HIV RESISTANCE TO INHIBITORS

5. • Luis Menéndez-Arias

RESISTANCE TO (MK-0518, Isentress)

Raltegravir was the first to progress into Phase III clinical trials and became the first one to be approved for clinical use. Raltegravir is indicated in combination with other antiretroviral agents for the treatment of HIV-1 in adult patients. Its potency and tolerability have made it an important option for first-line therapy, the treatment of highly antiretroviral-experienced patients, and regimen simplification.

Clinical studies revealed two distinct pathways that confer resistance to this inhibitor (for a recent review on clinical trials and associated drug resistance data, see Blanco et al. J Infect Dis 2011; 203: 1204-1214). One of them involves the acquisition of N155H (sometimes associated with L74M, E92Q, T97A, G136R, or V151I). The key mutation for the other pathway is Q148K/R/H, usually associated with E138K or with G140A/S. A third possible resistance pathway included mutations Y143R/C, together with L74A/I, E92Q, T97A, I203M and S230R (Malet et al. Antimicrob Agents Chemother 2008; 52: 1351-1358; Cooper et al. N Engl J Med 2008; 359: 355-365; Sichtig et al. J Antimicrob Chemother 2009; 64: 25-32; Eron et al. Lancet Infect Dis 2013; 13: 587-596). Resistant clones containing G140S/Q148H were stable over time, while viral populations with N155H often switched to G140S/Q148H [Miller et al. Antivir Ther 2008; 13 (suppl. 3): A8]. G140S confers lower levels of resistance than Q148H, but increases viral fitness in the presence of Q148H (Delelis et al. Nucleic Acids Res 2009; 37: 1193-1201). E92Q appears as a raltegravir resistance-associated mutation that increases the effects of N155H on raltegravir susceptibility when both appear together in the same clone (Fransen et al. J Virol 2009; 83: 11440-11446).

In phenotypic assays, N155H increased raltegravir resistance by 14-fold, E92Q and G140S/Q148H decreased viral sensitivity to the inhibitor around 7-8 times (Malet et al. Antimicrob Agents Chemother 2008; 52: 1351-1358; Kobayashi et al. Antiviral Res 2008; 80: 213-222; Jones et al. Antimicrob Agents Chemother 2009; 53: 1194-1203; Fransen et al. J Virol 2012; 86: 7249-7255), and Q148R alone showed 22-fold reduced susceptibility to the inhibitor [Goethals et al. Antivir Ther 2008; 13 (suppl. 3): A11; Kobayashi et al. Antiviral Res 2008; 80: 213-222]. Combined mutations G140S and Q148R reduced raltegravir susceptibility >150-fold (Kobayashi et al. Antiviral Res 2008; 80: 213-222; Fransen et al. J Virol 2012; 86: 7249-7255).

Biochemical analysis have demonstrated that HIV-1 group O integrase shows higher susceptibility to raltegravir than the prototypic HIV-1 group M subtype B integrase

369 (Depatureaux et al. Antimicrob Agents Chemother 2014; 58: 7141-7150). Raltegravir is also an effective inhibitor of primate lentiviruses (Koh et al. J Virol 2011; 85: 3677-3682) and human T-cell leukemia virus type I (HTLV-I) (Seegulam & Ratner. Antimicrob Agents Chemother 2011; 55: 2011-2017). In HIV-2, raltegravir resistance appears to be associated with the development of N155H (Garrett et al. AIDS 2008; 22: 1091; Xu et al. AIDS Res Hum Retroviruses 2009; 25: 843-847; Salgado et al. J Clin Virol 2009; 46: 173-175) or Q148R (Roquebert et al. AIDS 2008; 22: 2045-2046). Resistance genetic pathways are similar in HIV-1 (groups M and O) and in HIV-2 (Charpentier et al. Antimicrob Agents Chemother 2011; 55: 1293-1295; Alessandri-Gradt et al. AIDS 2015; 29: 1271-1273).

HIV-2 resistant variants involving the Y143C pathway have been found in patients who previously developed N155H (Xu et al. AIDS Res Hum Retroviruses 2009; 25: 843-847), but the effect of Y143C on raltegravir susceptibility is not significant (Smith et al. AIDS 2011; 25: 2235-2241), except when associated with other resistance-related mutations such as for example, E92Q (Ni et al. Retrovirology 2011; 8: 68). Interestingly, in vitro studies demonstrated that Y143C counteracts the effects of N155H on reduced drug susceptibility (Ni et al. Retrovirology 2011; 8: 68).

An HIV-2ROD variant carrying mutations Q91R/I175M has been selected in vitro in the presence of raltegravir (Perez Bercoff et al. Retrovirology 2010; 7: 98). This variant showed a 13-fold increase in the IC50 for the inhibitor.

INTEGRASE 1 FLDGIDKAQD EHEKYHSNWR AMASDFNLPP VVAKEIVASC DKCQLKGEAM 51 HGQVDCSPGI WQLDCTHLEG KVILVAVHVA SGYIEAEVIP AETGQETAYF 101 LLKLAGRWPV KTIHTDNGSN FTGATVRAAC WWAGIKQEFG IPYNPQSQGV 151 VESMNKELKK IIGQVRDQAE HLKTAVQMAV FIHNFKRKGG IGGYSAGERI 201 VDIIATDIQT KELQKQITKI QNFRVYYRDS RNPLWKGPAK LLWKGEGAVV 251 IQDNSDIKVV PRRKAKIIRD YGKQMAGDDC VASRQDED

Figure 5.1. Amino acid sequence of the HIV-1 integrase (strain HXB2). GenBank accession number K03455. Underlined sequence corresponds to the core domain of the .

370 Figure 5.2. Structural location of amino acids involved in resistance to raltegravir, in the core domain of HIV-1 integrase. The location of Mg2+ is indicated in magenta. Coordinates were taken from Protein Data Bank file 1BIU (Goldgur et al. Proc Natl Acad Sci USA 1998; 95: 9150-9154). Core domains are presented as a dimer, although integrase may also function as a tetramer. A detailed structural analysis of the contribution of resistance mutations and the role of conformational flexibility in binding to strand-transfer inhibitors has been recently published (Mouscadet et al. Drug Resist Updates 2010; 13: 139-150).

RESISTANCE TO (Stribild, GS-9137, JTK-303, 6-(3-chloro-2- fluorobenzyl)-1-[(2S)-1-hydroxy-3-methylbutan-2-yl]-7-methoxy-4-oxo-1,4- dihydroquinoline-3-carboxylic acid)

Elvitegravir showed an EC50 of 1.7 nM in cell-based assays, and was found to be well tolerated and efficacious in clinical trials (Klibanov. Curr Opin Invest Drugs 2009; 10: 190-200). However, it has to be co-formulated with a CYP3A inhibitor. In August 2012, elvitegravir was approved for clinical use by the U.S. Food and Drug Administration as a component of Stribild, a co-formulation also containing the reverse transcriptase inhibitors tenofovir and and the pharmacokinetic enhancer (DeJesus et al. Lancet 2012; 379: 2429-2438; Sax et al. Lancet 2012; 379: 2439-2448). Early studies involving 40 patients showed that there was no evidence of resistance after three weeks of therapy (DeJesus et al. J Acquir Immune Defic Syndr 2006; 43: 1-5) and was rare in patients treated for 144 weeks (Kulkarni et al. HIV Clin Trials 2014; 15: 218-230). HIV-1 variants containing T66I/Q95K/Q146P/S147G (with or without E138K) were selected in cell culture at high concentrations of the inhibitor, while H51Y/E92Q/S147G/E157Q was selected at low concentrations. T66I and E92Q were the key mutations for acquisition of elvitegravir resistance, and by themselves increased resistance around 35-fold (Shimura et al.

371 J Virol 2008; 82: 764-774). Cross-reactivity with other integrase inhibitors such as L-731,988, L-870,810 and S-1360 was also observed in those experiments.

E92Q, Q148K/H/R and/or N155H mutations have been detected in a significant number of patients showing virological failure to elvitegravir. In some patients, a secondary mutation (L68I or L68V) may also appear. These mutations are usually linked to E92Q. L68V confers low-level resistance to elvitegravir [Goodman et al. Antivir Ther 2008; 13 (suppl. 3): A15]. P145S, a mutation selected in one patient by raltegravir was found to confer resistance to elvitegravir [Kobayashi et al. Antiviral Res 2008; 80: 213- 222; Gallien et al. Antivir Ther 2010; 15 (suppl. 2): A15].

Amino acid substitutions T66I, E92Q and Q148R have been selected in vitro by both raltegravir and elvitegravir and can be considered resistance-associated mutations conferring resistance to multiple integrase inhibitors [Goethals et al. Antivir Ther 2008; 13 (suppl. 3): A11; Shimura et al. J Virol 2008; 82: 764-774; Jones et al. Antimicrob Agents Chemother 2009; 53: 1194-1203]. Recombinant viruses containing single amino acid substitutions selected under drug pressure showed reduced elvitegravir susceptibility: 92-fold for Q148R, 30-fold for N155H, 26-fold for E92Q, 10-fold for T66I, 4-fold for S147G, and 2-fold for T97A (Abram et al. Antimicrob Agents Chemother 2013; 57: 2654-2663).

Sustained antiviral response to elvitegravir has been reported for one treatment-naïve patient infected with HIV-2 (group B) (Zhang et al. AIDS 2014; 28: 2329-2331). The inhibitor has been found to be effective in vitro against primate and non-primate lentiviruses, showed intermediate efficiency on mouse mammary tumour virus and murine leukemia virus and was ineffective against the Rous sarcoma virus (Koh et al. J Virol 2011; 85: 3677-3682). Group O HIV-1 isolates were 10-fold less susceptible to elvitegravir inhibition than group M HIV-1 (Tebit et al. AIDS Res Hum Retroviruses 2016; 32: 676-688).

RESISTANCE TO (Tivicay, S/GSK1349572, (4R,12aS)-N-(2,4- difluorobenzyl)-7-hydroxy-4-methyl-6,8-dioxo-3,4,6,8,12,12a-hexahydro-2H- pyrido[1’,2’:4,5]pyrazino[2,1-b][1,3]oxazine-9-carboxamide)

Dolutegravir is a strand transfer inhibitor of HIV-1 integrase, developed from prototypical diketo acid structures (Hazuda. Curr Opin HIV AIDS 2012; 7: 383-389; Bailly & Cotelle.

Expert Opin Drug Discov 2015; 10: 1243-1253). It has an IC50 value of 2.7 nM in enzymatic assays and is active against HIV strains infecting PBMCs (EC50 of 0.5 nM). Improved pharmacodynamic properties of dolutegravir versus first-generation strand transfer inhibitors (i.e. higher dose-response curve slope) are largely responsible for its potent antiviral activity (Laskey & Siliciano. JCI Insight 2016; 1: e89810). HIV-2 group A and group B isolates showed similar susceptibility to dolutegravir than HIV-1 isolates (Smith et al. Retrovirology 2015; 12: 10). As observed with other diketo acid inhibitors of the viral integrase, S153F and S153Y were selected in HIV-1 passaged in vitro in the presence of the drug (Kobayashi et al. Antimicrob Agents Chemother 2011; 55: 813-821).

372 SPRING trials in treatment-naïve individuals showed that dolutegravir (taken at doses of 10, 25 and 50 mg twice a day) was an effective inhibitor of HIV replication, non inferior to (van Lunzen et al. Lancet Infect Dis 2012; 12: 111-118; Walmsley et al. J Acquir Immune Defic Syndr 2015; 70: 515-519; reviewed in Kandel & Walmsley. Drug Des Devel Ther 2015; 9: 3547-3555). SPRING-2 phase III clinical trials also showed at week 96 that once-daily dolutegravir was non-inferior to twice-daily raltegravir in naïve patients (Raffi et al. Lancet Infect Dis 2013; 13: 927-935). More recently, the SAILING study has shown that once-daily dolutegravir, in combination with two other antiretroviral drugs is well tolerated and shows more efficacy than twice-daily raltegravir in treatment- experienced patients (Cahn et al. Lancet 2013; 382: 700-708). Dolutegravir was approved by the U.S. Food and Drug Administration in August 2013 (for a review, see Ballantyne & Perry. Drugs 2013; 73: 1627-1637). The drug is currently used in antiretroviral naïve patients in association with / or emtricitabine/tenofovir. Dolutegravir has shown efficacy in vitro on raltegravir- and elvitegravir-resistant clinical isolates of HIV-1 containing mutations Y143R, Q148K, N155H and G140S/Q148H (Kobayashi et al. Antimicrob Agents Chemother 2011; 55: 813-821), although it has been reported that increased dolutegravir resistance via the Q148 pathway and secondary substitutions may occur in vitro at low concentrations of the drug (Seki et al. Antimicrob Agents Chemother 2015; 59: 2596-2606). However, VIKING I and II clinical trials showed that the use of dolutegravir in patients failing raltegravir therapy was effective only on those bearing HIV-1 integrase mutations Y143C/R or N115H, while only a third of those having Q148H/K/R mutations had a virological response to treatment with dolutegravir (Eron et al. J Infect Dis 2013; 207: 740-749; reviewed in Malet et al. Curr Opin Virol 2012; 2: 580-587). In these trials, virological failure due to resistance to dolutegravir was attributed to the accumulation of multiple integrase inhibitor resistance-related mutations (e.g. L74I/M, T97A, E138A/K, Q148H, N155H). Results from the Phase III VIKING study showed that the response to dolutegravir was significantly reduced in patients infected with HIV-1 variants containing the integrase mutation Q148H/K/R, plus two or more amino acid substitutions of the group formed by L74I, E138A/K/T and G140A/C/S (Castagna et al. J Infect Dis 2014; 210: 354-362).

Development of R263K is compatible with the presence of single amino-acid substitutions such as E92Q or N155H (Anstett et al. J Virol 2015; 89: 4681-4684). However, the combination of R266K with T66I, G140S, Y143R or Q148R is incompatible with high- level viral replication and the development of high-level resistance to dolutegravir (Anstett et al. J Virol 2015; 89: 4681-4684; Liang et al. J Virol 2015; 89: 11269-11274). The double-mutant E138K/Q148K showed high-level resistance to the drug in phenotypic assays (Figure 5.3). The Q148H/K/R mutations did not affect susceptibility to dolutegravir in HIV-1 subtype C and HIV-2 isolates (Kobayashi et al. Antimicrob Agents Chemother 2011; 55: 813-821; Garrido et al. Antiviral Res 2011; 90: 164-167; Malet et al. J Antimicrob Chemother 2011; 66: 1481-1483), although Q148K confers some resistance in the context of HIV-2ROD (Smith et al. Retrovirology 2015; 12: 10). Clinical studies have shown that dolutegravir-containing regimens provide substantial initial efficacy in heavily- treated HIV-2-infected patients with virus lacking mutations at codons 148 and 155 in the integrase-coding region. These data suggest that cross-resistance of raltegravir and

373 elvitegravir with dolutegravir is higher in HIV-2 than in HIV-1 (Descamps et al. Clin Infect Dis 2015; 60: 1521-1527). HIV-1 group O harboring Q148R and N155H shows higher resistance to dolutegravir compared with HIV-1 group M/subtype B (Depatureaux et al. J Acquir Immune Defic Syndr 2015; 70: 9-15). Selection experiments carried out in cord blood monocytic cells using HIV-1 clones of subtypes B, C and A/G yielded the R263K mutation (Quashie et al. J Virol 2012; 86: 2696- 2705). This mutation affecting a residue of the C-terminal domain of HIV-1 integrase conferred low-level resistance to the inhibitor. R263K was also identified in selection studies carried out with elvitegravir and confers low-level resistance to this inhibitor (Margot et al. Antiviral Res 2012; 93: 288-296). R263K has a relatively small effect on dolutegravir resistance when tested with the PhenoSense® Integrase assay (Monogram

Biosciences) (i.e. barely 2-fold increase in the IC50 relative to the wild-type HIV-1NL4-3) (Mesplède et al. Retrovirology 2013; 10: 22). Frequent HIV-1 integrase polymorphisms (e.g. L101I or T124A) have a minimal effect on dolutegravir susceptibility in phenotypic assays (Vavro et al. Antimicrob Agents Chemother 2013; 57: 1379-1384).

Secondary mutations such as M50I, H51Y, E138K or the polymorphism E157Q may appear in combination with R263K and increase resistance to dolutegravir [Mesplede et al. J Antimicrob Chemother 2014; 69: 2733-2740; Wainberg et al. J Int AIDS Soc 2014; 17 (suppl 3); 19518; Anstett et al. J Antimicrob Chemother 2016; 71: 2083-2088], although in the case of H51Y this increase is accompanied by a dramatic loss of enzymatic activity and viral replication capacity (Mesplède et al. Retrovirology 2013; 10: 22). However, M50I has a minimal impact on the viral replication capacity of the R263K mutant (Wares et al. Retrovirology 2014; 11: 7). R263K has a deleterious effect on HIV fitness that seems to be linked to the integration process. Prolonged with R263K-containing viruses lead to a gradual decrease in integrated DNA, without altering the levels of early and late reverse transcription products (Mesplède et al. mBio 2017; 8: e00157-17).

Other mutations associated with dolutegravir resistance occur at positions 118, 121 and 153. G118R and F121Y confer high level resistance to dolutegravir, raltegravir and elvitegravir in strains of HIV-1 subtypes CRF02_AG and B derived from patients treated with raltegravir and elvitegravir (Malet et al. J Antimicrob Chemother 2014; 69: 2118-

2122). In the wild-type HIV-1NL4-3 strain, single-mutants G118R and F121Y produced a 5- to 10-fold increase in resistance to dolutegravir. It has been also reported that mutations in the vicinity of the 3´-polypurine tract (3´PPT) could also confer high-level resistance to dolutegravir and other IN strand transfer inhibitors (Malet et al. Conference on Retroviruses and Opportunistic Infections 2017, Seattle, USA, Abstract 499).

RESISTANCE TO (GSK 1265744, GSK 744) AND (GS-9883)

Cabotegravir is a strand transfer inhibitor of HIV-1 integrase, with a carbamoyl pyridone structure similar to dolutegravir (Yoshinaga et al. Antimicrob Agents Chemother 2015; 59: 397-406). It can be administered as a long-acting parenteral nanosuspension

374 via intramuscular or subcutaneous infection. It has a half-life of 21-50 days under this presentation. It is currently being tested in phase II clinical trials as a maintenance therapy in combination with (Margolis et al. Lancet Infect Dis 2015; 15: 1145-1155). In those trials, cabotegravir showed a high barrier to resistance although the IN substitution Q148R was selected in one patient exposed to cabotegravir/rilpivirine.

Studies in vitro showed cabotegravir activity against raltegravir-resistant isolates. Mutant viruses containing the raltegravir-resistant Y143R, Q148K, N155H, and G140S/Q148H va­ riants had less than 6.1-fold increased cabotegravir inhibitory activity relative to the wild-type virus, while increases of 10 to >100-fold were observed for raltegravir inhibition (Yoshinaga et al. Antimicrob Agents Chemother 2015; 59: 397-406). Amino acid substitutions T124A, S153Y and Q146L in the HIV-1 IN were selected after successive passages of the virus in presence of drug. Studies with SIV-infected macaques treated with cabotegravir revealed the presence of IN mutations E92G/Q and G118R in virus found in rectal and vaginal fluids after 8 weeks of treatment (Radzio et al. Conference on Retroviruses and Opportunistic Infections 2017, Seattle, USA, Abstract 84).

Bictegravir is a structurally similar HIV-1 IN strand transfer inhibitor that like dolutegravir showed high efficacy up to 24 weeks, in phase II clinical trials, when given in combination with the RT inhibitors emtricitabine and (Sax et al. Lancet HIV 2017; 4: e154-e160). Bictegravir has potent activity against raltegravir- and elvitegravir-resistant strains. Its resistance profile is similar to that of dolutegravir with IN amino acid substitutions M50I and R263K, being selected after successive passages of HIV-1 in the presence of drug (Tsiang et al. Antimicrob Agents Chemother 2016; 60: 7086-7097). Currently, it is being developed as a single tablet coformulated with RT inhibitors tenofovir alafenamide and emtricitabine. In clinical trials, resistance was not detected after 48 weeks of treatment (Paul et al. Conference on Retroviruses and Opportunistic Infections 2017, Seattle, USA, Abstract 41). In phenotypic assays, HIV-1 IN mutants Y143R, N155H, R263K, M50I/R263K, E138K/R263K, and G140S/Q148H remained susceptible to bictegravir (≤3-fold increase in the EC50), but H51Y/R263K conferred low-level resistance to the drug (~8-fold) (Hassounah et al. Conference on Retroviruses and Opportunistic Infections 2017, Seattle, USA, Abstract 498).

375 Table 5.1. AMINO ACID SUBSTITUTIONS LOCATED IN THE HIV INTEGRASE (IN) ASSOCIATED WITH RESISTANCE TO ANTIRETROVIRAL DRUGS

Amino acid In vivo / Comments and references substitution In vitro S17N In vitro Polymorphism associated with decreased replication capacity ex vivo (Capel et al. Virology 2013; 444: 274-281). L45Q In vitro Suggested as minor changes with a phenotypic impact on the L45V susceptibility to elvitegravir, in the absence of major resistance mutations (Garrido et al. Antimicrob Agents Chemother 2012; 56: 2873-2878). A49P In vivo Implicated in an unusual mutational pathway leading to dolutegravir resistance, observed in a heavily-treated patient who previously developed the N155H mutation in response to raltegravir therapy (Hardy et al. J Antimicrob Chemother 2015; 70: 405-411). M50I In vitro Found in association with R263K in HIV-1 subtype B strains. Alone, it produces a 5.5-fold increase in elvitegravir resistance. In combination with R263K, it decreases the HIV-1 susceptibility to raltegravir, elvitegravir and dolutegravir without improving the viral replication capacity of the R263K mutant (Wares et al. Retrovirology 2014; 11: 7). Selected in vitro after exposure to increasing concentrations of bictegravir (GS-9883) (Tsiang et al. Antimicrob Agents Chemother 2016; 60: 7086-7097). H51Y In vitro Confers low-level resistance to elvitegravir (Margot et al. Antiviral Res 2012; 93: 288-296), and enhances dolutegravir resistance mediated by the R263K mutation (Mesplède et al. Retrovirology 2013; 10: 22). The double-mutant H51Y/R263K shows 15-fold decreased susceptibility to dolutegravir, relative to the wild-type virus, but shows a significant reduction in its replication capacity. See also R262K. V54I In vitro Selected as a compensatory mutation in virus containing the Q148R mutation and grown in the presence of raltegravir (Goethals et al. Virology 2010; 402: 338-346). T66A In vivo Appears early during treatment with elvitegravir (Winters et al. PLoS One 2012; 7: e40514). In vitro Confers low-level resistance to elvitegravir in phenotypic assays (Kobayashi et al. Antiviral Res 2008; 80: 213-222; Margot et al. Antiviral Res 2012; 93: 288-296). T66I In vivo Primary resistance mutation emerging during treatment with QUAD (elvitegravir/cobicistat/emtricitabine/tenofovir) and showing cross-resistance to raltegravir [White et al. Antivir Ther 2012; 17 (suppl 1): A12].

376 Amino acid In vivo / Comments and references substitution In vitro In vitro Selected after passaging the virus in the presence of diketoacid integrase (IN) inhibitors (e.g. L-708,906, L-731,988 and their derivatives) (Hazuda et al. Science 2000; 287: 646-650; Fikkert et al. J Virol 2003; 77: 11459-11470), the 1,3 propanedione derivative S-1360 (Fikkert et al. AIDS 2004; 18: 2019- 2028), and with GS-9137 (Jones et al. 14th Conference on Retroviruses and Opportunistic Infections 2007, Los Angeles, USA, Abstract 627). T66I produces moderate (4- to 7-fold) resistance to L-708,906 and L-731,988 (Hazuda et al. Science 2000; 287: 646-650; Fikkert et al. J Virol 2003; 77: 11459- 11470), and high-level resistance to L-chicoric acid (Lee & Robinson. J Virol 2004; 78: 5835-5847), S-1360 (Fikkert et al. AIDS 2004; 18: 2019-2028) and elvitegravir (GS- 9137) (Shimura et al. J Virol 2008; 82: 764-774; Jones et al. Antimicrob Agents Chemother 2009; 53: 1194-1203). T66I does not compromise the activity of dolutegravir but prevents the generation of R263K, supporting the use of dolutegravir in second-line therapy for individuals who experience treatment failure with elvitegravir due to the presence of the T66I substitution (Liang et al. J Virol 2015; 89: 11269-11274). T66I confers hypersusceptibility to dolutegravir in phenotypic assays (Liang et al. J Virol 2015; 89: 11269-11274). T66K In vivo Appears early during treatment with elvitegravir (Winters et al. PLoS One 2012; 7: e40514). In vitro Confers significant to high-level resistance to L-870,810, S-1360, S/GSK-364735, raltegravir and elvitegravir (Kobayashi et al. Antiviral Res 2008; 80: 213-222). L68I In vivo Secondary mutations that appeared in patients treated with L68V elvitegravir. L68V confers low-level resistance to elvitegravir in phenotypic assays [Goethals et al. Antivir Ther 2008; 13 (suppl. 3): A11]. V72I In vitro Selected as an accompanying mutation in virus resistant to L-870, 810 (a naphthyridine carboxamide IN inhibitor) (Hazuda et al. Proc Natl Acad Sci USA 2004; 101: 11233-1238). Common polymorphism detected during early acute HIV infection (Low et al. Antimicrob Agents Chemother 2009; 53: 4275-4282). Related to increased viral replication capacity ex vivo (Capel et al. Virology 2013; 444: 274-281).

377 Amino acid In vivo / Comments and references substitution In vitro L74M In vivo Accessory mutation that accompanies N155H in virus resistant to raltegravir (Cooper et al. N Engl J Med 2008; 359: 355-365). Major raltegravir resistance-associated mutation in patients infected with CRF02_AG HIV-1 subtype isolates (Malet et al. J Antimicrob Chemother 2011; 66: 2827-2830). In vitro Selected in cell culture after passage of HIV-1 (IIIB strain) in the presence of L-870,810 (Hombrouck et al. Antimicrob Agents Chemother 2008; 52: 2069-2078), L-708,906 (Fikkert et al. J Virol 2003; 77: 11459-11470) or S-1360 (Fikkert et al. AIDS 2004; 18: 2019-2028). E92G In vivo Elvitegravir resistance mutation rarely found as a minority variant in the viral quasispecies of naïve patients (Ceccherini-Silberstein et al. Antimicrob Agents Chemother 2010; 54: 3938-3948). In vitro Confers resistance to elvitegravir in phenotypic assays (Margot et al. Antiviral Res 2012; 93: 288-296). E92I In vitro Confers moderate resistance to elvitegravir and low-level resistance to L-870,810 and S-1360 (Kobayashi et al. Antiviral Res 2008; 80: 213-222). E92Q In vivo Secondary mutation associated with N155H or Y143C/R in patients failing treatment with raltegravir (Malet et al. Antimicrob Agents Chemother 2008; 52: 1351-1358; Cooper et al. N Engl J Med 2008; 359: 355-365). Also identified in patients with virological failure to elvitegravir [Goodman et al. Antivir Ther 2008; 13 (suppl. 3): A15; White et al. Antivir Ther 2012; 17 (suppl. 1): A12; Winters et al. PLoS One 2012; 7: e40514]. In vitro Selected in cell culture in the presence of elvitegravir. Confers high-level resistance to elvitegravir, L-870,810 and GS-9160 (Shimura et al. J Virol 2008; 82: 764-774; Jones et al. Antimicrob Agents Chemother 2009; 53: 1194-1203), and moderate cross- reactivity with other IN inhibitors (i.e. L-731,988, S-1360, S/GSK-364735 and raltegravir) (Shimura et al. J Virol 2008; 82: 764-774; Kobayashi et al. Antiviral Res 2008; 80: 213-222). E92V In vitro Confers moderate resistance to elvitegravir (Kobayashi et al. Antiviral Res 2008; 80: 213-222), and low-level resistance to GS-9160 (Jones et al. Antimicrob Agents Chemother 2009; 53: 1194-1203).

378 Amino acid In vivo / Comments and references substitution In vitro Q95K In vitro Together with other mutations, it appeared in virus selected in cell culture in the presence of elvitegravir (Shimura et al. J Virol 2008; 82: 764-774). It enhances N155H-mediated raltegravir resistance and improves viral replication capacity (Fun et al. J Antimicrob Chemother 2010; 65: 2300-2304). T97A In vivo Rarely-found polymorphism identified in elvitegravir-resistant HIV clones (Ceccherini-Silberstein et al. Antimicrob Agents Chemother 2010; 54: 3938-3948). In combination with Y143C/R, it has been also found in patients failing raltegravir-containing regimens (Reigadas et al. PLoS One 2010; 5: e10311).

In vitro In strand transfer assays carried out with recombinant IN, the T97A mutation produces a severe reduction of the susceptibility to raltegravir conferred by Y143C/R, while rescuing the catalytic defect due to the 143 mutation (Reigadas et al. Antimicrob Agents Chemother 2011; 55: 3187-3194). However, phenotypic studies with site-directed mutants and clinical isolates showed that it confers only low-level resistance to elvitegravir and raltegravir (Abram et al. Antimicrob Agents Chemother 2013; 57: 2654-2663; Abram et al. PLoS One 2017; 12: e0172206). Y99H In vitro Selected in the presence of BI-D, a non-catalytic IN inhibitor, together with mutations L102F, H171T and N222K [Fenwick et al. Antivir Ther 2011; 16 (suppl. 1): A9]. L101I In vitro Secondary mutation selected with dolutegravir (Kobayashi et al. Antimicrob Agents Chemother 2011; 55: 813-821). Found in raltegravir-treated patients usually accompanied by T124A (Malet et al. J Antimicrob Chemother 2011; 66: 1481-1483). Polymorphism that is more frequent in HIV-1 non-B subtypes than in subtype B isolates (Garrido et al. Antiviral Res 2011; 90: 164-167), particularly in subtype C where it is found in >85% of the isolates [Oliveira et al. Antivir Ther 2012; 17 (suppl. 1): A132]. However, its effects on resistance to dolutegravir or raltegravir are almost undetectable in phenotypic assays (Vavro et al. Antimicrob Agents Chemother 2013; 57: 1379-1384). L102F In vitro Major mutation associated with resistance to allosteric IN in- hibitors, and selected in vitro together with Y99H, H171T and N222K. Alone, L102F confers 61-fold decreased susceptibility to BI-224436 (Fenwick et al. Antimicrob Agents Chemother 2014; 58: 3233-3244).

379 Amino acid In vivo / Comments and references substitution In vitro G118R In vivo Identified in patients failing raltegravir treatment (Munir et al. J Antimicrob Chemother 2015; 70: 739-749). In combination with L74M, this amino acid substitution emerges as a major mutation associated with raltegravir resistance in patients infected with CRF02_AG HIV-1 strains (Malet et al. J Antimicrob Chemother 2011; 66: 2827-2830). Emergence of G118R has been reported after a switch to dolutegravir monotherapy in patients previously treated with elvitegravir-containing therapies (Brenner et al. J Antimicrob Chemother 2016; 71: 1948-1953). In vitro Confers high-level resistance to L-870,810, S-1360, MK-0536, S/GSK-364735, raltegravir and elvitegravir in phenotypic assays (Kobayashi et al. Antiviral Res 2008; 80: 213-222; Malet et al. J Antimicrob Chemother 2011; 66: 2827-2830; Métifiot et al. Antimicrob Agents Chemother 2011; 55: 5127-5133; Malet et al. J Antimicrob Chemother 2014; 69: 2118-2122). Selected in the presence of the second-generation IN inhibitor MK-2048, usually together with E138K (Bar-Magen et al. J Virol 2010; 84: 9210- 9216). G118R alone or in combination with H51Y or E138K confers low-level resistance to dolutegravir, elvitegravir and raltegravir in cell-based assays [Quashie et al. Antimicrob Agents Chemother 2013; 57: 6223-6235; 2014; 58: 3580 (erratum); Munir et al. J Antimicrob Chemother 2015; 70: 739-749]. The impacts of H51Y and E138K on resistance and enzyme efficiency, when present with G118R, were highly dependent on viral subtype. Thus, subtype B and circulating recombinant form CRF02_AG encoding that contained G118R were severely restricted in their viral replication capacity, but the impact of the mutation in subtype C integrases was very small (Quashie et al. J VIrol 2015; 89: 3163-3175). S119G In vitro These natural polymorphisms have been associated with reduced S119P replication capacity ex vivo (Brockman et al. J Virol 2012; 86: S119R 6913-6923; Capel et al. Virology 2013; 444: 274-281). S118R has S119T been identified as an escape mutation associated with a protective HLA-C*05 allele (Brockman et al. J Virol 2012; 86: 6913-6923). This polymorphism is frequently observed in combination with Q148H/R or N155H in treated patients and enhances the level of resistance to integrase strand transfer inhibitors produced by Y143C, Q148H or N155H. Other polymorphisms at this codon (e.g. S119G/P/T) do not have an impact on drug susceptibility (Hachiya et al. Antiviral Res 2015; 119: 84-88).

380 Amino acid In vivo / Comments and references substitution In vitro F121Y In vivo Identified in patients failing raltegravir treatment (Cavalcanti et al. Antiviral Res 2012; 95: 9-11; Munir et al. J Antimicrob Chemother 2015; 70: 739-749). In vitro Appears together with changes at positions 72 and 125 in virus selected in the presence of L-870,810 (Hazuda et al. Proc Natl Acad Sci USA 2004; 101: 11233-11238). Secondary mutation emerging in virus selected in the presence of S/GSK-364735 (Garvey et al. Antimicrob Agents Chemother 2008; 52: 901-908). It confers moderate to high-level resistance to S-1360, S/GSK- 364735 and elvitegravir, and low-level resistance to L-870,810 and raltegravir (Kobayashi et al. Antiviral Res 2008; 80: 213-222; Malet et al. J Antimicrob Chemother 2014; 69: 2118-2122; Munir et al. J Antimicrob Chemother 2015; 70: 739-749). T122I In vivo This polymorphism has been associated with poor virological response to raltegravir in a large European cohort [Armenia et al. Antivir Ther 2011; 16 (suppl. 1): A69]. T124A In vitro Selected in vitro with S/GSK-364735 (Garvey et al. Antimicrob Agents Chemother 2008; 52: 901-908), dolutegravir (Kobayashi et al. Antimicrob Agents Chemother 2011; 55: 813-821), and cabotegravir (GSK1265744) (Yoshinaga et al. Antimicrob Agents Chemother 2015; 59: 397-406). However, it does not affect IN inhibitor susceptibility in phenotypic assays (Kobayashi et al. Antiviral Res 2008; 80: 213-222; Vavro et al. Antimicrob Agents Chemother 2013; 57: 1379-1384). Frequent in IN inhibitor- naïve patients infected with non-B HIV-1 strains (Garrido et al. Antiviral Res 2011; 90: 164-167), and in raltegravir-treated individuals, sometimes in combination with L101I (Malet et al. J Antimicrob Chemother 2011; 66: 1481-1483). T124N In vitro Associated with resistance to KF116, a pyridine-based multimerization IN inhibitor. Confers high-level resistance to KF116 in combination with V165I/T174I (Hoyte et al. Conference on Retroviruses and Opportunistic Infections 2017, Seattle, USA. Abstract 157). T125K In vitro Involved in resistance to L-870,810 (Hazuda et al. Proc Natl Acad Sci USA 2004; 101: 11233-1238). It decreases drug susceptibility to raltegravir and L-870,810 of HIV-1 bearing IN mutation F121Y (Hazuda et al. Proc Natl Acad Sci USA 2004; 101: 11233- 11238; Kobayashi et al. Antiviral Res 2008; 80: 213-222).

381 Amino acid In vivo / substitution In vitro Comments and references A128N In vitro Selected in the presence of BI-224436, an allosteric IN inhibitor. A128T A128N and A128T confer 64-fold and 2.9-fold decreased susceptibility to the inhibitor. However, those mutations had no effect on resistance to raltegravir or elvitegravir (Fenwick et al. Antimicrob Agents Chemother 2014; 58: 3233-3244). Ala128 locates in the LEDGF/p75 binding pocket of the HIV-1 IN, at the BI-224436 binding site (Fader et al. ACS Med Chem Lett 2014; 5: 422-427). A128T confers marked resistance to allosteric inhibitors by suppressing IN multimerization mediated by those drugs (Feng et al. J Biol Chem 2013; 288: 15813-15820; reviewed in Engelman et al. Curr Opin Chem Biol 2013; 17: 339-345). E138A In vivo Appears in patients treated with elvitegravir, but combined with mutations at position 148 (Winters et al. PLoS One 2012; 7: e40514). E138K In vivo Secondary mutation associated with Q148H/K/R in patients treated with elvitegravir (Winters et al. PLoS One 2012; 7: e40514), associated with partial recovery in viral infectivity and/or resistance to integrase inhibitors (Seki et al. Antimicrob Agents Chemother 2015; 59: 2596-2606). In vitro Selected in vitro in the presence of raltegravir (Wai et al. 14th Conference on Retroviruses and Opportunistic Infections 2007, Los Angeles, USA, Abstract 87), elvitegravir (Shimura et al. J Virol 2008; 82: 764-774), S-1360 (Fikkert et al. AIDS 2004; 18: 2019-2028), S/GSK-364735 (Nakahara et al. Antiviral Res 2009; 81: 141-146) and MK-2048 (Bar-Magen et al. J Virol 2010; 84: 9210-9216). On the other hand, it has a minor influence on phenotypic resistance to those inhibitors, but produces a significant increase of resistance to L-870,810, raltegravir, elvitegravir and GS-9160 when the Q148K mutation is present (Jones et al. Antimicrob Agents Chemother 2009; 53: 1194-1203). E138K restores viral replication capacity after the emergence of G118R (Bar-Magen et al. J Virol 2010; 84: 9210- 9216), but has no effect on the low-level resistance produced by G118R [Quashie et al. Antimicrob Agents Chemother 2013; 57: 6223-6235; 2014; 58: 3580 (erratum)]. G140A In vitro Unlike G140S, this substitution confers high-level resistance to raltegravir when associated with Q148K. It decreases viral replicative capacity in vitro (Fransen et al. J Virol 2009; 83: 11440-11446).

382 Amino acid In vivo / substitution In vitro Comments and references G140C In vivo Secondary mutation associated with Q148H/K/R in patients treated with elvitegravir (Winters et al. PLoS One 2012; 7: e40514). G140S In vivo Appears as a secondary mutation in patients failing treatment with raltegravir or elvitegravir usually associated with Q148H/K/R (Cooper et al. N Engl J Med 2008; 359: 355- 365; Winters et al. PLoS One 2012; 7: e40514). It is less common in non-B clades of HIV-1 where it shows a higher genetic barrier (Doyle et al. J Antimicrob Chemother 2015; 70: 3080-3086). In vitro Arises in vitro in selection experiments with L-chicoric acid, and confers >100-fold increased resistance to this inhibitor (King & Robinson. J Virol 1998; 72: 8420-8424). In phenotypic assays, it confers low-level resistance to raltegravir, elvitegravir and S/GSK-364735 (Delelis et al. Nucleic Acids Res 2009; 37: 1193-1201; Nakahara et al. Antiviral Res 2009; 81: 141-146; Ceccherini-Silberstein et al. Antimicrob Agents Chemother 2010; 54: 3938-3948). Y143C In vivo Observed in patients failing raltegravir therapy (Cooper et al. N Y143R Engl J Med 2008; 359: 355-365; Canducci et al. AIDS 2009; 23: 455-460). It represents an alternative resistance pathway to N155H and Q148H/K/R in HIV-1. Y143C has a minor impact on raltegravir susceptibility in HIV-2 isolates (Ni et al. Retrovirology 2011; 8: 68; Smith et al. AIDS 2011; 25: 2235-2241). In vitro Both amino acid substitutions confer strong resistance to raltegravir in vitro, although Y143R confers greater reduction in susceptibility as a single substitution (Huang et al. Antimicrob Agents Chemother 2013; 57: 4105-4113). Y143C and Y143R confer higher levels of resistance than G140S/Q148H (Delelis et al. Antimicrob Agents Chemother 2010; 54: 491-501). However, elvitegravir remains fully active against mutant IN bearing Y143R, as well as against recombinant viruses having this mutation (Métifot et al. AIDS 2011; 25: 1175-1178). Y143A In vitro Their impact on raltegravir susceptibility is similar to that Y143G shown by Y143C. Usually, they require multiple secondary Y143H substitutions to develop large reductions in raltegravir Y143S susceptibility (Huang et al. Antimicrob Agents Chemother 2013; 57: 4105-4113). Patient-derived viruses containing Y143 substitutions exhibit cross-resistance to elvitegravir.

383 Amino acid In vivo / Comments and references substitution In vitro P145S In vivo Selected in one patient treated with raltegravir, in the context of a clinical trial including 49 individuals (Gallien et al. AIDS 2011; 25: 665-669). In vitro Confers high-level resistance to elvitegravir (Kobayashi et al. Antiviral Res 2008; 80: 213-222). Q146L In vitro Selected in vitro with cabotegravir (GSK1265744), it confers 2.1-fold decreased susceptibility to the drug (Yoshinaga et al. Antimicrob Agents Chemother 2015; 59: 397-406). Q146P In vitro Found in vitro in selection experiments carried out with elvitegravir (Shimura et al. J Virol 2008; 82: 764-774). Q146R In vitro Secondary mutation selected in vitro with S/GSK-364735 (Garvey et al. Antimicrob Agents Chemother 2008; 52: 901- 908). It has a minor effect on susceptibility to most IN inhibitors, as determined in phenotypic assays (Kobayashi et al. Antiviral Res 2008; 80: 213-222). S147G In vivo Amino acid substitution found in patients treated with elvitegravir who already developed Q148H/K/R (Winters et al. PLoS One 2012; 7: e40514). In vitro Appears together with other mutations (e.g. T66I, Q95K and Q146P, or H51Y, E92Q and E157Q) in viral isolates selected in vitro with elvitegravir (Shimura et al. J Virol 2008; 82: 764-774). Confers 9.5-fold increased resistance to elvitegravir, but has no effect on raltegravir resistance (Margot et al. Antiviral Res 2012; 93: 288-296). Q148H In vivo Major raltegravir resistance mutation (Malet et al. Antimicrob Agents Chemother 2008; 52: 1351-1358; Cooper et al. N Engl J Med 2008; 359: 355-365), and common in patients failing treatment with elvitegravir [Goodman et al. Antivir Ther 2008; 13 (suppl. 3): A15]. In vitro Confers high-level resistance to L-870,810, S-1360 and raltegravir, and moderate resistance to elvitegravir in phenotypic assays (Kobayashi et al. Antiviral Res 2008; 80: 213-222).

384 Amino acid In vivo / substitution In vitro Comments and references Q148K In vivo Major raltegravir resistance mutation (Malet et al. Antimicrob Agents Chemother 2008; 52: 1351-1358; Cooper et al. N Engl J Med 2008; 359: 355-365), and common in patients failing treatment with elvitegravir [Goodman et al. Antivir Ther 2008; 13 (suppl. 3): A15]. In vitro Confers high-level resistance to L-870,810, S-1360, raltegravir, elvitegravir, S/GSK-364735, and GS-9160 (Kobayashi et al. Antiviral Res 2008; 80: 213-222; Jones et al. Antimicrob Agents Chemother 2009; 53: 1194-1203; Nakahara et al. Antiviral Res 2009; 81: 141-146). It has a larger impact on raltegravir and elvitegravir resistance than Q148R. Q148N In vitro Q148N alone or in combination with G140S confers 2.4- 4.5-fold reduced susceptibility to elvitegravir depending on the viral genetic context, but had no effect on resistance to raltegravir and dolutegravir (Varghese et al. AIDS Res Hum Retroviruses 2016; 32: 702-704). Q148R In vivo Major raltegravir resistance mutation (Malet et al. Antimicrob Agents Chemother 2008; 52: 1351-1358; Cooper et al. N Engl J Med 2008; 359: 355-365; Canducci et al. AIDS 2009; 23: 455- 460), and common in patients failing treatment with elvitegravir [Goodman et al. Antivir Ther 2008; 13 (suppl. 3): A15; White et al. Antivir Ther 2012; 17 (suppl. 1): A12; Winters et al. PLoS One 2012; 7: e40514]. Found as a minority variant in HIV-infected patients naïve for IN inhibitors (Charpentier et al. AIDS 2010; 24: 867-873). Also found in patients treated with cabotegravir plus rilpivirine, in phase 2b clinical trials (Margolis et al. Lancet Infect Dis 2015; 15: 1145-1155). In vitro Confers high-level resistance to L-870,810, S-1360, raltegravir, elvitegravir, and S/GSK-364735 (Kobayashi et al. Antiviral Res 2008; 80: 213-222). I151L In vitro It confers resistance to L-870,810, S-1360 and elvitegravir (Kobayashi et al. Antiviral Res 2008; 80: 213-222). V151A In vitro Selected with GS-278012, an analogue of GS-9160. Confers some resistance to elvitegravir and enhances resistance to various IN inhibitors when mutations E92Q or E92Q/G140S are present (Jones et al. Antimicrob Agents Chemother 2009; 53: 1194-1203).

385 Amino acid In vivo / Comments and references substitution In vitro V151I In vivo Secondary mutation, associated with N155H, in patients failing raltegravir therapy (Malet et al. Antimicrob Agents Chemother 2008; 52: 1351-1358; Cooper et al. N Engl J Med 2008; 359: 355-365). Associated with resistance to integrase inhibitors in HIV-2 (Cavaco-Silva et al. PLoS One 2014; 9: e92747). In vitro Emerges as a secondary mutation in viral isolates selected in vitro with L-870,810 (Hazuda et al. Proc Natl Acad Sci USA 2004; 101: 11233-11238). S153F In vitro Selected in vitro dolutegravir (Kobayashi et al. Antimicrob Agents Chemother 2011; 55: 813-821). Produces a 5.2-fold increase in resistance to elvitegravir (Margot et al. Antiviral Res 2012; 93: 288-296). S153Y In vitro Selected in vitro with L-708,906 and other diketo acids, with elvitegravir and with dolutegravir (Hazuda et al. Science 2000; 287: 646-650; Shimura et al. J Virol 2008; 82: 764-774; Zahm et al Antimicrob Agents Chemother 2008; 52: 3358-3368; Kobayashi et al. Antimicrob Agents Chemother 2011; 55: 813- 821; Margot et al. Antiviral Res 2012; 93: 288-296). However, it has a minor effect on phenotypic resistance to those inhibitors (Kobayashi et al. Antiviral Res 2008; 80: 213-222). Selected in vitro with cabotegravir (GSK1265744) (Yoshinaga et al. Antimicrob Agents Chemother 2015; 59: 397-406). M154I In vivo Polymorphism found at a frequency of 6% in untreated patients and 21.3% in antiretroviral-treated patients (Ceccherini- Silberstein et al. J Antimicrob Chemother 2010; 65: 2305-2318). In vitro Selected in vitro with L-708,906 and L-731,988. The viral strains harboring mutations T66I/M154I or S153Y/M154I were 10 to 12 times more resistant to the inhibitor than the reference virus (Hazuda et al. Science 2000; 287: 646-650; Lee & Robinson. J Virol 2004; 78: 5835-5847). M154L In vivo Absent in naïve patients, reaches a prevalence of 5.7% in treated patients. This mutation is positively associated with the RT resistance mutations F227L and T215Y (Ceccherini-Silberstein et al. J Antimicrob Chemother 2010; 65: 2305-2318).

386 Amino acid In vivo / Comments and references substitution In vitro N155H In vivo Major raltegravir resistance mutation (Malet et al. Antimicrob Agents Chemother 2008; 52: 1351-1358; Cooper et al. N Engl J Med 2008; 359: 355-365; Canducci et al. AIDS 2009; 23: 455-465; Ferns et al. AIDS 2009; 23: 2159-2164), and common in patients failing treatment with elvitegravir [Goodman et al. Antivir Ther 2008; 13 (suppl. 3): A15; White et al. Antivir Ther 2012; 17 (suppl 1): A12; Winters et al. PLoS One 2012; 7: e40514]. N155H produces a larger decrease in viral replication capacity in comparison with other major raltegravir resistance mutations (e.g. Y143R, Q148H or Q148R). The 155 substitution renders the virus more susceptible to the inhibitor, while continued drug exposure leads to the outgrowth of N155H variants by isolates containing changes at positions 143 or 148 (Fransen et al. J Virol 2012; 86: 7249-7255). In vitro Confers high-level resistance to raltegravir, elvitegravir, L-870,810 and GS-9160, and significant resistance to S-1360, S/GSK- 364735, MK-2048 and compound G (Kobayashi et al. Antiviral Res 2008; 80: 213-222; Jones et al. Antimicrob Agents Chemother 2009; 53: 1194-1203; Van Wesenbeeck et al. Antimicrob Agents Chemother 2011; 55: 321-325). It confers the highest selective ad- vantage for raltegravir resistance (in comparison with E92Q and Q148H), as determined in fitness and drug susceptibility assays [Dam et al. Antivir Ther 2009; 14 (suppl. 1): A28]. N155S In vitro Selected in vitro with L-708,906 (Hazuda et al. Science 2000; 287: 646-650), and with some of its derivatives [Hazuda et al. Antivir Ther 2001; 6 (suppl. 1): 7-8]. Confers moderate to high-level resistance to L-870,810, S-1360, elvitegravir, S/GSK-364735, and GS-9160 (Kobayashi et al. Antiviral Res 2008; 80: 213-222; Jones et al. Antimicrob Agents Chemother 2009; 53: 1194-1203). N155T In vitro Confers high-level resistance to S-1360, elvitegravir and S/GSK-364735 (Kobayashi et al. Antiviral Res 2008; 80: 213-222). E157Q In vivo Identified in one patient treated in France with raltegravir. This mutation persisted after exposure to dolutegravir, and conferred some resistance to this drug in enzymatic assays (Danion et al. J Antimicrob Chemother 2015; 70: 1921-1923). Furthermore, E157Q also acts as a compensatory mutation for R263K, increasing dolutegravir resistance by 10-fold in comparison with R263K alone (Anstett et al. J Antimicrob Chemother 2016; 71: 2083-2088).

387 Amino acid In vivo / Comments and references substitution In vitro In vitro Selected in vitro with elvitegravir and raltegravir, but in the presence of additional mutations (e.g. H51Y, E92Q and S147G) (Shimura et al. J Virol 2008; 82: 764-774; Malet et al. Antimicrob Agents Chemother 2008; 52: 1351-1358). There is also one report on its rapid selection associated with increased viremia, in patients treated with raltegravir (Ghosn et al. J Antimicrob Chemother 2009; 64: 433-434). However, this amino acid substitution was detected in 4.5% of the patients during early acute HIV infection, and could be considered as a polymorphism influencing the response to IN inhibitors (Low et al. Antimicrob Agents Chemother 2009; 53: 4275-4282). G163E In vivo Selected after 20 days on therapy in one patient with a high viral load, who initiated treatment with fumarate/emtricitabine plus dolutegravir. Its significance is uncertain (Fulcher et al. Conference on Retroviruses and Opportunistic Infections 2017, Seattle, USA, Abstract 500). G163R In vivo Found in three patients failing raltegravir therapy, although its significance for resistance is not known (Cooper et al. N Engl J Med 2008; 359: 355-365). V165I In vitro See T124N. H171T In vitro Major mutation associated with resistance to BI-D, a non- catalytic IN inhibitor. It has been selected in vitro together with Y99H, L102F and N222K [Fenwick et al. Antivir Ther 2011; 16 (suppl. 1): A9]. Virus containing the H171T substitution showed around 68-fold resistance to BI-D, despite affecting only modestly IN-LEDGF/p75 binding (Slaughter et al. Retrovirology 2014; 11: 100). T174I In vitro Associated with resistance to non-catalytic site IN inhibitors, such as KF116 (see T124N) and GS-9695 (Mitchell et al. Conference on Retroviruses and Opportunistic Infections 2017, Seattle, USA. Abstract 434).

I203M In vivo Found in three patients failing raltegravir therapy, although its significance for resistance is not known (Cooper et al. N Engl J Med 2008; 359: 355-365).

388 Amino acid In vivo / Comments and references substitution In vitro T206S In vitro In vitro, it appears together with S230N in virus selected with the pyranodipyrimidine V-165 (Hombrouck et al. J Antimicrob Chemother 2007; 59: 1084-1095). An association between the presence of T206S in the baseline genotype and the absence of the primary Q148H/R mutation or any secondary mutation accompanying the N155H has been detected following raltegravir failure (Sichtig et al. J Antimicrob Chemother 2009; 64: 25-32).

N222K In vitro Minor mutation associated with resistance to the allosteric IN

inhibitor BI-224436. N222K increases 2.8 times the IC50 of the drug (Fenwick et al. Antimicrob Agents Chemother 2014; 58: 3233-3244).

S230N In vitro Selected in vitro in the presence of the pyranodipyrimidine V-165 (Hombrouck et al. J Antimicrob Chemother 2007; 59: 1084-1095). Found in one patient (out of 51) undergoing treatment with raltegravir-containing regimens (da Silva et al. J Antimicrob Chemother 2010; 65: 1262-1269). S230R In vitro Appears together with T66I and L74M in virus isolated in the presence of L-708,906 (Fikkert et al. J Virol 2003; 77: 11459- 11470). These variants were also resistant to S-1360 (Fikkert et al. AIDS 2004; 18: 2019-2028), but were susceptible to V-165 (Fikkert et al. J Virol 2003; 77: 11459-11470). D232N In vivo Associated with resistance to integrase inhibitors in HIV-2 (Cavaco-Silva et al. PLoS One 2014; 9: e92747). L234V In vitro Implicated in a rare mutational pathway leading to dolutegravir resistance, observed in a heavily-treated patient who previously developed the N155H mutation in response to raltegravir therapy (Hardy et al. J Antimicrob Chemother 2015; 70: 405-411). D256E In vitro Associated with lower ex vivo viral replication capacity (Capel et al. Virology 2013; 444: 274-281). R262K In vitro Found together with H51Y in virus selected in vitro with dolutegravir. The combination of both substitutions had a modest effect on dolutegravir susceptibility (Cutillas et al. Antimicrob Agents Chemother 2015; 59: 310-316).

389 Amino acid In vivo / Comments and references substitution In vitro R263K In vivo Developed in patients infected with HIV-1 group M subtypes B and C treated with dolutegravir in clinical trials, but conferred low levels of resistance to the drug (Cahn et al. Lancet 2013; 382: 700-708). In vitro Confers low-level resistance to elvitegravir (Margot et al. Antiviral Res 2012; 93: 288-296; Garrido et al. Antimicrob Agents Chemother 2012; 56: 2873-2878). In addition, R263K was the most common mutation emerging in selection experiments carried out in the presence of dolutegravir with viruses of subtypes B, C and A/G. This mutation produces an

11.2-fold increase in the IC50 for dolutegravir in comparison with that obtained with wild-type HIV-1NL4-3 (Quashie et al. J Virol 2012; 86: 2696-2705), although reported increases of the

IC50 seem to be smaller when assayed with the PhenoSense® Integrase assay (Mesplède et al. Retrovirology 2013; 10: 22). R263K delays the emergence of resistance against raltegravir, but the presence of secondary mutations such as H51Y or E138K mitigates this inhibitory effect (Oliveira et al. AIDS 2015; 29: 2255-2260). The R263K substitution in HIV-1 subtype C is more deleterious for IN enzymatic function and viral replication than in subtype B (Mesplède et al. AIDS 2015; 29: 1459-1466). It was also selected in vitro after exposure to increasing concentrations of bictegravir (GS-9883), usually in combination with M50I (Tsiang et al. Antimicrob Agents Chemother 2016; 60: 7086-7097). C280Y In vitro Selected in vitro with the styrylquinoline derivative FZ41 (Bonnenfant et al. J Virol 2004; 78: 5728-5736). It reduces drug susceptibility by 5- to 10-fold, in phenotypic assays.

390 Figure 5.3. Summary of phenotypic drug susceptibility data obtained from relevant mutations and combinations of amino acid substitutions in the HIV-1 IN, involved in resistance to IN inhibitors. High-level (>100-fold increase of the IC50 for the inhibitor), moderate (10- to 100-fold increase) and low-level (3- to 10-fold increase) resistance are represented by solid, hatched and grey boxes, respectively. Open boxes indicate susceptible viruses (<3-fold increase in the IC50 for the inhibitor). Data shown have been taken from Kobayashi et al. Antiviral Res 2008; 80: 213-222; Shimura et al. J Virol 2008; 82: 764-774; Fransen et al. J Virol 2009; 83: 11440-11446; Jones et al. Antimicrob

391 Agents Chemother 2009; 53: 1194-1203; Nakahara et al. Antiviral Res 2009; 81: 141-146; Delelis et al. Antimicrob Agents Chemother 2010; 54: 491-501; Goethals et al. Virology 2010; 402: 338-346; Ceccherini-Silberstein et al. Antimicrob Agents Chemother 2010; 54: 3938-3948; Abram et al. Antimicrob Agents Chemother 2013; 57: 2654-2663; Huang et al. Antimicrob Agents Chemother 2013; 57: 4105-4113; Fenwick et al. Antimicrob Agents Chemother 2014; 58: 3233-3244; Andreatta et al. Antimicrob Agents Chemother 2015; 59: 3441-3449. References for additional data: (i) dolutegravir (also known as S/GSK1349572 or soltegravir) (Kobayashi et al. Antimicrob Agents Chemother 2011; 55: 813-821; Hightower et al. Antimicrob Agents Chemother 2011; 55: 4552-4559; Eron et al. J Infect Dis 2013; 207: 740-748; Pollicita et al. J Antimicrob Chemother 2014; 69: 2412-2419; Anstett et al. J Virol 2015; 89: 4681-4684; Anstett et al. J Virol 2015; 89: 10482-10488; Liang et al. J Virol 2015; 89: 11269-11274); (ii) MK-2048 (Van Wesenbeeck et al. Antimicrob Agents Chemother 2011; 55: 321-325; Goethals et al. Antiviral Res 2011; 91: 167-176); (iii) cabotegravir (GSK1265744, GSK744) (Yoshinaga et al. Antimicrob Agents Chemother 2015; 59: 397-406); and (iv) bictegravir (GS-9883) (Tsiang et al. Antimicrob Agents Chemother 2016; 60: 7086-7097). Additional data are shown for mutants G118R, H51Y/G118R and G118R/E138K [Kobayashi et al. Antivir Ther 2011; 16 (suppl. 1): A49; Quashie et al. Antimicrob Agents Chemother 2013; 57: 6223-6235; 2014; 58: 3580 (erratum)]; Q148N (Varghese et al. AIDS Res Hum Retroviruses 2016; 32: 702-704); E157Q/R263K (Anstett et al. J Antimicrob Chemother 2016; 71: 2083-2088); and H51Y and/or R263K (Mesplède et al. Retrovirology 2013; 10: 22; Wares et al. Retrovirology 2014; 11: 7).

Table 5.2. OVERVIEW OF GENOTYPIC RESISTANCE TO INTEGRASE INHIBITORS

Integrase Selection Integrase mutations Refs. inhibitor process Bictegravir In vitro M50I, R263K 1 (GS-9883) Dolutegravir In vitro T124A/S153Y 2 (S/GSK1349572) L101I/T124A/S153F (in HIV-1) R263K; M50I/R263K; H51Y/R263K; E138K/R263K 3-5 E92Q/G193E; Q148K/R/H (± E138K, G140S, T97A) 6 T97A/Y143C; G140S/Q148R; 7 G140T/Q148R/N155H (in HIV-2) In patients R263K 8 G118R (± E138K), F121Y 9 G118R, Q148R/H, N155H 10 Cabotegravir In patients Q148R 11 (GSK1265744, GSK744)

392 Integrase Selection Integrase mutations Refs. inhibitor process Elvitegravir In vitro E92Q (+ H51Y, S147G, and E157Q) 12, 13 (GS-9137) T66I (+ R263K or + S153Y and F121Y) 12, 14 In patients T66A/K 15 E92Q (+ N155H) Q148R (+E138A/K or +G140C/S or +S147G) N155H GS-9160 In vitro L74M/E92V 16 L-708,906 In vitro T66I/M154I 17 T66I/S153Y 17 N155S 18 T66I/L74M/S230R 19 L-870,810 In vitro V72I/F121Y/T125K/V151I 18 L74M/E92Q/S230N 20 MK-2048 In vitro G118R/E138K 21 N155H 22 Raltegravir In vitro E138A/G140A/Q148K 23 (MK-0518) In patients a Major pathways: 23-28 1) N155H (+ L74M, E92Q, V151I, T97A, G136R) 2) Q148K/R/H (+ G140A/S and E138K) b Other possible pathways: Y143C/R (+ L74A/I, E92Q, I203M, and S230R) L74M/G118R (in CRF02_AG HIV-1 subtype) Most common pathway: G140S/Q148H S-1360 In vitro T66I/L74M/A128T/E138K/Q146K/S153A 29 K160D/V165I/V201I 29 T66I/T124A/N155S 30 S/GSK-364735 In vitro T66K/Q95R/Q146R/Q148R 30 F121Y/T124A E138K/Q148R G140S/Q148R a Resistance genetic pathways in HIV-2 include mutations E92Q/Y143R/N155H, T97A/Y143C, T97A/N155H, G140S/Q148R, Q148K and Q148R (Charpentier et al.

393 Antimicrob Agents Chemother 2011; 55: 1293-1295). Other mutations associated with resistance to HIV-1 IN inhibitors found in HIV-2 are V151I and D232N (Cavaco Silva et al. PLoS One 2014; 9: e92747). The substitutions Y143C/N155H and Q148R/N155H seem to define mutually exclusive pathways of raltegravir resistance in HIV-2 (Ni et al. Retrovirology 2011; 8: 68; Smith et al. AIDS 2011; 25: 2235-2241) b Deep sequencing analysis of virus isolated from three patients showed transitions from the N155H pathway to the G140S/Q148H pathway during raltegravir treatment (Mukherjee et al. AIDS 2011; 25: 1951-1959).

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