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

Transcription

1 National Cancer Institute Development of sero-assays to screen for XMRV antibodies in clinical samples Rachel K. Bagni, Ph.D. September 8, 2010

2 Xentropic murine leukaemia virus-related virus - XMRV The short course:» Gammaretrovirus» Linked to prostate cancer and chronic fatigue syndrome» Detected in controls» Possibility of association with other cancers» Conflicting results

3 Development of reagents 9 XMRV gene products (VP62) gag (MA, p12, NC, CA) pol (PR, RT, IN) env (SU, TM) Multiple clones Purify First strategy Performance in assay

4 XMRV clone setup 3 forms of sequence-verified Gateway Entry clones RBS-His6-ORF (for E. coli expression) tev-orf (for N-terminal fusions in any host, cleavable with TEV protease) Kozak-ORF-nostop (for C-terminal fusions in eukaryotic hosts) 4 types of protein Expression clones RBS-His6-ORF (E. coli) His6-MBP-tev-ORF (E. coli, solubility tag can be removed) His6-MBP-tev-ORF (baculovirus, solubility tag can be removed) His6-eGFP-tev-ORF (mammalian, expression monitored by fluorescence)\ Clones for protein secretion SU (native signal sequence) SU (honeybee mellitin signal sequence for insect cells) Env (native signal sequence) Env (honeybee mellitin signal sequence for insect cells)

5 XMRV protein production Initial Screening E. coli, baculovirus, mammalian small-scale microscale purification screening some clones may produce enough material for initial assay development Small-scale production choose best host for each protein scale-up expression optimize purification (native vs inclusion bodies for E. coli) goal is to produce 1-2 mg of each protein for assay development Final production identify optimal targets for serological assay scale-up expression/purification to make mg of each protein verify reproducibility of expression/purification protocols

6 XMRV antigen scale-up results MA p12 CA NC PR RT Int SU TM 75.9 kda 45.9 kda 48.1 kda 63.4 kda 31.6 kda 15.1 kda 9.7 kda 14.3 kda 7.7 kda 19.9 kda 5 μg each protein

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

8 Cross-reactivity with an antibody directed against anti-xeno MLV MA p12 CA NC PR RT IN SU TM SU 220 kda 120 kda 100 kda 80 kda 60 kda 50 kda 40 kda 30 kda 32 kda 220 kda 120 kda 100 kda 80 kda 60 kda 50 kda 40 kda 30 kda α-xeno: S. Ruscetti

9 Reagents available NIH AIDS Research and Reference Reagent Program 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

10 Serological Assay Platform: Meso Scale Discovery (MSD) Ruthenium (II) Sulfo-tris-bipyridine NHS ester

11 Development of XMRV serological 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

12 Strategy 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

13 Limitations Cut-offs established from one possible disease Assumption of sero-status Immune profile after infection unknown Requires validation using a bona fide, pedigreed antibody positive clinical control

14 Receiver Operator Characteristic 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 IN RT Area under ROC curve: 0.46 ( ) Area under ROC curve: 0.49 ( )

16 ROC Curves: CA, TM and SU CA TM Area under ROC curve: 0.66 ( ) SU Area under ROC curve: 0.72 ( ) Area under ROC curve: 0.86 ( )

17 Detection Rates Training Set Subjects ( pos ) Donors ( neg ) N = 39 (34/39) N = 77 (10/77)

18 Lo and Alter PNAS Study SU SU ECL Units Nucleic Acid: Not Tested Nucleic Acid: Detected

19 Summary ELISA-based assays to multiple XMRV antigens distinguish between donors and subjects Reactivity with SU, TM and/or CA Some subjects are reactive to p12, MA and NC Inclusion of antigens reactive in human sera into a positivity algorithm

20 Ongoing efforts Refinement of optimal cut-points Establish positivity algorithm Confirmatory assays: WB, NA tests Transmission Lymphoma, prostate, other solid tumor and CFS cohorts

21 SAIC-Protein Expression Lab James Hartley Dominic Esposito COG Troy Taylor MEG Ralph Hopkins EEG Bill Gillette PPG SAIC-Molecular Detection Group Katie Beam William Burgan Vijaya Gowda Joe Huguelet Allison Meade Cass Peluso Vanessa Wall - COG Acknowledgements NCI-CCR Bob Wiltrout Stuart Le Grice Frank Ruscetti Kathy Jones NIH Harvey Alter FDA Shyh-Ching Lo Cornell University Maureen Hanson SAIC-ACVP Denise Whitby Nazzarena Labo Whittemore-Peterson Institute Judy Mikovits