partial escape of hiv-1 from cytotoxic t lymphocytes during

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

[email protected]. (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

on April 14, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/


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


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/


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


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/


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


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/


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


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/


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


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/


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


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/


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


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/


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


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/


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


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/


REFERENCES 208

209

1. Ali, A., B. D. Jamieson, and O. O. Yang. 2003. Half-genome human immunodeficiency 210

virus type 1 constructs for rapid production of reporter viruses. J Virol Methods 110:137-211

42. 212

2. Ali, A., and O. O. Yang. 2006. A novel small reporter gene and HIV-1 fitness assay. J 213

Virol Methods 133:41-7. 214

3. Allen, T. M., M. Altfeld, S. C. Geer, E. T. Kalife, C. Moore, M. O'Sullivan K, I. 215

Desouza, M. E. Feeney, R. L. Eldridge, E. L. Maier, D. E. Kaufmann, M. P. Lahaie, 216

L. Reyor, G. Tanzi, M. N. Johnston, C. Brander, R. Draenert, J. K. Rockstroh, H. 217

Jessen, E. S. Rosenberg, S. A. Mallal, and B. D. Walker. 2005. Selective escape from 218

CD8+ T-cell responses represents a major driving force of human immunodeficiency 219

virus type 1 (HIV-1) sequence diversity and reveals constraints on HIV-1 evolution. J 220

Virol 79:13239-49. 221

4. Altfeld, M., T. M. Allen, X. G. Yu, M. N. Johnston, D. Agrawal, B. T. Korber, D. C. 222

Montefiori, D. H. O'Connor, B. T. Davis, P. K. Lee, E. L. Maier, J. Harlow, P. J. 223

Goulder, C. Brander, E. S. Rosenberg, and B. D. Walker. 2002. HIV-1 superinfection 224

despite broad CD8+ T-cell responses containing replication of the primary virus. Nature 225

420:434-9. 226

5. Balamurugan, A., M. J. Lewis, C. M. Kitchen, M. N. Robertson, J. W. Shiver, E. S. 227

Daar, J. Pitt, A. Ali, H. L. Ng, J. S. Currier, and O. O. Yang. 2008. Primary human 228

immunodeficiency virus type 1 (HIV-1) infection during HIV-1 Gag vaccination. J Virol 229

82:2784-91. 230


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/


6. Borrow, P., H. Lewicki, X. Wei, M. S. Horwitz, N. Peffer, H. Meyers, J. A. Nelson, J. 231

E. Gairin, B. H. Hahn, M. B. Oldstone, and G. M. Shaw. 1997. Antiviral pressure 232

exerted by HIV-1-specific cytotoxic T lymphocytes (CTLs) during primary infection 233

demonstrated by rapid selection of CTL escape virus. Nat Med 3:205-11. 234

7. Boutwell, C. L., C. F. Rowley, and M. Essex. 2009. Reduced viral replication capacity 235

of human immunodeficiency virus type 1 subtype C caused by cytotoxic-T-lymphocyte 236

escape mutations in HLA-B57 epitopes of capsid protein. J Virol 83:2460-8. 237

8. Coffin, J. M. 1995. HIV population dynamics in vivo: implications for genetic variation, 238

pathogenesis, and therapy. Science 267:483-9. 239

9. Crawford, H., J. G. Prado, A. Leslie, S. Hue, I. Honeyborne, S. Reddy, M. van der 240

Stok, Z. Mncube, C. Brander, C. Rousseau, J. I. Mullins, R. Kaslow, P. Goepfert, S. 241

Allen, E. Hunter, J. Mulenga, P. Kiepiela, B. D. Walker, and P. J. Goulder. 2007. 242

Compensatory mutation partially restores fitness and delays reversion of escape mutation 243

within the immunodominant HLA-B*5703-restricted Gag epitope in chronic human 244

immunodeficiency virus type 1 infection. J Virol 81:8346-51. 245

10. Draenert, R., S. Le Gall, K. J. Pfafferott, A. J. Leslie, P. Chetty, C. Brander, E. C. 246

Holmes, S. C. Chang, M. E. Feeney, M. M. Addo, L. Ruiz, D. Ramduth, P. Jeena, M. 247

Altfeld, S. Thomas, Y. Tang, C. L. Verrill, C. Dixon, J. G. Prado, P. Kiepiela, J. 248

Martinez-Picado, B. D. Walker, and P. J. Goulder. 2004. Immune selection for altered 249

antigen processing leads to cytotoxic T lymphocyte escape in chronic HIV-1 infection. J 250

Exp Med 199:905-15. 251

11. Felsenstein, J. 1989. PHYLIP-Phylogeny Inference Package (Version 3.2). Cladistics 5. 252


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/


12. Goulder, P. J., R. E. Phillips, R. A. Colbert, S. McAdam, G. Ogg, M. A. Nowak, P. 253

Giangrande, G. Luzzi, B. Morgan, A. Edwards, A. J. McMichael, and S. Rowland-254

Jones. 1997. Late escape from an immunodominant cytotoxic T-lymphocyte response 255

associated with progression to AIDS. Nat Med 3:212-7. 256

13. Jamieson, B. D., O. O. Yang, L. Hultin, M. A. Hausner, P. Hultin, J. Matud, K. 257

Kunstman, S. Killian, J. Altman, K. Kommander, B. Korber, J. Giorgi, and S. 258

Wolinsky. 2003. Epitope escape mutation and decay of human immunodeficiency virus 259

type 1-specific CTL responses. J Immunol 171:5372-9. 260

14. Jones, N. A., X. Wei, D. R. Flower, M. Wong, F. Michor, M. S. Saag, B. H. Hahn, M. 261

A. Nowak, G. M. Shaw, and P. Borrow. 2004. Determinants of human 262

immunodeficiency virus type 1 escape from the primary CD8+ cytotoxic T lymphocyte 263

response. J Exp Med 200:1243-56. 264

15. Kelleher, A. D., C. Long, E. C. Holmes, R. L. Allen, J. Wilson, C. Conlon, C. 265

Workman, S. Shaunak, K. Olson, P. Goulder, C. Brander, G. Ogg, J. S. Sullivan, W. 266

Dyer, I. Jones, A. J. McMichael, S. Rowland-Jones, and R. E. Phillips. 2001. 267

Clustered mutations in HIV-1 gag are consistently required for escape from HLA-B27-268

restricted cytotoxic T lymphocyte responses. J Exp Med 193:375-86. 269

16. Koibuchi, T., T. M. Allen, M. Lichterfeld, S. K. Mui, K. M. O'Sullivan, A. Trocha, 270

S. A. Kalams, R. P. Johnson, and B. D. Walker. 2005. Limited sequence evolution 271

within persistently targeted CD8 epitopes in chronic human immunodeficiency virus type 272

1 infection. J Virol 79:8171-81. 273

17. Leslie, A., D. Kavanagh, I. Honeyborne, K. Pfafferott, C. Edwards, T. Pillay, L. 274

Hilton, C. Thobakgale, D. Ramduth, R. Draenert, S. Le Gall, G. Luzzi, A. Edwards, 275


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/


C. Brander, A. K. Sewell, S. Moore, J. Mullins, C. Moore, S. Mallal, N. Bhardwaj, 276

K. Yusim, R. Phillips, P. Klenerman, B. Korber, P. Kiepiela, B. Walker, and P. 277

Goulder. 2005. Transmission and accumulation of CTL escape variants drive negative 278

associations between HIV polymorphisms and HLA. J Exp Med 201:891-902. 279

18. Liu, Y., J. McNevin, J. Cao, H. Zhao, I. Genowati, K. Wong, S. McLaughlin, M. D. 280

McSweyn, K. Diem, C. E. Stevens, J. Maenza, H. He, D. C. Nickle, D. Shriner, S. E. 281

Holte, A. C. Collier, L. Corey, M. J. McElrath, and J. I. Mullins. 2006. Selection on 282

the human immunodeficiency virus type 1 proteome following primary infection. J Virol 283

80:9519-29. 284

19. Martinez-Picado, J., J. G. Prado, E. E. Fry, K. Pfafferott, A. Leslie, S. Chetty, C. 285

Thobakgale, I. Honeyborne, H. Crawford, P. Matthews, T. Pillay, C. Rousseau, J. I. 286

Mullins, C. Brander, B. D. Walker, D. I. Stuart, P. Kiepiela, and P. Goulder. 2006. 287

Fitness cost of escape mutations in p24 Gag in association with control of human 288

immunodeficiency virus type 1. J Virol 80:3617-23. 289

20. Matthews, P. C., A. J. Leslie, A. Katzourakis, H. Crawford, R. Payne, A. 290

Prendergast, K. Power, A. D. Kelleher, P. Klenerman, J. Carlson, D. Heckerman, T. 291

Ndung'u, B. D. Walker, T. M. Allen, O. G. Pybus, and P. J. Goulder. 2009. HLA 292

footprints on human immunodeficiency virus type 1 are associated with interclade 293

polymorphisms and intraclade phylogenetic clustering. J Virol 83:4605-15. 294

21. Miura, T., C. J. Brumme, M. A. Brockman, Z. L. Brumme, F. Pereyra, B. L. Block, 295

A. Trocha, M. John, S. Mallal, P. R. Harrigan, and B. D. Walker. 2009. HLA-296

associated viral mutations are common in human immunodeficiency virus type 1 elite 297

controllers. J Virol 83:3407-12. 298


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/


22. Moore, C. B., M. John, I. R. James, F. T. Christiansen, C. S. Witt, and S. A. Mallal. 299

2002. Evidence of HIV-1 adaptation to HLA-restricted immune responses at a population 300

level. Science 296:1439-43. 301

23. Nei, M., and L. Jin. 1989. Variances of the average numbers of nucleotide substitutions 302

within and between populations. Mol Biol Evol 6:290-300. 303

24. Phillips, R. E., S. Rowland-Jones, D. F. Nixon, F. M. Gotch, J. P. Edwards, A. O. 304

Ogunlesi, J. G. Elvin, J. A. Rothbard, C. R. Bangham, C. R. Rizza, and et al. 1991. 305

Human immunodeficiency virus genetic variation that can escape cytotoxic T cell 306

recognition. Nature 354:453-9. 307

25. Pond, S. L., S. D. Frost, and S. V. Muse. 2005. HyPhy: hypothesis testing using 308

phylogenies. Bioinformatics 21:676-9. 309

26. Prado, J. G., I. Honeyborne, I. Brierley, M. C. Puertas, J. Martinez-Picado, and P. 310

J. Goulder. 2009. Functional consequences of human immunodeficiency virus escape 311

from an HLA-B*13-restricted CD8+ T-cell epitope in p1 Gag protein. J Virol 83:1018-312

25. 313

27. Schneidewind, A., M. A. Brockman, J. Sidney, Y. E. Wang, H. Chen, T. J. 314

Suscovich, B. Li, R. I. Adam, R. L. Allgaier, B. R. Mothe, T. Kuntzen, C. Oniangue-315

Ndza, A. Trocha, X. G. Yu, C. Brander, A. Sette, B. D. Walker, and T. M. Allen. 316

2008. Structural and functional constraints limit options for cytotoxic T-lymphocyte 317

escape in the immunodominant HLA-B27-restricted epitope in human immunodeficiency 318

virus type 1 capsid. J Virol 82:5594-605. 319

28. Troyer, R. M., J. McNevin, Y. Liu, S. C. Zhang, R. W. Krizan, A. Abraha, D. M. 320

Tebit, H. Zhao, S. Avila, M. A. Lobritz, M. J. McElrath, S. Le Gall, J. I. Mullins, 321


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/


and E. J. Arts. 2009. Variable fitness impact of HIV-1 escape mutations to cytotoxic T 322

lymphocyte (CTL) response. PLoS Pathog 5:e1000365. 323

29. Wong, J. T., and R. B. Colvin. 1987. Bi-specific monoclonal antibodies: selective 324

binding and complement fixation to cells that express two different surface antigens. J 325

Immunol 139:1369-74. 326

30. Wong, J. T., and R. B. Colvin. 1991. Selective reduction and proliferation of the CD4+ 327

and CD8+ T cell subsets with bispecific monoclonal antibodies: evidence for inter-T cell-328

mediated cytolysis. Clin Immunol Immunopathol 58:236-50. 329

31. Yang, O. O., E. S. Daar, B. D. Jamieson, A. Balamurugan, D. M. Smith, J. A. Pitt, C. 330

J. Petropoulos, D. D. Richman, S. J. Little, and A. J. Brown. 2005. Human 331

immunodeficiency virus type 1 clade B superinfection: evidence for differential immune 332

containment of distinct clade B strains. J Virol 79:860-8. 333

32. Yang, O. O., E. S. Daar, H. L. Ng, R. Shih, and B. D. Jamieson. Increasing CTL 334

targeting of conserved sequences during early HIV-1 infection is correlated to decreasing 335

viremia. AIDS Res Hum Retroviruses 27:391-8. 336

33. Yang, O. O., S. A. Kalams, A. Trocha, H. Cao, A. Luster, R. P. Johnson, and B. D. 337

Walker. 1997. Suppression of human immunodeficiency virus type 1 replication by 338

CD8+ cells: evidence for HLA class I-restricted triggering of cytolytic and noncytolytic 339

mechanisms. J Virol 71:3120-8. 340

34. Yang, O. O., P. T. Sarkis, A. Ali, J. D. Harlow, C. Brander, S. A. Kalams, and B. D. 341

Walker. 2003. Determinant of HIV-1 mutational escape from cytotoxic T lymphocytes. J 342

Exp Med 197:1365-75. 343


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/


35. Yang, O. O., A. C. Tran, S. A. Kalams, R. P. Johnson, M. R. Roberts, and B. D. 344

Walker. 1997. Lysis of HIV-1-infected cells and inhibition of viral replication by 345

universal receptor T cells. Proc Natl Acad Sci U S A 94:11478-83. 346

36. Yang, Z. 1997. PAML: a program package for phylogenetic analysis by maximum 347

likelihood. Comput Appl Biosci 13:555-6. 348

37. Yang, Z., R. Nielsen, N. Goldman, and A. M. Pedersen. 2000. Codon-substitution 349

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

mechanisms from CTL immune pressure in human immunodeficiency virus type 1 354

infection. J Virol 78:1324-32. 355

356

357

358


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/


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


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/


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


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/


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


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/


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


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

partial escape of hiv-1 from cytotoxic t lymphocytes during

Documents