Actualització en tropisme

Actualització en tropisme Roger Paredes IrsiCaixa i Lluita contra la SIDA Hospital Germans Trias i Pujol

Predicció tropisme CD4 Binding Co-receptor Binding Virus-Cell Fusion gp41 gp120 V3 loop CD4 Cell Membrane CCR5/CXCR4 (R5/X4) Adapted from Doms et al. Genes Dev. 2000;14:2677-2688.

Predicció tropisme X4 (SI) R5 (NSI) Dual tropic CCR5 CD4 CXCR4 Primary lymphocytes Monocyte/macrophages T-cell lines CD4 Naïve CD4 memory

Predicció tropisme Koot M, et al: Prognostic Value of HIV-1 Syncytium-Inducing Phenotype for Rate of CD4+ Cell Depletion and Progression to AIDS. Annals Int Med 1993

Predicció tropisme • Conclusions MERIT Trial • Twice-daily maraviroc was not noninferior to efavirenz at <50 copies/mL in the primary analysis. • However, 15% of patients would have been ineligible for inclusion by a more sensitive screening assay. • Their retrospective exclusion resulted in similar response rates in both arms Cooper D et al. JID 2010 Maraviroc no está aprobado en España para el tratamiento de pacientes naïve

Predicció tropisme Subject 18 Subject 07 Week Week Subject 19 Subject 47 Week Week Tsibris et al. PLoS One 2009

Predicció tropisme

Tests fenotípics ESTA Car, lent, no útil a CV < 1000 copies/mL MT2 Difícil de recuperar virus infecciosos, Restringit a laboratoris especialitzats Tests genotípics X4 preexisteixen a virtualment tots els pacients  origen de la majoria de fracassos a antagonistes del CCR5  Males eines diagnòstiques generen incertesa al clínic  Mercat potencial dels antagonistes CCR5 reduït No validats Seqüenciació Poblacional Poc sensible  Se’ns escapen X4 454(deep sequencing) En desenvolupament  Detecta X4 a tothom  Punt de tall? Plasma vs. PBMC Són equivalents pel diagnòstic? Problemes clínics

Tests fenotípics ESTA a PBMCs Tests genotípics Tropisme a DNA episomal Milloria de la predicció fenotípica a partir del genotip Tests genotípics de tropisme més exactes Tropisme genotípic i resposta a maraviroc Temes tractats

Tropism was successfully determined in all 20 PBMC samples 10/10 viremic  PBMC and plasma tropism concordant (8 R5, 2 DM). 10/ WITH low or undetectable viral load 6 R5 and 4 DM. Extensive homology of plasma and PBMC Env sequences. No phylogenetic distinctions between plasma and PBMC samples from the 5-paired samples. Tropism of clones derived from plasma and PBMC were concordant: one DM had solely of dual-tropic variants the other DM virus population was comprised predominantly of R5 variants with a minor subpopulation of dual-tropic variants 3 R5 virus populations were comprised of R5 variants Toma J et al -. Determining HIV-1 Coreceptor Tropism Using PBMC Proviral DNA Derived from Aviremic Blood Samples. Poster 541

Babic D et al. - Predicting HIV-1 Co-receptor usage and Response to CCR5 Inhibitor Therapy through Episomal cDNA. Poster 540 • PATIENTS AND METHODS: • 14 patients from ACTG 5211 study who failed vicriviroc therapy • 5 with emergence of DM virus • PBMCs collected from study entry through week 48 • Co-receptor usage determined based on phenotypes conferred by episomal full-length env genes or V3 genotypes

Babic D et al. - Predicting HIV-1 Co-receptor usage and Response to CCR5 Inhibitor Therapy through Episomal cDNA. Poster 540 Episomal env sequences were amplified in 81% (74/91) of PBMC samples.  Mostly in subjects with high VL

Babic D et al. - Predicting HIV-1 Co-receptor usage and Response to CCR5 Inhibitor Therapy through Episomal cDNA. Poster 540 • CONCLUSIONS • The Trofile assay and phenotypic assay based on episomal env sequences were largely concordant. • In two patients, episomal sequence analysis predicted the emergence of X4 or dual-tropic virus in plasma viral RNA. • These results support the use of episomal cDNA as an additional approach to predict the virologic response to entry inhibitors. • However, subjects were essentially viremic through the study follow-up. The efficiency of episomal DNA recovery in aviremic subjects might be lower than 80%

159 patients enrolled 170 phenotype / genotype samples Victoria Sánchez et al. A highly sensitive and specific model for predicting HIV-1 tropism in treatment-experienced patients combining interpretation of V3 loop sequences and clinical parameters. Poster 543

Victoria Sánchez et al. A highly sensitive and specific model for predicting HIV-1 tropism in treatment-experienced patients combining interpretation of V3 loop sequences and clinical parameters. Poster 543 V3 sequence Patients characteristics Clinical parameters Binary multivariate regression analyses • Treatment-experienced patients (Nagelkerke R square: 0.937) • Geno2pheno clinical model 15% prediction • Geno2pheno clonal model 1% prediction • Net charge prediction • Nadir CD4 • Exposure >3 and >4 ART classes • AIDS development

Victoria Sánchez et al. A highly sensitive and specific model for predicting HIV-1 tropism in treatment-experienced patients combining interpretation of V3 loop sequences and clinical parameters. Poster 543 V3 sequence Patients characteristics Clinical parameters Binary multivariate regression analyses • Treatment-experienced patients (Nagelkerke R square: 0.937) • Geno2pheno clinical model 15% prediction • Geno2pheno clonal model 1% prediction • Net charge prediction • Nadir CD4 • Exposure >3 and >4 ART classes • AIDS development • FPR score geno2pheno clinical • Years since HIV diagnosis • Log HIV-1 viral load

Victoria Sánchez et al. A highly sensitive and specific model for predicting HIV-1 tropism in treatment-experienced patients combining interpretation of V3 loop sequences and clinical parameters. Poster 543 Results (6): Predictive modelling of the co-receptor usage • P= - Log10 (1 / 1+e-y) • y = a1x1+a2x2+a3x3+a4x4+a5x5+a6x6 • a1 - a6: Regression coefficients of the variables in the model • (a1: -12; a2: -8; a3: -14; a4: +0.055; a5: -0.087 and a6: +3.6) • x1 - x6: Variables included in the equation: • X1: geno2pheno 15% FPR clinical model prediction • X2: V3 net charge prediction • X3: Exposure to > 3 antiretroviral classes • X4: FPR score of geno2pheno-clinical model • X5: Years since HIV infection diagnosis • X6: log HIV-1 RNA viral load • AUC: 0.966 (CI 95% 0.930-1.000, p<0.001) • Cut-off ≥ 5.75: CXCR4 (S: 96.6%, E: 92.3%)

Pou C et al. High Resolution Tropism Kinetics by Quantitative Deep Sequencing in HIV-1 Infected Subjects Initiating Suppressive First-Line ART. Poster 544 Study design and per-subject tropism prediction * FPR 10% ** FPR 10%, cut-off X4: 1%

Significance relative to ESTA: • * P<0.05 • ** P<0.01 • *** P<0.001 * * *** ** * Pou C et al. High Resolution Tropism Kinetics by Quantitative Deep Sequencing in HIV-1 Infected Subjects Initiating Suppressive First-Line ART. Poster 544 ROC curves for different G2P FPRs X4 prevalence in different compartments Phylogeny reconstruction for one subject Test characteristics * FPR 10% ** FPR 10%, cut-off X4: 1%

Pou C et al. High Resolution Tropism Kinetics by Quantitative Deep Sequencing in HIV-1 Infected Subjects Initiating Suppressive First-Line ART. Poster 544 C O N C L U S I O N S • V3-loop QDS is the most accurate tropism test, followed by V3-loop PS. • The most reliable settings to explore the ability of V3-loop QDS to predict clinical outcomes are: • Using a G2P[coreceptor] FPR ≤10% to define “predicted CXCR4 coreceptor usage”. • Defining an “X4” result as the presence of predicted X4 viruses in at least 1% of the virus population. • In the absence of CCR5 antagonist pressure, X4 viruses from plasma and PBMCs tend to cluster together and there are no clear evidences of viral evolution over time. These findings suggest that either plasma or PBMCs, and current or stored samples, could be used in most subjects for tropism testing. • However, discrepancies in X4 detection between plasma and PBMCs indicate that each tropism assay should be specifically validated in clinical trials assessing outcomes of CCR5 antagonist therapy.

To retrospectively evaluate virological response to maraviroc in treatment-naïve individuals where viral tropism was predicted using population-based sequencing of the HIV-1 V3 loop Patient eligibility criteria: • HIV-1RNA ≥ 2,000 copies/mL • No evidence of resistance to EFV, ZDV, or 3TC • ≥ 16 years of age • Treatment naive • R5HIV-1 infection McGovern RA et al. Population-based Sequencing of the V3-loop is Comparable to the Enhanced Sensitivity Trofile Assay (ESTA) in Predicting Virologic Response to Maraviroc of Treatment-naïve Patients in the MERIT Trial. ORAL 92 Efavirenz (EFV 600 mg QD) + Combivir™ (ZDV+3TC) Randomization 1:1:1 Maraviroc (MVC 300 mg BID) + Combivir (ZDV+3TC) Maraviroc (MVC 300 mg QD) + Combivir (ZDV+3TC) Original Trofile Screening (6 weeks) 96 wk 0 48 wk Primary analysis Maraviroc no está aprobado en España para el tratamiento de pacientes naïve Saag M, et al. 4th IAS 2007; Sydney, Australia. Abstract WESS104.

Plasma samples from the MERIT study (N=705) were amplified in triplicate using RT-PCR and sequenced using an ABI 3730 Automated base-calling was performed using custom ReCall software without manual review Tropism prediction using “geno2Pheno” co-receptor algorithm (g2P) with 5.75% FPR blinded to ESTA results and clinical outcome Outcome measures include: change in plasma viral load (pVL), percent undetectable (<50 copies/mL) and time to tropism change Trofile “X4” and “D/M” results were combined into a single “non-R5” category McGovern RA et al. Population-based Sequencing of the V3-loop is Comparable to the Enhanced Sensitivity Trofile Assay (ESTA) in Predicting Virologic Response to Maraviroc of Treatment-naïve Patients in the MERIT Trial. ORAL 92 Maraviroc no está aprobado en España para el tratamiento de pacientes naïve

McGovern RA et al. Population-based Sequencing of the V3-loop is Comparable to the Enhanced Sensitivity Trofile Assay (ESTA) in Predicting Virologic Response to Maraviroc of Treatment-naïve Patients in the MERIT Trial. ORAL 92 Maraviroc no está aprobado en España para el tratamiento de pacientes naïve

ESTA R5, Geno nonR5 (N=20) McGovern RA et al. Population-based Sequencing of the V3-loop is Comparable to the Enhanced Sensitivity Trofile Assay (ESTA) in Predicting Virologic Response to Maraviroc of Treatment-naïve Patients in the MERIT Trial. ORAL 92 0 -1 Concordant R5 (N=283) Concordant nonR5 (N=9) Log Viral Load Change -2 ESTA nonR5, Geno R5 (N=39) -3 -4 0 8 16 24 32 40 48 60 72 84 96 Week Maraviroc BID; LOCF Maraviroc no está aprobado en España para el tratamiento de pacientes naïve

Concordant nonR5 (N=9) Concordant R5 (N=283) ESTA R5, Geno nonR5 (N=20) McGovern RA et al. Population-based Sequencing of the V3-loop is Comparable to the Enhanced Sensitivity Trofile Assay (ESTA) in Predicting Virologic Response to Maraviroc of Treatment-naïve Patients in the MERIT Trial. ORAL 92 1.0 0.8 0.6 Percent copies <50 ESTA nonR5, Geno R5 (N=39) 0.4 0.2 0.0 0 8 16 24 32 40 48 60 72 84 96 Week Maraviroc BID; Missing=Failure Maraviroc no está aprobado en España para el tratamiento de pacientes naïve

McGovern RA et al. Population-based Sequencing of the V3-loop is Comparable to the Enhanced Sensitivity Trofile Assay (ESTA) in Predicting Virologic Response to Maraviroc of Treatment-naïve Patients in the MERIT Trial. ORAL 92 1.0 Genotype R5 (n= 322) 0.8 Genotype nonR5(n= 29) 0.6 Percent with R5 by Original Trofile 0.4 0.2 0.0 0 8 16 24 32 40 48 60 72 84 96 Week Maraviroc BID Maraviroc no está aprobado en España para el tratamiento de pacientes naïve

McGovern RA et al. Population-based Sequencing of the V3-loop is Comparable to the Enhanced Sensitivity Trofile Assay (ESTA) in Predicting Virologic Response to Maraviroc of Treatment-naïve Patients in the MERIT Trial. ORAL 92 1.0 ESTA R5 Genotype R5 0.8 Genotype nonR5 ESTA nonR5 0.6 Percent with R5 by Original Trofile 0.4 0.2 0.0 0 8 16 24 32 40 48 60 72 84 96 Week Maraviroc BID Maraviroc no está aprobado en España para el tratamiento de pacientes naïve

CONCLUSIONS In MERIT patients re-screened as R5 by population-based V3 sequence analysis, MVC BID was non-inferior to EFV, confirming previous results with ESTA Performance of population-based sequencing is similar to that of Enhanced Sensitivity Trofile assay where results were discordant Patients randomized to either MVC BID and QD called R5 by population-based sequencing saw greater virological success than those called non-R5 These results were also obtained with “deep” sequence analysis of V3 (see Abstract M-268) McGovern RA et al. Population-based Sequencing of the V3-loop is Comparable to the Enhanced Sensitivity Trofile Assay (ESTA) in Predicting Virologic Response to Maraviroc of Treatment-naïve Patients in the MERIT Trial. ORAL 92 Maraviroc no está aprobado en España para el tratamiento de pacientes naïve

Screening plasma samples from the MVC arms of trials in treatment-naïve “TN” (MERIT) and treatment-experienced patients “TE” (MOTIVATE-1 & -2 and A4001029) were examined. The EFV arm was also examined for comparison. Patient eligibility criteria: • HIV-1RNA ≥ 2,000 copies/mL • No evidence of resistance to EFV, ZDV, or 3TC • ≥ 16 years of age • Treatment naive • R5HIV-1 infection Swenson et al. Large-Scale Application of “Deep” Sequencing Using 454 Technology to HIV Tropism Screening. Poster 545 Efavirenz (EFV 600 mg QD) + Combivir™ (ZDV+3TC) Randomization 1:1:1 Maraviroc (MVC 300 mg BID) + Combivir (ZDV+3TC) Maraviroc (MVC 300 mg QD) + Combivir (ZDV+3TC) Original Trofile Screening (6 weeks) 96 wk 0 48 wk Primary analysis ESTA vs 454 Maraviroc no está aprobado en España para el tratamiento de pacientes naïve Saag M, et al. 4th IAS 2007; Sydney, Australia. Abstract WESS104.

Swenson et al. Large-Scale Application of “Deep” Sequencing Using 454 Technology to HIV Tropism Screening. Poster 545 Maraviroc no está aprobado en España para el tratamiento de pacientes naïve

Swenson et al. Large-Scale Application of “Deep” Sequencing Using 454 Technology to HIV Tropism Screening. Poster 545 Conclusions “Deep” sequence analysis of the HIV V3-loop gives improved prediction of virological response to MVC relative to the original Trofile assay, and similar to the currently-available Trofile assay (ESTA). Maraviroc no está aprobado en España para el tratamiento de pacientes naïve

Alerta! La seqüenciació poblacional d’V3 es va fer en pacients que van entrar a l’estudi, és a dir, després de l’screening amb Trofile El reanàlisi amb ESTA i 454 es va fer a les mostres d’screening Efavirenz (EFV 600 mg QD) + Combivir™ (ZDV+3TC) Randomization 1:1:1 Maraviroc (MVC 300 mg BID) + Combivir (ZDV+3TC) Maraviroc (MVC 300 mg QD) + Combivir (ZDV+3TC) Original Trofile Screening (6 weeks) 96 wk 0 48 wk Primary analysis Maraviroc no está aprobado en España para el tratamiento de pacientes naïve

Tests fenotípics Possible realitzar-los a PBMCs  De moment DNA total >> episomal Tests genotípics La seqüenciació poblacional d’V3 + screening previ amb Trofile és equiparable a ESTA 454 és equiparable a ESTA a mostres pre-screening  454 és la millor 454 és l’eina genotípica més exacta però no disponible a molts laboratoris En desenvolupament tècnic / bioinformàtic El clínic pot sentir-se força tranquil fent genotip convencional (useu FPR altes (>20%)) Ambdós possibles a PBMCs o plasma a mostres actuals o guardades Conclusions

Actualització en tropisme

Actualització en tropisme

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