HIV variants and US licensed assays

Indira Hewlett, Ph.DChief, Lab. of Molecular Virology

CBER/FDAXIX SOGAT, 2006

HIV genetic diversity: subtypes and homology

HIV-2

A B70%

50%HIV-1

M

A BC

D

EFG

H

I

J

70%

HIV-1O

60%

HIV-1N SIV

60%cpz

75%mac/SM

Adapted from Thomson et al. Lancet Infect Dis 2002.

Worldwide distribution of predominant HIV-1 group M subtypes and CRFs

CRF14_BG

CRF01_AE

B

Diagnostic implications• New serologic and NAT assays have

limited representation of viral epitopes and sequences

• Potential impact on sensitivity for new variants HIV variants

• CBER initiated collaboration with Cameroonian Ministry of Health to study HIV diversity and test performance

• Cameroon has all known subtypes and new variants

Study goals

Evaluate sensitivity of existing and new blood screening, rapid and other diagnostic tests for diverse subtypes

Characterize and genotype HIV variants in a region of high genetic diversity

Identify samples to serve as candidate reference reagents

Study Plan and Methods

• Blood samples (240 samples) collected from sites around Yaounde tested by a rapid HIV assay used to screen blood donors in Cameroon.

• Samples tested by 9 FDA licensed assays• 4 HIV antibody EIAs, 1 p24 antigen, one IFA, one

Wblot, 2 qualitative and one quanitative nucleic acid tests (NAT)

• Discordant samples analyzed by in-house test for group O

• Genotype analysis performed on HIV positive samples

Results

• 149/240 were found to be reactive by the test used in Cameroon

• 133/149 of samples were confirmed as positive on the basis of reactivity on all tests

• 5/149 were negative on all tests• 9/149 were discordant among assays• 2/149 were HIV-2 reactive • 3/149 samples positive by p24 assays

Results – con’t

• 91/240 were negative according to tests in Cameroon• 60 negative on all tests; 2/91 were positive• 17/91 were discordant amongst all assays• 12/91 were reactive on HIV-2 assay• 25 samples from both previously screened antibody

positive and negative samples were discordant between assays

• No HIV group O was detected in discordant samples using an in-house ELISA.

SUMMARY

CRF04, 2, 2.2%

New ISRs, 11, 12.4%

56, 62.9%

A, 1, 1.1%

F2, 2, 2.2%G, 5, 5.6%

CRF06, 1, 1.1%

CRF11, 5, 5.6%

CRF13, 1, 1.1%

D, 1, 1.1%

CRF01_AE, 4, 4.5%

D

CRF01_AE

A

G

F2

CRF02_AG

CRF04

CRF06

CRF11

CRF13

New ISRs

Summary

• Current licensed HIV NAT and Ab were able to detect most subtypes and recombinant HIV variants

However a small number of CRF02 AG were not detected by at least one manufacturer’s assay

• CRF02_AG most prevalent viral strain in Cameroon (62.9%)

• New ISRs identified in this study; reactive in NAT assays.

• High reactive rates seen with HIV negative Cameroonian samples

Regulatory implications

• HIV genetic diversity appears to be evolving globally at a fairly rapid rate

• Different rates of disease progression for different subtypes recently reported

• Continued surveillance for existing and new emerging variants and development of reference reagents may be warranted

• HIV variant Samples should be included in the evaluation of HIV and other human retroviral tests

Current PHS efforts

• Continued FDA surveillance for viral variants and screening and diagnostic assays

• PHS working group formed to monitor emerging natural and drug resistant HIV variants in global setting

• Evaluate implications for diagnosis (conventional and rapid), blood screening, therapy and vaccine development

• Develop repositories to aid in the evaluation of new diagnostic and blood screening tests, vaccines and new therapies

Application of Nanotechnology to diagnostics

Nano-Scale Diagnostics

• Nanotechnology offers some potentially unique features based on the size (1-100nm scale) and properties that could permit rapid, sensitive detection of multiple pathogens and analytes simultaneously

• Nanotechnology-based approaches could potentially provide a new generation of diagnostic assays

• Nano-scale detection could permit miniaturization allowing small volumes of sample to be tested with a high degree of sensitivity

Nanoparticle-Based Bio-Barcode Amplification (BCA) Assay

• Based on chemical probes) labeled on the nanoparticles (NPs) and magnetic microparticles (MMPs)

• Use barcode DNA-modified NPs for signal amplification and MMPs for easy separation

• Particle-initiated Ag developing technique for signal enhancement

• High sensitivity but without enzymatic amplification

• Microarray format (or could be adapted to ELISA format)

• Multiplex assay system for rapid and sensitive detection

Application of BCA to HIV detection

• BCA had been applied to detection of PSA

• Applicability of BCA to infectious disease testing was explored using HIV p24 antigen as proof of concept

• Potential use in settings where NAT is less available

Modified BCA Assay for p24 Detection Using Antibody-Coated Microplate

HIV-1 p24

Barcodes releaseand detection

Streptavidin-Coated Gold Nanoparticle

Step 1. Incubate target with ab-coated microplate (1 hour)

Step 2. Add 2nd biotin labeledantibody to the tube (30 min).

Step 3. After wash (2X), add streptavidin-NP (30min)

Step 4. After wash (2X), add barcode (30min)

Step 5. After wash (8X), Elute barcode (5min)

Biotin-labelledBarcode DNA

15 nm SA-Au

NP

Monoclonal Anti-p24 Ab

Biotin-labelledanti-p24 Ab

Immuno Microplate

IC untreated

IC treated

Negative

Positive (25pg/ml)

Detection of Immune Complex treated with Glycine/Tris

Without serum 50% serum P24 (pg/ml)

50

5

0.5

0

Detection of p24 by BCA down to 0.5 pg/mlin the Presence of 50% Human Serum

A B

ELISA BCA

Linearity in p24 Detection by ELISA and BCA

HIV-1 p24 in Seroconversion Samples

Samples tested

+ - Total

+ 14 1 15

- 1 30 31

Total 15 31 46

BCA

Sensitivity = 14/15 = 93.3%

Specificity = 30/31 = 96.7%

Concordance = 44/46 = 95.7%

Preliminary data on performance of BCA with clinical samples

Conclusion

• BCA assay could detect 0.5 pg/ml of HIV-1 p24 antigen compared with 10 ~ 50 pg of conventional p24 antigen capture assays (ELISA).

• There is a linear relationship between concentration of p24 and the signal intensities at the range of 0.5 ~ 500 pg / ml.

• BCA may be approx. 20 ~ 100 fold more sensitive than ELISA.

• 22 HIV negative samples tested were non reactive when tested by BCA

• In seroconversion panels, BCA detected HIV p24 earlier than current p24 assay and at the same time as PCR

Cameroon Ministry of Health

Leopold Zekeng

Bih Awazi

CBER/FDA

Ana Machuca Jinjie Hu

Shixing Tang Arindam Dhar

Owen Wood Sherwin Lee

Steve Kerby Maria Rios

NIH/NHLBI G. Nemo L. Harvath

Northwestern UniversityChad Mirkin

Stephen Wolinsky

NanosphereJames Storhoff

Blood Systems Research InstitutePhilip Norris