HIV Translational Research Unit - HTRU

1. Clinical studies set-upCharacterization of HIV viral reservoir and quantification of HIV DNA and RNA in patients >12 months on Combined Antiretroviral Therapy LinosVandekerckhove, University Ghent HIV Translational Research Unit - HTRU

2. Contents • Background • Study hypothesis • Study design • Study goal • 1ry and 2ry study objectives • Inclusion and exclusion criteria • Laboratory testing • Duration of the study • Confidentiality • Funding for clinical studies

3. Study’s hypothesis We hypothesize that in patients under cART, there are differences in: • episomalHIV-DNA forms, • cell associated HIV RNA • integrated HIV DNA copy number and/or when receiving different treatment regimens.

4. Background • With current combined antiretroviral therapy (cART) HIV viral load declines to undetectable levels. • However, with ultra-sensitive techniques residual levels of VL can be detected. • a link between residual plasma viremia and the size of the HIV viral reservoir has been suggested

5. HIV replication MATURATION BUDDING HIV virion CELL ENTRY cytoplasma TRANSLATION genomic RNA spliced RNA UNCOATING nucleus TRANSCRIPTION viral mRNA viral RNA REVERSE TRANSCRIPTION INTEGRATION Human DNA proviral DNA

7. Integrated proviral DNA LEDGF/p75 OH strand transfer OH integrase

8. HIV replication HIV virion CELL ENTRY cytoplasma UNCOATING nucleus viral RNA REVERSE TRANSCRIPTION INTEGRATION INTEGRATION Human DNA proviral DNA 1 LTR & 2 LTR episomal viral DNA

9. Background • Recent studies, demonstrated that Nevirapine has a distinct virological advantage. ¹’² • Its suppressing the viral load to levels below 1 HIV RNA copy/ ml. • Its assumed is because of greater penetration in extra-vascular compartments. ¹’² 1) Sharmati & Andreoni et al. SocAntimicrobChemo 2012 2) Haim Boukobza et al. AIDS 2011

10. Background • Additional data suggested an ongoing replication in patients under PI regimen, which may refuel the latent reservoir. ³ • Its assumed that the therapy regiment has impact on the size of the latent reservoir and on immune activation. 3) Buzon et al, Nat Medicine 2010

11. Study goal To evaluate whether there is a relation between the dynamics (and size) of the latent HIV reservoir in HIV-positive patients and the cART treatment regimen • Dynamics of the latent HIV reservoir = increase in ongoing viral replication measured by a difference of 50% • the cART treatment: Nevirapine versus PI

12. Study design • Cross-sectional matched case-control study • Study in which we evaluate all patients under cART, with different treatment regimen

13. 1ry and 2ry objectives Primary objectives • Evaluation of 2LTR HIV DNA, and CA HIV RNA copy number in patients on different cART regimen (PI + 2NRTI’s versus Nevirapine + 2NRTI’s). Secondary objectives • Evaluation of the integrated copy number and size of latent reservoir. • Evaluation of total HIV and integratedHIVDNA . • To assess change immune activation by determination of a lymphocyte activation markers. • To evaluate CD4/CD8 ratio’s in the patient cohort.

14. Inclusion and exclusion criteria • Include all patients on cARVsince at least one year which are/have: • Male or female, aged 18-80 years • Confirmed HIV-1 seropositive documented in the past • Patient s willing and able to give written informed consent • Patients on cARTfor conventional/medical reasons with an established undetectable viral load since at least 1 year. • The regimen of cARTshould be: • 2NRTI’s + boosted PI or • 2NRTI’s + NVP • Exclude all patients which have: • Confirmed HIV-2 seropositive • Positive pregnancy test

15. Laboratory testing • Blood sample of 60ml will be taken at one time point during normal follow-up of the patient. • The following laboratory testing will be performed: • Ficoll gradient centrifugation to isolate PBMC from blood • DNA, 2LTR and RNA extraction from PBMC • qPCR (Roche LC480 platform) and/or digital droplet PCR (ddPCR, Bio-Rad) quantification of: • (1), Cell associated RNA • (2) 2LTR episomal DNA and • (3) total HIV DNA • Ultrasensitive viral RNA quantification on plasma fraction

16. Statistical analysis Sample size calculation • By assuming: • a statistical power of 80%, • type I error of 0.05, • predicted failure rate of 10%, • using 2-sided significance testing, • a 10% of dropout patients rate, and • a difference of 50% in copies of episomal HIV DNA or CA HIV RNA between groups total sample size of 60would be needed in each treatment category group.

17. Duration of the study Total duration of the study is 3 years with: • 48 weeks for patient blood collection • 2 years for performance of all laboratory tests • 1 year for all data analysis and reporting

18. Confidentiality/ethics • In accordance with countries law for private life protection and patient’s rights, all information collected in this study is protected. • The participants have the right to request information of their own personal data at any time. • Representatives of the study have granted direct access to original medical records for verification of the clinical trial procedures. • From patients agreeing to participate, personal data and clinical information is coded.

19. Funding for clinical studies • National funding: • FWO (scientific Research Fund, Flanders) • IWT (Agency for Innovation by Science and Technology) • King Bouduin Foundation

21. Preliminary results for cell associated HIV RNA There is no statistically significant difference between groups; p for usRNA assay is p = 0,47 and for the msRNA assay is p = 0,140

22. Preliminary results for cell associated HIV RNA There is (significant) statistical difference between groups p <0.038 There is no statistically significant difference between groups p = 0.97

23. Limitations • Retrospective nature of the cohorts (systematic error?) • Sample size calculation is difficult as the end point is not yet validate • Funding is not always possible • Changes in the reservoir might be missed in PBMC’s • No longitudinal follow up in this study • Study is not powered for subgroup analysis