1. An Update on FDA/OBRR XMRV activities BPAC meeting, July 26, 2010 Indira Hewlett, Ph. D Chief, Laboratory of Molecular Virology DETTD/CBER/FDA

2. Current XMRV studies l Establish and develop highly sensitive and specific assays for XMRV, including PCR and immunoassays; evaluate assays using well characterized panels l Test samples from blood donors and HIV positive individuals from US and Africa (a geographically distinct region not yet studied) using these assays. l Develop FDA reference panels for future lot release of assays, if necessary l Study transfusion transmission of XMRV using appropriate rhesus macaque model l Study XMRV tropism, infectivity and pathogenesis.

3. PCR Primers for XMRV Detection 1 8185 nt538 598 nt1010 1148 nt4552 4572 4653 4673 nt5922 5942 6200 6273 gag pol env 352bp 612bp 413bp 419 1154 XMRV gag 1 st PCR XMRV gag 2 nd PCR 10 1 0.1 0.01 0 M 10 1 0.1 0.01 0 fg of XMRV DNA

4. qPCR: 2 x Master buffer12.5ul 25pmol/ul primer0.5 x 3 (4552F, 4653R, 4673R) Probe 100uM1ul for 9 reactions XMRV DNA 1ul H 2 O10ul Total25ul 50°C 2’, 95°C 10’ 95°C 15”, 60°C 60” 40cycles XMRV DNACt (copies/ml) 6.7 x 10 5 16.43 6.7 x 10 4 22.38 666725.99 667 29.90 66.7 33.74 6.737.06 0.7negative Real-time PCR for XMRV using plasmid DNA

5. 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.

6. Protocol for XMRV detection in plasma and cells l 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 1 st round of PCR, 35 cycles. — 2ul of 1 st PCR products for 2 nd PCR, 35 cycles. — 5ul of PCR products to run agarose gel. l 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.

7. M 1 2 3 4 5 6 7 8 9 10 (-) 2.0 1.6 1.0 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:1ul 10mM dNTPs1ul RNA8ul 10 x RT buffer2ul 25mM MgCl24ul 0.1M DTT2ul Rnase OUT1ul RT 200u/ul1ul 65C 5’  50C 50’ RNase H1ul 37C 20’ 1 st PCR: 2x Master buffer15ul 25pmol/ul GAG-O-F0.5ul 25pmol/ul GAG-O-R0.5ul XMRV cDNA2ul H 2 O12ul 94°C 5’ 94°C 30”, 55°C 30”, 72°C 45” 35cycles 72°C 7’ 2 nd PCR: 2x Master buffer15ul 25pmol/ul GAG-I-F0.5ul 25pmol/ul GAG-I-R0.5ul 1 ST PCR products2ul H 2 O12ul 94°C 5’ 94°C 30”, 55°C 30”, 72°C 45” 35cycles 72°C 7’

8. Detection of XMRV RNA/DNA and HIV-1 RNA in Blood Donors and HIV positive individuals from Africa SampleNo. tested PCR positive (%) HIV-1XMRV Blood donors (Cameroon) Plasma10554 (51)0 (0) PBMCs198 (42)0 (0) PBMC culture supernatants 5050 (100)0 (0) HIV patients ( Uganda) Plasma9449 (52)0 (0) Total268161 (60)0 (0)

9. Summary of Results and Future Studies l RT-PCR, qPCR assays for XMRV detection were established. Ongoing assay improvements for whole blood and plasma are underway. l 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. l Preliminary data show no evidence of detectable XMRV in the limited set of specimens from the select African countries in the study. l Additional blood donor PBMC and plasma, including US blood donor specimens are being tested for XMRV DNA and RNA respectively. l Testing of well pedigreed CFS patient samples is planned for the future.

10. FDA Lot release panel development efforts l FDA Lot release panels are used to establish standards for licensure of assays and post market surveillance of licensed assays. l 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 56 0 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.

11. Copy Number of 22Rv1 XMRV Pool Samples have copy numbers equivalent to 1 x10 10 copies/ml 1 st PCR 2 nd PCR -6 -7 -8 -9 -10 -11 -12 -13 -14 -C- C+ 22Rv1 Virus

12. FDA Lot Release Panel Development Efforts – con’t l XMRV NAT – DNA for whole blood testing l Aliquots of 22Rv1 and DU145 clone 7 cells will be sent to participating labs for copy number assignment based on consensus values. l Subsequently, cells will be spiked into whole blood at different copy numbers. l Frozen aliquots will be sent to labs for copy number determination. l Consensus values of cell copy numbers will be obtained prior to formulation.

13. Future Serologic Panel and Assay Development l Efforts are underway to obtain XMRV positive control specimens including human and/or animal sera for FDA panel development. l In-house serologic assays (EIA or Western blot) are under development using DU145 Clone 7 whole viral lysate. l Assays will be standardized using SFFV antisera and known XMRV positive human and animal sera. l 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.

14. Infectivity, tropism and transfusion transmission l Evaluate transfusion transmission using the rhesus macaque model; study viremia, viral kinetics, host responses, seroconversion, antibody profiles. l Evaluate secondary transmission by blood transfusion using blood collected during the pre-and post seroconversion period. l 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.

15. 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