Interpretation tools for coreceptor usage Rolf Kaiser Institute of - - PowerPoint PPT Presentation

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Interpretation tools for coreceptor usage Rolf Kaiser Institute of - - PowerPoint PPT Presentation

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Interpretation tools for coreceptor usage Rolf Kaiser Institute of Virology University of Cologne Coreceptor Usage 2 Chemokinreceptors are used in vivo as coreceptors: CCR5 & CXCR4 CCR5 only (R5-Virus) CXCR4 only (X4-Virus)

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Interpretation tools for coreceptor usage

Rolf Kaiser Institute of Virology University of Cologne

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Coreceptor Usage

2 Chemokinreceptors are used in vivo as coreceptors: CCR5 & CXCR4 CCR5 only (R5-Virus) CXCR4 only (X4-Virus) Viruses using CCR5 and CXCR4 (R5/X4-Virus, dualtropic) Mixtures of R5- and X4-Viruses in patients plasma (Mixed) For clinical purpose 2 classes: CXCR4 can be used CXCR4 cannot be used => R5 viruses that can be inhibited by R5-blockers

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HIV Entry Cell Assay (Trofile)

Adapted from Petropoulos CJ, et al. Antimicrob Agents Chemother. 2000;44:920-8.

Transfection

HIV env expression vector

+ + +

HIV genomic luc vector CD4 + CXCR4 +

Infection

Pseudovirus

  • capable of a single

round of replication

R5/X4 Tropic HIV

CCR5 and CXCR4 antagonists are used to confirm tropism

CD4 + CCR5 +

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SLIDE 4
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deCIPhR for Entry inhibitors

β-Gal

Pr

X4 Env T20 TF141-1

  • Cpd. conc. (nM)

0.1 10 1000

  • Av. %Inh.
  • 20

20 40 60 80 100 X4-3026 AMD3100

  • Cpd. conc. (nM)

0.01 1 100

  • 20

20 40 60 80 100 SCH417690 TAK779

  • Cpd. conc. (nM)

0.01 1 100 % virus inhibition

  • 20

20 40 60 80 100

Fusion Inhibitors CXCR4-antagonists CCR5-antagonists

T20 TF141-1

  • Cpd. conc. (nM)

0.1 10 1000

  • Av. %Inh.
  • 20

20 40 60 80 100 SCH417690 TAK779

  • Cpd. conc. (nM)

0.01 1 100

  • Av. %Inh.
  • 20

20 40 60 80 100 AMD3100 X4-3026

  • Cpd. conc. (nM)

0.1 10 1000

  • Av. %Inh.
  • 20

20 40 60 80 100

β-Gal

Pr

R5 Env

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HIV-1 PHENOSCRIPT-ENV

EUROFINS-TRT Data presented from JL Faudon

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Comparison of different phenotypic assays

Monogram-Trofile Eurofins-TRT R5 X4 R5 n=42 (56,8%) n=3 (4.1%) X4 n=8 (10%) n=21 (28,4%)

Skrabal et al J. Clin. Microbiol, 2007

14% discordant; different cell lines, other differences?

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Questions

Predicting coreceptor usage by genotypic analyses? Can we simply adapt our experience from resistance testing of RT and Protease? What gene(-region) to sequence? aa position 11 and 25 in env-V3 are predictive enough for R5/X4 (SI/NSI) (Fouchier et al 1995)

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SLIDE 9

6558 TGGGATCAAAGCCTAAAGCCATGTG

GGGTTCTTRGGAGCAGCAGGAAGCACT 7785

6957 GTACAATGTACACATGGAAT

TAATTGTGGAGGGGAATTTT 7371

V3 Izopet et al. Lehmann et al.

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1110 matched genotype (V3 region)-phenotype pairs:

Genotypic Monitoring of Coreceptor Usage

CTRPNNNTRRSISIGPGRAFYATGDIIGDIRQAHC R5 CTRPNNNTRKGIHMGPGS-FYVTGEIIGDIRQAHC R5

phenotyping genotyping

CSRPNNNTRKSVHIGPGQAFYATGDVIGDIRQAHC X4

X=(X1, …, Xi, …, Xn) Y

multiple alignment (against fixed reference alignment) statistical learning

T.Sing

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Internet-Tools for genotype based predictions

Web-PSSM (Mark Jensen et al),http://ubik.microbiol.washington.edu/computing/pssm/ PSSMs for Subtyp B vs. C* WetCat: (Pillai et al.)http://genomiac2.ucsd.edu:8080/wetcat/v3.html geno2pheno[coreceptor] http://www.genafor.org

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geno2pheno[coreceptor]

Current geno2pheno[coreceptor]: Specificity 90%, Sensitivity 76% for X4 trained on 1100 Los Alamos data

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Geno2pheno[coreceptor]: Output

Alignment relative to HXB2 Predicted coreceptor usage

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Question

Other attributes ?

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Gp120 and V3 region

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geno2pheno[coreceptor] For 90% Spec. for X4 Sensitivities are SVM-Seq: 79.4%

SVM-struct. descriptors 84,7% SVM-Seq+ struct. descriptors 86,4%. Poster #66 – O. Sander

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Genotyping results

  • Salvage Patients Uni Köln,

longitudinal study over 3 years

C.Lehmann, G.Fätkenheuer, M.Däumer, R.Kaiser, Journal of Clinical Virology 37 (2006) 300–304

  • Successful Amplification in 45 /45 patients (100%)
  • Interpretation with geno2pheno in 42/45

25 patients (56%) contiunously displayed an R5-Virus 12 patients (27%) contiunously displayed an X4-Virus In1 patient HIV ‚shifted‘ from R5 to X4 In 2 patients HIV ‚shifted‘ from X4 to R5 In 1 patient HIV ‚shifted‘ from X4 to R5 to X4 In 1 patient HIV ‚shifted‘ from R5 to X4 to R5

  • In Motivate Studies 8% switched between screening and start of

MVC-Therapy

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Genotyping results

Presentation in Dublin from the “ANRS” - group: Cathia Soulié, Anne Derache, Catherine Aimé, Anne-Geneviève Marcelin, Guislaine Carcelain, Anne Simon, Christine Katlama, Vincent Calvez

  • Successful Amplifikation in 83,1% of n=69 patients samples
  • Interpretation with geno2pheno in 66/69 cases, with PSSM in 58/69

cases (No Alignment Tool in PSSM) „mean of both systems“ ART naive: R5 71,2%, X4 28,8% ART experienced R5 58,8%, X4 41,2%

  • Comparable numbers with results from phenotyic studies?
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Percentage of HIV Co-receptor Usage

Study/Source Population N Homer cohort1 Naive 979 C & W cohort2 Naive 402 81% <1% 19% TORO 1/24 Experienced 612 62% 4% 34% ViroLogic5 Experienced >2000 48% 2% 50% ACTG 52116 Experienced 391 49% 4% 47% Demarest3 Naive 299 88% 0% 12% 82% <1% 18% R5 X4 R5/X4

1Brumme ZL, et al. J Infect Dis. 2005;192:466-474. 2Moyle GJ, et al. J Infect Dis. 2005;191:866-872. 3Demarest J, et al. ICAAC 2004. Abstract H-1136.

*This table may not include all available reported data. Majority of data are generated in the developed world (subtype B)

4Whitcomb JM, et al. CROI 2003. Abstract 557. 5Paxinos EE, et al. ICAAC 2002. Abstract 2040. 6Wilkin T, et al. CROI 2006. Abstract 655.

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Phenotypic and genotypic tests came to comparable numbers for coreceptor usage in therapy naives and therapy-experienced patient cohorts

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Minorities do they always exist? Do they always play a clinicilly relevant role?

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Are Coreceptor blockers dangerous and drive coreceptor switch?

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MOTIVATE 1 and 2: Change in CD4 Cell Count from Baseline by Tropism Result at Time of Failure

Mean change in CD4 count from baseline, cells/mm3 in patients with treatment failure Tropism result, Baseline → Treatment Failure Placebo + OBT N = 209 MVC QD + OBT N = 414 MVC BID + OBT N = 426 All treatment failures +14 (n = 97) R5 → R5 +15 (n = 80) +61 (n = 18) +138 (n = 17) +67 (n = 4) +49 (n = 68) +71 (n = 77) R5 → D/M or X4 +37 (n = 31) +56 (n = 32)

  • Approximately 8% of patients had a change in tropism result between screening and

baseline, demonstrating the change in background tropism over a 4–6 week period in this population

  • Response to maraviroc treatment in these patients was consistent with the results
  • f study A4001029 in patients with D/M-tropic virus (XVI IAC 2006, Abs THLB0215)

Data excludes patients who had no tropism result at time

  • f failure and patients with non-R5 virus at baseline

MOTIVATE 1 & 2-Week 24

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Resistance to Coreceptor blockers?

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HIV-1 isolate from a Δ32/wt CCR5 patient with unusual broad coreceptor usage

Gorry et al., Virology, Jan 2007

region

V1 V2 V3 C4 isolate

unusual long region Y308 I317 D321 K442 E444

(compensatory)

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Mutations under MVC pressure during cell culture passage

Passage V1 V2 V3 C3 C4 V4

CC1/ 85

CTRPNNNTRKSI HI GPGRAFYTTGDI I GDI RQAHC

st art T163 A316 T319 I323I/ V N335N/ P/ K/ Y S405S/ A/ T/ Esuscept ible number 16 T163K A316T/ S T319T/ S I323V N355Y S405A resistent revert ant A316 I323V suscept ible RU570

CTRPNNNTRKSI SFGPGQAI YTTGNI I GDI RQAHC

st art S132 QAI 315- 317 T319 N322 suscept ible number 18 (8) S132N ΔQAI N322D N442K D389N resistent number 18 (2) N322D suscept ible revert ant T319K/ T N322D suscept ible

Westby et al., Journal of Virology, Mar. 2007

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Clinical data that may improve the prediction

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Comparison of genotypic and or phenotypic data see Posters #53 N. Chueca and #82 E. Poveda Minorities are there but not always a problem X4 under immune control? We canot simply transfer our tools from RT- and Protease resistance to coreceptor prediction tools

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Institute of Virology, University of Cologne Martin Däumer Saleta Sierra-Aragon Eva Heger Claudia Müller Dörte Hammerschmidt Jens Verheyen Ibrahim Boussaad Eugen Schülter Herbert Pfister Rolf Kaiser MPI for Informatics, Saarbrücken Tobias Sing Achim Buech Andre Altmann Alexander Thielen Oliver Sander Thomas Lengauer

  • Dept. of Mathematics, UC Berkeley, CA, USA

Niko Beerenwinkel University of Essen; caesar, Bonn Daniel Hoffmann caesar, Bonn Ulrike Schuldenzucker MPl of Molecular Plant Physiology, Golm Joachim Selbig Institute of Clinical and Molecular Virology, German National Reference Center for Retroviruses, University of Erlangen-Nürnberg Barbara Schmidt Hauke Walter Klaus Korn

  • Dept. of Gastroenterology, University of Düsseldorf

Mark Oette

  • Dept. of Internal Medicine I, University of Cologne

Gerd Fätkenheuer Clara Lehmann

  • Dept. of Internal Medicine I, University of Bonn

Jürgen Rockstroh Medical Laboratory, Berlin Thomas Berg PZB, Aachen Heribert Knechten, Patrick Braun and the Resina study group

geno2pheno

www.geno2pheno.org / www.genafor.org

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Collaborative Trials for Phenotypic Assays Collaborative Trials for Genotypic Assays => ENVA?

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TG_66

day 0

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TG_66

day 28

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TG_66

day 56

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TG_66

3068 3164 3015

  • X4-Virus as minor variant at day 0.
  • Day 0 sample showed a T215D revertant
  • Primary resisant virus
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TG_66

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SI/NSI phenotype

  • Calculate the overall charge of the V3 peptide:

if V3-charge > +4 ⇒ SI (64/11) if V3-charge ≤ +4 ⇒ NSI (125/11)

V3 charge

SI NSI

On Cologne-Bonn training data: error 11,6 % leave-one-out 11,6 %