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Development of sero-assays to screen for

XMRV antibodies in clinical samples

Development of sero-assays to screen for

XMRV antibodies in clinical samples

Rachel K. Bagni, Ph.D.

December 14, 2010

OutlineOutline

• Reagent development

• Availability of reagents

• Assay development strategy

• Preliminary observations

• Path forward

Development of reagentsDevelopment of reagents

9 XMRV gene products (VP62)► gag (MAtrix, p12, NucleoCapsid, CApsid)

► pol (PRotease, Rev Transcriptase, INtegrase)

► env (Surface Unit, TransMembrane)

Image courtesy of Stig Jensen, NCI

SUTM

RTMA

p12CAPRRNA

NC

IN

XMRV Reagent DevelopmentXMRV Reagent Development

Clone Set-up

• 3 forms of sequence-verified Gateway Entry clones

• 4 types of protein expression clones

• Clones for protein secretion

Recombinant Antigen Production

• Initial screening

• Small-scale production

• Final production

XMRV antigen resultsXMRV antigen results

5 μg each protein

MA p12 CA NC PR RT Int SU TM

15.1 kDA 9.7 kDA

31.6 kDA

7.7 kDa

14.3 kDA

75.9 kDA

45.9 kDA 48.1 kDA

19.9 kDA

63.4 kDA

Cross-reactivity with antibodies directed against MLV proteins Cross-reactivity with antibodies directed against MLV proteins

20 kDa

30 kDa

40 kDa

50 kDa

120 kDa100 kDa

60 kDa

80 kDa

α-p30S. Ruscetti

20 kDa

30 kDa

40 kDa

50 kDa

80 kDa

120 kDa100 kDa

60 kDa

CA RT

α-RTM. Roth

SU

α-gp70S. Ruscetti

20 kDa

30 kDa

40 kDa

50 kDa

80 kDa

120 kDa100 kDa

60 kDa

120 kDa100 kDa

80 kDa

60 kDa

50 kDa

40 kDa

30 kDa

SU

α-env (SFFV):S. Ruscetti

Reagents available…Reagents available…

The following DNAs are deposited (64 clones):• E. coli expression clones (His6 and His6-MBP) – 20

• baculovirus clones (His6-MBP) – 10

• baculovirus secreted clones – 2

• analogous Entry (Gateway) clones – 32

NIH AIDS Research and Reference Reagent Program

NCI CPTC: Antibody Characterization Laboratory

https://www.aidsreagent.org

Serological Assay Platform:

Meso Scale Discovery (MSD)Serological Assay Platform:

Meso Scale Discovery (MSD)

Ruthenium (II) Sulfo-tris-bipyridine NHS ester

Antigen

Abs in serum

Labeled 2°

Electric Current

Qualification of XMRV recombinant antigens for use in sero-assaysQualification of XMRV recombinant antigens for use in sero-assays

• Unknown: prevalence of the virus in general population– Positive subjects in ‘normal’ donor population?

• Unknown: positive and negative samples

• Unknown: levels of antibodies in XMRV+ subjects

LimitationsLimitations

• Preliminary assay characteristics calculated from a small sample number

• Assumption of sero-status

• Immune profile after infection unknown

• Requires validation using bona fide, pedigreed antibody clinical controls

Titration of SFFV Env MAbTitration of SFFV Env MAb

Titration of anti-Capsid Ab (MLV)Titration of anti-Capsid Ab (MLV)

Qualification of XMRV recombinant antigens for use in sero-assaysQualification of XMRV recombinant antigens for use in sero-assays

• Define ‘training set’– 77 donors

• NCI-Frederick RDP

• donor plasma (1990s, BBI Diagnostics)

– 39 XMRV+ subjects (WPI-CFS)

• Assay to XMRV antigens:– SU, TM, MA, CA, p12,

NC, PR, RT and IN

• Use statistical analyses to determine utility of antigen in a screening assay

Receiver Operator Characteristic (ROC) CurvesReceiver Operator Characteristic (ROC) Curves

• Sensitivity– proportion of patients with the virus that will be

reactive on the test(s)

• Specificity– proportion of subjects without the virus that

will be non-reactive on the test(s)

• True reactive rate (Sensitivity) is plotted as a function of the false reactive rate (100-Specificity).

ROC Curves: IN and RTROC Curves: IN and RT

IN RT

Area under ROC curve: 0.46 (0.35-0.57) Area under ROC curve: 0.49 (0.39-0.61)

ROC Curves: CA, TM and SUROC Curves: CA, TM and SU

Area under ROC curve: 0.72 (0.63-0.81)

Area under ROC curve: 0.86 (0.79-0.92)

Area under ROC curve: 0.66 (0.56-0.75)

CA

SU

TM

Training Set for Assay DevelopmentTraining Set for Assay Development

2

2 3

12

Donors

N = 77 (10/77)

Subjects

10 1

10

4 5

N = 39 (34/39)

Used to adjust criteria in the absence of pedigreed clinical controls.

4

Donor samplesDonor samples

• 1000 donor samples– 500 NIH donors

– 500 CNMC donors

• Assayed for CA, TM and SU– SU re-testing planned

Preliminary findings (raw non-calibrated data):

NA

5.5%

SummarySummary

• Multiple XMRV recombinant antigens have been used in sero-assay development

• Detect reactivity to CA, TM and/or SU

• Some subjects are reactive to p12, MA and NC

• Inclusion of antigens reactive in human sera into a ‘positivity algorithm’

Ongoing effortsOngoing efforts

• Continued development efforts of sero-assays required

• Refinement of optimal cut-points

• Establish positivity algorithm

• Secondary assays: WB, NA tests

• Identification of pedigreed clinical controls

• Samples from experimentally infected animal models

Large Scale XMRV Production - ACVPLarge Scale XMRV Production - ACVP

Purified XMRV Virions

SDS/PAGE Immunological Analysis

HPLC Fractionation

a-MLV gp70a-MLV CA p30

M M MX X X

ConsiderationsConsiderations

• Preliminary assay characteristics calculated from a small sample number

Required:

• Analytical performance panels (antibody)

• Validation using bona fide, pedigreed antibody clinical controls

AcknowledgementsAcknowledgements

Katie BeamWilliam BurganVijaya GowdaJoe HugueletAllison Meade

Vanessa Wall - COG

James HartleyDominic Esposito – COG

Troy Taylor – MEG Ralph Hopkins – EEG

Bill Gillette – PPG

SAIC-Molecular Detection Group

SAIC-Protein Expression Lab

NCI-CCRBob Wiltrout

Stuart Le GriceFrank RuscettiKathy Jones

NIHHarvey Alter

Whittemore-Peterson InstituteJudy Mikovits

SAIC-ACVPJeff Lifson

Denise WhitbyNazzarena Labo

ConsiderationsConsiderations

• Preliminary assay characteristics calculated from a small sample number

Required:

• Analytical performance panels (antibody)

• Validation using bona fide, pedigreed antibody clinical controls