Xenotropic Murine Leukemia

Virus-Related Retrovirus:

CCR Assay Development

XMRV Publications – theDebate Continues

2006

1

2007

2

2008

6

2010

32

ProstateCancer

2009

11

ChronicFatigue

Syndrome

Source: PubMed (NLM)

Keywords: “Abstracts” & “XMRV”

NCI XMRV Assay Development

• July 2009 Meeting of extramural & NCI scientists to discuss public health impact of XMRV infection

• Oct. 2009 NCI XMRV Planning Committee established - meets on ad hoc basis

• Nov. 2009 Construction of 40 recombinant clones expressing XMRV antigens

Development of ultrasensitive assay to quantify XMRV DNA

• Dec. 2009 6 XMRV proteins purified to homogeneity by PEL/SAIC for diagnostic development

Development of assay to quantify XMRV RNA from plasma

• Jan. 2010 Serological XMRV testing initiated using NCI-produced CA antigen

Sensitive XMRV indicator cell line established (DERSE)

• Feb. 2010 Evaluation of XMRV antigens for serological testing

• Mar. 2010 Large-scale production of XMRV virions as a source of antigen

• Apr. 2010 64 XMRV expression clones delivered from NCI to NIH AIDS Reagent Repository

NCI XMRV Effort Builds onStrengths in Human Retrovirology

Laboratory ofExperimentalImmunology

Viral Technology Laboratory (SAIC)

HIV Drug ResistanceProgram

Protein Expression Laboratory (SAIC)

AIDS and CancerVirus Program

XMRVDiagnosisVirus Production Antigen Preparation

Serological Detection Nucleic Acid DetectionXMRV Infectivity Assay

XMRV InfectivityAssay

X-SCA: Single Copy XMRV DNA or RNA Detection – HIV DRP

• Three ultrasensitive nucleic acid testing assays (X-SCAs)

• Assay versions available for whole blood, PBMCs or plasma

• Quantitative polymerase chain reaction (PCR) assay design

• Detects XMRV at single copy level

• Detects XMRV DNA or RNA

Cro

ss

ing

Po

int

Log ConcentrationCycles

Flu

ore

scen

ce Amplification Curves Standard Curve

106 1Virus Copy#

X-SCA: Single Copy XMRV DNA or RNA Detection – HIV DRP

Current status:

72 blinded samples of donor plasma, spiked with known quantities of XMRV DNA or RNA, were tested using the X-SCA assay

• XMRV detected with SINGLE COPY sensitivity

• XMRV detected in plasma and whole blood with 100% accuracy

• No false positives or negatives

In progress

HVIU Virology Core will use X-SCAs to detect XMRV nucleic acid in a panel of samples tested across multiple platforms for comparative analyses

Purification of XMRV AntigensPEL/SAIC-Frederick

CA

NCPR

RT

IN

MA p12

SU

Oct 23 Dec 6

64 XMRV expression clones prepared for distribution

Large-Scale XMRV ProductionACVP, NCI-Frederick

Purified XMRV Virions

SDS/PAGE Immunological Analysis

HPLC Fractionation

-MLV gp70-MLV CA p30Coomassie

Stained

M M MX X X

XMRV Antigen/Serological AssayDevelopment - VTL/SAIC-Frederick

• Questions to be answered:

• XMRV prevalence in general population

• Reactive and non-reactive samples

• Levels of antibodies in XMRV-reactive subjects

• Define a ‘training set’:

• NCI-Frederick Research Donor Program

• Donor plasma (1990s, BBI Diagnostics)

• Subjects from Lombardi et al. study

• Assess utility of recombinant XMRV antigens:

• SU TM MA CA p12 NC PR RT IN

XMRV Serological Assay PlatformMeso Scale Discovery (MSD)

XMRV CA

EC

L U

nit

s

XMRV TM XMRV SU

www.mesoscale.com

XMRV Antigen/Serological AssayDevelopment - VTL/SAIC-Frederick

Current status:

• ELISA-based assay to multiple (9) XMRV antigens demonstrates:

• Reactivity with CA, SU and/or TM

• Reduced reactivity to p12, MA and NC

In progress:

• Inclusion of antigens reactive to antibodies in human sera into a ‘scoring algorithm’

• Confirmation required by Western blot and/or PCR

Cell Culture Analysis of XMRVInfection - HIV DRP

• Lombardi et al. - recovery of infectious XMRV from blood of human subjects

• Detectors of Exogenous Retroviral Sequence Elements (DERSE) indicator cells, using XMRV susceptible HEK293T and LNCaP lines, developed

• DERSE cells permit detection of infectious XMRV from patient samples in one week (vs. 3+ weeks by other methods)

• Under optimization to detect XMRV in EDTA- and heparin-containing samples

• Available for distribution via NIH AIDS Reagent Repository in Fall 2010

Cell Culture Analysis of XMRVInfection - HIV DRP

XMRV infection of LNCaP or 293T derived indicator cells

Propagation of wt XMRV or GFP reporter virus captured by XMRV results in signal amplification via GFP transduction of neighboring cells

Infection of DERSE L-iG cl.6 Cells with XMRV

Fluorescence &Light Microscopy

3 Days Post-Infection

FluorescenceMicroscopy

Comparative Evaluation ofNCI XMRV Assays

Nucleic AcidDNA

Nucleic AcidRNA

Serological AnalysisELISA

Serological AnalysisWestern Blot

Virus Culture-DERSE

Immunohistochemistry

NCI Cancer Panel

Acknowledgements

• G. Fanning-Heidecker, HIV DRP• M. Kearney, HIV DRP• V. KewalRamani, HIV DRP• K.-E. Lee, HIV DRP• Rein, HIV DRP

• R. Bagni, VTL/SAIC-Frederick• D. Esposito, PEL/SAIC-Frederick• J. Hartley, PEL/SAIC-Frederick

• J. Bess, ACVP/SAIC-Frederick• E. Chertova, ACVP/SAIC-Frederick• J. Lifson, ACVP/SAIC-Frederick

• K. Jones, LEI/SAIC-Frederick• F. Ruscetti, LEI