xenotropic murine leukemia virus-related retrovirus: ccr assay development
TRANSCRIPT
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