A HIGHER FRACTION OF DRUG RESISTANT PROVIRUSES EXPRESS UNSPLICED HIV RNA DURING ART COMPARED TO THE ARCHIVAL WILD-TYPE PROVIRUSES THAT COMPRISE THE HIV-1 RESERVOIR Mary Kearney NCI - Frederick HIV Dynamics and Replication Program

Conflict of Interest No conflicts of Interest to declare

Background We previously reported that about 5% of cells within clonally expanded populations are “actively-infected” (actively transcribing unspliced HIV RNA) and >90% are latently-infected (Musick, et al. CROI 2017). The fractions were not different for clones that carry replication-competent proviruses vs. those that have defective proviruses. Question How does HIV RNA expression in clones differ from expression in cells infected with proviruses that are resistant to the ART regimen?

Evaluated Proviral Expression in Patient #1 (Maldarelli, et al Evaluated Proviral Expression in Patient #1 (Maldarelli, et al. & Hughes, Science, 2014 and Simonetti, et al. & Maldarelli PNAS, 2016) SGS on plasma virus > 50 copies/mL < 50 copies/mL ABC+3TC+EFV Treatment Interruptions Treatment Failure AMBI-1 Single integration sites Small clones Large clones Results from Integration Sites Assay (ISA) What fraction of cells with drug resistant proviruses express HIV RNA during ART? How does this compare to cells with WT proviruses in the same sample?

Mean fraction of HIV expressing cells Higher Fraction of Expressing Cells in Drug Resistant Populations vs. Wild-Type Clones The fraction of cells that were actively-infected vs. latently infected was determined using the CARD-SGS assay (Wiegand, et al. & Kearney PNAS, 2017)  Population Type Mean fraction of HIV expressing cells p valuea Cells with WT proviruses 4.5% 8 x 10-14 Cells with DR proviruses 19.2% a Mann-Whitney test

Higher Levels of Expression from Drug Resistant Proviruses vs Higher Levels of Expression from Drug Resistant Proviruses vs. Wild-Type Clones Low levels of expression in cells with WT proviruses p value = 0.006 2-tail Fisher Exact test High levels of expression in some cells that have drug resistant proviruses

Conclusions The fraction and levels of proviral expression were significantly higher in cells with drug resistant variants than those with wild-type proviruses These findings show that ART can select both for cells infected before ART initiation that either do not express HIV RNA or express at low levels and for cells infected recently with drug-resistant viruses that express higher levels of HIV RNA.

Thank you to the NIH participant and his family! Acknowledgements University of Pittsburgh John Mellors Michele Sobolewski Francis Hong Joshua Cyktor Lou Halvas National Cancer Institute Andrew Musick Jon Spindler Frank Maldarelli Francesco Simonetti Steve Hughes William McManus Ann Wiegand Val Boltz Michael Bale Thank you to the NIH participant and his family! Leidos BioMedical Wei Shao Brandon Keele Carolyn Reid Joseph Meyer Tufts University John Coffin NCI - Frederick HIV Dynamics and Replication Program

10,450 Infected Cells Analyzed Proviral Expression Is Not Different in Clones with Replication-Competent vs. Defective Proviruses Replication Competent Mean fraction expressing = 4% Defective Mean fraction expressing = 4% No Outgrowth Mean fraction expressing = 12% % of cells within each clone that contain ca-HIV RNA Clone ID 10,450 Infected Cells Analyzed