CROI 2016 Abstract eBook
336 Sustained HIV Release by Single Persisting CD4+ T Cells During Latency Disruption
Jason M. Hataye 1 ; Joseph P. Casazza 2 ; David Ambrozak 1 ; Eli Boritz 2 ;TakuyaYamamoto 3 ; Daniel C. Douek 2 ; Alan S. Perelson 4 ; Richard A. Koup 2 1 NIH, Bethesda, MD, USA; 2 VRC, NIAID, NIH, Bethesda, MD, USA; 3 Osaka Univ, Osaka, Japan; 4 Los Alamos Natl Lab, Los Alamos, NM, USA
Background: While the magnitude, timing, persistence, and quality of HIV released from the source CD4+ T cells undergoing latency disruption are unknown, both multi-day sustained HIV release and instantaneous burst stochastic models of HIV replication have been proposed. Which model best represents latency disruption may have important implications for detecting replication-competent virus. Methods: We used HIV gag RNA RT-PCR to quantify HIV release from isolated primary CD4+ T cells undergoing latency disruption in limiting dilution cultures. Viral inhibition cultures with efavirenz revealed released virus without new rounds of infection and were compared to respective viral outgrowth cultures. Sustained release and instantaneous burst stochastic models were implemented using the Gillespie algorithmwith parameters derived from applying the deterministic model to experimental data. Results: While many outgrowth wells accumulated high amounts of virus, there were also many with declining virus that was nevertheless replication-competent, confirmed by virus transfer from the original wells to new outgrowth wells. Culture wells with >77% probability of being seeded by a single virus-producing cell were analyzed for viral release. Half of cells releasing virus did so in the first 4 days of culture. The accumulated virus for a well varied from less than 120 to 30,000 HIV gag RNA copies. Although a low amplitude instantaneous burst dynamic pattern was observed, the vast majority of accumulated HIV RNA was attributed to a higher amplitude virus release pattern sustained for 2-6 days. The intrinsic decay half-life of virus in cultures in which infection was blocked was 3 days. Applying this result to the deterministic model, we estimated an average total sustained release per cell of 5500 HIV RNA copies. Conclusions: Our results are consistent with a multi-day sustained virus release stochastic model that predicts, compared to an instantaneous burst model, higher virus extinction probabilities. Because extinction probabilities are highest at extremely low infected cell numbers, such as single infected cells at limiting dilution, the sustained release stochastic model predicts, and our results show, that replication-competent virus will be present in cultures that are negative for viral outgrowth. Our results further demonstrate that single virus-producing cells can survive and produce virus over several days without succumbing to viral cytopathic effects. 337 Analysis of HIV Proviruses in Clonally Expanded Cells In Vivo Francesco R. Simonetti 1 ; Jonathan Spindler 2 ; XiaolinWu 3 ; Shawn Hill 2 ;Wei Shao 4 ; JohnW. Mellors 5 ; Stephen H. Hughes 6 ; Mary F. Kearney 6 ; Frank Maldarelli 6 ; John M. Coffin 7 1 Univ of Milan, Milan, Italy; 2 HIV Dynamics and Replication Prog, NCI, Frederick, MD, USA; 3 Frederick Natl Lab, Frederick, MD, USA; 4 Leidos Biomed Rsr, Inc, Frederick, MD, USA; 5 Univ of Pittsburgh, Pittsburgh, PA, USA; 6 NCI, Frederick, MD, USA; 7 Tufts Univ, Boston, MA, USA Background: Recently, we and others found that clonally expanded populations of HIV infected cells persist during cART. Some cells were highly expanded; a clone with an integrant in the HORMAD2 gene accounted for c. 50% of all of the infected cells. Integration into specific introns of BACH2 or MKL2 increased the expansion and/or survival of infected cells. The proviruses in these clonally expanded cells have not been adequately characterized. One highly expanded provirus was found to be replication-competent (Simonetti et al. CROI 2015). We now report the characterization of other proviruses in highly expanded clones, including those integrated into the HORMAD2 and MKL2 genes. Methods: Longitudinal PBMC samples were obtained from two volunteers with chronic HIV infection. Integration sites were determined as previously described (Science 345: 179, 2014). Proviruses integrated in HORMAD2, MKL2 , and an intergenic region of the X chromosome were selectively amplified using specific primers for HIV and the flanking host DNA. Results: The clone carrying the integrant in an intron of the HORMAD2 was present both pre- and on-cART therapy and accounted for 50-80% of all of the infected cells after 7-8 y on cART, implying that expansion of the clone started early in infection. Proviral sequence analysis revealed a 675 nt single LTR with intact promoter elements. Similarly, analysis of the proviral integrant in an intergenic region of the X chromosome from the second donor showed it was a solo LTR. Seven MKL2 integrants in introns 4 or 6 of MKL2 in the same orientation as MKL2 transcription were also characterized from the second donor. One of the proviruses had a large pol- U3 internal deletion; of the remaining, 4 proviruses showed evidence G to A mutation leading to multiple stop codons and 3 were found to lack intact tat or rev . All seven, however, had intact LTR promoter elements and retained the major splice donor sequence. Conclusions: Proviruses in expanded clones can be intact, hypermutated, partially deleted or consist of only a single LTR. The proviruses that affect the function of the MKL2 gene to promote cell survival have intact LTR regulatory elements but some lack tat, which is normally required for transcription of the HIV genome. 338 Viral Evolution Analysis for the Distinction of True HIV-1 Elite-Controller Patients Maria Pernas ; Concepcción Casado; Isabel Olivares; Cecilio López-Galindez; for the HIV Controllers Consortium of the AIDS Spanish Network Inst de Salud Carlos III, Madrid, Spain Background: Elite controllers (EC) are a sub-set of HIV-infected patients able to spontaneously maintain viral loads below the limit of detection without therapy. Some EC remain with undetectable viral load, and they could be considered as the true elite controllers, while other undergoes events of viral replication. The distinction between both groups of EC is of major importance . In this work, we propose to study viral evolution as a marker to distinguish true EC. Methods: Serial samples of 15 EC, HIV-1 sero-positive individuals without antiretroviral therapy for at least one year and with 2 viral loads determinations < 50 copies / ml were included. After one year of follow-up, 5 maintained the virological control whereas 10 patients showed an increase in viral load > 50 copies/ml. For virological analysis, we compared samples when viral load was <50 copies/ml (t=0) in both group of patients. Proviral DNA was extracted from PBMC for amplification and sequencing of viral quasispecies by limiting dilution PCR in env and gag genes. Phylogenetic trees were constructed and calculations of diversity and viral dating (Casado et al., 2000) in both groups were performed. We also analyzed the presence of escape mutations in the gag gene. Results: Viral diversity in the env C2-V5 region in t=0 was statistically superior (p = 0.004) in patients who lost control (mean = 0.03 ± 0.01) than in those who maintained control (mean = 0.0028 ± 0.003). Viral sequences in patients without loss of control dated close to the time of primary infection, while viral sequences from patients who lost control corresponded to the year of the sample. Overall, no escape mutations in gag related to the loss of control were observed. However, when escape mutations (T242N for example) were detected, these mutations pre-existed in the t=0 samples. Conclusions: Viral evolution analysis allowed the distinction of two evolutionary patterns in patients with the same viral control phenotype. EC patients with virological control showed no signs of evolution (evolutionary stasis), suggesting that the virus has not replicated since primo-infection. In the other group of EC patients, we detected viral diversification even with undetectable viral load, indicating viral replication along the infection. Thus, lack of viral evolution could be used as a diagnostic marker for the identification of true EC. These EC patients could be potentially considered as functionally cured. Casado et al JAIDS 2000 23:68-74 339 Optimization of PCR Amplicons to Predict Clonality of Full-Length HIV-1 Sequences Sarah B. Laskey 1 ; Katherine M. Bruner 2 ; Robert Siliciano 3 1 Johns Hopkins Univ, Baltimore, MD, USA; 2 Johns Hopkins Univ Sch of Med, Baltimore, MD, USA; 3 Howard Hughes Med Inst, Baltimore, MD, USA Background: Virus-driven clonal expansion of latently infected memory CD4 + T cells may provide a mechanism of HIV-1 persistence in the presence of suppressive antiretroviral therapy. The only conclusive way to prove the presence of a clonally expanded infected cell is to sequence complete, identical viral genomes and integration sites multiple times in independent experiments. Due to the prohibitive difficulty of this measurement, many studies assume clonality when independent viral sequences are identical in short PCR amplicons of ~500-1500bp. In this study, we analyzed previously published full-length HIV-1 sequence alignments from 28 patients to determine which PCR amplicons, if any, are sufficient to demonstrate the clonality of an entire viral genome.
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