Background: Fostemsavir (FTV) is a member of a new drug class currently under investigation for highly drug-experienced HIV-1 infected patients. This antiretroviral is an attachment inhibitor that binds HIV-1 gp120, by blocking HIV attachment to host CD4+ T-cells. To date, mutations at seven positions (L116, A204, S375, M426, M434, S475 and V506) of gp120 are known to reduce FTV susceptibility both in vitro and in vivo. In this study, we evaluated the natural prevalence of such FTV-resistance mutations and their potential association with other HIV-1 gp120 polymorphisms.
Material and methods: The study was conducted on 1,997 HIV-1 gp120 sequences of B subtype obtained from plasma samples of individuals naïve to FTV from Los Alamos HIV Database. Sequences were aligned to HXB2 reference and manually edited. Prevalence of mutations at the FTV associated positions was evaluated in overall population and stratified according to HIV-1 tropism (Geno2Pheno algorithm, FPR=10%). A covariation analysis was performed to evaluate potential association between the FTV-resistance mutations and other gp120 polymorphisms present with a prevalence ≥5%. An average linkage hierarchical agglomerative clustering was also performed, analysing FTV-resistance mutations associated with each mutation alongside all the 511 gp120 amino acid positions.
Results: The following FTV mutations were detected: L116Q (0.05%), S375H (0.6%), S375M (1.4%), S375T (17.7%), M426L (7.6%), M434I (4.2%), M475I (1.7%), while mutations L116P, A204D and V506M were completely absent. Among other natural polymorphisms at FTV resistance positions, only the mutation M426R was found with a prevalence >5% (16.3%). Generally, no specific association between viral tropism and FTV mutations prevalence was found, with the exception for S375M (R5 vs. X4: 0.7% vs. 3.9%, p=0.009) and S375T (16.6% vs. 22.1%, p=0.03). By the covariation analysis, specific gp120 mutations were positively correlated with FTV-resistance positions. In particular, S375T correlated with I371V (phi=0.21; p=9.8x10^-15); S375M correlated with the three mutations L134W (phi=0.21; p=2.6x10^-8), I154V (phi=0.20; p=4.5x10^-8), and I323T (phi=0.24; p=9.6x10^-10); and M475I correlated with K322A (phi=0.24; p=1.8x10^-10). Finally, the polymorphism M426R strongly correlated with the mutations G167N (phi=0.33; p=1.6x10^-35), K192T (phi=0.47; p=1.5x10^-66), and S195N (phi=0.24; p=3.6x10^-22). The topology of the dendrogram revealed the existence of two distinct clusters and two pairs of mutations (bootstrap ≥0.98) that confirmed the involvement of divergent pathways of gp120 mutations potentially associated with FTV-resistance. Interestingly, all FTV mutations and polymorphisms present in the clusters are localized in class I/II–restricted T-cell epitopes and antibody epitopes (according to Los Alamos Immunology Database lists, available at https://www.hiv.lanl.gov/content/immunology/products.html), suggesting a potential role in HIV escape from immune response.
Conclusions: Despite the high variability of gp120, FTV-resistance mutations were found with a low prevalence in sequences from individuals FTV-naïve infected with HIV-1 B subtype. The potential contribution of some of these mutations with other specific gp120 polymorphisms to the development of a synergistic effect of resistance to FTV may have viro-immunological implications, thus deserving further in-depth in vitro and structural investigation.

Background: Protease is an effective therapeutic target for the treatment of HIV-1. However, drug resistance mutations challenge the long-term efficacy of protease inhibitors.
Methods: Protease sequences, collected between 1986 and 2017, were downloaded from Los Alamos database, from subtypes A-C, F, G and CRF02_AG. Transmitted Drug Resistance (TDR) was estimated using the World Health Organization 2009 surveillance drug resistance mutation (SDRM) list and genotypic resistance to antiretroviral drugs (ARV) was evaluated using the Standford HIVdb v8.4. Statistical analysis was performed using SPSS software.
Results: 62,676 HIV-1 protease sequences collected from patients were analysed. Of these, 39078 were drug naïve (DN) and 23598 were treated (TR). Among the DN, 836 (2.1%) presented TDR while 7085 (30%) of TR presented ADR. TDR was 3 times higher in men who have sex with men (MSM) than in heterosexuals (HET) (p<0.001) and was higher in subtype F1 (3.1%), followed by B (2.7%), A (1.7%), C (0.9%), CRF02_AG (1.2%) and G (1.1%), respectively (p<0.0001). Patients from Eastern Europe (5.6%), Oceania (3.1%), North America (2.9%), and Western Europe (2.7%) had higher TDR than patients from Asia (2.4%), South America (2.2%), and Africa (1.1%) (p<0.001). ADR was higher in subtypes F, B and G (47%, 44% and 19%, respectively), as other subtypes presented levels lower than 10% (p<0.0001). The ADR rate was higher in Western Europe (55%), South America (43%), Central and North America (34%) than in Oceania (18%), Asia (16%), Eastern Europe (9.5%) and Africa (6%) (p<0.0001). Concerning risk factor analyses the same profile was observed as for TDR: the rate of ADR among MSM was 3 times higher than HET group (p<0.0001). The simple logistic regression models showed that gender, risk factor, subtypes and geographic region could explain the presence of TDR and ADR. Among DN patients, TDR to Darunavir (DRV) (0.18% - 95 IC 0.14%-0.22%) was 5 times lower than resistance to Atazanavir (0.9% - 95 IC 0.8%-0.99%) and almost 10 times lower compared to Lopinavir (1.7% - 95 IC 1.4%-1.83%) (p<0.05).
Conclusions: Overall, TDR and ADR to Protease inhibitors is low, but substantially different when comparing risk groups, subtypes and geographic regions. DRV presents very low drug resistance levels and should be considered for first line regimens. Moreover, the findings indicated a higher TDR rate in Eastern Europe and Oceania but lower ADR when compared to other continents.

*Presenting on behalf of the authors.

Background: DAWNING is a non-inferiority study comparing dolutegravir (DTG) + 2 nucleoside reverse transcriptase inhibitors (NRTIs) with lopinavir/ritonavir (LPV/r) + 2 NRTIs in HIV-1 infected adults failing first-line therapy (HIV-1 RNA ≥400 c/mL) of a non-nucleoside reverse transcriptase inhibitor + 2 NRTIs.

Methods: Participants were randomized (1:1, stratified by Screening HIV-1 RNA and number of fully active NRTIs) to 52 weeks of open-label treatment with DTG or LPV/r + 2 investigator-selected NRTIs, including ≥1 fully active NRTI based on Screening resistance testing. The primary endpoint was the proportion of participants with HIV‑1 RNA <50 c/mL at Week 48 (Snapshot algorithm). Post-hoc efficacy analyses were performed based on baseline NRTI resistance profile and NRTI use in the second-line background regimen (BR).

Results: Of 624 participants randomized and treated, 499 (80%) received <2 active NRTIs at baseline. Overall, 84% (261/312) of participants on DTG versus 70% (219/312) on LPV/r achieved HIV-1 RNA <50 c/mL at Week 48 (adjusted difference 13.8%, 95% confidence interval [CI]: 7.3-20.3; P<0.001 for superiority). This difference was consistent regardless of the use of <2 or 2 fully active NRTIs in the BR. NRTI resistance was present in 561 participants (90%) at baseline, M184V/I (alone or plus additional NRTI resistance-associated mutations [RAMs]) in 513 (82%), K65R in 187 (30%), and ≥1 thymidine-analogue mutations (TAMs) in 152 participants (24%). Of participants with M184V/I alone or plus ≥1 NRTI RAMs, 430 participants (84%) took lamivudine (3TC) or emtricitabine (FTC) as part of their BR. Tenofovir disoproxil fumarate (TDF) was included in BR in the presence of K65R in 15 participants, whereas 86 participants with ≥1 TAMs took zidovudine (AZT). Among participants receiving 3TC or FTC in the presence of M184V/I, 85% (187/220) of participants on DTG versus 72% (152/210) on LPV/r had HIV-1 RNA <50 c/mL at Week 48 (difference 12.6%, 95% CI: 4.9-20.3). High responses were also observed in the DTG arm, when AZT or TDF were included in the BR in the presence of TAMs or K65R, respectively; however, participants numbers in these subgroups were small.

Conclusions: In DAWNING, response rates were high in participants receiving DTG + 2 NRTIs regardless of pre-existing resistance to one of the NRTIs in the BR, including in participants using 3TC or FTC in the presence of M184V/I. In World Health Organization interim guidance on HIV treatment, DTG + 2 NRTIs is now a recommended second-line treatment option for patients failing a non-NRTI-based regimen.

Data included in this abstract have been previously presented at the Conference on Retroviruses and Opportunistic Infections; March 4-7, 2019; Seattle, WA, USA.

BACKGROUND: Pre-existing resistance can affect the efficacy of switching antiretroviral regimens in HIV-infected individuals. Studies 1878 and 1844 demonstrated the non-inferior efficacy of switching stably suppressed HIV-1-infected adults to bictegravir/emtricitabine/tenofovir alafenamide (B/F/TAF) versus continuing boosted protease inhibitor (b/PI)-based triple regimens or dolutegravir/abacavir/lamivudine (DTG/ABC/3TC) through 48 weeks. After Week 48, all participants received open-label B/F/TAF. We retrospectively investigated pre-existing M184V/I and thymidine analog resistance mutations (TAMs), frequency of viral blips, and virologic outcomes through >2 years of B/F/TAF treatment.

MATERIALS AND METHODS: Participants included in the analysis had ≥1 post-baseline HIV-1 RNA measurement through September 15, 2018. Pre-existing HIV-1 drug resistance was first assessed by historical genotypes, and documented M184V/I or K65R at screening was exclusionary. Proviral DNA genotyping (GenoSure Archive® assay, Monogram Biosciences) was conducted retrospectively on samples from the baseline visit. Participants with resistance substitutions detected by proviral genotyping after randomization were allowed to continue on study. Transient viremia (blip; one HIV-1 RNA ≥50 copies/mL measurement preceded and followed by HIV-1 RNA <50 copies/mL) was assessed. Virologic outcomes were based on last available on-treatment HIV-1 RNA.

RESULTS: Altogether, 570 participants switched to B/F/TAF on Day 1 and were treated for a median of 116 weeks (IQR 108-120 weeks). Cumulative baseline genotypic data from historical and/or proviral genotypes were available for 78% (445/570) of B/F/TAF-treated participants: 36% (159/445) had ≥1 pre-existing primary resistance substitution to any antiretroviral drug at baseline. M184V/I was uncovered by proviral genotyping in 10% (44/445) of participants, and 1-2 TAMs were detected in 6.1% (27/445). Through Week 48, there were 74 viral blip events: 26 occurred in 4.4% (25/570) of B/F/TAF-treated participants compared to 31 in 8.1% (23/285) of participants in the b/PI group and 17 in 5.3% (15/281) of participants in the DTG/ABC/3TC group. After Week 48, there were 22 blip events in the B/F/TAF group: 18 occurred in 17 participants who experienced their first blip post-Week 48 and 4 occurred in 4 of those who also experienced a blip before Week 48. In total, 48 blips were observed in 7.4% (42/570) of B/F/TAF-treated participants through >2 years of follow-up. When stratified by baseline resistance, blips were observed in 4.5% (2/44) with M184V/I (both occurred before Week 48) and in 11% (3/27) with 1-2 TAMs (all occurred post-Week 48). At the time of analysis, 98% (561/570) of all B/F/TAF-treated participants were suppressed (HIV-1 RNA <50 copies/mL), including 98% (41/42) who had a viral blip on study, 95% (42/44) with M184V/I, and 93% (25/27) with 1-2 TAMs. No B/F/TAF-treated participant developed drug resistance.

CONCLUSIONS: In studies 1878 and 1844, high frequencies of previously unidentified baseline M184V/I and TAMs were detected among suppressed patients who were enrolled. In participants with or without pre-existing resistance, viral blips were infrequent and high rates of virologic suppression were maintained through >2 years of B/F/TAF treatment. Long-term suppression and the absence of treatment-emergent resistance indicate that B/F/TAF may be a treatment option for suppressed patients with documented or unidentified M184V/I and/or 1-2 TAMs.

BACKGROUND:
The World Health Organization recommends dolutegravir in treatment of HIV which can result in increased resistance to integrase inhibitors. Data on drug resistance to dolutegravir is predominantly available from subtype B, whereas in low- and middle-income countries subtypes other than B are the most common. The genetic barrier, defined as the number of mutations required to overcome drug-selective pressure, is a key factor in the development of HIV drug resistance. Because of high variability in codon usage between subtypes, particular HIV-1 subtypes could have different genetic barriers for drug resistance mutations. The aim of this study is to compare codon usage and the calculated genetic barrier for resistance between subtypes.

METHODS:
We considered the major drug resistance associated mutations (T66A/I/K, E92G/Q/V, G118R, E138A/K/T, G140A/C/S, Y143A/C/G/H/K/R/S, P145S, Q148H/K/N/R/S, N155D/H/S/T, R263K) and accessory drug resistance associated mutations (H51Y, L75F/I/M, T97A, F121Y, Q146P, V151A/I/L, S153F/Y, E157Q, G163K/R, S230R). Drug resistance associated mutations occurring outside of the integrase region were not considered. Codon usage was compared between subtypes at positions where drug resistance associated mutations occurred. The genetic barrier, calculated as the sum of transitions (scored as 1, or 0.2 for the G to A hypermutation) and/or transversions (2.5) required for evolution to any drug resistance substitution, was compared using the Kruskal-Wallis test and Mann-Whitney tests using a false discovery rate of 0.01 to correct for multiple comparisons.

RESULTS:
13,694 integrase sequences from integrase inhibitors naïve individuals (subtypes A-D, F, G, CRF01_AE and CRF02_AG) were selected from the Stanford HIV drug resistance database. The most frequent subtypes were B (6644 sequences/ 49%), C (2517/18%), and CRF01_AE (1824/13%). Several accessory mutations were present as polymorphism in <10% across subtypes (L74M, T97A, E157Q, G163R). L74I occurred as polymorphism in >10% of subtype A (21%) and CRF02_AG (12%). The codon usage was different between subtypes at major drug resistance positions 92 (GAG in a majority of subtype B versus GAA in other subtypes), 118 (GGT in CRF02_AE versus GGC), 140 (GGC in B versus GGA), 148 (CAG in C versus CAA), 263 (AGG in C versus AGA), and at accessory mutations position 151 (GTG in CRF01_AE and subtype A versus GTA), 153 (TCC in D versus TCT), 157 (GAG in subtype G versus GAA), and 163 (extensive differences across subtypes). An increased genetic barrier was only observed for the major mutation G140C in subtype B (p<0.0001 versus other subtypes). Compared to subtype B a different calculated genetic barrier was calculated for mutations L74F/I/M, T97A, V151A/I, E157Q, G163K/R and S230R (p<0.0001). Using different scores for transitions and transversions did not impact the results of the statistical analysis.

CONCLUSIONS:
Differences in codon usage between subtypes are common and predominantly affect the calculated genetic barrier of accessory drug resistance mutations and only one major drug resistance mutation, indicating that the rate of development of drug resistance will most likely be similar between subtypes. The impact of differences in codon usage between subtypes on the genetic barrier and drug resistance development has to be confirmed in in-vitro and/or in-vivo studies.

Background:
Widespread access to highly effective combination antiretroviral therapy (cART), despite wide range of the benefits, entails the risk of increasing emergence of HIV-1 drug resistance mutations related to the treatment failure. Transmitted drug resistance mutations (TDRM) are still severe problem because, when occur, can spread in the population intensively limiting the available therapeutic options. Long term surveillance of TDRM clinical and epidemiological aspects are crucial in planning strategy of effective initial therapy regimens to avoid spread of TDR. Public health interventions assuming reducing the spread of HIV-1 epidemy requires up-to-date knowledge about trends in TDRM prevalence over time. The aim of the study was to analyse the distribution of: HIV-1 subtypes, the prevalence of TDRM and evaluate the potential phylogenetic relationship of TDRM and its impact on the baseline susceptibility in newly diagnosed patients in Central Poland between 2012 and 2018.

Materials and Methods:
One thousand seven hundred and seventy four plasma samples obtained from antiretroviral treatment naïve HIV-1 positive patients, newly diagnosed in our Hospital during the period 2012-2018, were analysed. In the studied cohort dominated men –1648/1774(92,9%). Viral RNA isolation, PR-RT coding region amplification and sequencing were performed using ViroSeq HIV-1 Genotyping_Kit (Celera) and 3130-Avant Genetic Analyzer (Life_Technologies). Surveillance drug resistance mutations (SDRMs) were examined according to the Stanford Genotypic Resistance calibrated population resistance (CPR) tool version 6.0 based on the WHO surveillance transmitted drug resistance mutation list of 2009. For HIV-1 subtype determination REGA HIV-1&2 Subtyping Tool was applied. Phylogenetic analysis was based on maximum likelihood method with aLTR.

Results:
The percentage of subtype B infections is decreasing in studied period from 84,7% in 2012 to 65,22% in 2018. Opposite trend in prevalence of subtype A (with dominating A-1FSU) was determined: increase from 6,55% in 2012 to 24,4% in 2018. Prevalence of TDRM fluctuated in the years 2012-2018: 10,16% in 2012, decreased to 3,5% in 2015 and finally reached 5,5% in 2018. According to drug classes, mean resistance prevalence was: NRTI–4,07%, PI–1,4% and NNRTI–1,14%. The most frequent mutations were: NRTI–T215* (56,18%), NNRTI–K103N (47,83%) and PI–L90M (40%). Single cases of infection with variants resistant to two (NRTI and NNRTI) and three (NRTI, NNRTI, PI) drug classes were detected. Phylogenetic analysis revealed transmission clusters in the analysed populations.

Conclusions:
Presented data shows dynamic, epidemiologically interesting and clinically important, growth in prevalence of infections with non-B subtypes in particular with predominance of A-1FSU strains. Intense economical migration, mainly from Former Soviet Union Republics, enriches genetic heterogeneity of HIV-1 variants in Poland. Phylogenetic analysis revealed multiple, independent introductions of HIV-1 A-1FSU, followed by local transmissions. Detected prevalence of TDRM among Polish naïve patients stabilized on level 5,50% in 2018. Determined prevalence is low when compared to this observed during the first studies performed in the years 2000-2007, when TDR reached 20-15%. These data probably are a result good cooperation between clinics and Laboratory as well as documents high adherence of the patients and high antiviral potency of the new drugs.

*This work was in part sponsored by Foundation_for_Research_Development in Hospital_for_Infectious_Diseases,_Warsaw

INTRODUCTION: SAILING was a Phase3 clinical trial evaluating DTG 50mg once daily vs. raltegravir (RAL) 400mg twice daily in ART-experienced, integrase inhibitor (INSTI)-naïve, HIV-1 infected participants. Previous Week(WK) 48 results reported fewer participants meeting protocol defined virologic failure (PDVF) in the DTG arm: 21 PDVFs versus 45 in the RAL arm. 4/354 (1%) failed with integrase(INI)-genotypic or phenotypic resistance on DTG versus 17/361 (5%) on RAL (p=0.003) through WK48. Three additional participants receiving DTG in the open-label phase had emergent INSTI resistance through WK132. Here we present post hoc, longitudinal assessment of PDVF and viral resistance in patients remaining on DTG up to 360 weeks.

METHODS: PDVF required confirmed HIV-1 RNA >400 c/mL (Abbott RealTime assay). PDVF non-response was <1 log10 c/mL decrease by WK16, unless <400 c/mL, OR ≥400 c/mL on or after WK24. PDVF rebound was ≥400 c/mL after confirmed <400 c/mL, OR >1 log10 c/mL above nadir of ≥400 c/mL. Data was collected through 08/28/2018 for this instream work. Results were generated from observed data. Population-based genotypic and phenotypic testing was performed on baseline and PDVF timepoint samples by Monogram BioSciences.

RESULTS: Low rate of PDVF was maintained for 295 participants entering the open-label continuation phase and remaining on DTG arm following WK48 through WK360, with 25 additional participants meeting PDVF criteria: 2(0.7%) at WK60, 2(0.7%) at WK70, 8(2.9%) at WK84, 4(1.6%) at WK96, 2(0.9%) at WK108, 2(0.9%) at WK120, 1(0.5%) at WK132, 2(1.2%) at WK156, 1(2%) at WK264 and 1(3%) at WK324. Overall INSTI emergent resistance through 360 weeks showed that one participant added E138T/A+T97A to baseline Q148H+E138A+G140S, one with polymorphic V151V/I, three with R263K, and two with N155H. For the R263K cases, DTG fold-change (FC) ranged from 1.12 (R263R/K) and 1.93 (R263K) to 3.82 (A49G+S230R+R263K); for V151V/I, DTG FC was 0.92; for the two participants who developed N155H, DTG FCs were 1.8 (N155H) and 2.4 (I60L+T97A+N155H). An assessment of post-baseline emergent resistance to the background regimen (BR) was performed for DTG participants experiencing PDVF. No additional treatment-emergent NRTI resistance was observed in participants receiving DTG+2 NRTIs, even without full backbone activity. 3/7 PDVF participants with emergent INSTI resistance showed treatment emergent resistance to the BR: 2 had NNRTI resistance and one with pre-existing RAL resistance Q148+>2 (G140S+E138A) pathway developed PI resistance mutations V32V/I+I50L. For those 39/46 PDVF participants without emergent INSTI resistance, only one exhibited treatment emergent PI resistance mutation G48G/I/R/V to the BR.

CONCLUSIONS: The post-WK48 open-label phase evaluation of participants remaining on DTG continues to demonstrate low PDVF rate and low frequency of treatment emergent resistance to DTG and BR. Observed INSTI resistance had overall low impact on DTG FC. DTG+2 NRTIs was shown to be associated with few cases of emergent resistance to the BR. The cumulative 8+ years of SAILING resistance data provides additional evidence to support the DTG barrier to resistance and supports WHO interim guidelines with updated recommendations for including DTG+2 NRTIs as a recommended second-line treatment option for patients failing an NNRTI or PI first-line ARV regimen.