CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

Codons) saturation mutagenesis. We created a library of viruses carrying every possible amino acid in an Env region downstream from the CD4 binding loop. Following fitness competition in PBMCs in the absence of immune pressure, we identified several mutations that facilitate trimer opening. Methods: We used EMPIRIC saturation mutagenesis to create a plasmid library containing all possible mutations for aa 371-380 of LN40 Env in NL4-3 full length virus. We recovered viruses from transfected 293T cells and then infected PBMCs. To quantify competitive fitness, we measured the abundance of each mutant before and after PBMC infection using deep sequencing. We then studied 14 mutations that were selected at wt levels or higher in PBMCs. We evaluated changes in Env structure by testing the sensitivity of Env+ pseudovirions to neutralization by sCD4 and mabs b6, b12, 447-52D and 2G12. Results: Substitutions at aa380 conferred sensitivity to CD4bs mab, b6 and V3 mab, 447-52D, which recognize more open trimers e.g. on laboratory strains. Substitutions at aa373, 375 in addition to 380 increased sensitivity to sCD4 compared to wt. Several substitutions conferred increased sensitivity to the CD4bs mab b12, with changes in residues 373 and 375 affecting b12, sCD4, 447-52D and 2G12 sensitivity together. Our data indicate that the residues identified play an important role in enhancing CD4 binding and/or regulating trimer opening. Conclusions: We identified specific amino acids in a region of Env proximal to the CD4 binding loop that facilitate trimer opening, increasing the exposure of neutralizing epitopes and sensitivity to sCD4. The identification of specific positions in gp120 that affect trimer opening will help the design of stably closed trimers for use in vaccines. Background: Mucosally acquired HIV-1 infection results from a limited number of variants, and these infecting strains potentially have unique properties, such as increased susceptibility to entry blockers, relative interferon-alpha (IFN- α ) resistance, and replication differences in some primary cells. There is no data about the phenotypic properties of HIV-1 envelope variants found early after acquisition among subjects infected through injection drug use (IDU). Methods: We compared the characteristics of virus envelopes among injection drug users sampled prior to seroconversion (HIV RNA+/Ab-), within 1 year (early), and more than 2 years (chronic) after estimated acquisition. Full-length HIV-1 envelopes generated from pooling multiple bulk PCRs were incorporated into a HIV-1 NL4-3 backbone to create replication competent recombinant viruses. The envelopes were examined for various phenotypes, such as replication kinetics in primary cells, gut homing receptor, α 4 β 7, reactivity, and sensitivity to IFN- α . Summary phenotypic characteristics were compared using the Wilcoxon rank-sum test. Results: Virus envelopes from 7 HIV RNA+/Ab- subjects possessed lower genetic diversity (p = 0.05) and divergence (p = 0.05) compared to 7 unrelated individuals sampled during the chronic phase of disease. The HIV RNA+/Ab- as compared to the chronic phase envelopes were significantly more sensitive to a CCR5 receptor inhibitor (p = 0.03) and IFN- α (p = 0.008) and showed a statistical trend toward greater sensitivity to a fusion blocker (p = 0.07). The early as compared to chronic infection envelopes also demonstrated a statistical trend or significantly greater sensitivity to CCR5 (p = 0.06) and fusion inhibitor (p = 0.01) and IFN- α (p = 0.1). The HIV RNA+/Ab- as compared to chronic envelope viruses replicated to a lower extent in mature monocyte derived dendritic cells – CD4+ T cell co-cultures (p = 0.03), but there were no significant replication differences in other primary cells, including those expressing high levels of the α 4 β 7 integrin, among the viruses with envelopes from the 3 different stages of infection. Conclusions: Similar to mucosal acquisition, HIV-1 envelope quasispecies present in injection drug users prior to seroconversion have unique phenotypic properties compared to those circulating during the chronic phase of disease. This suggests that there is selection for specific HIV-1 variants during injection drug use. 218 Phenotypic Characterization of Transmitted/Founder Virus in HIV-1 Transmission Pairs Corinna S. Oberle 1 ; Beda Joos 1 ; Nottania K. Campbell 1 ; David Beauparlant 1 ; Herbert Kuster 1 ; Corinne Schenkel 1 ; Peter Rusert 1 ; AlexandraTrkola 1 ; Karin J. Metzner 1 ; Huldrych F. Günthard 1 1 University Hospital Zurich, Zurich, Switzerland; 2 Institute of Medical Virology, Zurich, Switzerland Background: In 60-90% of mucosal HIV-1 transmissions a single transmitted/founder (T/F) virus from a genetically diverse virus population of the transmitter infects the recipient. Whether HIV-1 transmission is a stochastic process or the T/F viruses have beneficial features facilitating transmission and infection is controversially discussed. Here, we investigated the phenotypes of viruses isolated from transmission pairs in order to discover properties that might favor transmission. Methods: Based on phylogenetic analyses of HIV-1 polymerase and envelope sequences and clinical data from patients enrolled in the Zurich Primary HIV-1 Infection Study (ZPHI) and Swiss HIV Cohort Study (SHCS), 9 potential transmission pairs of subtype B were identified and primary virus isolates of transmitter and recipient (acutely infected patients with single T/F virus) at the nearest time point of transmission were generated. Virus isolates were characterized in respect to replication capacity in peripheral blood mononuclear cells (PBMCs) in the absence and presence of IFN- α and in monocyte-derived macrophages (MDMs). Furthermore, sensitivity to different entry inhibitors (Maraviroc, DARPin 57.2, soluble CD4, T-20) and neutralizing antibodies (2G12, 4E10, 2F5), and the entry kinetics of these virus isolates were studied. Results: All virus isolates replicated efficiently in PBMCs and 15 of 18 virus isolates were capable to replicate in MDMs, however, to a lesser extent. No clear pattern could be observed: In some transmission pairs, the virus isolate from the transmitter replicated more efficiently in MDMs and/or PBMCs and vice versa in other pairs. All virus isolates were sensitive to IFN- α ; yet the degree to which replication was reduced varied within and between transmission pairs. In terms of entry, virus isolates from the same transmission pair were inhibited to similar degrees by different entry inhibitors and neutralizing antibodies. Moreover, virus obtained from transmitter and recipient showed similar entry kinetics. Conclusions: For some transmission pairs, differences in replication capacities in both PBMCs in the absence or presence of IFN- α and MDMs were detected, yet we could not identify a common property shared by T/F viruses. T/F viruses and the virus population of the transmitter showed similar sensitivity to entry inhibitors/neutralizing antibodies and similar entry kinetics. Hence, according to the investigated parameters no signifying phenotypic pattern could be attributed to T/F viruses. 219 HIV Coreceptor Tropism Switching Is CorrelatedWith Binding Affinity to CXCR4 Homero Vazquez 1 ; Antoine Chaillon 2 ; Douglas D. Richman 3 ; Sara GianellaWeibel 1 ; GabrielWagner 1 ; David M. Smith 3 1 University of California San Diego, San Diego, CA, US; 2 Inserm UMR U966, Tours, France; 3 Veterans Affairs Medical Center, San Diego, CA, US Background: The switch of HIV-1 co-receptor tropism from CCR5 (R5) to CXCR4 (X4) during the course of infection is associated with changes in the V3 region; however, little is known about the structural binding between V3 and R5 and X4 co-receptors during tropism switching. We used an in-silico approach to model the physical properties of the gp120- CD4-R5/X4 complexes during these changes. Methods: A molecular dynamics (MD) and continuum-electrostatics poison-Boltzmann approach was used for generation of interaction models and calculation of binding affinity (BA). Since no experimentally derived structure is available, we first mapped well-characterized R5 and X4 strains onto the backbone of V3, followed by molecular dynamic simulations and docking of simulated structures into R5 and X4 to generate a gp120-CD4-R5/X4 complex interaction model. A database of viral sequences (n= 42 sequences, 38 frommacaques, and 4 from patients) that switched over time from R5 to X4 tropismwas then utilized to investigate changes in BA during tropism switching. All sequences were modeled onto the backbone structures of the interaction models of both R5 and X4; and BA amongst gp120, CD4, and R5/X4 was also calculated. 217 Characterization of HIV-1 Envelopes in Acutely and Chronically Infected Injection Drug Users Behzad Etemad 1 ; Oscar A. Gonzalez 1 ; Laura F.White 2 ; Oliver B. Laeyendecker 3 ; Greg D. Kirk 3 ; Shruti Mehta 3 ; Manish Sagar 1 1 Boston University School of Medicine, Boston, MA, US; 2 Boston University, Boston, MA, US; 3 Johns Hopkins University, Baltimore, MD, US

Poster Abstracts

209

CROI 2015