Sophie Sayon

Impact of Y143 HIV-1 Integrase Mutations on Resistance to Raltegravir In Vitro and In Vivo

ABSTRACT Integrase (IN), the HIV-1 enzyme responsible for the integration of the viral genome into the chromosomes of infected cells, is the target of the recently approved antiviral raltegravir (RAL). Despite this drugs activity against viruses resistant to other antiretrovirals, failures of raltegravir therapy were observed, in association with the emergence of resistance due to mutations in the integrase coding region. Two pathways involving primary mutations on residues N155 and Q148 have been characterized. It was suggested that mutations at residue Y143 might constitute a third primary pathway for resistance. The aims of this study were to investigate the susceptibility of HIV-1 Y143R/C mutants to raltegravir and to determine the effects of these mutations on the IN-mediated reactions. Our observations demonstrate that Y143R/C mutants are strongly impaired for both of these activities in vitro. However, Y143R/C activity can be kinetically restored, thereby reproducing the effect of the secondary G140S mutation that rescues the defect associated with the Q148R/H mutants. A molecular modeling study confirmed that Y143R/C mutations play a role similar to that determined for Q148R/H mutations. In the viral replicative context, this defect leads to a partial block of integration responsible for a weak replicative capacity. Nevertheless, the Y143 mutant presented a high level of resistance to raltegravir. Furthermore, the 50% effective concentration (EC50) determined for Y143R/C mutants was significantly higher than that obtained with G140S/Q148R mutants. Altogether our results not only show that the mutation at position Y143 is one of the mechanisms conferring resistance to RAL but also explain the delayed emergence of this mutation.

Partially active HIV-1 Vif alleles facilitate viral escape from specific antiretrovirals.

Background:The HIV-1 Vif protein counteracts the antiviral activity of the cytidine deaminases APOBEC3G and APOBEC3F. Natural variation in Vif may result in reduced efficacy against APOBEC3 proteins and in increased HIV-1 diversity. We speculated that this mechanism could facilitate viral escape from certain antiretroviral drugs. Methods and results:We analyzed the protease, reverse transcriptase and Vif sequences of viruses from plasma obtained from 92 HIV-1-infected individuals failing antiretroviral treatment and 65 antiretroviral-naive patients. Mutation K22H in Vif was more frequent in patients failing to antiretroviral compared to antiretroviral-naive patients. In-vitro experiments showed that mutant K22H failed to completely neutralize APOBEC3G. Upon infection of MT-2 cells, most of the K22H proviral clones encoded increased numbers of G-to-A mutations. Among these mutations, the lamivudine drug-resistance-associated mutation M184I in reverse transcriptase was detected in 25% of clones in the absence of any lamivudine exposure. In our population, among pretreated patients, 72% of K22H viruses versus 42% in WT K22 viruses harbored at least two drug-resistance-associated mutations in a GA/GG dinucleotide context. More specifically, K22H viruses harbored significantly more G16E and M36I in protease than in those isolated from pretreated patients harboring WT K22 viruses. Conclusions:This study provides evidence that patients experiencing virological failure frequently harbor Vif point mutants (i.e. K22H). Such Vif alleles lose their ability to counteract APOBEC3 proteins, leading to an increase of G-to-A viral mutations that can facilitate the emergence of some antiretroviral resistance mutations.

Higher efficacy of nevirapine than efavirenz to achieve Hiv-1 plasma viral load below 1 copy/ml

Objectives:To compare the level of HIV-1 residual viremia, defined by a viral load below 50 copies/ml in patients receiving a tenofovir/emtricitabine and nevirapine (NVP) or efavirenz (EFV)-containing regimen. Design:One hundred and sixty-five HIV-1-infected patients were retrospectively included since they achieved virological suppression (viral load <50 copies/ml) for at least 6 months with a tenofovir/emtricitabine and non-nucleoside reverse transcriptase inhibitor-containing regimen (NVP, n = 75 and EFV, n = 90). Methods:Residual plasma viremia was measured using an ultrasensitive assay with a limit of quantification of 1 copy/ml. A Fishers exact test was used to compare the percentage of patients with HIV-1 RNA below 1 copy/ml between the two treatment groups. Logistic regression was used to search for factors associated with a viral load below 1 copy/ml among the different patient characteristics. Results:Patients in the NVP group had more frequently a viral load below 1 copy/ml than patients in the EFV group (81.3 vs. 55.6%, P < 0.001). In multivariate analysis, only NVP vs EFV (P = 0.005) and duration of viral suppression under antiretroviral treatment (P = 0.005) were independently associated with viral load below 1 copy/ml. Conclusions:It is well known that NVP has a good penetration in anatomic compartments that could explain a deep control of virus replication in some compartments and consequently decrease the residual level of viral load. The clinical relevance of having a viral load below 1 copy/ml has now to be studied for example on systemic inflammatory or immune activation markers.

The HIV-1 integrase G118R mutation confers raltegravir resistance to the CRF02_AG HIV-1 subtype

BACKGROUNDnMost of the previous studies that explored the molecular basis of raltegravir resistance were conducted studying the HIV-1 B subtype. It has been shown that the CRF02_AG subtype in relation to its natural integrase (IN) sequence could develop different genetic pathways associated with raltegravir resistance. The aim of this study was to explore resistance pathways preferably used by CRF02_AG viruses compared with subtype B.nnnMETHODSnTwenty-five HIV-1 CRF02_AG-infected patients failing a raltegravir-containing regimen were studied. IN gene sequences were examined for the presence of previously described IN inhibitor (raltegravir, elvitegravir, dolutegravir and MK-2048) resistance mutations at 20 amino acid positions.nnnRESULTSnAmong the 25 studied patients, 7 showed viruses harbouring major raltegravir resistance mutations mainly associated with the 155 genetic pathways and 18 showed viruses harbouring none of them; however, for 1 patient, we found a 118R mutation, associated with MK-2048 in vitro resistance, in a 74M background. For this patient, the phenotypic analysis showed that addition of only the G118R mutation conferred a high level of resistance to raltegravir (fold change = 25.5) and elvitegravir (fold change = 9.2).nnnCONCLUSIONSnThis study confirmed that mutation pathways for raltegravir resistance could be different between the two subtypes CRF02_AG and B with a preferential use of the 155 mutation in non-B subtypes. A new genetic pathway associated with raltegravir resistance, including the 118R mutation, has also been identified. This new genetic pathway, never described in subtype B, should be further evaluated for phenotypic susceptibility to dolutegravir and MK-2048.

E138K and M184I mutations in HIV-1 reverse transcriptase coemerge as a result of APOBEC3 editing in the absence of drug exposure.

Background:Recent clinical trials with rilpivirine combined with emtricitabine and tenofovir revealed that patients failing treatment, frequently, harbored viruses encoding resistance-associated mutations in the HIV-1 reverse transcriptase at position E138K and M184I. We show here that APOBEC3 proteins play a role in the emergence of these drug resistance mutations. Methods:We used a Vif mutant that has suboptimal activity against APOBEC3 to assess the in-vitro frequency of APOBEC3-induced resistance mutations in reverse transcriptase. To assess the degree of in-vivo G-to-A viral hypermutation, a large amount of data of HIV-1 RT proviral sequences from peripheral blood mononuclear cells (PBMCs) recovered from infected patients under HAART was analyzed. Results:In-vitro replication experiments in cell lines with and without APOBEC3 expression suggest that APOBEC3-driven mutagenesis contributes to the generation of both M184I and E138K within HIV proviral repository in the absence of drug exposure. Additionally, analysis of 601 patients PBMCs sequences revealed that the copresence of mutations E138K and M184I were never detected in nonhypermutated sequences, whereas these mutations were found at a high frequency (24%) in the context of APOBEC3 editing and in the absence of exposure to etravirine–rilpivirine. Conclusion:We demonstrate using in-vitro experiments and analyzing patients PBMCs sequences that M184I and E138K resistance-associated mutations may pre-exist in proviral reservoir at a high frequency prior to drug exposure, as a result of APOBEC3 editing. Thus, incomplete neutralization of one or more APOBEC3 proteins may favor viral escape to rilpivirine-emtricitabine.

Prevalence of resistance mutations related to integrase inhibitor S/GSK1349572 in HIV-1 subtype B raltegravir-naive and -treated patients

OBJECTIVESnTo compare the frequency of previously in vitro-selected integrase mutations (T124A, T124A/S153F, S153Y, T124A/S153Y and L101I/T124A/S153Y) conferring resistance to S/GSK1349572 between HIV-1 subtype B integrase inhibitor (INI)-naive and raltegravir-treated patients.nnnMETHODSnIntegrase sequences from 650 INI-naive patients and 84 raltegravir-treated patients were analysed.nnnRESULTSnThe T124A mutation alone and the combination T124A/L101I were more frequent in raltegravir-failing patients than in INI-naive patients (39.3% versus 24.5%, respectively, P = 0.005 for T124A and 20.2% versus 10.0%, respectively, P = 0.008 for T124A/L101I). The S153Y/F mutations were not detected in any integrase sequence (except for S153F alone, only detected in one INI-naive patient).nnnCONCLUSIONSnT124A and T124A/L101I, more frequent in raltegravir-treated patients, could have some effect on raltegravir response and their presence could play a role in the selection of other mutations conferring S/GSK1349572 resistance. The impact of raltegravir-mediated changes such as these on the virological response to S/GSK1349572 should be studied further.

TLR-2 Recognizes Propionibacterium acnes CAMP Factor 1 from Highly Inflammatory Strains

Background Propionibacterium acnes (P. acnes) is an anaerobic, Gram-positive bacteria encountered in inflammatory acne lesions, particularly in the pilosebaceous follicle. P. acnes triggers a strong immune response involving keratinocytes, sebocytes and monocytes, the target cells during acne development. Lipoteicoic acid and peptidoglycan induce the inflammatory reaction, but no P. acnes surface protein interacting with Toll-like receptors has been identified. P. acnes surface proteins have been extracted by lithium stripping and shown to induce CXCL8 production by keratinocytes. Methodology and principal findings Far-western blotting identified two surface proteins, of 24.5- and 27.5-kDa in size, specifically recognized by TLR2. These proteins were characterized, by LC-MS/MS, as CAMP factor 1 devoid of its signal peptide sequence, as shown by N-terminal sequencing. Purified CAMP factor 1 induces CXCL8 production by activating the CXCL8 gene promoter, triggering the synthesis of CXCL8 mRNA. Antibodies against TLR2 significantly decreased the CXCL8 response. For the 27 P. acnes strains used in this study, CAMP1-TLR2 binding intensity was modulated and appeared to be strong in type IB and II strains, which produced large amounts of CXCL8, whereas most of the type IA1 and IA2 strains presented little or no CAMP1-TLR2 binding and low levels of CXCL8 production. The nucleotide sequence of CAMP factor displays a major polymorphism, defining two distinct genetic groups corresponding to CAMP factor 1 with 14 amino-acid changes from strains phylotyped II with moderate and high levels of CAMP1-TLR2 binding activity, and CAMP factor 1 containing 0, 1 or 2 amino-acid changes from strains phylotyped IA1, IA2, or IB presenting no, weak or moderate CAMP1-TLR2 binding. Conclusions Our findings indicate that CAMP factor 1 may contribute to P. acnes virulence, by amplifying the inflammation reaction through direct interaction with TLR2.

Usefulness of an HIV DNA resistance genotypic test in patients who are candidates for a switch to the rilpivirine/emtricitabine/tenofovir disoproxil fumarate combination

OBJECTIVESnIn the context of a rilpivirine/emtricitabine/tenofovir disoproxil fumarate switch in HIV-1-infected patients with at least 1 year of virological success, we determined whether proviral DNA is an alternative to plasma HIV RNA for resistance genotyping.nnnMETHODSnResistance-associated mutations (RAMs) in DNA after at least 1 year of virological success [viral load (VL) <50 copies/mL] were compared with those identified in the last plasma RNA genotype available. Rilpivirine/emtricitabine/tenofovir disoproxil fumarate RAMs studied were K65R, L100I, K101E/P, E138A/G/K/R/Q, V179L, Y181C/I/V, M184V/I, Y188L, H221Y, F227C and M230I/L in the RT. We studied patients without virological failure (VF) and with at least 1 VF (two consecutive VLs >50 copies/mL). Kappas coefficient was used to measure agreement between the DNA and RNA genotypes.nnnRESULTSnIn patients without VF (nu200a=u200a130) and with VF (nu200a=u200a114), RNA and DNA showed resistance to at least one drug of the rilpivirine/emtricitabine/tenofovir disoproxil fumarate combination in 8% and 9% and in 60% and 45%, respectively. For rilpivirine RAMs, correlation between RNA and DNA was higher in patients without VF than in patients with VF (kappau200a=u200a0.60 versus 0.19, Pu200a=u200a0.026). Overall, the prevalence of RAMs was lower in DNA than in RNA.nnnCONCLUSIONSnIncomplete information provided by the DNA genotypic test is more notable in patients with VF, suggesting that all resistance mutations associated with prior VF have not been archived in the proviral DNA or decreased to a level below the threshold of detection. In the case where no historical plasma genotypic test is available, DNA testing might be useful to rule out switching to rilpivirine/emtricitabine/tenofovir disoproxil fumarate.

Virological factors associated with outcome of dual maraviroc/raltegravir therapy (ANRS-157 trial)

OBJECTIVESnROCnRAL ANRS-157 was a single-arm study designed to evaluate a switch to a maraviroc (300 mg twice a day) plus raltegravir (400 mg twice a day) regimen in virologically suppressed HIV-1-infected patients (ClinicalTrials.gov: NCT01420523). The aim of this work was to investigate the factors associated with virological failure (VF) (5/44 patients) or virological rebound defined as one viral load (VL) >50 copies/mL or VL >1 copy/mL.nnnMETHODSnAt baseline (BL), ultradeep sequencing (UDS) of DNA gp120 V3 and integrase regions and quantification of HIV DNA were performed in PBMCs. Tropism, VL, BL ultrasensitive HIV RNA VL, BL HIV DNA VL, subtype, age, ethnicity, transmission group, AIDS status, nadir CD4 and BL CD4 cell count, time since HIV diagnosis, duration of ART and suppressed viraemia, VL zenith, CD4/CD8 ratio and BL CD8 cell count were investigated as potential factors associated with virological rebound.nnnRESULTSnThe proportion of patients with VL <1 copy/mL did not evolve over time. Among the 44 included patients, 3 had minority X4-tropic viruses determined by UDS at BL and one of them presented VF. Minority resistant variants in the integrase gene were detected at BL at two positions (E138 and G140) for three patients who did not have VF. Among all studied factors, none was associated with virological rebound.nnnCONCLUSIONSnMaraviroc plus raltegravir failed to maintain virological suppression in virologically suppressed HIV-1-infected patients. However, neither minority viral variants nor ultrasensitive viraemia was found to be a predictive factor of VF or virological rebound in this context.

Disparities in HIV-1 transmitted drug resistance detected by ultradeep sequencing between men who have sex with men and heterosexual populations

Transmitted drug resistance (TDR) can impair the response to first‐line antiretroviral therapy. In treatment‐naïve patients chronically infected with HIV type 1 (HIV‐1), it was previously shown through Sanger sequencing that TDR was more common in men who have sex with men (MSM) than in other transmission risk groups. We aimed to compare two HIV‐1 transmission groups in terms of the presence of TDR mutations.