CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

Methods: Here, we generated fully CD4i viruses derived from the WT and the N7 mutant of SHIV 89.6 and isolated Env clones that are replication competent in CD4-negative cells. CD4-independece was confirmed by evaluation of cloned Envs in cell fusion assay and on infectious viruses when cloned back into SHIV backbone. Then we generated recombinant vaccinia viruses expressing CD4i 89.6WT (i89.6WT) and N7 (i89.6N7) mutant Envs. Antigenicity of these Envs on cell surface was analyzed using flow cytometry. Neutralization sensitivity of CD4i SHIV viruses was tested on TZM-bl cells. Biological function of these CD4i 89.6 Envs was tested by cell fusion assay. Results: We isolated two i89.6 WT Env clones (A2 and B1) and one i89.6N7 Env clone (D4t), which were derived, respectively, fromWT and N7 versions of SHIV 89.6. i89.6 N7-D4t Env maintains an intact CD4 binding site (bs), but CD4bs of i89.6-A2 and -B1 Envs are mutated. Cytoplasmic tails of all three Envs are truncated. In contrast to parental 89.6 WT and N7 Envs, i89.6 WT and N7 Envs can medicate cell-cell fusion in CD4-negative CCR5- and/or CXCR4-expressing cells. Only SHIV i89.6 WT and N7 viruses replicate in those CD4- negative cells. Recombinant vaccinia viruses expressing i89.6 WT and N7 were generated. Full length i89.6WT and N7 Envs can be expressed, glycosylated and processed into gp120 and gp41. i89.6WT and N7 Envs expressed by vaccinia virus vectors mediate cell fusion and exhibit antigenic properties expected of native Env trimers. They preserve epitopes of most broadly neutralizing Abs tested, but they also show altered antigenicity compared to parental Envs. Conclusions: Acquisition of the CD4i phenotype is accompanied by changes in the receptor/coreceptor binding properties of the Env and its antigenicity. How these changes may impact the immunogenicity of Env is being examined. 349 Phenotypic Neutralization Sieve Analysis of an SIV Nonhuman Primate Vaccine Challenge Study Fang-Hua Lee 1 ; Brandon F. Keele 3 ; Robert Doms 1 ; George M. Shaw 1 ; Mario Roederer 2 ; Katharine J. Bar 1 1 University of Pennsylvania, Philadelphia, PA, US; 2 Vaccine Research Center, NIAID, NIH, Bethesda, MD, US; 3 National Cancer Institute, Frederick, MD, US Background: We previously characterized the neutralization sensitivity of the nonhuman primate (NHP) challenge stock SIVsmE660 and found the isolate was generally highly neutralization sensitive with a minor population of resistant variants (Lopker et al ., J Virol 2013). We hypothesized that incomplete vaccine-mediated protection may result from protection against only the neutralization sensitive fraction of SIVsmE660. Subsequently, a large NHP vaccine study demonstrated incomplete protection from SIVsmE660 challenge and identified genotypic signatures associated with neutralization resistance (Roederer et al ., Nature 2014). Here, we perform a phenotypic neutralization sieve analysis of the breakthrough TF viruses of this NHP vaccine study. Methods: TF Env clones were derived by single genome sequencing of peak plasma vRNA from the breakthrough infections in heterologous Env-immunized animals (n=18 TF Envs) and controls (n=35 TF Envs). Neutralization sensitivity was assessed in a TZM-bl assay with SIV-infected macaque plasmas and a panel of 10 monoclonal antibodies (mAb) targeting V1, V2, V3, V4, CD4bs and CD4i epitopes. Results: The breakthrough TF Envs from immunized animals were significantly more neutralization resistant to immune plasma and mAbs targeting diverse gp120 epitopes than those from control animals. The maximum reductions in infectivity from plasma were 74.6% in control Envs, and 40.3% in vaccine Envs (p<0.0001, Mann-Whitney). The majority of vaccine TF Envs (13/18, 72%) were highly neutralization resistant (IC50<1:10), compared with the fraction comprising the control TF Envs (6/35, 17%, p=0.0002) and the SIVsmE660 isolate (10%, p=0.0002). The genotypic signatures of resistance identified in the Roederer study were highly predictive of phenotypic neutralization sensitivity. Conclusions: We demonstrate a phenotypic neutralization sieve effect in a large NHP vaccine challenge study with substantial enrichment for neutralization resistant viruses in the breakthrough infections of vaccinated animals. These findings support our hypothesis that incomplete vaccine-mediated protection results from selection against the highly neutralization sensitive component of the SIVsmE660 isolate. Further, we corroborate the genetic signatures of SIVsmE660 neutralization sensitivity predicted in the parent study. Together, these findings demonstrate proof-of-concept that phenotypic neutralization sieve analyses may be useful in assessment of vaccine trial outcomes and correlates analysis. 350 AvFc, a Novel Fc Fusion Protein Targeting Env High-Mannose Glycans Nobuyuki Matoba ; Adam Husk; J. Calvin Kouokam; Krystal Hamorsky;Tiffany Grooms-Williams; Garima Mahajan University of Louisville James Graham Brown Cancer Center, Owensboro, KY, US Background: AvFc is a recombinant fusion protein consisting of the high-mannose glycan-binding lectin Avaren and the Fc region of a human IgG1. We have previously shown that AvFc exhibits sub to low-nanomolar neutralization activity against a broad spectrum of HIV-1 and -2 primary strains and HCV genotypes. The objective of this study is to investigate the feasibility of AvFc for use in HIV prevention and/or treatment. Methods: Combinations of AvFc and other HIV inhibitors were evaluated in HIV-1 neutralization assays. Surface plasmon resonance (SPR) and flow cytometry were used to analyze binding characteristics of the Fc region of AvFc. Fc γ RIIIa receptor activation was analyzed using an Env-expressing and a Jurkat reporter cell lines. Flow cytometry, multiplex cytokine assay and quantitative PCR were used to test inflammatory potential in human peripheral blood mononuclear cells (PBMC). Rabbit and mouse vaginal irritation models were used to assess vaginal toxicity, while mice and rats were used to examine immunogenicity and systemic toxicity, respectively. Results: AvFc exhibited synergismwith Tenofovir, Maraviroc and VRC01 (combination index << 0.8, CalcuSyn software) against multiple HIV-1 strains. AvFc bound to Fc γ RI, Fc γ RIIIa, FcRn and C1q with affinities similar to those of human IgG 1 . Furthermore, the Fc γ RIIIa activation assay indicated that AvFc is capable of inducing antibody-dependent cell- mediated cytotoxicity. Unlike concanavalin A, AvFc had little cytotoxicity, mitogenicity or proinflammatory activity in human PBMC up to 100 μ g/ml, which is >1,000 times median anti-HIV IC 50 . In addition, AvFc did not induce any discernible toxic or inflammatory effects in both vaginal models. Repeated injection of Avaren with an adjuvant did not elicit any significant anti-Avaren IgG response in mice, which is indicative of low immunogenicity; being a small protein (12.5 kDa) with three tandem sequence repeats, Avaren may contain few, if any, T cell epitopes. A single intravenous infusion of 4 mg/kg AvFc in rats did not show any sign of toxicity; no weight loss or behavioral change was observed. Additionally, the complete blood count and serum chemistry of terminal blood samples appeared to be normal. Conclusions: Our data suggested that AvFc is a unique anti-HIV agent exhibiting broad HIV-neutralizing activity and Fc-mediated functions, and appears to be safe for mucosal and/or systemic administration. 351 DARPins as Entry Inhibitor Alternative to HIV-1 Broadly Neutralizing Antibodies Emanuel Stiegeler ; Nikolas Friedrich;Thomas Reinberg; Mylène Morin;YufanWu; JonasV. Schaefer; Peter Rusert; John Robinson; Andreas Plückthun; AlexandraTrkola University of Zurich, Zurich, Switzerland Background: Development of drugs that share the properties of broadly neutralizing antibodies (BNAbs) is highly desirable. Here we utilize the Designed Ankyrin Repeat Protein (DARPin) technology to generate HIV-1 inhibitors with broad neutralization activity directed against the V3 loop. DARPins, small synthetic binding proteins with high target affinities and specificities, have interesting properties as anti-HIV inhibitors in particular as they can recognize their target in a structure dependent manner (Mann A, J Virol 2013). Methods: V3 specific DARPins were selected from 1st and 2nd generation high diversity molecular DARPin libraries, the latter featuring additional randomized positions in the binding surface and a higher overall stability of the DARPin scaffold. Recombinant gp120 and a structurally arrested V3 epitope mimetic were used as panning targets during

Poster Abstracts

269

CROI 2015