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Keywords:

  • HIV protease gene;
  • HIV reservoir;
  • HIV-1 therapy;
  • resistance mutation;
  • ritonavir-boosted darunavir monotherapy

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Objective

To assess the changes on the HIV protease gene in plasma and peripheral blood mononuclear cell (PBMC) compartments during viral replication episodes in patients on boosted-darunavir monotherapy (mtDRV/rtv).

Methods

A prospective study was carried out in which adult HIV-1-infected patients who started mtDRV/rtv after viral suppression for ≥6 months with no major darunavir-related resistance mutations were enrolled. Patients with two consecutive plasma HIV RNA measurements > 200 HIV-1 RNA copies/mL were considered as having virological failure (VF), while patients with two consecutive plasma HIV RNA measurements > 50 copies/mL without meeting the VF criteria were considered to have virological rebound (VR). HIV protease genotypic profiles from plasma and PBMCs were performed at baseline and at VF and VR episodes.

Results

One hundred and fifty patients were included in the study, with overall VF and VR rates of 14% (n = 21) and 14.7% (n = 22), respectively. No major darunavir resistance mutations were observed in the plasma or PBMC samples. Circulating and cell-associated viruses showed a wild-type protease gene sequence in 54% and 23% of patients, respectively while the remainder patients only harboured minor protease inhibitor-associated mutations. Full concordance between plasma RNA and PBMC DNA protease genotypes was found in 23% of the sequences.

Conclusions

No darunavir-related mutations were found in patients with VF or VR, either in plasma or in PBMCs; thus, simplification to mtDRV/rtv does not comprise future antiretroviral treatment options.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Protease inhibitor (PI) simplification strategies, despite having demonstrated similar or only slightly lower efficacy compared with standard triple therapy in long-term virologically suppressed patients [1], remain controversial. The major concern with this option is whether the higher incidences of blips and low-level viraemia episodes observed are associated with a higher risk of the emergence of resistance mutations and thus the loss of future therapeutic options [2-5]. Moreover, whether the long-lived cellular HIV reservoir is responsible for these transient viraemic episodes during PI monotherapy is still unexplored. It has been suggested that transient viraemia arises from long-lived productively infected cells infected prior to therapy initiation, while low-level replication for a certain length of time could replenish and be conserved in the cellular reservoir [6, 7]. To date, most PI monotherapy studies have evaluated the emergence of resistance mutations at virological failure (VF) in plasma [8, 9]; no studies have examined paired cell-free and cell-associated viruses at both baseline and VF. Thus, our aim was to evaluate the changes that viral replication episodes induce in the HIV protease gene (HIV-pr) in both the plasma and the peripheral blood mononuclear cell (PBMC) compartments, and to assess whether the cellular reservoir might be compromised for future treatment options after failed boosted darunavir monotherapy (mtDRV/rtv) or even after any viral rebound (VR).

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Study population and design

The prospective MonDAR cohort study (ClinicalTrials.gov:NTC01606722) has consecutively included adult HIV-1-infected patients starting mtDRV/rtv who were followed for 2 years. At baseline, all patients had been virally suppressed (< 50 HIV-1 RNA copies/mL) for at least 6 months and did not have previous VF on PI-based regimens or a genotypic resistance test showing either a major or more than three minor resistance mutations to DRV/rtv [10].

Clinical and analytical assessments were performed at baseline, at 1 month and quarterly thereafter, and at each VR. HIV RNA was measured using the COBAS HIV-1 Test v2.0 (Roche Diagnostics, Barcelona, Spain). The HIV DNA proviral load was determined as previously described [11]. Patients with VF, defined as two consecutive viral loads > 200 copies/mL, or VR episodes, defined as two consecutive measurements > 50 copies/mL without the VF criteria, were selected. Paired blood samples were collected at baseline and at each visit. The study was designed and conducted according to the Declaration of Helsinki, approved by the Coordinating Committee on Ethics in Biomedical Research of Andalucía and the Spanish Agency for Medicines and Healthcare Products. All patients provided informed consent.

HIV-1 protease genotyping assays

Changes in HIV-pr were analysed at VF and VR in both cell-free and cell-associated viruses. HIV-pr amplification from RNA was performed by one-step reverse transcriptase polymerase chain reaction (retro-PCR; Super-Script III; Invitrogen, Madrid, Spain), with the following primers: forward, 5′-AAAGGGCTGTTGGAAATGTG-3′ and reverse, 5′-AAGTGCAACCAATCTGAGTC-3′. Retro-PCR conditions consisted of an initial step of 30 min at 45°C and 2 min at 94°C, followed by 40 cycles of 15 s at 94°C, 30 s at 55°C and 90 s at 68°C, and a final step of 5 min at 68°C.

For HIV DNA genotyping, after total DNA isolation from PBMCs, HIV-pr amplification was assessed by nested PCR (Expand High Fidelity PCR System; Roche Diagnostics). The first PCR was performed with the following primers: forward, 5′-GCCAAAAATTGCAGGGCCCCTAGGA-3′ and reverse, 5′-CATGCCATGGCTGGCTTTAATTTTACTGGTACAGTC-3′. A second PCR round was then performed using the same primers as used in the retro-PCR. The temperature programme consisted of 2 min at 94°C, followed by 35 cycles of 15 s at 94°C, 30 s at 53°C and 45 s at 72°C, and a final extension step of 7 min at 72°C.

The products from both retro-PCR and nested PCR were purified using the QIAquik Spin Kit (Qiagen, Valencia, CA, USA) and sequenced (Secugen, Madrid, Spain). HIV-pr sequences were analysed using the Stanford HIV-1 Drug Resistance Database (Standford University, Standford, CA, USA) and protease resistance mutations were selected according to the 2009 guidelines of the International AIDS Society [10].

Statistical analyses

Results were expressed as median and interquartile range (IQR; quartiles 1 to 3). Comparisons between groups were performed using the Kruskal−Wallis test or analysis of variance (anova) for continuous variables and the χ2 or Fisher's exact test for categorical variables. Statistical analyses were performed with spss software (v. 15.0; IBM, Madrid, Spain).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Study population

A total of 150 patients were classified as having VF (n = 21), VR (n = 22) or success on mtDRV/rtv (SMt; n = 107). Baseline characteristics were similar among the three groups, including the rate of previous VF on PI-based treatment and time on treatment containing PIs (data not shown). No patient presented previous VF on regimens containing DRV/rtv.

Changes in the protease gene in HIV RNA and HIV DNA at VF

At baseline, as the median duration of viral suppression before mtDRV/rtv initiation was 44 months (IQR 24−89 months), only 10 plasma protease genotypic tests were available (Table 1). HIV-pr from proviral DNA was characterized at baseline in 19 of 21 subjects with VF; two additional samples were not amplified as no HIV DNA was detected by real-time PCR (Table 1). Only four patients (23.5%) presented a wild-type (wt) protease genotype in their proviral genomes; L63P (n = 9) and V77I (n = 5) were the most frequent minor mutations detected. No major mutations were found in the protease gene in provirus.

Table 1. Genotypic resistance test for the HIV-1 protease gene at virological failure in plasma and peripheral blood mononuclear cells (PBMCs)
BaselineVirological failure
PatientCirculating virus (HIV RNA)Cell-associated virus (HIV DNA)Circulating virus (HIV RNA)Cell-associated virus (HIV DNA)Viral load (copies/mL)
  1. Viral loads are expressed as copies/mL.

  2. wt, wild-type gene; na, no amplification.

  3. *No sample available.

  4. Mean of two consecutive viral loads > 200 copies/mL.

 1L63PwtI64VI64V457
 2*M36II62VM36I943
 3*wtwtI64V1707
 4*L63P, V77IL63P, I64VL10V, I64V303
 5*M36I, I64VwtI64V9154
 6L10I, D30NwtI64VI64V54 785
 7*I62V, L63P, I64L, V77IL63P, V77IL63P,V77I17 125
 8*L63P, V77I*I62V, L63P, A71V, V77I4615
 9I62V, L63P, I93LI62VI62VI62V62 550
10L63P, A71T, G73S, L90ML10V, L63P, I64LwtL10V, L63P, I64L557
11wtI64VwtI64V6429
12wtnaL63PL63P498
13**wtM36I, I64V258
14**wtM36I4414
15*L63PwtL63P14 922
16L10I, D60E, I62V, L90M, I93LwtL63P, A71VD60E, I62V, L63P, I64L, A71V297
17L63PI62V, L63P, I64Vwtwt385
18L63Pnawtwt769
19*L63PwtL63P248
20L63PK20R, L63P, V77IwtD60E, L63P, V77I367
21*L63P, V77IwtL63P, V77I1379

At VF, total HIV DNA levels increased by a median of 142 (IQR 59−386) copies/106 PBMCs. At this time, 20 of 21 patients had a genotypic resistance test available for plasma HIV RNA; 60% of these patients (n = 12) harboured wt HIV-pr (Table 1). The remaining eight patients did not present any major PI resistance mutations, although minor mutations were detected, mainly L63P (n = 4) and I64V (n = 3). In contrast, 19 patients (90.5%) presented at least one minor resistance mutation or polymorphism in the protease gene of provirus at VF, the most frequent changes being found at positions 63 (n = 9) and 64 (n = 8) (Table 1).

Furthermore, a concordance rate of 35% (seven of 20) was found for the protease genotype between plasma HIV RNA and proviral HIV DNA samples at VF, while the concordance rate for the proviral HIV DNA protease sequence between baseline and VF was 39% (seven of 17).

Changes in the protease gene in HIV RNA and HIV DNA at VR

Of 22 patients with VR, 13 patients (59.1%) had at least one plasma genotypic resistance test before mtDRV/rtv, with L63P (n = 5) and M36I (n = 4) being the most frequent minor mutations observed. Additionally, 20 of 22 baseline PBMC samples were available for HIV DNA sequencing, which was performed successfully in 14 of these samples, no HIV DNA being detected in six samples. We found the L63P minor mutation in 85.7% of the analysed genotypes (n = 12) at baseline (Table 2).

Table 2. Genotypic resistance test for the HIV-1 protease gene at viral rebound in plasma and peripheral blood mononuclear cells (PBMCs)
BaselineViral rebound
PatientCirculating virus (HIV RNA)Cell-associated virus (HIV DNA)Circulating virus (HIV RNA)Cell-associated virus (HIV DNA)Viral load (copies/mL)
  1. Viral loads are expressed as copies/mL.

  2. wt, wild-type gene; na, no amplification.

  3. *No sample available.

  4. Mean of two consecutive viral loads > 50 copies/mL but at least one < 200 copies/mL.

22M36InaV77IG16E, M36I, L63S68
23*wtwtwt356
24*L63PL63PI62V, M36I695
25*nawtwt674
26M36I, L63PM36I, L63PwtM36I, L63P96
27*I62V, L63P, I64V, G73SL63P, V82FM36I, D60E, I62V, L63P, V77I519
28L63PnaV77Iwt121
29wtL63PV77IL10I, M36I, I62V, I64V175
30wtL63P, I64V, V82AwtL63P, V77I64
31*I62V, L63P, I64VL10V, I64VI62V, L63P, I64V341
32L63PL63PI64Vwt307
33*L63PI64Vwt298
34*M36I, L63PwtM36I, L63P240
35*L63PI64Vna126
36L10V, M46L, L90M*wtI62V, L63P, I64V126
37V77IL63Pwtwt177
38M36I, L63PL63PwtM36I, I62V, L63P243
39**NAL63P1621
40L10V M46I V77Inawtna82
41M36InaD60E, L63P, V77Iwt7168
42wtI62V, L63P, I64VV77I*137
43L63PnawtV77I397

At the time of VR, HIV RNA plasma samples from all patients were available for sequencing, which showed that a total of 10 subjects (47.6%) had wt HIV-pr in circulating plasma viruses. The remainder of the patients did not present any major PI resistance mutations, and the most frequent new minor mutations that emerged were I64V (n = 4) and V77I (n = 5). Similarly, in HIV DNA, seven (36.8%) patients had wt HIV-pr, and the most frequent new minor mutation or polymorphism found was L63P (n = 8). Overall, the proviral load after these VRs increased by a median of 109 (IQR 20-204) copies/106 PBMCs.

The concordance rate found between plasma HIV RNA and proviral HIV DNA protease genotypes in this group of patients at VR was 16.7% (three of 18), while this percentage increased to 30.8% (four of 13) when baseline and VR protease genotype profiles from PBMC samples were compared.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In our MonDAR cohort, only 14% of patients failed mtDRV/rtv, while 14.6% showed VR, without the emergence of major darunavir resistance mutations. Indeed, during these episodes, cell-free viruses and cell-associated viruses showed wt HIV-pr in 54% and 23% of patients, respectively. Only a few polymorphisms or minor mutations emerged, which did not comprise susceptibility to any PI, which is in agreement with the findings of previous studies [8, 9].

However, HIV-pr viruses from the plasma and PBMC compartments presented a very low concordance (23%), as observed in other studies [12-14]. Higher concordance rates would have been expected in patients with higher levels of viraemia, as the cellular reservoir in patients experiencing high replication rates of resistant strains would be replenished by these viruses, thus increasing the concordance rates between cell-free and cell-associated viruses. Indeed, identical HIV-pr sequences were observed in both compartments in the three subjects with the highest viral loads at VF.

Our findings support the hypothesis that, at the early stages of VR, the resistant strains in plasma differ from those associated with PBMCs, which other authors have demonstrated to be the case in the 2 years following VR [15]. In fact, the increases observed in total HIV DNA levels support the idea of an increase in the rates of integration and replenishment of the cellular reservoir in these patients [11], and would also explain the low concordance between archived and circulating viruses.

To summarize, the particularly strong binding of darunavir to HIV-pr probably results in a higher intrinsic genetic barrier than that observed with other boosted PIs [16, 17]. This simplification strategy therefore does not jeopardize future treatment options after VF, or even VR, as no major or minor resistance mutations associated with reduced susceptibility to darunavir or other PIs emerged either in plasma HIV RNA or in cellular HIV DNA.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We acknowledge in particular the participation of the patients in this study. We are indebted to M. Rodriguez, F. Cano and R. Martin for their help with specimen processing.

Conflicts of interest: LFL-C and PV have received unrestricted grants for research and honoraria for speaking at symposia from Abbott Laboratories (Spain), Bristol-Myers Squibb, GlaxoSmithKline, Gilead Sciences, Janssen-Cilag España, Merck Sharp & Dohme España, Roche Pharma SA, and ViiV Healthcare. All other authors have no conflicts of interest to declare.

Financial disclosure: This work was supported by a grant from the Dirección Gerencia del Servicio Andaluz de Salud, Consejería de Salud, Junta de Andalucía (exp. SAS 111237).

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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