Thomas Hope - US grants

[unreadable] DESCRIPTION (provided by applicant): The Filoviruses Ebola and Marburg are highly pathogenic and contagious viruses making them a potential bioweapon. The lethal nature of these agents has made them difficult to study because of the requirement for biosafety level 4 containment. However, individual components of these virions can be safely studied in isolation. Envelope function can be studied in the context of pseudotypes and virus-like particles can be generated by the expression of the matrix protein. Recently, we have developed methods that allow the visualization of HIV using fluorescent deconvolution microscopy. With these tools it is possible to directly observe virus binding and entry into target cells in real time. These direct observations of particle behavior have provided important new insights into how HIV interacts with target cells. The goal of this application is to adapt these methods to study filovirus entry. Envelope function will be studied in two contexts. First, fluorescently labeled HIV will be pseudotyped with filovirus envelope. These studies should provide new insights into the entry pathway for Ebola and Marburg. Secondly, we will develop a system using a green fluorescent protein (GFP) tagged matrix protein (VP40) to visualize VLPs that contain the filovirus envelope. The VLP system will require extensive development and validation but offers the opportunity to study the interaction of replication defective filovirus particles with target cells. Direct visualization of these viruses will provide important new insights into how these agents bind to and enter target cells. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Unsuccessful attempts to develop a vaccine against HIV have led to a great need for new preventative strategies, the most encouraging of which are microbicides. An effective microbicide would decrease the severity of the AIDS epidemic by decreasing the rate of sexual transmission. A number of models systems have been developed to provide insights into the mechanisms of male-to-female sexual transmission including explant cultures and the rhesus macaque vaginal transmission model. These systems have been successfully used to evaluate and identify candidate microbicides. There has been much less progress in the development of systems to evaluate female-to-male sexual transmission. There are currently no model systems of female-to-male sexual transmission that can determine whether potential microbicides are capable of preventing infection via the penis. To fill this gap, this application seeks to develop an explant culture system using human penile tissue. This will enable the efficacy and safety of candidate microbicides that prevent the sexual transmission of HIV to males to be evaluated. Additionally, my laboratory has recently developed methodology that allows the detection of individual virions in tissue. This system will be further developed to determine how HIV normally interacts with human penile tissue. In the R21 component of this application we will optimize these systems to determine the normal interaction of HIV with intact tissue and subsequent viral replication. In the R33 phase of the application we will evaluate the potential of different candidate microbicides, individually and in combination, to alter HIV interaction with the tissue and prevent HIV infection and replication. We will also determine if exposure to the microbicide causes any changes in the tissue that may have deleterious effects on normal cell function. The application seeks to develop new methods that determine whether chemicals can be used to prevent the sexual transmission of HIV. These methods will be used to determine the potency of protection from HIV infection, and the safety of the compounds before they are tested in humans. It is hoped that preventing HIV infection will decrease the number of people in the world infected with HIV and slow the AIDS epidemic.

Overview The laboratory for the Virus Imaging Core is currently located on the third floor of the Tarry Building on the Chicago Campus of Northwestern University. The laboratory will relocate to the state-of-the-art Lurie Biomedical Research Building in the fall of 2007. The Hope laboratory currently houses 3 DeltaVision RT deconvolution microscopes. Importantly, these microscopes are in a biocontained room located close to our HIV culture facility, allowing the safe observation of infectious HIV. Deconvolution microscopy uses computer processing to compensate for distortion caused by the optical path. Through the observation of fluorescent beads, an algorithm is developed which corrects the distortion caused by lenses and removes out-of-focus light163. The associated digital camera detects weak signals enabling great sensitivity and resolution, which is optimal for imaging viral particles in cells. Image capture with minimal illumination minimizes photobleaching and avoids phototoxicity, which can stress the cells during live cell observation. Two stand-alone workstations contain the complete Softworx software for deconvolutions and image processing, and off line image processing facilitates efficient instrument use. The systems are configured with filter sets allowing imaging of different types of fluorescent proteins including blue, cyan, green, yellow, and red fluorescent proteins. This permits simultaneous imaging of up to four different fluorescent proteins in live cells and 5 different fluors in fixed cell imaging. Importantly, two of these microscopes have environmental control chambers, including 5% CO2, allowing extended live cell timelapse microscopy. One of the microscopes with environmental control in equipped with a Photonics Mosaic digital diaphragm that allows precise sample illumination for photobleaching and photoactivation experiments. Additionally, this system has an ultrasensitive digital camera allowing maximal photon detection and signal amplification.

[unreadable] DESCRIPTION (provided by applicant): HIV infection of target cells requires at least three different molecules, the HIV receptor DC4, a coreceptor such as CXCR4 or CCR5, and the viral envelope protein. Much is known about the structure, biochemistry, and molecular biology of these essential proteins. Unfortunately, our understanding of the cell biology of these molecules and HIV entry limits our ability to understand how these proteins interact over time to mediate fusion between the viral and cellular membranes. To gain insights into this process, we will utilize a system recently developed in my laboratory, which allows individual virions of HIV to be imaged using fluorescent microscopy. The studies outlined here will combine our ability to visualize HIV interaction with living cells with the methods of modern cell biology to gain new insights into the process of HIV entry. Our preliminary studies reveal that the HIV receptor and coreceptor(s) are specifically localized on the cell surface to regions of active membrane activity. This observation suggests that the localization of these molecules is regulated. Using fluorescent protein fusions to CD4 and coreceptor(s) we will determine how the presence of HIV effects the cell biology of these cell surface proteins during infection. Using methods which allow the fusion of viral and cellular membranes to be detected visually we will determine the spatial interactions between the viral envelope protein and receptor/coreceptor(s) required for virion entry. Finally, we will utilize fluorescent HIV to characterize the role of DC-SIGN. The studies proposed here are significant for completing our understanding of HIV entry into cells because they will provide an important' context for the wealth of details which are currently known about HIV entry. This understanding can in turn be useful for the current push to bring compounds which specifically target, and disrupt, HIV entry to the clinic. [unreadable] [unreadable] [unreadable]

Twenty-five years since the discovery of HIV-1, the underlying biological mechanism(s) of male-to-female sexual transmission remain unclear. To initiate infection, HIV must traverse the protective outer genital epithelial barriers, establishing infection in underlying immunocompetent target cells. To study the nteractions of individual HIV virions with mucosal epithelial barriers, we developed a novel system comprising genital tissue explants and fluorescent microscopy. A difficult aspect of fluorescent microscopic analysis of tissue sections is the high background autofluorescence. To avoid this problem, we utilize HIV abeled with a photoactivatable form of green fluorescent protein (GFP). Tissue sections are initially scanned to define background autofluorecence. The field of view is then photoactivated, followed by a second scan. Viral particles appear as new signals observed only after photoactivation. Using this system we find that HIV can enter both the squamous ectocervical epithelium and the columnar endocervical epithelium in human explants. Future studies will analyze vaginal and uterine tissue. Virions are observed penetrating to depths that would facilitate contact with HIV targets such as Langerhans cells, macrophages, dendritic cells and CD4+ T cells. We find that the virus is primarily localized in interstitial spaces between cells in the squamous epithelium. This suggests that virus has the ability to move within the intact mucosal epithelia to encounter potential target cells of infection. Related studies will determine how cell-associated virus interacts with the tissues of the female genital tract. The specific aims seek to 1) define the interaction of HIV with the intact squamous epithelium of the ectocervix and vagina, 2) define the interaction of HIV with the intact columnar epithelium of the endocervix and uterus, 3) characterize the interaction of HIV with cervical mucous, and 4) determine how exogenous factors such as inflammation and other local environmental factors modulate the interaction of HIV with the mucosal epithelium leading to increased or decreased infection. The completion of these aims will increase our understanding of the underlying mechanism(s) of HIV sexual transmission. This information can then be utilized in the development of vaccines and microbicides designed to prevent HIV. RELEVANCE (See instructions): There is currently a large effort to develop microbicides and vaccines to prevent the sexual transmission of HIV. Yet we currently do not understand in much detail how HIV is sexually transmitted. The studies propose here seek to reveal novel details of how HIV interacts with the human female genital tract leading to infection. This information will be invaluable in the development of strategies to prevent HIV transmission.

DESCRIPTION (Provided by the Applicant): To develop a functional microbicide it is critical to know how it will interact within HIV in the context of the female genital tract. This is a critical issue as previous clinical trials have indicated that microbicides do not function as expected in the presence of semen. Likewise, other factors, such as cervical/vaginal mucus, might also modulate microbicide function. To date, little is known about how HIV interacts with these fluids and how the interaction of these fluids changes the local environment. Even less is known about how microbicides interact with HIV within this milieu. For example, the vehicle delivering the microbicide might interact with the biological fluids of sexual transmission to either increase or inhibit HIV acquisition or microbicide potency. The Hope laboratory has recently developed methods that allow the transport of HIV with cervical and cervical/vaginal mucus to be analyzed and quantified. These studies have revealed that mucus can perturb HIV transport and is pH sensitive. At acidic pH, as is found in the lactobacilli influenced environment of the vaginal vault, HIV transport is greatly reduced. At neutral pH, such as when semen is introduced into the system, HIV transport is reduced 10-15 fold relative to what is observed in media (water). Additionally, we have found, but not yet published, that virus-binding antibodies can further reduce transport in neutral pH cervical mucus. These antibodies do not need to be neutralizing as any antibody binding to the virus can decrease virus transport. Semen also contains mucins and other components that have the potential to alter HIV transport as we have observed in cervical mucus. How HIV is transported within semen and how this changes when mixed with mucus or microbicides is not defined. How this process influences HIV transport and interaction with mucosal barriers is not understood. In the first phase (R21) of this proposal we will define how HIV is transported in semen alone and mixed with mucus and/or microbicide vehicles such as carbopol gel and hydroxy ethyl cellulose (HEC). In the second phase (R33) of this proposal we will extend our studies into the environment of the rhesus macaque female genital tract to determine how biological fluids and microbicide vehicles alter the way that virus interacts with the mucosal barriers of this environment and how these changes can increase or decrease SIV acquisition. These studies will lead to a better understanding of how virus interacts with biological fluids and how these interactions might alter microbicide efficacy.

Vaginal and rectal transmission models of SIV/SHIV in rhesus macaques offer potential insights into the mechanisms by which systemic viral acquisition is established after mucosal exposure to HIV during sexual transmission. The nature of target cells infected and the sites of transmission after vaginal challenge remain only partially defined, and even less is known about early events of rectal transmission. To gain insights into the initial targets of infection, we have developed a single-round dual reporter system that can specifically identify the cells infected by the challenge inoculum. Further, we have recently reported that macaque vaginal challenge with a mixture of the dual reporter vector and replication competent SIV enables us to identify small, early foci of SIV replication 48 hours post-challenge. As illustrated in preliminary data included in the application, we have also been able to adapt the single-round dual reporter system to identify the first cells infected after atraumatic rectal challenge. Interestingly, our studies of vaginal and rectal early transmission with the a single-round dual reporter pseudotyped with M-tropic JRFL and the T-tropic SIVmac239 48 hours post-challenge all show a similar preference for the same early target cells, with the majority of cells infected being Th17 cells, and the immature DC being a minor population. This observation suggests that the available target cells to initiate mucosal acquisition may be limited. To extend these insights, we will examine how different envelope proteins and the SIV protein Vpx influence early tropism. We know that later during pathogenesis and general immune activation, multiple cell types can be infected and depleted after infection becomes systemic. Therefore, we will determine early changes in the SIV/SHIV target cells by examining the kinetics of viral spread as the initial foci of infection expands from 2 to 4 days. Finally, as early as 48 hours post-vaginal challenge with SIVmac239, we observe evidence of host responses to mucosal infection including apoptotic infected cells, lysed infected cells, and phagocytosed infected cells. Likewise, preliminary RNA-Seq analysis revealed changes in gene expression associated with the 48 hour foci of infection. By combining microscopic analyses of tissue sections and detecting the cells associated with host gene expression changes using fluorescent antibodies and RNA probes, we will be able to visualize the first wave of host responses to the virus. We will define the who, where, and when of which cells are infected, which cells are generating virus specific alarms, and which cells are responding to these alarms. Collectively, completion of these studies will result in a great increase in our understanding of the cascade of the earliest events of vaginal and rectal transmission including the location and phenotype of infected cells and the innate host responses to infection within the first few days of infection. A better understanding of the earliest events of mucosal transmission has the potential to advance efforts in HIV prevention science. Knowledge of the cell type and location of the earliest targets of transmission will reveal where the intervention must be targeted for maximal impact.

DESCRIPTION (provided by applicant): There has been a renaissance in the use of cell biology techniques to study biological functions and mechanisms. This advance has been driven by the development of new fluorescent probes, more sensitive imaging systems and new techniques. Increased information has led to the realization that the cell is highly organized in ways that facilitate the interaction of different cellular factors to mediate biological function. These studies have greatly enhanced our basic understanding of the biology of infectious diseases, cancer, and development. Traditionally, the resolution obtainable by light based microscopy techniques is limited by diffraction to about half of the wavelength of the light utilized. More recently, several advancements in microscope technologies have allowed this diffraction limit to be overcome. To take advantage of these recent technological advances, we propose to purchase an OMX super-resolution microscope system built by Applied Precision Instruments of Issaquah, Washington. This system utilizes structured illumination to overcome the diffraction limits of normal light microscopy. At Northwestern University, the NIH supported research of many scientists requires fluorescent microscopy to characterize small viral and cellular structures within cells, which can mediate key biological functions. Therefore, it is critcal that the components of these structures and their relationships to one another be defined in the highest possible detail. As demonstrated with preliminary data included in the application, the OMX can deliver new insights into the spatial details of such structures including the nuclear lamins, desmosomes, and intracellular viral complexes. The OMX super-resolution microscope system will be housed in the outstanding Cell Imaging Core Facility at Northwestern, giving widespread access to this revolutionary imaging technology. There is currently nothing like this state-of-the-art technology in our Cell Imaging Core Facility or in the greater Chicagoland area. Therefore, this system will greatly enhance the NIH supported research of the Northwestern user group and similar types of research here and at nearby institutions. PUBLIC HEALTH RELEVANCE: Access to a super-resolution microscope will increase the capabilities and facilitate the cell biology research of the user group at Northwestern University and others in the region. This in turn will facilitate a greater level of productivity and advance f knowledge from NIH sponsored research at Northwestern University and the greater Chicago area.

DESCRIPTION (provided by applicant): Male circumcision has been shown to be clinically effective in reducing HIV acquisition rates in African men. The scientific rationale for the reduction in HIV transmission remains unknown, and work in our laboratory has shown that the mechanism is much more complex than those theorized early on (regarding keratin thicknesses and number of potential HIV target cells). We have found that more dynamic properties of the penile epithelia, such as permeability and inflammatory responses, may play more important roles. This proposal endeavors to further explore changes in penile epithelia after male circumcision, which relate to the skin's barrier function, and to compare them to previously circumcised and uncircumcised cohorts. Adjunct studies will focus on studies of baseline junctional protein expression in the foreskin and clinical factors that may affect HIV susceptibiliy in men. With these studies, we hope to gain a better understanding of how HIV is sexually transmitted in the male genital tract, how male circumcision works to prevent this, and how to develop effective future strategies in HIV prevention.

PROJECT SUMMARY THIRD COAST CFAR CORE E: VIRAL PATHOGENESIS CORE The Viral Pathogenesis (VP) Core seeks to bring the tools, services and reagents of biological HIV research to the interdisciplinary research teams of the TC-CFAR. We will also provide training to facilitate the ability of diverse teams spanning the breadth of HIV research at the University of Chicago and Northwestern University. The TC-CFAR has set the goal of starting new trans-disciplinary collaborations to reduce the rate of new acquisition of HIV in the high-risk YMSM populations in Chicago. Accomplishing this will require novel interactions between the clinical, behavioral/social, and biomedical HIV researchers at the two institutions. The specific aims of the VP Core are: 1) provide virological assays and genomics support; 2) provide BSL2+ access and the reagents necessary for virological research including reporter and other cell lines, virus stocks, plasmids, mucosal samples, and humanized mice; 3) provide training and support for cutting-edge HIV and tissue imaging; and 4) provide education in biological HIV research and develop new services/technologies through seminars and brainstorming sessions. To achieve these aims, the VP Core will leverage existing strengths at the two institutions to stimulate new research studies that advance efforts to decrease HIV acquisition. Through providing cutting edge approaches and stimulating new collaborations, the VP Core will provide support that will bridge these diverse groups. The Director of the VP Core, Tom Hope, brings 25 years of HIV research experience to lead this effort. Rich Longnecker, Co-Director of Virology, adds basic virology knowledge and years of experience gained by leading research teams at Northwestern University. Yoav Gilad, Co-director of Genomics, brings critical experience and capabilities in genomic science to support the effort. Together, this leadership team will support the ability of the researchers of the TC-CFAR to expand their research horizons and drive our science forward towards the goal of ending the HIV epidemic.

? DESCRIPTION (provided by applicant): The concept of preventing sexual transmission of the human immunodeficiency virus (HIV) infection via pre- exposure prophylaxis (PrEP) with antiretroviral drugs (ARV) has been clinically proven, yet its implementation remains difficult, as poor adherence has repeatedly confounded clinical progress. Individuals at high risk for HIV infection would benefit from ARV drug delivery systems with long duration and sustained/controlled drug release. Technological achievements in pharmaceutics and medicinal chemistry allow us to envision combining clinically advanced drug delivery technologies and extremely potent and long-acting ARV into drug delivery systems that could protect users from HIV for many months, and perhaps as long as a year. The objective of the Sustained Long-Acting Protection from HIV (SLAP-HIV) program is to test and clinically develop a long-acting drug delivery system of one of the following fourth generation ARV: cabotegravir, rilpivirine, tenofovir alafenamide fumarate, or the tenofovir analog CMX-157. Several parenteral drug delivery systems will be made and tested by three competing teams exploring three unique drug delivery platforms: reservoir implants, degradable implants, and controlled release injectables. We will compare our systems functionally for stability, manufacturability, duration, and pharmacokinetic and safety endpoints. We will work in parallel with high-risk groups to develop user based design input and criteria to inform the clinical development of the drug delivery systems. We will also investigate the acceptability of a wide variety of designs in high-risk individuals. A single lead formulation from each drug delivery project will be selected for further study of pharmacokinetics and pharmacodynamics in non-human primates. From these studies, a single lead formulation will be selected for clinical development, based on achieving a well-defined target product profile. Next, we will focus on studies required for an investigational new drug application (IND) along with the transfer of manufacturing methods to a contract research organization. We will conduct pharmacokinetics and pharmacodynamics studies in macaques to further study the prophylactic potential of the lead product and provide non-GLP support data for the IND. Finally, a Phase 1 clinical study will conclude the program to examine the safety, pharmacokinetics and acceptability of the lead ARV eluting drug delivery system. In total, these studies will advance our understanding of long-acting drug delivery systems and their ability to disrupt mechanisms of transmission. We have designed the program to ensure success and will be aided by the inclusion of many leading HIV experts, whose combined expertise spans drug delivery research, pharmaceutical development and HIV science. The impact of the product platforms developed from this research will extend beyond HIV PrEP and will advance our understanding of how to implement long-acting HIV therapy for those already infected, and to other therapeutic areas where long-acting systems are needed.

Summary: Current HIV vaccine induced antibody responses are known to generate virus binding antibodies, but a vaccine that generates broadly neutralizing antibodies has yet to be identified. Alternative approaches need to be developed to gain enhanced vaccine function by optimizing the effector functions of the antibodies generated. Through antibody-cell surface mucin interactions, the antibodies would facilitate trapping of HIV before it could reach and interact with underlying columnar epithelial cells, which would effectively neutralize the virus. We have identified a specific interaction between a cell-associated mucin, MUC16, and IgG. This interaction appears to be an effector function regulated during the immune response because 1) the interaction between IgG and MUC16 is increased during chronic HIV infection, 2) the MUC16 associating IgGs are enriched for binding to gp41, but not gp120, and 3) the MUC16 associating IgGs are depleted for ADCC activity. In the proposed work, we will dissect and define the interaction between MUC16 and IgG, define the ability of MUC16 to trap HIV via antibody specific interactions, and identify immune responses that are optimal for directing vaccine induced antibodies to increase MUC16-IgG interactions. Exploring the interaction of antibody-mucin interactions to enhance vaccine function represents an excellent opportunity to tune and optimize current vaccines to prevent acquisition. Importantly, MUC16 covers the columnar epithelium of the upper female reproductive tract and the digestive tract. Enhancing barrier function of these mucosal sites of HIV transmission with a vaccine induced antibody response could prove to increase the efficacy of current vaccine regimens. Knowing which type of IgG subtype(s) and glycoform(s) are involved in MUC16 association could also become an important aspect of vaccine development. For example, different adjuvants, delivery systems, and vaccination regimens could be considered for optimal mucin interacting antibody responses in small trials. This information could inform challenge studies in rhesus macaques and then eventually in a large clinical trial.

? DESCRIPTION (provided by the applicant): The purpose of this grant application is to request funds to partially support planning, organizing and hosting of the only international scientific meeting dedicated to biomedical HIV prevention. The conference, designated HIV Research for Prevention 2016 (HIV R4P 2016) will be held October 18-21, 2016, in Chicago Illinois. HIV R4P 2016 will continue the excellence that was achieved in Cape Town, South Africa at the inaugural meeting in 2014. By all measures, the meeting was a tremendous success bringing together HIV researchers focused on vaccines, microbicides, and pre-exposure prophylaxis (PrEP). The post-conference survey revealed that over 98% of attendees were satisfied with the conference. The primary goals of HIV R4P 2016 are to help create opportunities for trans-disciplinary interactions in biomedical prevention research; expand discussions of cross-cutting issues as new HIV prevention options are developed, and increase coordination and communication among international groups. The pursuit of effective biomedical HIV prevention options has progressed further and faster in the last few years than at any time since the epidemic began. Recent breakthroughs in vaccines, ARV-based microbicides, PrEP, treatment for prevention and other prevention technologies create new opportunities to reduce the impact of the global epidemic. This progress also brings opportunity for scientific debate and creates new challenges as we continue to move HIV biomedical prevention research forward. This international conference will bring together researchers, advocates, clinicians, policy makers, private sector partners and public health experts to present, discuss, and debate the latest research in ARV-based prevention, vaccines, and related areas of HIV prevention. It will also provide a forum to address cross-cutting issues, emphasizing the importance of combination prevention, and it will break down any existing research siloes between different prevention technologies. HIV R4P 2016 will therefore have a significant impact on the advancement of scientific research in unique prevention areas with the potential to make significant progress in the prevention of HIV infection. The conference will attract between 1,200-1,500 members of the HIV prevention community and the requested funding will provide 220 full scholarships, support the participation of 58 speakers, and provide necessary infrastructure to support the meeting. Other funding sources will provide additional support, as well as subsidized registration rates for early-career and lower-middle income country (LMIC) scientists. Information presented at the conference will be disseminated to give free, worldwide access to all who are unable to attend in person. Peer-reviewed abstracts will be published online in an open access manner, and posters will be available online as e-posters. All sessions will be available as webcasts, including free downloads of slides and audio on the conference web site making the information presented available to all interested scientists and the general public.

ABSTRACT This Project is designed to characterize the early post-entry steps of HIV-1 replication, core evolution, and TRIM5? restriction in structural and mechanistic detail. Following virus-cell fusion, the viral genomic RNA (gRNA) is reverse transcribed1-6, the core interacts with host factors and loses its capsid (?uncoating?)7-11, and the host restriction factor TRIM5? can recognize the capsid and block these transformations.12-16 To characterize these processes, we will determine 3D structures of replication initiation complexes that comprise reverse transcriptase (RT), the host tRNALys,3 primer, and various gRNA templates. We will study the conformational changes that accompany replication (Aim 1), develop and apply new single particle assays for studying reverse transcription within isolated viral cores (Aim 2), image transduced cells using fluorescent microscopy and correlated light and electron cryotomography (CLEM-ECT) to visualize and characterize core trafficking, reverse transcription, uncoating and restriction (Aim 3), and reconstitute, image and model TRIM5?- capsid complexes to learn how TRIM5? inactivates the core and stimulates innate immune responses (Aim 4).

PROJECT SUMMARY/ABSTRACT Young women are highly vulnerable to HIV infection, particularly in endemic areas, with young adults making up 30-40% of new HIV infections globally. However, there is very little understanding of why there is discordant HIV infection susceptibility in adolescents versus adults. Furthermore, there is currently no established pre- clinical model to assess any prevention or therapeutic interventions with consideration of age. Here we aim to create the first nonhuman primate model of SIV infection and susceptibility in adolescents. We will assess the mucosal environment of the female genital tract (FGT) of adult compared to adolescent pigtail macaques (Macaca nemestrina). We will determine whether there are baseline differences across the menstrual cycle in adolescent versus adults, and will also determine whether adolescent pigtail macaques are more susceptible to SIV challenge. We will use state-of the art proteomic, microbiome, immunological and in vivo imaging techniques to take advantage of the in depth sampling (multiple tissue and longitudinal) capabilities of the nonhuman primate model. We will determine whether signatures associated with SIV/HIV susceptibility exist in adolescents compared to adults. We will also validate these studies using human cervicovaginal samples from adolescent and adult women. Furthermore, we will assess the mechanisms underlying an altered mucosal environment in adolescents. This will provide the first pre-clinical macaque model of HIV infection in adolescents that can delineate infection susceptibility, correlates of infection, and potential mechanisms underlying altered female genital tract in adolescents. These studies will be a critical advance in understanding of the increased susceptibility of adolescents to HIV infection, and provide the first model for testing interventions in young adults.

The Hope laboratory has recently published the identification and characterization of the earliest cells infected by SIV in the mucosa 48 hours after vaginal and rectal inoculation. In developing this system, the Hope laboratory has developed critical methods and expertise allowing the study of SIV and immune target cell populations within the mucosal barriers. These capabilities will be leveraged along with a strategy to specifically seed infection in tissue so that the latent reservoir will be generated within a discrete area at these mucosal sites (and other body sites). The specific seeding of the reservoir can be validated and complemented by the unique PET/CT imaging expertise/infrastructure pioneered by Francois Villinger to detect foci of active virus infection. With the knowledge of mucosal reservoir localization, we will also track SIV rebound from latency in the early reservoir cells as it cascades to viremia when early, suppressive ART is withdrawn. Identification of these early events will be aided by bioluminescent reporter viruses and whole animal PET/CT. These approaches will provide critical new insights into latently infected cells and facilitate the development of a cure to HIV infection. The proposed studies will achieve the following specific aims: (1) Define and characterize the mucosal virus reservoir cells (cells harboring SIV that remain persistent/latent during suppressive ART). This will specifically characterize the cells with persistent virus that are established during the earliest aspects of acute SIV infection of macaques. This aim will determine the phenotypes, locations, viral production status, and persistence within the mucosa of these reservoirs established with the first days of infection before starting suppressive ART. (2) Define and characterize the spread of virus within and beyond the mucosal foci of SIV infected cells, and from which rebound viremia originates, after stopping early, suppressive ART. This aim uses using both PET/CT and reporter viruses, and will also evaluate host responses and biomarkers. (3) Determine the stability of the mucosal reservoir over time and evaluate the effects of catalytic mTOR inhibitors (given before and temporarily after stopping ART) on the SIV viremia rebound from mucosa, following 24 weeks of viral suppression by ART that started in the first days after mucosal inoculation of SIV. We will utilize recently developed methods and expertise to observe the formation of the mucosal reservoir formed in the first few days after virus acquisition. By seeding the reservoir in specific areas we should be able to observe the rebound from latency. A better understanding of the localization and cell type of the reservoir cells will facilitate the development of a cure for HIV.

Project Summary One of the central limitations of PET/MRI is the lack of accurate means of attenuation correction, which leads to inaccurate and inconsistent quantification of radiotracer uptake. The inconsistency of PET quantification prevents the widespread adoption of PET/MRI in clinical trials, where a precise and reproducible measurement of radiotracer uptake is required. We propose in this application to develop a standard methodology to evaluate MR based attenuation correction techniques and the related PET quantitation accuracy in order to qualify PET/MRI scanners for clinical trials and clinical work. This work will be done in three specific aims. First, we will evaluate materials and create a physical phantom for evaluation of MR based attenuation correction techniques. We will subsequently validate the phantom using a patient study to demonstrate that we can accurately estimate deviations of measured activity using the PET/MRI phantom. Finally, we will harmonize the reconstruction parameters on the two available PET/MRI scanners and evaluate the reproducibility of the quantitative accuracy across 12 PET/ MRI sites split between the two PET/MRI manufacturers. Finally we will incorporate the PET/MRI harmonization data into a global harmonization study that includes both PET/MRI and PET/CT. Through the work described, we aim to create a standardized approach to evaluate the accuracy of PET quantification in the setting of PET/MRI. A standardized methodology will incent vendors to develop attenuation correction methods that result in more consistent uptake estimates across systems, and the ability to qualify scanners will help the adoption of PET/MRI into clinical trials and clinical care.

HIV acquisition in men through penile exposure is one of the least studied aspects of HIV transmission. Medical Male Circumcision (MMC) has been shown in clinical trials to reduce the risk of HIV infection in heterosexual men. It is not fully known how MMC works to decrease male heterosexual transmission. One possible explanation is that the urethral and skin mucosal barriers of the penis change after MMC to strengthen its defenses against HIV. Another possibility is that the foreskin itself is particularly vulnerable to HIV, having weaker defenses allow the virus to more easily reach susceptible immune target cells. This might especially be true when an uncircumcised man has a sexually transmitted infection (STI) would generate a local immune response and recruitment of susceptible HIV target cells. These possibilities form the basis of this study which will determine how the penile mucosal barriers change after MMC. We will recruit several cohorts of sexually active males between 18-35 years old receiving MMC in Chicago and Cape Town and from an STI clinic in Cape Town. To address the potential role of STIs, we will also enroll males in Cape Town who are asymptomatically positive for Chlamydia Trachimonas (CT) or Human Papilloma Virus (HPV). To monitor a changing local environment, we will follow these participants for 2-6 months after CT treatment or MMC. We will measure changes in penile skin integrity using in vivo monitoring of trans epithelial water loss (TEWL) and collect foreskins to use in laboratory-based investigations aimed at identifying factors that may lead to increased HIV susceptibility. We will also characterize the penile skin and urethral microbiome and characterize inflammation levels in the urethra. We will explore possible mechanisms for how MMC works to decrease HIV acquisition in men through three specific aims. Aim 1 will determine how circumcision and asymptomatic STI (CT and HPV) influence the urethral immune environment and microbiome. In Aim 2, we will compare differences in the coronal sulcus (CS) microbiome and TEWL pre and post MMC, and assess the potential impact of asymptomatic CT and HPV infections on these measurements. These findings will be linked to microbiome and immune changes in the urethra. Finally, in Aim 3, we will compare target cells, barrier function, structural barrier protein expression, and virus interactions between inner and outer foreskin and determine if infection with an asymptomatic STI can alter the local mucosal environment. We therefore hypothesize that the inner foreskin: 1) has greater permeability, 2) has reduced expression of skin integrity proteins, 3) contains more HIV-1 immune cells and 4) allows for greater HIV attachment and penetration, than the outer foreskin. These interactions may be influences by the presence of asymptomatic CT and HPV. Collectively, our results will provide fundamental knowledge to inform alternative HIV prevention strategies.

PROJECT SUMMARY / ABSTRACT The goal of this proposal is to develop a novel approach using Prostate Specific Membrane Antigen (PSMA)- directed radiotherapy as an immunogenic stimulus in combination with the immune checkpoint inhibitor pembrolizumab to provide durable tumor regressions in patients with PSMA-avid metastatic prostate cancer. This will be accomplished by using 68Ga-PSMA-11 PET to select patients for treatment, as well as utilizing robust immunologic assays developed in Dr. Lawrence Fong's laboratory at UCSF to quantify the magnitude and breadth of immunogenic stimulus provided by Lu-PSMA radioligand priming. The majority of metastatic CRPC tumors are avid for PSMA expression, and small molecule-based radioligand therapies targeting PSMA have demonstrated impressive response rates in heavily pre-treated patients. However, response durations are quite limited in duration. This is attributable to acquired radioresistance, progression of non-PSMA avid tumors, and cumulative toxicity limiting dose intensity. To overcome these limitations, we aim to shift the paradigm of PSMA-directed radiotherapy, utilizing a single dose of 177Lu-PSMA- 617 to achieve an initial tumor response and immunogenic priming, sustained by subsequent treatment with immune checkpoint inhibition. We hypothesize that a priming dose of 177Lu-PSMA-617 will result in rapid tumor response in PSMA-avid lesions that will induce antigen spread and host anti-tumor response that will be sustained with long-term immune checkpoint inhibition, leading to durable tumor regressions in both PSMA-avid and-negative prostate tumors. Our grant leverages the growing body of literature supporting radiation as an immunogenic priming stimulus, via induction of tumor infiltrating lymphocytes and expansion of T cell clonotypes, supporting the feasibility of this approach. To accomplish this important project, we have assembled an exceptional team of investigators with complimentary expertise in early phase drug development/clinical cancer research (Dr. Aggarwal), PSMA imaging and radioligand therapy (Dr. Hope), prostate cancer immunology and biology (Dr. Fong), and histopathology (Dr. Vandeneberg). In the current proposal, we aim to: Determine the recommended phase 2 dose and preliminary efficacy of 177Lu-PSMA-617 in combination with pembrolizumab in patients with PSMA-avid metastatic prostate cancer (Aim 1); Quantify the immunogenic priming effect of Lu-PSMA RLT (Aim 2). Successful results of our novel proof-of-concept study will form the strong justification for the subsequent evaluation of the combination in a randomized clinical trial, and ultimately may form the basis for a new treatment to improve disease outcomes for men with advanced prostate cancer.

Summary: The goal of the Inter-CFAR collaborative symposium entitled ?HIV and Women 2019? is to share cutting edge research, extend knowledge and foster action to address gaps toward understanding the complex clinical, biological and social aspects of HIV in women. In contrast to other conferences, HIV and Women 2019 focuses on all aspects of HIV in the context of women including structural and behavioral factors impacting their vulnerability while serving as a venue for highlighting the work of early stage investigators including underrepresented minorities. Very few national conferences feature representatives of these groups in such a prominent role, drawing together researchers who may not otherwise have such opportunities. The format of this meeting will enable considerable dialogue among the stakeholders to maximize the effectiveness of strategies to advance the field and promote mentoring and development of early stage and under-represented investigators. HIV and Women 2019 will continue to provide major support to provide for scholarships, networking support and opportunities early stage investigators and underrepresented minorities to attend as participants through travel early stage investigator focused oral and poster presentations, mentoring activities, and sessions with senior leaders in the field. Women and HIV 2019is hosted by the Chicago-based Third Coast CFAR (TC-CFAR), which is located at Northwestern University and the University of Chicago. The 3 research themes of HIV and Women 2019 will focus on 1) acquisition; transgender The impact of hormonal contraceptive on HIV 2) Sex differences in long-acting ART formulations; and 3) Optimizing PrEP for men, women, and populations.All sessions will integrate areas of basic, clinical and social and behavioral sciences in invited talks. Panel discussions will be held at the end of the invited talks (30 minutes) and selected abstracts (15 minutes). To keep the sessions on track and guide the panel discussions, a senior investigator will be paired with a junior level investigator and a community representative to lead each session. The panels will clarify key questions and develop future research strategies that will advance the field. Opportunities for informal networking between junior and senior investigators in the field will be provided throughout the meeting including networking and mentoring lunch with breakout topics, a networking mixer/dinner with the poster session on the first evening. These networking opportunities will be complemented by a mentoring focused session that will highlight grant preparation advice, current NIH funding priorities and opportunities, and CFAR- related resources available to facilitate women's focused research. Outcomes from past conferences include new collaborations and innovative approaches, faculty appointments/promotions and funded grant applications. This is the 6th version of this successful symposia organized by the Inter-CFAR Collaboration on HIV Research in Women working group that will guide women new strategies for future research on HIV-related issues unique to while promoting career development among early career investigators.

Project Abstract Castrate resistant prostate cancer is a uniformly lethal disease and, although there have been a number of new therapeutic agents approved, there is still a large need for more effective treatments in this patient population. In this proposal, we will study a radioligand therapy (RLT) that targets the prostate specific membrane antigen (PSMA), a molecule that is expressed on the majority of prostate cancer cells. In multiple small studies, PSMA RLT has shown to have remarkable efficacy in heavily pretreated patients, with over 40% of patients having decreased PSA of over 50%. PSMA RLT relies on two basic premises: targeting the radiation to PSMA and the radiosensitivity of the tissue to the deposited radiation. First, we will understand how PSMA expression is regulated, allowing us to pharmacologically target cancer cells to increase their PSMA expression. Second, we will understand whether tumors respond because of intrinsic sensitivity to radiation or high doses of deposited radiation. This knowledge will be critical to determine if patients need higher expression of PSMA or increased tumor cell sensitivity in order to improve the response to this therapy. We will test treatments capable of increasing the sensitivity of tumors to radiation as well as potential therapies that can increase expression of PSMA. This work will be used to inform the development of a clinical trial that will involve PSMA RLT and a co-administered therapy to increase its already promising efficacy.

Project Summary/Abstract The mucosal system of the female reproductive tract (FRT) is multifactorial, combining epithelial and mucus barriers, a highly specialized immunology, the microbiome, and hormonal fluctuations to protect this complex and essential organ from attack by pathogen?s such as HIV. Perturbation of any of the components of this system has the potential to decrease barrier function while potentially increasing a woman?s vulnerability to HIV sexual acquisition. Recent studies have revealed that an increase in the presence of multiple (more than 3) pro-inflammatory cytokines are a strong signature of increased HIV acquisition in women. This increased inflammatory environment is consistent with the dysfunction of normal mucosal barrier function. A number of factors have been implicated in this inflammatory state including hormones, the microbiome, and epithelial barrier disruption. However, the origin of these inflammatory cytokines and the mechanism of how they are related to increased HIV acquisition is not understood. To advance HIV prevention science, we need a better understanding of the FRT mucosal system. In this project we will examine hysterectomy derived cervical tissues and mucus donated by high-risk populations in Nairobi, Kenya to gain insight into the changes in the mucosal system that alters epithelial and mucus barrier function. The increased Inflammatory cytokine production can directly or indirectly influence tissue resident HIV target cells to increase the probability of sexual transmission. Our hypothesis is that target cells become more susceptible to HIV infection by infiltrating into the squamous epithelium of the FRT in response to the increased inflammatory cytokines. We will also examine how antibodies can potentially enhance the mucus barrier function potentially revealing a novel strategy for HIV vaccine development.

ABSTRACT: The early phase of the HIV lifecycle encompasses the steps from virus fusion to provirus integration and represents a critical therapeutic target. Small molecule inhibitors of HIV encoded reverse transcriptase and integrase are central components of many therapeutic treatment regimens and pre-exposure prophylaxis. Despite the therapeutic importance of these steps, the field still lacks consensus on several outstanding questions including how trafficking and uncoating are linked to reverse transcription, how and in what state the provirus transits through the nuclear pore, what host factors are involved in these processes, and what distinguishes between a virus that will establish successful infection and one that will fail. Due to the inefficient and relatively stochastic nature of early phase replication, only a small percentage (~15%) of particles that enter the cytoplasm after fusion will result in successful provirus integration. As a result, population-based assays that measure what most viruses do may or may not actually capture what successful viruses do. Nevertheless, technical limitations have historically mandated a reliance on population-based assays, immortalized cell line models, and indirect measurements of biological processes whose underlying assumptions don?t necessarily reflect the biological priors. Only recently have innovations in single-particle tracking, molecular imaging, gene editing, and structural determination allowed for researchers to overcome these limitations, but these specialized technologies have not yet been brought together to answer these critical questions in HIV biology. Here, we assemble a team of HIV researchers with complementary expertise in these powerful approaches to dissect and define the interactions, kinetics, and dynamics between fusion and integration that result in productive infection. We propose to leverage a newly optimized toolbox of molecular labeling methods, a technique collectively termed Infectious Virion Tracking (IVT), to image and track the behavior of individual viral components, ultimately separating individual virions that result in successful infection from those that enter the cell non-productively. Additional specialized technologies including primary cell CRISPR-Cas9 gene editing and cryogenic electron microscopy will be leveraged to interrogate the structure and function of individual components along the route to productive infection. Wielding this novel and innovative series of tools, approaches, and equipment, we aim to: 1) Define the infectious pathway of HIV from fusion to integration in optimized cell culture models and primary human target cells; 2) Determine the role of host permissivity factors and viral components in the processes of the early phase of the HIV life-cycle; and 3) Visualize and define the structure of the viral based machines associated with the HIV genome as it progresses through reverse transcription, traffics through the cytoplasm, enters the nucleus, and ultimately integrates in the host chromosomal DNA. As a collaborative team with complementary specialties that address critical limitations in the field, we are in a unique position to make significant contributions to our current understanding of the early phases of HIV replication.

PROJECT SUMMARY/ABSTRACT Young women are highly vulnerable to HIV infection, particularly in endemic areas, with young adults making up 30-40% of new HIV infections globally. However, there is very little understanding of why there is discordant HIV infection susceptibility in adolescents versus adults. Furthermore, there is currently no established pre- clinical model to assess any prevention or therapeutic interventions with consideration of age. Here we aim to create the first nonhuman primate model of SIV infection and susceptibility in adolescents. We will assess the mucosal environment of the female genital tract (FGT) of adult compared to adolescent pigtail macaques (Macaca nemestrina). We will determine whether there are baseline differences across the menstrual cycle in adolescent versus adults, and will also determine whether adolescent pigtail macaques are more susceptible to SIV challenge. We will use state-of the art proteomic, microbiome, immunological and in vivo imaging techniques to take advantage of the in depth sampling (multiple tissue and longitudinal) capabilities of the nonhuman primate model. We will determine whether signatures associated with SIV/HIV susceptibility exist in adolescents compared to adults. We will also validate these studies using human cervicovaginal samples from adolescent and adult women. Furthermore, we will assess the mechanisms underlying an altered mucosal environment in adolescents. This will provide the first pre-clinical macaque model of HIV infection in adolescents that can delineate infection susceptibility, correlates of infection, and potential mechanisms underlying altered female genital tract in adolescents. These studies will be a critical advance in understanding of the increased susceptibility of adolescents to HIV infection, and provide the first model for testing interventions in young adults.

PROJECT SUMMARY THIRD COAST CFAR CORE E: VIRAL PATHOGENESIS CORE (VPC) The Viral Pathogenesis Core (VPC) brings highly sophisticated tools, services and reagents of biological HIV research to the interdisciplinary research teams of the TC CFAR. The largest number of VPC users address Overall aim 3: to discover/develop scientific innovations that better prevent HIV and improve treatment of all conditions for persons living with HIV (PLWH). We provide, and are enhancing, consultation and training to facilitate the ability of our diverse teams spanning the breadth of HIV research disciplines at the University of Chicago and Northwestern University to contribute to development of new biological interventions that will reduce transmission of HIV, address the increased risk of chronic diseases of aging in the HIV+ population, and advance HIV cure research. VPC has already strongly grown opportunities for adding biological tools to the work of clinical and behavioral HIV researchers addressing Overall aims 1 (improving HIV prevention and care continuums) and 2 (non-AIDS co-morbidities). New activities will add engagement of laboratory researchers studying aging biology and aging-related disorders who are interested in collaborations on aging in HIV, in support of Overall aim 2. The specific aims of the VP Core are: 1) provide an expanding menu of virological, imaging and immunological assays, as well as consultation and navigation to other disciplines and technologies, that will advance innovative, interdisciplinary HIV research; 2) provide expert design/performance of HIV experiments, as well as consultation/navigation on imaging, systems biology, -omics; 3) strengthen information exchange and partnerships with other HIV research disciplines and with laboratory / translational researchers new to HIV who are interested in non-AIDS comorbidities. The VPC?s cutting edge tools for studying HIV at the molecular, cell, tissue, and organismal levels include highly sophisticated instrumentation; experienced technical support and customized training; and expert consultation / navigation for new technologies, creative experimental designs and innovative pathogenic hypotheses. A primary objective of the VPC is to make these approaches equally available to the molecular virologist, the clinical/translational researcher, and the behavioral/social scientist. VPC adds value and the necessary rigor in all experimentation, including for projects that previously had limited or no access to approaches such as multiplex biomarker assays and virologic assessments. The Data Insight Group (DIG) is expanding consultation and navigation to specialized multi-omics approaches to studies of this virus, as well. The DIG brings together an exciting group of young investigators utilizing advanced technologies in their own research programs as a mechanism to extend incorporation of these approaches into other TC CFAR members? research. Customized mentorship, navigation and training will also build and strengthen partnerships and collaborations. In this way, the VPC will advance the mission of the TC CFAR to catalyze scientific innovations that will slow, and eventually stop, the HIV epidemic.