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International Workshop on HIV & Hepatitis Virus Drug Resistance and Curative Strategies - June 7- 11, Cabo, Mexico
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Written for NATAP by David Margolis, MD,Univ of North Carolina
"Boehringer Ingelheim discussion of new integrase inhibitors under study at Boehringer........ GlaxoSmithKline presented a great deal of data on mutations seen and RC measured in RAL resistant isolates recovered from patients undergoing treatment with the 2nd-generation INSTI dolutegravir (DTG, formerly Shionogi/GSK1349572) in the Viking 1 and Viking 3 studies.......Tibotec presented data on TMC310911, a novel human immunodeficiency virus type 1 protease inhibitor (abstr. 3). In cell culture experiments, TMC310 had more activity against multi-protease inhibitor-resistant viruses than darunavir..........observations around resistance to TMC278.......David Wyles (UCSD) plenary talk on HCV drug resistance. reviewed the progress in the development of direct acting antiviral (DAA), such as the NS3 protease inhibitors, the NS5B nucleoside and non-nucleoside polymerase inhibitors, and NS5A antagonists.......goal in HCV therapy is cure.......3 HCV polymerase resistance mutation patterns, again suggesting that combinations that avoid resistance may be approachable....... cyclophin inhibitors (eg. alisporivir or Debio 025) act by blocking the interaction with NS5a and NS5b, and may increase sustained viral response when added to Peg-interferon/ribavirin therapy. As cyclophilin inhibitors target a host factor, resistance may be slower to develop.......detection of resistance in HCV is challenging as mutant that exist at a frequency of less than 0.1% could not be detected by even ultradeep sequencing, and given the replicative capacity of HCV this could still mean that there are a billion mutant virions in the patient!.........Raymond Schinazi discussed the synthesis and testing of potent inhibitors of HCV NS5A.........Chris Petropoulos (Monogram)presented a new assay in development at Monogram that characterized the activity of HCV NS5A and NS3/4A protease inhibitors.....TMC435 monotherapy (200 mg once daily). The drug was potent in pateints with genotypes 4, 5, and 6 with HCV RNA declines after 3 to 8 days of therapy of more than 2-4 logs."
This was the 20th HIV Drug Resistance workshop (abstracts to be published in Antiviral Therapy 2011; vol. 16 Suppl 1), which has recently broadened its scope in recognition of the changing issues in the treatment of HIV and other chronic human viral diseases. The poster session included meeting photographs going back to 1992, where many of the attendees could be vaguely recognized as pictures of their more youthful selves.
Resistance to new antiretroviral agents
Alan Engleman of Harvard Medical School, USA (abstr. P1) opened the Workshop with a plenary talk entitled "Structural insights into integrase function and inhibitor resistance." He discussed the insights that structural biochemistry studies of HIV integrase have given to the biology of HIV integration and the action of integrase inhibitors. Alan reviewed molecular mechanism by which HIV integrase stitches HIV DNA in to the human genome. He described the "intasome" - the complex of HIV DNA, HIV integrase, and other proteins that insert HIV DNA in to the genome of the infected cell.
As the study of HIV integrase structure and the linkage of integrase structure to function initially proved to be difficult, examination of enzyme orthologs in other related animal viruses provided critical insights. The construction and characterization of prototype Primate Foamy virus (PFV) intasomes (Hare Nature 2010) allowed the first detailed study of a retroviral integrase that is related to HIV. It was found that the PFV intasome is tetramer of integrase with shared active site of four catalytic core domains that face each other. This arrangement was then also found in another mammal retrovirus, MMV. As the integration process proceeds, target DNA is found to lie in between two molecules of integrase, and Engleman described the integrase acting "like a snowplow, plowing into double helix..." of human DNA, splitting it so that HIV DNA can react and insert.
Engleman then characterized the molecular interactions that were relevant to the evolution of drug resistance to the current inhibitors of HIV integrase. Current drug inhibitors sit in the catalytic site pocket of HIV integrase, and eject DNA from the site to prevent the integration reaction. Mutations at Y143 remove an interaction that raltegravir (RAL) requires to remain in the catalytic pocket. Mutations at Q148 alone make it difficult for RAL to access the pocket, and access is more restricted when other integrase inhibitor resistance mutations are present. Mutations at N155 alter the how a metal ion is held within the integrase enzyme, decreasing the ability of RAL to hold on the integrase.
Craig Fenwick of Boehringer Ingelheim Canada (abstr. 1) opened the session devoted to new antiretrovirals with a discussion of new integrase inhibitors under study at Boehringer that inhibit by binding outside of the catalytic site. A high-throughput drug screening campaign was described that sought molecules that induced alterations in 3' DNA processing by integrase. The first "hit" molecule with this type of antiviral activity that was identified inthis screen was named BI-B. The molecule was shown to inhibit the binding of the key human cellular factor LEDGF to the intasome, and (perhaps because of this) HIV DNA binding to the intasome. BI-C, a more potent derivative of BI-B was also described. These antiviral molecules displayed potency that was more than 3 times that of RAL in some HIV replication assays, and these potent effects were thought to be due to tight, cooperative binding of the inhibitors within the intasome.
Resistance selection experiments found the selection of a novel A128T mutation, but further selection resulted in resistance mutations that all appeared in the allosteric binding pocket (L102F; A124D; H171T). Fenwick termed this type of inhibitor NCINI (noncompetitive integrase inhibitor) in contrast to inhibitors of strand transfer (INSTI; eg. RAL). Of significant interest, there was no cross-resistance seen: NCINI mutations gave no resistance to INSTIs, and vice versa, raising the possibility of combination therapy in the future. In laboratory replication competence (RC) assays, NCINI mutations reduced RC from 30% (A128) to less than 1% (later mutations). Fenwick reported an NCINI drug is now in phase I testing.
Mark Underwood of GlaxoSmithKline (abstr. 2) very rapidly presented a great deal of data on mutations seen and RC measured in RAL resistant isolates recovered from patients undergoing treatment with the 2nd-generation INSTI dolutegravir (DTG, formerly Shionogi/GSK1349572) in the Viking 1 and Viking 3 studies. In Viking 1, patients were DTG naïve and received drug daily. In Viking 3, 15 patients were INSTI-experienced, had a Q148 and at least one other relevant RAL mutation, and received DTG twice daily. In these early studies, patients received DTG in replacement for RAL for 11 days (functional monotherapy period) and then optimized background therapy. 41% of patients achieved <50 c/ml at week 24; 57% in patients who were using 1 other effective drug by PSS score, and 75% if using >1 other effective drug by PSS score. There was very limited data on acquired resistance in 5 patients who failed DTG. 2 of these 5 patients acquired the N155H mutation, and other data from these 5 patients suggested that the acquisition of new mutations was causally linked to failure of therapy.
Inge Dierynck of Tibotec presented data on TMC310911, a novel human immunodeficiency virus type 1 protease inhibitor (abstr. 3). In cell culture experiments, TMC310 had more activity against multi-protease inhibitor-resistant viruses than darunavir. 79% of isolates that were resistant to tipranavir in vitro were sensitive to TMC310. The addition of up to 8 primary PI mutations gave only a 2.3-fold increase in resistance to TMC310. Similarly, with 6 mutations associated with darunavir resistance, there was 3.2 fold increase in resistance to TMC310, and 4.3 fold resistance with 8 darunavir mutations. It was difficult to select resistance in vitro to TMC310 due to the poor replication capacity of viruses carrying multiple PI mutations. While these findings are promising, they will obviously require confirmation in clinical studies.
The next session was then entirely devoted to observations around resistance to TMC278, recently licensed as ripilvirine (RPV). Laurence Rimsky of Tibotec (abstr. 9) presented 48 weeks genotypic and phenotypic data on HIV-1 isolates obtained from patients failing RPV in the Phase III studies ECHO and THRIVE. As has been reported, a pooled analysis of the results of ECHO and THRIVE at 48 weeks showed an equivalent frequency of failure of therapy in patients given RPV when compared to patients treated with efavirenz (EFV) when the baseline HIV RNA was 100,000 c/ml, but an increased frequency of failure in the RPV arm when baseline HIV RNA was > 100,000 c/ml. There was no differences in success of therapy in subtype B compared to non-subtype B virus. Two points were especially of note: 1) although the frequency of NNRTI resistance in failure patients was equivalent in the EFV and RPV arms, the rate of NRTI resistance was higher in the PRPV arm, and 2) in RPV failure the E138K and K101E mutations predominated regardless of baseline HIV RNA levels.
Laura Napolitano from Monogram Biosciences (abstr. 10) then discussed the impact of E138 mutations in HIV reverse transcriptase on RPV drug susceptibility. Phenosense assays were performed on 7257 wild-type samples of HIV subtype B. These assays established the biological cutoff (BCO) for RPV sensitivity, the drug concentration at which the growth of 99% of wild-type viruses is 50% inhibited by RPV. A 2.5 fold decrease in RPV sensitivity was therefore initially set as the BCO. Previous studies have identified K101E/P, Y181C/I/V, H221Y, and the unique E138G/K/Q/R mutations as RPV resistance-associated mutations (RAMs). Excluding viruses with these known RAMs, rare viruses were found above the BCO, and found to encode have E138A. The effect of E138A was found to be similar to that of E138G, K, Q, or R. 179I, 227C, 230M/L were also found to confer some resistance to RPV. Excluding all these mutations, the BCO of RPV was found to be 2.0-fold. Continued monitoring of databases will provide further information of novel mutations that may influence drug susceptibility to RPV.
Mark Wainberg of McGill (abstr. 11) then presented finding from his virology laboratory on the effects of the E138K RPV resistance mutation. The lab's findings showed quite clearly that the long-recognized fitness defect suffered by HIV as a cost for the acquisition of the M184V or M184I resistance mutation to 3TC or FTC was eliminated in these laboratory assays by the acquisition of the E138K mutation. Wainberg showed convincing primer extension assays, the direct measurement of products of HIV reverse transcription produced in vitro by HIV RT encoding the 184 and/or 138 mutations, clearly demonstrating short, inefficient reverse transcripts RTs with either of the M184 mutations, but restoration of wild-type levels of HIV DNA production when the E138K mutation was engineered into either of these weakened RTs.
Corroborating these findings, in the following presentation Zixin Hu from the Kuritzkes lab at Harvard (abstr. 12) showed that the combination of the E138K and M184I mutations results in increased RT activity and confers a relative replication advantage as compared with the E138K/M184V double mutant, explaining the selection of the E138K/M184I combination in the presence of RPV. In engineered laboratory viruses, fitness by growth competition and single cycle replication assays showed that either M184 mutation increased RPV resistance in the presence of the E138K mutation from 2-fold to 3-fold. There was no difference in fitness, or growth in the presence of 3TC, of the M184V virus when compared to the M184I mutation in presence of the E138K mutation. However, the growth the E138K/M184I in presence of EFV or RPV surpassed that of E138K/M184V, consistent with the frequent occurrence of E138K/M184I mutants in patients with virological failure of RPV.
Inexplicably, these findings were not replicated by Kirsten White and colleagues at Gilead (abstr. 13). In very similar studies to those of Wainberg and Hu, White engineered mutations into a laboratory HIV strain. She found that adding M184I to a virus that carried E138K slightly increased resistance to RPV, while adding M184V slightly decreased resistance to RPV. The extent of these changes varied slightly using different laboratory virus strains and different techniques of measuring resistance, although the general trends were the same in the Gilead studies. Molecular modeling studies suggested that these changes were explained by changes in physical interactions between the altered RT and RPV. White concluded that the predominance of the E138K/M184I dual mutant in RPV failures was driven by greater resistance to RPV, and not by fitness effects. In the end, it seemed most likely that the discrepant results presented in this session were related to different laboratory systems. But more studies will be required to fully explain the mechanisms of RPV and 2nd-generation NNRTI drug resistance, and the role (if any) of replicative fitness in drug resistance in therapy that includes RPV or other 2nd-generation NNRTIs.
Resistance to HCV antiviral agents
On the second day of the meeting David Wyles (UCSD) opened with a plenary talk on HCV drug resistance (abstr. P2). He reviewed the progress in the development of direct acting antiviral (DAA), such as the NS3 protease inhibitors, the NS5B nucleoside and non-nucleoside polymerase inhibitors, and NS5A antagonists. HCV protease inhibitors when used with Peg-interferon/ribavirin have a 10-20% failure rate, and in these cases resistance mutations are detected about 95% of the time. Cross-resistance among current HCV PIs is widespread, although some new classes with a higher barrier to resistance (eg. MK-5177) are emerging.
HCV RNA nucleoside/tide polymerase inhibitors are thwarted with mutations that induce a low fold change in EC50, but also create a large decline in apparent replicative fitness, distinct resistance patterns, and the opportunity for combination therapy. The barrier to developing viruses that are replicatively fit but encode drug-resistance mutations appears relatively high; up to 7 mutations are needed to return drug-resistant HCV 50% fitness in one in vitro study.
So far there appear to be at least 3 HCV polymerase resistance mutation patterns, again suggesting that combinations that avoid resistance may be approachable. Further, cyclophin inhibitors (eg. alisporivir or Debio 025) act by blocking the interaction with NS5a and NS5b, and may increase sustained viral response when added to Peg-interferon/ribavirin therapy. As cyclophilin inhibitors target a host factor, resistance may be slower to develop.
Wyles then pointed out key differences between HIV and HCV therapy. He noted the need for further studies of the loss of resistant variants after failure of therapy, and that some evidence suggests that this may differ between HCV subtype 1a and 1b. Unlike HIV, as there is no persistent reservoir of HCV infection, the decay of resistant HCV species is critical, as it may return the patient to the drug-naïve state after failure of therapy. In one study using ultradeep sequencing, 3 years after 14 day drug exposure, 1 in 15 pt had a 5% residual population of virus with a drug resistant mutation, but in a study of patients exposed to Tegobuvir, resistant mutations appeared to persist. This may also affect the HIV-derived paradigm that cautions against continued exposure to failing therapy: although there has been one observation of accumulation of mutations during telapravir failure, at least at the level of standard sequencing all of these resistant viruse disappeared immediately after stopping therapy. However, the detection of resistance in HCV is challenging as mutant that exist at a frequency of less than 0.1% could not be detected by even ultradeep sequencing, and given the replicative capacity of HCV this could still mean that there are a billion mutant virions in the patient!
Further, Wyles pointed out that the immunological aspect of HCV therapy added another level of complexity. A patient's intrinsic ability to respond to Peg-interferon/ribavirin therapy, clouds the impact of drug resistance, as little response may allow failure founded by a small population of resistant viruses, while a good immune response may prevent failure despite numerous resistant viruses. Finally Wyles provided an excellent and memorable quotable phrase --- "HCV therapy is a sprint not a marathon." This is especially important for those used to treating HIV to remember: there is no persistent reservoir to worry about in HCV, and so the goal in HCV therapy is cure, the sooner, the faster, the better.
The discussion of new antivirals for HCV began as Raymond Schinazi of Emory (Abstr. 14) then discussed the synthesis and testing of potent inhibitors of HCV NS5A. The group began by examining the structure of a known inhibitor from BMS, BMS-790052, and dividing it into 5 domain s by chemical structure. The structure of each of these domains were then altered, and 200 analogs with various combinations of altered domains then tested for antiviral activity in an HCV replicon assay. Inhibitor with activity in the picomolar range were found, and shown not to significantly inhibit Cyp3A4, be stable in liver microsome preparations, and have a wide therapeutic window (toxic concentration 10,000-fold higher than therapeutic concentration). These pilot compounds retained activity against all 5 HCV genotypes. Drug resistance mutations could be selected, and when present were found in the NS5A sequence, but mutations were sometimes found in NS4b, NS5b, NS3 and sites related to resistance to the cyclophlin inhibitor Debio. Although 3 advanced compounds tested had no cross-resistance with the parent BMS drug, Schinazi advised caution about the potential for cross-resistance with out inhibitors, given the changes induced outside of NS5A seen in some cases.
Chris Petropoulos (Monogram)presented a new assay in development at Monogram that characterized the activity of HCV NS5A and NS3/4A protease inhibitors of using a recombinant replicon reporter assays (Abstr. 21). Optimized luciferase reporter replicons were engineered to accommodate the efficient insertion of patient-derived NS3 and NS5A sequences derived by RT-PCR from HCV positive patient plasma. Assay performance was evaluated using reference replicons engineered to contain NS3 protease and NS5A mutations that are associated with reductions in drug susceptibility, as well as replicons containing patient-derived NS5A amplification products. The assay appears robust, with a dynamic range of >4 log10.
Angela Lam from Pharmasset (Abstr. 15) discussed the activity of two guanosine analog HCV NS5b polymerase inhibitors, PSI-352938 and PSI-353661 tested in HCV genotype 2a (J6/JFH-1) replicon cells. The chain terminator nucleotides were active against replicons encoding the S69T and S282T resistance mutations, and although resistance mutations could be selected against a HCV genotype 2 replicon, the HCV genotype 1 replicon could did not develop resistance to these compounds in passage experiments. In general, multiple mutations were required to develop resistance, usually including a mutation at C233H. No cross resistance to other NS5b inhibitors was seen.
To extend findings reported earlier of the potent once-daily HCV NS3/4A protease inhibitor in Phase III development in studies of patients with HCV genotype 1, Oliver Lenz (Tibotec, abstr. 16) reported findings on the activity of TMC435 against HCV genotypes 2-6 from Tibotec study TMC435-C202. Thirty-seven treatment-naive patients infected with HCV G2 (n=6), G3 (n=8), G4 (n=8), G5 (n=7) and G6 (n=8) received seven days of TMC435 monotherapy (200 mg once daily). The drug was potent in pateints with genotypes 4, 5, and 6 with HCV RNA declines after 3 to 8 days of therapy of more than 2-4 logs. Potent declines of RNA (>2 logs) was seen in 3 of 6 patients with genotype 2, but the drug was poorly active in the patients studied with genotype 3 HCV, likely due to D168Q mutations observed. The resistance pathways of TMC435 in these genotypes appear similar to that observed in genotype 1.
Sandra De Meyer (Tibotec, abstr. 17) studied the genotypes of HCV isolated in patients with virologic failure and emergence of resistance with or without a Peg-IFN/RBV lead-in period in the phase III REALIZE study of the recently license NS3/4A protease inhibitor telapravir. HCV NS3/4A population sequencing was performed at baseline, during treatment, and at follow-up visits.
Telaprevir-resistant variants were classified into lower-level resistance (3-25-fold: V36A/M, T54A/S, R155I/K/M/T and A156S) and higher-level resistance (more than 25-fold: V36M+R155K and A156T/V). On-treatment virologic failure (VF) was defined as discontinuation due to a virologic stopping rule and/or having viral breakthrough. Relapse was evaluated in patients who completed their assigned treatment regimen. With or without a lead-in period, there was about a 60% rate of SVR, and about a 20% rate of VF on therapy. VF was more frequent in patients with genotype 1a, and in prior non-responders to Peg-IFN/RBV, but not in patient with prior relapse after Peg-IFN/RBV. There was no difference in the rate to telapravir resistance seen in patients treated with or without a lead-in period. After VF, telapravir resistance could not be detected by population sequencing in 58% of patients after telapravir was stopped, and the median time to loss of detectable variant was 4 to 15 months for different mutations. The emergence, types, and persistence of resistant variants appeared (preliminarily) similar in Peg-IFN/RBV experienced patients when compared to therapy naïve patients.
In parallel, Julie Strizki of Merck reported the association of boceprevir resistance-associated amino acid variants (RAVs) at baseline to treatment outcomes in the SPRINT-2 and RESPOND-2 studies (abstr. 18). The results here were less clear. Various RAVs were seen at baseline, but infrequently, and while those associated with failure tended to confer a higher fold-increase in bocepravir resistance, this effect was not dramatic. It was unclear if this was due to an effect of the assays used to measure resistance (that is a relatively small numerical shift in the assay confer significant clinical resistance) or that treatement failures were largely driven by other factors, such as the immunological response engendered by the co-administered interferon/ribavirin therapy. This highlighted a clinical challenge in assessing drug resistance in HCV therapy, in that the ability to precisely measure the immune contribution to blockade of HCV replication is imprecise, but in contrast to HIV therapy the immunological effect can be very potent (as demonstrated by spontaneous clearance of HCV in some untreated patients). For example, in patients who had a good virological respons to the lead-in period prior to bocepravir, the SVR rate was 79% regardless of the presence of RAVS, but in poor responders the SVR rate was only 34% in the absence of RAVs and 23% in the presence of RAVs. Further in the presence of four specific mutations that appeared highly associated with virologic failure (V36M, T54S, V55A and/or R155K), zero of 7 patients with a poor lead-in response achieved SVR. It appears that we must accumulate a great deal more information on resistance and immune response to accurately predict therapy success, or alternatively we must find combinations of drugs that are so good that these facts become irrelevant. Patients and providers in the future would likely prefer the latter option, but many patients today may not be able to wait for that.
Tara Kieffer (Vertex) then outlined the development of drug resistance in the Phase III telapravir studies in genotype 1 patients (abstr. 19). Telaprevir, when added to peginterferon and ribavirin, significantly increased SVR rates in Phase 3 studies of treatment-naive (ADVANCE and ILLUMINATE) and treatment-experienced (REALIZE) genotype 1 HCV patients. Population sequence analysis of the NS3-4A region was performed in all patients at baseline, and during treatment and follow-up in patients who did not achieve an SVR. At baseline more than 2.7-fold resistance was rarely seen. But at failure of therapy or relapse/rebound of HCV viremia, about 75% had resistant HCV. The responsible resistant mutations were diverse, but the largest population had V36M/R155K high-level resistance. Different resistance mutations were associated with failure in genotype 1a compared to genotype 1b. Again, interferon responsiveness played an important role, as only 1 to 7% of naïve or relapsing patients failed therapy, but 36% of patients with prior nonresponse to Ifn/RBV failed telapravir combination therapy. On the other hand, 50 of 56 failing patients no resistance found at a median follow-up of 25 months, by a limited clonal analysis.
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