Quer 23 24abril14

8/12/2019 Quer 23 24abril14

1/81

1

Vall dHebron Institut de Recerca (VHIR)Hospital Universitari Vall dHebron (HUVH)

Institut dInvestigaci Sanitria del Instituto de Salud Carlos III (ISCIII)

Lab. Malalties Hptiques 006. VHIR-HUVH

Ciber Enfermedades Hepticas y Digestivas (Ciberehd) del Instituto de Salud Carlos III

Universitat Autnoma de Barcelona

Josep Quer PhD

josep .q uer@v hir.org

Uso de la Secuenciacin masiva para el estudio del Virus

de la Hepatitis C

8/12/2019 Quer 23 24abril14

2/81

2

Viruses that infect the Liver (Hepatotropic)

-Epstein Barr virus (Herpesviridae) dsDNA 185kb-Citomegalovirus (Herpesviridae) dsDNA 229kb-Yellow fever virus (Flaviviridae) ssRNA 11kb-Varicela Zoster (Herpesviridae) dsDNA 125kb-Herpes Simplex (Herpesviridae) dsDNA 152kb-Coxsackie virus (Picornaviridae) ssRNA+ 7.5kb-Lassa fever (Arenaviridae) ssRNA bisegm (7.2kb and 3.5kb).

-Marburg (Filovirus) ssRNA- 19.1kb-Ebola (Filovirus) ssRNA- 18.9kb-Rift valley fever (Bunyaviridae) ssRNA- trisegmented-Measles (Paramyxovirus) ssRNA- 16kb

-HEPATITIS A (HAV)-HEPATITIS B (HBV)-HEPATITIS C (HCV)-HEPATITIS delta D (HDV)-HEPATITIS E (HEV)

Viruses that primarily infect the liver causinga clinically relevant and widely extended

lesion:

VIRAL HEPATITIS

8/12/2019 Quer 23 24abril14

3/81

8/12/2019 Quer 23 24abril14

4/81

4

8/12/2019 Quer 23 24abril14

5/81

5

H V

HBV

HCV

HDV

37bp

5 3

27bp68bp

1 7194ORF3(328)

ORF1(5079)ORF2(1980)

CAPSIDAPEPTIDO SEALMT Proteasa Helicasa Replicasa

NO ESTRUCTURAL ESTRUCTURAL

ESTRUCTURAL

p73NCR5NCR

ESTRUCTURAL NO ESTRUCTURAL

C E1 E2 2 3 4B 5A 5B4Ap7

NO ESTRUCTURALVP2 VP3VP45NCR

VpgVP1 2A 2B 2C 3Cpro 3Dpol3A 3B

3NCR

AAAA

HEV

GENOMES. High level of variation in natural isolates.

Cellular RNA polimerase II

Polymerase with Reverse

Transcriptase activity

HCV RNA DependentRNA Polymerase

HIV Reverse Transcriptase

8/12/2019 Quer 23 24abril14

6/81

6Simmonds P & Smith D. Chapter 43: Evolution of hepatitis viruses pp575-586 in Viral Hepatitis. 4th ed.

Ed.Thomas, Lok, Locarnini & Zuckerman. John Wiley & sons. Oxford 2014.

Simmonds et al. Hepatology 2005

HIV

8/12/2019 Quer 23 24abril14

7/81

7

High level of replication: HBV 1011// HCV 1012// HIV 1.03x1010particles/day

LACK OF PROOFREADING MECHANISMS.

Rate of accumulation (fixation) of mutations (rate of evolution) = substitutions/base/year

HAV: 1x10-3 -10-4

HBV: 1.4-7.9 105

HCV: 1.5x10-3

HDV: 3.05.0x10-3

HEV: 1.401.72x10-3

Mutation rate =substitution/base/replication cycle

HIV 3.4 x 10-5

~106-fold higher

than for cellu lar

DNA

DNA VIRUS

10-7to 10-9

Human Herpesvirus (HHV): 1,58x10-8

Mean: 0,003 mutations per genome (Dr ake&Hwang Genetics2005:170:969-970)

2.-VARIABILITY

8/12/2019 Quer 23 24abril14

8/81

8

CONSENSUS

MUTANTESPECTRA

**

* *

* **

* **

*

*

* **

* *

**

*

*

*

*

QUASIESPECIES

Holland JJ et al. Science 1982; 215(4540):1577-1585Domingo E & Holland JJ. Evolutionary biology of viruses. 1994Martell et al. J.Virol. 1992; 66(5):3225-3229Vignuzzi Nature 2006; 439:344-348Vignuzzi M, et al. Nature 2005.

QUASIESPECIESSTRUCTURE

Any RNA viral population is composed of a

complex mixture of different but closelyrelated genomes, known as QUASISPECIES.

Quasispecies composition is subjected tocontinuous change following Principles ofDarwinian evolution

Reproduction with genetic variationCompetitionSelectionInteraction and Cooperation

8/12/2019 Quer 23 24abril14

9/81

9

HCV prevalence (%) in General Population

< 0.5%

0.5-1%

1.1-1.5%

1.6-2.5%

2.6-3.5%

3.6-5%

>5%

14,9%

Chronically infected patients

2,3% general population

160 millions

HCV

Quer J& Esteban JI. In ViralHepatitis 4th edition. 2013.

8/12/2019 Quer 23 24abril14

10/81

10

2.5%

1.3%

2%

3%

5%

1%

1%

2%

2%2%

1.5%

1%1%

2%

3%

0.2%

0.6%

2%3%

0.8%

0.2%

0.2%

0.5%

0.2%

3%

1%

0.1%

1%

1%

1.3%

1.5%

2.5%

2.5%

0.2%

< 0.5%

0.5-1%

1.1-1.5%

1.6-2.5%

2.6-3.5%

> 3.6%

Ucrania 4%

3%

JI.Esteban, S.Sauleda, J.Quer J.Hepatol.2008;48:148-162

PREVALENCE OF HCV CHRONIC INFECTION IN EUROPE

HVC GENOTYPES AND SUBTYPES

8/12/2019 Quer 23 24abril14

11/81

11

Smith D, Bukh J, et al. Expanded classification of HCV 7genotypes and 67 subtypes, updated criteria and assignment web

source.Hepatology 2013 Oct 1. doi: 10.1002/hep.26744.

HVC GENOTYPES AND SUBTYPES

7a

NS5B *Complete genome

*Adaptation H.T. Hraber et al.Virology Journal 2006;3:103-

8/12/2019 Quer 23 24abril14

12/81

12

World DISTRIBUTION of HCV Genotypes

http://hcv.lanl.gov/components/sequence/HCV/geo/geo.comp

5

6

4

8/12/2019 Quer 23 24abril14

13/81

13

Hraber HT et al Virology Journal 2006; 3:103Zein NN ClinMicrobRev 2000;13:223-235 (review)Simmonds et al. Hepatology. 2005

8/12/2019 Quer 23 24abril14

14/81

14

HCV

NATURAL HISTORY

Acute

infection

Chronic

infection

Compensated

cirrosis

Decompensated

cirrosis

Hepatocarcinoma

Hemorrhagic

varices

Hepatic

encephalopathy

AscitisCuration

Antiviral treatment Death

Liver

Transplant

6 weeks 1-2 years to 10-20 years 20-30 years

8/12/2019 Quer 23 24abril14

15/81

15

Follow-up patients / year assocated to Liver damage % Patients(estimation)

Moderate Liver Damage (F0-F3) 308# 70

Compensated Cirrhosis 1.831* 15

Decompensated Cirrhosis 21.688* 10

Hepatocelular Carcinoma 34.595* 3

Liver Transplant (Mean value) 104.542# 2

Postransplant Follow-up 29.911**adaptation in Razavi et al Hepatol. 2013; 57(6):2164-2170. doi: 10.1002/hep.26218

# data from Diari Oficial Generalitat (DOG 6326 - 1.3.2013) CVE-DOGC-A-13051031-2013. RESOL.

SLT/353/2013

SOCIAL & ECONOMICAL COST. NOT TO TREAT.

8/12/2019 Quer 23 24abril14

16/81

16

SVR=Sustained Virologic Response

HCV-RNA neg. 24weeks after stopping Treatment.

THERAPY Non-cirrhotic Patients

SVR G1 G2,3,5,6 G4

pegIFN+RBV (24weeks) 38% 75-80% 38%

pegIFN+RBV (48weeks)42% 70%

TREATMENT of CHRONIC HCV

STANDARD-OF-CARE pegIFN alfa 2a/2b + RIBAVIRINA

ANTI HCV TREATMENT What next?

8/12/2019 Quer 23 24abril14

17/81

17

SOC

pIFN + RBV

(~40% SVR in G1)

SOC 1

pIFN + RBV + DAA

(30% increase SVR in G1)

2011 2011-2013

ANTI-HCV TREATMENT. What next?

2011-2012. Federal Drug Administration (FDA) & European Medicines Agency (EMA) approved two NS3

protease inhibitors (to be used in Triple Therapy with pIFN+RBV):

Telaprevir(VX-950). (INCIVO. Tibotec. Janssen. J&J /// INCIVEK. VertexN.America-). EMA Sep 20, 2011

Boceprevir(SCH503034). (VICTRELIS. Merck Sharp & Dohme). 2012.

TRIPLE THERAPY G1 PATIENTS (Naive v Treatment Experienced patients (TE)

SVR NAIVE Relapsers Partial Responders Null-Responders

pegIFN+RBV 38% 29% 7% 5%

pegIFN+RBV+BOC 63-66% 75% 52% 40%

pegIFN+RBV+TVR 72-75% 83-88% 54-59% 29-33%

pegIF+RBV+BOC:Bacon BR. et al. N Engl J Med 2011; 364:1207-1217. // Poordad F et al. N Engl J Med 2011: 364: 1195-1206 // Bronowicki JP. etal. J Hepatol 2012: 56: S6

pegIFN+RBV+TVR:Sherman KE et al. N Engl J Med 2011; 365: 1014-1024. // Jacobson IM et al. N Engl J Med 2011; 364 : 2405-16. // Zeuzem S. etal. N Engl J Med 2011;364:241728

8/12/2019 Quer 23 24abril14

18/81

18

CUPIC TRIALPATIENTS Liver fibrosis stage F3/F4.SVR to pegIFN+Rbv: 17-26%

56%

Summary from EASL 2013 for Hepatitis C - New HCV DAAs on their way soon: what do the phase III studies tell us?

ITT = intent to treat

ANTI-HCV TREATMENT What next?

8/12/2019 Quer 23 24abril14

19/81

19

SOC

pIFN + RBV

(~40% SVR in G1)

SOC 1

pIFN + RBV + DAA

(Goal: 65%-80% SVR in G1)

Future SOC 2

Improved pIFN(?) + RBV(?) + DAA(Goal: 80% SVR in G1)

DAA(s) + RBV only

(Spare pIFN)

DAA Combination

Spare IFN and RBV

pIFN + RBV + DAAs

(Quad therapy)

2011 2014 & Next2011-2013

pIFN + DAAs

(Spare RBV)

ANTI HCV TREATMENT. What next?

COMBINATION of DAAsIFN-free therapies

NEW oral DAAs approval to be used in triple therapywith pegIFN/Rbv:

Simeprevir- OLYSIOby Janssen(NS3 2nd gen. DAAmacrocyclic) to treat Genotype 1.

Approved by FDA Nov. 23rd 2013Approved by EMA 2014

Sofosbuvir-Sovalsiby Gilead. (NS5B inhibitor Ni) pan-genotyping activity to treat Genotypes 1, 2, 3 and 4Approved by FDA Dec. 6th 2013Approved by EMA January 25th 2014

AASLD. The Liver Meeting 2013. Nov. Washington USA.

SOCIAL COST. TREATMENT.

8/12/2019 Quer 23 24abril14

20/81

20Jacobson I, et al. AASLD 2010, Abstract 211

% of patients with

Telaprevir 12 wks +

pegIFN/RBV

N=363

Telaprevir 8 wks +

pegIFN/RBV

N=364

PegIFN/RBV

(control)

N=361

Any adverse event* 99 99 98

Fatigue 57 56 57

Pruritis 50 45 36

Headache 41 43 39

Nausea 43 40 31

Rash 37 35 24

Anemia 37 39 19

Insomnia 32 32 31

Diarrhoea 28 32 22

Influenza-like illness 28 29 28

Pyrexia 26 30 24

ADVERSE EVENTS

ECONOMICAL COST. TREATMENTS.

8/12/2019 Quer 23 24abril14

21/81

21

Treatment COST. CATSalut.

DRUGTELAPREVIR (T) BOCEPREVIR (B)Unit price (Lab sales price) 52,60 9Dosage 750 mg/8h 800 mg/8hCost per day 315,6(6 tablets/day) 108(12 tablets/day)

Cost complet treatment

12weeks T + PR24 weeks 31145 (26510 (T) + 4635 (PR24)) ...

12w T + PR48 w 35781(26510(T) + 9271(PR48)) ...24w B + PR28 w ... 23551(18144 (B) + 5407(PR28))32w B + PR48 w ... 33463(24192 (B) + 9271(PR48))44w B + PR48 w (cirrhosis) ... 42535(33264 (B) + 9271(PR48))

PR=pegIFN+Ribavirin

Estimated COST.

Therapy SOF 12w (G1 & G2) 60.000

Therapy SOF 24w (G3) 120.000

Triple therapy pegIFN+RBV+SMV 60.000

COST TREATMENT + FOLLOW-

UP / PATIENT / YEAR

pegIFN+RBV 9.371,88

8/12/2019 Quer 23 24abril14

22/81

22

Response factorsIFN + RBV + DAA

orDAAs-IFNfree

Treatment regimen

PegIFN exposureRBV exposureDAA exposure

Host factors

Age, gender, race,obesity, ETOH,

Genetic factors (IL28B)

Disease featuresFibrosis, steatosis,

coinfection (HBV, HIV)

Viral factorsGenotype

HCV RNA level

Quasispecies(baseline resistance)

PREDICTORS OF ANTI-HCV RESPONSE

Efectiveness of anti-HCV therapy depens on:

8/12/2019 Quer 23 24abril14

23/81

23

1.- Quasispecies diversity. Viral load.

py p

ACUTEPHASE

CHRONICPHASE

STANDARD TREATMENTpegIFN alfa + RBV

80-90%

G145% SVR

56% Non response

G2 & 3)

80% SVR

20% Non response

SVR=Sustained virologic response

HCV-RNA neg. 24 weeks after stopping treatment.

40-50%

HCV


8/12/2019 Quer 23 24abril14

24/81

24


Efectiveness of anti HCV therapy depens on:

2. Resistance mutations to DAAs

8/12/2019 Quer 23 24abril14

25/81

25

HCV GENOME.

8/12/2019 Quer 23 24abril14

26/81

26

1 190

191-383 384-746

747-809

810-1026

1027-1657

1658-1711

1712-1972

1973-24202421-3011

Core

protein

Envelope

glycoprotein 1

Envelope

glycoprotein 2

Putative ionchannel

Autoprotease

Helicase and

Serine protease

NS3 co-

factor

RNA-dependent RNA

polymeraseFormation of

replication

complex

Phosphoprotein.

Regulation

replication

-PREVIR-ASVIR

-BUVIR

RESISTANCE MUTATIONS to NS3.

8/12/2019 Quer 23 24abril14

27/81

27

NS3 (67kDa): 631 aa 1893 nts

PROTEINASE 189aa (567nts) / HELICASE 442aa (1326nts)

Faldaprevir=FDVSimeprevir=SMV

TVR

BOC

RESISTANCE MUTATIONS to NS3.

Sarrazin C, Hzode C, Zeuzem S & Pawlotsky JM J.Hepatol 2012; S88-S100

RESISTANCE MUTATIONS to NS5A.

8/12/2019 Quer 23 24abril14

28/81

28

N5A: 1344 nts // 448 aa

1 (6248) to 1344 (7601)NT1 (1973) to 448 (2420)

aa

GS-5885

G1a

G1b

RESISTANCE MUTATIONS to NS5A.

M28 R30 L31M Q54HP58 E62 Y93H

E62D

Q30R-E62D Galmozzi 2012 Hepatol.

Q54H-Y93HGalmozzi 2012 Hepatol.

RESISTANCE MUTATIONS to NS5B.

8/12/2019 Quer 23 24abril14

29/81

29

1 (7602) to 1776 (9377)NT 1 (2421) to 591 (3011)aa

NUCLEOS(T)IDIC INHIBITORS

NON NUCLEOS(T)IDIC INHIBITORS

Sofosbuvir GS-7977

BMS791325

Deleobuvir

8/12/2019 Quer 23 24abril14

30/81


8/12/2019 Quer 23 24abril14

31/81

31


2.- Presence of resistance mutations to DAAs.3.- HCV genotypeis the most important viral factor

predicting IFN treatment responsiveness.

G1 = SVR 38-41%

G2 = SVR 93%

G3 = SVR 79%

G4 = Difficult-to-treat virus. SVR rates similar than G1.

G5 = Easy-to-treat virus. SVR rates similar than G2 & 3 (67% SVR).

G6 = Intermediate level of response between 1 and 2.

Higher SVR Rates With BOC in Pts With HCV

8/12/2019 Quer 23 24abril14

32/81

32

Genotype 1b vs 1a

BOC RGT

5950

Genotype 1aGenotype 1b

Treatment Naive[1]BOC/PR48 BOC/PR48BOC RGT

Treatment Experienced[2]

1. Poordad F, et al. N Engl J Med. 2011;364:1195-1206.2. Bacon BR, et al. N Engl J Med. 2011;364:1207-1217.

66 63

70

6165

73

0

20

40

60

80

100

SV

R(%)

RGT= response guided-therapy

4. Subtype-dependent efficacy of DAAs

8/12/2019 Quer 23 24abril14

33/81

33

FDV= FaldaprevirSMV= Simeprevir=TMC435SOF=SofosbuvirDCV=Daclatasvir

PI = Protease inhibitor

Sarrazin et al., Gastroenterology 2010; Sullivan, et al. EASL 2011;Jacobson et al., NEJM 2011; Poordad et al., NEJM 2011; Manns et al.,EASL 2013; Jacobson et al., EASL 2013; Ferenci et al., EASL 2013

pegIFN+RBV+PI (NS3)NS5A NS3 +

NS5B(Ni)NS5B

SVR rateNAIVE patients

TVR BOC SMV FDV DCV Faldaprevir+Deleobuvir

SOF+RBV

G1a 71% 59% 75% 69% 58% 11%84%(naive)

10% (noresponse prio

therapy)G1b 79% 66% 85% 84% 87% 57%

EFFICACYG1b - G1a 8% 7% 10% 15% 29% 46%

G1

TURQUOISE-II study by ABBVIE.Compensated cirrhosis

8/12/2019 Quer 23 24abril14

34/81

34

Compensated cirrhosis

RITONAVIR [ABT-450](NS3)+

OMBITASVIR [ABT-267](NS5A)

+DASABUVIR [ABT-333](NS5B)+

Rbv

DASABUVIR [ABT-333](NS5B) is not active on Genotype 4 (PEARL-I study Hezode et al)

Phase II ION-1and ION-2 study by GILEAD:

LEDIPASVIR [GS 5885] (NS3)

8/12/2019 Quer 23 24abril14

35/81

35

LEDIPASVIR [GS-5885] (NS3)+

SOFOSBUVIR [GS7977] (NS5B) pan-genotyping

SVR LDV+SOF:12w= 99%24w= 99%

SVR LDV+SOF+Rbv:No further benefit

Very mild AE.

SVR LDV+SOF:12w= 94

24w= 98%

SVR LDV+SOF+Rbv:No further benefit

COST SOFOSBUVIR 12w= $84000 ($1000/tablet) Addition of LDV will increase the cost.

Egypt 1Mpeople to be treated Cost SOF 12w= 900.

ION-1 n=440 ION-2 n=865

G1a & G1b

PHASE III STUDIES FISSION, POSITRON & VALENCE by GILEAD:

SOFOSBUVIR [GS7977] (NS5B) pan genotyping

8/12/2019 Quer 23 24abril14

36/81

36

Relapse with RAVmutation to NS5B =S282T is G2b patient

SOFOSBUVIR [GS7977] (NS5B) pan-genotyping+

RIBAVIRIN (Rbv)Approved FDA / EMA

G2 & G3

1 Quasispecies diversity Viral load


8/12/2019 Quer 23 24abril14

37/81

37


2.- Presence of resistance mutations to DAAs.

3.- HCV genotype is the most important viral factor


4.- HCV subtypeis as a key determinant of the efficacy of

direct antiviral therapy.


8/12/2019 Quer 23 24abril14

38/81

38


2.- Presence resistance mutations to DAAs.

3.- HCV genotypeis the most important viral factor


4.- HCV subtypeis as a key determinant of the efficacy ofdirect antiviral therapy.

5.- Infection with more than one subtypeat the same time.

8/12/2019 Quer 23 24abril14

39/81

39

CLASSIC (VIRUS):

CULTURE:In most cases it is not possible (HCV, HBV, ...). Problems of falsenegatives.

SEROLOGY:Antibodies Detection:

- Cross reactions

- Limited specificity to identify genotypes/types/subtypes.

Antigen detection: In most cases it is inaccessible or has low sensitivity.

MOLECULAR BIOLOGY:

DNA and RNA detection and sequencing, using PCR, is the most sensitive andefficient method.

DIAGNOSTIC TECHNIQUES

AVAILABLE TECHNOLOGY in ROUTINE DIAGNOSTICSLABORATORY.

8/12/2019 Quer 23 24abril14

40/81

40

Reverse hybridising assay (Versant HCV Genotype 2.0 System (LiPA) (Siemens). Lmit de detecci >5%. Usesimmobilised biotinylated oligonucleotides specific to different genotypes and subtypes. Indentify genotypes 1-

6 and differentiate 1a and 1b subtypes but not others. Study of 5-core.

Direct sequence analysis (Trugene HCV 5NC genotyping kit) (Siemens). Determines the HCV genotype andsubtype by direct analysis of the nucleotide sequence. The accuracy of subtyping is poor because of theexclusive analyses of the 5UTR. 20% erroneous classification (17%1a as 1b and 3% 1b as 1a.Chevaliez PlosONE 2009; 4:e82009).

Real-time PCR technology (RealTime HCV Genotype II assay) (Abbott) . Genotype 1a and 1b were wellclassified, but single genotype 2, 3, 4, and 6 isolates were misclassified at the genotype level. Study of NS5B.

None of these technologies allows:high-throughput and confident subtypingdetection of mixed infections

detection of resistance mutations

or:

indication of recombinant genomes

presence of compensatory / multiple mutations in the same

genome.

Cloningand Sanger Sequencing. Difficult/impossible to be implemented for routine diagnostics lab.

Vall dHebronInstitut de Recerca (VHIR)Hospital Universitari Vall dHebron(HUVH)Inst i tut dInvestigaci Sanitria d el Ins titut o d e Salud Carlos III (ISCIII)

8/12/2019 Quer 23 24abril14

41/81

41

Inst i tut d InvestigaciSanitria d el Ins titut o d e Salud Carlos III (ISCIII)

454 / GS-Junior

-100.000 reads- 800nts 454 / GS-FLX+

-1.000.000 reads- 800-900nts

High resolution HCV subtyping MANUAL

8/12/2019 Quer 23 24abril14

42/81

42

High resolution HCV subtyping . MANUAL

BLOOD EXTRACTION (5-10mL). SERUM

High resolution HCV subtyping MANUAL

8/12/2019 Quer 23 24abril14

43/81

43

SERUM SAMPLE


High resolution HCV subtyping

8/12/2019 Quer 23 24abril14

44/81

44

High resolution HCV subtyping

RNA EXTRACTION

HCV STUDIES. UDPS

8/12/2019 Quer 23 24abril14

45/81

45

RT-PCR-NESTED

HCV 3NCR5NCRSTRUCTURAL NO STRUCTURAL

C E1 E2 7 NS2 NS3 NS5A NS5BNS4B4A

8/12/2019 Quer 23 24abril14

46/81

genome 3 NCR5 NCRNS5B (HCV RNA Dependent

RNA Polymerase)

8254 8707

RT-PCR (35 cycles)

Hemi-Nested M13 (30 cycles)

ReNested MID (15 cycles)

M13f M13r

M13f M13r

3 Specific primer + 5Universal M13 forward

3 Specific primer + 5Universal M13 reverse

Primer 1

Primer 1

Primer 3

Primer 2

MID MID

C E1 E2 p7 NS2 NS3 NS5A NS5BNS4B4A

M13fOligo A+ TCAG MID Primer 1 M13f TCAG+Oligo BMIDPrimer 3

Sanger sequencing High-resolution HCVsubtyping (454/GS-Junior)

454nts

428nts

499nts

Oligo A+ TCAG TCAG+Oligo B

339nts

339nts


8/12/2019 Quer 23 24abril14

47/81

47

AGAROSE GEL PURIFICATION


HIGH-RESOLUTION HCV SUBTYPING 454/GS-Junior

8/12/2019 Quer 23 24abril14

48/81

48

Original file (GS-Junior software)

RAW DATA includes 150.000 reads (sequences)

HIGH RESOLUTION HCV SUBTYPING

8/12/2019 Quer 23 24abril14

49/81

49

Approved January 2014: 7 Genotypes and 67 subtypes.

8/12/2019 Quer 23 24abril14

50/81

50

Smith D, Bukh J, et al. Expanded classification of HCV 7genotypes and 67 subtypes, updated criteria and assignment web

source.Hepatology 2013 Oct 1. doi: 10.1002/hep.26744.

pp y yp yp

References are continuously updated.

If classification changes, sequences can be easly re-subtyped.

G1 (7): a,b,c,e,g,h,lG2 (11): a,b,c,d,e,i,j,k,m,q,rG3 (6): a,b,g,h,i,kG4 (17): a,b,c,d,f,g,k,l,m,n,o,p,q,r,t,v,wG5 (1): aG6 (24): a,b,c,d,e,f,g,h,i,j,k,l,m,n,o,p,q,r,s,t,u,v,w,xaG7 (1): a

HCV genotypes and subtypes. Region Discriminating power.

8/12/2019 Quer 23 24abril14

51/81

51

While the NS5B region is highlyvariable and discriminates betterbetween subtypes, the 5 core ismore stable and better suited forsampling from patients.

The table besides gives the nearestsubtype to each subtype, both innumber of differences and in % identity.

Pos: 5core 168:469,NS5B 8283:8618


8/12/2019 Quer 23 24abril14

52/81

52


8/12/2019 Quer 23 24abril14

53/81

53G1b

Infection with more than one subtype at the same time.


8/12/2019 Quer 23 24abril14

54/81

54

PATIENT CASE:

Infection with more than one subtypeat the same time.

8/12/2019 Quer 23 24abril14

55/81

Infection with more than one subtypeat the same time


8/12/2019 Quer 23 24abril14

56/81

56

yp

G1a



8/12/2019 Quer 23 24abril14

57/81

57

PATIENT CASE:

yp



8/12/2019 Quer 23 24abril14

58/81

58G4d

Infection with more than one subtypeat the same time = Mixed infection.

5HIGH RESOLUTION HCV SUBTYPING

8/12/2019 Quer 23 24abril14

59/81

59

PATIENT CASE:

Patient 37 is infected by:

G1a (1506 reads -60.53%-)+ G4d (982 reads -39.47%)

COMPARATIVE study

8/12/2019 Quer 23 24abril14

60/81

60

Sangersequencing

NS5BHigh-resolution HCVsubtyping GS-Junior

NS5B

Versant HCVGenotype 2.0LiPA Siemens

5-core

Real Time HCVGenotype II. Abbott

5-coreNS5B (1a/1b)

Patient code

Direct NS5B

sequencing

Versant HCV Genotype

2.0

Abbott Real time HCV

genotype II.

UDPS HCV Subtyping

454/GS-Junior*P1-P17 1a 1a 1a 1aP18-61 1b 1b 1b 1b

P62 1a 1 1 1aP63 1a 1a 1 1a

8/12/2019 Quer 23 24abril14

61/81

61

G1 samples(n=82)

P63 1a 1a 1 1aP64 1a 1 1a 1aP65 1a 1a 1 1aP66 1a 1 1 1aP67 1a 1 1 1a

P68 1a 1 1 1aP69 1a 1a 1 1aP70 1b 1 1b 1bP71 1b 1 1 1bP72 1b 1 1b 1bP73 1b 1 1b 1bP74 1b 1 1b 1bP75 1b 1 1 1bP76 1b 1b 1 1b

P77 1b 1b 1b 1b (43%)+3a(35%)+1a (22%)P78 1b 1b 1 1b

P79 1b 1/1a 1b 1b

P80 1b 1 1b 1bP81 1b 1b 1 1bP82 1b 1b 1 1b

Table summary Untyped G1 samplesNumber of patients Versant/Siemens Rt-PCR/Abbott

G1a 25 5 (20%) 7 (28%)G1b 57 8 (14%) 6 (10%)

Total 82 13 (16%) 13 (16%)

16 %of Genotype 1 samples cannot be subtyped by the commercial techniques used in routine labs.

Non-G1 samples

8/12/2019 Quer 23 24abril14

62/81

62

Patient CodeDirect PCR Sanger

sequencing

Versant HCV Genotype 2.0

LiPA. Siemens

Real time HCV genotype II.

Abbott

High-resolution HCV

subtyping 454/GS-Junior

P-IND-1 2c 2a/c 2 2c

P-IND-2 2c 2a/c 2 2c

P-IND-3 3a 3 3 3a

P-IND-4 3a 3a ND 3a

P-IND-5 3a 3a 3 3a

P-IND-6 3a 3a 3 3a

P-IND-7 4a 4 4 4a

P-IND-8 4a 4a/c/d 4 4a

P IND 9 4d 4 4 4d

Non-G1 samples

8/12/2019 Quer 23 24abril14

63/81

63

P-IND-9 4d 4 4 4d

P-IND-10 4d 4 4 4d

P-IND-11 4d 4a/c/d 4 4d

P-IND-12 4d 4a/c/d 4 4d

P-IND-13 4f 4f 4 + 5* 4f

P-IND-14 4f 4f 4 + 5* 4f

P-IND-15 4f Ind 4 + 5* 4f

P-IND-16 4f Ind 4 + 5* 4f

P-IND-17 4f Ind 4 + 5* 4f

P-IND-18 4f Ind 4 + 5* 4f

P-IND-19 4f Ind 4 + 5* 4f

P-IND-20 4f Ind 4 + 5* 4f P-IND-21 4f Ind 4 + 5* 4f

P-IND-22 4f Ind 5 4f




P-IND-26 4r Ind 1 + 4 4r

P-IND-27 5a 5a 5 5a

P-IND-28 6c neg 5 6c

* 4 or 5

Mixed InfectionsMixed infections

8/12/2019 Quer 23 24abril14

64/81

64

P-IND-29 4d Ind 4 4d (72%)+1a (28%)

P-IND-30 2j 2 ND 2j (95%)+4f (5%)

P-IND-31 4p Ind 3 + 4 4p (53%)+3a (47%)P-IND-32 4d Ind 1a + 4 4d (87%) +1a (13%)

2,52 %mixed infections in patients

visited in the Hospitaloutpatient clinics

HCV SUBTYPE PREVALENCE HUVH BARCELONA 2012-13.

24%

56%

9%

7%1% 3%

1a

1b

3a

4d

4f

Other

109 patients analyzed by 4th March 2013

SPECIAL CASE:


8/12/2019 Quer 23 24abril14

65/81

65

PATIENT INFECTED with 1b + 3a + 2c + 4d + 1a.

8/12/2019 Quer 23 24abril14

66/81

Patient treated two years ago with TVR: Alignment of the 48 different amplified nucleotide sequencesHere we show 150 from total 443 nts.

8/12/2019 Quer 23 24abril14

67/81

67

Patient treated two years ago w ith TVR: Al ignment of 48 amino acid sequences

8/12/2019 Quer 23 24abril14

68/81

68V36A = 0.2% Q80R = 0.27%

NS3 (67kDa): 631 aa 1893 nts

PROTEINASE 189aa (567nts) / HELICASE 442aa (1326nts)

8/12/2019 Quer 23 24abril14

69/81

69

Faldaprevir=FDV

Simeprevir=SMV

TVR

BOC

Sarrazin C, Hzode C, Zeuzem S & Pawlotsky JM J.Hepatol 2012; S88-S100

8/12/2019 Quer 23 24abril14

70/81

70

Kwong AD, Najera I, et al. Gastroenterology 2011Mar;140(3):755-60. doi:10.1053/j.gastro.2011.01.029

SUMMARY

8/12/2019 Quer 23 24abril14

71/81

71

SUMMARY

1.- Massive sequencing allows high-resolution studies on viral infections. Variabilitystudies. Detection of minority mutants some of them can be clinically relevant.

2.- HCV subtype is a key determinant of the efficacy of direct antiviral therapy. High-

resolution HCV subtyping based in massive sequencing and phylogeny represents a

maximum precision methodology for HCV subtyping and for quantification of mixedinfections.

3.- High resolution HCV sequencing allows detection of resistance mutations.

Antonio MadejnJavier Garca-Samaniego F i R d F

Ricardo MorenoPaloma Sanz-Cameno

8/12/2019 Quer 23 24abril14

72/81

Javier Garca-Samaniego Francisco Rodrguez-FrasMara Buti

David Garca-Cehic

Rosario CasillasMara BlasiMara Homs

David TaberneroLeo Nieto

Mara CuberoSilvia Sauleda

Marta BesAndrea CaballeroRafael EstebanJaume Guardia

Juan Ignacio Esteban

Sofa Prez del Pulgar

Xavier Forns

Esteban DomingoCelia PeralesJulie Sheldon

Carlos Briones

Manolo Romero-GmezJose Antonio del Campo

Josep GregoriMiguel Alvrez-TejadoJos Manuel MuozJordi Gmez

Javier SalmernPaloma Muoz de Rueda

Rosa Quiles-Prezngela Extremera

Rosa Lpez-Rodrguez

8/12/2019 Quer 23 24abril14

73/81

73

THANK YOU !

Why genotypes have different sensitivity to pegIFN?

8/12/2019 Quer 23 24abril14

74/81

74

IL28B (=IFN3),IL28A (= IFN2)i IL29 (=IFN1)

8/12/2019 Quer 23 24abril14

75/81

75

IL28B(=IFN3)

rs12979860(chr19:39738787)

IL28A(=IFN2)

IL29(=IFN1)

CC

SVR topegIFNbased

treatments

Favorable

rs368234815(chr19:39739154)

-G

IL28B(=IFN3)

rs12979860(chr19:39738787)

IL28A(=IFN2)

IL29(=IFN1)

SVR topegIFNbased

treatments

Unfavorable CT / TT

rs368234815(chr19:39739154)

TT

IFN4

Carlos Fernndez-Carrillo, P.Gonzlez, G.Crespo, M.Coto-Llerena, M.Navasa, X.Forns, Sofia Prez del PulgarXXXIX Congreso AEEH Madrid, 19-21 Febrero 2014

8/12/2019 Quer 23 24abril14

76/81

8/12/2019 Quer 23 24abril14

77/81

77

NS3 V36A/M/L Q41 F43C/S T54A/S V55A Q80R/K R155K/T/Q A156T A156S/V/I V158I D168A/V V170A/T

Telaprevir

Boceprevir

Vaniprevir

ITMN-191

BILN-2061

TMC-435

BI-201335

Low level resistance 100 x

Not done

NS3 resistance mutation (protease-helicase)

8/12/2019 Quer 23 24abril14

78/81

78

Identification of non-primate hepaciviruses (NPHV)

8/12/2019 Quer 23 24abril14

79/81

A novel hepacivirus identified by high-throughput

sequencing of respiratory samples from dogs

Animals from respiratory disease outbreaks in two sheltersfound positive

The novel virus is the closest known homologue to HCV

Kapoor PNAS 2011

High prevalence of NPHV in horses

Serology identified high prevalence (35%) of NPHV specific

antibodies in horse sera

8 (22%) of immuno-reactive samples were positive for NPHV

by RT-PCR

One equine isolate was nearly identical to the canineviruses!!!

Epidemiology, disease association, transmission?

New hepacivirus isolates in rodents and bats!

Burbelo JVI 2012 Kapoor mBio 2013 Quan PNAS 2013

Import 2012 % pes relatiu/total

Taula 102. Import dels medicaments dispensats a pacients en rgim ambulatori als hospitals de Catalunya

concertats i de lICS, 2012

8/12/2019 Quer 23 24abril14

80/81

Import 2012 % pes relatiu/total

Antiretrovirals 153.286.409 23,6

Citosttics 150.838.716 23,3

Immunosupressors selectius 99.378.048 15,3Altres medicaments d's hospitalari 83.793.753 12,9

Esclerosi mltiple 41.061.364 6,3

Eritropoetina 26.327.776 4,1

Hormona de creixement 18.723.454 2,9

Factors antihemoflics 18.724.676 2,9

Nutrici enteral (NE) per sonda a domicili ms material NE 18.659.082 2,9

Tractaments hepatitis C 15.095.863 2,3Immunoglobines humanes inespecfiques 11.891.718 1,8

Factors d'estimulants de colnies 6.731.840 1

Medicaci estrangera 2.973.335 0,5

Medicaci i solucions per a la nutrici parenteral (NP) intravenosa 656.824 0,1

Medicaments per al tractament de la fibrosi qustica 223.953 0

Total 648.366.813 100

Font: Gerncia dAtenci Farmacutica i Prestacions Complementries.

Pagina 210. Memoria 2012 CatSalut.

8/12/2019 Quer 23 24abril14

81/81

Quer 23 24abril14

Documents

Transcript of Quer 23 24abril14