1. 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

2. Conflict of Interest
No conflicts of Interest to declare

3. 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?

4. 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?

5. 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

6. 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

7. 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.

8. 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

9. 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