Nonnucleoside Reverse Transcriptase Inhibitor Resistance Mutations Research Articles

Portable Nanopore sequencing solution for next-generation HIV drug resistance testing

BackgroundTackling HIV drug resistance is one of major challenges for ending AIDS epidemic, but the elevated expense of cutting-edge genomics hampers the advancement of HIV genotype testing for clinical care. MethodsWe developed a HIV genotype testing pipeline that centers on a cost-efficient portable Nanopore sequencer. Accuracy verification was conducted through comparison with parallel data obtained via fixed-site Pacbio sequencing. Our complete pol-gene sequencing strategy coupled with portable high-throughput sequencing was applied to identify drug resistance mutations across 58 samples sourced from the ART-treated Los Angeles General Medical Center Rand Schrader Clinic (LARSC) cohort (7 samples from 7 individuals) and the ART-naïve Center for HIV/AIDS Vaccine Immunology (CHAVI) cohort (51 samples from 38 individuals). ResultsA total of 472 HIV consensus sequences, each tagged with a unique molecular identifier, were produced from over 1.4 million bases acquired through portable Nanopore sequencing, which matched those obtained independently via Pacbio sequencing. With this desirable accuracy, we first documented the linkage of multidrug cross-resistance mutations across Integrase Strand Transfer inhibitors (INSTIs) and Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) from an individual failing a second-generation INSTI regimen. By producing more than 500 full-length HIV pol gene sequences in a single portable sequencing run, we detected Protease Inhibitor (PI), Nucleoside Reverse Transcriptase Inhibitor (NRTI), NNRTI and INSTI resistance mutations. All drug resistance mutations identified through portable sequencing were cross-validated using fixed-site Pacbio sequencing. ConclusionsOur accurate and affordable HIV drug resistance testing solution is adaptable for both individual patient care and large-scale surveillance initiatives.

APOBEC3 selects V179I in HIV-1 reverse transcriptase to provide selective advantage for non-nucleoside reverse transcriptase inhibitor-resistant mutants

The V179I substitution in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is selected in humans or mouse models treated with certain nonnucleoside reverse transcriptase inhibitors (NNRTIs). While it is often observed together with other NNRTI resistance mutations, V179I does not confer drug resistance. To understand how V179I arises during NNRTI treatment, we characterized it in HIV-1 molecular clones with or without the NNRTI resistance mutations Y181C or Y181V. While V179I alone did not confer resistance to any NNRTIs tested, when present with Y181C/V it enhanced drug resistance to some NNRTIs by 3- to 8-fold. In replication competition experiments in the presence of the NNRTI rilpivirine (RPV), V179I modestly enhanced Y181C HIV-1 or Y181V HIV-1 replication compared to viruses without V179I. As V179I arises from a G to A mutation, we evaluated whether it could arise due to host APOBEC3 deaminase activity and be maintained in the presence of a NNRTI to provide a selective advantage for the virus. V179I was detected in some humanized mice treated with RPV and was associated with G to A mutations characteristic of APOBEC3 activity. In RPV selection experiments, the frequency of V179I in HIV-1 was accelerated in CD4+ T cells expressing higher APOBEC3F and APOBEC3G levels. Our results provide evidence that V179I in HIV-1 RT can arise due to APOBEC-mediated G to A hypermutation and can confer a selective advantage to drug-resistant HIV-1 isolates in the presence of some NNRTIs.

Development of HIV Drug Resistance in a Cohort of Adults on First-Line Antiretroviral Therapy in Tanzania during the Stavudine Era

As more HIV patients start combination antiretroviral therapy (cART), the emergence of HIV drug resistance (HIVDR) is inevitable. This will have consequences for the transmission of HIVDR, the success of ART, and the nature and trend of the epidemic. We recruited a cohort of 223 patients starting or continuing their first-line cART in Tanzania towards the end of the stavudine era in 2010. Patients were then followed for one year. Of those with a viral load test at baseline and follow-up time, 34% had a detectable viral load at the one-year endpoint. For 41 patients, protease and reverse transcriptase genotyping were successful. Eighteen samples were from cART-naïve patients, and 23 samples were taken under therapy either at baseline for cART-experienced patients or from follow-up samples for both cART–naïve and cART–experienced patients. The isolates were subtype A, followed by C and D in 41.5%, 22%, and 12.2% of the patients, respectively. No transmitted HIVDR was detected, as scored using the surveillance drug resistance mutations (DRMs) list. However, in 3 of the 18 samples from cART-naïve patients, the clinical Rega interpretation algorithm scored 44D or 138A as non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance-associated polymorphisms. The most observed nucleoside reverse transcriptase inhibitor (NRTI) mutation was 184V. The mutation was found in 16 patients, causing resistance to lamivudine and emtricitabine. Nineteen patients had NNRTI resistance mutations, the most common of which was 103N, observed in eight patients. These high levels of resistance call for regular drug resistance surveillance in Tanzania to inform the control of the emergence and transmission of HIVDR.

HSV-2 Infection as a Potential Cofactor for HIV Disease Progression and Selection of Drug Resistance Mutations in Adults under WHO-Recommended First-Line Antiretroviral Therapy: A Multicentric, Cross-Sectional Study in Cameroon, Central African Republic, Chad, and Gabon.

Although herpes simplex virus-2 (HSV-2) infection is a known cofactor for HIV transmission in Central Africa, its role in HIV disease progression is unclear. The aim of this study was to examine the potential link between HSV-2 infection and HIV disease progression, in addition to identifying the presence of genes conferring HIV antiretroviral resistance mutations. This was a cross-sectional study involving 302 HIV-infected adults in Central Africa with virological failure (viral load >1000 copies/mL) on first-line antiretroviral therapy from four different countries. The seroprevalence of HSV-2 was 32% (96/302). Amongst the HIV-infected individuals who were HSV-2 seropositive, the mean HIV viral load and CD4 count were 4.82 ± 0.83 log copies/mL and 243 ± 144 cells/microliter, respectively. Among the HIV-infected individuals who were HSV-2-seronegative, the mean HIV viral load and CD4 count were 3.48 ± 0.44 log copies/mL and 646 ± 212 cells/microliter, respectively (p < 0.001). There was a statistically significant relationship (p < 0.001) between HSV-2 seropositivity and the presence of resistance mutations to antiretrovirals (ARV), non-nucleoside reverse transcriptase inhibitors (NNRTI), and nucleoside reverse transcriptase inhibitors (NRTI) with odds ratios of 9.7, 10, and 11.9, respectively. There was no link between HSV-2 serostatus and protease inhibitor (PI) resistance mutations. There was a substantial accumulation of resistance mutations in HSV-2-seropositive compared to -seronegative patients. These findings support the link between HIV disease progression and HSV-2 infection. An association was observed between the presence of NNRTI and NRTI resistance mutations and HSV-2 seropositivity.

Frequent cross-resistance to rilpivirine among subtype C HIV-1 from first-line antiretroviral therapy failures in South Africa.

BackgroundRilpivirine (TMC278LA) is a promising drug for pre-exposure prophylaxis of HIV-1 because of its sub-nanomolar potency and long-acting formulation; however, increasing transmission of non-nucleoside reverse transcriptase inhibitor-resistant HIV-1 with potential cross-resistance to rilpivirine could reduce its preventive efficacy. This study investigated rilpivirine cross-resistance among recombinant subtype C HIV-1 derived from 100 individuals failing on first-line non-nucleoside reverse transcriptase inhibitor-containing antiretroviral therapy in South Africa whose samples were sent for routine HIV-1 drug resistance testing to Lancet Laboratories (Johannesburg, South Africa).MethodsPlasma samples were selected from individuals with HIV-1 RNA > 10,000 copies/ml and ≥1 non-nucleoside reverse transcriptase inhibitor-resistance mutation in reverse transcriptase. Recombinant HIV-1LAI-containing bulk-cloned full-length reverse transcriptase sequences from plasma were assayed for susceptibility to nevirapine (NVP), efavirenz (EFV) and rilpivirine in TZM-bl cells. Fold-change (FC) decreases in drug susceptibility were calculated against a mean IC50 from 12 subtype C HIV-1 samples from treatment-naïve individuals in South Africa. Cross-resistance was evaluated based on biological cutoffs established for rilpivirine (2.5-FC) and the effect of mutation combinations on rilpivirine phenotype.ResultsOf the 100 samples from individuals on failing antiretroviral therapy, 69 had 2.5- to 75-fold decreased susceptibility to rilpivirine and 11 had >75-fold resistance. Rilpivirine resistance was strongly associated with K103N especially in combination with other rilpivirine-associated mutations.ConclusionThe frequently observed cross-resistance of HIV-1 suggests that the preventive efficacy of TMC278LA pre-exposure prophylaxis could be compromised by transmission of HIV-1 from individuals with failure of first-line non-nucleoside reverse transcriptase inhibitor-containing antiretroviral therapy.

Transmission Dynamics of HIV-1 Drug Resistance among Treatment-Naïve Individuals in Greece: The Added Value of Molecular Epidemiology to Public Health

The presence of human immunodeficiency virus type 1 (HIV-1) drug resistance among drug-naïve patients remains stable, although the proportion of patients with virological failure to therapy is decreasing. The dynamics of transmitted resistance among drug-naïve patients remains largely unknown. The prevalence of non-nucleoside reverse transcriptase inhibitors (NNRTI) resistance was 16.9% among treatment-naïve individuals in Greece. We aimed to investigate the transmission dynamics and the effective reproductive number (Re) of the locally transmitted NNRTI resistance. We analyzed sequences with dominant NNRTI resistance mutations (E138A and K103N) found within monophyletic clusters (local transmission networks (LTNs)) from patients in Greece. For the K103N LTN, the Re was >1 between 2008 and the first half of 2013. For all E138A LTNs, the Re was >1 between 1998 and 2015, except the most recent one (E138A_4), where the Re was >1 between 2006 and 2011 and approximately equal to 1 thereafter. K103N and E138A_4 showed similar characteristics with a more recent origin, higher Re during the first years of the sub-epidemics, and a declining trend in the number of transmissions during the last two years. In the remaining LTNs the epidemic was still expanding. Our study highlights the added value of molecular epidemiology to public health.

Drug resistance mutations and its associated factors among 579 HIV/AIDS patients experiencing failure of antiretroviral therapy in Jiangsu Province, China

Objective: To understand the incidence and related factors of HIV-1 drug resistance among HIV/AIDS patients experiencing treatment failure in Jiangsu province, China. Methods: The HIV/AIDS integrated prevention and control data information management system of China were used to collect the basic data of patients, blood specimens were collected from patients who had antiretroviral therapy (ART) failure with ≥12 months and older than 18 years in 2016 in Jiangsu, excluding cases with missing information, 713 cases were enrolled in this study. HIV-1 RNA was extracted, and then pol gene region was amplified and sequenced. The obtain sequences were submitted to Stanford University HIV Drug Resistance Database to interpret and analyse HIV-1 drug resistance and sub-types. Multivariate logistic regression model was used to explore the related factors of drug resistance. Results: A total of 579 subjects were amplified successfully, male accounted for 85.66% (496 cases), and the median age was 39 years old. The main route of infection was sexual transmission (553 cases, 95.51%). A total of 331 patients with drug resistance gene mutation were detected, drug resistance mutation rate was 57.18%. Compared with patients with baseline CD4(+)T cell count >500 cells/μl, patients with CD4(+)T cell count in 201-500 cells/mm(3) and ≤200 cells/μl had a higher incidence of genetic drug resistance, the odds ratio was 3.33 and 6.87, respectively. Compared with patients with treatment less than 24 months, patients treated for 25-48 months had a higher incidence of drug resistance, the odds ratio was 1.88. Compared with patients infected by CRF07_BC strains, patients infected by CRF01_AE strains were associated with higher incidence of drug resistance, the odds ratio was 2.22 and 3.32, respectively. Protease inhibitor (PI) resistance mutations, nucleoside reverse transcriptase inhibitor (NRTI) and non-nucleoside reverse transcriptase inhibitor (NNRTI) were found in 3.80%, 33.16% and 53.37% of patients, respectively. 31.95% of patients harbored NRTI and NNRTI resistance mutations simultaneously. M184V/I and K103N/Q were the highest frequency of NRTI and NNRTI resistance mutation, the prevalence of M184V/I and K103N/Q were 28.15% and 22.28%, respectively. Conclusion: The status of HIV-1 drug resistance mutations are complex and diverse among patients experiencing failure of ART in Jiangsu. Patients with lower baseline CD4(+)T cell count, longer treatment time and HIV-1 CRF01_AE and B strains infection were associated with higher incidence of drug resistance mutation.

MIV-150 and zinc acetate combination provides potent and broad activity against HIV-1.

We previously showed that the combination of the non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 with zinc acetate (ZA) formulated in a carrageenan (CG; MZC) gel provided macaques significant protection against vaginal simian-human immunodeficiency virus-RT (SHIV-RT) challenge, better than either MIV-150/CG or ZA/CG. The MZC gel was shown to be safe in a phase 1 clinical trial. Herein, we used in vitro approaches to study the antiviral properties of ZA and the MIV-150/ZA combination, compared to other NNRTIs. Like other NNRTIs, MIV-150 has EC50 values in the subnanomolar to nanomolar range against wild type and NNRTI or RT-resistant HIVs. While less potent than NNRTIs, ZA was shown to be active in primary cells against laboratory-adapted and primary HIV-1 isolates and HIV-1 isolates/clones with NNRTI and RT resistance mutations, with EC50 values between 20 and 110μM. The MIV-150/ZA combination had a potent and broad antiviral activity in primary cells. In vitro resistance selection studies revealed that previously described NNRTI-resistant mutations were selected by MIV-150. ZA-resistant virus retained susceptibility to MIV-150 (and other RTIs) and MIV-150-selected virus remained sensitive to ZA. Notably, resistant virus was not selected when cultured in the presence of both ZA and MIV-150. This underscores the potency and breadth of the MIV-150/ZA combination, supporting preclinical macaque studies and the advancement of MZC microbicides into clinical testing.

Safety and Efficacy of a Dapivirine Vaginal Ring for HIV Prevention in Women

BackgroundThe incidence of human immunodeficiency virus (HIV) infection remains high among women in sub-Saharan Africa. We evaluated the safety and efficacy of extended use of a vaginal ring containing dapivirine for the prevention of HIV infection in 1959 healthy, sexually active women, 18 to 45 years of age, from seven communities in South Africa and Uganda.MethodsIn this randomized, double-blind, placebo-controlled, phase 3 trial, we randomly assigned participants in a 2:1 ratio to receive vaginal rings containing either 25 mg of dapivirine or placebo. Participants inserted the rings themselves every 4 weeks for up to 24 months. The primary efficacy end point was the rate of HIV type 1 (HIV-1) seroconversion.ResultsA total of 77 participants in the dapivirine group underwent HIV-1 seroconversion during 1888 person-years of follow-up (4.1 seroconversions per 100 person-years), as compared with 56 in the placebo group who underwent HIV-1 seroconversion during 917 person-years of follow-up (6.1 seroconversions per 100 person-years). The incidence of HIV-1 infection was 31% lower in the dapivirine group than in the placebo group (hazard ratio, 0.69; 95% confidence interval [CI], 0.49 to 0.99; P=0.04). There was no significant difference in efficacy of the dapivirine ring among women older than 21 years of age (hazard ratio for infection, 0.63; 95% CI, 0.41 to 0.97) and those 21 years of age or younger (hazard ratio, 0.85; 95% CI, 0.45 to 1.60; P=0.43 for treatment-by-age interaction). Among participants with HIV-1 infection, nonnucleoside reverse-transcriptase inhibitor resistance mutations were detected in 14 of 77 participants in the dapivirine group (18.2%) and in 9 of 56 (16.1%) in the placebo group. Serious adverse events occurred more often in the dapivirine group (in 38 participants [2.9%]) than in the placebo group (in 6 [0.9%]). However, no clear pattern was identified.ConclusionsAmong women in sub-Saharan Africa, the dapivirine ring was not associated with any safety concerns and was associated with a rate of acquisition of HIV-1 infection that was lower than the rate with placebo. (Funded by the International Partnership for Microbicides; ClinicalTrials.gov number, NCT01539226.)

Rilpivirine and Doravirine Have Complementary Efficacies Against NNRTI-Resistant HIV-1 Mutants.

Rilpivirine (RPV) is the latest non-nucleoside reverse transcriptase inhibitor (NNRTI) to be approved by Food and Drug Administration to combat HIV-1 infections. NNRTIs inhibit the chemical step in viral DNA synthesis by binding to an allosteric site located about 10 Å from the polymerase active site of reverse transcriptase (RT). Although NNRTIs potently inhibit the replication of wild-type HIV-1, the binding site is not conserved, and mutations arise in the binding pocket. Doravirine (DOR) is a new NNRTI in phase III clinical trials. Using a single round HIV-1 infection assay, we tested RPV and DOR against a broad panel of NNRTI-resistant mutants to determine their respective activities. We also used molecular modeling to determine if the susceptibility profile of each compound was related to how they bind RT. Several mutants displayed decreased susceptibility to DOR. However, with the exception of E138K, our data suggest that the mutations that reduce the potency of DOR and RPV are non-overlapping. Thus, these 2 NNRTIs have the potential to be used together in combination therapy. We also show that the location at which DOR and RPV bind with the NNRTI binding pocket of RT correlates with the differences in their respective susceptibility to the panel of NNRTI-resistance mutations. This shows that (1) DOR is susceptible to a number of well-known NNRTI resistance mutations and (2) an understanding of the mutational susceptibilities and binding interactions of NNRTIs with RT could be used to develop pairs of compounds with non-overlapping mutational susceptibilities.

The prevalence of primary HIV-1 drug resistance in newly reported HIV infections in Henan

To study the prevalence of primary HIV-1 drug resistance in newly reported HIV infected individuals receiving no antiviral treatment in Henan. Network direct reporting information of newly reported HIV infection cases in six cities of Henan during January to June, 2013 and January to June, 2014 were collected, and blood samples were collected from the cases to conduct genotypic drug resistance test and sequence analysis. Primary HIV-1 drug resistance was detected in 45 of 624 newly reported HIV infection cases, the prevalence of primary HIV-1 drug resistance was 7.21%, which was classified as moderate. Univariate analysis revealed that the prevalence of primary HIV-1 drug resistance was higher in females(χ(2)=11.463, P = 0.001), in age group <20 years(χ(2)=8.969, P=0.011), in illiterates(χ(2)=18.072, P=0.001)and in cases of HIV subtype B infection(χ(2)=9.897, P=0.019). Multi-univariate analysis revealed that the risk of primary HIV-1 drug resistance was high in females(OR=2.194, 95%CI: 1.111-4.331). Non-nucleoside reverse transcriptase inhibitor(NNRTI), Nucleoside reverse transcriptase inhibitor(NRTI)and Protease inhibitor(PI)resistance mutations were found in 4.97%, 3.53% and 1.12% of the cases, respectively. M184V/I(2.08%)and K103N/S(2.88%)were the most commonly emerged NRTI and NNRTI resistance mutation. Multiple NRTI resistance mutation was found in four cases. HIV subtype B infections accounted for largest proportion(51.76%, 323/624), followed by CRF07_BC cases(23.72%, 148/624)and CRF01_AE cases(19.71%, 123/624). The prevalence of primary HIV-1 drug resistance was moderate in the newly reported HIV infected individuals in Henan. The surveillance for HIV-1 drug resistance transmission should be strengthened and drug resistance test before the antiviral treatment should be given.

HIV CRF08_BC AND X4 STRAIN ARE ASSOCIATED WITH POSING MAJOR NUCLEOSIDE AND NON-NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITORS RESISTANCE MUTATIONS

In HIV treatment, surveillance of mutations inducing resistance to NRTIs (Nucleoside Reverse Transcriptase Inhibitors) and NNRTIs (Non-Nucleoside Reverse Transcriptase Inhibitors) is important. This study analyzed 2,071 HIV-1 genomic sequences directed to detection of major NRTIs and NNRTIs resistance mutations and viral tropism. CRF08_BC and X4 strain more likely had major NRTIs (adjusted odds ratio (aOR) 3.0, 95% CI 1.182-7.820, p=0.021 and aOR 1.7, 95% CI 1.233-2.368, p=0.001; respectively) and NNRTIs (aOR 3.3, 95% CI 1.281-8.365, p=0.013 and aOR 1.4, 95% CI 1.037-1.977, p=0.029, respectively) resistance mutations. Subtype A1 (aOR 0.2, 95% CI 0.069-0.702, p=0.011) and C (aOR 0.4, 95% CI 0.241-0.648, p&lt;0.001) were associated with major NRTIs resistance mutations. The occurrence of major NRTIs and NNRTIs resistance mutations in HIV subtype A1, C, CRF08_BC, and X4 viruses should be a particular concern.

Analysis of HIV-1 drug resistance among 1 922 individuals experiencing virological failure of first-line antiretroviral therapy in Henan province

To study the condition of HIV-1 drug resistance mutation among failures of first-line antiretroviral therapy in Henan province. The sub platform of China's legal infectious disease monitoring information reporting system-HIV/AIDS integrated prevention and control data information management system was used to collect the information of patients experiencing first-line antiretroviral treatment failure (virus load ≥ 1 000 copies/ml) more than one year among nine cities of Henan in 2011. A total of 40 cases with no information and 212 cases with incomplete drug resistance results were deleted, and 1 922 cases were included in this study and genotype resistance testing was carried out. Non-conditional logistic regression analysis was used to analyse the influencing factors of drug resistance mutation. A total of 1 922 cases were included in the analysis. 1 039 cases were males, 833 cases were females, the age was (45.7 ± 12.1) years, 82.73% (1 590) were married, and 87.93% (1 690) were transmitted by blood. 64.20% (1 234) patients acquired drug resistance. Nucleoside reverse transcriptase inhibitor (NRTI), non-nucleoside reverse transcriptase inhibitor (NNRTI) and protease inhibitor (PI) resistance mutations were found in 62.59% (1 203), 49.74% (956) and 0.94% (18) of subjects, respectively. 42.09% (809) of patients harbored NRTI and NNRTI resistance mutations synchronously. ≥ 1TAM was the most commonly emerged NRTI resistance mutation (41.94% (806)), the prevalences of TAM-1 and TAM-2 were 8.48% (163) and 4.24% (81), respectively. K65R/N and Q151M complex existed in 23 and 4 patients, respectively. K103N/S was the most commonly emerged NNRTI resistance mutation (34.32% (659)). Non-conditional logistic regression analysis showed that, factors associated with high drug resistance were the following: transmitted by mother to child (OR = 9.05, 95% CI: 1.14-72.12), clinical stage was IV (OR = 1.70, 95% CI: 1.09-2.66) and 5-year-treated (OR = 1.59, 95% CI: 1.03-2.47). Factors associated with low drug resistance were the following: 1-year-treated (OR = 0.19, 95% CI: 0.13-0.27). Complex patterns of HIV-1 drug resistance mutations were identified among individuals experiencing failure of first-line antiretroviral therapy in Henan province. Factors associated with high drug resistance were lived in Luohe, Shangqiu, Nanyang, Xinyang, transmitted by mother to child, clinical stage was IV, and 5-year-treated.

Identification of mechanistically distinct inhibitors of HIV-1 reverse transcriptase through fragment screening

Fragment-based screening methods can be used to discover novel active site or allosteric inhibitors for therapeutic intervention. Using saturation transfer difference (STD) NMR and in vitro activity assays, we have identified fragment-sized inhibitors of HIV-1 reverse transcriptase (RT) with distinct chemical scaffolds and mechanisms compared to nonnucleoside RT inhibitors (NNRTIs) and nucleoside/nucleotide RT inhibitors (NRTIs). Three compounds were found to inhibit RNA- and DNA-dependent DNA polymerase activity of HIV-1 RT in the micromolar range while retaining potency against RT variants carrying one of three major NNRTI resistance mutations: K103N, Y181C, or G190A. These compounds also inhibit Moloney murine leukemia virus RT but not the Klenow fragment of Escherichia coli DNA polymerase I. Steady-state kinetic analyses demonstrate that one of these fragments is a competitive inhibitor of HIV-1 RT with respect to deoxyribonucleoside triphosphate (dNTP) substrate, whereas a second compound is a competitive inhibitor of RT polymerase activity with respect to the DNA template/primer (T/P), and consequently also inhibits RNase H activity. The dNTP competing RT inhibitor retains activity against the NRTI-resistant mutants K65R and M184V, demonstrating a drug resistance profile distinct from the nucleotide competing RT inhibitors indolopyridone-1 (INDOPY-1) and 4-dimethylamino-6-vinylpyrimidine-1 (DAVP-1). In antiviral assays, the T/P competing compound inhibits HIV-1 replication at a step consistent with an RT inhibitor. Screening of additional structurally related compounds to the three fragments led to the discovery of molecules with improved potency against HIV-1 RT. These fragment inhibitors represent previously unidentified scaffolds for development of novel drugs for HIV-1 prevention or treatment.

96-week resistance analyses of the STaR study: rilpivirine/emtricitabine/tenofovir DF versus efavirenz/emtricitabine/tenofovir DF in antiretroviral-naive, HIV-1-infected subjects

Background:STaR (GS-US-264-0110) was a 96-week phase 3b study evaluating the safety and efficacy of two single-tablet regimens, rilpivirine/emtricitabine/tenofovir DF (RPV/FTC/TDF) and efavirenz/emtricitabine/tenofovir DF (EFV/FTC/TDF) in treatment-naive, HIV-1-infected subjects.Methods:Genotypic analyses (population sequencing) of HIV-1 protease (PR) and reverse transcriptase (RT) were performed at screening; subjects with pre-existing resistance to study drugs were excluded. The protocol-defined resistance analysis population had genotypic/phenotypic analyses at failure and baseline for PR and RT.Results:Through week 96, the resistance analysis population included 24/394 subjects (6.1%) receiving RPV/FTC/TDF and 9/392 subjects (2.3%) receiving EFV/FTC/TDF. In the RPV/FTC/TDF arm, HIV-1 isolates from 21/394 subjects (5.3%) developed non-nucleoside reverse transcriptase inhibitor (NNRTI) and/or nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations and 20/21 isolates had both NNRTI and NRTI genotypic and/or phenotypic resistance. In the EFV/FTC/TDF arm, isolates from 4/392 subjects (1.0%) developed NNRTI and/or NRTI resistance mutations. Resistance development after week 48 was infrequent (1.0% RPV/FTC/TDF; 0.3% EFV/FTC/TDF). When stratified by baseline HIV-1 RNA ≤ or >100 000 copies/ml, 9/260 (3.5%) versus 12/134 (9.0%) RPV/FTC/TDF-treated subjects and 3/250 (1.2%) versus 1/142 (0.7%) EFV/FTC/TDF-treated subjects developed resistant isolates, respectively. Pre-existing NRTI- and NNRTI-associated resistance mutations (not related to study drugs) did not impact treatment response to either regimen.Conclusions:Resistance development to RPV/FTC/TDF consisted of NNRTI and NRTI mutations and was more frequent than resistance development to EFV/FTC/TDF through week 96. Emergent resistance after week 48 was infrequent in both arms. Within the RPV/FTC/TDF arm, resistance development was more frequent in subjects with baseline HIV-1 RNA >100 000 copies/ml compared to baseline HIV-1 RNA ≤ 100 000 copies/ml.

Limited clinical benefit of minority K103N and Y181C-variant detection in addition to routine genotypic resistance testing in antiretroviral therapy-naive patients.

The presence of minority nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV-1 variants prior to antiretroviral therapy (ART) has been linked to virologic failure in treatment-naive patients. We performed a large retrospective study to determine the number of treatment failures that could have been prevented by implementing minority drug-resistant HIV-1 variant analyses in ART-naïve patients in whom no NNRTI resistance mutations were detected by routine resistance testing. Of 1608 patients in the Swiss HIV Cohort Study, who have initiated first-line ART with two nucleoside reverse transcriptase inhibitors (NRTIs) and one NNRTI before July 2008, 519 patients were eligible by means of HIV-1 subtype, viral load and sample availability. Key NNRTI drug resistance mutations K103N and Y181C were measured by allele-specific PCR in 208 of 519 randomly chosen patients. Minority K103N and Y181C drug resistance mutations were detected in five out of 190 (2.6%) and 10 out of 201 (5%) patients, respectively. Focusing on 183 patients for whom virologic success or failure could be examined, virologic failure occurred in seven out of 183 (3.8%) patients; minority K103N and/or Y181C variants were present prior to ART initiation in only two of those patients. The NNRTI-containing, first-line ART was effective in 10 patients with preexisting minority NNRTI-resistant HIV-1 variant. As revealed in settings of case-control studies, minority NNRTI-resistant HIV-1 variants can have an impact on ART. However, the implementation of minority NNRTI-resistant HIV-1 variant analysis in addition to genotypic resistance testing (GRT) cannot be recommended in routine clinical settings. Additional associated risk factors need to be discovered.

Low-frequency NNRTI-resistant HIV-1 variants and relationship to mutational load in antiretroviral-naïve subjects.

Low-frequency HIV variants possessing resistance mutations against non‑nucleoside reverse transcriptase inhibitors (NNRTI), especially at HIV reverse transcriptase (RT) amino acid (aa) positions K103 and Y181, have been shown to adversely affect treatment response. Therapeutic failure correlates with both the mutant viral variant frequency and the mutational load. We determined the prevalence of NNRTI resistance mutations at several RT aa positions in viruses from 204 antiretroviral (ARV)-naïve HIV-infected individuals using deep sequencing, and examined the relationship between mutant variant frequency and mutational load for those variants. Deep sequencing to ≥0.4% levels found variants with major NNRTI-resistance mutations having a Stanford-HIVdb algorithm value ≥30 for efavirenz and/or nevirapine in 52/204 (25.5%) ARV-naïve HIV-infected persons. Eighteen different major NNRTI mutations were identified at 11 different positions, with the majority of variants being at frequency >1%. The frequency of these variants correlated strongly with the mutational load, but this correlation weakened at low frequencies. Deep sequencing detected additional major NNRTI-resistant viral variants in treatment-naïve HIV-infected individuals. Our study suggests the significance of screening for mutations at all RT aa positions (in addition to K103 and Y181) to estimate the true burden of pre-treatment NNRTI-resistance. An important finding was that variants at low frequency had a wide range of mutational loads (>100-fold) suggesting that frequency alone may underestimate the impact of specific NNRTI-resistant variants. We recommend further evaluation of all low-frequency NNRTI-drug resistant variants with special attention given to the impact of mutational loads of these variants on treatment outcomes.

Characterization of HIV-1 Drug Resistance Development Through Week 48 in Antiretroviral Naive Subjects on Rilpivirine/Emtricitabine/Tenofovir DF or Efavirenz/Emtricitabine/Tenofovir DF in the STaR Study (GS-US-264-0110)

The Single-Tablet Regimen (STaR) study (GS-US-264-0110) is a 96-week phase 3b study evaluating the safety and efficacy of 2 single-tablet regimens, rilpivirine/emtricitabine/tenofovir DF (RPV/FTC/TDF), and efavirenz/emtricitabine/tenofovir DF (EFV/FTC/TDF) in treatment-naive HIV-1-infected subjects. Genotypic analyses (population sequencing) of HIV-1 protease and reverse transcriptase were performed at screening; subjects with pre-existing resistance to study drugs were excluded. The primary protocol-defined resistance analysis population (RAP) had genotypic/phenotypic analyses at failure and baseline for protease and reverse transcriptase. At week 48, the primary RAP included 20/394 subjects (5.1%) receiving RPV/FTC/TDF and 7/392 subjects (1.8%) receiving EFV/FTC/TDF. In the RPV/FTC/TDF arm, isolates from 17/394 subjects (4.3%) developed nonnucleoside reverse transcriptase inhibitor (NNRTI) and/or nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations, and 16/17 isolates had both NNRTI and NRTI resistance mutations. In the EFV/FTC/TDF arm, isolates from 3/392 subjects (0.8%) developed NNRTI and/or NRTI resistance mutations. When stratified by baseline viral load of either ≤ 100,000 or >100,000 copies/mL, 5/260 (1.9%) versus 12/134 (9.0%) RPV/FTC/TDF-treated subjects and 2/250 (0.8%) versus 1/142 (0.7%) EFV/FTC/TDF-treated subjects developed resistant isolates, respectively. The presence of pre-existing NRTI- and NNRTI-associated resistance mutations not excluded at screening (not related to study drugs) did not impact treatment response to either regimen. Among subjects in the primary RAP, resistance development to RPV/FTC/TDF consisted of NNRTI and NRTI mutations and was more frequent than resistance development to EFV/FTC/TDF. In subjects with baseline viral load ≤ 100,000 copies/mL, resistance development was low (<2%) for both RPV/FTC/TDF and EFV/FTC/TDF arms and less frequent compared with subjects with baseline viral load >100,000 copies/mL, for RPV/FTC/TDF.

Drug resistance in children at virological failure in a rural KwaZulu-Natal, South Africa, cohort

BackgroundBetter understanding of drug resistance patterns in HIV-infected children on antiretroviral therapy (ART) is required to inform public health policies in high prevalence settings. The aim of this study was to characterise the acquired drug resistance in HIV-infected children failing first-line ART in a decentralised rural HIV programme.MethodsPlasma samples were collected from 101 paediatric patients (≤15 yrs of age) identified as failing ART. RNA was extracted from the plasma, reverse transcribed and a 1.3 kb region of the pol gene was amplified and sequenced using Sanger sequencing protocols. Sequences were edited in Geneious and drug resistance mutations were identified using the RegaDB and the Stanford resistance algorithms. The prevalence and frequency of mutations were analysed together with selected clinical and demographic data in STATA v11.ResultsA total of 101 children were enrolled and 89 (88%) were successfully genotyped; 73 on a non-nucleoside reverse-transcriptase inhibitor (NNRTI)-based regimen and 16 on a protease inhibitor (PI)-based regimen at the time of genotyping. The majority of patients on an NNRTI regimen (80%) had both nucleoside reverse-transcriptase inhibitor (NRTI) and NNRTI resistance mutations. M184V and K103N were the most common mutations amongst children on NNRTI-based and M184V among children on PI-based regimens. 30.1% had one or more thymidine analogue mutation (TAM) and 6% had ≥3 TAMs. Only one child on a PI-based regimen harboured a major PI resistance mutation.ConclusionsWhilst the patterns of resistance were largely predictable, the few complex resistance patterns seen with NNRTI-based regimens and the absence of major PI mutations in children failing PI-based regimens suggest the need for wider access to genotypic resistance testing in this setting.

A Gripping New Mechanism of Drug Resistance in HIV-1 Reverse Transcriptase

HIV-1 Reverse Transcriptase (RT) converts viral RNA into dsDNA that is integrated into the human genome. Nonnucleoside RT inhibitors (NNRTIs) are highly effective in the treatment and prevention of HIV. NNRTI resistance mutations arise from therapy, yet the mechanism(s) of how these mutations inhibit polymerization by RT is unclear. We examine the role of NNRTI on the dynamics between RT and its Template/Primer (T/P) substrate using a combination of molecular modeling, single-molecule and bulk fluorescence techniques and provide an unprecedented glimpse into the dynamics of RT-T/P interaction as well as the intramolecular conformation of RT itself while bound to its substrate. The data suggests a unique mechanism of resistance that is mediated by interplay between intramolecular conformational changes in RT and intermolecular dynamics of the RT-template/primer-dNTP complex.