The Journal of Experimental Medicine
JEM © The Rockefeller University Press
Vol. 201, No. 6, March 21, 2005 891–902
Transmission and accumulation of CTL
escape variants drive negative associations
between HIV polymorphisms and HLA
Sylvie Le Gall,
Andrew K. Sewell,
and Philip Goulder
Peter Medawar Building, University of Oxford, Oxford OX13SY, UK
Partners AIDS Research Center, Massachusetts General Hospital, Charlestown, MA 02129
HPP, The Doris Duke Medical Research Institute, University of Natal, Durban 4015, South Africa
Department of Genitourinary Medicine, High Wycombe General Hospital, Buckinghamshire HP11 2TT, UK
The Harrison Clinic, Radcliffe Infirmary, Oxford OX2 6HE, UK
Department of Microbiology, University of Washington School of Medicine, Seattle, WA 98195
Centre for Clinical Immunology and Biomedical Statistics, Royal Perth Hospital, Perth WA 6000, Australia
Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM 87545
Human immunodeficiency virus (HIV)-1 amino acid sequence polymorphisms associated with
expression of specific human histocompatibility leukocyte antigen (HLA) class I alleles suggest
sites of cytotoxic T lymphocyte (CTL)-mediated selection pressure and immune escape.
The associations most frequently observed are between expression of an HLA class I molecule
and variation from the consensus sequence. However, a substantial number of sites have been
identified in which particular HLA class I allele expression is associated with preservation of
the consensus sequence. The mechanism behind this is so far unexplained. The current studies,
focusing on two examples of “negatively associated” or apparently preserved epitopes,
suggest an explanation for this phenomenon: negative associations can arise as a result of
positive selection of an escape mutation, which is stable on transmission and therefore
accumulates in the population to the point at which it defines the consensus sequence.
Such negative associations may only be in evidence transiently, because the statistical power
to detect them diminishes as the mutations accumulate. If an escape variant reaches fixation
in the population, the epitope will be lost as a potential target to the immune system. These
data help to explain how HIV is evolving at a population level. Understanding the direction of
HIV evolution has important implications for vaccine development.
It is well established that CTLs exert a strong
positive selection pressure on HIV, resulting in
the appearance of escape mutations that allow
the virus to evade these responses (1–7). CTLs
are HLA class I restricted, as they only recognize
their cognate peptide when it is presented in
the context of the appropriate HLA molecule.
A recent study by Moore et al. (3) exploited
this property to show, via the identification of
associations between particular HLA alleles
and sequence polymorphism within the RT
region, that escape mutation in HIV is common
and restricted by a wide array of different HLA
molecules. Unexpectedly, 25 “negative associ-
ations” were also identified, linking the pres-
ence of a particular allele with preservation of
the consensus sequence. This phenomenon
was termed “negative selection pressure,” to
describe selection pressure that favored the
conservation of WT virus in vivo (3). One
suggested mechanism by which negative asso-
ciations arise is through selection for the pres-
ervation of epitopes targeted by ineffective
CTLs, whose presence is favored by the virus
(8). An alternative hypothesis is that sites of
negative selection in HIV represent residues in
Abbreviations used: BCL,
B-lymphoblastoid cell line; ICS,
intracellular cytokine staining;
NEGATIVE HLA ASSOCIATIONS IN HIV | Leslie et al.
which common HLA types have already selected a series of
“optimized” mutations by passage through many infected
individuals of the same HLA type (8). In this scenario, the
consensus sequence would represent adaptation to high fre-
quency alleles, with the positively selected escape mutations
that are driven by those alleles embedded within it (3). Indeed,
this mechanism has been proposed to contribute to clade-
specific HIV-1 sequence differences (9).
To better understand the consequences of selection pres-
sure exerted on HIV by CTLs, we have focused on three al-
5801, and B
with effective suppression of viremia (10–15) and therefore
likely to impose strong selection pressure on the virus. HLA-
57 and B
5801 are closely related, targeting many of the
same epitopes (16) and selecting for the same escape muta-
tions (17). Through large-scale population sequencing of
both C-clade and B-clade HIV, we identified two examples
of negative associations: one associated with HLA-B
5801 and the other with HLA-B
cess by which these phenomena arose and discuss their im-
plications with regard to the evolutionary fate of CTL
epitopes in HIV-1 infection.
51, which are associated
51. We describe the pro-
HLA-B*57/5801 is associated with the conservation Nef
We initially analyzed proviral DNA sequences from 117
HIV-1 C-clade–infected study subjects recruited from Dur-
ban, South Africa, to seek sequence polymorphisms that
were linked with expression of either HLA-B
The phenotypic frequencies of HLA-B
the Durban population are 6.4 and 10.1%, respectively. To
increase the likelihood of identifying HLA-B
ciated sequence polymorphisms, a proportionately greater
number of subjects expressing HLA-B
gether, 46 out of 117, 39%) were included in the study
group. We initially focused on HIV-1 Nef, one of the most
immunogenic regions of the HIV-1 proteome (18, 19). The
sequence polymorphisms we identified in association with
expression of HLA-B
57/5801 included a single strong asso-
ciation between HLA-B
57/5801 and conservation of the
consensus sequence Gly, representing both the local consen-
sus, generated from all proviral DNA and RNA sequences,
and the Los Alamos HIV database C-clade consensus (http:
//www.hiv.lanl.gov) at residue 83 (Table I and Fig. 1 A).
From 12 out of 18 (67%) HLA-B57
of 28 (96%) HLA-B
5801 individuals, we isolated proviral
DNA sequences encoding the consensus Gly at residue 83 in
Nef, compared with 25 out of the 71 (35%) HLA-B
5801individuals sequenced (P
ity of viruses not encoding Gly at residue 83 instead ex-
As the function of HLA class I molecules is to present
peptides on the cell surface for recognition by CTLs, we
hypothesized that the association between HLA-B
57 or B
57 and of B
57 or B
individuals and 27 out
). The major-
5801 and Nef-83-Gly was linked to CTL activity. How-
ever, Nef-83 does not lie within any published HLA-
57/5801-restricted epitope. Therefore, we used the
BIMAS epitope prediction software (http://bimas.cit.nih.
gov/molbio/hla_bind) to identify 8–11-mer peptides in
this region capable of binding HLA-B
and this revealed a candidate epitope in the 9-mer
KGAFDLSFF (KF9, Nef residues 82–90). Residue 83 lies
at position 2 within this putative epitope, which is a pri-
mary anchor position for HLA-B
cording to elution data, the preferred residues at this posi-
tion for HLA-B
57/5801 are Ala, Thr, and Ser, rather than
Gly (16). Indeed, using the same software, KF9 with Ala at
position 2 (KAAFDLSFF) has a 10-fold higher binding
score than KGAFDLSFF, the putative epitope derived
from the consensus sequence. This suggested a mechanism
by which the observed “negative HLA association” might
have arisen: that residue 83 lies within the HLA-B
5801-restricted epitope, KAAFDLSFF, and positive selec-
tion pressure, exerted through recognition by KF9-specific
CTLs, induces escape mutation at this residue, from the
WT Ala to Gly.
57 and B
57/5801. However, ac-
C-clade consensus at residue 83 in Nef
HLA-B57/5801 is associated with conservation of the
JEM VOL. 201, March 21, 2005
Positive selection for the A
mediated by HLA-B*57/5801
We first sought to confirm our data by sequencing viral
RNA from an additional 61 individuals of the same cohort.
8 out of 9 HLA-B
57/5801individuals expressed a variant
from Nef-83-Ala compared with 30 out of 52 individuals
lacking these alleles (Fig. 1 B), confirming the association
57/5801 and sequence polymorphism at
residue 83. These sequences were from RNA, showing the
association holds true for both circulating virus and proviral
DNA. However, in this small cohort the differences were
not significant (P
0.074). The samples used were chosen at
random, with no bias toward individuals expressing HLA-
57/5801. This highlights the fact that even for alleles with
a high phenotypic frequency, unless large cohorts are evalu-
ated, it is difficult to detect associations between HLA and
G mutation at Nef-83
sequence polymorphism where there is a high background
of polymorphism within a population.
Next, we studied mother-to-child transmission (MTCT)
pairs to determine whether the hypothesized events of an es-
cape mutation at residue 83 in the face of HLA-B
in fact occur (Table II). Four MTCT pairs were identified in
which an HLA-B
57/5801mother transmitted HIV to her
57/5801child. In each case, the maternal virus en-
codes Ala at residue 83 (KF9/83A), which mutates to Gly
(KF9/83G) in the child. For the S30 MTCT pair, in whom
transmission occurred between 6 wk and 3 mo postpartum,
maternal virus was sequenced both before and after transmis-
sion, with all viral RNA clones expressing KF9/83A. How-
ever, in the child sequenced at 9 mo postpartum,
ter transmission, all 10 RNA clones expressed KF9/83G.
These data show that HLA-B
mutation at Nef-83 and furthermore that this change occurs
rapidly after transmission. Therefore, Nef-83 is in fact sub-
jected to positive rather than negative selection pressure, op-
erating through HLA-B
57/5801, leading in the majority of
cases to an Ala
8 mo af-
57/5801 selects for the A
KF9/83G persists in the absence of HLA-B*57/5801
To understand how Gly might have replaced Ala as the con-
sensus sequence at residue 83, we next sought to determine
B57/5801. (A) Association between the expression of HLA-B57/5801 and
conservation of the C-clade consensus AA (Gly). Sequencing of Durban
subjects done by population sequencing of proviral DNA. (B) Sequencing
of viral RNA from Durban cohort reveals the same association. (C) The
same association revealed in RNA sequence data from a separate, B-clade–
Selection pressure exerted on residue Nef 83 by HLA-
is stable on transmission
A83G mutation is selected for by HLA-B57/5801 and
KAAFDLSFF No. of clones
6 yrDNA pop. seq.
unknown DNA pop. seq.
4 yr DNApop. seq.
unknown DNApop. seq.
DNApop. seq.6 wk
pop. seq.1 yr
unknown DNApop. seq.
NEGATIVE HLA ASSOCIATIONS IN HIV | Leslie et al.
if KF9/83G is stable in the absence of HLA-B*57/5801. We
identified one previously published horizontal transmission
pair (17) in which the HLA-B*5801? subject 6007 transmit-
ted HIV to the HLA-B*57/5801? subject 6008. In both in-
dividuals, virus encodes the KF9/83G mutation (Table II).
In addition, we identified two MTCT pairs, 1043 and 1060,
in which neither mother nor child possesses HLA-B*57/
5801. In both cases, both mother and child display virus en-
coding KF9/83G, which is seen to be stable in the child for
at least 2 yr after transmission (Table II), despite the absence
of HLA-B*57/5801. In a final MTCT pair, 1043, both ex-
press HLA-B*5801 and both carry virus encoding KF9/83G.
These data, combined with the fact that we see a high fre-
quency of KF9/83G in individuals that lack HLA-B*57/
5801 (Fig. 1), show that KF9/83G, selected for by HLA-
B*57/5801, is transmitted and is then stable in the absence of
the selection pressure that drove it. Data from a cohort of
B-clade–infected subjects in Perth, Australia, demonstrate
the same effect (Fig. 1 C), with 52% of the cohort as a whole
expressing KF9/83G as the consensus compared with 86%
(12 out of 14) of HLA-B*57/5801? subjects that express
KF9/83G (P ? 0.009).
KF9 is an HLA-B*57/5801-restricted epitope
Next, we sought to confirm that KF9 is indeed an HLA-
B*57/5801-restricted epitope, and further, that KF9/83G
represents an escape mutation. In a subsequent screening of
HLA-B*57/5801? subjects with the KF9/83A 9-mer, 20 out
of 51 showed detectable responses by ex vivo ELISPOT
(median magnitude of 300 sfu/million PBMCs; not de-
and restriction of B-clade KF9 (KAAVDLSHF) using fresh PBMCs isolated
from the B-clade–infected 9092850 (B*5801/1517) by IFN-? ELISPOT.
(C–E) Recognition of KF9/83A and KF9/83G peptides by: (C) KF9-specific
CTL line from B-clade–infected 0001726HW (B*57/7) by ELISPOT, (D) fresh
PBMCs from 9092850 by ELISPOT, and (E) KF9-specific CTL line from the
KF9 is an HLA-B*57/5801 epitope. (A and B) Optimization
C-clade–infected SK059 (B*5801/-) by ICS. (F) Ala to Gly substitution at
position 2 increases off-rate of KF9. Recognition of HLA-B*5801? BCLs,
pulsed with either KF9/83A or KF9/83G for 1, 6, or 24 h, by KF9-specific
CTL line (SK059) by ICS. Background staining of unpulsed BCLs at each
time point was subtracted.
JEM VOL. 201, March 21, 2005
picted). In addition, we identified one B-clade–infected in-
dividual, 9092850-RI, whose PBMCs recognized the
B-clade version of KF9 (KAAVDLSHF). Furthermore, by
peptide pulsing fresh PBMCs from HLA-B*57/5801? indi-
viduals who do not make ex vivo responses to KF9 and cul-
turing in the presence of cytokines, we generated KF9-
specific CTLs from one B-clade–infected, HLA-B*57?
individual. Using fresh PBMCs from 9092850-RI, we con-
firmed that KF9 is the optimal epitope (Fig. 2 A) and that
the restriction element was indeed HLA-B*5801 (Fig. 2 B).
These data show that KF9 is an HLA-B*57/5801-restricted
epitope and that KF9/83G occurs at the primary anchor po-
sition 2 within it.
An Ala→Gly change at position 2 affects peptide off-rate
To determine whether KF9/83G is an escape mutation, we
titrated out KF9 peptides containing either Ala (KF9/83A)
or Gly (KF9/83G) at position 2, using either fresh PBMCs
or cultured cells from each subject, and tested recognition
via IFN-? production by ELISPOT assay or intracellular cy-
tokine staining (ICS; Fig. 2, C–E). In each case, the KF9/
83A peptide was slightly better recognized at low peptide
concentrations than the KF9/83G peptide, consistent with
the hypothesis that KF9/83G represents an escape mutation
in this epitope. However, in the three individuals tested in
this way, the KF9/83G variant appears to be recognized to
some degree. It remains unclear as to whether such differ-
ences represent escape mutation or cross-recognition of vari-
ant epitopes (20). Given that Gly is not a preferred residue at
position 2 in the B*57/5801 peptide-binding motif (16), we
hypothesize that this change would affect peptide binding af-
finity or off-rate, effects that might not be detected by
ELISPOT or ICS, as these are systems in which the present-
ing cells and peptide are normally coincubated for the dura-
tion of the assay. Therefore, we conducted further assays to
determine the effect of the Ala→Gly change at position 2 on
the binding of KF9 to HLA-B*57/5801. EBV-transformed
B cells (BCLs) expressing HLA-B*5801 were pulsed for 1 h
with either the KF9/83A or KF9/83G peptides. The cells
were then washed and tested for recognition by a B*5801-
restricted KF9-specific CTL line via ICS. In contrast to the
direct ICS/ELISPOT assays, although the BCLs pulsed with
KF9/83A were strongly targeted, recognition of those
pulsed with the KF9/83G variant was completely ablated
(Fig. 2 F). When the cells were incubated for an additional 5
or 23 h, rewashed, and tested, the BCLs pulsed with KF9/
83A were still recognized, although the magnitude of re-
sponse was seen to decrease in a time-dependent manner. As
expected, after further incubation, BCLs pulsed with the
KF9/83G variant peptide remained untargeted. These data
suggest that the Ala→Gly change at position 2 within KF9
affects peptide off-rate, with the KF9/83G variant form dis-
associating from HLA-B*5801 more readily than the KF9/
To confirm the impact of this Ala→Gly substitution at
residue 2 within HLA-B*57/5801-binding peptides, we
next focused on the Gag epitope KAFSPEVIPMF (KF11),
which dominates the HLA-B*57–restricted HIV-specific
CTL response during chronic infection (13, 14). We have
identified an identical Ala→Gly mutation selected at posi-
tion 2 in KF11. Sequencing of HLA-B*57? individuals from
the Durban cohort reveals positive selection for a mutation
at position 2 within KF11 (Gag residue 163), the majority of
which involve an Ala→Gly substitution (KF11/163G; Table
III and Fig. 3 A). 9 out of 37 HLA-B*57? individuals ex-
pressed KF11/163G compared with 8 out of 149 HLA-
B*57? subjects (P ? 0.0002). All four viruses isolated from
HLA-B*57/5801? individuals that expressed KF11/163G
also displayed at least one other known footprint of this allele
(A146P, I147L. H219Q, and G248X; reference 17) com-
pared with 30 out of 105 that express these footprints but
lack the KF11/163G mutation (P ? 0.008; references 17
and 21; not depicted). This indicates that the KF11/163G
mutation in these individuals owes its origin to HLA-B*57.
Hence, we hypothesize that like KF9/83G, KF11/163G is
transmitted and stable in the absence of HLA-B*57.
As observed for KF9, titration of the KF11/163A and
KF11/163G peptides in an IFN-? ELISPOT assay reveals
only minor differences in response to these peptides (Fig. 3 B).
However, we hypothesized that KF11/163G also represents
an escape mutation, as identical mutations observed in two
separate HLA-B*57–restricted epitopes are unlikely to be
selected unless they both provide a selective advantage.
Therefore, we conducted the same binding assay for KF11,
using HLA-B*57? BCLs pulsed with either KF11/163A or
Table III. Escape mutation in HLA-B57–restricted epitope KF11
NEGATIVE HLA ASSOCIATIONS IN HIV | Leslie et al.
KF11/163G peptides, with recognition by a KF11-specific
CTL line tested via ELISPOT. Unlike KF9, after 1 h, both
the KF11/163A- and KF11/163G-pulsed BCLs were recog-
nized, indicating that both peptides were still bound to
HLA-B*57 on the surface of the BCLs (Fig. 4 A). However,
when we incubated the cells for an additional 11 h, washed,
and retested them, recognition of the KF11/163G-pulsed
BCLs was almost completely ablated, with only a very small
response observed at the highest E/T ratio (Fig. 4 B). In
contrast, the KF11/163A-pulsed cells were still well recog-
nized. Indeed, even after 36 h, KF11/163A-pulsed cells re-
mained well recognized, whereas there was no response de-
tected to the KF11/163G-pulsed BCLs (Fig. 4 C). The same
results were observed when the experiment was repeated us-
ing a KF11 clone derived from a separate HLA-B*57? indi-
vidual (Fig. 4, D and E). In this case, recognition of pulsed
BCLs was determined in a chromium release assay, showing
that the KF11/163G mutation affects killing of target cells
and not only IFN-? production. Although these assays not
only measure peptide off-rate and include the contribution
of TCR sensitivity to WT and variant peptide forms, they
also demonstrate clear-cut differences in recognition that
were not apparent when peptides were coincubated for the
duration of the assay with target cells. These data indicate
that an Ala→Gly change at position 2 within two separate
HLA-B*57–restricted epitopes has the same effect on pep-
tide recognition, with the Gly variant form being less well
recognized than the WT Ala form. We hypothesize this is
due to an increase in peptide off-rate.
Even relatively small changes in off-rate can greatly affect
the ability of a ligand to trigger T cells (22). Therefore, sub-
tle changes in peptide off-rate associated with an Ala→Gly
substitution at position 2 within B*57-restricted epitopes
might be expected to reduce recognition of the epitope on
the surface of an infected cell. To test this, we transfected an
HLA-B*57? BCL line with synthetic mRNA constructs en-
coding the KF11/163A epitope or the KF11/163G or
KF11/163N variants (Table III). Constructs were derived
from a 162-residue fragment of p24 from the HIV molecular
clone NL4-3, expressing either WT KF11/163A or variant
KF11 epitope at the NH2 terminus and tagged with the
B*57-restricted epitope ISW9 (ISPRTLNAW, Gag 147–
155) at the COOH terminus as a positive control. Trans-
fected BCLs were used to stimulate KF11- or ISW9-specific
CTL clones in an IFN-? ELISPOT assay (Fig. 5 A). Both
ISW9 and KF11 clones recognized BCLs transfected with
WT KF11/163A mRNA. However, KF11 clones failed to
epitope, KF11. (A) Association between expression of HLA-B57 and an
Ala to Gly substitution at position 2 within KF11 (KF11/163G). (B) Recogni-
tion of KF11/163A and KF11/163G peptides by KF11-specific CTL lines
(9308196RI) by IFN-? ELISPOT.
Identical changes observed in a second HLA-B57–restricted
rate of KF11. Recognition of HLA-B*57? BCLs pulsed with either KF11/163A
or KF11/163G peptides (A–C) for 1, 12, and 36 h using a KF11-specific CTL
(9308196RI) measured by IFN-? ELISPOT. At each time point, effector cells
Ala to Gly substitution at position 2 also increases off-
were titrated out and tested for recognition of 25,000 pulsed BCLs (D and E)
for 1 and 12 h using a KF11 clone measured by percent of killing in a 51Cr
assay. For all assays, background staining of unpulsed BCLs at each time
point was subtracted.
JEM VOL. 201, March 21, 2005
recognize BCLs transfected with either KF11/163G or
KF11/163N variant mRNA. As expected, recognition of
ISW9 was unaffected by these distal mutations in the KF11
epitope. Therefore, the consequence of the Ala→Gly substi-
tution at position 2 within KF11 is that the peptide is no
longer presented at sufficient levels on the cell surface to in-
duce an IFN-? response. Similar assays were performed us-
ing KF9-specific CTLs and mRNA transcripts encoding
WT KF9/83A or variant KF9/83G, and these showed the
equivalent result (Fig. 5 B). These data show that the KF9/
83G and KF11/163G variants are indeed escape mutations
that would allow the virus to evade these CTL responses.
Ile→Val at integrase-31 is an HLA-B*51 negatively
associated B-clade polymorphism
To determine whether the mechanism by which the nega-
tive association between HLA-B*57/5801 and Nef-83 arose
is a more general phenomenon, we next sought other exam-
ples of associations between HLA alleles and conservation of
the consensus. We focused on another allele, HLA-B*51,
because, like HLA-B*57/5801, it is associated with slow
progression in B-clade infection (11, 12) and hence likely to
impose strong selection pressure on the virus. Initially, we
studied a B-clade–infected horizontal transmission pair, in
which the donor expressed HLA-B*51 and the recipient did
not, to follow escape mutations arising in the former that
were transmitted and stable in the latter. We observed such a
mutation at position 4 in the integrase epitope, LPPVVAKEI
(“LI9”: residues 28–36; reference 23), involving an Ile→Val
substitution at residue 31 (LI9/31V), where Val is the
B-clade consensus amino acid in this position (http://
www.hiv.lanl.gov). 22 out of 22 clones sequenced from viral
RNA at the first time point in the B*51? donor encoded Ile
at residue 31 (LI9/31I), but at the time of transmission 18
mo later, 11 out of 11 clones sequenced now encoded Val.
This LI9/31V mutation was transmitted to the B*51? recipi-
ent and was still found in 100% of the clones isolated from
the latest sampling point 8 mo after transmission (Fig. 6 A).
In a second similar transmission pair, the B*51? recipient
continued to carry virus expressing the same LI9/31V muta-
tion at least 14 mo after transmission (not depicted).
To determine whether there is an association between
the occurrence of Val at this position and B*51 expression,
we compared viral sequences in 40 B*51? and 246 B*51?
B-clade–infected persons and found that although the clear
majority (61%) of the B*51? subjects carry virus expressing
LI9/31V, a significantly greater proportion (88%) of B*51?
persons carry virus expressing LI9/31V (P ? 0.0011). This
suggests that as for KF9/83G, the Ile→Val substitution that
now forms the consensus in B-clade infection is driven by
CTL escape in B*51? subjects. To further test this hypothe-
sis, we examined the situation in the C-clade–infected cohort
in Durban, in which B*51 is rare. In contrast to B-clade virus,
the C-clade consensus amino acid at integrase-31 is Ile, with
68% of HLA-B*51? subjects from this cohort expressing
LI9/31I (Fig. 6 B). We sequenced the virus in six B*51?
C-clade–infected subjects, and in each case the LI9/31V
mutation was present (P ? 0.0003). Thus, we have observed
a second clear case in which the negative association with an
HLA allele and an amino acid occurs as a result of the posi-
tive selection of an escape mutation that is transmitted and
apparently stable in the absence of the selecting HLA allele.
A substantial number of the described links between HLA
class I expression and HIV sequence polymorphism involve
associations for conservation of the consensus sequence
rather than variation away from it. The mechanism that op-
erates to produce these associations has been unclear. The
term “negative selection pressure,” which was proposed to
explain their existence, suggests that selection pressure is op-
erating against sequence change at these positions. However,
of endogenously processed peptides. (A) KF11 clone tested against either
HLA-B57? BCLs transformed with mRNA containing either the KF11/163A
or KF11/163G epitope, and a second B57 restricted epitope ISW9, no mRNA
(Mock), or untransformed BCLs. (B) KF9-specific CTL line (SK059), tested
against either HLA-B5801? BCLs transformed with mRNA containing
either KF9/83A or KF9/83G and ISW9, or untransformed BCLs. In all cases,
recognition was measured by IFN-? production in an ELISPOT assay.
Ala to Gly substitution at position 2 affects presentation
epitope LPPVVAKEI (LI9). (A) A mutation from Ile to Val (LI9/31V) at
position 4 within in LI9 is selected for in B*51? donor and is transmitted
to B*51? recipient. LI9/31V is stable in the recipient, still being found in
100% of the RNA clones isolated 8 mo after transmission. (B) LI9/31V is
associated with the expression of HLA-B*51 in both B-clade and C-clade.
In B-clade, this is seen as a negative association, as LI9/31V has become
the consensus. However, in the C-clade epidemic in Durban, where
HLA-B*51 is rare, LI9/31I is still the consensus and hence this is seen as a
Negative association in a second, HLA-B*51–restricted
NEGATIVE HLA ASSOCIATIONS IN HIV | Leslie et al.
the data presented here show that KF9/83G, which forms
the consensus sequence, actually represents an escape muta-
tion from the WT KF9/83A, driven by targeting of the pre-
viously undescribed HLA-B*57/5801-restricted epitope,
KF9. This KF9/83G mutation is transmitted and persists in
the absence of HLA-B*57/5801, and as a result, its subse-
quent accumulation at the population level has lead to re-
placement of Ala by Gly as the consensus sequence. Simi-
larly, the HLA-B*51–associated escape mutant LI9/31V in
the epitope LI9 is transmitted and persists after transmission
to the point where the escape variant LI9/31V now repre-
sents the consensus sequence in B-clade infection. The same
escape mutation is observed in the C-clade–infected popula-
tion in Durban, with all HLA-B*51? individuals sequenced
expressing LI9/31V. However, in this C-clade population,
in which HLA-B*51 is rare, LI9/31I remains the consensus.
Thus, the same LI9/31V variant is identified as a “positive
association” in C-clade infection, and as a “negative associa-
tion” in B-clade infection.
These data suggest that, like the more typically ob-
served “positive” associations, “negative” HLA associations
with sequence polymorphism can also be the result of pos-
itive selection. The apparent differences are only evident
because sequences are viewed as an evolutionary snapshot
and relate simply to the extent to which an escape muta-
tion has accumulated in the population. An escape muta-
tion that does not undergo reversion (17, 24, 25), but
which has yet to accumulate in the population to the ex-
tent that the consensus sequence has switched to the vari-
ant form, as in the case of LI9/31V in C-clade infection, is
seen as a positive HLA association. The same mutation an-
alyzed later in the epidemic will be seen as a negative HLA
association because the variant will have become the con-
sensus sequence (Fig. 7, A and B). We hypothesize that
this is a primary mechanism by which associations between
the expression of particular HLA alleles and sequence con-
servation, such as those identified by Moore et al. (3),
might arise. Therefore, these negative associations repre-
sent the genetic footprints of immune responses, and their
presence supports the hypothesis that HLA-restricted CTL
responses are driving HIV evolution at the population level
(3, 8, 17, 26).
Recent studies have demonstrated that not all escape
mutations will increase in frequency in the population over
time. Escape mutations that occur at a fitness cost to the vi-
rus revert to WT on transmission to individuals lacking the
HLA allele that drove their selection (17, 24, 27). Only mu-
tations that have a minimal effect on viral fitness, and are
therefore selectively neutral, are likely to persist in the ab-
sence of the relevant HLA allele and thereby accumulate at
the population level (21). A recently described example is
the HLA-B*57/5801-associated processing escape mutation
A146P within p24 Gag (Fig. 7 A). The frequency of this es-
cape mutation is increasing over the course of the epidemic,
and in vitro competition experiments comparing the fitness
of viruses that were isogenic other than for the change en-
5801-restricted epitopes and one B*51-restricted epitope in which escape
mutation is transmitted and stable in the absence of the selecting allele.
This is observed as a positive association between the selecting allele and
sequence polymorphism or a negative association between the selecting
allele and sequence conservation depending on whether or not the escape
mutation has spread in the population to the point at which it has replaced
the consensus sequence. The B*57/5801-restricted TW10 reverts on trans-
mission and hence will not spread at the population level. Mutations in KF11,
KF9, and IVW9 (B-clade only; all occurring at primary anchor positions)
and in ISW9 (which affect processing) remove the epitope as a potential
target and hence might be expected reach fixation. Conversely, mutations
in HW9 and LI9 occur at nonanchor residues and are likely to remain as
The evolutionary fate of CTL epitopes. (A) Five HLA-B*57/
targets to the immune system. (B) Three potential outcomes of CTL escape
mutation are: (a) escape mutations that revert on transmission: frequency
of escape mutation will not exceed the phenotypic frequency of the se-
lecting allele. The epitope will remain a potential target to the immune
system and positive selection pressure will always be evident. (b) Escape
mutation that is stable but does not affect processing/presentation. Fre-
quency of mutation will equilibrate at 50%, the epitope will remain a
target but there will be no clear consensus and evidence of selection
pressure maybe lost. (c) Escape mutation that is stable and affects epitope
processing/presentation. Epitope will become extinct as mutation reaches
fixation, when all evidence of selection pressure and indeed the epitope
itself will be lost.
JEM VOL. 201, March 21, 2005
coding the A146P substitution demonstrated no reduction in
replicative capacity resulting from this mutation (21).
A further important feature of transmitted escape mutants
is that the immunological significance and pattern of accu-
mulation of these variants will vary. Escape mutations that
interfere with TCR recognition likely only represent escape
at the individual and not the population level (17, 28). There
is a vast array of unique TCRs that can be generated by
TCR gene rearrangement and N-region substitutions (29),
which in combination with a high degree of cross reactivity,
suggest that the T cell repertoire is broad enough to respond
to essentially all foreign peptides that contain appropriate
MHC anchor residues (30). Therefore, although variation in
the TCR contact residues may result in loss of peptide rec-
ognition in an individual, it is likely that if transmitted to
other individuals sharing the restricting HLA allele, they will
be able to generate a response to this variant form. This is
perhaps best exemplified by escape mutation in the HLA-
A*02–restricted Gag epitope SLYNTVATL (SL9), in which
responses have been observed to all of the commonly ob-
served polymorphisms (31, 32), and, although HLA-A*02?
individuals are often infected with a virus encoding variants
of SL9 at positions 3, 6, and 8 in the epitope, their ability to
generate primary responses to SL9 is unaffected (33). Thus,
despite persistent selection of stable escape mutations, SL9
remains a frequently targeted epitope. Conversely, epitopes
in which mutation disrupts either the processing or presenta-
tion are effectively removed as possible CTL targets (4, 21,
34) and hence, if the escape does not revert, individuals sub-
sequently infected with this virus will no longer be able to
generate a response. If such mutations spread up to the point
of fixation, the epitope can be defined as “extinct,” as it no
longer exists as a potential target to the immune system. This
principle has been demonstrated in EBV, in which an im-
munodominant HLA*A11-restricted epitope has been lost in
viral strains isolated from communities in New Guinea, in
which HLA*A11 is highly prevalent (phenotypic frequencies
of 25–50%; reference 35).
Consideration of these factors should allow one to pre-
dict the evolutionary fate of a given CTL epitope (Fig. 7,
A and B). Epitopes such as the HLA-B*57/5801-restricted
TW10 in which the escape mutation reverts (17) will remain
targets to the immune system. The frequency of the escape
mutation in the population is likely to stabilize at some level
below the phenotypic frequency of the selecting allele, de-
pending on how rapidly and consistently escape occurs, and
the positive selection operating will always remain apparent.
Conversely, epitopes such as KF9, KF11, the HLA-B*57/
5801-restricted Gag epitope, ISW9 (21), and RT epitope
IVW9 (RT 244–252), in which the escape affects processing
and/or presentation and does not revert, are likely to be
those that tend toward extinction over time. In between
these two extremes, there are epitopes such as SL9, in which
the escape mutation does not revert but where the epitope is
still presented and thus remains a target for the immune sys-
tem. Potential examples of such epitopes from our data in-
clude the HLA-B*57/5801-restricted Nef epitope HW9
(Nef 116–124) and the HLA-B*51–restricted LI9. In both of
these the escape mutation is stable in the absence of the se-
lecting allele but does not occur at a primary anchor residue.
Under such circumstances, one might expect the frequency
of the mutation in the population to stabilize at ?50%, with
no clear consensus discernible.
The precise frequency in the population of any given
mutation will depend on several factors. The frequency of
the selecting allele may have an effect, and indeed it has
been proposed that high frequency alleles are more likely to
drive escape variants that are embedded in the consensus as
“negative HLA associations” (3). However, the degree to
which different alleles drive immune escape in HIV infec-
tion differs substantially and is not simply related to the al-
lele frequency (15). In particular, HLA-B alleles as a whole
tend to drive escape more than HLA-A alleles, and there are
likely to be differences between individual alleles in this re-
spect, just as there are between the individual alleles and vi-
ral set point (15). Other HLA-dependent factors that will
influence the frequency of an escape variant would include
duration of epidemic, timing of generation of the escape
variant within the infection, and likelihood of transmission
of the variant (related to HLA-associated viral load). Fur-
thermore, the clustering of epitopes restricted by many dif-
ferent alleles into regions of high immunogenicity means
that the frequency of KF9/83G and LI9/31V are likely to
be affected not only by HLA-B*57/5801 and B*51, respec-
tively, but also by other alleles, the identity of which are
likely to vary from one population to another. Finally, for
mutations such as KF9/83G and LI9/31V that are essentially
selectively neutral, there are important HLA-independent
influences, such as genetic drift and founder effects, which
are likely to have a strong impact on the frequency of these
variants within different populations. These latter influences
of genetic drift and founder effects would not themselves
explain the negative HLA associations observed here and
previously (3), although they may influence the observed
frequency of particular amino acids and therefore contribute
to the determination of a consensus sequence.
An additional discussion point that emerges from these
data concerns the inadequacy of standard assays, involving
coincubation of peptide with target cells for the duration of
the assay, for use in analyzing variant recognition. The use of
assays that include assessment of peptide off-rate appear to be
a more faithful representation of the in vivo recognition of
variants, as assessed by minigene transfection of targets (36)
or, in these studies, by the mRNA transfection assays de-
scribed. These indicate that the off-rate assay can reveal
clear-cut differences in recognition between variants that are
debatable when viewed from the perspective of the standard
assay. Peptide optimization occurring within the ER ensures
that only ligands with slow HLA dissociation rates are se-
lected (37, 38). It is therefore likely that the increase in pep-
NEGATIVE HLA ASSOCIATIONS IN HIV | Leslie et al.
tide off-rate associated with A→G substitution at position 2
in B*57/5801-restricted epitopes is such that this variant is
out-competed in the ER by alternative B*57/5801-restricted
peptides. These studies thus highlight the limitations of com-
monly used assays, which rely on coincubation of targets
with peptides for the duration of the assay and may greatly
underestimate the true level of escape mutation being driven
by CTL selection pressure.
The importance of understanding the direction of HIV
evolution in the development of HIV vaccines is clear. A
vaccine that induces CTL response to epitopes no longer in
circulation will be redundant and perhaps even harmful in
inducing immunodominant responses incapable of recog-
nizing the circulating forms of the virus. This concept has
been considered in the context of whether a consensus
based on circulating forms might have advantages over an
ancestral reconstruction for vaccine antigen design. The ob-
servation of negative HLA associations, which are positively
selected escape variants that might be in the process of dis-
appearing by becoming embedded within consensus se-
quence, indicates that the process of adaptation of HIV to
host HLA alleles is certainly occurring. This suggests that
even epitopes such as KF11, which is presently the domi-
nant HLA-B*57–restricted response in chronic infection,
may become extinct as the epidemic evolves. In considering
the epitopes that should be incorporated into today’s HIV
vaccine, one could argue that only those epitopes that re-
vert after transmission should be included, because these not
only incur a cost to viral replicative capacity when escape
occurs, but they also are likely to remain in circulation and
accessible as epitopes many years into the epidemic. Epitopes
with negative HLA associations, on the other hand, might
be the epitopes least likely to make an effective contribu-
tion to a vaccine, because, in cases where epitope process-
ing and presentation has been affected, the challenge virus
will not be recognized by corresponding CTLs. Addition-
ally, even if the variant epitope remains a target, the cost to
viral fitness of any subsequent escape mutation induced is
likely to be low. Thus, understanding the evolutionary events
subsequent to transmission is of direct relevance to HIV
MATERIALS AND METHODS
Subjects studied. C-clade HIV samples were collected from Cato Manor
antenatal clinic, Durban, South Africa. All subjects are antiretroviral therapy
naive. B-clade samples were collected from cohorts encompassing Europe
and North America, with the majority also being antiretroviral therapy na-
ive at the time of analysis. Additional B-clade samples were obtained from
the Hemophilia Growth and Development Study in the United States (39).
This study was approved by the University of Natal Review Board, the
Oxford Research Ethics Committee, and the MGH Review Board. All in-
dividuals gave informed consent for participating in this study. HLA class I
typing was performed on genomic DNA by PCR single-stranded confor-
mation polymorphism (40).
Sequencing of proviral DNA and viral RNA. Genomic DNA was
extracted from whole blood via the Puregene DNA isolation kit (Gentra).
We amplified HIV gag sequences by nested PCR as described previously
(17), using the following primers: Gag-specific primers and Nef-specific
primers: 5?-TTCAGCTACCACCGATTGAGA-3?, 5?-TGAGGGTTG-
GCCACTCC-3? for first round PCR and 5?-TTCAGCTACCAC-
CGATTGAGA-3?, 5?-TGAGGGTTGGCCACTCC-3? for second round
PCR; RT-specific primers: 5?-GAAGGACAGTACTAAGTGGAG-3?,
5?-CTGGCTACATGGACTGCTAC-3? and 5?-ACTGCATTCACC-
ATACCTAG-3?, 5?-TTATGTGCTGGTACCCATGA-3?; and INT
primers: 5?-CATGGTGGACAGACTATTGGC-3?, 5?-CCTGCCATC-
TGTTTTCCATA-3? and 5?-CCTCCCCTAGTAAAATTATGG-3?, 5?-
GCTGTCTCTGTAATAAACCCG-3?. PCR product was purified by
PEG precipitation and then either directly sequenced (population sequenc-
ing) or cloned as described previously (41) using Topo TA cloning kit (In-
vitrogen). Viral RNA was obtained from plasma using the Nucleospin
RNA extraction kit (Mackery-Nagel). This was converted to cDNA, am-
plified, and sequenced as described previously (17). All sequencing used the
BigDye ready reaction termination mix V3 (Applied Biosystems) for Gag
using Gag-specific primers as described previously (17) and for Nef, RT,
and INT using the two second round PCR primers and the following four
additional primers: Nef: 5?-ACCTGAGGTCTGACTGGAAAG-3?, 5?-
TCCG-3?, and 5?-ACCTCAGGTGCCTTTAAGAC-3?; RT: 5?-TGG-
AACTGAA-3?, 5?-CATAGAAAGTTTCTGCTCCT-3?, and 5?-CTC-
CCCTAGTAAAATTATGGTA-3?; and INT: 5?-TCACTAGCCATT-
GCTCTCC-3?, 5?-ATTGGAGGAAATGAACAAGTAGA-3?, 5?-GCC-
CACCAACAGGCTGC-3?, and 5?-GAAACAGGACAAGAAACAGC-
3?. All sequences were analyzed on the ABI 3700 DNA analyzer. All resi-
due numbers are from HXB2 reference sequence. Total number HIV-1
viruses sequenced as follows: Gag, 194 C-clade; RT, 113 B-clade; INT,
286 B-clade and 66 C-clade; Nef, B-clade 81 and C-clade 117. We con-
structed neighbor-joining trees of all the sequences together with reference
sequences from the Los Alamos Database (http://www.hiv.lanl.gov) to
verify the viral clades and to exclude the possibility of contamination with
laboratory HIV strains.
Cells lines. KF11-specific CTL lines were generated as described previ-
ously (42) and maintained in R10 medium (RPMI 1640 [Sigma-Aldrich],
10% fetal calf serum, 10% L-glutamate, and 10% penicillin/streptavadin)
with the addition of 25 ng/ml human recombinant IL-15 (PreproTech).
KF9 lines were generated by peptide pulsing 5 ? 10?6 PBMCs with 100
mM KF9 peptide for 1 h and resuspended in R10. After 1 wk in culture (at
37?C, 5% CO2), the medium was replaced two to three times a week with
R10 containing 50 U/ml IL-2 and 5% T-stim (Becton Dickinson). KF11
and ISW9 clones were generated by limiting dilution, and characterized and
maintained as described previously (43).
Definition of KF9 epitope. KF9 optimization and restriction was con-
firmed (14) using fresh PBMCs from the HIV B-clade–infected subject
9092850 (HLA-B*57/1517). Responses were tested by IFN-? ELISPOT
(42). Fresh PBMCs isolated from HLA-B*57/5801? individuals from the
Durban cohort were tested for recognition of the optimal KF9 peptide by
IFN-? ELISPOT. Fresh PBMCs or KF9-specific CTL lines were tested for
recognition of KF9/83A and KF9/83G by IFN-? ICS (42).
Peptide off-rate assay. For KF9, HLA-B*5801? BCLs were pulsed with
0.1 ?M KF9/83A or KF9/83G peptide, incubated for 1 h (at 37?C, 5%
CO2), and washed three times with PBS. The pulsed BCLs were split into
aliquots and either tested for recognition by KF9-specific CTL via ICS or
incubated for an additional 5–23 h, rewashed, and then tested. For KF11,
HLA-B*57? BCLs were pulsed with 0.1 ?M WT or A163G KF11 peptide,
incubated, and washed as described above. Pulsed BCLs were split into ali-
quots and either tested for recognition at different E/T ratios by KF11-spe-
cific CTLs via IFN-? ELISOT (in triplicate), or incubated for an additional
11–36 h, rewashed, and tested. The experiment was repeated using a KF11
clone, with recognition tested by 51Cr release (21).
JEM VOL. 201, March 21, 2005
Processing and presentation. BCLs were transfected with synthetic
mRNA transcribed from a PCR product template. For KF9, the forward
primer was 5?-TAATACGACTCACTATAGGGAGAGCCACCATGG-
CTGGCAAGTGGTC-3? and the reverse primer was 5?-TTACCATG-
AAGCTXCC-3?, where X ? G for KF9/83A or C for KF9/83G. The
PCR template was a synthetic gene encoding the 2001 consensus B Nef
sequence (http://www.hiv.lanl.gov) purchased as a plasmid from DNA 2.0.
For KF11, the forward primer was 5?-TAATACGACTCACTATAG-
XY ? GC for KF11/163A, GG for KF11/163G, and AA for KF11/163N,
and the reverse primer was 5?-TTACCATGCATTTAAAGTTCTAGGT-
GATATAGCTTGCTCGGCTCTTAGAGT-3? and the PCR template
was HIV clone NL4.3. Synthetic mRNA was transcribed from PCR
template using the Message Machine transcription kit (Ambion), and a 3?
poly A tail was added with the Poly(A) Tailing Kit (Ambion). 106 APCs
were transfected (300 V/0.5 mS, square wave) in a 0.2-cm cuvette with
synthetic RNA using a GenePulserII electroporator (Bio-Rad Laborato-
ries) using 10 ug mRNA. Transfected LCLs were immediately mixed
with CTLs at a ratio of 1:1 and incubated overnight in an IFN-?
We thank Ms. Sherzana Sunderji for technical assistance. We thank Patricia D’Souza
for discussions and for greatly facilitating these collaborative studies.
This work was supported by the National Institutes of Health (contract N01-Al-
15422 [“HLA typing and CTL epitope mapping to guide HIV vaccine development”]
and AI46995-01A1), the Wellcome Trust (to P. Goulder and A. Leslie), the Elizabeth
Glaser Pediatric AIDS Foundation (to P. Goulder), and the Doris Duke Charitable
Foundation. N. Bhardwaj, P. Goulder, and B. Korber are Elizabeth Glaser Scientists
and B. Walker is a Doris Duke Distinguished Clinical Science Professor.
The authors have no conflicting financial interest.
Submitted: 20 July 2004
Accepted: 24 January 2005
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