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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.

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an update on fda obrr xmrv activities

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
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.
slide3

1 8185

gag pol env

419 1154

nt538 598 nt1010 1148 nt4552 4572 4653 4673 nt5922 5942 6200 6273

612bp

413bp

352bp

PCR Primers for XMRV Detection

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

slide4

Real-time PCR for XMRV using plasmid DNA

  • XMRV DNA Ct
  • (copies/ml)
  • 6.7 x 105 16.43
  • 6.7 x 104 22.38
  • 6667 25.99
  • 667 29.90
  • 66.7 33.74
  • 6.7 37.06
  • 0.7 negative

qPCR:

2 x Master buffer 12.5ul

25pmol/ul primer 0.5 x 3 (4552F, 4653R, 4673R)

Probe 100uM 1ul for 9 reactions

XMRV DNA 1ul

H2O 10ul

Total 25ul

50°C 2’, 95°C 10’

95°C 15”, 60°C 60” 40cycles

slide5

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
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.
slide7

XMRV testing of plasma from HIV +ve and –ve blood donors from Cameroon

M 1 2 3 4 5 6 7 8 9 10 (-)

2.0

1.6

1.0

0.5

XMRV

(gag)

HIV-1

(gp41)

RT:

Random primer 5ng/ml: 1ul

10mM dNTPs 1ul

RNA 8ul

10 x RT buffer 2ul

25mM MgCl2 4ul

0.1M DTT 2ul

Rnase OUT 1ul

RT 200u/ul 1ul

65C 5’  50C 50’

RNase H 1ul

37C 20’

1st PCR:

2x Master buffer 15ul

25pmol/ul GAG-O-F 0.5ul

25pmol/ul GAG-O-R 0.5ul

XMRV cDNA 2ul

H2O 12ul

94°C 5’

94°C 30”, 55°C 30”, 72°C 45” 35cycles

72°C 7’

2nd PCR:

2x Master buffer 15ul

25pmol/ul GAG-I-F 0.5ul

25pmol/ul GAG-I-R 0.5ul

1ST PCR products 2ul

H2O 12ul

94°C 5’

94°C 30”, 55°C 30”, 72°C 45” 35cycles

72°C 7’

slide8

Detection of XMRV RNA/DNA and HIV-1 RNA

in Blood Donors and HIV positive individuals from Africa

summary of results and future studies
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.

FDA Lot release panel development efforts
copy number of 22rv1 xmrv pool

-6 -7 -8 -9 -10 -11 -12 -13 -14 -C- C+

1st PCR 2nd PCR

22Rv1 Virus

Copy Number of 22Rv1 XMRV Pool

Samples have copy numbers equivalent to 1 x1010 copies/ml

fda lot release panel development efforts con t
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
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
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.

acknowledgments
Acknowledgments

CBER/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