Post on 02-Jan-2016
transcript
An Update on FDA/OBRR XMRV activities
BPAC meeting, July 26, 2010
Indira Hewlett, Ph. D
Chief, Laboratory of Molecular Virology
DETTD/CBER/FDA
Current XMRV studies Establish and develop highly sensitive and specific assays for
XMRV, including PCR and immunoassays; evaluate assays using well characterized panels
Test samples from blood donors and HIV positive individuals from US and Africa (a geographically distinct region not yet studied) using these assays.
Develop FDA reference panels for future lot release of assays, if necessary
Study transfusion transmission of XMRV using appropriate rhesus macaque model
Study XMRV tropism, infectivity and pathogenesis.
PCR Primers for XMRV Detection1 8185
nt538 598 nt1010 1148 nt4552 4572 4653 4673 nt5922 5942 6200 6273
gag pol env
352bp
612bp
413bp
419 1154
XMRV gag 1st PCR XMRV gag 2nd PCR
10 1 0.1 0.01 0 M 10 1 0.1 0.01 0 fg of XMRV DNA
qPCR:2 x Master buffer 12.5ul25pmol/ul primer 0.5 x 3 (4552F, 4653R, 4673R)Probe 100uM 1ul for 9 reactionsXMRV DNA 1ulH2O 10ulTotal 25ul
50°C 2’, 95°C 10’ 95°C 15”, 60°C 60” 40cycles
XMRV DNACt (copies/ml)
6.7 x 105 16.436.7 x 104 22.386667 25.99667 29.9066.7 33.746.7 37.060.7 negative
Real-time PCR for XMRV using plasmid DNA
Therefore, •one round of PCR and qPCR could achieve a detection limit around 10 copies of XMRV plasmid DNA per reaction
•while nested PCR could detect 1 copy of XMRV DNA under assay conditions.
Protocol for XMRV detection in plasma and cells
Plasma:— Extract RNA from 140ul of plasma with QIAamp Viral RNA kit.— 8ul of RNA for reverse transcription with random primer.— 5ul of cDNA for 1st round of PCR, 35 cycles.— 2ul of 1st PCR products for 2nd PCR, 35 cycles.— 5ul of PCR products to run agarose gel.
PBMC:— Cells extracted with QIAamp DNA blood Mini kit.— 0.5 ug used for PCR amplification.— Nested PCR using gag primers.— PCR products analyzed on agarose gel.
M 1 2 3 4 5 6 7 8 9 10 (-)
2.01.61.0
0.5
XMRV testing of plasma from HIV +ve and –ve blood donors from Cameroon
XMRV(gag)
HIV-1(gp41)
RT:Random primer 5ng/ml: 1ul10mM dNTPs 1ulRNA 8ul10 x RT buffer2ul25mM MgCl2 4ul0.1M DTT 2ulRnase OUT 1ulRT 200u/ul 1ul65C 5’ 50C 50’RNase H 1ul37C 20’
1st PCR:2x Master buffer 15ul25pmol/ul GAG-O-F 0.5ul25pmol/ul GAG-O-R 0.5ulXMRV cDNA 2ulH2O 12ul
94°C 5’ 94°C 30”, 55°C 30”, 72°C 45” 35cycles72°C 7’
2nd PCR:2x Master buffer 15ul25pmol/ul GAG-I-F 0.5ul25pmol/ul GAG-I-R 0.5ul1ST PCR products 2ulH2O 12ul
94°C 5’ 94°C 30”, 55°C 30”, 72°C 45” 35cycles72°C 7’
Detection of XMRV RNA/DNA and HIV-1 RNAin Blood Donors and HIV positive individuals from Africa
Sample No.tested
PCR positive (%)
HIV-1 XMRV
Blood donors (Cameroon)
Plasma 105 54 (51) 0 (0)
PBMCs 19 8 (42) 0 (0)
PBMC culture supernatants
50 50 (100) 0 (0)
HIV patients ( Uganda)
Plasma 94 49 (52) 0 (0)
Total 268 161 (60) 0 (0)
Summary of Results and Future Studies
RT-PCR, qPCR assays for XMRV detection were established. Ongoing assay improvements for whole blood and plasma are underway.
Using our current assay, XMRV was not detected in a total of 268 samples of plasma or PBMC and PBMC cell supernatants from blood donors in Cameroon, and HIV positive patients in Uganda.
Preliminary data show no evidence of detectable XMRV in the limited set of specimens from the select African countries in the study.
Additional blood donor PBMC and plasma, including US blood donor specimens are being tested for XMRV DNA and RNA respectively.
Testing of well pedigreed CFS patient samples is planned for the future.
FDA Lot release panel development efforts FDA Lot release panels are used to establish standards for licensure of
assays and post market surveillance of licensed assays. XMRV NAT - RNA for plasma testing
— Culture supernatant was prepared from 22Rv-1 cells or DU145 Clone 7 cells
– viral RNA copy numbers estimated using PCR assays targeting the gag region. — Virus stocks were heat inactivated at 560 C for 60 min;
– No infectious virus detected – no significant effect on copy numbers
— Titered, inactivated culture supernatants:– RNA copy number determination by multi-lab
– Copy number value assigned based on consensus.
— Panel will consist of varying copy numbers of virus stocks spiked into negative plasma.
Copy Number of 22Rv1 XMRV Pool
Samples have copy numbers equivalent to 1 x1010 copies/ml
1st P
CR
2nd P
CR
-6 -7 -8 -9 -10 -11 -12 -13 -14 -C- C+
22R
v1 V
iru
s
FDA Lot Release Panel Development Efforts – con’t
XMRV NAT – DNA for whole blood testing Aliquots of 22Rv1 and DU145 clone 7 cells will be sent to
participating labs for copy number assignment based on consensus values.
Subsequently, cells will be spiked into whole blood at different copy numbers.
Frozen aliquots will be sent to labs for copy number determination.
Consensus values of cell copy numbers will be obtained prior to formulation.
Future Serologic Panel and Assay Development
Efforts are underway to obtain XMRV positive control specimens including human and/or animal sera for FDA panel development.
In-house serologic assays (EIA or Western blot) are under development using DU145 Clone 7 whole viral lysate.
Assays will be standardized using SFFV antisera and known XMRV positive human and animal sera.
Serologic assays will be used to characterize materials for future FDA serology panels, if needed and evaluate immune responses, seroconversion in future rhesus macaque infectivity studies.
Infectivity, tropism and transfusion transmission
Evaluate transfusion transmission using the rhesus macaque model; study viremia, viral kinetics, host responses, seroconversion, antibody profiles.
Evaluate secondary transmission by blood transfusion using blood collected during the pre-and post seroconversion period.
Evaluate XMRV infectivity for cells of lymphoid, epithelial origin; study host factors and signaling pathways to identify biomarkers of infection by genomics and proteomics based approaches.
AcknowledgmentsCBER/FDA
Shixing Tang
Jiangqin Zhao
Krishnakumar Devadas
Ragupathy Viswanath
Owen Wood
Sherwin Lee
Phil Snoy
Joel Beren
Steve Kerby
Chintamani Atreya
Hira Nakhasi
Other Collaborators
Robert Silverman, Cleveland Clinic
Jaydeep Das, Cleveland Clinic
Kathryn Jones, NCI
Francis Ruscetti, NCI
Phillipe Nyambi, NYU
Thomas Quinn, JHU
Andrew Redd, JHU
Blood XMRV Scientific Working Group