Clin Microbiol Infect 2011; 17: 928–934
The objective of this study was to address the evolution of human immunodeficiency virus type 1 (HIV-1) mutations resistant to the integrase inhibitor raltegravir after drug interruption. Thirteen HIV-1 infected patients undergoing virological failure due to the selection of raltegravir-resistant variants, who had interrupted raltegravir treatment, were enrolled. For all patients, the virological failure was associated with the selection of variants, with mutations conferring resistance to all of the drugs present in their regimens. Patients were prospectively monitored at baseline (raltegravir interruption) and every 4–24 weeks for clinical, virological and immunological parameters, including HIV-1 viraemia, CD4+ T-cell counts, and sequence analysis of the HIV-1 integrase sequence. Reversion to the wild-type HIV-1 integrase sequence genotype was observed between 4 and 36 weeks after raltegravir withdrawal in eight out of the 13 patients. Reversion was not observed in three patients. In two patients, reversion was partial at week 24 from raltegravir interruption. These results highlight that in eight out of 13 patients under treatment with raltegravir and experiencing a virological failure, HIV-1 variants harbouring mutations associated with raltegravir resistance become undetectable after drug interruption within a few weeks (in some cases, very rapidly). This occurs under different therapy regimens and in patients receiving 3TC mono-therapy. In the other patients, complete reversion of the integrase sequence is not observed, and either primary or secondary resistance mutations are fixed in the replication competent viral population in vivo also for long time, suggesting that other factors may influence this dynamic process.
Raltegravir, the prototype of a new class of anti-retroviral compounds (integrase inhibitors; INI), has been demonstrated to be an effective drug in the treatment of either naïve or experienced human immunodeficiency virus type 1 (HIV-1) infected subjects [1–4]. As for other classes of anti-retroviral drugs, the selection of variants carrying drug-resistance-associated mutations in the HIV-1 integrase sequence has been described in patients not responding to raltegravir including regimens . Moreover, in subjects maintaining raltegravir in their regimen despite the selection of resistance mutations, a detectable increase in HIV-1 resistance levels to raltegravir driven by the continuous evolution of the viral integrase sequence has been documented [5–7]. Finally, comparative analyses of the geno-phenotypic features of resistant variants selected during raltegravir treatment in vivo have indicated that the replication capacity (RC) of resistant clones is compromised in INI-resistant HIV-1 variants [8–12].
In the past two decades, studies of different classes of anti-retroviral compounds have demonstrated that viral reversion from highly resistant to fully susceptible wild-type viral variants is generally observed within a variable time in cell-free plasma virus after interruption of the compounds [13,14]. It is presently believed that the lower relative fitness of resistant variants allows the reversion to wild-type archival provirus sequences in the absence of drug-selection pressure. Of note, secondary (or compensatory) and rarely primary mutations (such as the 103N in the reverse transcriptase HIV-1 sequence) not associated with reduction of viral RC  are maintained in some cases after drug interruption, thus remaining as genotypic scars in the replicating virus. These footprints of previous failing regimens may be useful from a diagnostic point of view in either naïve or treatment-exposed patients, facilitating the determination of the most reliable historical GSS score . Recently, several studies have evaluated the genotypic and phenotypic patterns of raltegravir-resistant variants selected in vivo, elucidating the role of drug resistance and RC in patients not responding to regimens including raltegravir [8–12]. However, data on the genotypic monitoring of the evolution of raltegravir-associated resistance mutations after drug interruption are still limited. Due to the importance of increasing the therapeutic options for HIV-1 infected subjects, these results could be central to the understanding of the biology of raltegravir-resistant variants and to the diagnostic management of patients treated with raltegravir.
Patients and Methods
This research was conducted in accordance with the Declaration of Helsinki and national and institutional standards and was approved by the San Raffaele Ethical Committee.
Genotypic analyses, virological and immunological evaluation
Thirteen patients (age, 49 ± 11 years; 11 male, 2 female), who underwent virological failure due to the selection of HIV-1 (subtype B) resistant variants, and who had interrupted raltegravir treatment, were enrolled in the present study. All patients had a long history of treatment (on average 16 years of documented antiretroviral therapy), with an average duration of HIV-1 infection of 19 years.
Patients were prospectively monitored at baseline (time of raltegravir interruption) and every 4–32 weeks for clinical, virological and immunological parameters, including HIV-1 viraemia (Versant HIV-qRNA 3.0 Assay; Siemens Healthcare Diagnostics, Deerfield, IL, USA), and CD4+ T-cell counts (Fig. 1 and Table 1).
|Patient||Weeks after RAL start||Weeks after RAL interruption||RAL-associated resistance mutations||Therapy||CD4+ T cells/mm3||Copies HIV RNA/mL|
|P1||0||7||2 35 613|
|4||T206S,V72I||RAL + ETV + TDF + FTC||8||1 49 893|
|8||T206S,V72I||RAL + ETV + TDF + FTC||24||47 291|
|16||T206S,V72I||RAL + ETV + TDF + FTC||130||34 832|
|24||Y143R V165I,T206S||RAL + ETV + TDF + FTC||13||1 00 000|
|36||Y143H/C/R, V165I, V201V/I, T206S||RAL + ETV + TDF + FTC||62||91 579|
|52||Y143YH/C/R, V165I, V201V/I, T206S||19||84 372|
|56||4||V165I, V201V/I, T206S||37||2 68 771|
|P2||0||TDF + FTC||224||74|
|56||G140G/S, Q148R||RAL + ENF + TDF + FTC||206||1574|
|60||G140G/S, Q148R||RAL + ENF + TDF + FTC||213||2152|
|64||4||WT||TDF + FTC + DRV/r + ETV||223||7593|
|4||E138K, Y143K, Q148R||RAL + MVC + TDF + FTC||159||30 035|
|8||G140S,Y143R||RAL + MVC + TDF + FTC||179||51 439|
|16||T97A,E138A,Y143K||RAL + MVC + TDF + FTC||106||84 658|
|20||G140S,Q148H||RAL + DRV/r + MVC + TDF + FTC||135||32 935|
|28||G140S,Q148H||RAL + DRV/r + MVC + TDF + FTC||179||25 157|
|84||E138A,G140S,Y143H,Q148H,K156N||RAL + DRV/r + MVC + TDF + FTC||96||1 02 180|
|96||12||E138A,G140S,Y143H,Q148H,K156N||DRV/r + TDF + FTC||78||94 080|
|P4||0||TDF + FTC + ENF + LPV/r||101||8785|
|24||G140S,Q148H||RAL + ATV + ENF + MVC||22||75 391|
|48||G140S,Q148H||RAL + ATV + ENF + MVC||38||2 15 980|
|72||12||T125A,G140S,Q148H/Q||DRV/r + TDF + FTC||10||90 170|
|96||36||WT,T125A||TDF + FTC + DRV/ + ENF||4||2 75 751|
|P5||0||RAL + MVC + DRV/r||142||82 000|
|4||G140S, Q148R, G163R||RAL + MVC + DRV/r||136||72 072|
|44||G140S,Q148R,G163R||RAL + MVC + DRV/r||323||1 55 000|
|68||G140S, Q148H, G163R||RAL + DRV/r||198||2 34 901|
|104||12||WT||ENF + DRV/r + ETV + MVC||48||1 09 500|
|P6||0||70||3 93 022|
|20||T97A, Y143C||RAL + TDF + FTC + ENF||68||38 883|
|28||T97A, Y143C||RAL + TDF + FTC + ENF||85 735|
|32||L74M, T97A, Y143C/G||RAL + TDF + FTC + ENF||96||6 39 595|
|36||T97A, Y143C||RAL + TDF + FTC + DRV/r + MVC + ENF||209||642|
|52||T97A, Y143R||RAL + TDF + FTC + DRV/r + MVC + ENF||288||240|
|64||L74M, T97A, E138A,Y143C||RAL + TDF + FTC + DRV/r + MVC + ENF||281||836|
|76||L74M, T97A, E138A,Y143C||RAL + TDF + FTC + DRV/r + MVC + ENF||213||3116|
|88||L74M, T97A, E138A,Y143C||RAL + TDF + FTC + DRV/r + MVC + ENF||306||32 190|
|92||12||T97A, Y143C||3TC||180||2 77 300|
|16||G140S,Q148R||RAL + FTC + MVC + TDF||451||14 070|
|24||Y143R||RAL + ETV + 3TC||359||6497|
|32||T97A, Y143R||RAL + ETV + 3TC||376||6527|
|44||T97A, Y143R||RAL + ETV + 3TC||418||7728|
|84||4||L74M, T97A, Y143R||TDF + FTC + DRV/r||270||986|
|92||12||WT||TDF + FTC + DRV/r||272||472|
|24||T125A,E138K,G140S, Q148H||RAL + ATV + TDF + FTC||364||22 281|
|76||52||G140S, Q148H||ETV + DRV/r + MVC||494||49|
|112||88||T125A,G140S,Q148H||ETV + DRV/r + MVC||371||49|
|120||96||T125A,G140S,Q148H||ETV + DRV/r + MVC||328||2593|
|44||G140S, Q148H||RAL + DRV/r + ETV + 3TC||416||7871|
|80||G140S,Q148H,G163E||RAL + DRV/r + ETV + 3TC||372||4693|
|84||4||G140S,Q148H,G163E||TDF + FTC + DRV/r||302||2213|
|92||10||G140S,Q148H,G163E||TDF + FTC + DRV/r||370||5511|
|8||V201I,Y143S||RAL + ABC + 3TC + fAPV||620||8484|
|16||V125A,V151I,N155H,V201I||RAL + ABC + 3TC + fAPV||634||26 448|
|32||T97A,V125A,V151I,N155H,V201I||RAL + ABC + 3TC + fAPV||597||22 129|
|64||32||T97A,G143S||ETV + 3TC + DRV/r||580||569|
|96||64||G163Q||ETV + 3TC + DRV/r||970||57|
|112||80||G163Q||ETV + 3TC + DRV/r||1012||45|
|P12||0||RAL + TPV/r + 3TC||260||7220|
|24||L74M, T97AT, Y143R, V165I||RAL + TPV/r + 3TC||92||80 700|
|52||24||L74M, Y143R, V165I||DRV/r + 3TC||286||203|
|P13||0||RAL + TDF + FTC + LPV/r + AZT||250||48 616|
|36||G140S,Q148H||RAL + ETV + DRV/r||335||1 73 753|
|112||40||G140S,Q148H||TDF + FTC + DRV/r||311||43 970|
Genotypic analyses of the reverse transcriptase and protease sequences and of the env gene to evaluate the presence of mutations associated with drug resistance or changes in viral tropism, were also performed at the beginning of the raltegravir-including regimen, at virological rebound and during maintenance of raltegravir despite failure [5,9]. After raltegravir interruption (the baseline point for this study) pol and env genotypic analyses were performed at multiple time-points (every 4–40 weeks) (for Methods see Ref. [5 and 9]).
Amplification of HIV-1 integrase sequence
Viral RNA was purified using the QIAmp viral RNA mini kit (Qiagen, Valencia, CA, USA). Only one sample at each time point was processed, and clinical samples and all amplification steps were carried out using a limiting dilution strategy to minimize artificial recombination events. The integrase region spanning codons 1–288 was targeted, using the following nested-RT-PCR using primers Int1F, 5′- CAT GGG TAC CAG CAC ACA CAA AGG-3′ and Int1R, 5′-CCA TGT TCT AAT CCT CAT CCT GTC -3′ for the first PCR round, while primers Int2F 5′-GGA ATT GGA GGA AAT GAA CAA GTA GAT -3′ and Int2R 5′GCC ACA CAA TCA TCA CCT GCC ATC-3′ were used in the second PCR round. The first nested-RT-PCR reaction was performed in 50 μL using the SuperScriptTM III Platinum High-Fidelity One-Step qRT-PCR System (Invitrogen, Carlsbad, CA, USA) with the following thermal profile: 30 min at 50°C and 10 min at 95°C for 1 cycle, 1 min at 95°C, 1 min at 52°C and 1 min and 10 s at 72°C for 50 cycles followed by 10 min at 72°C. The nested PCR reaction was performed in 100 μL using the PCR SuperMix High Fidelity (Invitrogen) with the same thermal profile. Direct sequencing was performed using an ABI PRISM 3100 Genetic Analyzer® (Applied Biosystem, Foster City, CA, USA). Resistance to raltegravir was evaluated according to the Stanford database report and published ex vivo phenotypic data [8–12]. As evaluated by serial dilutions of TA-cloned (Invitrogen) reference (NL4-3 derived) amplicones spiked in plasma samples obtained from HIV-1-negative patients, this assay was demonstrated to have an analytical sensitivity of about 240 copies with subtype B variants [9,17]. The electropherogram was analysed manually by a trained virologist to guarantee that resistant variants present in a proportion higher than 5–10% in the viral population could be identified.
Results and Discussion
In the present study, 13 HIV-1 infected patients not responding to a raltegravir-containing regimen were enrolled. Virological and immunological parameters as well as the therapeutic regimens are shown in Fig. 1 and Table 1. For all patients, virological failure was associated with selection of variants with mutations conferring resistance to all of the drugs present in their regimens (data not shown). In particular, failure of response to raltegravir was associated with the selection of variants displaying different combinations of primary and secondary mutations after 4–56 weeks (Table 1). In many of them, especially in those where failure was associated with signature mutations other than the G140S + Q148H combination, a dynamic evolution of the viral population was documented, as previously described [5,9]. Despite the low number of patients and the variability in the sampling times as well as in the length of the follow-ups between patients, several important observations could be made on the evolution of the viral population after raltegravir interruption.
After failure of the raltegravir-including regimen, one patient (#1) underwent antiretroviral therapy (ART) interruption. Due to the absence of other therapeutic options two patients (patients #6 and #7) were maintained under 3TC monotherapy , while all the others switched to the best possible raltegravir-sparing regimen based on the results of genotype, viral tropism and clinical history.
In eight out of the 13 patients (Table 1, patients # 1, 2, 4, 5, 6, 7, 8 and 11), reversion to the wild-type HIV-1 integrase sequence genotype was observed after 4–36 weeks from raltegravir withdrawal. In contrast, reversion was not observed in four patients (patients # 3, 9, 10 and 13). In one patient (patient # 12) reversion was partial at week 24 from raltegravir interruption, with the Y143R mutation still present at the genotypic evaluation. In detail, both primary and secondary mutations acquired at drug failure disappeared if not present at baseline in all subjects, with the exceptions of patient # 1 (V165I not present at baseline), patient # 4 (T125A) and patient # 11 (G163Q). While the first two variants are more frequently found as polymorphisms, mutations in position 163 are rare in subtype B viruses . Although we can hypothesize that many secondary mutations underwent carryover negative selection driven by the reduction of viral fitness conferred by primary mutations, we cannot exclude that some of them have per se a direct impact on integrase efficiency and viral RC, and are very infrequently found in the circulating viruses worldwide . Additionally, a progressive substitution of the resistant variants by the wild-type viruses occurred in multiple steps in two patients (patients #6 and #11). Moreover, amino acidic reversion was associated with the reappearance of pre-raltegravir nucleotide sequences and the speed of reversion of each resistance-associated codon combination was not proportional to the number of nucleotide changes needed to revert to the wild-type integrase sequence.
We must underline, however, that the analysis of integrase region was performed by direct sequencing, thus we could describe only major variations in the composition of the replicating viral population and resistant variants may have remained undetected if present below 5–10% in the quasispecies.
The individual differences in time required to revert to a wild-type genotype may be related to different conditions or factors. An important factor could be the efficacy of ART. For patient # 9, the switch to ETV DRV/r and MVC appeared to be effective and viraemia dropped to low levels within 8 weeks. This could have influenced virus evolution, being the reason for the lack of reversion after 96 weeks of raltegravir withdrawal. On the contrary, a successful therapy (although with a slower viral decay) did not give similar results in the HIV-1 integrase sequence evolution of patient # 11; in this patient, a progressive, slow and multistep reversion to wild type was observed. Interestingly, the G143S mutation (+T97A), which has no effect on viral RC, replaced other variants associated with a lower relative RC (N155H) , before a wild-type variant still containing the G163Q mutation was selected. A similar observation can be made for patient # 6, who remained on 3TC monotherapy; in this patient, the circulating Y143C + T97A + L74M + E138A variant was rapidly substituted by the Y143C + T97A variant selected at week 24, and not with the Y143R + T97A combination, previously present and associated with lower RC .
The lack of reversion observed in other patients, including patients # 3 and # 10, where the resistant variants remained as the main circulating populations, may be due to the limited time (12 and 10 weeks, respectively) elapsed between raltegravir withdrawal and the last available genotype. However, in some subjects with identical patterns of resistance mutations, variants carrying resistance mutation became undetectable very rapidly after raltegravir interruption. As a further proof of the heterogeneous effect on HIV-1 biology of resistance-associated mutations, in one patient (patient #13) genotypic reversion to wild type was not observed even 40 weeks after raltegravir withdrawal, suggesting that in this particular patient the G140S + Q148H combination may not be associated with a reduced viral RC.
Of note, despite the modest viral RC reduction associated with raltegravir-resistant mutations in vitro [8–12], INI resistant variants became undetectable after raltegravir interruption in eight out of 13 patients under study, suggesting that the fitness reduction is sufficient to favour wild-type sequences in vivo. In this scenario, several reasons may explain the differences in time necessary to obtain a complete genotypic reversion. Firstly, the influence on viral RC associated with raltegravir-resistant mutations may differ among patients, being modulated by the genetic background of the circulating integrase sequences. Secondly, the overall HIV-1 fitness in vivo may also be influenced by changes of viral proteins other than the integrase due to multiple resistance mutations, especially in patients with a long history of antiretroviral treatment, such as those enrolled in this study. In fact, the complete or partial interruption of the drugs present in the raltegravir-including regimen, or alternatively, the complete switch to a different regimen (associated with resistance mutations located in distinct viral genes), may theoretically influence the composition of the viral population and the reselection of the variants carrying the wild-type integrase sequence. However, during the present study, all the patients that were maintained under a combination of RT and PR inhibitors did not document major variations in the reverse transcriptase and protease HIV-1 sequences (data not shown). Failure to respond to Maraviroc was associated with changes in tropism (data not shown). In those patients that received the 3TC monotherapy and in patient #8, partial reversion was documented in the protease sequence with loss of mutations associated with viral RC reduction  (in patient # 6, V32I and I47V; in patient # 7 and #8, I54L).
In conclusion, this study highlights that in patients under treatment with raltegravir and experiencing virological failure, circulating HIV-1 variants harbouring mutations associated with raltegravir resistance may rapidly be substituted by wild-type viruses after drug interruption. The reversion occurs within a few weeks (in same cases, very rapidly) and under different therapy regimens, including patients receiving 3TC monotherapy. This underlines the need for prompt and frequent genotype monitoring in patients failing to respond to raltegravir-containing regimens in order to plan future therapies. In a few patients, however, complete reversion of the integrase sequence is not observed, and either primary or secondary resistance mutations are fixed in the main circulating viral population in vivo also for a long time. Specific studies of the relative RC of these variants and analyses of the overall viral fitness in these HIV-1-infected patients with a long history of treatment could be useful in fully clarifying all the aspects of raltegravir resistance.
This was partially supported by grants from the Italian Ministry of University and Research.
Conflicts of interest: nothing to declare. FC and MC designed the study; BB, EC, MS, FC and AG carried out the experiments; FC, MC, AC, AL, AC, NG and EB analysed the data; VS, NG, AC, AL, SN and FC followed-up patients; FC and MC wrote the manuscript. All the authors revised and approved the actual version of the manuscript.