CROI 2015 Program and Abstracts

Abstract Listing

Oral Abstracts

55LB HVTN505 Breakthrough Sequences Show HIV Vaccine-Associated Differences in Env-gp120 Morgane Rolland 1 ; Allan deCamp 2 ; Breana M. Hall 3 ; SodsaiTovanabutra 1 ; Mario Roederer 4 ; Scott M. Hammer 5 ; Magdalena E. Sobieszczyk 5 ; Peter B. Gilbert 2 ; Jerome H. Kim 6 ; James Mullins 3 The HVTN505 Sieve Analysis Group 1 US Military HIV Research Program; Henry M. Jackson Foundation for the Advancement of Military Medicine Inc, Silver Spring, MD, US; 2 Fred Hutchinson Cancer Research Center, Seattle, WA, US; 3 University of Washington, Seattle, WA, US; 4 Vaccine Research Center, Bethesda, MD, US; 5 Columbia University, New York, NY, US; 6 US Military HIV Research Program, Silver Spring, MD, US Background: The HVTN505 HIV vaccine study enrolled 2,504 US participants in a phase IIb test-of-concept efficacy trial of a DNA/rAd5 vaccine expressing Gag/Pol/Nef from subtype B and Env proteins from subtypes A/B/C. Although the vaccine showed no efficacy with respect to HIV acquisition or post-infection viral load (Hammer, NEJM, 2013), we analyzed breakthrough HIV isolates to evaluate potential sieve effects. Methods: Sequencing was performed following PCR amplification of near full-length single HIV genomes derived from plasma specimens at HIV diagnosis. Phylogenetic and statistical analysis methods assessed individual genes, k-mers, and amino acid (AA) sites to compare breakthrough sequences from vaccine and placebo recipients, with multiplicity adjustment procedures (20% false discovery rate). Results: We characterized 480 HIV genomes from 27 vaccine and 20 placebo recipients. All infections were with subtype B. Infections were established by multiple founder variants in 5 of 26 vaccine recipients (19%) and in 8 of 20 placebo recipients (40%) (Fisher’s exact test, p= 0.19). Two vaccine recipients were infected by two phylogenetically- unrelated viruses ( env diversity= 8.9% vs 0.3% for other subjects). Intra-host diversity was significantly lower in vaccine than placebo recipients for Gag, Pol, Env-gp120 and Vif proteins (p ≤ 0.04, q ≤ 0.09). The distance between founder and vaccine sequences was measured for each participant and compared across treatment groups. While differences in other proteins were not significant, Env-gp120 sequences from vaccine recipients were significantly more distant from the subtype B vaccine insert than sequences from placebo recipients (median = 0.30 vs 0.29; Mann-Whitney test, p= 0.01, q= 0.12). Vaccine/placebo AA differences in Env-gp120 were identified at six 9-mers starting at HXB2 positions 28, 86, 193, 365, 427, and 467, and at site 133 (p< 0.005, q< 0.2). Mapping of the 9-mer starting at AA193 on a trimer structure showed these residues in contact with the tip of the V3 loop of another monomer, thus possibly affecting the binding of V3- specific antibodies (Fig.1) – an interesting finding as the HVTN505 vaccine regimen elicited strong anti-V3 responses.

Oral Abstracts

Figure 1. Env-gp120 residues corresponding to the 9-mer starting at HXB2 position 193 that distinguished the vaccine and placebo groups are figured with circles on the BG505. SOSIP structure (PDB ID: 4NCO). The start and end residues of the 9-mer (AA193 and 201) are numbered on the monomer on the left, the residues are colored in green and turquoise with a mirror image on another monomer. At the tip of the V3 loop, GPG is in magenta, with the 5 atoms in the proline’s ring shown as spheres (the N atom being a blue sphere, carbon atoms in magenta). Conclusions: Our results suggest an HVTN505 vaccine-driven sieve effect against Env-gp120. Analysis of breakthrough HIV isolates in vaccine efficacy trials is important to assess whether a vaccine elicited a selective pressure in vivo and to identify which responses are associated with efficacy and which are not. 258 PCR-Free Full Genome Characterization of Diverse HIV-1 Strains by Nextgen Sequencing Viswanath Ragupathy 1 ; Feng Gao 2 ; Ana Sanchez 2 ; Marco Schito 3 ;Thomas Denny 2 ; Michael Busch 4 ; Jiangqin Zhao 1 ; Christelle Mbondji 1 ; SaiVikramVemula 1 ; Indira Hewlett 1 1 US Food and Drug Administration, Silver Spring, MD, US; 2 Duke Human Vaccine Institute and Departments of Medicine, Duke University Medical Center, Durham, NC, US; 3 Henry Jackson Foundation, DAIDS, NIAID, Bethesda, MD, US; 4 Blood Systems Research Institute/University of California San Francisco, San Francisco, CA, US Background: HIV-1 genotyping is an important tool for clinical and epidemiological studies. High level of genetic variation, recombination and mutations pose difficulty in successful PCR amplification of HIV-1 genomes. In addition, new emerging subtypes may not be detected with standard PCR primers. Here, we report a novel PCR-free multiplex method for characterization of full length HIV-1 genomes (~9.7kb) using the Nextgen RNA Seq approach. Methods: A total of 27 diverse HIV-1 strains representing subtypes A-G, CRFs, URFs and Group O were obtained from the Global HIV-1 diversity panel that was assembled at the Duke EQAPOL. Viral RNA was extracted, reverse transcribed as described in the Illumina Truseq RNA Kit and sequenced using the MiSeq platform. Sequence reads were quality filtered and reference mapped using CLC genomic work bench software v6.0.4. Consensus sequences were generated for each virus and used for phylogenetic tree analysis using the neighbor-joining method based on the Kimura two-parameter substitution model and recombination patterns were determined using Simplot. Drug resistance was inferred from the Stanford HIV drug resistance program, and co-receptor usage was determined using the Geno2Pheno (g2p) 5-10% FPR. Results: The multiplex RNA sequencing approach yielded >10000x coverage for each of the viral genomes. Pools of viral isolates were de-multiplexed and discriminated using bio-informatics. After filtering reads specific for HIV-1, each position in the viral genome had >1000x coverage. This approach enabled reconstruction of whole genome HIV-1 haplotypes accurately including flanking LTRs. Analysis of full HIV-1 genome sequences using Simplot correctly identified 15 pure subtypes, one Group O virus, and recombination patterns of 8 CRFs and 3 URFs. All these HIV subtypes identified were comparable to Sanger sequencing. In addition, this approach revealed NNRTI, integrase and protease drug- specific minor variants and drug resistance mutations with >1000x coverage. The g2p analysis predicted 89% of isolates as being R5 tropic, and the remaining were identified as X4 tropic. Conclusions: We have developed a reliable, PCR-free and multiplexing approach to characterize whole HIV-1 genome sequences. This novel PCR-free method can be used for characterization of new, emerging unknown subtypes or recombinants and to reduce PCR-derived sequence errors. The multiplexing approach makes this NGS method more cost- effective and less labor-intensive than conventional methods.

104

CROI 2015