CROI 2016 Abstract eBook

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Poster Abstracts

selection at DRM sites compared to other sites (Exact p >0.05), and 3) no association between AM and DRM. Longitudinal analyses within recipients revealed a significant decrease in the frequency of minority DRM over time, while overall viral diversity increased (p<0.05). Conclusions: Using data from transmission pairs, we found no evidence of sexual transmission of minority DRM. The presence of minority DRM only during early infection, when effective population size is low, is consistent with the mutation-selection balance hypothesis, in which deleterious mutations (i.e. DRMs) are more efficiently purged from the population later in infection when the larger effective population size allows more efficient selection. 488 Transmitted Drug Resistance in HIV-1 Subtype C Hyperacute Infection Urisha Singh 1 ; Avashna Singh 1 ; Marc Noguera-Julian 2 ; Manjeetha Jaggernath 1 ; Amber Moodley 1 ; Krista Dong 3 ; Bruce D.Walker 3 ;Thumbi P. Ndungu 1 ; Michelle Gordon 4 1 Univ of KwaZulu-Natal, Durban, South Africa; 2 IrsiCaixa Inst for AIDS Rsr, Badalona, Spain; 3 Ragon Inst of MGH, MIT, and Harvard, Cambridge, MA, USA; 4 Nelson R Mandela Sch of Med, Univ of Kwazulu-Natal, Durban, South Africa Background: Transmitted Drug Resistance (TDR) in HIV-1 presents a risk for the future success and longevity of Highly Active Antiretroviral Therapy (HAART) especially in resource limited settings in which no genotypic testing is conducted prior to treatment initiation and options for treatment and salvage therapy is limited. For HIV-1 subtype C, the most prevalent subtype in sub-Saharan Africa, there is limited information available on the prevalence of TDR and the persistence of TDR associated mutations, aspects which are integral in informing decisions on future treatment strategies for HIV-1. We investigated the prevalence and persistence of low frequency TDR associated mutations in HIV-1 subtype C hyperacute infection. Methods: HIV uninfected women at high risk for HIV infection were followed and sampled twice weekly as part of a comprehensive prevention and empowerment programme. Plasma samples from 14 participants, who were infected with HIV-1 during study follow-up, were obtained within one week of onset of plasma viremia (OPV) and at regular intervals thereafter and subject to ultra-deep pyrosequencing (454 Life Sciences, Roche Diagnostics). Data was analysed using the amplicon variant analyser where the cut-off for low frequency drug resistance mutations (DRMs) was 1%. Results: UDPS identified low frequency DRMs in 8 out of 14 participants (57%). The K65R nucleotide reverse transcriptase inhibitor (NRTI) associated DRM was the most prevalent low frequency DRM detected (6 out of 14 participants, 47%). Other DRMs detected included the D67N (3.88%) NRTI-associated DRM, the F53L (17.6%) and M46L (6.3%) protease inhibitor (PI) associated DRMs and the T97A integrase strand transfer inhibitor (InSti) associated DRM. The K103N NNRTI-associated DRM was detected in high frequency. Whilst the K103N DRM persisted at 1 year after OPV, the F53L and M46L DRMs reverted as early as 7 days after OPV. Conclusions: Our results showed that low frequency mutations are common in HIV-1 subtype C hyperacute infection. Most of these mutations reverted rapidly and thus would be undetectable by routine methods used in the surveillance of TDR. The possible storage of TDR associated mutations in latent reservoirs raises concern for their reemergence under drug selection pressure and their subsequent impact on treatment outcomes. Further work to identify viral species in latent reservoirs is suggested for this cohort. 489 Integrase Inhibitors-Transmitted Drug Resistance Detected by UltraDeep Sequencing Eve Todesco 1 ; Jeremy Jaffre 1 ; Cathia Soulié 1 ; Daniele Armenia 2 ; MarcWirden 3 ; Sidonie Lambert 1 ; Christine Katlama 1 ; Francesca Ceccherini-Silberstein 2 ;Vincent Calvez 1 ; Anne-Geneviève Marcelin 1 1 Sorbonne Univs, Paris, France; 2 Univ of Rome Tor Vergata, Rome, Italy; 3 APHP, Pitie Salpêtrière Hosp, Paris, France Background: Transmitted Drug Resistance (TDR) can impair first-line antiretroviral therapy response. Moreover, HIV-1 minority resistant variants (MRV) can be a source of virological failure if they are present before antiretroviral treatment: it was mainly shown for non nucleoside reverse transcriptase inhibitors first line based regimens. Few data are available for TDR Integrase Strand Transfer Inhibitors (ISTIs). In this work, we have studied resistance mutations in integrase gene by Sanger sequencing and UltraDeep Sequencing (UDS) in ISTI-naive patients. Methods: Integrase genotypic analysis was performed by Sanger sequencing and by UDS. Plasma samples of 65 treatment-naïve Men having Sex with Men (MSM) patients were analyzed from the amino acid 53 to 281. GS Amplicon Variant Analyzer was used to analyze the UDS data, with a detection threshold of MRV of 1% (forward and reverse). Resistance was interpreted according to the last version of ANRS algorithm ( www.hivfrenchresistance.org ). Results: Among the 65 patients, 60% of themwere infected by B subtype. Viruses of six patients harbored majority resistant mutations by Sanger sequencing (four L74I and two E157Q mutations). Three viruses harbored MRV detected by UDS only: two R263K (at a rate of 9.7%, mutational load: 7099 copies/mL; and 13.5 %, 8345 copies/mL) and one E138K mutations (at 4.8%, 111 copies/mL). All these mutations were retrieved among B subtype viruses. Conclusions: None of the three classical ISTIs signature resistance mutations (at positions 143, 148 and 155) were retrieved. However, in this population of MSM naive-treatment patients, the prevalence of ISTI-resistance mutations, mainly related to polymorphisms, seems to be relatively high (9.2% by Sanger and 13.8% by UDS). In conclusion, with the increase use of ISTIs in clinical practice, TDR for this therapeutic class should be carefully monitored in the future, as well as the impact of these MRV on the virological response. 490 Background: Targeted next generation sequencing (NGS) is a powerful tool for detecting low frequency, HIV-1 drug-resistant mutations but frequent in vitro recombination prevents accurate detection of linked mutations and assessment of phylogenetic relationships. We developed a new NGS-based ultrasensitive single-genome sequencing (uSGS) assay that eliminates in vitro recombinants and reduces PCR errors to investigate linkage of resistance mutations and the phylogenetics of variants in plasma samples. Methods: NGS uSGS libraries were generated by tagging cDNA molecules from plasma RNA with primer IDs, optimizing PCR, and generating overhangs on amplicons for efficient ligation of Illumina adaptors. Libraries were sequenced with paired-end Illumina MiSeq technology. A modified version of the algorithm of Zhou, et. al. was used to remove sequences whose primer IDs contained PCR/sequencing errors. The uSGS pipeline further eliminated PCR recombinants and PCR/sequencing errors by applying a “>80%majority rule” to each site in alignments of reads with the same primer ID. uSGS data were analyzed for unique linkage patterns and for phylogenetics using neighbor-joining (NJ) analyses. Results: Using the uSGS assay, a median of 1227 SGS were obtained from each of 3 plasma samples from 2 ART-experienced donors. The presence of clusters of resistant variants on independent nodes of NJ trees implied that resistant variants emerged independently and diversified primarily due to stochastic changes rather than from in vivo recombination with other variants. Within clusters of variants, rare, linked resistance mutations were detected in each sample. In the 1st sample from one donor, a single variant was detected with linked mutations in RT at codons 106, 108, and 101 (0.06% frequency) on a background of 67N (98.9% aac). In a 2 nd sample from the same donor obtained 2 weeks later, a different 67N mutation (aat, 1.1%) was linked to the previously detected rare mutations at codons 106 and 101. Similarly, in plasma from the second donor, rare (0.08% frequency) linked mutations were detected at codons 70, 108, and 184. Conclusions: The new ultrasensitive SGS assay described here can detect rare, linked mutations at drug resistance sites and permits accurate phylogenetic analyses of HIV variants. This capability will improve the understanding of resistance evolution and could help identify individuals at risk of treatment failure because of linked resistance mutations. Linkage of Rare Drug Resistance Mutations Detected by New Ultrasensitive SGS Valerie F. Boltz 1 ; Junko Hattori 1 ;Wei Shao 2 ; John M. Coffin 3 ; Frank Maldarelli 1 ; Mary F. Kearney 1 1 NCI, Frederick, MD, USA; 2 Leidos Biomed Rsr, Inc, Frederick, MD, USA; 3 Tufts Univ, Boston, MA, USA

Poster Abstracts

190

CROI 2016