HIV variants and US licensed assays
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HIV variants and US licensed assays
Indira Hewlett, Ph.DChief, Lab. of Molecular Virology
CBER/FDAXIX SOGAT, 2006
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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
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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
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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
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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
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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
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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
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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.
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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
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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
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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
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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
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Application of Nanotechnology to diagnostics
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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
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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
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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
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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
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IC untreated
IC treated
Negative
Positive (25pg/ml)
Detection of Immune Complex treated with Glycine/Tris
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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
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ELISA BCA
Linearity in p24 Detection by ELISA and BCA
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HIV-1 p24 in Seroconversion Samples
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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
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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
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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