Long-term ART initiated during primary HIV-1 infection limits the HIV-1 reservoir size but not to levels found in LTNPs Eva Malatinkova, Ward De Spiegelaere, Pawel Bonczkowski, Maja Kiselinova, Karen Vervisch, Wim Trypsteen, Margaret Johnson, Chris Verhofstede, Danny de Looze, Charles Murray, Sabine Kinloch-de Loes, Linos Vandekerckhove HIV Translational Research Unit (HTRU) Ghent University, Ghent, Belgium 8th IAS Conference on HIV Pathogenesis, Treatment and Prevention, Vancouver, Canada, July 2015

Background: HIV-1 reservoir Persistent long-lived reservoir: one of the major obstacles to achieve an HIV-1 cure Reservoir: integrated HIV-1 DNA that is replication competent copies ART HIV-1 reservoir Time Viral load ART stop = Viral rebound

Early initiation of ART: is associated with reduced HIV-1 reservoir may lead to post-treatment viral control (VISCONTI controllers 1 ) START study: early ART: lower risk of developing AIDS or other serious ilnesses In a treatment interruption study (SPARTAC 2 ), total HIV-1 DNA correlates with time to viral rebound Better understanding of HIV-1 reservoir can guide therapeutic strategies towards a functional cure 1 Saez-Cirion et al., Plos Pathog Williams et al., eLife Background: Early ART

To assess the long-term impact of early ART on HIV-1 reservoir in blood and gut mucosa To compare the reservoirs between: early or late treated patients LTNPs, representing virological control without ART acute seroconverters as early stage viremic patients Can a decade of ART initiated during primary HIV-1 infection reduce the HIV-1 reservoir to the levels found in LTNPs? Aim of the study

PHI: Primary HIV-1 infection A cross-sectional, observational study Patients consisted of 4 defined cohorts in 2 clinical centers: PHI ART * Early ART PHI Late ART ART PHI LTNP no ART PHI no ART (n=25) (n=17) (n=32) (n=10) ART no ART Patient cohorts years (total=84) 10 years * Fiebig: II-IV (n=10), V (n=15)

Value for cohort* PHILate ARTEarly ARTLTNP n=10N=32n=25n=17 Clinical characteristics Age (years)39 (30-46)48 (31-53)44 (34-53)49 (31-51) Number of females (%)1 (10)5 (15.6)0 (0)7 (41.2) Total ART duration (years)09.8 ( )10.8 ( )0 Viremia zenith (log 10 HIV-1 c/ml)6.2 ( )4.9 ( )5.5 ( )2.5 ( ) CD4 count, nadir (cells/mm 3 )440 ( )155 (0-266)390 ( )624 ( ) CD4 count at sampling (cells/ mm 3 )440 ( )624.5 ( )714 ( )793 ( ) CD4/CD8 ratio0.62 ( )0.74 ( )1.10 ( )0.91 ( ) * Values are reported as median (IQR) Study participant characteristics

HIV-1 reservoir size integrated HIV-1 DNA total HIV-1 DNA Ongoing replication (2-LTR circles) Transcriptional activity (HIV-1 usRNA) HIV-1 reservoir size and dynamics

Materials and methods HIV-1 reservoir size and dynamics: newly-developed PCR- based quantification methods: Total HIV-1 DNA, 2-LTR 1 and usRNA 2 measured by ddPCR Integrated HIV-1 DNA by repetitive sampling Alu-HIV PCR 3 Differences between cohorts: Wilcoxon signed-rank test Correlations: Linear regression 1 Malatinkova et al., J Clin Microbiol Kiselinova et al., PLoS One De Spiegelaere et al., Clin Chem. 2014

Results: HIV-1 DNA reservoir size in blood

Reduced total HIV-1 DNA in early vs late, however not reaching LTNP levels Early ART (n=25) PHI (n=10) LTNP (n=17) Late ART (n=32) p=0.015* p<0.001* p=0.041* p<0.001*

Results: HIV-1 DNA reservoir size in blood

Reduced integrated HIV-1 DNA in early vs late, however not reaching LTNP levels Early ART (n=25) PHI (n=10) LTNP (n=17) Late ART (n=32) p=0.021* p<0.001* p=0.003* p=0.006*p=0.002*p=0.104

Results: HIV-1 reservoir dynamics in blood

Early ART (n=25) PHI (n=10) LTNP (n=17) Late ART (n=32) Episomal 2-LTR circles high in recent ART-naïve seroconverters p=0.595 p=0.743 p=0.259 p=0.002* p<0.001*

Results: HIV-1 reservoir dynamics in blood

Cell-associated HIV-1 usRNA lower in early vs late treated patients and not different from LTNP levels Early ART (n=24) PHI (n=10) LTNP (n=17) Late ART (n=30) p=0.615 p=0.027* p=0.007* p=0.117p=0.092p=0.426

Results: CD4/CD8 ratio

Higher CD4/CD8 ratio in LTNPs and early vs late treated patients Early ART (n=25) LTNP (n=17) PHI (n=10) Late ART (n=32) p=0.978 p=0.036* p=0.009* p=0.007*p=0.048*p=0.448

Negative correlation: CD4/CD8 ratio and integrated HIV-1 DNA in blood R 2 =0.14, p<0.01

Results: HIV-1 DNA reservoir size in rectal biopsies

Total HIV-1 DNA in rectal biopsies not different in early or late treated patients and LTNP No correlation of total HIV-1 DNA measured in blood and rectal biopsies Early ART (n=14) LTNP (n=8) Late ART (n=29) p=0.375 p=0.337 p=0.259

Cross-sectional study design PCR-based assays for HIV-1 reservoir quantification, not measured replication-competent reservoir (e.g. QVOA) Inadequate power to examine influence of Fiebig stage Qualitative differences in HIV-1 sequence not examined Limitations of the study

Conclusions ↓total and integrated HIV-1 DNA Late treated Reduced reservoir size in blood LTNP ↓usRNA↑ CD4/CD8 ratio LTNP Fast seeding of reservoir and other immunological aspects Late treated Lower transcriptional activity and enhanced immune restoration Late treated Early treated

We thank the study participants for their essential contribution UCL London Sabine Kinloch-de Loes Margaret Jonhson Charles Murray AIDS Reference Laboratory Chris Verhofstede HTRU Linos Vandekerckhove Ward De Spiegelaere Pawel Bonczkowski Maja Kiselinova Wim Trypsteen Sofie Rutsaert Marie-Angélique De Scheerder Clarissa Van Hecke Karen Vervisch UZ Ghent Dirk Vogelaers Steven Callens Erica Sermijn Jolanda Pelgrom Beatrijs Van Der Gucht Filip Van Wanzeele Els Caluwé Danny De Looze Acknowledgements

Thank you!