Vironostika® HIV-1 Plus O Microelisa System Main Features Assay configuration and procedures for S/P and DBS Solid Phase Antigens Comparison to the current licensed Vironostika® HIV-1

Main Features Vironostika® HIV-1 Plus O Mircoelisa System is an ELISA for the qualitative detection of antibodies to HIV-1 including Group O, in human specimens collected as serum, plasma, or dried blood spots on filter paper. It is not intended for use in screening blood donors. Solid phase contains HIV-1 viral lysate, viral envelope and Group O peptide. The system contains Sample Addition Monitoring, Color Coded Reagents, liquid controls and Substrate, and an improved ease of use. The assay is designed to improve the overall sensitivity while maintaining the specificity as compared to the current licensed Vironostika® HIV-1.

Kit Components

Overview of Vironostika® HIV-1 Plus O Microelisa System WASH WASH WASH WASH STOP STOP Solid phase: Solid phase: Anti - HIV in Sample Goat anti - human Ig /HRP conjugate Chromogenic substrate (ABTS) NaF substrate (ABTS) Viral Lysate Viral env Group O Peptide Sample diluent contains sample addition monitor Incubation 37°C One-step preparation Incubation 37°C Ready to use format Incubation 15-30°C Read (405nm)

Assay Procedures for Serum/Plasma (S/P) and Dried Blood Spots (DBS) 120 min, 37°C 60 min, 37°C S/P 10µl SAMPLE 200µl DILSIM III (1:21) Conj 150µl Wash READ At 405 nm 10-13 min, RT DBS Elute with 150µl of DBS Elution Medium 25µl ELUTE 125µl DILSIM III (1:6) [S] STOP

Sample Addition Monitoring

Solid Phase Antigen HIV-1 Viral Lysate Prepared from H9/HIV-1IIIB Same viral lysate as in the current Vironostika HIV-1 Group O peptide RRETLLQNQQLLSLWGCKGKLVCYT Synthetic peptide of 23 amino acids with Arg-Arg added to the N-terminal The sequence derived from ANT70 gp41 regions (Journal of Virology, 1990,1207-1216) Conjugated to BSA for plate coating Native HIV-1 gp160

Development of Native HIV-1 gp160 Advanced Bioscience Laboratories developed a cell culture system that secreted gp160 which contained the gp41 moiety (minus a small truncation at the C-terminus). The native gp160 is highly soluble and exists with carbohydrate moiety. Kalyanaraman et al., AIDS Res. Hum. Retroviruses, 1990; Kalyanaraman et al., Virology, 1990. Shown to be an excellent diagnostic antigen. Nair et al., J. Cln. Microbiol., 1994

Immunodominant Domains in HIV-1 env Proteins

Serum EIA Reactivity of HIV-1 gp160 10 100 1000 False Positive Very Weak Positive Medium Positive 1 10 100 1000 False Postive Very Weak Positive Medium Positive 1.2 1.2 1.0 1.0 0.8 0.8 450 450 0.6 0.6 A A 0.4 0.4 0.2 0.2 0.0 0.0 1000 100 10 1 1000 100 10 1 gp160 ( gp160 ( ng ng /well) /well)

The Vironostika® HIV-1 Plus O Trial Summary 18 geographically diverse sites participated. 4 kit lots evaluated. 36,000+ tests performed (Serum/Plasma + DBS). Studies included: Reproducibility Sensitivity Known HIV-1 positive samples High risk populations Seroconversion panels Dilution panels Specificity Low risk populations Samples containing potentially interfering substances

Specificity in Low Specificity in Low - - Risk Populations (n=6019) 1000 2000 3000 4000 5000 < . 1 - 9 2 3 4 5 6 7 8 > = FREQUENCY SIGNAL TO CUTOFF RATIO HIV 1 Plus O VirHIV

Known HIV-1 Specimens DBS HIV-1 Clade A – 100% (10/10) Serum/Plasma HIV-1 Clade B – 100% (12/12) HIV-1 Clade B/D – 100% (1/1) HIV-1 Clade C – 100% (10/10) HIV-1 Clade C/E – 100% (1/1) HIV-1 Clade D – 100% (10/10) HIV-1 Clade E – 100% (7/7) HIV-1 Clade E/A – 100% (3/3) HIV-1 Clade E/C – 100% (1/1) HIV-1 Clade E/F – 100% (1/1) HIV-1 Clade F – 100% (10/10) HIV-1 Clade G – 100% (4/4) HIV-1 Clade H – 100% (2/2) HIV-2 – 100% (20/20) HIV-O – 100% (11/11) Serum/Plasma HIV-1 Clade A – 100% (10/10) HIV-1 Clade B – 100% (12/12) HIV-1 Clade B/D – 100% (1/1) HIV-1 Clade C – 100% (10/10) HIV-1 Clade C/E – 100% (1/1) HIV-1 Clade D – 100% (10/10) HIV-1 Clade E – 100% (7/7) HIV-1 Clade E/A – 100% (3/3) HIV-1 Clade E/C – 100% (1/1) HIV-1 Clade E/F – 100% (1/1) HIV-1 Clade F – 100% (10/10) HIV-1 Clade G – 100% (4/4) HIV-1 Clade H – 100% (2/2) HIV-2 – 100% (20/20) HIV-O – 100% (11/11)

Sensitivity All serum/plasma specimens and dried blood spot specimens were repeatedly reactive with Vironostika® HIV-1 Plus O Mircoelisa System. Therefore, the sensitivity for both specimen types in this study was 100% (95% CI: 99.64 – 100%).

High-Risk Populations Prison Inmates, STD, Hosp ER Pts, and HIV Outreach Clinic Pts The specificity of the Vironostika® HIV-1 Plus O assay in this study of high-risk populations was calculated to be 1,392/1,398 = 99.57% (95% CI = 99.07% - 99.84%). Specimen Type Population Number of specimens Initially Reactive Repeatedly Western Blot Positive 1 251 16 14 8 2 513 13 3 500 73 68 4 250 28 27 Serum or Plasma Total 1,514 130 122 116 N/A Dried Blood Spots 750 95

Seroconversion Panel Testing Panel ID Sample Collection Days Vironostika HIV-1 Plus O Comparative Test1 924 8, 10, 26, 33, 35, 40 33 40 927 0, 28, 33, 35, 40 931 9, 15, 28, 33, 35, 42 28 932 0, 3, 13, 27, 34, 50, 78, 163, 194 34 NR2 940 0, 7, 11, 15, 18, 22, 25, 29 15 22 071 1, 3, 17, 22, 28 17 111 1, 2, 8, 16, 20, 22, 27 8 16 241 1, 7, 9, 15, 17, 22, 24 321 1, 8, 12, 15, 21 21 341 1, 7, 10, 21, 23, 28 351 1, 8, 11, 15 NR 361 1, 3, 9, 11, 16, 18 18 1The licensed Vironostika HIV-1 Microelisa System 2None of the specimens in this panel was reactive

Reactivity of H&L vs  Specific Conjugate Seroconversion Panels 0.5 1 1.5 2 2.5 3 3.5 4 4.5 PRB 924-4 PRB 924-5 PRB 924-6 PRB 924-7 PRB 924-8 PRB 940-2 PRB 940-3 PRB 940-4 PRB 940-5 SV0241-C SV0241-D SV0241-E SV0341-C SV0341-D SV0341-E SV0341-F SV0321-A SV0321-B SV0321-C SV0321-D SV0321-E S/CO H&L  Specific

Dilution Panel Testing First dilutions with non-reactive test results for each dilution series.   Sample ID Clade Type SERUM Vironostika HIV-1 Plus O System Comparative Test 1 5805 HIV-1 Clade B 1:1,920 1:240 H629 1:32,000 1:8,000 MD-O HIV-1 Group O 1:25,600 1:400 302-1 1:1,600 NR 2 301-42 HIV-1 Clade A 1:12,800 302-18 HIV-1 Clade C 1:6,000 1:1,500 301-24 HIV-1 Clade D 1:48,000 1:3,000 302-23 HIV-1 Clade E 1:24,000 302-28 HIV-1 Clade F 302-17 1:4,000 1:250 ( 1 ) The licensed Vironostika HIV-1 Microelisa System ( 2 ) Non-reactive

Detection of HIV-1 Group O Specimens Vironostika HIV - 1 Plus O System Comparative Test 1 Specimen ID Sample Dilution Mean SCR 2 1:100 6.1 2.0 1:50 4.0 0.4 3 7.4 1.6 4 5.6 1.1 5 7.7 6 7.2 7 5.3 0.5 8 7.6 2.2 9 5.9 10 1:5 3.9 11 4.5 Total Repeatedly Reactive 11/11 7/11 The licensed Vironostika HIV 1 Microelisa System SCR = Signal to Cutoff Ratio

Detection of HIV-2 Positive Specimens Specimen ID Sample Dilution Mean SCR* 1 1:10 3.3 2 5.8 3 1:500 1.5 4 4.3 5 1:50 1.7 6 5.5 7 2.5 8 5.0 9 1:100 3.7 10 1:1,000 1.8 11 1:200 1.3 12 1.4 13 1.9 14 15 4.9 16 3.6 17 18 2.7 19 2.0 20 *Specimens with SCR equal to or greater than 1.0 are considered reactive with the test.

Conclusions Vironostika® HIV-1 Plus O is intended for use as an aid in diagnosis of infection with HIV-1 and not for use in screening blood donors. This assay uses three solid phase antigens; inactivated, purified HIV-1 viral lysate proteins, a purified viral envelope protein (native gp160), and a synthetic group O peptide. Assay procedure similar to the current licensed Vironostika® HIV with added features to improve ease of use. The assay design sought to improve overall sensitivity to Group M and Group O while maintaining the specificity compared to the current licensed Vironostika® HIV-1. Detection of HIV-1 Group M antibodies was demonstrated with subtypes A, B, B/D, C, C/E, D, E, E/A, E/C, E/F, F, G and H. The assay exhibited higher analytical sensitivity with dilutional panels from clinical specimens for both HIV-1 Group M and HIV-1 Group O specimens compared to the current licensed Vironostika® HIV-1.