1. Update on Assay Development George J. Dawson, Ph.D. Infectious Diseases: Core R & D Abbott Laboratories West Nile Virus

2. West Nile Virus Strategies Determine marker profile The overlap between RNA, antigen, and IgM detection Markers associated with acute, symptomatic infection Develop prototype IgM class antibody test for detection of antibodies to WNV Recombinant proteins have been obtained from two laboratories (Material Transfer Agreement’s executed) EIA’s are being evaluated for IgM detection Primer pairs have been selected for in-house RT-PCR Evaluate utility of IgM test Screening blood donors Reinstatement of WNV RNA positive donors Diagnosis of acute infection

3. Derived from core, preM regions 5' 3' Structural Nonstructural CprME12A 2B34A4B5 423924 2427 348341764569 642668737638 10353 55 501aa 228 899 E prM C Degenerate primers Degenerate primers selected for PCR studies West Nile specific primers Primer pairs were selected for in-house studies to determine overlap between viremia and IgM West Nile Virus PCR Primers

4. 5' 3' StructuralNonstructural CprME12A 2B34A4B5 423924 2427 348341764569 642668737638 10353 55 501aa. Protein 1: Envelope gene (truncated) expressed in eukaryotic cells E E prM Signal Sequence Protein 2: PreM/M/Env expressed in eukaryotic cells as vesicles and secreted Signal Sequence West Nile Virus Strain HNY1999

5. WNV Ag coated solid phase Serum Added Indirect Assay – WNV IgM/IgG Test Protein 1 Anti-IgG Conjugate Added Anti-IgM Conjugate Added IgM Format IgG Format Anti-gamma added to specimen diluent in IgM format

6. Anti-IgM Coated Solid Phase Serum Added IgM Capture EIA Protein 2 Antigen Added Conjugate Added

7. IgM and IgG assays were developed with protein 1 and 2; WNV RNA testing was performed on repeatably reactive human specimens and on all monkey sera West Nile Virus Serologic Studies Sample CategoryNumber of Specimens Volunteer blood donors241 Confirmed positive specimens: IgM only IgM/IgG positives 8989 Paired human samples12 pairs CDC Proficiency Panel20 Experimentally Infected Rhesus Monkeys study in progress 5 animals

8. West Nile Virus Serologic Studies Rationale for Assay Format Selection(s) The original data demonstrating the utility of Protein 1 for WNV diagnostics utilized an indirect assay format Data generated in-house for Protein 1 with an IgM Capture format was not adequate The original data demonstrating the utility of Protein 2 for WNV diagnostics utilized an IgM Capture assay format In-house studies indicated that performance of Protein 2 in an indirect assay format was not adequate Additional studies are planned

9. 7 Repeat Reactives All were RNA Negative and Negative by IgM Capture EIA Utilizing Protein 2 Indirect IgM Assay Utilizing Protein 1

10. Volunteer Blood Donors (N=241 ) 37 182 10 5 1 222 0 20 40 60 80 100 120 140 160 180 200 0.51.01.52.02.5 3.03.5 4.04.55.05.56.06.57.07.58.08.59.09.5 10.0 S/N Value Frequency Provisional Cutoff at : 7SD + population mean No repeatably reactive specimens were noted IgM Capture of WNV Antibodies in Serum Samples

11. Cutoff. Note: All 8 specimens reactive using protein 2; only 5 specimens were reactive with EIA utilizing protein 1 Confirmed IgM positive and IgG positive IgM Detection of WNV Antibodies in Serum Samples

12. Confirmed IgM positive and IgG negative Cutoff Note: All 9 specimens reactive using protein 2; only 3 specimens were reactive with EIA utilizing protein 1. Sample 11 was WNV RNA positive IgM Detection of WNV Antibodies in Serum Samples

13. Indirect Assay using Protein 1 11 21 7 11 9 57 5 19 2 62 2 43 6 24 7 26 6 20 50 3 21 12 55 Cutoff The first sample from 5 of 12 patients was reactive in the IgM Indirect EIA using protein 1 5 of 12 follow-up bleed samples were reactive IgM Detection of WNV Antibodies in Paired Serum Samples Numbers at the top of the columns indicate days post onset of symptoms

14. Paired Specimens The first sample from all 12 patients was reactive in the IgM Capture EIA using protein 2 11 of 12 follow-up bleed samples were reactive IgM Capture EIA using Protein 2 0 10 20 30 40 50 60 70 Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Patient 9 Patient 10 Patient 11 Patient 12 S/N 11 21 7119 57 5 19 2 2 43 6 24 7 26 6 20 50 3 21 12 55 62 Cutoff IgM Detection of WNV Antibodies in Paired Serum Samples Numbers at the top of the columns indicate days post onset of symptoms Bleed 1 Bleed 2

15. Note: Abbott’s IgM Capture EIA results using Protein 2 on the CDC Proficiency Panel generated equivalent or greater S/N values than those provided by CDC. IgG Reactivity + + + - + + - + + + + + + I + - + + I - IgM Capture EIA using Protein 2 "N" denotes known negative specimens "I" denotes indeterminate IgG only unconfirmed

16. SUMMARY Protein 2 detected more acute phase WNV samples than Protein 1 Data suggest that conformational epitopes present in Protein 2 are important for early IgM detection The cell line expressing Protein 2 is being scaled-up in-house Preliminary evaluation of IgM testing indicates: For blood screening – additional studies are required The IgM response was robust in most symptomatic individuals IgM in conjunction with IgG could be part of the donor reinstatement algorithm For diagnostics, IgM is the preferred method of detection Prototype assay formats are being investigated EIA’s on polystyrene beads Microparticle based assays on AxSYM and IMx

17. Studies Planned or In Progress Optimization of assay Incubation times, sample volume Concentration of rare reagents, washing steps Processing steps for rare reagents Confirmatory strategies RT-PCR on early phase specimens IgG test with Protein 2 IgM and IgG tests with alternate protein Plaque reduction neutralization tests ( in collaboration with CDC or state labs) External Studies Serologic studies on quarantined units from 2002 epidemic Donor reinstatement studies as companion to WNV NAT efforts Testing at state and/or hospital laboratories during 2003 WNV season Additional proteins are being expressed in-house to determine their potential utility