CROI 2016 Abstract eBook

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Poster Abstracts

viral outgrowth assay (qVOA) was performed. Subsequently, a nested substudy of 10 patients was set-up to analyse HIV-1 integration sites and tat/rev induced limiting dilutions assay (TILDA). The selected patients had low level of total HIV-1 DNA (<250 cp/10 6 PBMCs) or high level of qVOA (> 2 IUPM). The percentage of integration site clonality was estimated based on results from the integration site analysis, and the frequency of cells with inducible HIV RNA transcription were estimated using maximum likelihood method. Results: Integrated and total HIV-1 DNA were detected in all patients, and both measures correlated well (p=0.002, R²=0.85). Replication-competent virus was detected in 80% of patients by the qVOA and this correlated with integrated and total HIV-1 DNA (p=0.05, R²=0.44; p = 0.019, R²=0.54; respectively). In total 317 integration sites were analysed. A wide range of percentage of clonality (25.0-91.4%) was observed between patients. The majority of patients had an average of 30% clonality, which on average comprised of 3.4 predominant clonal integration sites per patient. Patients with higher clonal diversity (≥ 6 clones) had higher estimate for TILDA (frequency of cells with inducible msRNA > 200 cells /10 6 PBMCs) and higher CD4 nadir count (p=0.048, R²=0.46). Interestingly, one patient had an extremely high clonality (>90%), which was represented by a single clone. This patient also had no inducible virus in both VOA and TILDA, suggesting that the clonal provirus in this patient is replication incompetent. The single predominant clone was associated with the extremely CD4 nadir count in this patient (3 cells/ul.) Conclusions: We observed a good correlation between VOA and integrated and total HIV-1 DNA. Integration site sequencing revealed that most of the ART treated patients in this study had an HIV DNA reservoir consisting of 30% clonally expanded cells. These levels of clonal provirus did not affect the amount of inducible virus, except for the one patient with >90% clonality. 362 Measurement of Integrated HIV DNA by Pulse-Field Gel Electrophoresis and ddPCR Steven Lada ; Caroline Ignacio; Douglas D. Richman; Matt F. Strain Univ of California San Diego, San Diego, CA, USA Background: Assays that measure the size of the latent reservoir with high sensitivity, precision and throughput are needed for clinical studies directed toward eradication. Droplet digital PCR (ddPCR) provides an efficient, standard-independent method to measure HIV DNA, but distinguishing between integrated and unintegrated molecular forms remains a challenge. Previous attempts to remove contaminating episomal and linear unintegrated DNA have been limited by low sensitivity or assay throughput. We sought to develop an assay specific for integrated HIV DNA by initial isolation of high-molecular weight DNA via automated pulse-field gel electrophoresis (PFGE) with the BluePippin system followed by ddPCR. Methods: 8E5 cells were grown in culture, while CD4 cells were enriched from PBMCs and infected with NL43 HIV for 7 days. DNA was extracted by Qiagen QIAamp DNA Blood Mini Kit. 8E5 DNA was loaded at 1000ng or 100ng while CD4 DNA was loaded at 500ng or 50ng per well into Pippin Gel 0.75% DF Cassettes, and separated using a PFGE protocol. After collection, DNA samples were assayed by ddPCR to measure HIV 2-LTR junction, HIV gag, and cellular RPP30. Recovery efficiencies were calculated by assuming that measured copy numbers were Poisson-distributed. Results: Preliminary PFGE protocols resulted in poor recovery of high molecular weight DNA (3-10%). Further optimization, including a custom high-pass PFGE protocol greatly improved recovery of high molecular weight DNA; 29-38% for 8E5 DNA, and 24-61% for CD4 DNA. Efficient recovery of high molecular weight DNA increased with the amount of loaded DNA. Contaminating episomal 2-LTR HIV DNA in infected CD4s was reduced by 98% for 500ng DNA (CI: 97-99%) and 97% for 50ng DNA (CI: 93-100%) as measured by ddPCR. In 8E5, which lack lowmolecular weight HIV DNA, HIV gag and RPP30 were recovered with similar efficiency. The coefficient of variation of the normalized HIV gag copy number per cell for the combined PFGE-ddPCR protocol was 14%. Conclusions: Automated PFGE to remove episomal and linear species of HIV DNA permits the recovery of chromosomal HIV DNA with high purity and efficient recovery. This technology provides a sensitive and precise approach to the measurement of integrated HIV DNA with sufficient throughput for translational research studies. 363 Inhibition of the IN-LEDGF/p75 Interplay by LEDGINs Reduces Reactivation From Latency Background: Despite the success of cART, HIV-1 persists in a latent state in patients. Persistence is a consequence of proviral integration. LEDGF/p75 is the pivotal chromatin tethering factor targeting HIV integration into active transcription units through its interaction with HIV-1 integrase. We investigated the role of integration site selection in the establishment of HIV persistence employing LEDGF/p75 knockout cells and LEDGINs interfering with the LEDGF/p75-IN interplay. Methods: To evaluate whether the reactivation potential of the quiescent proviral reservoir is dependent on integration site selection during reservoir establishment, LEDGF/ p75 WT and KD/KO cell lines were infected with HIV NL4.3-tCD34 or a double reporter virus that allows direct visualization of the quiescent pool. Formation of the quiescent pool in primary CD 4+ T-cells was studied in the presence and absence of LEDGINs during primary infection. Cells were reseeded 2 weeks post infection and reactivation by different latency reversing agents was monitored by FACS analysis. Integration sites were amplified using LM-PCR. Results: LEDGF/p75 depletion hampers HIV-1 reactivation by latency reversing agents in cell culture. LEDGIN-mediated inhibition of the LEDGF/p75-IN interaction shifts integration out of transcription units and relocalizes the 3D nuclear location of the provirus away from the nuclear rim, comparable with previous observations for LEDGF/p75 depletion. Interestingly, LEDGIN treatment increases the relative fraction of quiescent provirus and blocks HIV reactivation from latency, both in a dose-dependent manner (IC 50 CX014442 ≈ 7.24 μM). This approach may represent a viable alternative to the shock-and-kill strategies for functional eradication of HIV in patients as LEDGIN treatment apparently succeeds in rendering almost 100% of the virus into a quiescent state refractory to reactivation. Conclusions: LEDGF/p75 determines the integration site pattern of HIV. The presented data in cell lines and in primary cells support our hypothesis that LEDGINs, small molecules that inhibit the interaction between integrase and LEDGF/p75, retarget integration away from the nuclear rim and in genomic regions that are less sensitive to reactivation. If further corroborated in clinical trials, this strategy may pave the way towards a functional cure. Pushing the provirus into quiescence could drive the basic reproduction number of HIV below a threshold required for sustained infection even after treatment interruption. 364 Treatment of HIV and AML by Allogeneic CCR5-d32 Blood Stem-Cell Transplantation Guido Kobbe 1 ; Rolf Kaiser 2 ; Elena Knops 3 ; Nadine Luebke 4 ; Gabor A. Dunay 5 ; Johannes Fischer 6 ; Falk Huettig 1 ; Rainer Haas 1 ; Dieter Haeussinger 1 ; Bjoern-Erik O. Jensen 1 1 Duesseldorf Univ Hosp, Heinrich-Heine Univ Duesseldorf, Duesseldorf, Germany; 2 Inst of Virology, Univ of Cologne, Cologne, Germany; 3 Univ Clinic of Cologne, Cologne, Germany; 4 Inst for Virology, Heinrich-Heine-Univ, Univ Hosp, Duesseldorf, Germany; 5 Heinrich Pette Inst, Hamburg, Germany; 6 Inst of Transplantation Diagnostics and Cell Therapeutics, Heinrich-Heine Univ Duesseldorf, Duesseldorf, Germany Background: The Berlin patient is presumed to be the only person cured from HIV-infection by hematopoietic stem cell transplantation (HSCT) from a homozygous CCR5-d32 unrelated donor. Attempts to reproduce cure by HSCT have failed because of either viral rebound or death due to the underlying malignancy. We here report a patient alive, well and negative for proviral DNA 900 days after allogeneic CCR5-d32 HSCT. Methods: A 41y old HIV-infected male patient was diagnosed acute myeloid leukemia (AML, inv16, CBF-MYH11) in 01/2011. Since the diagnosis of HIV-infection in 10/2010 he had been treated with TDF/FTC+ DRV (01/2011 VL 44 cop/mL; CD4 + 474 cells/µl). To avoid interactions with chemotherapy DRV was switched to RAL in 03/2011. He achieved CR of the AML after 1 induction course (ICE) and received a 2 nd induction and 3 consolidation courses according to AML-SG 07/04. In 09/2012 AML relapsed and he was treated with A-HAM and a 2 nd cycle high-dose cytarabine. While in 2 nd CR he received 8.74x10E6/kg unmodified peripheral blood stem cells from a female 10/10 CCR5-d32 DKMS-donor after conditioning with fludarabine and treosulfan in 02/2013. Before transplant HIV resistance analysis was performed for PR, RT, IN and viral tropismwas determined. Results: There were no significant resistance mutations and the coreceptor-usage was predicted as R5-tropic (Sanger sequencing: FPR 44.5%; NGS: 0.14% X4 at 3.5% FPR; geno2pheno). The proviral DNA load was 29400 cop/mL and in the western blot all anticipated bands could be detected. During transplant and until today the patient remained on Zeger Debyser 1 ; LenardVranckx 1 ; Jonas Demeulemeester 1 ; Annegret Boll 2 ; Rik Schrijvers 1 ; EricVerdin 3 ; Suha Saleh 1 ; Frauke Christ 1 ; Rik Gijsbers 1 1 Katholieke Universiteit Leuven, Leuven, Belgium; 2 Univ of Trento, Trento, Italy; 3 Gladstone Inst of Virology and Immunology, San Francisco, CA, USA

Poster Abstracts

138

CROI 2016