CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

Conclusions: We demonstrate that SAMHD1 expression is decreased during CD4 + T cell activation and proliferation. Also, CD4 + T-cell subsets known to be more susceptible to HIV-1 infection, e.g. Th17 and Tfh cells, display lower levels of SAMHD1. These results pin point a role for SAMHD1 expression in HIV-1 infection and the concomitant depletion of CD4 + T cells. 207 SAMHD1 Partially Blocks Lentiviral Gene Transfer Into Hematopoietic Stem Cells Duo Li 1 ; Erika Schlaepfer 1 ; Annette Audigé 1 ; Baek Kim 2 ; Roberto Speck 1 1 University Hospital Zurich, Zurich, Switzerland; 2 Department of Pediatrics Emory School of Medicine, Atlanta, GA, US Background: Understanding how to achieve efficient transduction of hematopoietic stem cells (HSCs), while preserving their self-renewing capacity, is key for applying lentivirus-based gene engineering methods in Phase I/II clinical trials. The sterile alpha motif (SAM) domain and HD domain-containing protein 1 (SAMHD1) was recently identified as a HIV-1 restriction factor in myeloid cells and resting CD4 + T cells that interferes with reverse transcription by decreasing the nucleotide pools or by its RNase activity. HIV-2 and SIV have evolved to counteract the effects of SAMHD1 by their accessory protein Vpx, which targets SAMHD1 for proteasomal degradation. We hypothesized that SAMHD1 also interferes with HIV-1 vector-based HSCs transduction. Methods: Expression of SAMHD1 in HSCs was quantified by western blotting and real-time quantitative PCR (qPCR). For HSCs transduction, we used HR-GFP-Vpx-/+ lentivirus, which carries Vpx and encodes GFP. Integrated provirus and viral DNA intermediates in HSCs and monocyte-derived macrophages (MDMs) were quantified by qPCR. Transduction efficiency was assessed by flow cytometry. Results: Our results show that SAMHD1 is highly expressed in HSCs already at 2 hours culturing in a medium enriched with cytokines conventionally used for transduction of HSCs. In contrast, fresh HSCs have poor SAMHD1 expression. Expression levels of SAMHD1 in cultured HSCs are comparable to those found in MDMs. Following lentiviral based transduction with HR-GFP-Vpx+, we did not observe any increase of proviral DNA in HSCs while there was a significant one in MDMs which served as positive control for the assay. Similarly, there was a less than 3-fold increase of DNA intermediates in HSCs a vigorous one in MDMs. HSCs exposed to HR-GFP-Vpx+ showed a minor but significant increase in the number of GFP + cells which was associated with a decrease in SAMHD1 expression. GFP + cells were detected mostly within the population of cells containing low amounts of SAMHD1. There was an significant increase of the percentage of GFP + SAMHD1 - cells, while GFP + SAMHD1 + cells decreased 7 days post infection when compared to cells exposed to HR-GFP-Vpx- viral like particles. Conclusions: HSCs cultured in cytokine-enriched medium, unlike uncultured cells, express high levels of SAMHD1. Vpx-mediated decreases of SAMHD1 expression levels enhances transduction rate in a minor sub-population of HSCs. The data imply that blocks mainly at cell entry are the major limiting step for efficient transduction. Jan Munch 3 ; Onofrio Zirafi 3 ; Shariq Usmani 3 ; Kyeong-Ae Kim 3 ; Frank Kirchhoff 3 ; Christopher D. Pilcher 1 ; Haichuan Liu 1 ; H. EwaWitkowska 1 ;Warner C. Greene 2 ; Nadia R. Roan 1 1 University of California San Francisco, San Francisco, CA, US; 2 The J. David Gladstone Institutes, San Francisco, CA, US; 3 Ulm University Medical Center, Ulm, Germany Background: Semen, the most common vehicle for HIV transmission, enhances HIV infection in vitro . Previously, naturally-occurring peptides derived from the semen proteins prostatic acid phosphatase (PAP) and semenogelin (SEM) were shown to assemble into amyloid fibrils that markedly enhance HIV infection. Here, we investigated whether fresh semen samples contain these amyloids and affect the antiviral efficacy of various classes of microbicides. Methods: Confocal microscopy, electron microscopy (EM), atomic force microscopy (AFM), quantitative mass spectrometry, ELISAs, and infection assays were used to detect, quantitate, and characterize endogenous HIV-enhancing amyloids in semen and to determine how semen affects the efficacy of microbicides. Results: Endogenous PAP and SEM amyloids were detected in unmanipulated semen by immunogold EM, confocal microscopy, and AFM. These amyloids are present in semen from uninfected and HIV-infected individuals and directly bind HIV virions. The endogenous levels of these amyloidogenic fragments correlated significantly with the HIV enhancing activity of the semen samples, suggesting that they may be responsible for the ability of semen to enhance HIV infection. We also found that semen reduced the antiviral efficacy, by 10 to 20-fold, of multiple classes of antiretrovirals (ARVs) including neutralizing antibodies, NRTI’s and NNRTI’s, and inhibitors against Integrase and Protease. In striking contrast, semen deficient in amyloids did not enhance HIV infection or impair the antiviral activity of these ARVs. Notably, the sole microbicide that retained full activity in the presence of semen was Maraviroc (MVC), an HIV entry inhibitor that was the only microbicide investigated which targeted a host component instead of the virus itself. Conclusions: Our results demonstrate that amyloids are abundant in fresh semen and that the ability of semen to enhance HIV infection markedly impairs the activity of microbicides targeting viral components. In contrast, MVC, which targets the cellular CCR5 entry cofactor, retained full activity in the presence of semen. Thus, compounds targeting cellular components may be advantageous for microbicide development. Our results may explain why most microbicides largely failed in clinical trials and suggest that inclusion of compounds that antagonize seminal amyloids will greatly enhance the efficacy of antiretroviral microbicides. 209 Semen’s HIV Enhancing Activity Is an Individual Characteristic Independent of VL Christopher D. Pilcher ;Teri Liegler; Jason Neidleman; H. EwaWitkowska;Wendy Hartogensis; Kara Marson; Peter Bacchetti; Frederick M. Hecht;Warner C. Greene; Nadia R. Roan University of California San Francisco, San Francisco, CA, US Background: Semen enhances HIV infection in vitro , and semen-derived amyloids made up of a peptide from the abundant protein semenogelin 1 (SEM1) are sufficient to mediate this activity. We sought to compare the infectivity-enhancing activity of semen among HIV infected men, and examine its relationship to concentrations of amyloidogenic peptides. Methods: We studied 188 paired seminal and blood plasma samples from 113 individual UCSF Options study subjects at least four months after acute HIV infection. 20 subjects were studied at multiple timepoints prior to ART; 20 were studied before and after suppressive ART and 20 before and after ART interruption. Quantitative mass spectrometry and ELISAs were used to quantify amyloidogenic SEM peptide fragments. Semen-mediated enhancement of HIV infection was measured by mixing serial dilutions of each sample with HIV prior to adding it to TZM-bl reporter cells, and assessing infection levels by luminescence 3 days later. Seminal fluid VL was quantified using Abbott RealTime PCR. THURSDAY, FEBRUARY 26, 2015 Session P-A4 Poster Session Poster Hall 2:30 pm– 4:00 pm Enhancers and Inhibitors of Viral Infectivity and Entry 208 Fresh Semen Harbors HIV-Enhancing Amyloids and Decreases the Efficacy of Microbicides

Poster Abstracts

205

CROI 2015