Xenotropic Murine Leukemia Virus-Related Retrovirus: CCR Assay Development

1. Xenotropic Murine LeukemiaVirus-Related Retrovirus:CCR Assay Development

2. 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

3. 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

4. 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)

5. 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

6. 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

7. 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

8. 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

9. 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

10. www.mesoscale.com XMRV Serological Assay PlatformMeso Scale Discovery (MSD) XMRV CA ECL Units XMRV SU XMRV TM

11. 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

12. 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

13. 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

14. 3 Days Post-Infection Fluorescence Microscopy Fluorescence & Light Microscopy Infection of DERSE L-iG cl.6 Cells with XMRV

15. 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

16. 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