CROI 2015 Program and Abstracts

Abstract Listing

Oral Abstracts

Conclusions: ABX464 represents a novel class of anti-HIV molecules with unique properties. ABX464 has a long lasting effect in humanized mice and neutralizes the expression of HIV-1 proviral genome of infected immune cells including reservoirs and it is therefore a promising drug toward HIV cure. 105 Residual Viremia Caused by Clonally Expanded Tumor-Infiltrating CD4+ Cells Francesco R. Simonetti 1 ; Michele D. Sobolowski 2 ; Shawn Hill 1 ;Wei Shao 3 ; Elizabeth Fyne 2 ; XiaolinWu 1 ; John M. Coffin 4 ; Stephen H. Hughes 1 ; JohnW. Mellors 2 ; Frank Maldarelli 1 1 National Cancer Institute (NCI), Frederick, MD, US; 2 University of Pittsburgh, Pittsburg, PA, US; 3 Frederick National Laboratory for Cancer Research, Frederick, MD, US; 4 Tufts University, Boston, MA, US; 5 National Cancer Institute (NCI), Frederick, MD, US Background: Clonal expansion of infected cells can contribute to HIV-1 persistence. We recently reported that a clonally-expanded population of HIV-infected cells was responsible for persistent viremia despite ART in a patient with metastatic squamous cell carcinoma (Maldarelli, et al., 2014). We conducted extensive ante- and post-mortem analysis of HIV-infected cells from this patient to investigate the organ and tumor distribution of this clonal variant. Methods: Plasma and peripheral blood mononuclear cells (PBMC) were obtained ante mortem, and samples from spleen, ileum, rectum, 5 lymph nodes, and 5 tumor metastases were obtained at autopsy. Tissues were characterized by histo- and immunohistochemistry and qPCR of the genomic DNA was used to measure the levels of HIV gag sequences. A new single genome sequencing assay was performed to amplify the 800bp nef-U3 region from blood and tissues. Integration site flanking sequences were used to PCR amplify the full length of expanded proviruses. An ex-vivo infectious virus recovery assay was developed to characterize inducible HIV variants from PBMC-derived CD4+ cells. Sequences were subjected to phylogenetic and statistical analyses. Results: HIV sequences (N=317) were recovered from plasma, PBMC, spleen, lymph nodes, and tumor tissues, which were infiltrated with both CD4+ and CD8+ T cells. In general, HIV variants were well mixed across blood and tissues and there was no evidence of localized replication. One provirus from a highly amplified clone (AMB-1), known to produce the majority of the HIV RNA detected in plasma, was enriched in tumor tissues. AMB-1 proviruses were present in each tumor metastasis, with an overall abundance in tumor tissue significantly (3.5-fold) greater than that detected in lymphoid tissues (p=0.0005). The full length AMB-1 sequence revealed an intact provirus and no drug resistance mutations. AMB-1 RNA was recovered in day 7 culture supernatants after stimulation (PHA, irradiated blasts) and co-culture of CD4+ T-cells with CD8-depleted allogeneic blasts but declined with continued culture associated temporally with outgrowth of other variants. Conclusions: This study is the first report of residual viremia caused by an expanded cell carrying a specific intact HIV provirus. Cells from this clone accumulated specifically in cancer metastases. These observations suggest that immune stimuli, like tumor antigens, can contribute to cell expansion, and perhaps to the activation of the provirus and release of virions into plasma. 106 Analysis of HIV RNA in Single Cells Reveals Clonal Expansions and Defective Genomes Background: Little is known about HIV-1 provirus expression in patients on suppressive ART. Characterizing cell-associated RNA (CAR) sequences in single cells can reveal their relationship to proviral populations and to persistent viremia. Here, we describe a new, sensitive method to assess the genetics of HIV CAR in viremic and virologically suppressed patients. Methods: HIV-1 CAR was extracted from 4-6 separate aliquots of PBMCs from 3 patients using methods that were verified (by spiking known quantities of ACH2 cells into HIV negative PBMCs) to have high recovery of HIV-1 DNA and RNA. DNase-treatment and cDNA synthesis were optimized to ensure complete degradation of HIV DNA and high efficiency of long cDNA synthesis. Products were diluted to <1 HIV cDNA molecule/reaction, amplified, and sequenced (CAR-SGS). In patients who initiated ART with high HIV diversity, identical RNA sequences from the same aliquot of PBMCs were assumed to be derived from the same cell expressing HIV RNA, while identical sequences in different aliquots were assumed to be derived from clonally expanded, expressing cells. Single-genome sequences of proviral DNA and plasma HIV-1 RNA were compared to CAR sequences using standard bioinformatics methods. Results: We developed and optimized methods for CAR-SGS to be used to profile cellular HIV-1 RNA expression in patients. 3 patients were studied: 1 was untreated and viremic and 2 were suppressed on ART. An average of 41 single cells per patient was analyzed. We found 53% of the HIV expressing cells in the viremic patient to be “high producers” (more than one RNA molecule detected) and 30% to be high producers in the suppressed patients. The diversity of the total RNA populations both before and during ART was ~1% in pro-pol , and G to A hypermutants were detected in the suppressed patients at similar levels to their proviral DNA populations (20% and 22%). We also detected HIV-1 expression from clonally expanded populations in one suppressed patient. Conclusions: A newmethod to characterize the expression of HIV-1 proviruses that persist during ART reveals high levels of expression of RNA from infected individuals as well as clonally expanded cells. Further studies are needed to determine if HIV-1 expression results from spontaneous reactivation from latency or continuous low-level virus transcription and if these cells can be the source of viral rebound when ART is interrupted. 107 Low-Level HIV Viremias Originate in Part From Infected Proliferating Cells Marta E. Bull 1 ; Sherry McLaughlin 2 ; Hannah Huang 2 ; Sheila Styrchak 2 ; Jaime Soria 3 ; EduardoTicona 3 ; Alberto La Rosa 4 ; Caroline Mitchell 5 ; James Mullins 1 ; Lisa Frenkel 1 1 University of Washington, Seattle, WA, US; 2 Seattle Children’s Research Institute, Seattle, WA, US; 3 Hospital Nacional dos de Mayo, Lima, Peru; 4 Investigaciones Médicas en Salud (INMENSA), Lima, Peru; 5 Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, US; 6 University of Washington, Seattle, WA, US Background: Understanding mechanisms that allow HIV infection to persist despite suppressive antiretroviral therapy ( ART) may help develop cure strategies. We observed that cells with HIV integrated into genes associated with cell proliferation or cancer form persistent clones, defined as multiple cells with identical proviruses (HIV env C2-V5) and with identical integration sites ( IS ). We hypothesized that these cell clones can produce virions, evident as low-level viremias ( LLV ). Methods: A subset of HIV-infected ART-naïve subjects, enrolled into a 2-year observational study just before the initiation of nevirapine-based ART, were selected for this study based on detection of LLV (40-500 copies/mL) after one-year of suppressive (<40 copies/mL) ART. Samples were collected every 3 months over the 2 year study. HIV env (C2-V5) sequences were derived from peripheral blood mononuclear cells ( PBMC ) and plasma prior to starting ART and at a single LLV visit by single-genome-analysis ( SGA ). Multiple chromosomal IS and associated HIV env sequences were generated using an integration site looping assay ( ISLA ) (PMID25011556) from PBMC at the LLV visit that was diluted to a median single infected cell. IS were aligned using Bowtie and the location in the human genome determined using BLAT. HIV env sequences from LLV and PBMC were aligned with MUSCLE, and phylogenetic trees generated using DIVEIN. The proportion of LLV with env sequences identical to those in infected proliferating PBMC were evaluated by Fisher’s exact test and distances frommost recent common ancestor ( MRCA) were evaluated by Wilcoxon signed rank test. Results: Eight participants had LLV on 21/64 study visits. A median of 21 env sequences (range 19-28) were generated from a single LLV, and 90%were identical within a subject. A total of 287 IS sequences were generated from PBMC samples at the LLV visit (median 42 IS/subject, range 27-47). Proliferation of infected cells, evident frommultiple cells with identical IS, was observed in 6/8 subjects. Two of these six subjects had proliferating clones with env sequences identical to those in LLV. Three of the six subjects with LLV sequences without linkage to proliferating PBMC had evidence of ongoing viral evolution, and hence continuing viral replication. AnnWiegand 1 ; Jonathan Spindler 1 ;Wei Shao 2 ; Feiyu Hong 3 ; Anthony R. Cillo 3 ; Elias Halvas 3 ; Elizabeth Fyne 3 ; John M. Coffin 4 ; JohnW. Mellors 3 ; Mary F. Kearney 1 1 National Cancer Institute, Frederick, MD, MD, US; 2 Leidos, Frederick, MD, US; 3 University of Pittsburgh, Pittsburgh, PA, US; 4 Tufts University, Boston, MA, US

Oral Abstracts

138

CROI 2015