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VironostikaVironostika®® HIV-1 Plus O Microelisa System 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

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Main FeaturesMain 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.

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Kit ComponentsKit Components

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Overview of VironostikaOverview of Vironostika®® HIV-1 Plus O HIV-1 Plus O Microelisa SystemMicroelisa System

STOP

WASH WASH

Solid phase: NaFsubstrate (ABTS)

STOP

WASH WASH

Solid phase: Anti-HIV in SampleAnti-HIV in Sample Goat anti-human Ig /HRPconjugateGoat anti-human Ig /HRPconjugate

Chromogenicsubstrate (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)

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Assay Procedures for Serum/Plasma (S/P) and Assay Procedures for Serum/Plasma (S/P) and Dried Blood Spots (DBS)Dried Blood Spots (DBS)

120 min, 37°C

60 min, 37°CS/P

10µl SAMPLE

200µl DILSIM III

(1:21)

Conj 150µl

60 min, 37°C

Wash READAt 405 nm

10-13 min, RT

DBS

Elute with 150µl

of DBS ElutionMedium

25µl ELUTE

125µl DILSIM III

(1:6)

[S]150µl

Wash STOP 150µl

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Sample Addition MonitoringSample Addition Monitoring

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Solid Phase AntigenSolid 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

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Development of Native HIV-1 gp160Development 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

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Immunodominant Domains in HIV-1 Immunodominant Domains in HIV-1 envenvProteinsProteins

gp160

gp120

gp41

504-518

504-518

579-601

579-601

296-331

296-331

gp160

gp120

gp41

504-518

504-518

579-601

579-601

296-331

296-331

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Serum EIA Reactivity of HIV-1 gp160Serum EIA Reactivity of HIV-1 gp160

1101001000

False Postive

Very WeakPositive

Medium Positive

0.2

0.4

0.6

0.8

1.0

1.2

0.0

A4

50

gp160 (ng /well)

1000 100 10 11101001000

False Positive

Very WeakPositive

Medium Positive

0.2

0.4

0.6

0.8

1.0

1.2

0.0

A4

50

gp160 (ng /well)

1000 100 10 1

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The Vironostika® HIV-1 Plus O Trial Trial SummarySummary

• 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

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Specificity in LowSpecificity in Low--Risk Populations (n=6019)Risk Populations (n=6019)

0

1000

2000

3000

4000

5000

<0

.10

0.10

-0

.19

0.20

-0

.29

0.30

-0

.39

0.40

-0

.49

0.50

-0

.59

0.60

-0

.69

0.70

-0

.79

0.80

-0

.89

0.90

-0

.99

>=1

.00

FRE

QU

EN

CY

SIGNAL TO CUTOFF RATIO

HIV-1 Plus O

VirHIV

HIV-1 Plus O

VirHIV

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Known HIV-1 SpecimensKnown HIV-1 Specimens

Serum/PlasmaHIV-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)

DBS

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)

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SensitivitySensitivity

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%).

E s tim a tio n o f C lin ic a l S e n s itiv ity

S p e c im en

T yp e

C D 4 +

S tra tu m

N u m b e r o f s p e c im e n s

N u m b e r o f In itia lly

R e a c tive

N u m b e r o f R e p e a te d ly

R e a c tive

< 2 0 0 2 5 0 2 5 0 2 5 0 2 0 0 -4 99 3 8 5 3 8 5 3 8 5

> 4 9 9 3 7 5 3 7 5 3 7 5

S e ru m o r

P la s m a

T o ta l 1 ,0 1 0 1 ,0 1 0 1 ,0 1 0

< 2 0 0 2 5 0 2 5 0 2 5 0 2 0 0 -4 99 3 8 5 3 8 5 3 8 5

> 4 9 9 3 7 5 3 7 5 3 7 5

D rie d B lo o d

S p o ts

T o ta l 1 ,0 1 0 1 ,0 1 0 1 ,0 1 0

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High-Risk PopulationsHigh-Risk PopulationsPrison Inmates, STD, Hosp ER Pts, and HIV Outreach Clinic PtsPrison 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

Number of

Initially

Reactive

Number of

Repeatedly

Reactive

Western Blot

Positive

1 251 16 14 8

2 513 13 13 13

3 500 73 68 68

4 250 28 27 27

Serum or Plasma

Total 1,514 130 122 116

1 0 N/A N/A N/A

2 0 N/A N/A N/A

3 500 68 68 68

4 250 27 27 27

Dried Blood

Spots

Total 750 95 95 95

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Seroconversion Panel TestingSeroconversion Panel Testing

Panel ID Sample Collection Days Vironostika HIV-1 Plus O

Comparative Test1

924 8, 10, 26, 33, 35, 40 33 40927 0, 28, 33, 35, 40 33 40931 9, 15, 28, 33, 35, 42 28 33932 0, 3, 13, 27, 34, 50, 78, 163, 194 34 NR2

940 0, 7, 11, 15, 18, 22, 25, 29 15 22071 1, 3, 17, 22, 28 17 22111 1, 2, 8, 16, 20, 22, 27 8 16241 1, 7, 9, 15, 17, 22, 24 15 22321 1, 8, 12, 15, 21 15 21341 1, 7, 10, 21, 23, 28 21 28351 1, 8, 11, 15 15 NR361 1, 3, 9, 11, 16, 18 18 NR

1The licensed Vironostika HIV-1 Microelisa System

2None of the specimens in this panel was reactive

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Seroconversion Panels

0

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/C

O

H&L

Specific

Reactivity of H&L vs Reactivity of H&L vs Specific Conjugate Specific Conjugate

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Dilution Panel TestingDilution Panel Testing

First dilutions with non-reactive test results for each dilution series.

  

Sample ID

  

Clade Type

SERUM

Vironostika HIV-1Plus O System

 Comparative Test 1

5805 HIV-1 Clade B 1:1,920 1:240H629 HIV-1 Clade B 1:32,000 1:8,000MD-O HIV-1 Group O 1:25,600 1:400302-1 HIV-1 Group O 1:1,600 NR 2

301-42 HIV-1 Clade A 1:12,800 1:1,600302-18 HIV-1 Clade C 1:6,000 1:1,500301-24 HIV-1 Clade D 1:48,000 1:3,000302-23 HIV-1 Clade E 1:24,000 1:1,500302-28 HIV-1 Clade F 1:12,800 1:1,600302-17 HIV-1 Clade D 1:4,000 1:250

( 1 ) The licensed Vironostika HIV-1 Microelisa System

( 2 ) Non-reactive

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Detection of HIV-1 Group O SpecimensDetection of HIV-1 Group O Specimens

Vironostika HIV-1 Plus O System

Comparative Test

1

Specimen ID

Sample Dilution Mean SCR

2 Mean SCR

1 1:100 6.1 2.0 2 1:50 4.0 0.4 3 1:50 7.4 1.6 4 1:50 5.6 1.1 5 1:50 7.7 2.0 6 1:50 7.7 7.2 7 1:50 5.3 0.5 8 1:50 7.6 2.2 9 1:50 5.9 0.4 10 1:50 3.9 0.4 11 1:50 7.6 4.5

Total Repeatedly Reactive 11/11 7/11 1The licensed Vironostika HIV-1 Microelisa System

2SCR = Signal to Cutoff Ratio

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Detection of HIV-2 Positive SpecimensDetection of HIV-2 Positive Specimens

Specimen ID Sample Dilution Mean SCR*

1 1:10 3.32 1:10 5.83 1:500 1.54 1:10 4.35 1:50 1.76 1:10 5.57 1:10 2.58 1:10 5.09 1:100 3.710 1:1,000 1.811 1:200 1.312 1:200 1.413 1:10 1.914 1:50 1.315 4.916 1:1,000 3.617 1:50 1.718 1:1,000 2.719 1:10 2.020 1:10 1.8

*Specimens with SCR equal to or greater than 1.0 are considered reactive with the test.*Specimens with SCR equal to or greater than 1.0 are considered reactive with the test.*Specimens with SCR equal to or greater than 1.0 are considered reactive with the test.

1:1,000

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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.