonstrate rapid and dramatic HIV evolution in response to immune pressures that in general reflect established cytotoxic T-lymphocyte (CTL) response hierarchies in early infection. On a population level, HLA-driven evolution was observed in approximately 80% of published CTL epitopes. Five of the 10 most rapidly evolving epitopes were restricted by protective HLA alleles (HLA-B*13/B*51/B*57/B*5801; P = 0.01), supporting the importance of a strong early CTL response in HIV control. Consistent with known fitness costs of escape, B*57-associated mutations in Gag were among the most rapidly reverting positions upon transmission to non-B*57-expressing individuals, whereas many other HLA-associated polymorphisms displayed slow or negligible reversion. Overall, an estimated minimum of 30% of observed substitutions in Gag/Pol and 60% in Nef were attributable to HLA-associated escape and reversion events. Results underscore the dominant role of immune pressures in driving early within-host HIV evolution. Dramatic differences in escape and reversion rates across codons, genes, and HLA restrictions are observed, highlighting the complexity of viral adaptation to the host immune response.
JOURNAL OF VIROLOGY, Sept. 2008, p. 9216–9227 Vol. 82, No. 18
0022-538X/08/$08.000 doi:10.1128/JVI.01041-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Marked Epitope- and Allele-Specific Differences in Rates of Mutation
in Human Immunodeficiency Type 1 (HIV-1) Gag, Pol, and Nef
Cytotoxic T-Lymphocyte Epitopes in Acute/Early
HIV-1 Infection
Zabrina L. Brumme,
†* Chanson J. Brumme,
Jonathan Carlson,
Hendrik Streeck,
Mina John,
Quentin Eichbaum,
Brian L. Block,
Brett Baker,
Carl Kadie,
Martin Markowitz,
Heiko Jessen,
Anthony D. Kelleher,
Eric Rosenberg,
John Kaldor,
Yuko Yuki,
Mary Carrington,
Todd M. Allen,
Simon Mallal,
Marcus Altfeld,
David Heckerman,
and Bruce D. Walker
Partners AIDS Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts
; Microsoft Research,
Redmond, Washington
; Department of Computer Science, University of Washington, Seattle, Washington
; Centre for
Clinical Immunology and Biomedical Statistics, Royal Perth Hospital and Murdoch University, Perth, Australia
Aaron Diamond AIDS Research Center, New York, New York
; Jessen Praxis, Berlin, Germany
; National Centre in
HIV Epidemiology and Clinical Research, University of New South Wales, Sydney, Australia
; Cancer and
Inflammation Program, Laboratory of Experimental Immunology, SAIC-Frederick, Inc., NCI-Frederick,
Frederick, Maryland
; and Howard Hughes Medical Institute, Chevy Chase, Maryland
Received 18 May 2008/Accepted 1 July 2008
During acute human immunodeficiency virus type 1 (HIV-1) infection, early host cellular immune responses
drive viral evolution. The rates and extent of these mutations, however, remain incompletely characterized. In
a cohort of 98 individuals newly infected with HIV-1 subtype B, we longitudinally characterized the rates and
extent of HLA-mediated escape and reversion in Gag, Pol, and Nef using a rational definition of HLA-
attributable mutation based on the analysis of a large independent subtype B data set. We demonstrate rapid
and dramatic HIV evolution in response to immune pressures that in general reflect established cytotoxic
T-lymphocyte (CTL) response hierarchies in early infection. On a population level, HLA-driven evolution was
observed in 80% of published CTL epitopes. Five of the 10 most rapidly evolving epitopes were restricted by
protective HLA alleles (HLA-B*13/B*51/B*57/B*5801; P 0.01), supporting the importance of a strong early
CTL response in HIV control. Consistent with known fitness costs of escape, B*57-associated mutations in Gag
were among the most rapidly reverting positions upon transmission to non-B*57-expressing individuals,
whereas many other HLA-associated polymorphisms displayed slow or negligible reversion. Overall, an esti-
mated minimum of 30% of observed substitutions in Gag/Pol and 60% in Nef were attributable to HLA-
associated escape and reversion events. Results underscore the dominant role of immune pressures in driving
early within-host HIV evolution. Dramatic differences in escape and reversion rates across codons, genes, and
HLA restrictions are observed, highlighting the complexity of viral adaptation to the host immune response.
Cytotoxic T lymphocytes (CTL) recognizing HLA class-I-
restricted viral epitopes presented on the infected cell surface
are critical for the resolution of acute-phase plasma viremia
(13, 39, 53). However, durable human immunodeficiency virus
(HIV) immune control rarely is achieved, due in part to rapid
viral evolution within the new host. Indeed, the course of HIV
disease is influenced by the strength and specificity of the early
CTL response (7, 76), combined with the virus’ ability to adapt
to changing immune pressures through the selection of HLA-
restricted CTL escape mutations and the reversion of trans-
mitted escape mutations from the previous host (14, 22, 36, 38,
39, 52, 58, 59, 67, 68). Given the extent of CD4 T-cell destruc-
tion that occurs during the acute phase (24, 25), it is important
to achieve a deeper understanding of the interplay between
immune response and viral adaptation in early infection. Re-
cently, immunodominance hierarchies of CTL epitope target-
ing in early HIV infection have been characterized (7, 76);
however, a comprehensive population-based assessment of the
rates and extent of HLA-associated immune adaptation in
early HIV infection remains to be undertaken.
It is now understood that CTL escape occurs along generally
predictable pathways based on the host HLA profile (2, 17, 27),
and that escape and reversion represent major forces driving
viral evolution and diversity at both the individual and popu-
lation levels (2, 11, 14, 34, 39, 52, 57–59, 65, 67, 68, 70).
However, due in part to the lack of a consistent definition of
HLA-associated mutation, as well as the lack of large longitu-
dinal HIV sequence datasets (2, 9, 46, 61), HLA-driven viral
adaptation in early HIV infection remains incompletely char-
acterized. In this study, we employ a strict definition of HLA-
attributable substitution based on a comprehensive, predefined
list of HLA-associated polymorphisms in HIV type 1 (HIV-1)
* Corresponding author. Mailing address: Partners AIDS Research
Center, Massachusetts General Hospital, 149 13th St., Charlestown,
MA 02129-2000. Phone: (617) 643-2357. Fax: (617) 726-4691. E-mail:
† These authors contributed equally to this work.
Published ahead of print on 9 July 2008.
subtype B in order to estimate the proportion of viral evolution
attributable to HLA-associated selection pressures in Gag, Pol,
and Nef in the first year of HIV infection in a cohort of 98
untreated, subtype B-infected seroconverters. In addition, we
systematically compute the rates of escape and estimate the
rates of the reversion of these HLA-associated polymorphisms
in early HIV infection, revealing marked differences in the
rates of escape and reversion across codons, genes, and HLA
HIV seroconverter cohort. The HIV seroconverter cohort consisted of 98
untreated, HIV subtype B-infected individuals enrolled through a private med-
ical clinic (Jessen-Praxis) in Berlin, Germany (n 38), and three sites within the
Acute Infection and Early Disease Research Program (AIEDRP): Massachu-
setts General Hospital, Boston (n 25), Aaron Diamond AIDS Research
Center, New York, NY (n 24), and the National Centre in HIV Epidemiology
and Clinical Research, University of New South Wales, Sydney, Australia (n
11). Of these, 61 (62%) individuals were identified during acute infection as
defined by either documented positive HIV RNA (5,000 copies/ml) and either
(i) a negative HIV-1 enzyme immunoassay (EIA) or (ii) a positive EIA but a
negative or indeterminate Western blotting result (AIEDRP stage 1; n 53) or
by a detectable serum p24 antigen and either (i) a negative EIA or (ii) a positive
EIA but a negative or indeterminate Western blotting result (AIEDRP stage 2;
n 8). The time frame for acute infection as defined here ranges from 2 to 6
weeks following infection (31). The remaining 37 (38%) individuals were iden-
tified during early HIV infection, as defined by (i) a negative EIA during the
previous 6 months or (ii) a negative detuned HIV-1 EIA (Vironostika-LS EIA;
BioMerieux, Raleigh, NC) (44) at enrolment (AIEDRP stages 3a and 3b, re-
The date of HIV infection was estimated using clinical history (where avail-
able) by subtracting 4 weeks from the baseline (for stages 1a or 2a), by subtract-
ing 6 weeks from the baseline (for stages 1b or 2b), by calculating the midpoint
between the last negative and the first positive EIA (for stage 3a), or by sub-
tracting 4 months from the baseline (for stage 3b). Subjects were monitored for
a median of 425 days after the estimated infection date. The study was approved
by the respective institutional review boards and was conducted in accordance
with human experimentation guidelines of Massachusetts General Hospital. All
subjects provided written informed consent.
HIV RNA genotyping and HLA typing. HIV-1 RNA genotyping of gag, pol, and
nef was performed on serial plasma samples (median of 5 samples/patient). In
general, patients were monitored at baseline, 1 month, and every 2 or 3 months
thereafter, although in some cases more frequent sampling was performed.
HIV-1 gag (codons 1 to 500; HXB2 nucleotides [nt] 790 to 2289), a portion of pol
(protease codons 1 to 99 and reverse transcriptase [RT] codons 1 to 400; nt 2253
to 3749), and nef (codons 1 to 206; nt 8797 to 9414) were amplified by nested
reverse transcription-PCR from extracted plasma HIV RNA using gene-specific
primers. Population (bulk) sequencing was performed on an Applied Biosystems
3730 automated DNA sequencer. Data were analyzed using Sequencher (Gene-
codes). Nucleotide mixtures were assigned if the secondary peak height exceeded
25% of the dominant peak height. Data were aligned to the HIV-1 subtype B
reference HXB2 sequence (GenBank accession no. K03455) using a modified
NAP algorithm (42). All subjects harbored subtype B infections, as confirmed by
comparing gag/pol/nef sequences to all HIV subtype reference sequences (http:
// HLA class I typing was per-
formed by standard sequence-specific PCR or sequence-based typing.
Predefined list of HLA-associated polymorphisms. Previous studies of immune-
driven HIV evolution have lacked a consistent definition of HLA-attributable mu-
tation. We address this by defining HLA-associated escape and reversion based on
a predefined, comprehensive list of HLA-associated polymorphisms identified in a
cross-sectional analysis of 1,200 chronically infected, antiretroviral-naı¨ve individu-
als from three published cohort studies in Canada (17, 18), the United States (45),
and western Australia (11) using phylogenetically corrected methods and a q-value
correction for multiple tests (q 0.2, which translates to a 20% false discovery rate)
(60, 75). As described previously (17; D. Heckerman, C. J. Brumme, M. John,
J. Carlson, R. Haubrich, S. Riddler, L. Swenson, I. Tao, S. Szeto, D. Chan, C. Kadie,
N. Frahm, C. Brander, B. D. Walker, Z. L. Brumme, P. R. Harrigan, and S. Mallal,
presented at the 15th International Workshop on HIV Dynamics and Evolution,
Santa Fe, NM, 27 to 30 April 2008), HLA-associated polymorphisms are dichoto-
mized based on the presence or absence of selection pressure: escape amino acids
represent the residue most likely to emerge under HLA-restricted CTL selection
pressure, while reversion amino acids represent the immunologically susceptible
residues most likely to reemerge following transmission to an HLA-unmatched
individual. In general, the reversion amino acids at any particular HLA-associated
position tend to be consensus, while the escaped forms tend to be nonconsensus, but
this is not always the case. The list contains over 1,000 unique HLA-associated
polymorphisms occurring at 20% of codons in Gag/Pol and 50% of codons in
Nef (Heckerman et al., 15th International Workshop on HIV Dynamics and Evo-
lution). Of these, 30% map within optimally defined CTL epitopes (http://www (Fig. 1).
Of the 180 optimally defined epitopes in Gag, Pol, and Nef, specific sites and
pathways of HLA-driven substitutions were defined for 74 epitopes. Of these 74,
3 were restricted by HLA alleles not observed in the seroconverter cohort and
were excluded from analysis; the remaining 71 epitopes, along with their escape
sites, are listed in Fig. 1.
Data analysis and definitions. (i) Rates of escape and frequencies of epitope
recognition in early infection. The time to escape for each CTL epitope was
defined as the number of days that elapsed between the estimated infection date
and the first detection of a full or partial amino acid change consistent (for HLA,
codon, and the direction of amino acid selection) with the predefined list of
HLA-associated polymorphisms (Fig. 1). Note that this definition is independent
of whether an amino acid is consensus or nonconsensus: in the presence of the
restricting HLA, any observed full or partial amino acid change away from the
reversion form and/or toward the escaped form was considered escape. Note that
some epitopes (e.g., B*57 TW10) have more than one escapable site; in these
cases, we computed the time to the first escape event at any of these sites. In
addition, the baseline sequence was analyzed for the presence of HLA-associated
polymorphisms to identify cases where escape may have occurred very early (or,
indistinguishably, rare cases where an escaped variant may have been acquired at
transmission). When such a polymorphism was present at baseline in a person
with the restricting allele, it was counted as an escape mutation.
Corresponding epitope recognition frequencies were derived from an inde-
pendent, partially published, cross-sectional data set of 289 individuals enrolled
through the AIEDRP network who were screened for HIV-1-specific CTL re-
sponses against optimally defined epitopes using a gamma interferon (IFN-)
enzyme-linked immunospot (ELISpot) assay at a single time point 8 weeks
following the initial presentation with primary HIV infection, as described pre-
viously (7, 76); this effectively means that individuals were tested anywhere
between 3 and 8 months after the estimated infection date. In addition, a
subset of seroconverters for whom peripheral blood mononuclear cell samples
were available during study follow-up (n 8) were longitudinally investigated for
HLA-restricted CTL responses to optimally described epitopes using IFN-
ELISpot assays.
(ii) Estimating rates and frequencies of reversion, and the total proportion of
amino acid substitutions attributable to HLA-associated selection pressures.
Rates and frequencies of reversion were conservatively estimated using methods
similar to those used for escape, with one important difference. Since the vast
majority of reversions are toward the consensus, it was not possible to infer
reversion events occurring prior to the baseline. In this analysis, therefore, time
zero was defined as the estimated infection date, and the baseline sequence was
treated as the transmitted sequence. Reversion was defined as the presence of a
specific known HLA-associated polymorphism at the baseline time point in an
individual not bearing this HLA allele, followed by full or partial reversion
toward the predefined immunologically susceptible (usually the consensus)
amino acid during follow-up.
Similarly, the proportion of overall amino acid substitutions attributable to
HLA-associated selection pressures (escape and/or reversion) on a gene-wide
basis was conservatively calculated as the fraction of the total observed substi-
tutions that achieved an exact match to an HLA-associated polymorphism in the
predefined list.
Rates of recognition and escape in CTL epitopes. CTL es-
cape mutations were defined according to a comprehensive list
of known HLA-associated polymorphisms derived from com-
bined cohorts numbering 1,200 (Heckerman et al., 15th In-
ternational Workshop on HIV Dynamics and Evolution). Us-
ing this list, specific sites and pathways of HLA-driven
substitutions were defined for 74 of the 180 published opti-
mally defined epitopes in Gag, Pol, and Nef (Heckerman et al.,
15th International Workshop on HIV Dynamics and Evolu-
tion). Of these, 71 were restricted by HLA alleles observed in
the presently described seroconverter cohort (Fig. 1) and, thus,
were evaluated for rates of escape in the first year of infection.
Epitope escape rates were characterized using Kaplan-
Meier methods, where the time to escape was defined as the
time elapsed between the estimated infection date and the first
observation of a full or partial substitution that was consistent
with the predefined list of HLA-associated polymorphisms.
Evidence for escape was observed in 57 of 71 (80%) of these
epitopes at the 1-year time point (Fig. 2). Escaping epitopes
were relatively equally distributed across Gag, Pol, and Nef.
Among evolving epitopes, escape was generally rapid: 42
(74%) of evolving epitopes escaped within the first 6 months of
infection (an estimate that included cases where the specific
HLA-associated polymorphism was already present at baseline
in the context of the restricting allele).
The most rapidly escaping epitope was HLA-B*57 TW10 in
p24 Gag (Fig. 2) (58). All evaluable B*57-expressing individ-
uals in the cohort either already harbored the T242N and/or
G248A escape mutations (18, 58) at baseline (n 6) or se-
lected one or both within the first 6 months (n 1). Notably,
T242 is a highly conserved residue in clade B, with over 90% of
sequences in our clinically derived data set (n 1,200) exhib-
iting the consensus T, arguing against the likelihood of this
mutation having been transmitted. The single B*57-expressing
individual in whom escape was not documented was lost to
follow-up less than 2 months after infection. Thus, the crude
first-year epitope escape rate for TW10 was calculated as 38%/
person/month. Overall, first-year escape rates for all B*57-
restricted epitopes were relatively high and could be ranked in
the following order from most rapidly to slowest escaping:
TW10-Gag (escape rate, 38%/month) IW9-RT (18.5%/
month) YY9-Nef (9.2%/month) HW9-Nef (7.2%/
month) IW9-Gag (6.7%/month) KF11-Gag (1.6%/month)
(Fig. 2 and 3). Note that QW9 (Gag) was not assigned an
escape rate, as no B*57-associated polymorphisms within QW9
were predefined (Fig. 1). With the exception of KF11, all
B*57-restricted epitopes examined ranked within the top half
of the distribution of evolving CTL epitopes, consistently with
the observation that the B*57-restricted CTL response fea-
tures the broad, robust targeting of multiple epitopes in early
infection (7, 76).
Five of the 10 most rapidly evolving epitopes were restricted
by protective HLA-B alleles, including B*57, B*58(01), B*13,
and B*51 (21, 41) (P 0.01 by Fisher’s exact test). TW10
(Gag) also represented the most rapidly escaping epitope in
B*58-expressing individuals (and the third most rapidly escap-
ing epitope overall), while B*13-RI10 (Pol), B*57-IW9 (RT),
FIG. 1. Locations of HLA-associated substitutions within optimally
defined CTL epitopes. Published sequences of the 71 optimally defined
CTL epitopes harboring at least one HLA-associated polymorphic site,
based on analysis of over 1,200 persons with chronic infection, are
listed. HLA-associated polymorphic residues are indicated in red.
Overlapping and/or variant epitopes restricted by the same HLA allele
were removed to avoid double counting of escape mutations. A com-
plete list of all optimally defined epitopes is available at http://www
and B*51-TI8 (RT) represented the fifth, seventh, and ninth
most rapidly escaping epitopes, respectively (Fig. 2).
In order to characterize the relationship between the fre-
quency of CTL responses and the corresponding frequencies
of escape, we analyzed a data set of 289 individuals in primary
infection screened for CTL responses against optimally de-
fined epitopes using an IFN- ELISpot assay (7, 76). As ex-
pected, the rates of escape generally mirrored epitope-target-
ing frequencies (Fig. 3). For example, with the exception of
KF11, the escape order for B*57-restricted epitopes generally
reflected the established immunodominance hierarchies in pri-
mary infection (7), with TW10 representing the most fre-
quently targeted (70%) and most rapidly escaping B*57-re-
stricted epitope. HLA-A*11 and HLA-B*08 alleles also
illustrate this relationship (Fig. 3). However, for some alleles,
notably B*07, we observed a relatively poor relationship be-
tween the frequency of recognition and escape (Fig. 3).
Overall, we observed a robust positive correlation between
frequencies of epitope recognition and escape in Gag (Spear-
man’s r 0.5; P 0.01) and Pol (r 0.8; P 0.002) epitopes,
highlighting the ability of HIV to rapidly adapt to CTL-in-
duced selection pressures, even in more conserved regions of
the viral proteome (Fig. 4a, b). Of interest, B*27-KK10, the
most frequently (90%) targeted epitope in early infection, es-
caped at a slower rate relative to other frequently targeted
epitopes, consistently with previous reports (35, 39, 49). B*27-
KK10 ranked as the 20th most rapidly evolving epitope overall,
with 5 of 11 B*27-positive individuals selecting the L268M
mutation during follow-up and an additional two exhibiting
both R264K and L268M at the earliest time point (indicating
either the transmission of these mutations or very early escape
within this epitope [10]).
Notably, we identified a small number of epitopes that ex-
hibited little or no HLA-driven sequence evolution despite
relatively high (40%) frequencies of recognition during acute
infection. These included the well-characterized B*57-KF11
epitope (23, 63), A*25-EW10 (Gag) (51), and others (Fig. 4).
Of interest, the frequency of escape in A*02-SL9 (Gag) was
zero, despite it representing one of the most immunodominant
A*02-restricted responses in acute infection (with an 18% re-
sponse frequency) (7) and large numbers of A*02-expressing
persons to evaluate. In contrast to previous reports identifying
escape mutations at codons 3,6, and/or 8 of this epitope (29, 37,
43), in our predefined list of HLA-associated polymorphisms,
mutations at these codons were attributed to other HLA-re-
stricted epitopes that overlapped SL9 (A*29 and Cw*14) (Fig.
1). The only A*02-associated polymorphism preidentified in
SL9 occurred at position 7 (Gag codon 83) (Fig. 1). No sub-
stitutions at this position were observed in any A*02-express-
ing seroconverter during study follow-up, resulting in an es-
cape rate of zero.
We observed a number of cases where epitope escape fre-
quencies exceeded recognition frequencies in Gag and Pol (for
example, A*24-KW9 [Gag] and A*03-QR9 [RT], the second
and sixth most rapidly evolving epitopes overall, as well as a
number of B*07-restricted epitopes; Fig. 4). This type of dis-
cordance, however, was observed most frequently in Nef and
contributed to the poor correlation between recognition and
escape frequencies in this protein (r 0.09; P 0.7) (Fig. 4c).
FIG. 2. First-year rates of escape among HLA-restricted, optimally defined CTL epitopes. The rates of escape in optimally defined CTL
epitopes in Gag (blue diamonds), Pol (red squares), and Nef (green triangles) in the first year of infection are shown. Vertical bars indicate 95%
confidence intervals. Epitopes were restricted to those containing a predefined HLA-associated polymorphism (Fig. 1). Note that this analysis
incorporates an estimate of cases where the epitope may have escaped prior to baseline sampling, performed by analyzing each individual’s baseline
HIV sequence for known HLA-restricted escape mutations. Note that in doing so, we cannot discriminate cases of the very early selection of escape
mutations from rare cases where an escaped variant may have been acquired at transmission.
Rates of reversion in CTL epitopes. Reversion was conser-
vatively defined as the presence of a known HLA-associated
polymorphism at the baseline time point in an individual not
bearing this HLA allele, followed by full or partial reversion
toward the immunologically susceptible (usually the consen-
sus) amino acid during follow-up. Within the first year of
infection, reversions were observed at 29 (6%), 14 (3%), and
26 (13%) Gag, Pol and Nef codons, respectively, while an
additional 9, 7, and 10 sites reverted after the first year. The
majority of reverting codons (65%) were within published
HLA-restricted CTL epitopes. On average, reversions within
Gag epitopes restricted by protective HLA alleles (B*13, B*51,
B*57, and B*58) occurred more rapidly than reversions out-
side these regions (P 0.02); however, no such differences
were observed in Pol or Nef. The rates of reversion at known
escape sites within optimally described epitopes in the first
year of infection are summarized in Fig. 5. The most rapidly
reverting mutations within Gag CTL epitopes were B*57-as-
sociated escape mutations at codons 147 and 242 in the IW9
and TW10 epitopes, respectively, while the most rapidly re-
verting epitope-associated mutation within Nef was at codon
135 (residue 2 of A*24-RW8). Although it is important to
emphasize that the rates of escape and reversion are not di-
rectly comparable due to the differences in calculations (see
Materials and Methods and Discussion), it is nevertheless in-
teresting that these three mutations were relatively rapidly
escaping as well, implying a substantial fitness cost. However,
the factors that shape HIV evolution are complex and, thus,
one would not expect rates of escape to correlate with rates of
reversion in all cases. RT codon 135 (residue 8 of B*51-TI8),
for example, was not observed to revert despite the relatively
frequent transmission of the escaped variant (n 21), implying
a negligible fitness cost to this mutation.
Proportion of HIV evolution attributable to HLA-mediated
selection pressures. The proportion of overall evolution in Gag,
Pol, and Nef, within and outside published epitopes, was calcu-
lated by computing the proportion of the total observed amino
acid changes between the baseline sequence and the final se-
quence matching an HLA-associated polymorphism on the pre-
defined list. Means of six, four, and seven nonsynonymous sub-
stitutions per person were observed in Gag, protease/RT, and
Nef, respectively, corresponding to overall nonsynonymous sub-
stitution rates of 0.01, 0.008, and 0.03 substitutions/subject/codon.
A total of 36, 32, and 58% of observed substitutions in Gag, Pol,
and Nef were attributable to HLA-associated selection pressures
(P 0.0001); overall, reversions accounted for 70% of these
The resolution of acute-phase viremia is influenced by the
strength and repertoire of the cellular immune response and
the speed and efficiency by which the virus is able to adapt to
these responses. Previous studies have demonstrated substan-
tial viral evolution in acute infection (2, 9, 59); however, in
general, most longitudinal studies of HLA-mediated viral evo-
lution have been limited to small numbers of patients, have
been largely biased toward protective HLA alleles associated
with long-term viremic control, and have lacked a standardized
classification scheme for what constitutes an HLA-attributable
The current study overcomes many of the limitations of
previous investigations in this area, as it represents the first
large-scale study of HLA-driven HIV evolution in acute clade
B infection that employs a rational list of HLA-associated
polymorphisms (defined through the analysis of a large [n
1,200] independent data set) to systematically characterize the
rates of immune-driven evolution on a protein-wide basis in
the context of all HLA restrictions. The study focused on Gag,
Pol, and Nef, because these proteins have been featured in
candidate CTL vaccine design strategies, including the recent
failed STEP vaccine trial (74); thus, developing a more in-
depth understanding of CTL-driven evolution in these proteins
is of key importance. We estimate that a minimum of 30 to
35% of nonsynonymous substitutions in Gag/Pol and a mini-
mum of 60% of substitutions in Nef are attributable to HLA-
restricted immune pressures in the first year of infection (of
which the majority represent reversions), confirming dramati-
cally different levels of HLA-mediated adaptation in HIV pro-
teins (17).
Using Kaplan-Meier analysis, we systematically computed
the rates of escape in published CTL epitopes, revealing mark-
edly different kinetics of escape among them. Although we
were not able to evaluate CTL responses in this cohort due to
a lack of cryopreserved cells, we were able to integrate the
sequence data derived here with epitope recognition frequen-
cies characterized in a cohort of 289 persons with primary HIV
infection (7, 76). A robust correlation between the epitope
response frequencies and the rates of escape in Gag and Pol
was observed, illustrating that in general, if the immune re-
sponse mounts pressure against an epitope, in most cases
HLA-driven mutations will appear shortly thereafter. There
were relatively few examples of frequently targeted epitopes
that maintained their sequences for extended periods of time
without evidence of escape.
Of interest, protective HLA alleles (21, 41, 47) were over-
represented among rapidly escaping epitopes, consistent with
the observation that these alleles impose stronger in vivo im-
mune selection pressures than others (33, 72). In particular,
B*57, the strongest host genetic factor associated with protec-
tion against HIV progression (21), restricted the most rapidly
escaping epitope overall (TW10), and all but one of the re-
maining B*57-restricted epitopes displayed rapid escape as
FIG. 3. Rates of CTL escape generally reflect known immunodominance hierarchies of CTL epitope recognition in primary infection. The
recognition frequencies of specific HLA-restricted epitopes (assessed by IFN- ELISpot assays measuring CTL responses to optimally described
HIV peptides in a cohort of 289 individuals in primary HIV infection) (7, 76) are indicated in the left panels. Kaplan-Meier curves representing
the corresponding rates of escape are indicated in the right panels in matching colors. Note that there are two cases where Kaplan-Meier curves
for a pair of epitopes are superimposed so that only one curve is visible; these are B*08 EV9 and DL9 (which do not escape) and B*07 FL9 and
TM9 (which overlap and share a single escaping site; Fig. 1).
well. The somewhat paradoxical observation that certain al-
leles remain protective despite rapid and frequent escape may
be explained by the fact that a key correlate of protection is the
ability to mount a broad and robust CTL response very early in
infection (4, 64), which indirectly manifests itself as CTL-
driven sequence changes at these key sites. On average, B*57-
expressing individuals respond to a mean of 4.7 B*57-restricted
epitopes (genome-wide) in early infection, the highest number
for all HLA restrictions, compared to a mean of two or three
epitopes for the vast majority of other alleles (7, 76). From the
perspective of HLA imprinting on HIV sequences at the pop-
ulation level, HLA-B*57 represents one of the alleles with the
largest total number of identified HLA-associated polymor-
phic sites in Gag/Pol/Nef, with associations identified at 29
unique residues in these proteins (compared to 17 and 18 for
B*07 and B*08, respectively) (Heckerman et al., 15th Interna-
tional Workshop on HIV Dynamics and Evolution). Further-
more, the earliest CTL responses likely are the most potent
and possess the greatest antiviral potential; in no other stage of
disease does one observe such a dramatic decline in viremia
from the acute peak to set-point levels (19).
A number of additional reasons may explain why certain
alleles remain protective despite the rapid selection of HLA-
driven substitutions. First, not all of the identified HLA-asso-
ciated polymorphisms confer an absolute escape phenotype:
often, the variant will maintain at least some capacity to bind
HLA and/or be cross-recognized by CTL (77), and there is
evidence that B*57-restricted CTL have a superior ability to
cross-recognize peptide variants compared to that of less pro-
tective alleles (77). Furthermore, the ability to mount a de
novo CTL response against a selected variant (3, 30, 78) may
be greater in earlier disease while immune function is relatively
intact. Finally, recent data suggest that the breadth of CTL
responses to Gag contributes strongly to HIV control (1, 28,
50, 79), and that the fitness costs of escape mutations in Gag
are substantial (16, 23, 62, 73), suggesting that long-term pro-
tective effects are due to strong immune selective pressures
driving viral evolution toward less-fit forms (6). Indeed, the
observed rapid and frequent reversion of escape mutations
within B*57-TW10 and IW9 in Gag supports this hypothesis
(Fig. 4). Taken together, these results support the importance
of a strong early CTL response in the control of HIV viremia,
even if a consequence of this is the rapid selection of muta-
The observation of discordant cases where in vivo escape
frequencies exceeded in vitro recognition frequencies merits
discussion. The fact that epitope recognition (7, 76) and escape
were evaluated in independent cohorts accounts for a portion
of these discordances (escape frequencies in the seroconverter
cohort represent cumulative longitudinal frequencies calcu-
lated at 1 year postinfection, whereas recognition frequencies
represent measurements taken at a single time point between
3 and 8 months following the estimated date of infection). The
remainder may be attributed to an underestimation of CTL
FIG. 4. Correlation between frequencies of recognition and escape
by protein. Spearman’s rank correlation was used to characterize the
relationship between the frequencies of recognition of CTL epitopes in
Gag, Pol, and Nef with their corresponding frequencies of escape in
the first year of infection. A regression line was drawn to highlight
trends. All tested CTL epitopes are represented; those recognized
and/or evolving at frequencies of ⬎⬃40% are labeled with the epitope
name and HLA restriction.
responses and/or an overestimation of escape frequencies, as
follows. First, as CTL responses to wild-type peptides decline
following escape in the autologous viral sequence (2), the rec-
ognition frequencies of rapidly escaping epitopes could have
been underestimated if subjects were screened after escape
had occurred. Indeed, for the eight seroconverters for whom
longitudinal peripheral blood mononuclear cells were avail-
able, we observed a decline in responses to B*51 TI8-RT
following escape in both B*51-expressing individuals in this
subgroup (not shown). Similarly, CTL responses may be un-
derestimated in cases where the autologous viral sequence
differs from the tested epitope sequence (5, 62), a fact that
would more substantially affect variable proteins such as Nef.
Another contributing factor may be the overestimation of
escape. This is particularly an issue in cases where the identi-
fied escape variant for a given epitope represents a commonly
occurring (or in some cases, the consensus) residue. Both A*24
KW9-Gag and A*03 QR9-RT are examples of such cases: for
both epitopes, the immunologically susceptible form (position
1 of KW9 and position 9 of QR9, corresponding to Gag and
RT codons 28 and 277, respectively) represents a nonconsen-
sus amino acid, meaning that the subtype B consensus residue
is considered the escaped form. The fact that our analysis (Fig.
2 to 4) included subjects exhibiting an escaped residue at base-
line could have resulted in an overestimation of escape for
these epitopes if these mutations were present at transmission.
Finally, it is worth noting that in both cases (as well as for all
B*07-restricted epitopes examined here), the published
epitope sequence features at least one residue in its HLA-
associated escaped form. Work is ongoing to assess whether
these cases represent examples where the population HIV
consensus has adapted to frequently observed HLA alleles
(57); in any event, this observation merits consideration, as it
could result in the systematic underestimation of CTL re-
sponse frequencies to such epitopes.
There are a number of additional limitations that are im-
portant to discuss. Most importantly, in the absence of know-
ing the transmitted sequence, we cannot definitively identify
sequence changes occurring prior to baseline sampling. Al-
though the rates of escape analysis take into consideration
likely escape events occurring prior to baseline sampling, it is
not possible to infer reversion events (or to calculate the total
amount of evolution) occurring prior to baseline sampling.
Thus, not only does this limitation render the reversion rates to
be substantial underestimates of the true rates, it also renders
them not directly comparable to the escape rates. Neverthe-
less, the analysis still yields informative data regarding the
locations, frequencies, and estimated rates of reversion, which
illuminate potential positions at which mutations likely occur
at substantial costs to fitness. For example, it was interesting
that two of the most rapidly reverting codons were Gag 147
and 242 within B*57-TW10 and IW9 epitopes, respectively (39,
58), consistent with measurable costs to viral fitness associated
with escape in B*57-restricted Gag epitopes (16, 62). Con-
versely, the rapidly selected B*51-associated escape mutation
at RT codon 135 (residue 8 of B*51-TI8) is not observed to
FIG. 5. Locations and first-year rates of commonly observed reversions within published HLA-restricted epitopes in Gag, Pol, and Nef.
Conservative estimates of the rates of reversion of known HLA-associated escape mutations in the absence of the restricting HLA (expressed as
percent reversions/person-month) at Gag, Pol, and Nef codons within published HLA-restricted CTL epitopes are shown, along with the total
number of observations (transmitted mutations) at each codon. A minimum of five observed cases of the transmission of the escaped variant was
required for display. Note that in contrast to the escape analysis, this analysis does not take into consideration reversions that may have occurred
prior to the baseline sampling. Thus, estimated reversion rates represent considerable underestimates of the true rate and are not directly
comparable to epitope escape rates. PR, protease.
revert despite relatively frequent transmission, suggesting a
relatively minor fitness cost.
If the rates of reversion and/or escape differ substantially
before and after baseline sampling, this difference could con-
tribute to errors in our estimates. Note, however, that no sig-
nificant difference in the proportion of HLA-attributable evo-
lution was observed among individuals captured within 3
(n 61) or 3 to 6 months (n 37) after infection. The
inconsistent detection of minority variants below a threshold of
10 to 20% of the circulating species is a known limitation of
the bulk PCR and sequencing techniques employed (55, 56);
however, comprehensive clonal sequencing was not feasible in
a cohort of this size and length of follow-up.
In addition, there are some limitations associated with de-
fining HLA-associated substitutions through the analysis of an
independent large clade B data set. As with any method, there
will be both false-positive as well as false-negative results
within this list (20) (J. Carlson, submitted for publication);
thus, this list will not necessarily contain all escape mutations
previously reported in the literature. Potential differences in
the ethnic composition in the two cohorts also should be ac-
knowledged. Information on ethnicity was unavailable; how-
ever, the HLA composition of both cohorts reflected expected
allele frequencies among North American populations (not
shown). Finally, although many mutational pathways are
broadly predictable at the population level (17, 18, 65, 70),
unique HLA-driven escape, reversion, or secondary/compen-
satory changes also will occur in individual patients; thus, em-
ploying a population-level definition of HLA-associated sub-
stitution at the individual level may underestimate the amount
of evolution attributable to HLA. Nevertheless, the use of a
predefined list of HLA-associated substitutions allows the
comprehensive, systematic classification of amino acid substi-
tutions both within and outside CTL epitopes, as well as the
ability to investigate escape and reversion across entire pro-
teins and across all HLA restrictions. Indeed, a similar classi-
fication technique was employed in a recent study of transmit-
ted CTL escape mutations in HIV clade C (36). The current
study investigated HLA-associated polymorphisms in Gag, Pol,
and Nef only; however, as comprehensive lists of HLA-associ-
ated polymorphisms become available for additional genes, the
analysis of escape and reversion rates in other HIV proteins
will become possible. Finally, it is important to acknowledge
the incompleteness as well as potential bias toward common
and/or protective HLA alleles in the current published CTL
epitope lists. As new epitopes are discovered, however, the
rates of escape and reversion can easily be calculated using the
current data set.
The relatively short follow-up period limits our ability to
assess the long-term impact of early escape on HIV disease
outcomes. In addition, we were unable to confirm the results of
recent studies that demonstrate reduced viral loads in individ-
uals transmitting HLA-associated escape mutations (22, 36);
however, the lack of statistical power to detect these effects in
the current study must be noted.
Given these limitations, our estimates that 30 to 35% and
60% of overall substitutions in Gag/Pol and Nef, respectively,
are attributable to CTL escape and reversion confirm a substan-
tial role of HLA-associated immune pressures in driving early
within-host HIV evolution (2, 9, 39, 59). Selective forces respon-
sible for the non-HLA-attributable fraction may include CD4
T-cell responses (48, 71), adaptations to other host factors (12),
reversions of such mutations selected in previous hosts, or ran-
dom drift. A small number of substitutions in protease/RT (and
possibly Gag (26) could be due to the reversion of transmitted
resistance mutations. Indeed, a small number of resistance-asso-
ciated polymorphisms and surveillance mutations (54) were ob-
served in this cohort; note, however, that there was no overlap
between major resistance-associated and HLA-associated poly-
morphic sites in protease/RT.
Substantial variability in the degree to which different viral
proteins adapt to HLA-associated immune pressures during
early HIV infection underscores the importance of selecting
appropriate immunogens and determining how to best incor-
porate sequence diversity in vaccine design (5, 15, 32, 66, 69).
In particular, strongly targeted yet slowly escaping epitopes
(such as B*57-KF11 and A*25-EW10) may be of particular
interest, as may be strongly targeted epitopes that can escape
only at substantial fitness costs to the virus (such as B*57-
TW10) (16). Specifically, immunogenic viral regions exhibiting
high mutational barriers to escape may be good vaccine can-
didates, as they may mediate the effective long-term control of
HIV CTL (39). On the other hand, epitopes that escape at
considerable costs to viral replicative capacity also may be
relevant to vaccine design: a vaccine capable of inducing im-
mune responses that drive HIV toward crippled forms also
may be effective in reducing viral replication to levels that slow
disease progression (6). Indeed, the observation that HLA-
B*57-restricted TW10 and IW9 are among the fastest escaping
and the fastest reverting epitopes in Gag strongly supports the
fitness costs of CTL escape as a key mediator of long-term
viremia control in B*57-expressing individuals.
Taken together, our results confirm a substantial role of
HLA-associated selection pressures on early within-host HIV
evolution and support further research into CTL-based vaccine
strategies incorporating information on common escape path-
ways, despite recent setbacks in the HIV vaccine field (8, 74).
We thank the patients for their participation.
We thank Toshiyuki Miura, Mark Brockman, Nicole Frahm, and
Christian Brander for helpful discussions and Philip Goulder for the
critical reading of the manuscript.
Z.L.B. is supported by a postdoctoral fellowship from the Canadian
Institutes for Health Research (CIHR). H.S. is supported by a fellow-
ship from the Deutscher Akademischer Austauschdienst (DAAD).
D.H., J.C., and C.K. are funded by Microsoft Research. This study was
supported in part by the Howard Hughes Medical Institute, the Mark
and Lisa Schwartz Foundation, the Harvard University Center for
AIDS Research (CFAR), National Institutes of Health grant
R01AI50429, and Acute Infection Early Disease Research Network
(AIEDRP) grant U01AI052403. This project has been funded in part
with federal funds from the National Cancer Institute, National Insti-
tutes of Health, under contract N01-CO-12400. This research was
supported in part by the Intramural Research Program of the NIH,
National Cancer Institute, Center for Cancer Research.
Sources of support were not involved in the design and conduct of
the study, nor were they involved in the collection, analysis, and inter-
pretation of the data or in the preparation, review, or approval of the
The content of this publication does not necessarily reflect the views
or policies of the Department of Health and Human Services, nor does
the mention of trade names, commercial products, or organizations
imply endorsement by the U.S. government.
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    • "Transmitted/early viruses tend to exhibit particular Env characteristics, including shorter variable loops with fewer glycosylation sites (Derdeyn et al., 2004; Sagar, 2010) and near-exclusive CCR5 co-receptor usage (Sagar, 2010); however, there is little knowledge regarding whether Nef characteristics of early viruses may differ from those from later infection stages (Noviello et al., 2007). Similarly, envelope length and number of glycosylation sites may increase during the infection course (Sagar et al., 2006) (though this remains somewhat controversial (Novitsky et al., 2009a)); however, few studies have examined early sequence and/or functional changes in Nef in large numbers of patients, particularly in a subtype C context (Brumme et al., 2008; Kuang et al., 2014; Noviello et al., 2007). "
    [Show abstract] [Hide abstract] ABSTRACT: Nef plays a major role in HIV-1 pathogenicity. We studied HIV-1 subtype C infected individuals in acute/early (n=120) or chronic (n=207) infection to investigate the relationship between Nef-mediated CD4/HLA-I down-regulation activities and disease progression, and the influence of immune-driven sequence variation on these Nef functions. A single Nef sequence per individual was cloned into an expression plasmid, followed by transfection of a T cell line and measurement of CD4 and HLA-I expression. In early infection, a trend of higher CD4 down-regulation ability correlating with higher viral load set point was observed (r=0.19, p=0.05), and higher HLA-I down-regulation activity was significantly associated with faster rate of CD4 decline (p=0.02). HLA-I down-regulation function correlated inversely with the number HLA-associated polymorphisms previously associated with reversion in the absence of the selecting HLA allele (r=−0.21, p=0.0002). These data support consideration of certain Nef regions in HIV-1 vaccine strategies designed to attenuate the infection course.
    Full-text · Article · Nov 2014
    • "The early cohort was comprised of HIV-1 subtype B infected patients recruited through various observational seroconverter studies including the Acute Infection and Early Disease Research Program (AIEDRP) sites in Boston and New York (USA), Sydney (Australia), a private medical clinic in Berlin (Germany), and observational cohort studies in Montreal and Vancouver (Canada) [13,23,24]. Infection dates for the patients in the early cohort were estimated as described in [13,23]. "
    [Show abstract] [Hide abstract] ABSTRACT: Background The reproducible nature of HIV-1 escape from HLA-restricted CD8+ T-cell responses allows the identification of HLA-associated viral polymorphisms ¿at the population level¿ ¿ that is, via analysis of cross-sectional, linked HLA/HIV-1 genotypes by statistical association. However, elucidating their timing of selection traditionally requires detailed longitudinal studies, which are challenging to undertake on a large scale. We investigate whether the extent and relative timecourse of immune-driven HIV adaptation can be inferred via comparative cross-sectional analysis of independent early and chronic infection cohorts.ResultsSimilarly-powered datasets of linked HLA/HIV-1 genotypes from individuals with early (median¿<¿3 months) and chronic untreated HIV-1 subtype B infection, matched for size (N¿>¿200/dataset), HLA class I and HIV-1 Gag/Pol/Nef diversity, were established. These datasets were first used to define a list of 162 known HLA-associated polymorphisms detectable at the population level in cohorts of the present size and host/viral genetic composition. Of these 162 known HLA-associated polymorphisms, 15% (occurring at 14 Gag, Pol and Nef codons) were already detectable via statistical association in the early infection dataset at p¿¿¿0.01 (q¿<¿0.2) ¿ identifying them as the most consistently rapidly escaping sites in HIV-1. Among these were known rapidly-escaping sites (e.g. B*57-Gag-T242N) and others not previously appreciated to be reproducibly rapidly selected (e.g. A*31:01-associated adaptations at Gag codons 397, 401 and 403). Escape prevalence in early infection correlated strongly with first-year escape rates (Pearson¿s R¿=¿0.68, p¿=¿0.0001), supporting cross-sectional parameters as reliable indicators of longitudinally-derived measures. Comparative analysis of early and chronic datasets revealed that, on average, the prevalence of HLA-associated polymorphisms more than doubles between these two infection stages in persons harboring the relevant HLA (p¿<¿0.0001, consistent with frequent and reproducible escape), but remains relatively stable in persons lacking the HLA (p¿=¿0.15, consistent with slow reversion). Published HLA-specific Hazard Ratios for progression to AIDS correlated positively with average escape prevalence in early infection (Pearson¿s R¿=¿0.53, p¿=¿0.028), consistent with high early within-host HIV-1 adaptation (via rapid escape and/or frequent polymorphism transmission) as a correlate of progression.Conclusion Cross-sectional host/viral genotype datasets represent an underutilized resource to identify reproducible early pathways of HIV-1 adaptation and identify correlates of protective immunity.
    Full-text · Article · Aug 2014
    • "In the context of antigen presentation and Ctl responses, multiple studies have identified Hla-a*03-restricted HIv-1 epitopes, especially a conserved epitope (KK9/rK9) in Gag (p17) (Balamurugan et al. 2010; Brumme et al. 2008; Dinges et al. 2010; Goulder et al. 1997; Heath et al. 2011; Peretz et al. 2011; ). Other Hla-a*03- restricted Ctl epitopes have been mapped to env/gp120 (tW9 and ve12) (McKinnon et al. 2007; Peretz et al. 2011;), nef (aK9, Gl9, QK10, rK9, and rK10) (almeida et al. 2011; Balamurugan et al. 2010; Brumme et al. 2008; Peretz et al. 2011;), Pol (aK11, atK9, DI11, Ka9, and sK11) (Balamurugan et al. 2010; Brumme et al. 2008; Chen et al. 2009; Peretz et al. 2011; turnbull et al. 2009), rev (tY9) (Yang et al. 2005), and a cryptic antigen (rr9) encoded by an alternative open reading frame (Berger et al. 2010). none of these existing immunologic data have been stratified by sex. "
    [Show abstract] [Hide abstract] ABSTRACT: Research in the past two decades has generated unequivocal evidence that host genetic variations substantially account for the heterogeneous outcomes following human immunodeficiency virus type 1 (HIV-1) infection. In particular, genes encoding human leukocyte antigens (HLA) have various alleles, haplotypes, or specific motifs that can dictate the set-point (a relatively steady state) of plasma viral load (VL), although rapid viral evolution driven by innate and acquired immune responses can obscure the long-term relationships between HLA genotypes and HIV-1-related outcomes. In our analyses of VL data from 521 recent HIV-1 seroconverters enrolled from eastern and southern Africa, HLA-A*03:01 was strongly and persistently associated with low VL in women (frequency = 11.3 %, P < 0.0001) but not in men (frequency = 7.7 %, P = 0.66). This novel sex by HLA interaction (P = 0.003, q = 0.090) did not extend to other frequent HLA class I alleles (n = 34), although HLA-C*18:01 also showed a weak association with low VL in women only (frequency = 9.3 %, P = 0.042, q > 0.50). In a reduced multivariable model, age, sex, geography (clinical sites), previously identified HLA factors (HLA-B*18, B*45, B*53, and B*57), and the interaction term for female sex and HLA-A*03:01 collectively explained 17.0 % of the overall variance in geometric mean VL over a 3-year follow-up period (P < 0.0001). Multiple sensitivity analyses of longitudinal and cross-sectional VL data yielded consistent results. These findings can serve as a proof of principle that the gap of "missing heritability" in quantitative genetics can be partially bridged by a systematic evaluation of sex-specific associations.
    Full-text · Article · Jun 2014
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