Development of sero -assays to screen for XMRV antibodies in clinical samples

1. Development of sero-assays to screen forXMRV 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) SU TM p12 CA NC PR RT RNA MA IN Image courtesy of Stig Jensen, NCI

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

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

7. Reagents available… NIH AIDS Research and Reference Reagent Program https://www.aidsreagent.org 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 NCI CPTC: Antibody Characterization Laboratory

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

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 EnvMAb

12. Titration of anti-CapsidAb (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-reactiveon 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 RT IN 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 CA TM Area under ROC curve: 0.66 (0.56-0.75) Area under ROC curve: 0.72 (0.63-0.81) SU Area under ROC curve: 0.86 (0.79-0.92)

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

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 HPLC Fractionation SDS/PAGE Immunological Analysis X M M X M X a-MLV CA p30 a-MLV gp70

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

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