CROI 2024 Abstract eBook

Abstract eBook

Invited Session

INVITED SESSION PRESENTATION SUMMARIES

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Overview of the Scott M Hammer Workshop for New Investigators and Trainees Serena S. Spudich 1 , Katharine J. Bar 2 1 Yale University, New Haven, CT, USA, 2 University of Pennsylvania, Philadelphia, PA, USA Background: Over the past four decades, remarkable progress has been made in understanding HIV epidemiology, pathogenesis, treatment, and prevention from the combined efforts of community members, clinicians, investigators, and funding agencies worldwide. Yet more work and new approaches are needed to achieve the ambitious goal of ending the epidemic and ensuring optimal quality of life for those living with HIV. To encourage and stimulate the next generation of investigators, the CROI Program Committee organizes an annual Workshop for New Investigators and Trainees comprised of expert and comprehensible talks to introduce key topics in basic, clinical, and public health investigation into HIV and related infections and to highlight relevant work to be presented over the ensuing days at CROI. This year, the program will begin with a presentation by Mr Adam Castillejo , an advocate and activist who will provide a community perspective on HIV cure. Dr Frank Kirchhoff will provide an overview of the molecular virology of HIV-1 and SARS-CoV-2 and describe key related presentations at CROI. Dr Elizabeth Connick will cover the immune responses against HIV and SARS-CoV-2. Dr Afam A Okoye will review advances in preclinical and clinical approaches for HIV remission or eradication. Dr LaRon E. Nelson will address advances in different strategies for preventing HIV transmission. Dr Jennifer Jao will provide an overview of key topics in maternal-child HIV and highlight relevant work presented at CROI. The workshop will end with a presentation by Dr Jeanne M Marrazzo , Director of the National Institute of Allergy and Infectious Diseases at the NIH, who will discuss opportunities in and provide insights on careers in research and discovery, reflecting on her personal career journey to date. By completing the workshop, attendees will have achieved a head start toward maximizing the knowledge gained and research ideas as they navigate CROI 2024. Background: Electron microscopy polyclonal epitope mapping (EMPEM) is a powerful technique for rapidly mapping the epitopes of serum antibodies and providing a visual readout of immune responses. EMPEM is performed using blood samples from immunized animal models, human volunteers, or convalescent patients of recent viral infection. Recently, our group has applied high resolution cryoEM methods to the same samples (cryoEMPEM) to visualize the amino acid interactions between antibodies and antigen. This approach can also be used to predict the sequences of the observed antibodies. By integrating NGS data of B cell repertoires with cryoEMPEM data, we are able to rapidly identify and provide unprecedented molecular resolution of epitope specific polyclonal antibody responses. EMPEM studies are complementary to traditional serological approaches, which altogether inform on the types of epitopes targeted, the diversity of epitopes targeted, and the consistency of epitopes targeted between study volunteers. When applied to HIV vaccine research, the molecular details revealed by this approach can confirm whether epitope specific, on-target antibodies are present in the sera and if they contain any structural and genetic features of known broadly neutralizing antibody precursors. A Spotlight on HIV: Visualizing Post-Entry Events by Correlative Light and Electron Microscopy Barbara Müller University Hospital Heidelberg, Heidelberg, Germany Background: The post-entry phase of HIV-1 replication - from cytosolic entry of the viral capsid encasing the genomic RNA to the integration of Cryoelectron Microscopy-Based Polyclonal Epitope Mapping (cryoEMPEM) Gabriel Ozorowski The Scripps Research Institute, La Jolla, CA, USA

the reverse transcribed viral cDNA into the host cell genome - has long represented an enigmatic part of the viral replication cycle. During this phase, subviral particles need to undergo a complex sequence of transitions in composition and structure, which is challenging to unravel using traditional bulk virological and biochemical approaches. Direct visualization of incoming viral structures in the cytosol by electron microscopy has been hampered by the difficulty to detect and identify rare, small objects with unknown morphology, which are embedded in a vast and complex cytosolic environment of similar electron density. Correlative Light and Electron Microscopy (CLEM) addresses this problem: rare, defined objects of interest are localized within a complex environment via fluorescence-based detection and subsequently analyzed with high spatial resolution using electron microscopic approaches. Today, continuous methodological advancements allow us to analyze the morphology of virus-derived complexes within their intracellular environment in unprecedented detail. Application of this approach to HIV-1 post-entry yielded new insights that, in conjunction with other results, identify the mature capsid as a key organizer of post-entry events. The presentation will introduce the CLEM approach and highlight insights into post-entry events in HIV-1 replication, from the visualization of cytoplasmic reverse transcribing complexes in infected cells approximately 20 years ago to recent high resolution in situ imaging of capsid-like structures in transit through the nuclear pore. Single-Virus Tracking: Capturing Fast, Three-Dimensional Viral Dynamics in Live Tissue Models Kevin Welsher Duke University, Durham, NC, USA Background: The epithelium is a nessential first barrier against viral infection. Understanding the interactions that enable pathogens to cross this complex barrier is critical to treating a wide range of diseases, including the recent SARS-CoV-2 pandemic. Single-virus tracking (SVT) is a potentially powerful tool to capture the molecular scale details of viral infection in live cells. SVT typically relies on fluorescence microscopy and can provide different insights from ensemble bulk experiments. However, the reliance on traditional fluorescence microscopy techniques has limited the application of SVT to mono-layer cell cultures with poor temporal resolution upon expansion to three-dimensional tissue models. In this presentation, we will detail current SVT methods and their advantages and limitations. We will then introduce a new active-feedback SVT method for overcoming the current limitations of SVT. This new method, called 3D Tracking and Imaging (3D-TrIm), uses fast, real-time measurement to "lock on" to a single virion and measure its dynamics at kHz or faster sampling rates across large three-dimensional spatial scales in live cells. We will demonstrate how 3D-TrIm captures transient viral contacts with the cell surface with millisecond temporal resolution, and how this new technique can translate SVT from simple monolayer cell culture models to more complex three-dimensional tissue models. Single-Cell Multi-Omics of HIV Cellular Reservoirs Iain C Clark University of California Berkeley, Berkeley, CA, USA Background: HIV persists indefinitely within tissue and blood cell reservoirs, necessitating life-long antiretroviral therapy (ART). Single-cell omics technologies represent a promising approach to understanding cells that harbor provirus, including the unique cell-intrinsic mechanisms that promote cell survival, proliferation, immune evasion, or HIV silencing. However, despite technological advances in single-cell analysis, there are several unique challenges to using these tools in HIV cure research. First, HIV-infected cells are rare in vivo, which necessitates the single-cell sequencing of hundreds of thousands of cells per sample, at great expense. Second, HIV accumulates in heterochromatin and may not express HIV RNA. Methods like scATAC-seq or scRNA-seq therefore only capture HIV+ cells with transcriptionally active

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CROI 2024