1
Virological response after a short-term CCR5 antagonist exposure in HIV-infected patients: 1
Frequency of subjects with virological response and associated factors 2
3
Ezequiel Ruiz-Mateos1,2*, Alejandro González-Serna1,2, Miguel Genebat1, Kawthar Machmach1,2, 4
Francesc Vidal3, Mª Ángeles Muñoz-Fernández4, Sara Ferrando-Martinez1,4, Manuel Leal1*. 5
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1Laboratory of Immunovirology, Biomedicine Institute of Seville (IBIS), Service of Infectious 7
Diseases, Virgen del Rocío University Hospital, Seville, Spain. 8
2Department of Clinical Biochemistry, Biomedicine Institute of Seville (IBIS)/Virgen del Rocío 9
University Hospital, Seville, Spain. 10
3Hospital Universitari Joan XXIII, IISPV, Universitat Rovira I Virgili, Tarragona, Spain 11
4Laboratorio de Inmuno-Biología Molecular, Gregorio Marañón University Hospital, Madrid, 12
Spain. 13
Running title: Frequency and factors associated with MRV treatment 14
15
*Corresponding authors: Ezequiel Ruiz-Mateos PhD. [email protected] and 16
Manuel Leal MD. [email protected]. Laboratory of Immunovirology, Biomedicine Institute 17
of Seville (IBIS), Service of Infectious Diseases, Virgen del Rocío University Hospital, Seville, 18
Spain. Lab. 211. Planta 2ª. Avd. Manuel Siurot s/n. 41013- Seville – SPAIN. Tel. 0034 19
955923109. Fax. 0034 955013292. 20
21
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.00753-11 AAC Accepts, published online ahead of print on 1 August 2011
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ABSTRACT 22
23
The virological response after an eight day Maraviroc monotherapy has been proposed as an 24
alternative method to decide the prescription of CCR5 antagonist to HIV-infected patients. The 25
frequency of eligible patients for a combined antiretroviral therapy which includes Maraviroc 26
based on the result of this clinical test is not well known at the moment. In the same way, 27
clinical and immunovirological factors associated with the virological response after antagonist 28
exposure need to be determined. Ninety consecutive HIV-infected patients were exposed to 29
an eight day Maraviroc monotherapy. Virological response was considered positive if either a 30
reduction of 1 log10 HIV-RNA copies/mL or undetectable viral load (<40 HIV-RNA copies/mL), 31
was achieved. CXCR4 and CCR5 tropic virus levels were determined by using patients’ viral 32
isolates and multiple rounds of infection of indicator cell lines (U87-CXCR4 and U87-CCR5). The 33
frequency of patients with a positive virological response was 72.2% (94.7% and 66.2% for 34
naïve and pretreated patients, respectively). The positive response rates dramatically 35
decreased in patients with lower CD4+ T-cell counts. CXCR4-tropic virus level was the only 36
variable independently associated with the virological response after short-term Maraviroc 37
exposure. Lower CD4+ T-cell strata were associated with higher CXCR4-tropic virus levels. 38
These results support that CCR5 antagonists should be an early treatment option before the 39
expansion of CXCR4-tropic strains. 40
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INTRODUCTION 41
42
To gain entry into cells, HIV uses the CCR5 (R5) and/or CXCR4 (X4) co-receptor as well as the 43
CD4 receptor (1). The selective blocking of R5 by the first commercialized co-receptor 44
antagonist, Maraviroc (MRV) (4), makes the determination of HIV tropism essential before this 45
drug is prescribed to HIV-infected patients (10). To date, the most widely used co-receptor 46
tropism tests are the recombinant phenotypic Trofile assay (Trofile) (24) and its later version, 47
the Enhanced Sensitivity Trofile assay (ESTA) (25). 48
However, Trofile has some limitations such as: it requires samples with more than 1000 HIV-49
RNA copies/mL, around 20% of the results are non-reportable mainly due to low viral loads 50
and limitations regarding the reproducibility of this method have been described in different 51
studies (12, 19). Other methods, such as genotypic tropism test (3, 16, 17) has been proposed 52
as an alternative to the commercialized phenotypic method, however the main caveats of 53
these tests is the low sensitivity to detect Dual/Mixed (DM)/X4 tropic viruses (14). Due to 54
these limitations, a short-term exposure to MRV has been proposed as a method to assay the 55
sensitivity to R5 antagonists (Maraviroc Clinical Test, MCT) (7). This method has the advantage 56
that the outcome is a real time evidence of drug sensitivity and not a tropism result. However, 57
the frequency of subjects with a virological response after the short-term MRV exposure is not 58
well known at the moment. This frequency has been only partially published in smaller set of 59
patients (7, 9) and is unknown in naïve patients. On the other hand, it would be interesting to 60
know the factors that are associated with the virological response experienced after a short-61
term MRV exposure. In this sense, regarding Trofile, CD4 T-cell levels have been independently 62
associated with a DM/X4 tropic result that corresponds to subjects not eligible for R5 63
antagonist treatment (15). However, the clinical and inmunovirological factors that are 64
associated with virological response after MCT are unknown. 65
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Thus, the aims of this work were to analyze the frequency of subjects eligible for R5 antagonist 66
treatment based on MCT and study the clinical and inmunovirological factors associated with 67
the virological response after MCT. 68
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METHODS AND MATERIALS 70
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PATIENTS AND INTERVENTION 72
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This study was conducted in the Infectious Diseases Service at Virgen del Rocio University 74
Hospital and the Biomedicine Institute of Seville (IBiS) (Seville, Spain). Ninety consecutive HIV-75
infected patients who underwent MCT from July 2008 until March 2011 were included in the 76
present study. These patients with a median age of 42 [36–46] years had persistently 77
detectable plasma viral loads (>40 HIV-RNA copies/mL) and all of them were asymptomatic at 78
the time of the study. Patients, or legal guardians for those subjects under 18 years old, had 79
given written informed consent and the Ethical Committee of the Hospital approved the study. 80
MCT has been previously described (7). Briefly, patients were exposed to an eight day MRV 81
monotherapy and the subsequent virological response was analyzed. MCT was considered 82
positive if a significant viral load reduction, defined as a reduction ≥1 log10 HIV-RNA copies/mL 83
or undetectable viral load (<40 HIV-RNA copies/mL), was achieved on day eight after adding 84
MRV. Once the MCT result was obtained, a new HAART regimen was started according to the 85
following criteria: (i) previous genotype resistance testing results; (ii) previous antiretroviral 86
exposure; and (iii) response to MCT, in order to include MRV or not in the new HAART. 87
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METHODS 89
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X4 and R5 virus levels determination 91
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In a subgroup of these consecutive patients (n=57), the phenotypic tropism test TROCAI was 92
assayed at baseline as previously reported (9). Briefly, TROCAI is based on the production of 93
viral isolates from patients through a co-culture and multiple rounds of infection U87-X4 and 94
U87-R5 indicator cell lines. For the purpose of this study, results were expressed as Log viral 95
load (VL) in the U87-X4 and U87-R5 well supernatant (LogX4VL and LogR5VL, respectively). 96
97
Viral load quantification 98
99
HIV-1 RNA was measured in patients’ fresh plasma and in frozen samples of U87-X4 and U87-100
R5 cell-free supernatants by quantitative PCR (COBAS Ampliprep/COBAS Taqman HIV-1 test, 101
Roche molecular systems, Basel, Switzerland) according to the manufacturers’ instructions. 102
The lower detection limit was 40 HIV-1 RNA copies/mL. Plasma samples were tested for anti-103
Hepatitis C virus (HCV)-antibodies using HCV-ELISA (Siemens Healthcare Diagnosis, USA). A 104
qualitative PCR amplification was performed for plasma HCV RNA amplification (COBAS 105
Amplicor, Roche Diagnosis, Barcelona, Spain) with a lower detection limit of 15 IU/mL. 106
107
CD4-T cell quantification 108
109
CD4 T-cell counts were determined in fresh whole blood using the Epics XL-MCL (Beckman-110
Coulter Inc., California) flow cytometer according to the manufacturers’ instructions. Fresh 111
whole blood was stained with directly conjugated monoclonal antibodies, anti-CD3–112
phycoerytrin (PE) and anti–CD4-fluorescein isothiocyanate (FITC) (BD Bioscience). 113
114
Statistical analysis 115
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Statistical analyses were performed using the Statistical Package for the Social Sciences 117
software (SPSS 17.0, Chicago, IL, USA). Median and interquartile ranges were used to describe 118
continuous variables and a percentage for categorical ones. Pearson’s test was used to analyze 119
correlation between variables. Differences between groups were analyzed with Mann-Whitney 120
U test and a bivariate logistic or linear regression analysis when appropriate. To analyze the 121
independent factors associated with MCT response or VL changes after MCT, a multivariate 122
logistic or linear regression analysis, respectively, was assessed with variables showing a p 123
value <0.1 in the bivariate analysis. Variables showing a p value <0.05 were considered 124
statistically significant. 125
126
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RESULTS 128
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Frequency of virological response after MCT 130
Patients’ characteristics at baseline are shown in Table 1. In terms of antiretroviral treatment 131
21% of the patients were naïve. After MRV exposure, 72.2% of the overall patients 132
experienced a virological response (MCT positive subjects), which meant that around three 133
quarters of the patients could benefit from an antiretroviral treatment that includes MRV 134
(Figure 1a). However, we observed different response in pretreated and naïve patients. When 135
we split the populations in these two categories we observed how almost all naïve patients 136
were MCT positive (94.7%) as opposed to what happened in pretreated patients (66.2%) 137
(Figure 1a). Interestingly, when the virological response was analyzed depending on different 138
baseline CD4+ T-cell strata, higher CD4+ T-cell levels were associated with higher percentages 139
of MCT positive subjects. There was a dramatic decrease in MCT positive subjects when CD4+ 140
T-cell levels were below 200 CD4+ T-cells/µL (Figure 1b). Thus, the high percentage of MCT 141
positive naive patients could be associated with higher baseline CD4+ T-cell levels in this group 142
(Figure 1b). 143
144
Factors associated with the virological response after MCT 145
Our aim was to determine which factors were associated with virological response after MCT. 146
We observed how individuals with no virological response (MCT negative subjects) differed 147
from the MCT positive subjects in several variables; they were mostly pretreated, had been 148
diagnosed for a longer period of time, and the risk of transmission was mainly illicit drug use. 149
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According to results mentioned above, MCT negative subjects showed almost 3 fold lower 150
CD4+ T-cell levels (Table 2). In fact, when we adjusted for all these variables, CD4+ T-cell levels 151
was the only variable independently associated with the virological response after MCT (Table 152
2). Interestingly, in a subgroup of these patients (n=57) we analyzed X4 and R5 virus levels. We 153
expressed the results as the amount of X4/DM (from now on X4) and R5 viruses produced after 154
the infection by the patients’ viral isolates of indicator cell lines that express X4 or R5. We 155
observed that the risk of transmission, time since diagnosis, and CD4+ T-cell levels were 156
associated with virological response after MCT and, in addition, the amount of X4 virus levels 157
was also strongly associated. In this case, however, when all these variables were adjusted, the 158
amount of X4 virus was the only variable independently associated with the virological 159
response after MCT (Table 3). Of note that, despite the high differences observed in X4, R5 160
virus levels were extremely similar between both groups (Figure 2a-b). Remarkably, when 161
patients were grouped based on treatment category, the amount of X4 viruses tended to be 162
lower in naïve patients when compared to pretreated patients (Figure 2c). This trend was 163
similar regarding R5 virus levels (data not shown). Importantly, we also observed higher CD4+ 164
T-cells levels in naïve compared to pretreated patients (Figure 2d). In addition, when patients 165
were divided according to CD4+ T-cell strata, a dramatic increase in X4 virus levels were 166
observed in patients with CD4+ T-cell levels lower than 200 cells/µL and again there were no 167
differences in R5 virus levels based on different CD4+ T-cell levels (Figure 2e-f). 168
MCT result is categorical (MCT positive and negative subjects), but this result can be 169
transformed in a continuous variable that analyzes the viral load changes after eight days drug 170
exposure. We verified how using viral load changes (six subjects with <1000 HIV-RNA 171
copies/mL were excluded of the analysis, n=51) the sexual risk of transmission, CD4+ T-cell 172
counts and X4 virus levels were associated with the viral load changes experienced after the 173
clinical test. In the same way, the amount of X4 virus was the only variable independently 174
associated with the viral load changes after MCT (Table 4).175
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DISCUSSION 176
177
Results presented herein show that around three quarters of HIV-infected patients are eligible 178
for R5 antagonist therapy based on the virological response observed after a short-term 179
exposure to the drug. Furthermore, X4 tropic virus levels are the only variable independently 180
associated with this response. 181
MCT has been proposed as an alternative method to decide the prescription of R5 antagonist 182
treatment in HIV-infected patients. MCT does not give a tropism result but the sensitivity to 183
antiretroviral drug effect (7). MCT can have some potential limitations, as is the development 184
of resistance mutations to MRV during the test. Besides, during MCT the pressure of the R5 185
antagonist could lead to a X4 virus switch. However, no deleterious effects after administering 186
a rescue therapy based on the MCT result have been observed (8). Current studies to address 187
the safety of MCT and development of resistance are in progress at the moment. On the other 188
hand, MCT can overcome the limitations of phenotype and genotype tropism tests which, due 189
to high or low sensitivity in detecting X4-tropic strains, respectively, can render variable rates 190
of discordant results with the virological response after MRV exposure (6, 9). Besides, this 191
clinical approach is easy, cheap and can be performed on subjects with <1000 HIV-RNA 192
copies/mL. Another advantage is that a non-reportable result cannot be obtained when using 193
MCT. All these characteristics make MCT an attractive model to analyze the factors associated 194
with the response after a R5 antagonist exposure. 195
According to this model a high proportion of HIV-infected patients (73.3%) could be treated 196
with a combined antiretroviral therapy (cART) which includes MRV. These results together 197
with the different frequencies of response which depend on the treatment category (naïve 198
versus pretreated patients) have important clinical implications. The fact that the positive 199
response frequencies are higher in naïve subjects and are associated with higher CD4+ T-cells 200
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levels, strongly suggests the use of a cART that includes a R5 antagonist as a first line HIV-201
infection treatment, due to as it is well known R5 tropic virus predominate early in the 202
infection and X4 tropic virus appear in late infection in 50% of the patients (11). These results 203
agree with the clinical trials that show non-inferiority at undetectability (<50 HIV-RNA 204
copies/mL) of a cART which includes MRV versus Efavirenz in naïve patients (2, 20, 21). 205
On the other hand, to know what factors are associated with virological response after MRV 206
short-term exposure will be particularly interesting in order to optimize R5 antagonist 207
treatment and design strategies to revert factors associated with the absence of response. In a 208
first attempt, CD4+ T-cell levels was the only variable independently associated with the MCT 209
response. These results agree with previous studies that showed CD4+ T-cell levels were 210
associated with Trofile results (15, 23). In these studies, Trofile did not communicate the 211
amount of X4 viraemia, thus the extent to which the X4 viral load influenced the tropism result 212
was unknown. However, in a subgroup of consecutive patients where X4 and R5 virus levels 213
were available, only the X4 virus levels were associated with virological response after being 214
adjusted by CD4+ T-cell levels and type of transmission. This outcome was reproduced when 215
the MCT result was not considered as categorical but as a continuous variable expressed as 216
viral load changes after an eight day MRV exposure. This result may explain the association of 217
high frequencies of response in naïve patients, who tended to have lower X4 virus levels and 218
higher CD4 T-cells levels compared to pretreated patients and in general in those patients 219
with high CD4+ T-cell levels. In fact an inverse correlation was observed between CD4 T-cell 220
and X4 virus levels (r=-0.499; p<0.0001, Pearson test). These results agree with the classic 221
concept of syncitial inducing viral strains associated with the proportion of patients with 222
disease progression and lower CD4 T-cell levels (11). 223
One limitation of our results is the fact of what we have called X4 virus levels is not actually the 224
X4 viral load present in the patient’s peripheral blood but the viral load of the supernatant that 225
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is a result of multiple rounds of infection of the U87-X4 cell line with the patients’ virus isolate. 226
We believe that other methods such as ultra-deep sequencing should be informative in this 227
respect (22), as no quantitative information can be obtained from Trofile. However, the results 228
shown in this work suggest the use of the X4 viral load supernatant as a good surrogate marker 229
for X4 viraemia. It is important to note that no association was found between R5 virus levels 230
and the virological response after MCT. This indicates that the R5 tropic component of the 231
patient’s total viral load is not involved in virological response to R5 antagonists. This means 232
that a MCT positive patient will have very low X4 virus levels, and then the changes in viraemia 233
during MCT will resemble changes in R5 viraemia, the predominant one in this type of patients. 234
While a MCT negative patient has variable X4 virus levels, then the R5 viraemia decreases 235
during MCT will probably be accompanied by increases in X4 viraemia after the R5 antagonist 236
exposure, which will lead to no changes in the final viral load after the test. Thus factors 237
associated with the high levels of X4-tropic viruses at baseline could be associated with the 238
absence of virological response to R5 antagonists. Findings in previous works suggest the 239
importance of X4 density in this process (13). In fact, X4 density on CD4+ T-cell surface has 240
been associated with the emergence of X4-tropic strains during the course of HIV-infection (5). 241
These hypotheses also support the belief that the changes in T-cell subsets proliferation rates 242
which differentially express R5 and X4 favor X4 tropic virus expansion at lower CD4+ T-cell 243
levels (18). 244
In summary, our results demonstrate for the first time that X4 tropic virus levels are 245
independently associated with the absence of response to R5 antagonist therapy. The 246
association of high X4 tropic and low CD4+ T-cell levels agrees with the option of early R5 247
antagonist treatment. Further studies are needed to analyze which factors are associated with 248
the expansion of X4-tropic strains in order to optimize treatment with R5 antagonists and to 249
look for immunotherapeutic strategies to avoid the co-receptor switch during the course of 250
HIV infection. 251
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ACKNOWLEDGEMENTS 253
This study has been supported by Redes Temáticas de Investigación en SIDA (ISCIII RETIC 254
RD06/0006/0021, RD06/0006/0035 and RD06/0006/1004) and by Fondo de Investigación 255
Sanitaria grants PI07/0976 and PS09/01595; and by the Fondos Europeos para el Desarrollo 256
Regional (FEDER). SFM and ERM have grants from the Fondo de Investigaciones Sanitarias 257
(CD10/00382 and CP08/00172, respectively). 258
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REFERENCES 261
262
1. Berger, E. A., R. W. Doms, E. M. Fenyo, B. T. Korber, D. R. Littman, J. P. Moore, Q. J. 263 Sattentau, H. Schuitemaker, J. Sodroski, and R. A. Weiss. 1998. A new classification 264 for HIV-1. Nature 391:240. 265
2. Cooper, D. A., J. Heera, J. Goodrich, M. Tawadrous, M. Saag, E. Dejesus, N. Clumeck, 266 S. Walmsley, N. Ting, E. Coakley, J. D. Reeves, G. Reyes-Teran, M. Westby, E. Van Der 267 Ryst, P. Ive, L. Mohapi, H. Mingrone, A. Horban, F. Hackman, J. Sullivan, and H. 268 Mayer. 2010. Maraviroc versus efavirenz, both in combination with zidovudine-269 lamivudine, for the treatment of antiretroviral-naive subjects with CCR5-tropic HIV-1 270 infection. J Infect Dis 201:803-13. 271
3. Chueca, N., C. Garrido, M. Alvarez, E. Poveda, J. de Dios Luna, N. Zahonero, J. 272 Hernandez-Quero, V. Soriano, C. Maroto, C. de Mendoza, and F. Garcia. 2009. 273 Improvement in the determination of HIV-1 tropism using the V3 gene sequence and a 274 combination of bioinformatic tools. J Med Virol 81:763-7. 275
4. Dorr, P., M. Westby, S. Dobbs, P. Griffin, B. Irvine, M. Macartney, J. Mori, G. Rickett, 276 C. Smith-Burchnell, C. Napier, R. Webster, D. Armour, D. Price, B. Stammen, A. 277 Wood, and M. Perros. 2005. Maraviroc (UK-427,857), a potent, orally bioavailable, and 278 selective small-molecule inhibitor of chemokine receptor CCR5 with broad-spectrum 279 anti-human immunodeficiency virus type 1 activity. Antimicrob Agents Chemother 280 49:4721-32. 281
5. Fiser, A. L., T. Vincent, N. Brieu, Y. L. Lin, P. Portales, C. Mettling, J. Reynes, and P. 282 Corbeau.2010. High CD4+ T-Cell Surface CXCR4 Density as a Risk Factor for R5 to X4 283 Switch in the Course of HIV-1 Infection. J Acquir Immune Defic Syndr. [Epub ahead of 284 print]. 285
6. Genebat, M., E. Ruiz-Mateos, A. Gonzalez-Serna, I. Pulido, M. A. Munoz-Fernandez, 286 S. Ferrando-Martinez, and M. Leal. 2011. Discordance rates between Trofile test and 287 short-term virological response to maraviroc. Antiviral Res 89:182-5. 288
7. Genebat, M., E. Ruiz-Mateos, J. A. Leon, A. Gonzalez-Serna, I. Pulido, I. Rivas, S. 289 Ferrando-Martinez, B. Sanchez, M. A. Munoz-Fernandez, and M. Leal. 2009. 290 Correlation between the Trofile(R) test and virological response to a short-term 291 maraviroc exposure in HIV-infected patients. J Antimicrob Chemother 64:845-9 292
8. Genebat, M., E. Ruiz-Mateos, I. Pulido, A. Gonzalez-Serna, A. Garcia-Perganeda, G. 293 Mendez, M. C. Romero-Sanchez, S. Ferrando-Martinez, and M. Leal. 2010. Long-term 294 immunovirogical effect and tolerability of a maraviroc-containing regimen in routine 295 clinical practice. Curr HIV Res 8:482-6. 296
9. Gonzalez-Serna, A., M. Leal, M. Genebat, M. A. Abad, A. Garcia-Perganeda, S. 297 Ferrando-Martinez, and E. Ruiz-Mateos. 2010.TROCAI (tropism coreceptor assay 298 information): a new phenotypic tropism test and its correlation with Trofile enhanced 299 sensitivity and genotypic approaches. J Clin Microbiol 48:4453-8. 300
10. Hammer, S. M., J. J. Eron, Jr., P. Reiss, R. T. Schooley, M. A. Thompson, S. Walmsley, 301 P. Cahn, M. A. Fischl, J. M. Gatell, M. S. Hirsch, D. M. Jacobsen, J. S. Montaner, D. D. 302 Richman, P. G. Yeni, and P. A. Volberding. 2008. Antiretroviral treatment of adult HIV 303 infection: 2008 recommendations of the International AIDS Society-USA panel. Jama 304 300:555-70. 305
on March 25, 2018 by guest
http://aac.asm.org/
Dow
nloaded from
16
11. Koot, M., I. P. Keet, A. H. Vos, R. E. de Goede, M. T. Roos, R. A. Coutinho, F. 306 Miedema, P. T. Schellekens, and M. Tersmette. 1993. Prognostic value of HIV-1 307 syncytium-inducing phenotype for rate of CD4+ cell depletion and progression to AIDS. 308 Ann Intern Med 118:681-8. 309
12. Landovitz, R. J., J. B. Angel, C. Hoffmann, H. Horst, M. Opravil, J. Long, W. Greaves, 310 and G. Fatkenheuer. 2008. Phase II study of vicriviroc versus efavirenz (both with 311 zidovudine/lamivudine) in treatment-naive subjects with HIV-1 infection. J Infect Dis 312 198:1113-22. 313
13. Lin, Y. L., P. Portales, M. Segondy, V. Baillat, C. M. de Boever, V. Le Moing, B. Reant, 314 B. Montes, J. Clot, J. Reynes, and P. Corbeau. 2005. CXCR4 overexpression during the 315 course of HIV-1 infection correlates with the emergence of X4 strains. J Acquir Immune 316 Defic Syndr 39:530-6. 317
14. Low, A. J., W. Dong, D. Chan, T. Sing, R. Swanstrom, M. Jensen, S. Pillai, B. Good, and 318 P. R. Harrigan. 2007. Current V3 genotyping algorithms are inadequate for predicting 319 X4 co-receptor usage in clinical isolates. AIDS 21:F17-24. 320
15. Moreno, Clotet, Sarria, Ortega, Leal, A. Rodriguez, and R. Sanchez-de la. 2009. 321 Prevalence of CCR5-tropic HIV-1 among treatment-experienced individuals in Spain. 322 HIV Clin Trials 10:394-402. 323
16. Poveda, E., E. Seclen, M. Gonzalez Mdel, F. Garcia, N. Chueca, A. Aguilera, J. J. 324 Rodriguez, J. Gonzalez-Lahoz, and V. Soriano. 2009. Design and validation of new 325 genotypic tools for easy and reliable estimation of HIV tropism before using CCR5 326 antagonists. J Antimicrob Chemother 63:1006-10. 327
17. Raymond, S., P. Delobel, M. Mavigner, M. Cazabat, C. Souyris, K. Sandres-Saune, L. 328 Cuzin, B. Marchou, P. Massip, and J. Izopet. 2008. Correlation between genotypic 329 predictions based on V3 sequences and phenotypic determination of HIV-1 tropism. 330 AIDS 22:F11-6. 331
18. Ribeiro, R. M., M. D. Hazenberg, A. S. Perelson, and M. P. Davenport. 2006. Naive and 332 memory cell turnover as drivers of CCR5-to-CXCR4 tropism switch in human 333 immunodeficiency virus type 1: implications for therapy. J Virol 80:802-9. 334
19. Schurmann, D., G. Fatkenheuer, J. Reynes, C. Michelet, F. Raffi, J. van Lier, M. 335 Caceres, A. Keung, A. Sansone-Parsons, L. M. Dunkle, and C. Hoffmann. 2007. 336 Antiviral activity, pharmacokinetics and safety of vicriviroc, an oral CCR5 antagonist, 337 during 14-day monotherapy in HIV-infected adults. AIDS 21:1293-9. 338
20. Sierra-Madero, J., G. Di Perri, R. Wood, M. Saag, I. Frank, C. Craig, R. Burnside, J. 339 McCracken, D. Pontani, J. Goodrich, J. Heera, and H. Mayer. 2010. Efficacy and safety 340 of maraviroc versus efavirenz, both with zidovudine/lamivudine: 96-week results from 341 the MERIT study. HIV Clin Trials 11:125-32. 342
21 A. Mills, D. Mildvan, D. Podzamczer, G. Fätkenheuer, M. Leal, S. Than, S. Valluri, C. 343 Craig, J. Heera, S. Portsmouth. 2010. Once-daily maraviroc (MVC) + 344 atazanavir/ritonavir (ATV/r) vs. emtricitabine/tenofovir (TDF/FTC) + ATV/r in 345 treatment naïve patients: a week 24 planned interim analysis. XVIII International AIDS 346 Conference, Vienna, Austria. Abstract THLBB203. 347
22 Swenson, L. C., T. Mo, W. W. Dong, X. Zhong, C. K. Woods, M. A. Jensen, A. Thielen, 348 D. Chapman, M. Lewis, I. James, J. Heera, H. Valdez, and P. R. Harrigan. 2011. Deep 349 sequencing to infer HIV-1 co-receptor usage: application to three clinical trials of 350 maraviroc in treatment-experienced patients. J Infect Dis 203:237-45. 351
23. Waters, L., S. Mandalia, P. Randell, A. Wildfire, B. Gazzard, and G. Moyle. 2008. The 352 impact of HIV tropism on decreases in CD4 cell count, clinical progression, and 353 subsequent response to a first antiretroviral therapy regimen. Clin Infect Dis 46:1617-354 23. 355
24. Whitcomb, J. M., W. Huang, S. Fransen, K. Limoli, J. Toma, T. Wrin, C. Chappey, L. D. 356 Kiss, E. E. Paxinos, and C. J. Petropoulos. 2007. Development and characterization of a 357
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novel single-cycle recombinant-virus assay to determine human immunodeficiency 358 virus type 1 coreceptor tropism. Antimicrob Agents Chemother 51:566-75. 359
25. Wilkin, T. J., M. B. Goetz, R. Leduc, G. Skowron, Z. Su, E. S. Chan, J. Heera, D. 360 Chapman, J. Spritzler, J. D. Reeves, R. M. Gulick, and E. Coakley. 2011. Reanalysis of 361 Coreceptor Tropism in HIV-1-Infected Adults Using a Phenotypic Assay with Enhanced 362 Sensitivity. Clin Infect Dis 52:925-8. 363
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Table 1. Baseline characteristics of the patients (n=90)
Characteristic Value
Male sex, n (%) 71 (79)
Age (years) 42 [36–46]
Sexual transmission, n (%) 47 (52)
IDUa
transmission, n (%) 35 (39)
Naïve subjects, n (%) 19 (21)
Time since diagnosis (years) 15 [3-19]
HCV PCR+b, n (%) 31 (34)
CD4+ counts (cells/mL) 309 [175–459]
Plasma VLc (Log HIV-RNA copies/mL) 4.5 [3.7–5.0]
a IDU, intravenous drug use. b
Positive PCR for hepatitis C virus. c VL, Viral Load. Values other than number (percent) are expressed as 368
medians (interquartile range [IQR]). Values other than number (percent) are expressed as medians (interquartile range [IQR]). 369
370
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371
372
Table 2. Factors associated with the virological response after MCT in all the study subjects (n=90).
MCT positive
(n=65)
MCT negative
(n=25)
Bivariate analysis*
p; O.R. (C.I. 95%)
Multivariate analysis**
p; O.R. (C.I. 95%)
Male sex, n (%) 50/65 (77) 21/25 (84) 0.464; 0.635 (0.188–2.140) N.A.¤
Age (years) 42 [35 – 46] 43 [37 – 47] 0.677; 1.009 (0.967–1.054) N.A.
Naïve subjects, n (%) 18/65 (28) 1/25 (4) 0.036; 9.191 (1.157–73.048) 0.283; 3.857 (0.328–45.364)
Sexual transmission, n (%) 40/65 (62) 7/25 (28) 0.006; 4.114 (1.505–11.250) 0.406; 1.767 (0.462–6.765)
IDU† transmission, n (%) 22/65 (34) 13/25 (52) 0.117; 0.472 (0.185–1.206) N.A.
Time since diagnosis (years) 12 [2 – 19] 16 [12 – 19] 0.068; 0.944 (0.888–1.004) 0.890; 1.007(0.911–1.114)
HCV PCR+¥ , n (%) 22/65 (34) 9/25 (36) 0.847; 0.910 (0.347–2.387) N.A.
CD4+ counts (cells/mL) 355 [226 – 465] 149 [21 – 320] 0.001; 1.006 (1.002–1.009) 0.011; 1.004 (1.001–1.008)
Plasma VL¶ (Log HIV-RNA
cop./mL)
4.4 [3.7 – 4.9] 4.6 [3.5 – 5.2] 0.557; 0.861 (0.521–1.421) N.A.
*Bivariate and **multivariate analysis were performed using a logistic regression model, variables showing a p<0.1 in the bivariate analysis were included in the 373
multivariate analysis. In the multivariate analysis variables with a p<0.05 were considered statistically significant. ¤ Not applicable. †Injecting drug user. ¥PCR positive for 374
Hepatitis C Virus. ¶VL: Viral load. Values other than number (percent) are expressed as medians (interquartile range [IQR]). 375
376
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377
378
Table 3. Factors associated with the virological response after MCT (n=57).
MCT positive
(n=39)
MCT negative
(n=18)
Bivariate analysis*
p; O.R. (C.I. 95%)
Multivariate analysis**
p; O.R. (C.I. 95%)
Male sex, n (%) 31/39 (80) 15/18 (83) 0.733; 0.775 (0.179–3.348) N.A.¤
Age (years) 42 [36 – 45] 41 [27 – 46] 0.500; 1.017 (0.969–1.067) N.A.
Naïve subjects, n (%) 9/39 (23) 0/18 (0) N.D.€ N.A.
Sexual transmission, n (%) 22/39 (56) 4/18 (22) 0.021; 4.529 (1.261–16.271) 0.221; 19.832 (0.165–2383.7)
IDU† transmission, n (%) 14/39 (36) 9/18 (50) 0.315; 0.560 (0.181–1.737) N.A.
Time since diagnosis (years) 13 [2 – 19] 17 [12 – 18] 0.081; 0.929 (0.855–1.009) 0.428; 1.142 (0.822–1.587)
HCV PCR+¥, n (%) 11/39 (28) 6/18 (33) 0.694; 0.786 (0.236–2.616) N.A.
CD4+ counts (cells/mL) 349 [223 – 432] 105 [14 – 181] <0.0001; 1.010 (1.005–1.015) 0.920; 1.001 (0.989–1.012)
Plasma LogVL¶ (HIV-RNA
cop./mL)
4.6 [4.2 – 5.0] 4.9 [3.7 – 5.2] 0.626; 0.840 (0.415–1.696) N.A.
LogX4VL&
(HIV-RNA cop./mL) 2.8 [1.7 – 3.2] 6.6 [5.4 – 7.6] <0.0001; 0.115 (0.035–0.374) 0.007; 0.096 (0.017 – 0.535)
LogR5VL§ (HIV-RNA cop./mL) 6.6 [5.9 – 7.6] 6.7 [5.7 – 7.4] 0.939; 1.016 (0.675–1.529) N.A.
*Bivariate and **multivariate analysis were performed using a logistic regression model, variables showing a p<0.1 in the bivariate analysis were included in the 379
multivariate analysis. In the multivariate analysis variables with a p<0.05 were considered statistically significant. ¤ Not applicable. € Not determined; this contrast was 380
not possible due to the low number of patients. †Injecting drug user. ¥PCR positive for Hepatitis C Virus. ¶ VL: Viral load. & HIV-RNA copies/mL in the U87-CXCR4 cell line 381
well supernatant. § HIV-RNA copies/mL in the U87-CCR5 cell line well supernatant. In this analysis only patients with X4 and R5 virus levels available were included 382
(n=57). Values other than number (percent) are expressed as medians (interquartile range [IQR]). 383
384
385
386
387
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389
390
Table 4. Factors associated with viral load changes after MCT (n=51).
Changes in VL after MCT
Bivariate analysis*
p; B (C.I. 95%)
Multivariate analysis**
p; B (C.I. 95%)
Male sex, n (%) 0.698; -0.118 (-0.727 – 0.491) N.A. ¤
Age (years) 0.961; 0.000 (-0.019 – 0.018) N.A.
Naïve subjects, n (%) 0.297; 0.302 (-0.273 – 0.877) N.A.
Sexual transmission, n (%) 0.022; 0.469 (0.042 – 0.897) 0.672; 0.077 (-0.286 – 0.440)
IDU† transmission, n (%) 0.205; -0.289 (-0.742 – 0.164) N.A.
Time since diagnosis (years) 0.169; -0.021 (-0.050 – 0.009) N.A.
HCV PCR+¥, n (%) 0.243; -0.289 (-0.779 – 0.202) N.A.
CD4+ counts (cells/mL) 0.001; 0.002 (0.001 – 0.003) 0.346; 0.001 (-0.001 – 0.002)
Plasma LogVL¶ (HIV-RNA
cop./mL)
0.523; -0.120 (-0.497 – 0.256) N.A.
LogX4VL&
(HIV-RNA cop./mL) <0.0001; -0.263 (-0.338 – -0.187) <0.0001; -0.234 (-0.322 – -0.146)
LogR5VL§ (HIV-RNA cop./mL) 0.227; -0.089 (-0.253 – 0.074) N.A.
*Bivariate and **multivariate analysis were performed using a linear regression model, variables showing a p<0.1 in the bivariate analysis were 391
included in the multivariate analysis. In the multivariate analysis variables with a p<0.05 were considered statistically significant. ¤ Not applicable. 392
†Injecting drug user. ¥PCR positive for Hepatitis C Virus. ¶ VL: Viral load. & HIV-RNA copies/mL in the U87-CXCR4 cell line well supernatant. § HIV-RNA 393
copies/mL in the U87-CCR5 cell line well supernatant. In this analysis only patients with X4 and R5 virus levels available and with >1000 HIV RNA 394
copies/mL were included (n=51). Values other than number (percent) are expressed as medians (interquartile range [IQR]). 395
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397
Figure legends 398
399
Figure 1. Frequency of virological response after MCT. a) Percentage of subjects with a 400
virological response after a short-term MRV exposure (MCT positive). Black bar represents the 401
percentage of MCT positive subjects in the total population. The population was also split in 402
pretreated and naïve patients (grey bars). b) Percentage of MCT positive subjects depending 403
on different baseline CD4+ T-cell strata, values in the “X” axis represent number of cells/ µL. 404
Black bars depict the total number of subjects, while grey bars represent only naïve subjects. 405
We did not observed any naïve subjects in the lower CD4+ T-cell strata, n=0 (<100 and 100-199 406
cells/µL). 407
408
Figure 2. X4 and R5 virus levels and CD4+ T-cell levels depending on treatment category. Log 409
viral load in the U87-X4 and U87-R5 well supernatant were used to express X4 and R5 virus 410
levels depending on: a) MCT positive patients (n=39) presented significantly lower X4 virus 411
levels than MCT negative patients (n=18). b) R5 virus levels were similar in patients with 412
different response after MCT. c) Naïve patients (n=9) presented a trend to have lower X4 virus 413
levels than pretreated patients (n=48). d) In addition, naïve patients (n=19) showed higher 414
CD4+ T-cell levels compared to pretreated patients (n=71) e) Patients with the lower CD4+ T-415
cell strata (<100 (n=11) and 100-199 CD4+ T-cell/µL (n=11)) presented significantly higher X4 416
virus levels than patients with higher CD4+ T-cell strata (200-350 (n=15) and >350 (n=20)). f) 417
On the contrary, R5 virus levels were independent of the CD4+ T-cells strata. Black bar 418
represents the median. Mann-Whitney U-test was used. 419
420
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