1. National Cancer Institute Development of sero-assays to screen for XMRV antibodies in clinical samples Rachel K. Bagni, Ph.D. December 14, 2010

2. Outline Reagent development Availability of reagents Assay development strategy Preliminary observations Path forward

3. Development of reagents 9 XMRV gene products (VP62) ► gag (MAtrix, p12, NucleoCapsid, CApsid) ► pol (PRotease, Rev Transcriptase, INtegrase) ► env (Surface Unit, TransMembrane) Image courtesy of Stig Jensen, NCI SU TM RT MA p12 CA PR RNA NC IN

4. XMRV Reagent Development Clone Set-up 3 forms of sequence-verified Gateway Entry clones 4 types of protein expression clones Clones for protein secretion Recombinant Antigen Production Initial screening Small-scale production Final production

5. XMRV antigen results 5 μg each protein MA p12 CA NC PR RT Int SU TM 15.1 kDA 9.7 kDA 31.6 kDA 7.7 kDa 14.3 kDA 75.9 kDA 45.9 kDA 48.1 kDA 19.9 kDA 63.4 kDA

6. Cross-reactivity with antibodies directed against MLV proteins 20 kDa 30 kDa 40 kDa 50 kDa 120 kDa 100 kDa 60 kDa 80 kDa α-p30 S. Ruscetti 20 kDa 30 kDa 40 kDa 50 kDa 80 kDa 120 kDa 100 kDa 60 kDa CA RT α-RT M. Roth SU α-gp70 S. Ruscetti 20 kDa 30 kDa 40 kDa 50 kDa 80 kDa 120 kDa 100 kDa 60 kDa 120 kDa 100 kDa 80 kDa 60 kDa 50 kDa 40 kDa 30 kDa SU α-env (SFFV): S. Ruscetti

7. Reagents available… The following DNAs are deposited (64 clones): E. coli expression clones (His6 and His6-MBP) – 20 baculovirus clones (His6-MBP) – 10 baculovirus secreted clones – 2 analogous Entry (Gateway) clones – 32 NIH AIDS Research and Reference Reagent Program NCI CPTC: Antibody Characterization Laboratory https://www.aidsreagent.org

8. Serological Assay Platform: Meso Scale Discovery (MSD) Ruthenium (II) Sulfo-tris-bipyridine NHS ester Antigen Abs in serum Labeled 2° Electric Current

9. Qualification of XMRV recombinant antigens for use in sero-assays Unknown: prevalence of the virus in general population –Positive subjects in ‘normal’ donor population? Unknown: positive and negative samples Unknown: levels of antibodies in XMRV+ subjects

10. Limitations Preliminary assay characteristics calculated from a small sample number Assumption of sero-status Immune profile after infection unknown Requires validation using bona fide, pedigreed antibody clinical controls

11. Titration of SFFV Env MAb

12. Titration of anti-Capsid Ab (MLV)

13. Qualification of XMRV recombinant antigens for use in sero-assays Define ‘training set’ –77 donors NCI-Frederick RDP donor plasma (1990s, BBI Diagnostics) –39 XMRV+ subjects (WPI-CFS) Assay to XMRV antigens: –SU, TM, MA, CA, p12, NC, PR, RT and IN Use statistical analyses to determine utility of antigen in a screening assay

14. Receiver Operator Characteristic (ROC) Curves Sensitivity –proportion of patients with the virus that will be reactive on the test(s) Specificity –proportion of subjects without the virus that will be non-reactive on the test(s) True reactive rate (Sensitivity) is plotted as a function of the false reactive rate (100-Specificity).

15. ROC Curves: IN and RT INRT Area under ROC curve: 0.46 (0.35-0.57 ) Area under ROC curve: 0.49 (0.39-0.61)

16. ROC Curves: CA, TM and SU Area under ROC curve: 0.72 (0.63-0.81) Area under ROC curve: 0.86 (0.79-0.92) Area under ROC curve: 0.66 (0.56-0.75) CA SU TM

17. Training Set for Assay Development 2 2 3 1 2 Donors N = 77 (10/77) Subjects 10 1 4 5 N = 39 (34/39) Used to adjust criteria in the absence of pedigreed clinical controls. 4

18. Donor samples 1000 donor samples –500 NIH donors –500 CNMC donors Assayed for CA, TM and SU –SU re-testing planned Preliminary findings (raw non-calibrated data): NA 5.5%

19. Summary Multiple XMRV recombinant antigens have been used in sero-assay development Detect reactivity to CA, TM and/or SU Some subjects are reactive to p12, MA and NC Inclusion of antigens reactive in human sera into a ‘positivity algorithm’

20. Ongoing efforts Continued development efforts of sero- assays required Refinement of optimal cut-points Establish positivity algorithm Secondary assays: WB, NA tests Identification of pedigreed clinical controls Samples from experimentally infected animal models

21. Large Scale XMRV Production - ACVP Purified XMRV Virions SDS/PAGE Immunological Analysis HPLC Fractionation  -MLV gp70  -MLV CA p30 MMMXXX

22. Considerations Preliminary assay characteristics calculated from a small sample number Required: Analytical performance panels (antibody) Validation using bona fide, pedigreed antibody clinical controls

23. Acknowledgements Katie Beam William Burgan Vijaya Gowda Joe Huguelet Allison Meade Vanessa Wall - COG James Hartley Dominic Esposito – COG Troy Taylor – MEG Ralph Hopkins – EEG Bill Gillette – PPG SAIC-Molecular Detection Group SAIC-Protein Expression Lab NCI-CCR Bob Wiltrout Stuart Le Grice Frank Ruscetti Kathy Jones NIH Harvey Alter Whittemore-Peterson Institute Judy Mikovits SAIC-ACVP Jeff Lifson Denise Whitby Nazzarena Labo

24. Considerations Preliminary assay characteristics calculated from a small sample number Required: Analytical performance panels (antibody) Validation using bona fide, pedigreed antibody clinical controls