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Lewis et al 1 Partial Escape of HIV-1 from CTLs
1
Submitted as a Short-Form Paper (formerly Notes) 2
Partial Escape of HIV-1 From Cytotoxic T Lymphocytes 3
During Chronic Infection 4
5
Martha J. Lewis1,2*, Mirabelle Dagarag1, Basim Khan, Ayub Ali1,2, 6
Otto O. Yang1,2,3 7
8
1Division of Infectious Diseases, Department of Medicine, David Geffen School of Medicine at 9
University of California, Los Angeles 10
2AIDS Institute, University of California, Los Angeles 11
3Department of Microbiology, Immunology, and Medical Genetics, David Geffen School of 12
Medicine at University of California, Los Angeles 13
(Mirablle Dagarag is currently a senior research scientist at Diagnostic Products Corporation) 14
(Basim Khan was a medical student at the David Geffen School of Medicine at University of 15
California, Los Angeles) 16
17
Running title: Partial Escape of HIV-1 From CTLs 18
*Corresponding author: 10833 LeConte Ave., CHS 37-121, Los Angeles, CA, 90095. 19
malewis@mednet.ucla.edu. (310) 825-0205 (office); (310) 825-3632 (fax). 20
21
Keywords: Viral Escape Mutation, HIV-1, Cytotoxic T Lymphocytes, Viral Fitness 22
Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.06724-11 JVI Accepts, published online ahead of print on 2 May 2012
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Lewis et al 2 Partial Escape of HIV-1 from CTLs
ABSTRACT 23
24
Viral mutational escape from CD8+ cytotoxic T lymphocytes (CTLs) typically is considered to 25
be a dichotomous process, and uncommon during chronic HIV-1 infection. Ex vivo passaging of 26
HIV-1 from persons with chronic infection, however, revealed evolution of many fixed 27
substitutions within and around CTL-targeted regions, with an associated increase in replicative 28
capacity. This indicates evolution of mutations during chronic HIV-1 infection that trade 29
replicative fitness for incomplete evasion of CTLs, or “partial escape.” 30
31
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Lewis et al 3 Partial Escape of HIV-1 from CTLs
TEXT BODY 32
33
HIV-1-specific CD8+ cytotoxic T lymphocytes (CTLs) exert a major selective pressure 34
that shapes viral sequences (22), through evolution to avoid CTL antiviral activity (3, 18). 35
Mutational escape from CTLs generally is considered an “all-or-nothing” phenomenon. Because 36
persistence of a CTL response is driven by antigenic stimulation (allowing waning of responses 37
against cleared pathogens), mutational escape of an epitope typically has been associated with 38
decay of the CTL response against that epitope (6, 13). In line with this scenario, it has been 39
noted that rapid viral escape and CTL re-targeting occur during early infection (6, 14, 18), while 40
chronic infection is marked by stability of both epitope sequences and CTL targeting (16), or 41
very delayed epitope escape mutation (12, 13). 42
43
However, the generation of escape mutations can be limited by structural and functional 44
constraints on viral replicative capacity (RC), and increasing data indicate that the options for 45
evasion of CTLs targeting some epitopes are associated with substantial RC costs (7, 15, 19, 21, 46
26, 27). The reported examples of this phenomenon involve immunodominant epitopes 47
restricted by Human Leukocyte Antigen (HLA) alleles that are associated with superior immune 48
containment of HIV-1 (B*13, B*27, B*57), suggesting that both CTL antiviral pressure and RC 49
loss associated with escape contribute to the benefits of these HLA types. In these cases, escape 50
tends not to be associated with decay of the CTL response. Thus because these responses persist 51
during chronic infection, there appears to be a situation where the optimal balance for HIV-1 is a 52
tradeoff of maintaining RC for incomplete evasion of CTLs (driving CTL persistence). This 53
intermediate scenario represents “partial escape”. 54
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Lewis et al 4 Partial Escape of HIV-1 from CTLs
55
To investigate whether this phenomenon occurs for epitopes presented by other HLA 56
types, the evolution of HIV-1 from persons with chronic infection was examined after ex vivo 57
passaging in the absence of CTL selection, to observe whether chronically CTL-targeted 58
epitopes would “revert” as a reflection of reduced RC due to CTL pressure in vivo. HIV-1 59
cultures were established from four participants with chronic untreated HIV-1 infection and 60
viremia of at least 5 x 103 log RNA copies per ml plasma (range 7,500-19,000), who were 61
enrolled in a University of California, Los Angeles Institutional Review Board-approved study. 62
None had HLA B*13, B*27, or B*57 haplotypes. The average duration of infection of these 63
subjects was 14.3 years (median 15, range 6-21), with an average blood CD4+ T lymphocyte 64
count of 395 cells/ml (range 284-594). 65
66
HIV-1-specific targeting was defined by standard interferon-γ ELISpot assays using 15-67
mer peptides overlapping by 11 amino acids, spanning the entire HIV-1 consensus subtype B 68
sequence proteome (NIH AIDS Research and Reference Reagent Program), as previously 69
described (5, 31, 32). Recognized epitope regions were defined as singly targeted peptides, or 70
the region of overlap between targeted consecutive overlapping peptides; in most cases a likely 71
minimal epitope could be inferred from the HLA type of the subject and known epitopes 72
reported in the Los Alamos National Laboratory HIV Immunology Database (see Table 1). The 73
median number of epitope regions targeted by these four participants was 9.5, with a range of 9 74
to 17 (Figure 1). The most highly targeted protein was Pol (median 5.5 epitope regions, range 2 75
to 8), followed by Gag (median 3.5 epitope regions, range 2 to 7). 76
77
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Lewis et al 5 Partial Escape of HIV-1 from CTLs
Virus was recovered from peripheral blood mononuclear cells by expanding CD4+ T 78
lymphocytes using a CD3:CD8 bi-specific antibody (29, 30, 33, 35). Recovered viruses were 79
passaged weekly in freshly expanded CD4+ T lymphocytes from multiple, non-HLA matched, 80
healthy HIV-1-uninfected donors for 10 to 14 weeks (median 11). All cells were maintained in 81
RPMI 1640 medium supplemented with 10% fetal calf serum, L-glutamine, penicillin, 82
streptomycin, and recombinant human interleukin-2 at 50U/ml (NIH AIDS Research and 83
Reference Reagent Program). Sequencing of HIV-1 was performed on genomic DNA from the 84
infected cell cultures using standard PCR cycling conditions and primer pairs from a previously 85
described HIV-1 subtype B primer set; baseline sequences were obtained by plasma RT-PCR (4). 86
Multiple PCR products were cloned using the TOPO TA Kit (Invitrogen). Approximately 10 87
individual clones were sequenced (median 9) for each epitope region (Figure 1) pre-and post-88
passaging (GenBank Accession numbers pending). All sequences were aligned with the Los 89
Alamos National Laboratory HIV Sequence Database consensus sequences for subtype B using 90
BioEdit, and then analyzed for amino acid changes occurring after passaging. 91
92
Across all subjects, 26 amino acid changes within CTL-targeted epitope regions were 93
noted between the consensus sequences of baseline and passaged viruses (Table 1). One 94
participant (subject 25) had both the greatest numbers of amino acid changes and CTL responses 95
(Figure 1). Sequence changes were evaluated for statistical significance by Fisher’s exact test, as 96
well as analyzed for selection pressure using the PAML software program and the likelihood 97
method of Nielsen and Yang (36, 37). Of the 26 changes, 24 were statistically significant, and 7 98
showed evidence of significant positive selective pressure by ratio of the non-synonymous to 99
synonymous mutation rate ratio (dN/dS>1). 17 of the 26 substitutions (65%) were changes from 100
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Lewis et al 6 Partial Escape of HIV-1 from CTLs
non-consensus to the subtype B consensus amino acid, suggesting optimization of viral RC after 101
passaging ex vivo. 102
103
As a more global measurement of whether the sequence evolution after ex vivo passaging 104
reflected selective pressure, the diversity of pre- and post-passaging sequences were assessed 105
using SENDBS with the HKY model and 500 bootstrap replicates (23). Sequence diversity 106
tended to decrease after passaging, although this reached statistical significance only for four 107
regions (Figure 2). Overall, these results suggested the evolution of HIV-1 towards an optimized 108
fittest sequence in the absence of CTL pressure, therefore resulting in loss of diversity. 109
110
Several amino acids flanking the epitope regions also demonstrated change after 111
passaging. Because changes in neighboring amino acids can affect epitope processing (10, 14, 112
28, 38) or compensate for loss of RC from epitope escape mutations (19), the analysis was 113
broadened to examine available sequences flanking the epitope regions, at least 30nt and as 114
much as 750nt of flanking sequence was examined. Maximum likelihood and neighbor-joining 115
phylogenetic trees (with 500 bootstrap replicates) were constructed using PHYLIP 3.64 (11) to 116
determine the relationship between the initial and passaged viruses. For each subject, the 117
passaged viruses clustered with strong bootstrap support for 13 of the 21 regions, and did not 118
intermingle with the initial sequences, indicating significantly directed evolution (Figure 2B). 119
120
Next, the 13 regions demonstrating directed evolution were examined for differential 121
selective pressure between the initial in vivo and ex vivo passaged viruses using the Selection 122
LRT program of HyPhy (25). Four regions demonstrated significantly changed dN/dS, 123
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Lewis et al 7 Partial Escape of HIV-1 from CTLs
indicating that the in vivo viruses were under substantially different selective pressure than the ex 124
vivo passaged viruses. The dN/dS ratios for the in vivo clusters were < 1 in all cases (mean 0.41, 125
range 0.15 to 0.66), and the ratios along the branches separating the 2 groups were significantly 126
higher in all cases (mean 3.29, range 0.77 to 7.30). These results indicated that purifying 127
selection dominated in vivo, and evolution ex vivo without CTL pressure was dominated by 128
positive selection. 129
130
A site-by-site analysis for selection revealed 24 sites under positive selection during ex 131
vivo passaging (Table 2). Seven sites were within mapped targeted epitope regions for the 132
subjects, 5 corresponded to known HLA-associated polymorphisms for HLA types of the 133
subjects (provided by Dr. Simon Mallal, not shown), and the remaining 12 did not fall within 134
mapped epitope regions or correspond to known HLA-associated polymorphisms. The 135
observation of positively selected sites in regions outside of our defined epitope regions and the 136
overall changes in diversity described above were consistent with escape or compensatory 137
mutations outside the epitope regions, or other escape mutations within epitopes that were 138
missed in our CTL mapping. Overall, the data suggested that CTL shape the evolution of protein 139
regions via multiple changes within or flanking targeted epitopes, through direct escape 140
mutations and/or compensatory mutations. 141
142
Finally, in order to determine whether the changes observed during adaptation in culture 143
had a direct affect on viral fitness, the replication capacity of baseline and passaged viruses was 144
compared. The GagPol coding region from baseline and passaged samples was amplified and 145
cloned in bulk into an NL4-3 based proviral clone, each with a different reporter gene, then used 146
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Lewis et al 8 Partial Escape of HIV-1 from CTLs
to make recombinant reporter virus as previously described (1) (note, subject 19 was not tested 147
because no remaining baseline sample was available.). The gagpol region was selected since this 148
region contained most of the observed changes and is most likely to have a direct affect on the 149
virus’ ability to replicate in vitro. Reporter viruses were sequenced to confirm that they 150
contained the expected polymorphisms and substitutions and that they were phylogenetically 151
indistinguishable from the previously sequenced quasispecies at the respective time points. 152
Paired baseline and passaged viruses were co-cultured at low MOI and each reporter copy 153
number, normalized to β-actin copy number, was measured by qPCR on days 1, 3, and 5 as 154
previously described (2). Replication rates, expressed as increase in log10 copies 155
(normalized)/day, were compared (Figure 3); for each subject the passaged week 11 sample had 156
a higher replication rate relative to the baseline sample, with the differences for subjects 17 and 157
26 being highly statistically significant, p=0.002 and 0.0004, respectively. This demonstrates 158
that the sequence changes in GagPol observed after passage resulted in an increased replication 159
capacity and implies that the mutations associated with CTL selective pressure at baseline indeed 160
result in a diminished in vivo fitness. 161
162
Because CTL responses depend on antigenic stimulation, they decay when fully escaped. 163
Thus the observed stability of CTL responses during chronic infection demonstrates the lack of 164
complete escape (16), which by contrast is seen frequently during acute infection (6, 14, 24), 165
suggesting that the earliest escape mutations have low fitness costs that allow complete escape 166
with little fitness cost. In contrast, however, escape mutations in immunodominant epitopes 167
targeted during chronic infection by persons with the protective HLA types B*13, B*27, and 168
B*57 are not associated with the loss of CTL responses against those epitopes (7, 15, 19, 21, 26, 169
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Lewis et al 9 Partial Escape of HIV-1 from CTLs
27). In these cases, the high fitness costs for escape likely limit options to epitope variants that 170
are either still recognized well enough to drive CTL proliferation, or associated with such severe 171
loss in RC that competing levels of index virus persist. Thus, this appears to represent “partial 172
escape,” where HIV-1 does not appear to have the capability to generate an epitope mutant 173
where the loss of RC is completely outweighed by evasion of CTLs targeting that epitope. This 174
situation leads to a net loss of fitness, through a combination of residual CTL recognition and 175
loss of RC, and is believed to be a major mediator of the protective effects of these HLA types. 176
177
While escape from CTLs otherwise has been considered usually as a dichotomous 178
process, our results suggest this not to be the case, and suggest that escape during chronic 179
infection likely falls along a continuum of partial escape for common non-protective HLA types 180
as well. Once HIV-1 is removed from the selective pressure of CTLs in vivo, targeted regions 181
show amino acid substitutions and loss of diversity that indicate optimization of fitness through 182
reversion of escape mutations. The majority of substitutions correspond to consensus amino 183
acids for subtype B, further supporting evolution towards the presumed fittest sequence. Some 184
of these substitutions do not match the consensus sequence, suggesting either that the consensus 185
residue represents a common escape mutation shared by the majority of persons (17, 20), or that 186
the optimal amino acid is context-dependent. Formal fitness assessments demonstrate that the 187
observed sequence changes result in fitness gains, confirming that the evolution of sequences ex 188
vivo reflects selection of fitter variants, in agreement with predictions that fitter members rapidly 189
overgrow quasispecies (8). Thus it appears that partial escape commonly imposes a fitness cost 190
that contributes to CTL antiviral effects in chronic infection in the same manner as shown for 191
immunodominant responses mediated by HLA types associated with superior immune control. 192
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Lewis et al 10 Partial Escape of HIV-1 from CTLs
193
Although we observed consistent trends that strongly suggest the phenomenon of partial 194
escape, larger sample sizes with more subjects will be required to define the frequency of this 195
process. More data also will be required to explore the pathways for epitope escape and factors 196
determining the degree of fitness loss; in the context of escapes with high fitness costs for 197
protective HLA types, there are examples of changes in the T cell receptor binding residues (7, 198
19, 21) and HLA-binding anchor residue mutations (9, 26, 27), as well as flanking sequence 199
mutation (10). Finally, a caveat to these studies is that fitness costs of sequence polymorphisms 200
in vitro may differ from those in vivo, particularly for nonstructural proteins, e.g. Nef (34). Still, 201
our results provide support for the phenomenon of partial escape and its contribution to the 202
antiviral activity of CTLs. 203
204
We wish to thank our study subjects for their participation, and Dr. Simon Mallal for providing 205
data on HLA-associated sequence polymorphisms in HIV-1. This work was supported by 206
NIH/NIAID grant AI043203 (O.O.Y.). 207
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Lewis et al 11 Partial Escape of HIV-1 from CTLs
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models for heterogeneous selection pressure at amino acid sites. Genetics 155:431-49. 350
38. Yokomaku, Y., H. Miura, H. Tomiyama, A. Kawana-Tachikawa, M. Takiguchi, A. 351
Kojima, Y. Nagai, A. Iwamoto, Z. Matsuda, and K. Ariyoshi. 2004. Impaired 352
processing and presentation of cytotoxic-T-lymphocyte (CTL) epitopes are major escape 353
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infection. J Virol 78:1324-32. 355
356
357
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Lewis et al 18 Partial Escape of HIV-1 from CTLs
Table 1. Statistical analysis of amino acid changes within mapped CTL epitope regions 359
360
Subject: HLA
haplotype
Epitope (Amino Acid) a
HLA restriction
Amino acid substitutions
a
Fisher’s resultb
dN/dS c Reversion to consensus
00017: A02, 03;B44, 51;C04, 14
Integrase (26-36) B51 L28I p=0.0277 ns No
00019: A02, 02;B44, 50; C06, 16
Protease (69-83) Rev (17-31) Vpr (53-67)
A02 A02 A02
I77V F21S S63I
p=0.0294 p=0.0062p=0.0297
8.0 ND 6.4
Yes No Yes
00025: A02, 03; B15, 40; C02, 03
p17 (9-23)
p17 (21-35)
p17 (49-63) p17 (73-83)
p17 (81-95)
p24 (76-90)
p24 (136-146) Protease (69-83)
RT (282-304)
B40
A03
A02 A02
A02
A02, B40
B15 A02
B15
E11G K12E R26K Q28K R30K L34I I61L
R76K I82V R91K V94I R95K L83V P87H
M136L Y69H K70N I77V
I293V
p=0.0031p=0.0031p=0.0288p=0.0032p=0.0001p=0.0031p=0.0031
p<0.00001p<0.00001p<0.00001p<0.00001p<0.00001p<0.00001p=0.0006p=0.0067
p<0.00001p<0.00001p<0.00001p=0.0001
ns ns ns ns ns ns ns ns 5.7 ns ns ns ns 1.5 1.5 ns ns ns ND
Yes Yes Yes Yes Yes Yes Yes No No No No Yes No Yes Yes Yes No Yes No
00026: A01, 02; B08, 44; C05, 07
RT (82-92) Integrase (262-276) Integrase (278-288)
UnknownB15, B42Unknown
K83R X263R X281V
p<0.00001ns ns
ns 4.6 4.6
Yes Yes Yes
361 a numbering according to HXB2 reference sequence, X= any amino acid 362
b p value for Fisher’s exact test. ns-not significant, p value>0.05 363
c dN/dS – ratio of the rate (d) of non-synonymous (N) to synonymous (S) replacements. ns-not 364
significantly >1, ND not determined 365
366
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Lewis et al 19 Partial Escape of HIV-1 from CTLs
Table 2. Amino Acids Undergoing Positive Selection During Passaging 367
368
Subject #
Protein Amino acid change a
dN/dS b p value c
00017
Protease Vpr
X37A** X20L
51.5 7.8
<0.01 ns
00019
Protease
Vpr
T37S** E48G I77V* S63I*
8.0 8.0 8.0 6.4
<0.01 <0.01 <0.01
ns 00025
Gag p17 Gag p24
I82V* P87H* X102
M136L* T171V
5.7 1.5 1.5 1.5 1.5
ns ns ns ns ns
00026
Gag p24
RT
Integrase
Vif
I91V X120N** X171V D192N
V245T** T286X D324P I329X** Q334N D232X V281X* R263G* X147Y
3.4 3.4 3.4 4.1 4.1 4.1 4.1 4.1 4.1 4.6 4.6 4.6 1.4
ns ns ns
0.05 0.05 0.05 0.05 0.05 0.05 ns ns ns ns
369
a numbering according to HXB2 reference sequence, X = any amino acid 370
b dN/dS – ratio of the rate (d) of non-synonymous (N) to synonymous (S) replacements. 371
c ns-not significant 372
* Within a CTL epitope determined by ELISpot mapping. 373
** Although not within a CTL epitope as determined by ELISpot mapping, these mutations have been 374
reported as associated with HLA alleles present in the subject’s haplotype. 375
376
377
378
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Lewis et al 20 Partial Escape of HIV-1 from CTLs
FIGURE LEGENDS 379
380
Figure 1: Summary of CTL targeting and associated amino acid changes. The black arrowheads 381
mark the location of CTL responses within the HIV-1 coding regions for each subject as 382
determined by IFN-γ ELISpot mapping of CD8+ T lymphocytes using a consensus subtype B 383
peptide library (no responses were seen against Vpu or Tat). The regions shaded in gray were 384
sequenced before and after ex vivo passaging (see Figure 2 for the exact sequence regions). 385
Amino acid sites that changed with passaging are marked with open arrowheads. Amino acid 386
sites that displayed evidence of positive selection with passaging are marked with shaded 387
diamonds (see Tables 1 and 2 for the exact amino acid positions). 388
389
390
Figure 2: Changes in sequence diversity with passaging. A. The average diversity of the initial 391
in vivo (black bars) and ex vivo passaged (gray bars) virus sequences was calculated and 392
evaluated by 500 bootstrap replicates to give a mean and standard error. Significant differences 393
in pre- and post-passaging values are marked with an asterisk (t-test p<0.05). B. A 394
representative neighbor-joining phylogenetic tree for subject 25 p17 Gag (1-155) shows the 395
passaged sequences have less diversity and form an independent cluster with 100% bootstrap 396
support. 397
398
Figure 3. Comparison of the replication capacity of the ex vivo and passaged viruses. 399
Recombinant NL4-3-based reporter viruses carrying the GagPol coding region from baseline and 400
passaged viruses from subjects 17, 25 and 26 were created. Paired baseline and passaged 401
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Lewis et al 21 Partial Escape of HIV-1 from CTLs
samples, each with a different reporter, were grown simultaneously at low MOI in co-culture, 402
and viral copy number was measured by qPCR on days 1, 3 and 5. Mean and standard deviation 403
of the slope of the growth curve of each sample (log10 copies/day) was calculated based on 404
duplicate infections. P values for the difference between baseline and passaged virus replication 405
rates are shown above each group of paired samples. 406
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