CROI 2015 Program and Abstracts

Abstract Listing

Oral Abstracts

be overcome before routine use in cross-sectional settings: 1) streamlining the cost and workflow, 2) ensuring proper classification between multiple-founder recent infections and chronic infections. Methods: For enhancing the cost efficiency of the sequence-based assay, we developed a high-throughput next-generation sequencing platform; a signal-masking bioinformatics pipeline was devised to analyze 18,434 envelope gene segments (HXB2 7212-7601) obtained from 12 incident and 24 chronic patients. To give the assay power to appropriately discriminate multiple-founder recent infections from chronic infections, we formulated a mathematical model which posits the intersequence nucleotide base difference distribution of each subject’s sequence sample as a function of infection duration and the number of founder sequences. This model was tested by analyzing HIV subtype B and C samples from 40 incident subjects with multiple founder viruses. Results: First, the cost-effective pyrosequencing platform correctly classified all 12 incident subjects (100% sensitivity) and 23 out of 24 chronic subjects (96% specificity). Our signal-masking bioinformatics pipeline yielded a process error rate of 5.8 × 10 -4 per base. Sampling simulations showed that the biomarkers were tolerant of the two factors most likely to affect the accuracy: sequencing errors and template resampling. Second, a quantitative guideline for segregating viral lineages was provided by our mathematical model, enabling us to assess when each subject was infected. The infection periods obtained from our model estimates and from Fiebig laboratory staging showed a statistically significant linear relationship (p<0.0005), correctly identifying all 40 individuals with incident infections. Conclusions: The high-throughput platform permits the assay to be cost-effective, and when it is combined with our mathematical model, we can obtain recency signatures from the complex gene pool that arises frommultiple founder viruses. Our sequence-based approach marks significant progress towards accurate determination of HIV incidence from genomic readouts measured from cross-sectional samples from a single blood draw. 255 A Comprehensive Analysis of Primer IDs to Study Heterogenous HIV-1 Populations David Seifert 1 ; ArminTöpfer 1 ; Francesca Di Giallonardo 2 ; Stefan Schmutz 2 ; Huldrych F. Günthard 2 ;Volker Roth 3 ; Niko Beerenwinkel 1 ; Karin J. Metzner 2 1 ETH Zurich, Basel, Switzerland; 2 University Hospital Zurich, Zurich, Switzerland; 3 University of Basel, Basel, Switzerland Background: Haplotyping of HIV-1 populations is an essential step to better understand the evolutionary dynamics of the virus. With the advent of next-generation sequencing (NGS), haplotyping of viral populations has become feasible. Since HIV-1 is highly heterogeneous, several statistical methods have been devised to deal with error-prone NGS data, however, they often do not capture the population correctly. In order to correct for errors, the use of PrimerIDs (primer identifiers) has been proposed. Here, we used PrimerIDs to systematically estimate different enzymatic error rates and to comprehensively study the feasibility of PrimerIDs. Methods: Plasmids containing full-length genomes of 5 HIV-1 clones were separately amplified in bacteria and then transfected into 293T cells. Generated infectious HIV-1 particles were pooled, DNase treated, and a fragment of the pol gene was reverse transcribed with SuperScript III reverse transcriptase (RT) and primers containing random 10-mers. Reverse transcription was performed in six independent replicates. Subsequently, nested PCR was performed using Platinum Taq DNA Polymerase followed by adapter ligation and sequencing with Illumina MiSeq. Results: From an average number of 1.1 million reads, we called consensus sequences for PrimerIDs, each supported by at least 10 sequencing reads, to yield on average 11,000 consensus sequences per replicate. From these consensus sequences, we could call all mutant bases from the five reference viruses. We estimated a RT error rate of 6.23e-4 (95% CI: [6.13e-4, 6.32e-4]). We inferred the recombination rate of the RT to be 3.44e-5 (95% CI: [2.26e-5, 4.92e-5]). The PCR substitution rate of 1.18e-4 (95% CI: [1.14e-4, 1.22e-4]) was determined from those mutants having arisen in the first cycle of the PCR. We calculated the total number of transcribed RNAs to be on the order of 60,000 from the observed collision rate of 2%. We observed no sequence-specific bias in PrimerID frequencies, the same RT efficiencies as compared to commonly used short, specific RT primers, and no effects of primerIDs on the estimated distribution of the five viruses in the mix. Conclusions: PrimerIDs allow for determining error rates in RT-PCR-NGS protocols and are applicable to study HIV-1 heterogeneity when attention is paid to collision rates. Given these advantages, the protocol is still labor- and cost-intensive and does not significantly improve on the variance of frequency estimates. 593 Analysis of Resistance Haplotypes Using Primer IDs and Next Gen Sequencing of HIV RNA Valerie F. Boltz 1 ; Jason Rausch 1 ;Wei Shao 2 ; Charles Coomer 1 ; JohnW. Mellors 3 ; Mary Kearney 1 ; John M. Coffin 4 1 National Institutes of Health (NIH), Frederick, MD, US; 2 Leidos, Frederick, MD, US; 3 University of Pittsburgh, Pittsburgh, PA, US; 4 Tufts University, Boston, MA, US Background: Targeted sequencing technologies using primer IDs can result in more accurate representations of HIV-1 populations but PCR bias and recombination have hampered progress. Here we describe a newmethod for library construction that produces a larger number of tagged consensus sequences, increases sensitivity of haplotype determination, and reveals the sources of recombination. Methods: Each molecule of cDNA frommixtures of varying percentages of wild-type and mutant HIV-1 pol transcripts containing 14 drug resistance mutations was tagged uniquely using a gene-specific primer with primer IDs. cDNAs were then PCR amplified using two methods: (1) 90mer primers containing required MiSeq sequences; (2) 22mer primers containing uracil followed by digestion, cleavage and ligation to linkers containing MiSeq sequences. DNA was sequenced using paired-end MiSeq Illumina technology and consensus sequences were derived from a super-majority ( ≥ 80% consensus) for each unique ID. Consensus sequences were analyzed for PCR bias, errors, recombination, and sensitivity for detecting haplotypes. Results: Of the total cDNA molecules used as template, amplified cDNA with unique tags ranged from 3-19% for method 1 and from 15-52% for method 2. The average error rates for method 1 and 2 were 9.3x10 -5 and 1.4x10 -4 , respectively, both comparable to RT error rates. The PCR recombination rate for method 1 was 0.16% but only 0.01% for method 2. Method 1 was able to detect drug resistance mutations down to 0.01% and method 2 down to 0.001%. The sensitivity of haplotype detection was better for method 2: for samples containing 10% or 1%mutant, method 1 never detected linkage of all 14 mutations, whereas method 2 detected all 14 33-35% of the time. Method 2 always detected linkage of the 8 mutations nearest the 3’ end of the amplicon suggesting that PCR recombination is due to incomplete cDNA synthesis. Conclusions: A linker ligation method of amplifying tagged cDNA reduced both PCR bias and recombination rate compared to standard methods, and was superior at detecting haplotypes within 200bp of the 3’ end of the template. However, it correctly detected linkage across the entire 570bp amplicon in only 1/3 of sequences, suggesting that cDNA synthesis is typically incomplete leading to PCR recombination and thus limiting sensitivity for detection of linked mutations. Improved methods are needed for cDNA synthesis to increase the reliability of haplotype determination for HIV-1 populations. 300 Effect of CMV and HIV Replication on T-Cell Exhaustion and Senescence During ART Jennifer M. Dan 1 ; Marta Massanella 1 ; David M. Smith 1 ; Eric S. Daar 2 ; Michael P. Dube 3 ; Richard Haubrich 1 ; Sheldon Morris 1 ; Sara GianellaWeibel 1 1 University of California San Diego, La Jolla, CA, US; 2 Harbor–University of California Los Angeles Medical Center, Torrance, CA, US; 3 University of Southern California Keck School of Medicine, Los Angeles, CA, US Background: HIV-infected men who have sex with men (MSM) are nearly universally infected with CMV, and both viruses are associated with T-cell dysfunction and inflammation-related morbidities. The effect of asymptomatic CMV replication and persistent HIV transcription during suppressive ART on markers of T cell exhaustion and senescence is poorly defined.

Oral Abstracts

106

CROI 2015