Una O'Doherty - US grants

This proposal outlines a 5 year plan for the training of this candidate for an academic career as a Transfusion Medicine Virologist. She has completed 3 years of formal residency training in Clinical Pathology, and is now working in her sponsor's laboratory. Her research interests center upon HIV-1 molecular pathogenesis, in particular, the regulation of HIV-1 replication in resting and activated T cells. She has a strong background in cellular immunology, having received a Ph.D. in this field under Dr. Ralph M. Steinman in the Department of Cellular Physiology and Immunology at The Rockefeller University. This experience poises her to investigate viral infection in primary leukocytes; however, she lacks the experience in HIV molecular biology necessary to attack questions of regulation independently. An integrative research and training program for the Principal Investigator is being established within the Department of Pathology. The candidate's sponsors are Michael H. Malim and Francisco Gonzalez- Scarano, both recognized authorities on molecular HIV-1 pathogenesis. In addition, an advisory board of prominent scientists will insure the candidate's development. While all steps of the virus life cycle offer opportunities for regulation, it is her intention to examine events that occur between fusion at the plasma membrane and integration of viral DNA into cellular DNA. One reason for pursuing this path is that these aspects of HIV replication have received little attention to date. In addition, there is evidence that infection of T cells is blocked at an early step, since 1 in 100 T cells from infected individuals contain HIV DNA, but only 1 in 10,000 contain integrated HIV DNA. A greater understanding of these critical early steps is likely to identify novel virus-host cell interactions that may serve as future targets for therapeutic consideration. She describes three specific aims: (1) measuring HIV uptake by quiescent T cells; (2) examining the kinetics of viral transport to and through nuclear pore complexes in quiescent and activated T cells; and (3) determining the changing composition of post-entry nucleoprotein complexes in resting and activated T cells. These unprecedented studies promise to provide critical new insights into the HIV disease process. Concurrently, Dr. O'Doherty will acquire the expertise necessary for her to become an independent Transfusion Medicine Virologist.

HIV-1 infected individuals accumulate a reservoir of treatment-resistant, latenUy infected resting CD4+ T cells, even when successful combination therapy clears their viremia. A newly developed technique, polychromatic flow cytometry (PFC), has revealed that the body's population of resting T cells comprises a complex mixture of cells with a variety of different proteins on their surfaces, presumably all specialized for different tasks and in a range of activation states. Thus, T cell activation is far from an "all or none" process. We have begun to investigate the role(s) that the growing number of resting T cell subtypes and states of partial activation may play in the development of viral reservoirs. Using PFC and a quantitative assay for HIV-1 integration we plan to dissect the activation requirements for establishment of stable HIV-1 latency (versus complete productive infection) and the resting T cell subtypes comprising the latent reservoir in infected individuals. A number of pertinent variables will be examined, including: 1. The relative efficiency of integration vs production in naive, central and effector memory resting CD4+ T cells exposed to virus in vitro without activation. 2. The effect of antigen independent intercellular interactions on integration vs production in resting CD4+ T cells. 3. The effects of distinct stimuli alone and in combinations on the efficiency of integration vs production. Stimuli to be tested include individual cytokines and costimulators (e.g., CTLA-4) immobilized on artificial immunostimulatory beads 4. The relative frequency of integrated HIV-1 proviral DNA in memory and na'l've CD4+ T cells isolated from the blood of HIV-1 infected individuals, compared to the same populations purified from uninfected mononuclear cells and exposed to HIV-1 in vitro. Longterm Objectives: Because latently infected resting T cells are the barrier to curing HIV disease, we hope to better define the mechanisms underlying latency, including the cellular activation requirements for its efficient establishment. We will then apply what we learn from our in vitro studies to define more precisely the phenotype(s) of the CD4+ resting T cells that are latently infected in patients.

DESCRIPTION (provided by applicant): Studying the latent reservoir in HIV-1 infected individuals is critical because it is a major barrier to cure. The most clearly defined latently infected cells in vivo are resting CD4+ T cells. Latently infected resting CD4+ T cells are assumed to be the source of viral reactivation when antiretroviral therapy is interrupted. In HIV-1 infected individuals who are maximally suppressed on antiretroviral therapy, approximately 1 in 10(4) resting CD4+ T cells contain integrated proviral DNA, yet only about 1 in 10(6) cells produce virus upon stimulation- i.e. contain latent provirus. What explains the large discrepancy between the amount of integrated HIV-1 proviral DNA and the percentage of cells that are latently infected? What determines whether proviral integration leads to defective versus latent infection? These questions are difficult to answer using cells from HIV-1 infected individuals, because the frequency of latently infected cells is low. The mechanisms that drive a provirus toward defective versus latent infection are undefined. Understanding how proviruses are rendered defective may lead to therapies that decrease the size of HIV-1 reservoirs. Our ability to achieve a high frequency of latent infection and to separate defective from latently infected cells allows us to answer questions that cannot be studied using in vivo samples. We have developed an in vitro model of HIV-1 latency in resting CD4+ T cells that overcomes the low frequency of latent infection that is present in vivo and allows us to study mechanisms of proviral inactivation versus proviral latency. We propose to study three aspects of proviral integration in resting CD4+ T cells that may influence the defective and latent state. They are: (1) the frequency of multiply infected cells which gives the appearance of inactivated proviruses (2) the frequency of mutations in defective and latent proviruses (3) the integration site selection of defective and latent proviruses.

[unreadable] DESCRIPTION (provided by applicant): In my R01, I proposed to determine if HIV could integrate in resting CD4+ T cells and to study how activation of T cells affects that process. I showed that, contrary to dogma, integration does occur in resting cells. This work led to an in vitro model of HIV latency and formed the basis for a new paradigm of HIV reservoir formation - that reservoirs may form without T cell activation. As part of these studies, I developed a new, quantitative, highly sensitive assay for HIV integration. This assay is the first that is sensitive enough to measure integration in PBMCs from patients. Prior integration assays lack the necessary sensitivity, thus my assay is a significant advance. Here, I propose to use the new assay to measure integration in subsets of CD4+ cells in vivo (Aim 1). Comparing integration levels in patients off HAART to patients on HAART may provide another way to evaluate the efficacy of therapy. [unreadable] [unreadable] In the process of my R01 research, I also developed an interest and expertise in resting T cell restriction of HIV. Here, I propose to study the restriction of HIV in CD4+ cells, examining both the cellular and viral factors. My integration assay is sensitive enough for use in highly restricted cells. Using the tools that I developed during my R01 studies, I will characterize the restrictions to HIV infection in blood-derived CD4+ cells (Aim 2), and examine the role that auxiliary viral proteins play in overcoming the restriction in resting T cells (Aim 3). Identification of cellular pathways and viral proteins involved in restriction of HIV infection could provide new targets for antiretroviral therapies. [unreadable] [unreadable] My long-term goal is to be an independent physician-scientist who conducts basic research that has clinical relevance. My short-term goal is to obtain this award, because it will guarantee me 75% protected time, which I need in order to focus on my research and make significant contributions to the field. Since becoming an Assistant Professor, I am first or senior author on five publications. I have recently recruited one undergraduate student, two graduate students and a postdoc, with whom I have submitted manuscripts, thus my productivity is increasing. The ability to develop my own niche in HIV research and to obtain funding (6 grants, including an R01) demonstrate my potential for success as an independent physician-scientist. Finally, the letters of recommendation from my department chair, division chief, mentor, and collaborators convey the strong support that I receive at Penn. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): A challenge in gene therapy is the limited ability of vectors to transduce G0 resting CD4+ T cells and G0 stem cells. It is important to develop such vectors because transducing resting cells, as compared to activated cells, is more physiological, less expensive, less labor-intensive, possibly safer, and the gene-marked cells may be longer lasting in vivo. VSV-G protein is an envelope that is commonly used to pseudotype lentiviral vectors for transducing activated CD4+ T cells;however, lentiviral vectors pseudotyped with VSV-G do not transduce G0 CD4+ T cells efficiently. A long-term goal of our lab is to design a better envelope for transducing G0 resting CD4+ T cells. Therefore, we investigated why VSV-G pseudotypes do not transduce resting CD4+ T cells by measuring binding, fusion, reverse transcription, and integration in resting cells. Using this approach of assaying individual steps of HIV infection, we previously showed, contrary to dogma, that HIV transduces resting CD4+ T cells. When we compared lentiviral vectors pseudotyped with VSV-G or HIV envelope (Env), we found that HIV Env pseudotypes fused to resting CD4+ T cells 100 times more efficiently than VSV-G pseudotypes. Further investigation of this finding suggested that HIV Env fuses to resting cells much more efficiently because binding of HIV Env to co-receptor triggers signaling that induces cellular changes that enhance viral fusion. Toward the goal of designing a better envelope for transducing G0 resting CD4+ T cells, we will test the ability of other envelopes, singly and in combination with HIV Env, to fuse to resting T cells, and explore the mechanism underlying the ability of HIV Env to efficiently fuse to resting CD4+ T cells. PUBLIC HEALTH RELEVANCE: Gene therapy is developing into a viable alternative for the treatment of many diseases including immune system disorders, certain cancers, and even HIV infection. In gene therapy, a gene of interest is delivered to a target cell, which in some cases is a quiescent cell;however, to obtain efficient gene transfer it is often necessary to activate a quiescent cell to enter the cell cycle. An important but elusive goal is delivery of the gene of interest to certain quiescent cells without activating them, because cell cycling not only alters the function of the quiescent cell, but may also be a safety risk. In this grant we propose to identify methods that allow delivery of a gene to quiescent CD4+ T cells as the first step toward achieving the goal of gene delivery without cellular activation.

DESCRIPTION (provided by applicant): HIV-infected individuals accumulate a reservoir of treatment-resistant, latently infected resting CD4+ T cells, even when therapy is started shortly after exposure. It remains unclear how treatment resistant HIV viral reservoirs are maintained. One theory is that latently infected cells are established early in infection and that these cells persist in the presence of HAART. A second theory is that a very low level of HIV replication is required for viral persistence. The goal of this application is to determine if ongoing replication can be demonstrated to occur in the presence of HAART. If this can be demonstrated to occur then it should be considered as a potential mechanism for reservoir maintenance. Distinguishing between these theories is important because it will drive what approaches we should take if HIV infection is to be eradicated from infected patients. However, regardless of the mechanism(s) of reservoir maintenance or the approaches to eradication of HIV, assays that detect ongoing HIV replication would be useful for monitoring therapy. In an effort to assess if ongoing replication occurs in patients on HAART, we propose to monitor total and integrated HIV DNA over time after initiating HAART. The idea behind the proposal is that in the absence of ongoing replication total DNA should eventually equal integrated HIV DNA. We will measure total and integrated HIV DNA in mononuclear cells in both blood and the GI tract and test if an excess correlates with independent measures of ongoing replication (Aim1A). In addition, we will measure these intermediates within subsets of these cells (Aim1B). Finally, we will take a complementary approach to determine if ongoing replication occurs by testing for viral spread to short lived cells (Aim1C). We expect our experiments will provide information on whether ongoing replication occurs in HIV infected individuals on HAART and potentially new information on HIV pathogenesis. PUBLIC HEALTH RELEVANCE: It is unclear why HIV is treatable, but not curable. It appears that there is a treatment resistant reservoir, but how this reservoir is maintained in the presence of HAART is unclear. In this grant proposal, we try to address this question by determining if HIV replication is completely stopped with antiviral therapy. We propose experiments that attempt to answer this question by measuring viral DNA intermediates over time on antiviral therapy.

DESCRIPTION (provided by applicant): HIV-infected individuals accumulate a reservoir of treatment-resistant, latently infected resting CD4+ T cells. A special category of HIV infected individuals called elite suppressors have undetectable viral loads in the absence of highly active antiretroviral therapy (HAART) and have a smaller HIV reservoir. There is compelling evidence that elite suppressors (ES) have higher functioning CTL activity against HIV than treated and untreated chronic progressors. Our recent data provide evidence that CTL in ES have activity against latently infected resting CD4+ T cells in vivo. Specifically, we demonstrate for the first time that ES patients have dramatically lower levels of integrated HIV DNA and a relatively large excess of unintegrated HIV DNA compared to HAART patients. This is consistent with CTL pressure since HIV proteins are expressed more efficiently from integrated compared to unintegrated HIV DNA. In addition, we have preliminary data that latently infected resting CD4+ T cells express HIV proteins but do not permit spreading infection and thus should be susceptible to CTL pressure. At the same time, we show with our in vitro model of latency that CTL have activity against latently infected resting CD4+ T cells. Thus, the range of CTL targets is greater than previously thought since it appears that latently infected cells can be targets of CTL. In this application, we plan to test for further evidence of CTL pressure by assessing the frequency of HIV RNA+ cells among resting CD4+ T cells in ES vs HAART patients. We expect the frequency of HIV RNA+ cells normalized to integration to be lower in ES because we expect the majority of HIV RNA+ cells will express protein and be subjected to CTL lysis. Finally, we will determine if integration levels increase over time (as suggested by our preliminary data) in all or a subset of ES and if an increase in integration levels correlates with a loss of CTL function in ES. We also consider the alternate hypothesis that integration levels may increase over time in ES because defective proviruses accumulate.

DESCRIPTION (provided by applicant): Interferon-?? (IFN-?) is known for its ability to restrict ongoing HIV replication, but with the advent of ART, interest in IFN-?'s activity against HIV waned. Exciting new data from a recent trial renews our interest as it suggests IFN-? may have activity against HIV reservoirs since integrated HIV DNA (but not total HIV DNA) was reduced in a subset of patients treated with IFN-? (pegylated interferon-?2A). This R21 proposal addresses a role for CTL as driving the decrease in HIV integration found with IFN-? therapy. Our rationale that IFN-? may enhance immune clearance is derived from the fact that HIV proteins can be expressed more efficiently from integrated HIV DNA than from unintegrated HIV DNA, which would lead to preferential clearance of integrated over unintegrated HIV DNA-the results found in vivo in the prior IFN-a trial. These findings led us to our overarching hypothesis: that IFN-? enhances immune clearance of the reservoir. We will use our latent model to further explore if IFN-? increases immune clearance by either making the reservoir more visible or by enhancing effector function. Our general approach is to purify CD4s and CD8s from ART infected patients and to treat these cells individually with IFN-? before coculture and then assess if reservoirs are reduced. We will compare these results to HDACi which are known to enhance HIV expression and are currently being tested for activity against HIV reservoirs in clinical trials. The effects of HDACi on antigen presentation and HIV specific CTL are unknown. We will dissect if the effects are on CD4s or CD8s and if HIV antigen expression, presentation or CTL function are enhanced by IFN-a. We will also compare and contrast IFN-? Responders and Nonresponders in the original trial to elucidate the essential steps to reservoir reduction. We are in a unique position to contribute to this question because of our expertise in measuring integrated HIV DNA and our ability to detect small changes in integration levels. We envision our study will lead to future better trial design and new insights that will be required for HIV eradication. Our specific aims are: Aim 1: Determine if IFN-? enhances clearance of latent cells in vitro and ex vivo by increasing HIV expression, antigen presentation, and/or CTL effectiveness and compare to the effects of HDACi to understand how IFN-? might reduce reservoirs. Aim 2: Examine if in vivo evidence in the prior IFN-a trial supports our model of CTL- mediated clearance.

? DESCRIPTION: There is renewed interest in testing strategies to eradicate HIV. A preclinical model for testing potential eradication approaches avoids exposing patients to ineffective and potentially harmful agents. Moreover, an ex vivo model might allow us to characterize why the reservoir is resistant to clearance. We propose a new approach to evaluate the effect of potential therapies on CTL clearance of HIV ex vivo. This model represents an innovative approach to reservoir characterization because it uses CTL clearance as an indicator of reservoir protein expression, allowing us to take advantage of the sensitivity of the immune system to measure low-level protein expression. Our ex vivo model uses cells from ART patients to study the effectiveness of a candidate anti-reservoir therapy: interferon alpha (IFN-a). We find that patient-derived CD4+ T cells show a drop in levels of integrated HIV DNA after treatment with IFN-a and subsequent coculture with Gag- primed CD8+T cells. Using this system we will characterize the proviruses that are resistant to ex vivo clearance by sequence analysis (Aim 1) and expression studies (Aim 2). Using samples from an ongoing IFN-a trial as a proof of principal, we will test whether our ex vivo model can predict which patients respond to IFN-a therapy in vivo (Aim 3). Finally, we will study the proviral clones that are resistant to clearance to understand if lack of RNA or protein expression may explain lack of clearance (Aim 4). We also will determine whether repeat stimulation or combination stimulation leads to enhanced reservoir clearance in coculture. We hypothesize that the fraction of proviral DNA that can be induced to express HIV proteins (and is therefore susceptible to clearance) is higher than previously appreciated. If our hypothesis is correct, then replication defective viruses are often capable of expressing viral protein and both replication competent and defective viruses will be cleared in our assay. To test our hypothesis, we will determine whether the protein-expressing cells in our system contain replication competent proviruses. Notably, as long as replication competent viruses are cleared along with replication defective proviruses, then our approach will likely have utility in identifying effective therapies.

? DESCRIPTION (provided by applicant): A major hurdle in HIV cure research is the lack of robust high throughput assays to assess the character and size of the reservoir. This challenge is compounded by the need to distinguish between cells infected with replication competent and replication defective HIV. Objectives: We propose to distinguish replication competent from defective proviruses by counting the number of cells that contain HIV DNA that are capable of expressing HIV Gag+ and down regulating CD4. We achieve high throughput detection by adapting a new technology, called FAST (Fiber-optic Array Scanning Technology), to detect rare cells that express HIV Gag and down regulate CD4 (Gag+CD4dim cells). FAST is a proven technique that has been utilized to detect rare tumor cells in a high throughput fashion (e.g. ~1 tumor cell in 20 million normal cells in one minute). Our goal is to determine if FAST can be used to monitor therapies that target HIV reservoirs. Method: We first compare the FAST assay to a FACS sorting assay developed by Dr. O'Doherty to test if our FAST assay can specifically measure true Gag+CD4dim cells. In the FACS assay, Gag+CD4dim cells are sorted and the number of true positives is determined by measuring proviral DNA in the sorted population. Specificity is demonstrated by showing enrichment for HIV DNA only in the Gag+CD4dim and not in the Gagneg cells. In Aim 1, we test the specificity of FAST to identify Gag+CD4dim by comparing it to the number quantified by FACs sorting in patients at baseline. FAST provides additional specificity by visualizing the characteristic punctate staining of internalized CD4. In Aim 2, we compare FAST to FACS after stimulating negatively-selected CD4+ T cells. In Aims 1 and 2, we will determine if the Gag+CD4dim cell phenotype distinguishes replication competent from defective HIV by sequencing provirus from the sorted cells after limiting dilution PCR. We expect that hyper mutated proviruses will not express Gag or any HIV proteins and proviruses with massive deletions will either be disabled to express Gag and/or the proteins responsible for CD4 down regulation. Thus, by selecting for Gag+CD4dim we expect to eliminate most proviruses that are defective. In Aim 3, we correlate FAST measures of reservoir with QVOA and integration levels. Significance and relevance to public health: A high-throughput quantitative assay would allow more rapid evaluation of multiple therapies that target reservoirs. Our preliminary data suggest to us that the ability to express HIV Gag proteins and down regulate CD4 upon stimulation as in Aim 2 will be a strong correlate of replication competence. Moreover, we envision the utility of the assay will extend beyond reservoir measurement as it can be exploited to determine the dose response, efficacy and kinetics of multiple candidate therapies that are designed to induce HIV protein expression.

The advent of antiviral therapy (ART) revealed a treatment-resistant reservoir in CD4+ T cells capable of refueling HIV viremia when treatment is stopped. This reservoir is a major barrier to achieving a cure for HIV infection. Recent work suggests reservoir decay on ART is slower in some individuals due to proliferation of T cells containing intact proviruses since identical intact sequences are predominant after many years on ART in roughly half of subjects. These identical sequences likely represent clones of cells that have proliferated. The driving forces behind proviral clonal expansion remain mysterious, but integration into introns of oncogenes likely plays a role. To study this question, we combine proviral sequencing with integration site sequencing. Our innovation is recognizing that to properly assign proviruses we need longer stretches of HIV DNA than typically provided by current methodologies. We propose a new method to clone integration sites that is based on long-range PCR techniques previously utilized by our group. In Aim 1 we propose to develop a method that will capture unique junctions that are created in proviruses with large deletions. These deleted proviruses retain splicing ability and fall into two broad categories, those with strong and those with weak potential to express HIV proteins. In Aim 2, we propose a method to clone the integration site of intact proviruses which requires amplifying longer stretches of HIV DNA that are contiguous with the human DNA. With this in mind, we describe a systematic approach to identify the chromosomal address of the intact proviruses including intact proviral clones. We hypothesize that intact proviral clones are generally in introns and that this placement within the intron plays an important role in clonal expansion as it permits splicing of HIV to downstream exons of oncogenes. This in turn provides a new target for HIV eradication strategies. The premise of our proposal is largely based on preliminary data from our group showing that there are two counterbalancing forces that cause (1) proviral contraction through immune clearance and (2) proviral expansion through clonal proliferation after splicing to a downstream oncogene. We ask in this proposal if the same two forces act on both defective and intact replication-competent proviruses. The significance of our proposal include that it may contribute to growing evidence that the reservoir is more visible than previously realized. Our work suggests that perturbing these two forces by either enhancing immune clearance or targeting splicing or downstream exons of splicing may reduce reservoir size. We envision this work could lead to a larger study to understand if dysfunctional cytotoxic T cells have a role in intact proviral clonal expansion.

The advent of antiviral therapy (ART) revealed a treatment-resistant reservoir in CD4+ T cells capable of refueling HIV viremia when ART is stopped. This reservoir is a major barrier to achieving a cure for HIV infection. Our recent work suggests the inherent reservoir decay is more rapid than previously recognized. This decay is obscured due to an opposing force that results in proviral clonal expansion. The driving forces behind proviral clonal expansion remain mysterious, but integration into introns of oncogenes may play a role. Overall objective: In this proposal, we dissect the drivers of reservoir contraction (Aim 1) and expansion (Aim 2). Our approach will be to perturb both forces and to study the resulting effects. We will use proviral, integration site, and RNA sequencing to understand how perturbing these forces affects the genetic make-up of proviruses, their propensity to expand, and their expression. Our approach is to perform massive deep sequencing in a few individuals rather than large sample size. We believe our intriguing results validate our deliberate decision to limit sample size to obtain deeper sequence information within each individual. With this approach, we recently provided unprecedented depth and elucidated previously unknown selection pressures. Design and Methods: In Aim 1, we isolate the role of immune clearance by measuring reservoir contraction in vivo and in vitro. We also dissect the cytotoxicity induced by HIV proteins by mutating individuals Open Reading Frames before infecting CD4 T cells with a barcoded virus. In Aim 2, we dissect the drivers of clonal expansion, including HIV-driven cell division, by using a barcoded virus. We also use longitudinal integration site analysis to compare the rate of clonal expansion as well as the ?character? of the expanded proviral clones in elite controllers, acutely and chronically infected individuals on ART. The premise of our proposal is largely based on our work showing that there are two counterbalancing forces that cause (1) proviral contraction through viral cytotoxicity and immune clearance and (2) proviral expansion through clonal proliferation. The significance of our proposal includes that it may contribute to growing evidence that the reservoir is more visible than previously realized. We envision this work could lead to approaches that enhance immune clearance or target splicing to reduce reservoir size.