Reconstruction of the Intra-Host Evolution of HCV Mathieu Flinders Max Planck Institute for Informatics April 5, 2013
Data Source: Gastroenterology Clinic, JW Goethe University Hospital, Frankfurt 2/19 April 5, 2013
Data • 3 patients (AM/VX-8, CG/VX-1, WM/VX-9) received telaprevir (TPR) monotherapy Source: Gastroenterology Clinic, JW Goethe University Hospital, Frankfurt 3/19 April 5, 2013
Data • 3 patients (AM/VX-8, CG/VX-1, WM/VX-9) received telaprevir (TPR) monotherapy • 3 patients (DB, FP, MR) received boceprevir (BCP) monotherapy Source: Gastroenterology Clinic, JW Goethe University Hospital, Frankfurt 4/19 April 5, 2013
Data • 3 patients (AM/VX-8, CG/VX-1, WM/VX-9) received telaprevir (TPR) monotherapy • 3 patients (DB, FP, MR) received boceprevir (BCP) monotherapy • 3 patients (IM1, IM3, IM11) received danoprevir (DNP) monotherapy Source: Gastroenterology Clinic, JW Goethe University Hospital, Frankfurt 5/19 April 5, 2013
Data • 3 patients (AM/VX-8, CG/VX-1, WM/VX-9) received telaprevir (TPR) monotherapy • 3 patients (DB, FP, MR) received boceprevir (BCP) monotherapy • 3 patients (IM1, IM3, IM11) received danoprevir (DNP) monotherapy • treatment failed to eliminate HCV Source: Gastroenterology Clinic, JW Goethe University Hospital, Frankfurt 6/19 April 5, 2013
Data • 3 patients (AM/VX-8, CG/VX-1, WM/VX-9) received telaprevir (TPR) monotherapy • 3 patients (DB, FP, MR) received boceprevir (BCP) monotherapy • 3 patients (IM1, IM3, IM11) received danoprevir (DNP) monotherapy • treatment failed to eliminate HCV • 5–7 blood samples (A, B, C...) taken over a period of 8–50 months (4+ years) Source: Gastroenterology Clinic, JW Goethe University Hospital, Frankfurt 7/19 April 5, 2013
Data • 3 patients (AM/VX-8, CG/VX-1, WM/VX-9) received telaprevir (TPR) monotherapy • 3 patients (DB, FP, MR) received boceprevir (BCP) monotherapy • 3 patients (IM1, IM3, IM11) received danoprevir (DNP) monotherapy • treatment failed to eliminate HCV • 5–7 blood samples (A, B, C...) taken over a period of 8–50 months (4+ years) • HCV NS3 sequenced using Roche/454 FLX Source: Gastroenterology Clinic, JW Goethe University Hospital, Frankfurt 8/19 April 5, 2013
Danoprevir/r (RG7227) Class: protease inhibitor (ritonavir-boosted) Manufacturer: Hoffmann-La Roche/Genentech Dose: 100mg (boosted with 100mg of ritonavir), twice-daily Status: phase III, fixed-dose combination in phase I Population: genotypes 1 and 4 Daclatasvir (BMS-790052) Class: NS5a inhibitor Manufacturer: Bristol-Myers Squibb Dose: 60mg, once-daily Status: phase III Population: genotypes 1–4 Pipeline Report (2012) 9/19 April 5, 2013
Analysis • region of interest: nucleotide positions 106–522 (length 417bp; chosen to include known resistance mutations*) *geno2pheno[hcv] 10/19 April 5, 2013
Analysis • region of interest: nucleotide positions 106–522 (length 417bp; chosen to include known resistance mutations*) • agglomerative clustering with novel tree-cutting method for finding haplotypes: B8a *geno2pheno[hcv] 11/19 April 5, 2013
Analysis • region of interest: nucleotide positions 106–522 (length 417bp; chosen to include known resistance mutations*) • agglomerative clustering with novel tree-cutting method for finding haplotypes: sampling time (A, B, C...) B8a *geno2pheno[hcv] 12/19 April 5, 2013
Analysis • region of interest: nucleotide positions 106–522 (length 417bp; chosen to include known resistance mutations*) • agglomerative clustering with novel tree-cutting method for finding haplotypes: sampling time frequency in (A, B, C...) population (%) B8a *geno2pheno[hcv] 13/19 April 5, 2013
Analysis • region of interest: nucleotide positions 106–522 (length 417bp; chosen to include known resistance mutations*) • agglomerative clustering with novel tree-cutting method for finding haplotypes: sampling time frequency in (A, B, C...) population (%) B8a make name unique *geno2pheno[hcv] 14/19 April 5, 2013
Analysis • region of interest: nucleotide positions 106–522 (length 417bp; chosen to include known resistance mutations*) • agglomerative clustering with novel tree-cutting method for finding haplotypes: sampling time frequency in (A, B, C...) population (%) B8a make name unique • intra-patient evolutionary networks reconstructed by joining each haplotype to its nearest ancestor: A23 B8a C9 A13 B8b C8 *geno2pheno[hcv] 15/19 April 5, 2013
Analysis • region of interest: nucleotide positions 106–522 (length 417bp; chosen to include known resistance mutations*) • agglomerative clustering with novel tree-cutting method for finding haplotypes: sampling time frequency in (A, B, C...) population (%) B8a make name unique • intra-patient evolutionary networks reconstructed by joining each haplotype to its nearest ancestor: A23 B8a C9 A13 B8b C8 connect with nearest ancestor *geno2pheno[hcv] 16/19 April 5, 2013
Analysis • region of interest: nucleotide positions 106–522 (length 417bp; chosen to include known resistance mutations*) • agglomerative clustering with novel tree-cutting method for finding haplotypes: sampling time frequency in (A, B, C...) population (%) B8a make name unique • intra-patient evolutionary networks reconstructed by joining each haplotype to its nearest ancestor: A23 B8a C9 A13 B8b C8 connect with dashed lines for nearest ancestor interval > 12m *geno2pheno[hcv] 17/19 April 5, 2013
Sampling Times 18/19 April 5, 2013
BCP patient FP 19/19 April 5, 2013
BCP patient MR cf. Gray et al (2012) 20/19 April 5, 2013
TPR patient CG/VX-1 (1/2) 21/19 April 5, 2013
TPR patient CG/VX-1 (2/2) 22/19 April 5, 2013
TPR patient WM/VX-9 (1/2) 23/19 April 5, 2013
TPR patient WM/VX-9 (2/2) 24/19 April 5, 2013
Conclusions Broadly coherent structure in terms of propagation of resistance mutations and variant abundances Varied branching structures provide evidence for both strong and weak selection, consistent with the assumption that evolution will be weak - over short time intervals, and/or - in the absence of protease inhibitor, and otherwise strong. Reversion to wild-type ( i.e. the extinction of resistant strains) does occur and takes as little as 13 months Trials of HCV protease inhibitors are potentially valuable case studies of evolution with strong selection 25/19 April 5, 2013
References Resistance mutations: SE Schelhorn, AM Sikorski, M Zeidler, S Sierra-Aragon, B Beggel, J Büch, R Kaiser, T Lengauer, geno2pheno[hcv] (2011). Population structure: RR Gray, M Salemi, P Klenerman, OG Pybus, A New Evolutionary Model for Hepatitis C Virus Chronic Infection. PloS Pathogens (2012). 26/19 April 5, 2013
Acknowledgments Saarbrücken: Thomas Lengauer Glenn Lawyer Prabhav Kalaghatgi Bastian Beggel Sven-Eric Schelhorn Frankfurt: Christoph Sarrazin Stefan Zeuzem Simone Susser 27/19 April 5, 2013
Thank you! 28/19 April 5, 2013
DNP patient IM11 (Slide A) 29/19 April 5, 2013
DNP patient IM11 (Slide B) 30/19 April 5, 2013
Method Example phylogenetic tree with N = 24 sequences: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 31/19 April 5, 2013
Method 1. Join sequences bottom-up, noting sizes of clusters involved. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 32/19 April 5, 2013
Method 1. Join sequences bottom-up, noting sizes of clusters involved. 2. Where two clusters both ≥ 3 are joined... cut! 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 33/19 April 5, 2013
Method 1. Join sequences bottom-up, noting sizes of clusters involved. 2. Where two clusters both ≥ 3 are joined... cut! Note: If number of sequences N is large, use max {3, N *5%}. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 34/19 April 5, 2013
Method 1. Join sequences bottom-up, noting sizes of clusters involved. 2. Where two clusters both ≥ 3 are joined... cut! Note: If number of sequences N is large, use max {3, N *5%}. Q: How many clusters? 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 35/19 April 5, 2013
Method 1. Join sequences bottom-up, noting sizes of clusters involved. Step 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 1+1 1+1 1+1 1+1 1+1 1+1 1+1 1+1 1+1 all < 3 36/19 April 5, 2013
Method 1. Join sequences bottom-up, noting sizes of clusters involved. Step 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 2 + 1 2 + 1 2 + 2 2 + 2 2 + 1 2 + 1 all < 3 37/19 April 5, 2013
Method 2. Where two clusters both ≥ 3 are joined... cut! Step 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 3 + 3 4 + 4 3 + 1 3 + 2 38/19 April 5, 2013
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