CROI 2016 Abstract eBook

Abstract Listing

Poster Abstracts

378

Follicular Tregs Reduce HIV Replication and Reactivation From Latency Ex Vivo Brodie Miles ; Elizabeth Connick Univ of Colorado, Denver, CO, USA Background: Most HIV replication occurs within B cell follicles in follicular T helper cells (T FH

), which likely contribute substantially to the HIV reservoir in virologically suppressed

individuals. Follicular Tregs (T FR

), also reside in B cell follicles, yet little is known about the effects of T FR

regulation on HIV replication and HIV latency within T FH

. We hypothesized

that T

FR are able to suppress both HIV replication in T FH

and reactivation of HIV from latent infection of CD4 T cells during infection ex vivo .

Methods: Tonsil cells were spinoculated with X4- or R5- tropic GFP reporter viruses, or mock spinoculated, and T FH (CD3+CD8-CXCR5+CD25lo/-) were analyzed by flow cytometry after 2 days. In select experiments, CD25 cells were depleted prior to spinoculation. For latency experiments, isolated CD4 T cells were spinoculated with X4 HIV and cultured with CXCR4 antagonist (bicyclam) to prevent de novo infection in culture, sorted to remove GFP+ cells after 2 days, and cultured an additional 4 days to confirm stable latency in culture. During removal of GFP+ cells, T FR (CD3+CD8-CXCR5+CD25hi) were also isolated and added back to CD4 T cells at a 1:1 ratio. To stimulate reactivation of cells, cultures were treated with 500 nM SAHA for 24 hours at day 4. Cell phenotypes and GFP expression were determined by flow cytometry. Statistical analyses were performed by Wilcoxon matched-pair tests using GraphPad Prism 6. Results: Removal of CD25+ regulatory cells led to an increase in %GFP+ T FH (median 1.7 fold in X4, p=0.002, 2 fold in R5, p=0.004; n=10) and an increase of GFP intensity in T FH (median 1.2 fold in X4, p=0.002 1.5 fold in R5, p=0.002; n=10) compared to undepleted cultures. A natural and stable HIV reservoir is generated by spinoculation of unstimulated tonsillar CD4 T cells and removing productively infected (GFP+) cells during culture. Latently infected tonsil CD4 T cells are reactivated during SAHA treatment (median 2.38% p24+GFP+; n=3), but not in the presence of T FR (median 0.1% p24+GFP+; n=3). Conclusions: Regulatory T cells inhibit HIV replication in T FH and prevent HIV reactivation from latency in CD4 T cells ex vivo . They likely contribute to viral persistence in virologically suppressed individuals in vivo . Targeting the suppressive mechanisms of T FR that prevent HIV reactivation in latently infected cells may be an important therapeutic approach to efficiently activate and purge the latent reservoir in vivo . 379 A Subset of Infectious Proviruses Persist and Expand Following Activation Ex Vivo John K. Bui 1 ; Elias Halvas 2 ; Elizabeth Fyne 1 ; Michele D. Sobolewski 1 ; Dianna Koontz 2 ; Mary F. Kearney 3 ;Wei Shao 4 ; Feiyu Hong 1 ; JohnW. Mellors 1 1 Univ of Pittsburgh, Pittsburgh, PA, USA; 2 Univ of Pittsburgh Sch of Med, Pittsburgh, PA, USA; 3 NCI, Frederick, MD, USA; 4 Leidos Biomed Rsr, Inc, Frederick, MD, USA Background: The most effective latency reversing agents for HIV-1 are also potent T-cell activators (Cillo, PNAS 2014). Recent studies show that virus producing cells can persist and expand in vivo (Maldarelli, Science 2014). We hypothesized that activation of HIV-infected CD4 + T-cells could lead to clonal expansion of proviruses rather than their elimination. Methods: We established an ex vivo cell culture system involving stimulation of patient-derived CD4 + T cells with PMA/ionomycin (day 1-7), followed by rest (day 7-21), and then restimulation (day 21-28). Raltegravir (300 nM) and efavirenz (300 nM) were added to cultures to block virus spread throughout the experiments. Cell-associated HIV-1 DNA (CAD) and virion RNA in the supernatant were quantified by qPCR at weekly intervals. Single genome sequencing (SGS) was performed to characterize proviruses and virion RNA. The replication-competence of proviruses in cultured cells was determined by the viral outgrowth assay (VOA) at multiple time points. Results: Experiments were performed with purified CD4 + T-cells from five consecutive donors who had been suppressed on ART for > 2 years (median = 13.4 years). In all experiments, HIV-1 RNA levels in supernatant increased following initial stimulation, decreased or remained stable during the rest period, and increased again with restimulation. Cell-associated HIV-1 DNA levels did not show a consistent pattern of change. SGS revealed several different outcomes of proviral populations: 1) virus production following the first but not the second stimulation; 2) virus production following only the second stimulation; 3) virus production following both stimulations; 4) no virus production after either stimulation, 5) cellular expansion of non-induced proviruses; and importantly 6) persistence and likely expansion of induced proviruses, including replication-competent proviruses. This latter outcome was observed in 3 of 3 donors tested, as evidenced by identical sequence matches between p24 positive VOA wells and virion RNA sequences at multiple time points. Conclusions: These results indicate that reversal of HIV-1 latency with CD4 + T cell activation results in multiple outcomes of proviral populations, including persistence and expansion of replication-competent proviruses. These findings underscore the complexity of eliminating HIV reservoirs with latency-reversal agents and highlight the need for new strategies to kill HIV-infected cells before they can clonally expand. 380 Transcription of Novel HIV-1 RNA Species in the Setting of “Undetectable”Virus Hiromi Imamichi 1 ; Robin Dewar 2 ; Joseph A. Adelsberger 3 ; Catherine A. Rehm 4 ; Catherine Seamon 4 ; Gregg Roby 5 ; Alice Pau 4 ; H. Clifford Lane 4 1 NIAID, NIH, Frederick, MD, USA; 2 Frederick Cancer Rsr and Develop Cntr, Frederick, MD, USA; 3 Leidos Biomed Rsr, Inc, Frederick, MD, USA; 4 NIAID, NIH, Bethesda, MD, USA; 5 NIAID, NIH, Rockville, MD, USA Background: Proviruses in circulating PBMCs have been characterized as “defective” and are thought to represent a “graveyard” of viral sequences with little contribution to HIV-1 pathogenesis. We have previously demonstrated the ability of such defective provirus to transcribe viral RNA and its long-term persistence in vivo. In the present study, we demonstrate that the presence of transcription-competent “defective” proviruses is a consistent finding in patients with HIV-1 infection and examine the importance of their persistence in the context of the persistent immune activation seen in patients with prolonged viral suppression. Methods: Four HIV-infected patients (pts) with pVL ≥40 copies/ml (range 26,758-225,658) and four on suppressive cART with pVL <40 copies/ml for >6 yrs (range 6.1-11.9) were studied. Nuclear and cytoplasmic fractions of the same population of CD4 + T cells served as sources for HIV-DNA and RNA, respectively. Single genome amplicons generated via a U5-U5/R PCR were used to sequence full-length HIV-1 genomes. Western blots were performed using the Cambridge Biotech HIV-1 Western Blot kit. Results: Single-genome sequencing of 175 HIV-1 proviruses determined that 53% (48/90) in pVL≥40 pts and 99% (84/85) in pVL<40 pts were defective. The majority of defective proviruses could be characterized as truncated forms with gross internal deletions (87% in pVL≥40; and 93% in pVL<40). The remainder, while full-length, contained multiple lethal mutations (13% in pVL≥40; and 7% in pVL<40). Truncated proviruses were also found following in vitro infection of primary CD4 + T cells with the DH12 clone of HIV-1 as early as 24 h post infection with a frequency of 20% (4/20). A number of novel RNAs and corresponding truncated proviral DNAs (including 2 cases with evidence of clonal expansion) were identified in pVL<40 pts, suggesting ongoing transcription of these defective proviruses. These novel RNA sequences contained open reading frames not corresponding to known mRNA species. Western blots obtained from the sera of these patients showed persistence of viral bands in a variety of patterns suggesting an asynchronous decay in viral proteins in vivo. Conclusions: Cells harboring defective proviral DNA capable of transcribing novel RNAs with open reading frames can be seen following in vitro and in vivo infection. The proteins encoded by these “zombie” proviruses may explain the persistence of antibodies to HIV-1 and the persistent immune activation seen even in patients with “undetectable” virus.

Poster Abstracts

143

CROI 2016