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HIV variants and US licensed assays

HIV variants and US licensed assays. Indira Hewlett, Ph.D Chief, Lab. of Molecular Virology CBER/FDA XIX SOGAT, 2006. HIV genetic diversity: subtypes and homology. 70%. A. B. C. J. HIV-2. HIV-1 M. D. I. 50%. E. A. B. H. F. 70%. G. 75% mac/SM. 60%. HIV-1 O. HIV-1 N.

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HIV variants and US licensed assays

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  1. HIV variants and US licensed assays Indira Hewlett, Ph.D Chief, Lab. of Molecular Virology CBER/FDA XIX SOGAT, 2006

  2. HIV genetic diversity: subtypes and homology 70% A B C J HIV-2 HIV-1 M D I 50% E A B H F 70% G 75% mac/SM 60% HIV-1 O HIV-1 N SIV 60% cpz

  3. CRF01_AE Worldwide distribution of predominant HIV-1 group M subtypes and CRFs CRF14_BG B Adapted from Thomson et al. Lancet Infect Dis 2002.

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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.

  9. SUMMARY

  10. 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

  11. 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

  12. 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

  13. Application of Nanotechnology to diagnostics

  14. 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

  15. 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

  16. 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

  17. Modified BCA Assay for p24 Detection Using Antibody-Coated Microplate Monoclonal Anti-p24 Ab Biotin-labelled anti-p24 Ab Biotin-labelled Barcode DNA 15 nm SA-Au NP Barcodes release and detection Immuno Microplate HIV-1 p24 Streptavidin- Coated Gold Nanoparticle Step 1. Incubate target with ab-coated microplate (1 hour) Step 2. Add 2nd biotin labeled antibody 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)

  18. Detection of Immune Complex treated with Glycine/Tris IC untreated IC treated Negative Positive (25pg/ml)

  19. Detection of p24 by BCA down to 0.5 pg/ml in the Presence of 50% Human Serum Without serum 50% serum P24 (pg/ml) 50 5 0.5 0 A B

  20. Linearity in p24 Detection by ELISA and BCA ELISA BCA

  21. HIV-1 p24 in Seroconversion Samples

  22. Preliminary data on performance of BCA with clinical samples • Samples tested • + - Total • + 14 1 15 • - 1 30 31 • Total 15 31 46 Sensitivity = 14/15 = 93.3% Specificity = 30/31 = 96.7% Concordance = 44/46 = 95.7% BCA

  23. 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

  24. Acknowledgements Cameroon Ministry of Health Leopold Zekeng Bih Awazi NIH/NHLBI G. Nemo L. Harvath CBER/FDA Ana Machuca Jinjie Hu Shixing Tang Arindam Dhar Owen Wood Sherwin Lee Steve Kerby Maria Rios Northwestern University Chad Mirkin Stephen Wolinsky Nanosphere James Storhoff Blood Systems Research Institute Philip Norris

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