Xenotropic Murine Leukemia Virus-Related Retrovirus: CCR Assay Development

XMRV Publications – the Debate Continues Prostate Cancer Chronic Fatigue 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 NCI XMRV Planning Committee established - meets on ad hoc basis Nov Construction of 40 recombinant clones expressing XMRV antigens Development of ultrasensitive assay to quantify XMRV DNA Dec XMRV proteins purified to homogeneity by PEL/SAIC for diagnostic development Development of assay to quantify XMRV RNA from plasma Jan Serological XMRV testing initiated using NCI-produced CA antigen Sensitive XMRV indicator cell line established (DERSE) Feb Evaluation of XMRV antigens for serological testing Mar Large-scale production of XMRV virions as a source of antigen Apr XMRV expression clones delivered from NCI to NIH AIDS Reagent Repository

NCI XMRV Effort Builds on Strengths in Human Retrovirology Laboratory of Experimental Immunology Viral Technology Laboratory (SAIC) HIV Drug Resistance Program Protein Expression Laboratory (SAIC) AIDS and Cancer Virus Program XMRV Diagnosis Virus Production Antigen Preparation Serological DetectionNucleic Acid Detection XMRV Infectivity Assay XMRV Infectivity Assay

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 Crossing Point Log Concentration Cycles Fluorescence Amplification Curves Standard Curve Virus 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 Antigens PEL/SAIC-Frederick

Large-Scale XMRV Production ACVP, NCI-Frederick Purified XMRV Virions SDS/PAGE Immunological Analysis HPLC Fractionation  -MLV gp70  -MLV CA p30 Coomassie Stained MMMXXX

XMRV Antigen/Serological Assay Development - 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) XMRV CA ECL Units XMRV TM XMRV SU

XMRV Antigen/Serological Assay Development - 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 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 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 Fluorescence Microscopy

Comparative Evaluation of NCI XMRV Assays Nucleic Acid DNA Nucleic Acid RNA Serological Analysis ELISA Serological Analysis Western 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