Xenotropic murine leukemia virus related retrovirus ccr assay development
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Xenotropic Murine Leukemia Virus-Related Retrovirus: CCR Assay Development. XMRV Publications – the Debate Continues. Source: PubMed (NLM) Keywords: “Abstracts” & “XMRV”. 32. 11. 6. 2. 1. 2006. 2007. 2008. 2009. 2010. Chronic Fatigue Syndrome. Prostate Cancer.

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Xenotropic Murine Leukemia Virus-Related Retrovirus: CCR Assay Development

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Xenotropic murine leukemia virus related retrovirus ccr assay development

Xenotropic Murine LeukemiaVirus-Related Retrovirus:CCR Assay Development


Xmrv publications the debate continues

XMRV Publications – theDebate Continues

Source: PubMed (NLM)

Keywords: “Abstracts” & “XMRV”

32

11

6

2

1

2006

2007

2008

2009

2010

Chronic

Fatigue

Syndrome

Prostate

Cancer


Nci xmrv assay development

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 on strengths in human retrovirology

XMRV Infectivity

Assay

XMRV

Diagnosis

Antigen Preparation

Virus Production

Serological Detection

Nucleic Acid Detection

XMRV Infectivity Assay

NCI XMRV Effort Builds onStrengths in Human Retrovirology

HIV Drug Resistance

Program

Protein Expression

Laboratory (SAIC)

AIDS and Cancer

Virus Program

Laboratory of

Experimental

Immunology

Viral Technology

Laboratory (SAIC)


X sca single copy xmrv dna or rna detection hiv drp

Standard Curve

Amplification Curves

Crossing Point

Fluorescence

Cycles

Log Concentration

106

1

Virus Copy#

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


X sca single copy xmrv dna or rna detection hiv drp1

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 antigens pel saic frederick

Dec 6

Oct 23

RT

SU

IN

CA

PR

MA

p12

NC

64 XMRV expression clones prepared for distribution

Purification of XMRV AntigensPEL/SAIC-Frederick


Large scale xmrv production acvp nci frederick

Purified XMRV Virions

HPLC Fractionation

SDS/PAGE

Immunological Analysis

X

M

M

X

M

X

Coomassie

Stained

a-MLV CA p30

a-MLV gp70

Large-Scale XMRV ProductionACVP, NCI-Frederick


Xmrv antigen serological assay development vtl saic frederick

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 platform meso scale discovery msd

www.mesoscale.com

XMRV Serological Assay PlatformMeso Scale Discovery (MSD)

XMRV CA

ECL Units

XMRV SU

XMRV TM


Xmrv antigen serological assay development vtl saic frederick1

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 xmrv infection hiv drp

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 xmrv infection hiv drp1

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

3 Days Post-Infection

Fluorescence

Microscopy

Fluorescence &

Light Microscopy

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


Comparative evaluation of nci xmrv assays

Virus Culture-DERSE

Serological Analysis

ELISA

Nucleic Acid

DNA

NCI Cancer Panel

Serological Analysis

Western Blot

Nucleic Acid

RNA

Immunohistochemistry

Comparative Evaluation ofNCI XMRV Assays


Acknowledgements

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


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