The evolution of DRMs during selections with RAL and during therapy has been elucidated with the advent of new-generation sequencing methods [24,25▪▪,26,27,32,33]. In a comprehensive study to better define primary resistance variations in patients during continued RAL therapy, characterization of 200 RAL-resistant viruses was carried out. These specimens were derived from patients in the SCOPE cohort, RAL BENCHMRK phase III studies, as well as from patient samples submitted to the Monogram Clinical Reference Library for routine INI testing, with clonal analysis performed for select patient virus isolates [25▪▪]. They found that variants with Y143R or Q148H/R tended to have larger susceptibility fold changes than N155H containing viruses in line with previous studies [12,24,26,32–35]. There were also temporal shifts in subpopulation proportions of N155H, Y143R, and Q148H DRMs within the same patient. By using molecular clones from different patients, isolated at different time-points, they showed that N155H, under continued RAL therapy, is gradually replaced with Y143R or Q148HR, and that the pathway that eventually becomes the predominant species is determined by a specific amino acid substitution at residue 148 as well as by a secondary mutation in addition to Y143R [25▪▪]. In one patient, N155H was the first mutation to appear at week 8, and was then supplanted by the Y143R mutation that first appeared at week 16 and remained the dominant species at week 24; the maximum fold change measured in this patient was 54. Another patient had all three primary mutations present as subpopulations at week 11 (11% E92Q/Y143R; 21% wt; 21% E92Q/N155H; 56% G140S/Q148R), despite the fact that none of these mutations was detected at baseline. Y143R was present in all clones by week 28 (5% Y143R/E92Q; 5% Y143R; 90% Y143R/T97A). The fold change in this patient was greater than 150 at week 11 and remained high with only slight decreases in replicative capacity compared to baseline. In another patient harboring all three primary mutations at week 11 (5% N155H; 23% Q148H/G140S), the N155H mutation was lost by week 16 (15% Y143R; 85% Q148H/G140S) with Q148H/G140S being present in all viruses by week 24. Thus, the 148 pathway dominated when the mutation was Q148H/G140S and the Y143 pathway became dominant in a mixture of N155H and Y143R mutations in these three patients. In an individual in whom a mixture of all three primary mutations was present, the dominance of the 148 pathway was offset only when Y143R occurred in combination with E92Q [25▪▪].
An earlier study analyzed resistance in 23 patients who began a salvage therapy containing RAL. Despite an absence of the 143, 148, and 155 mutations at baseline (frequency <1%), the Y143R, Q148H, and N155H mutations appeared at virological failure under RAL therapy with increased resistance and viral fitness . The presence of secondary resistance mutations such as T97A, V151I, and G163R, despite being detected at very low levels, did not have any effect on the development of resistant variants at failure , suggesting that patterns of resistance development did not appear to be significantly affected by baseline mutations , though other baseline mutations in integrase and other proteins such as protease and reverse transcriptase may have an effect on levels of susceptibility even for variants with identical resistance profiles [28,33,36].
Treatment of patients with EVG has the potential to select for EVG resistance mutations, many with demonstrated cross-resistance to RAL [33,37–39]. Both the 148 and 155 resistance pathways cause a high fold change for EVG [6,12,28,33,39,40]. Mutations at position 143 do not affect the susceptibility of EVG , but EVG is associated with additional primary mutations at position 66 in conjunction with mutations that select for high resistance (fold changes >150) (Table 2[8,11–16,17▪,18,19▪,20–23]) . Additionally, the RAL secondary mutation E92Q is a primary resistance mutation for EVG [33,37,41]. In 10 patients, treated with EVG over 2 weeks, primary resistance mutations selected were T66A/K, E92Q, Q148R, and N155H . After 48 weeks of treatment, there was more resistance mutational diversity in the EVG-treated patients, with the most common double DRM combinations being G140CS/Q148HKR, E138AK/Q148HKR, S147G/Q148HKR, and E92Q/N155H, with a triple DRM combination E138K/S147G/Q148R being present in three patients. Despite having sequenced multiple clones at multiple time-points in the EVG-treated patients, primary DRMs were not detected at baseline. Moreover, the DRMs E138AK, G140C/S, and S147G were never identified alone and combinations of N155H/S together with S147G or G140C/S were not seen . In the RAL-treated arm, the most common combinations identified were G140S/Q148H, sometimes in conjunction with E138A or Y143C .
DTG has not yet been shown to select for resistance mutations in clinical trials [4,42▪,43], and appears to have a very high genetic barrier for the development of resistance. Cell culture work has, however, selected potential DRMs for DTG; the mutations T124A, S153Y, T124A/S153Y, and L101Y/T124A/S153Y were observed on passage with DTG up to day 112 [17▪] without, however, resulting in a large fold change for this drug [17▪,35]. Additionally, recent passage of DTG in multiple subtype B, CRF02_AG, and C viruses selected the moderate DTG DRM R263K in five of six subtype B viruses, and in one of two CRF02_AG viruses, but not in subtype C viruses [19▪]. Instead, the G118R mutation, which had previously been selected in a subtype-C virus with an INSTI termed MK-2048  and in a RAL-treated patient with a CRF02_AG virus , was also selected in one of two CRF02_AG and one of two subtype C viruses, while a H51Y mutation was selected in three of four non-B subtypes [19▪]. Other studies have started with RAL-resistant viruses and attempted to select additional resistance mutations using DTG [45,46]; to date, DTG has been shown to be efficacious against all primary RAL and EVG DRMs, with only reduced susceptibility (10 < fold changes < 20) toward viruses harboring the G140S plus Q148H/R/K mutations, regardless of whether additional mutations were present [45,46,47▪▪]. The clinical use of twice-daily DTG with other drugs may be able to suppress viruses containing RAL and EVG mutations .
As with other ARV DRMs, the primary driving force for the occurrence of integrase DRMs is the high error rate of the HIV reverse transcriptase enzyme , that leads to a diverse pool of reverse transcribed DNA and multiple virus subpopulations. In the presence of nonsuppressive INSTI-containing therapy, mutants that have partial resistance to the INSTI being used will out-compete the susceptible wild-type viruses. If, as in the case of N155H viruses, the initially selected mutants have low fitness, they may gradually develop additional mutations that increase fitness and resistance or become replaced by more resistant and fit viruses. There does not appear to be any known effect of baseline polymorphisms on the development of resistance pathways, and multiple studies have shown that INSTI resistance development is stochastic. Despite the fact that N155H is usually the first detected primary DRM in cases of INSTI resistance, its maintenance and replacement by other resistant viruses depends entirely on relative resistance/fitness ratios between the resistant viruses that are present and more fit viruses that gradually become dominant during long-term therapy. This underscores the importance of suppressing viral replication as a means of restricting viral subpopulations. Another driving force behind INSTI resistance is the amount of INSTI that may be bound to integrase at any given time. DTG binds to wild-type integrase significantly longer than either RAL (∼9×) or EVG (∼27×), and this may underscore its ability to resist the development of resistance as well as to retain activity against viruses that are resistant to RAL and EVG [48▪▪]. The long residence time of DTG in an integrase-bound state may also reduce the effect of another driving force of resistance, that is, adherence. This is because DTG binds longer to the protein (t1/2 = 72 h) [48▪▪] and remains bound to intracellular integrase long after it is no longer detectable in the plasma. This may provide continued inhibition, even in the case of low adherence to drugs, limiting the emergence of resistance phenotypes [4,43,48▪▪]. Another factor that may play a role in resistance is variability among viral subtypes [33,36,49]. There is an increasing body of literature to suggest that wild-type HIV proteins of various subtypes are similar in activity and drug susceptibility. This notwithstanding, some DRMs may result in different levels of susceptibility to different INSTIs in different subtypes [19▪,33,36,44,49]. This subject will need to be carefully monitored as INSTIs become more integrated into standard antiretroviral therapy.
INSTIs are ARV drugs that contribute positively to patient health and promise to become even more important in the future. New genotyping methods have confirmed the appearance of DRMs in patients failing therapy, underlining the importance of effective treatment. In this regard, single-pill regimens will positively contribute to viral suppression. If new drug combinations are tolerated by patients, the excellent tolerability of INSTIs will lead to a scenario in which combinations of INSTIs with non-nucleoside reverse transcriptase inhibitors (NNRTIs) and NRTIs will become more commonplace.
Papers of particular interest, published within the annual period of review, have been highlighted as:
Additional references related to this topic can also be found in the Current World Literature section in this issue (pp. 100–101).
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A longitudinal analysis of viral subpopulation under continued therapy showing superiority of Y143 and Q148 pathways relative to N155.
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One example of transmission of INSTI resistance.
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Reported transmission of INSTI resistance.
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Transmission of an INSTI-resistant HIV strain.
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The 48-week SPRING results.
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Activity of DTG against RAL-resistant viruses derived from treated patients.
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Correlation between INSTI dissociation rate from integrase–DNA complex and resistance.
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