CROI 2017 Abstract e-Book

Abstract eBook

Oral Abstracts

driven sampling (RDS) and social network sampling. In the field of HIV and STI research, RDS has been successfully utilized to recruit substance users, commercial sex workers, and men who have sex with men. In this workshop, we review of the evidence for the use of RDS as a reliable sampling methodology, assess whether many studies meet the assumptions for RDS in order to claim a representative sample, and identify systematic biases in RDS sampling. In addition, we will review recent studies that have compared RDS to other recruitment approaches or have modified RDS methods. The presentation will also discuss how social network informed sampling may be combined with RDS to improve sampling, what types of studies may be best suited for RDS and social network sampling approaches, and what might be accomplished by an RDS approach versus more conventional sampling strategies. 6 CRISPR/Cas9 gene editing strategies have revolutionized our ability to engineer the human genome for robust functional interrogation of complex biological processes. We have recently adapted this technology to primary human CD4+ T cells to generate a high-throughput platform for analyzing the role of host factors in HIV replication and pathogenesis. Unlike traditional RNA interference or complementation approaches, this technique generates permanent genetic changes to the host genome at high efficiency with only transient expression of the modifying agents. CRISPR/Cas9 ribonucleoproteins (crRNPs) are synthesized in vitro and delivered to activated primary human CD4+ T cells by electroporation. These cells are then expanded and parsed out for validation, cryopreservation, and infection. Our platform supports the arrayed generation of hundreds of specific gene manipulations in only a few hours time and is widely adaptable to an array of culturing conditions, infection protocols, and downstream applications. We first used this platform to perform proof-of-principle experiments targeting host factors with defined roles in HIV replication. As expected, CXCR4 or CCR5 knock-out primary T cells are resistant to HIV infection in a tropism-dependent manner, whereas knock-out of LEDGF or TNPO3 results in a tropism-independent reduction in infection. We next bridged this approach to other proteomic and genomic discovery platforms as a secondary screen for host factor functionality, identifying several candidate dependency and restriction factors for additional mechanistic study. Finally, we found that crRNP multiplexing allows for the editing of multiple genes simultaneously, enabling studies of functional redundancy or epistasis among multiple host and viral factors. This technology should prove useful for not only discovery-based scientific research, but may additionally accelerate target validation for pharmaceutical and cell-based therapies to cure HIV infection. We are currently adapting this technology to achieve efficient genome editing at the level of a single base pair, as well as adapting it to other primary cell types for the study of additional biological processes and disease states. Recent advances with CRISPR interference, CRISPR activation, BaseEditor technology, high-fidelity Cas9 enzymes, and Cas9 inhibitors will also be discussed. 7 IDENTIFYING AND PROFILING VIRUS-SPECIFIC T CELLS USING MASS CYTOMETRY EvanWilliamNewell, Singapore Immunol Network, Singapore Blood and tissue samples taken as part of clinical studies and trials can provide critical information on the roles of the immune response in patient outcome. However, the cellular compositions of these samples are often highly diverse, and important information can be lost if rare cells are overlooked. For instance, antigen specific T cells are critical initiators and orchestrators of the adaptive immune response, but cells specific for any given pathogen or cancer can be exceedingly rare, especially in blood. Here, the utility of high dimensional mass cytometry analysis together with rapidly evolving computational analysis tools will be discussed. A major advantage of this approach is the ability to directly analyze relationships between antigen-specificity and each cell’s phenotypic profile. This is particularly relevant for the study of T cells, whose phenotypic markers can be intuitively segregated into a number of categories such as antigen-specificity, differentiation state, functional capacity, and trafficking receptor profiles. All of these can be measured simultaneously on individual cells through the use of metal-labeled monoclonal antibodies and highly multiplexed combinatorially-coded peptide-MHC tetramers. Our ongoing analysis of T cell responses in chronic hepatitis B viral infection in human patients demonstrate the utility of this approach to identify and profile difficult to study HBV-specific T cells. Our results show that HBV-specific T cell responses are highly diverse in terms of epitopes being targeted and the phenotypes of the corresponding cells. We are investigating the potential utility of T cell phenotypes as biomarkers for patient outcomes. 8 QUANTIFYING HIV-1 mRNA STRUCTURE AND TRANSLATION EFFICIENCY IN CELLS Silvi Rouskin, Whitehead Inst, Cambridge, MA We have recently developed DMS-MaPseq, which allows for targeted RNA structure probing in vivo at single molecule and single nucleotide resolution. First, cells are treated with DMS, which rapidly modifies unpaired adenines and cytosines. Unlike other chemical reagents, DMS is a very small molecule specific to the nitrogen atoms involved in Watson- Crick base-paring, which enables probing of RNA structure in the presence of RNA binding proteins. In the DMS-MaPseq technique, we use DMS concentrations that result in multiple modifications per single molecule. We then use a newly commercialized high fidelity and processive thermostable group II reverse transcriptase (TGIRT) enzyme, which converts DMS modifications into mutations in the cDNA. The cDNA is converted to dsDNA by PCR and then sequenced on an Illumina platformwith long reads. Since the mutation background is negligible, the mutations within a single sequencing read correspond to the open bases within a single RNA molecule. There are two key advantages of using this approach compared to other chemical probing approaches: 1) we can selectively probe the structure of any RNA of interest, even at very low abundance, simply by using a RNA specific RT primer 2) we can analyze multiple DMS modification sites per RNA molecule, which allows us to distinguish heterogeneous RNA structure subpopulations in vivo. We use a combination of DMS-MaPseq and ribosome profiling to quantify the HIV-1 RNA structure and translation efficiency in cells. 9 INTERACTIVE CASE-BASED WORKSHOP ON HEPATITIS C Moderators: Susanna Naggie , Duke Univ, Durham, NC, USA, and DavidWyles , Denver Health, Denver, CO, USA This interactive case-based session is geared toward clinicians who are involved in HCV treatment. Speakers will present cases on: Staging and treatment of early stage HCV ( John D. Scott , Univ of Washington, Seattle, WA, USA), effect of drug resistance on DAA treatment and re-treatment of patients with chronic HCV infection ( Alessandra Mangia , Ospedale Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy), common drug interactions with HIV/HCV coinfection treatments ( Debika Bhattacharya , Univ of California Los Angeles, Los Angeles, CA, USA), and cirrhosis issues ( Sanjay Bhagani , Royal Free Hosp, London, United Kingdom). 10 BERNARD FIELDS LECTURE: INSIGHTS INTO HIV PREVENTION, PATHOGENESIS, AND TREATMENT FROM NONHUMAN PRIMATE MODELS Jeffrey D. Lifson, Frederick National Laboratory for Cancer Research, Frederick, MD, USA Infection of Asian macaques with Simian Immunodeficiency Viruses (SIV) endemic in African nonhuman primates (NHP) can lead to progressive pathogenesis/immunodeficiency that recapitulates key aspects of human HIV infection and AIDS. Different NHP models, employing different naturally occurring or engineered SIVs or related chimeric viruses, used to infect different macaque species, can be used to authentically model relevant features of human HIV infection. Experimental flexibility and control, and opportunities for extensive tissue sampling afforded by NHP models provide advantages for studying virus/host interactions. NHP models have yielded key insights into AIDS virus transmission, allowing characterization of the earliest stages of infection, pathways of early viral spread, and initial host responses, and permit the preclinical safety and activity evaluation of prophylactic vaccines and other prevention approaches. When matched to conditions of clinical vaccine evaluation, results from NHP vaccine studies have been largely congruent. NHP models have also informed our understanding of the pathogenesis of AIDS virus infection, including processes such as early, extensive depletion of intestinal CD4+ T cells, mucosal disruption, microbial translocation and persisting systemic immune activation, along with inflammation related fibrotic disruption of secondary lymphoid tissues. Comparison of SIV infections in Asian macaques and African “natural host” species, where infection can result in extensive viral replication but does not typically lead to progressive disease, highlight the role of host responses in pathogenesis. NHP models have also shed light on mechanisms of viral persistence despite apparently effective viral suppression by antiretroviral drug treatment or potent immune responses, through viral sequestration in relatively immune privileged sanctuary sites such as B cell follicles in HIGH-THROUGHPUT GENOME ENGINEERING IN PRIMARY CD4+ T CELLS Judd F. Hultquist, Univ of California San Francisco, San Francisco, CA, USA

Oral Abstracts

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CROI 2017