actualització en tropisme

Actualització en tropisme

Roger ParedesIrsiCaixa i Lluita contra la SIDAHospital Germans Trias i Pujol

Virus-CellFusion

gp41

gp120

V3 loop

CD4Binding

CD4

CellMembrane

Co-receptorBinding

CCR5/CXCR4(R5/X4)

Adapted from Doms et al. Genes Dev. 2000;14:2677-2688.

Predicció tropisme

X4 (SI) R5 (NSI)

CXCR4 CCR5CD4

T-cell lines Primary lymphocytes Monocyte/macrophages

CD4 Naïve CD4 memory

Dual tropic

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

Predicció tropisme

Cooper D et al. JID 2010

Conclusions MERIT Trial

1. Twice-daily maraviroc was not noninferior to efavirenz at <50 copies/mL in the primary analysis.

2. However, 15% of patients would have been ineligible for inclusion by a more sensitive screening assay.

3. Their retrospective exclusion resulted in similar response rates in both arms

Maraviroc no está aprobado en España para el tratamiento de pacientes naïve

Week

Subject 47

Week

Subject 18

Week

Subject 19

Week

Subject 07

Predicció tropisme

Tsibris et al. PLoS One 2009

Predicció tropisme

Problemes clínics• 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?

Temes tractats

• 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

• 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

Temes tractats



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

Episomal env sequences were amplified in 81% (74/91) of PBMC samples. Mostly in subjects with high VL

No. of patients (n=14)

Phenotype/Genotype (episomal cDNA)

Trofile assay(plasma RNA)

2 DM DM

8 R5 R5

1 X4 at baseline X4 at week 8

1 DM at week 2 DM at week 48

1 R5 DM

1 DM R5


CONCLUSIONS

1. The Trofile assay and phenotypic assay based on episomal env sequences were largely concordant.

2. In two patients, episomal sequence analysis predicted the emergence of X4 or dual-tropic virus in plasma viral RNA.

3. These results support the use of episomal cDNA as an additional approach to predict the virologic response to entry inhibitors.

4. However, subjects were essentially viremic through the study follow-up. The efficiency of episomal DNA recovery in aviremic subjects might be lower than 80%


Temes tractats



4 p a tien ts e n ro lled4 sa m p le s c o lle c te d

4 p a tien ts sc re en ed

M O T IV A T E 2

1 p a tien t e x c lu d e d

1 4 p a tie n ts e n ro lle d1 4 sa m p le s co lle c ted

1 5 p a tie n ts sc ree n ed

E A P m a rav iro c

1 6 p a tie n ts e x c lu d e d

1 4 1 p a tie n ts e n ro lle d1 5 2 sam p le s co lle c ted

1 5 7 p a tie n ts sc ree n e d

C lin ica l c o h o rt

159 patients enrolled170 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

Binary multivariate regression analyses Treatment-experienced patients (Nagelkerke R square: 0.937)

Geno2pheno clinical model 15% predictionGeno2pheno clonal model 1% predictionNet charge predictionNadir CD4 Exposure >3 and >4 ART classesAIDS development



PatientscharacteristicsV3 sequence

Clinical parameters

Treatment-experienced patients (Nagelkerke R square: 0.937)

Geno2pheno clinical model 15% predictionGeno2pheno clonal model 1% predictionNet charge predictionNadir CD4 Exposure >3 and >4 ART classesAIDS development

FPR score geno2pheno clinicalYears since HIV diagnosisLog HIV-1 viral load



Binary multivariate regression analyses

PatientscharacteristicsV3 sequence

Clinical parameters

1 - Specificity1,00,80,60,40,20,0

Sens

itiv

ity

1,0

0,8

0,6

0,4

0,2

0,0

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%)



Temes tractats



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%

ROC curves for different G2P FPRs

Test characteristicsPhylogeny reconstruction for one subject

X4 prevalence in different compartments

PLASMA PBMC

ESTA PS* QDS** PS* QDS** MT2

Sensitivity - 36.4 90 36.3 90.9 45.5

Specificity - 94.4 82.4 100 55.6 100

PPV - 80 75 100 55.6 100

NPV - 70.8 93.3 73.1 90.9 76

* FPR 10%** FPR 10%, cut-off X4: 1%

ESTA PS QDS MT20

10

20

30

40

50

60

70

80

90

100PlasmaPBMC T1PBMC T2

% s

ub

ject

s w

ith

pre

dic

ted

X4

Significance relative to ESTA: * P<0.05** P<0.01*** P<0.001

*

***

* *

**


1. V3-loop QDS is the most accurate tropism test, followed by V3-loop PS.

2. 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.

3. 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.

4. 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.

C O N C L U S I O N S


Temes tractats



• 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

Randomization 1:1:1

Maraviroc (MVC 300 mg BID) + Combivir (ZDV+3TC)

Efavirenz (EFV 600 mg QD) + Combivir™ (ZDV+3TC)

Primary analysis

0 48 wk 96 wkOriginal Trofile Screening (6 weeks)

Saag M, et al. 4th IAS 2007; Sydney, Australia. Abstract WESS104.

Maraviroc (MVC 300 mg QD) + Combivir (ZDV+3TC)

• HIV-1 RNA ≥ 2,000 copies/mL• No evidence of resistance to EFV, ZDV, or 3TC

• ≥ 16 years of age• Treatment naive• R5 HIV-1 infection

Patient eligibility criteria:

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




• 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


Maraviroc BID; LOCF

Concordant R5 (N=283)

Concordant nonR5 (N=9)

ESTA nonR5, Geno R5 (N=39)

ESTA R5, Geno nonR5 (N=20)

Week

Lo

g V

iral

Lo

ad C

ha

ng

e

-4-3

-2-1

0

0 8 16 24 32 40 48 60 72 84 96




Maraviroc BID; Missing=Failure

Concordant nonR5 (N=9)

Concordant R5 (N=283)

ESTA nonR5, Geno R5 (N=39)

ESTA R5, Geno nonR5 (N=20)Per

cen

t co

pie

s <

50

Week

0.0

0.2

0.4

0.6

0.8

1.0

0 8 16 24 32 40 48 60 72 84 96




Maraviroc BID

Genotype R5 (n= 322)

Perc

ent w

ith R

5 by

Orig

inal

Tro

file

Week

0.0

0.2

0.4

0.6

0.8

1.0

0 8 16 24 32 40 48 60 72 84 96

Genotype nonR5 (n= 29)




Genotype R5

Genotype nonR5

Per

cen

t w

ith

R5

by

Ori

gin

al T

rofi

le

Week

ESTA nonR5

ESTA R5

Maraviroc BID

0.0

0.2

0.4

0.6

0.8

1.0

0 8 16 24 32 40 48 60 72 84 96




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)




• 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.

Randomization 1:1:1



Primary analysis


Saag M, et al. 4th IAS 2007; Sydney, Australia. Abstract WESS104.


• HIV-1 RNA ≥ 2,000 copies/mL• No evidence of resistance to EFV, ZDV, or 3TC

• ≥ 16 years of age• Treatment naive• R5 HIV-1 infection

Patient eligibility criteria:

Swenson et al. Large-Scale Application of “Deep” Sequencing Using 454 Technology to HIV Tropism Screening. Poster 545

ESTA vs 454



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).


Alerta!

Randomization 1:1:1



Primary analysis



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


Conclusions• 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

actualització en tropisme

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