TMA Assay for Detection of West Nile Virus

BPAC Meeting - March 13, 2003

Cristina Giachetti, Ph.D.J.Linnen; A.Broulik; W. Wu; J.Cline; M.Lewis;

G.Dennis; M.Cass; M.Alden; V.Casas; and S.Miller

Gen-Probe Incorporated, San Diego, CA

Objectives of the West Nile Virus Assay Development Program

To develop and manufacture a TMA-based assay for detection of West Nile virus in blood, plasma and organ/tissue donor specimens Phase 1 clinical study to determine RNA reactivity in

archived linked samples collected in areas of potentially high WNV incidence rate during 2002

Phase 2 clinical protocol to allow for widespread donor screening. It is anticipated that this trial will be initiated July 2003.

WNV Assay Development Goals

Analytical sensitivity: at least 95% detection at 50 copies/mL

Detection of genetic variants of WNV (e.g Lineage 1, including Kunjin virus, and Lineage 2 strains) with similar sensitivity

Analytical specificity: > 99.5 %

Internal Control to validate each reaction

Completely compatible with Procleix® eSAS (semi-automated) and TIGRISTM (fully automated) instrument platforms

Amplification(TMA)

Amp Rgt.,Oil,

and Enzyme

Water baths

Procleix® Semi-Automated System: Assay Protocol

Pipetting

Calibrators,Specimens,

and TCR

TECAN

SampleProcessing

Viral Lysis,RNA Capture, and Washes

TCS

Detection(HPA)

ProbeHybridization

Selection

Water bath

Results

AutomaticRLU

Reading

Luminometer

TECAN Target Capture System (TCS) Luminometer

Specimen ProcessingTarget Capture/Magnetic Microparticle Separation

Viral Lysis Treat specimens with heat and detergent Release nucleic acid

Nucleic Acid Capture Hybridize target sequence to capture probes Hybridize capture probe to oligomer sequence bind to magnetic particle

Removal of unwanted specimen Apply magnetic field to separate target from residual sample Remove residual specimen by washing

Magnetic Microparticle

TTTTTTTTTTTTTT

TTTTTTTTTTTTTT

TTTTTTTTTTTTTT

Ma

gn

e t

3’ AAAAAA…AAACAUCUAAC…CGU 5’

5’ GUAGAUUG…GCA 3’

Capture Oligo

Target RNA orDNA

TT

TT

TT

TT

TT

TT

TT

TT

TT

TT

TT

TT

TT

TT

AmplificationTranscription-Mediated Amplification (TMA)

Utilizes two enzymes: Reverse

Transcriptase

T7 RNA Polymerase

Amplifies RNA or DNA

Produces RNA amplicon

Exponential amplification(> billion fold amplification in less than one hour)

Isothermal, simplifies automation

DetectionHybridization Protection Assay (HPA)

Utilizes Acridinium Ester (AE) labeled probes

Reaction Steps:

1. HybridizationAE-labeled probe hybridizes to target RNA in solution

2. SelectionLabel on unhybridized probe is hydrolyzed, label on hybridized probe is protected

3. DetectionLabel on protected hybridized probe is detected by chemiluminescence

Detection (cont.)Dual Kinetics Analysis (DKA)

Used to differentiate Internal Control (IC) signal from target signal

Utilizes Acridinium Ester (AE) labeled probes with differential kinetics of light-off

Ortho Fluoro Acridinium Ester labeled probe = flasher probe, hybridizes to IC

2’Methyl Acridinium Ester labeled probes = Glower probes, hybridize to West Nile virus amplicon

ETF Algorithm deconvolutes light-off and calculates each signal

0

2000

4000

6000

8000

10000

12000

14000

16000

1 4 7 10

13

16

19

22

25

28

31

34

37

40

43

46

49

Interval

RLU

Flasher

Background

Glower

Current Performance of WNV Assay

Specificity in normal blood donor specimens Specificity in the presence of other blood

borne viruses Analytical sensitivity Clinical sensitivity

Specificity in normal blood donor specimens

Frozenplasmas

Freshplasmas

Combinedresults

Number tested 475 100 575Initial reactive rate (%) 0 0 0

Repeat reactive rate (%) 0 0 0

Initial IC failures 4 0 4

Initial IC failure rate (%) 0.84 0 0.69

Repeat IC failure rate 0 0 0

Specificity in the presence of other blood borne viruses

Blood borne viruses Number tested Result

HAV 5 Non-reactiveCMV 7 Non-reactiveEBV 7 Non-reactiveHBV 4 Non-reactiveHCV 2 Non-reactiveHIV-1 1 Non-reactiveHTLV I/II 7 Non-reactive

Normal populationn=575

Blood borne virusesN=33

Average s/co IC 1.99 2.1Average s/co analyte 0.01 0.00Initial/repeat reactive rate 0 0Specificity 100% 100%

Analytical SensitivityLimit of detection using in vitro transcript

WNV RNA(copies/mL)

AnalyteAve. s/co

% positiven=40

95% detectionlimit [95% CI]

50% detectionlimit [95% CI]

300 31.8 100100 31.1 100

30 26.0 100

10 18.1 97.5

3 12.1 55.0

1 11.7 35.0

0.3 2.9 10.8

0.1 0.08 0

0 0.08 0

8.2[6.6-11.5]

copies/mL

2.9[2.2-3.9]

copies/mL

Analytical SensitivityDetection of WNV in CDC viral panel*

WNV PFU/mL Positivity (%)N= 20

95% DetectionLimit [95% CI]

50% DetectionLimit [95% CI]

1.1 100

0.37 100

0.12 100

0.04 100

0.013 100

0.004 70

0.0015 55

0 0

0.008[0.005-0.042]

PFU/mL

0.001[-0.009-0.003]

PFU/mL

* Provided by Dr. Robert Lanciotti, CDC-DVBID

Analytical SensitivityDetection of WNV in Qualification Panel QWN701 from BBI

Panel Member WNV copies/mL % Positive (n=4)

QWN701.01 100 100QWN701.02 0 0QWN701.03 30 100QWN701.04 1,000 100QWN701.05 300 100QWN701.06 100 100QWN701.07 0 0QWN701.08 30 100QWN701.09 1,000 100QWN701.10 100 100QWN701.11 0 0QWN701.12 30 100QWN701.13 300 100

Analytical SensitivityLimit of detection in Lineage 2 Panel

WNV RNA(copies/mL)

AnalyteAve. s/co

% positiven=20

95% detectionlimit [95% CI]

50% detectionlimit [95% CI]

300 31.2 100100 24.7 100

30 16.4 100

10 7.57 95.0

3 8.45 45.0

1 1.59 5.0

0.3 0.2 0

0.1 0.02 0

0 0.02 0

9.0[7.1-12.8]

copies/mL

4.5[3.5-6.1]

copies/mL

Clinical SensitivityDetection of WNV in Specimens from CDC

Case Investigations

* Reactive in either plasma or RBC segment; ** IgM positive; *** Reactive with increased sample volume

Procleix WNV Assay TMA Confirmatory AssayCase IDNeat 1:8 1:16 Neat 1:8 1:16

CDC TaqManAssay*

IA-1 ++ +++ +++ ++ +++ +++ +IN-3 ++ +++ +++ ++ +++ +++ +IN-6 ++ +++ +++ ++ +++ +++ +MI-11 ++ +++ +++ ++ +++ +++ +MI-1 ++ +++ +++ ++ +++ +++ +MI-4 ++ +++ +++ ++ +++ +++ +OH-3 ++ +++ +++ ++ +++ +++ +IN-4 ++ +++ +++ ++ +++ +++ +GA-1 ++ +++ +++ ++ +++ +++ -/+***IL-4 ++ +++ +++ ++ +++ +++ -/+***NE-1** ++ -----+ +++--- ++ --- +-- -

Summary

Specificity

No cross-reactivity with other blood borne viruses

Initial reactive rate in normal blood donor population was 0% [n=575], for 100% specificity

Summary

Analytical Sensitivity 95% Detection rate at

6.6-11.5 copies/mL Lineage 1 transcript 7.1-12.8 copies/mL Lineage 2 virus 0.005-0.042 pfu/mL CDC virus panel

50% Detection rate at 2.2-3.9 copies/mL Lineage 1 transcript 3.5-6.1 copies/mL Lineage 2 virus 0.001-0.003 PFU/mL CDC virus panel

Summary

Clinical Sensitivity

Procleix WNV assay has a clinical sensitivity equal or better than CDC Taqman Assay

All reactive results with Procleix WNV Assay were confirmed with TMA assay using an alternative amplification region

Conclusions

Performance to date meets design goals for specificity and sensitivity

Results support feasibility of pool testing

Acknowledgments

This project is funded in part with federal funds from the National Heart, Lung and Blood

Institute under Contract