Diagnosis and pathogenesis of HIV variants: A progress report

1. Diagnosis and pathogenesis of HIV variants: A progress report Indira Hewlett, Ph.D Molecular Virology Lab

2. Topics of investigation • Diagnostic studies of HIV variants • Genetic characterization of variants to understand virus evolution and diversity • Pathogenesis of variants – HIV-1 subtypes, HIV-2, multi-drug resistant strains • Reference reagents for HIV subtypes for NAT assays used for 1) blood screening and 2) to monitor therapy and vaccine trials • Smallpox vaccination and blood safety

3. HIV variants: Studygoals • Evaluate sensitivity of existing and new blood assays for diverse subtypes with sera from Cameroon known to harbor diverse HIV strains • Characterize and genotype HIV variants • Investigate pathogenesis of major variants • Identify samples to serve as candidate reference reagents

4. Conclusions and Future Directions • FDA licensed tests detected additional HIV positive specimens than those identified in Cameroon. EIA results from different manufactures did not always agree with each other, WB may not resolve some discordant results. • CRF02_AG was the most prevalent viral strain found in Cameroon samples (70%) • New CRFs AC, AF and GF were identified in this study • Analysis of discordant and negative samples with degenerate primers, PCR-RT assay to identify emerging retroviruses in collaboration with OVRR • Microarray for identification of HIV genotypes, subtypes, new viruses, validate by comparison with sequencing data • Investigation of tropism by molecular cloning of env genes of specific isolates

5. Objectives of studies on drug resistant HIV studies To study virologic characteristics: • replication • cellular tropism • genotype • apoptosis • chemokine and cytokine production To examine whether there was correlation between genotype, co-receptor usage and virus replication

6. CONCLUSIONS AND FUTURE DIRECTIONS • Viruses from patients on prolonged antiretroviral therapy continued to use classical chemokine co-receptors. • Tropism was not apparently related to CD4 cell count, Viral load, and genotypic mutation in RT, Protease and/or env-C2V3 regions. • Future studies on cytokine regulation, apoptosis, differential gene expression using gene grids

7. Standardization of NAT: CBER HIV-1 subtype NAT panel • Need analytical standards for • Quality control, quality assurance and global standardization of NAT • Licensure and post-market surveillance through lot release testing • 7 subtypes of HIV-1 group M: A, B, C, D, E, F, G ; group N and group O • Pilot-scale prototype panels tested in collaborative study involving 5 NAT manufacturers at various dilutions • Data analyzed at FDA and consensus values assigned to viral stocks and final panel formulated • HIV-2 panel under development

8. Smallpox vaccination: Concerns for Blood donation • Vaccinees could present as blood donors • Potential viremia in vaccinated individuals and concerns due to lack of data on virus transmission by blood • Studies needed to determine appropriate deferral period for vaccinated donors • Concerns raised about potential, false positive reactions with sera from Smallpox vaccinees similar to flu vaccination • Impact on blood availability due to increased numbers of donors deferred as a result

9. Conclusions Safety: • Plasma from vaccinees may not pose a significant risk for virus transmission • Additional studies to evaluate cellular viremia by culture and Taqman assay are underway Availability: • Vaccination did not cause significant interference with most donor screening assays • Significant interference observed with a few assays

10. Summary • Molecular epidemiology of HIV strains in country with high viral diversity • Diagnostic impact of HIV variants • Virologic characteristics of drug resistant variants • Development of reference materials for standardization of viral subtypes • Impact of Smallpox vaccination on blood safety