1. HIV-1/HIV-2 PLUS O To detect HIV Antibody Groups M & O Genetic Systems™ HIV-1/HIV-2 PLUS O EIA   HIV Subtypes and Variants   Description of HIV-1/HIV-2 PLUS O EIA Format, components, QC criteria Detection of HIV-1 (M & O), and HIV-2 Detection of Seroconversions Specificity   Confirmation

2. HIV Subtypes and Variants B ABDABDBCEBCE B C C BCFBCF F ABCDABCD DEDE Group O ? BEBE HIV-1: ABDEFGHIJ…O HIV-2: ABCDEFG DHDH G BOBO HIV-1 Subtype B is predominant in the U.S., but non-B subtypes are found in 2% of HIV-Positive U.S. blood donors, and increasing. (Delwart et al., AIDS Research and Human Retroviruses 19:1065-1070, 2003.) HIV-2 HIV-1

3. HIV Subtypes and Variants Most Problematic for Antibody Detection  HIV-2: HIV-2 testing mandated for blood screening June 1, 1992. 1 st : Genetic Systems™ HIV-1/HIV-2 EIA (viral lysate) 2 nd : Genetic Systems™ HIV-1/HIV-2 Peptide EIA  HIV-1 Group O: FDA asked U.S. manufacturers to modify HIV kits (7/31/96 and 7/30/97). 3 rd : Genetic Systems™ HIV-1/HIV-2 PLUS O EIA licensed 8/5/03

4. Principle of the Test: Direct Antibody Sandwich Immunoglobulins from the sample bind simultaneously to antigens (4 total) on the microplate and to similar HRP-conjugated antigens (5 total) in solution. HRP IgG Rec. gp160 (HIV-1 M) Pep. env (HIV-2) Pep env (HIV-1 O) 4 Plate Antigens Rec. p24 (HIV-1 M) 5 HRP-Antigen Conjugates 2 HIV-1 M peptides HIV-1 M rec. p24 HIV-1 O peptide HIV-2 peptide

5. Principle of the Test: Direct Antibody Sandwich Binding of IgM to the microplate is stabilized by multiple attachments, and the signal is amplified by multiple HRP-Antigen Conjugate binding sites. HRP HRP HRPHRP HRP HRP IgM 5 HRP-Antigen Conjugates 2 HIV-1 M peptides HIV-1 M rec. p24 HIV-1 O peptide HIV-2 peptide Rec. gp160 (HIV-1 M) Pep. env (HIV-2) Pep env (HIV-1 O) 4 Plate Antigens Rec. p24 (HIV-1 M)

6. Earlier IgM Detection…a Benefit of the Antibody Sandwich Format Seroconversion Panel PRB 940 Antibody Sandwich: HIV-1/HIV-2 PLUS O EIA Abbott HIVAb HIV-1/HIV-2 Indirect Antibody: HIV-1/HIV-2 Peptide EIA Cutoff ~Day 8 ~Day 14

7. Kit Components Three kit sizes: 480/960/4800 Tests Direct Antibody Sandwich Format Unique Kit Components: -R1Microwell Strip Plates (8X12) -R3Specimen Diluent -C0 Negative Control -C1 HIV-1 Positive Control -C2 HIV-2 Positive Control -C3 HIV-1 Group O Positive Control -R4Conjugate Concentrate (11X) -R5Conjugate Diluent “Shared” Components: 30X Wash (R2), Substrate Buffer (R8), TMB Chromogen 11X (R9), Stopping Solution (R10)

8. Procedure (60’ 30’ 30’) Add 25µl Specimen Diluent + 75µl Control or Sample to each well.* Cover and incubate 60 +/- 5 min. at 37 +/- 2ºC. Wash a minimum of 5 times with 30-60 second soaks. Add 100 µl Working Conjugate to each well.* Cover and incubate 30 +/- 5 min. at 37 +/- 2ºC. Wash a minimum of 5 times with 30-60 second soaks. Add 100 µl Working TMB to each well. Cover and incubate 30 +/- 5 min. at RT. Add 100 µl Stopping Solution to each well. Read within 30 minutes at 450nm, with the 615-630nm filter as a reference. *Optional O.D. readings may be taken to verify addition of specimen or reagent. Color indicates differences in the procedure from HIV-1/HIV-2 Peptide EIA.

9. Procedure Monitoring  Sample Dispensing  Conjugate Dispensing  T M B  Stopping solution Before After After incubation of a positive sample Sample OD 630 > 0.150 Conjugate OD 630 > 0.100 OptionalVerification

10. Quality Control: Validation of Results Kit Controls (6 wells total): C0 Negative Control (3 wells) Each value A 450 =0.001-0.150 (one may be discarded) C1 HIV-1 Positive Control (1 well) A 450 >0.700 C2 HIV-2 Positive Control (1 well) A 450 >0.700 C3 HIV-1 Group O Positive Control (1 well) A 450 >0.700 Cutoff = xNC + 0.250 Color indicates differences in the procedure from the HIV-1/HIV-2 Peptide EIA.

11. Performance Results: HIV-1 Group M Sensitivity Reactivity in HIV-1 Known Positive Samples Results Obtained with Genetic Systems™ HIV-1/HIV-2 PLUS O EIA Licensed HIV-1/HIV-2 EIA GroupNumber Repeatedly Reactive AIDS (N=313)313 (100.00%) 313 (100.00%) Known HIV-1 Positive, U.S. (N=490) 490 (100.00%) 490 (100.00%) Known HIV-1 Positive, Non-U.S. (N=199*) 199 (100.00%) 199 (100.00%) TOTAL:1002 (100.00%) 1002 (100.00%) * Australia, New S. Wales (N=36) Central African Republic (N=40) Ghana (N=5) Kenya (N=3) Nigeria (N=46) Sierra Leone (N=40) Thailand (N=21) Zimbabwe (N=8)

12. Performance Results: HIV Variant/Low Titer Samples BBI Performance Panels (N=130 Positives) Mixed Titer PRB203 (N=23 Positives) Low Titer PRB105 (N=14 Positives) African HIV Series AfrRB1 (N=46 Positives) Worldwide WWRB301 (N=47 Positives) HIV-1/HIV-2 Peptide EIA 124/130 (95.4%) Abbott HIVAb HIV-1/HIV-2 128/130 (98.5%) Genetic Systems™ HIV-1/HIV-2 PLUS O EIA 130/130 (100.0%)

13. Performance Results: HIV-2 Sensitivity Results Obtained from Known Positive HIV-2 Samples with Genetic Systems™ HIV-1/HIV-2 PLUS O EIA Number TestedInitially ReactiveRepeatedly Reactive Pos. by HIV-2 Western blot * 302 (100.00%) 302 (100.00%) 302 (100.00%) * *HIV-2 samples were repeatedly reactive on an HIV-2 EIA, positive on an HIV-2 Western blot, and indeterminate or negative on an HIV-1 Western blot.

14. Performance Results: HIV-1 Group O Sensitivity Results Obtained from Known Positive HIV-1 Group O Samples with Genetic Systems™ HIV-1/HIV-2 PLUS O EIA Number TestedInitially ReactiveRepeatedly Reactive 77*77 (100.00%) 76** (100.00%) *Known HIV-1 Group O samples were obtained from individuals living in Cameroon (N=70), the United States (N=2), Spain (N=2) and France (N=3). **One initially reactive Group O specimen was not available in sufficient volume for repeat testing.

15. Performance Results: Seroconversions Reactivity with HIV-1 on 50 Commercial Seroconversion Panels HIV-1/HIV-2 PLUS O Equivalent HIV-1/HIV-2 PLUS O More Sensitive HIV-1/HIV-2 PLUS O Less Sensitive vs. HIV-1/HIV-2 Peptide EIA 12/46* (26%) 34/46* (74%) 0 (0%) vs. Abbott HIVAb HIV-1/HIV-2 35/50 (70%) 9/50 (18%) 6/50 (12%) vs. Licensed Western Blot 13/50 (26%) 37/50 (74%) 0 (0%) Four of the 50 seroconversion panels did not have test results with the licensed HIV-1/HIV-2 Peptide EIA and are no longer available for testing.

16. Performance Results: Seroconversions HIV-1/HIV-2 PLUS O: 21 Days Earlier cutoff

17. Performance Results: U.S. Blood Donors Results Obtained from Random Donors tested with Genetic Systems™ HIV-1/HIV-2 PLUS O EIA Sample Type Number TestedNon-Reactive Initially Reactive Repeatedly Reactive HIV-2 EIA Repeatedly Reactive Pos. by HIV-1 Western blot alone Serum (Sites 1, 2) 6103 (100.00%) 6097 (99.90%) 6 (0.10%) 6 (0.10%) 00 Plasma (Sites 2, 3) 5056 (100.00%) 5044 (99.76%) 12 (0.24%) 6 (0.12%) 00 TOTAL11,159 (100.00%) 11,141 (99.84%) 18 (0.16%) 12 (0.11%) 00 Specificity 99.89% (95% confidence interval 99.83-99.96)

18. Subtypes/Variants Tested by HIV-1 Western Blot Worldwide HIV Panel WWRB302 (members 1-13) All HIV+ samples in the panel (N=28) exhibited A450/A630>3.000 in testing with Genetic Systems™ HIV-1/HIV-2 PLUS O EIA GHIV-2GANegHIV-2CA PCLPC NC NC Group 0 AGGA

19. SUMMARY  Dissemination of HIV-1 non-B subtypes and variants is a growing concern in the U.S.  Reliable detection of HIV-1 Group O or HIV-2 antibody requires the use of specific antigens in the test kit.  HIV-1/HIV-2 PLUS O EIA performance was demonstrated with human serum, plasma, and cadaveric serum samples.  100% detection of known HIV-1 (N=1002), HIV-2 (N=302), and HIV-1 group O (N=77) specimens.  100% detection of HIV-1 low titer samples and HIV subtypes of worldwide origin (N=130) from four panels.  Detection of HIV-1 seroconversion panels (N=50) better than licensed competitors.  Specificity 99.89% (95% C.I. 99.83-99.96) in blood and plasma donors at three sites (N=11,159).  Current confirmatory algorithms will detect most HIV-1 group O samples; unique banding may assist identification.