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 HIV-2 AB 70% 50% HIV-1 M AB C D E F G H I J 70% HIV-1 O 60% HIV-1 N SIV 60% cpz 75% mac/SM

3. 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

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 HIV-1 p24 Barcodes release and detection Streptavidin- Coated Gold Nanoparticle Step 1. Incubate target with ab-coated microplate (1 hour) Step 2. Add 2 nd 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) Biotin-labelled Barcode DNA 15 nm SA-Au NP Monoclonal Anti-p24 Ab Biotin-labelled anti-p24 Ab Immuno Microplate

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

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

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

21. HIV-1 p24 in Seroconversion Samples

22. 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

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. 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 University Chad Mirkin Stephen Wolinsky Nanosphere James Storhoff Blood Systems Research Institute Philip Norris