CROI 2017 Abstract e-Book

Abstract eBook

Poster and Themed Discussion Abstracts

Methods: We examined the HIV-1 proviral landscape in virally suppressed HIV-1-infected individuals. We then examined whether defective HIV-1 proviruses can be recognized by CTLs in vitro and ex vivo. Results: In the HIV-1 proviral landscape of virally suppressed HIV-1-infected individuals, we found that the frequency of hypermutated proviruses correlates negatively with the duration of infection, while the frequency of proviruses containing large internal deletions correlates positively with the duration of infection, implying a dynamic process. To examine whether defective HIV-1 proviruses can be recognized by CTLs in vitro, we co-cultured CD4+ T cells transfected with defective HIV-1 proviruses with autologous CTL clones known to recognize specific CTL epitopes. CTL recognition of cells containing defective HIV-1 proviruses were measured by CD107a loading. Surprisingly, we found that cells containing hypermutated proviruses which have a premature stop codon or mutated start codon 5’ to an intact CTL epitope can be recognized by CTLs, while cells containing large internal deletions cannot be recognized by CTLs. To understand whether hypermutated HIV-1 proviruses may be recognized by CTLs ex vivo, we used targeted deep-sequencing to examine the frequency of APOBEC-mediated nonsense mutations in gag. We compared resting and activated CD4+ T cells treated with and without autologous prestimuated CTLs. We found that the frequency of HIV-1 RNA containing premature stop codons 5’ to the CTL recognition site decreased upon CTL co-culture, indicating that cells containing hypermutated proviruses can be eliminated by CTLs ex vivo. Conclusion: We found that hypermutated HIV-1 proviruses may be targeted by CTLs. HIV-1 DNA measurement may in part reflect the effect of HIV-1 elimination strategies. HIV-1 proviral landscape may be shaped, at least partially, by CTL selection pressure. 297 DYNAMICS OF HIV CLONAL EXPANSION AND PERSISTENCE DURING ART Elizabeth M Anderson 1 , Shawn Hill 1 , Jennifer Bell 1 , Francesco R. Simonetti 2 , Cathy Rehm 3 , Sara Jones 4 , Rob Gorelick 4 , John Coffin 5 , Frank Maldarelli 1 1 NCI, Frederick, MD, USA, 2 The Johns Hopkins Univ, Baltimore, MD, USA, 3 NIAIDs, Bethesda, MD, USA, 4 Leidos Biomed Rsr, Inc., Frederick, MD, USA, 5 Tufts Univ, Boston, MA, USA Background: HIV persistence during antiretroviral therapy (ART) is a substantial obstacle to HIV cure. HIV infected cells can undergo clonal expansion and specific clones may be highly expanded. We previously reported one provirus integrated in the HORMAD2 gene that accounted for 20-40% of all proviruses in the patient. Although c. 98% of proviruses are defective, deleted clones may continue to express gag. Mechanisms by which clones emerge and persist over time are uncertain. To investigate the dynamics of HIV clonal expansion during ART we developed multiplexed droplet digital approaches (ddPCR) to quantify HIV proviruses, including specific integrants, prior to and following prolonged ART Methods: HIV infected ART-naïve individuals (N=5) underwent ART and were followed for a median of 9.8 yrs (range 2.8-16.4y). Cell associated DNA (CA-DNA) was recovered from PBMCs pre-ART, during first phase viral decay, and during long term ART. CA-DNA was quantified using multiplexed ddPCR assays targeting HIV gag, the HIV LTR (RU5), as well as a host gene (CCR5). We designed a specific ddPCR primer set using oligos overlapping the host-HIV junction sequence to quantify the integrant in HORMAD2 Results: All patients had successful suppression of HIV RNA on ART to <50 c/mL plasma within 5 mos and HIV DNA/1e6 CD4+ cells decreased for both gag (avg 12.6-fold, range 8-19) and LTR (avg 8.2-fold, range 4-11). In 3 of the 5 patients the ratio of LTR to gag increased progressively between the first phase decay and long term time points (avg 6.5-fold, paired T-test p<0.01) demonstrating loss of full length proviruses; in 2/5 patients, LTR/gag ratios remained stable. The HORMAD2 integrant was not detectable at pre-ART, 1 mo, and 2 mos on ART (<1 cp in total of 500,000 infected cells). After 1 year on suppressive ART, however, the HORMAD2 integrant was present at a frequency of 30% of all infected cells and persisted for 6 yrs on ART Conclusion: HIV populations change during ART with progressive gag deletion in most, but not all patients. Clonal expansion of HIV infected cells can be rapid and sustained at stable levels during prolonged ART, suggesting both antigen induced clonal expansion and homeostatic proliferation maintain HIV populations. Multiplexed quantitative assays suitable for single cell analyses can be modified to specifically quantify individual integrated proviruses. Tracking specific integrants over time sheds light on the dynamics of HIV infected cells clonal expansion and mechanisms of their persistence 298 CLONES OF HIV-INFECTED CELLS ARISE IN VIVO IN THE FIRST FEWWEEKS FOLLOWING INFECTION David Wells 1 , Jennifer M. Zerbato 2 , Joann D. Kuruc 3 , Joseph J. Eron 3 , Mary F. Kearney 4 , John Coffin 5 , Frank Maldarelli 4 , Xiaolin Wu 1 , John W. Mellors 2 , Stephen H. Hughes 4 1 Liedos Biomed Rsr, Inc, Frederick, MD, USA, 2 Univ of Pittsburgh, Pittsburgh, PA, USA, 3 Univ of North Carolina at Chapel Hill, Chapel Hill, NC, USA, 4 NCI, Frederick, MD, USA, 5 Tufts Univ, Boston, MA, USA Background: In HIV-infected individuals on successful long-term antiretroviral therapy (ART), a significant fraction (>40%) of the infected cells are in expanded cell clones. Most of the HIV proviruses in infected cells that persist after long-term ART are defective, including most of the proviruses in clonally expanded cells. However, Simonetti et al. (Proc. Natl. Acad. Sci. USA, 2016 113: 1883-1888) showed that a large clone carried an intact provirus and released detectable levels of infectious virus into the blood. Thus, infected cell clones can make up a part of the HIV reservoir that has made it impossible to cure HIV infections with the currently available therapies. Given the importance of clonal expansion of infected cells in those on ART, we wanted to determine how soon after an individual is infected can infected clones grow large enough to be readily detectable in our integration site assay (clone size >105 cells). Methods: Methods: We determined the HIV integration sites (using the technology described in Maldarelli et al., 2014 Science 345: 179-183) in samples of peripheral blood mononuclear cells taken at the time of HIV infection diagnosis and Fiebig staging. These individuals were placed on ART at the time of diagnosis, and the integration sites were also determined in follow-up samples, taken after 2-3.5 years of suppressive ART. Results: Results: We have analyzed 3797 independent integration sites in samples from 7 patients: 2 from stage III, 1 from stage IV-V, and 3 from stage V. We are analyzing samples from additional stage IV and stage V patients. The integration site data (see appended table) showed that some HIV infected clones can grow large enough to be detectable as early as ~3-4 weeks after infection is detectable and that some of the early clones that arise early persist for years after ART initiation. Conclusion: Conclusions: Clones of HIV infected cells can be detected within ~3-4 weeks following HIV infection. This finding may help explain how the HIV reservoir is established early in infection.

Poster and Themed Discussion Abstracts

CROI 2017 117