PPG: Integrating Mechanistic Insights from Diverse Models to Prevent Reactivation of CMV following Transplantation

Cytomegalovirus (CMV) is a member of the herpesvirus family that latently infects the majority of the population. Reactivation of latent CMV causes significant infectious complications in immunocompromised hosts, including recipients of solid organ transplants. Current anti-viral therapies are associated with toxicity and development of resistant strains. Furthermore, these therapies target replication of the virus after reactivation has occurred. The goal of this project is to better understand the molecular mechanisms that control repression of viral gene expression in latency, and to identify factors that lead to activation of viral gene expression in response to transplantation. This knowledge may lead to the development of novel therapies that prevent reactivation, rather than treating it after it has occurred. There are two overall themes: 1) that epigenetic factors control both repression of viral gene expression in latency and activation of gene expression in reactivation; 2) that an inflammatory immune response triggers reprogramming of viral chromatin to activate viral gene expression in transplant recipients.

This program project is comprised of three sub-projects and three cores. In Project 1, Dr. Zhang will investigate molecular pathways leading to reactivation of murine CMV (MCMV) using both in vivo models that closely mimic the clinical setting of reactivation in immunocompromised transplant recipients and recently developed in vitro models. In Project 2, Dr. Hummel will investigate epigenetic factors that control latency and reactivation of HCMV in hematopoietic progenitor cells, which are sites of latency in vivo. In Project 3, Dr. Thorp will investigate MCMV infection, latency and reactivation in transplantation, specifically using donor-specific transplant tolerance.

Projects and Cores

Faculty Associated with the Program Project: Integrating Mechanistic Insights from Diverse Models to Prevent Reactivation of CMV following Transplantation
PPG Program Project Director	Mary Hummel, PhD
Project 1 and Core C Leader	Jenny Zhang, MD
Project 2 Leader	Mary Hummel, PhD
Project 3 Leader	Edward B Thorp, PhD
Core A Leader	Jenny Zhang, MD
Core B Leader	Qing C Chen, MD, PhD
Core C Leader	Lihui Zhao, PhD

Project 1: In Vivo and In Vitro Models for Mechanistic Insights into MCMV Reactivation

Complementary In Vivo and In Vitro Models Provide Mechanistic Insights into MCMV Reactivation in a Clinically Relevant Mouse Kidney Transplant Mode

Reactivation of latent human cytomegalovirus (HCMV) remains a significant cause of morbidity and mortality in transplant recipients, despite the use of antiviral drugs. Therefore, new approaches are required to reduce the complications from this pathogen. Due to the species specificity of HCMV, we have developed a clinically relevant transplant model using the highly related murine CMV (MCMV) as a model to study CMV latency and reactivation in the context of organ transplantation. In this model, MCMV latently infected kidneys are transplanted into B6 mice, inducing transcriptional reactivation of Immediate Early (IE) genes. Addition of a clinically relevant immunosuppression regimen results in reactivation of latent virus in the donor kidney, which disseminates into other organs of the recipient. The central hypothesis of this proposal is that the inflammatory response elicited by the transplanted kidney results a signaling cascade that stimulates epigenetic reprogramming of latent viral genomes, transcriptional reactivation of IE genes, and in immunosuppressed recipients, re-entry of latent virus into the lytic replication program. To test this hypothesis, in Aim 1 we will investigate the requirement for candidate factors identified in our preliminary studies, and their downstream signaling intermediates, in inducing reactivation. We have also shown that transplant-induced reactivation of IE expression is associated with epigenetic reprogramming. In Aim 2 we will investigate promising therapeutic interventions that target the epigenome for their ability to prevent reactivation of latent MCMV in our model. In Aim 3 we will investigate a new in vitro model of differentiation- induced reactivation of latent virus in bone-marrow derived murine hematopoietic progenitor cells This complementary in vitro model will be exceptionally useful for defining molecular mechanisms in the absence of the complexity of our in vivo model, and for corroborating data from in vitro studies of HCMV latency and reactivation. Upon completion, our studies will have identified signaling pathways that lead to epigenetic reprogramming of viral chromatin to reactivate latent MCMV and potential therapeutic targets. This project has considerable synergy with Project 2, which will investigate inflammatory and epigenetic factors that control HCMV latency and reactivation, and Project 3, which will investigate complications due MCMV infection and inflammation on tolerance to donor-specific antigens.

Faculty

Xue Feng Liu, PhD Project Leader

Project 2: Epigenetic Control of HCMV Latency and Reactivation

Previous studies have shown that HCMV establishes latency in myeloid lineage cells, including CD34+ hematopoietic progenitor cells (HPCs). These pluripotent cells have a unique epigenetic environment in which HDAC expression is low, and many genes are transcriptionally inactive, but carry bivalent chromatin marks (repressive H3K27me3 and activating H3K4me3) characteristic of facultative chromatin. We hypothesize that HCMV exploits the unique epigenetic environment of HSCs, so that most lytic genes are repressed through bivalent histone modification, but the viral genome is poised to reactivate under appropriate stimulation. The major immediate early (IE) genes encode transcriptional regulatory proteins required to activate lytic infection. Expression of these genes is controlled by the major immediate early promoter (MIEP), which carries binding sites for both activating and repressive transcription factors. We further hypothesize that repressive cellular transcription factors bind to the MIEP to mediate heterochromatinization of the genome in HPCs, and that HCMV reactivation requires 1) a switch in factors binding to the MIEP, from repressive to activating transcription factors; 2) recruitment of co-activator complexes through interaction with DNA-binding partners; 3) reprogramming of viral chromatin by enzymes that mediate histone modifications. We will investigate these hypotheses in Aims 1 and 2 using experimentally infected CD34+ cells as a model for latency and differentiation to a dendritic cell phenotype as a model for reactivation. In Aim 3 we use a mutant virus that conditionally expresses IE proteins to investigate the role of the IE proteins in reprogramming viral chromatin to activate lytic replication. Through the use of state-of-the art epigenetics, proteomics, and functional analyses, our studies will have determined mechanisms that regulate viral chromatin in latency and reactivation, and identified potential new targets for therapeutic intervention to prevent reactivation of CMV. This project synergizes with Projects 1 and 3, which will investigate the roles of inflammation and epigenetics in control of MCMV in latency and reactivation.

Faculty

Mary Hummel PhD, Project Leader

Project 3: MCMV Infection, Latency and Reactivation in Transplantation Tolerance

A high prevalence of CMV seropositivity in both organ donors and organ recipients makes CMV reactivation following transplantation a significant cause for morbidity and mortality and a contributor to poor graft outcome. An emerging concept in CMV reactivation points to transplant-induced inflammation as an early trigger for CMV transcriptional reactivation. Administration of immunosuppressants further suppresses viral-specific immunity, leading to the eventual completion of lytic viral replication. We hypothesize that achieving robust donor-specific transplant tolerance will inhibit transplant-associated inflammation, therefore remove a critical early trigger for epigenetic reprogramming and transcriptional reactivation of the CMV viral genome, and consequently prevent CMV reactivation. We propose to test this hypothesis in a forward-thinking, highly clinically relevant donor-specific transplant tolerance model using a strategy of pre-transplant donor “negative vaccination” by delivery of donor cells treated with the chemical crosslinker 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI). Our studies in murine models of transplantation tolerance have already led to ongoing pre-clinical studies in non-human primate models of allogeneic and xenogeneic transplantation. Moreover, a first-in-human clinical trial based on the same principle using peptide-coupled autologous cells in patients with multiple sclerosis recently established the clinical feasibility, tolerability, and safety of this novel tolerance strategy. Our preliminary data using MCMV demonstrated that tolerance by this approach prevented immediate early (IE) gene transcriptional reactivation from latent MCMV. Conversely, acute MCMV infection impaired attempted tolerance induction and destabilized established tolerance. Therefore, in the current application (Project 3), we propose to examine the following three areas using murine transplant models with MCMV infection and ECDI-donor cell tolerance strategy: (1) the effects of donor-specific tolerance on MCMV acute infection, establishment of latency, and reactivation from latency; (2) the effects of MCMV infection on the induction and stability of donor-specific tolerance; (3) the cellular mechanisms underlying the reciprocal interactions between MCMV infection and donor-specific tolerance. Our long-term goal is to determine therapeutic targets for prevention of CMV reactivation while establishing and maintaining stable donor-specific transplant tolerance.

Faculty

Edward B. Thorp, PhD, Project Leader

Core A: Microsurgery & Histopathology Core

Investigation of the molecular pathways that lead to CMV reactivation in transplant recipients has been hindered by the lack of appropriate animal models due to the species specificity of HCMV. The mouse transplant models are essential for us to continue investigating inflammatory mediators and epigenetic processes that regulate CMV reactivation following transplantation. The Microsurgery and Histopathology Core (Core A) combines histopathologic expertise and techniques with the existing microsurgery core at NUCTC (NUCTC) and will provide the necessary skill sets, expertise and experience for the use of mouse transplant models to test the hypotheses and aims proposed by individual Project leaders while guaranteeing technical success and efficiencies. The long-term goal of Core A is to serve the Program Project as a central resource with state-of-the-art microsurgical techniques and histological methodologies to support technical and analytical needs of all proposed studies.

Core A is responsible for performing the mouse kidney and heart transplant procedures proposed in project 1 and 3 with quality, consistency and efficiency, providing immediate post-transplant animal care and drug administration. The Core also provides technical and analytical support for histopathological evaluation such as renal function monitoring, tissue sample collection/preservation/processing for light and immunofluorescence microscopy analyses. In addition, the core supports and facilitates collaborations with Core B, Projects 1 and 3. Core A will provide the necessary support for storage of extra transplant samples and maintaining the archive of tissue samples for further analyses. Core A will coordinate experiments among projects and ensure maximal utilization of tissues samples from each transplant, and will be responsible for distribution of tissue samples to the relevant investigators for their research applications, Core B-specific activities, and off-campus collaborations, as proposed by the Project PIs.

Core A is staffed by experienced microsurgeons and lab technologists, and is unique in that it has the capability and flexibility to address highly specialized technical needs of both animal surgical models and histologic analyses, as well as to facilitate interactions among projects and investigators. With combined expertise in transplant microsurgery and histopathology, as well as state-of the art microsurgery and histology facilities, Core A is well positioned to achieve its long-term goal.

Faculty

Jenny Zhang MD, Core Leader

Yashpal Kanwar MD, PhD, Co-investigator

Core B: Precision Cell Isolation & Analysis Core

The precision cell isolation and analysis core, designated as Core B, is a merging of core facilities to support the technical and analytical needs of all studies in the program as they relate to cell isolation and molecular analysis.

Core B includes the Robert H. Lurie Comprehensive Cancer Center Flow Cytometry Core Facility and the Northwestern Proteomics Center of Excellence and Core Facility. Both core facilities provide state-of-the-art instrumentation. The flow core facility houses multiple cell sorting and analysis instruments including 6-laser instruments capable of up to 18 parameters. The proteomics facility houses numerous instruments capable of the most advanced levels of targeted quantitative bottom-up proteomics and precision top-down proteomics.

The precision cell isolation and analysis core combines the knowledge and expertise of the flow cytometry and proteomics center personnel into a central resource. In addition, this core provides the support for collaborative efforts in nucleic acid analysis. Core B facilitates collaborations to utilize novel nucleic acid hybridization techniques to identify infected cells and coordinates samples and data for CHiP seq and RNA seq studies.

Core B synergizes and synchronizes the use of multiple cutting-edge technologies on specific populations of cells from an array of sources.

Faculty

Qing C Chen MD, PhD Core Leader
Neil Kelleher PhD, Co-investigator
Suchitra Swaminathan PhD, RHLCCC Flow Cytometry Core Facility

Core C: Administrative Services Core

The primary objective of the AdministrativeCore, designated as Core C is to support the administrative needs of the various Projects and Scientific Cores as they relate to the organization and administrative management of the overall program, coordination and communication within the program, internal quality controls, management of day-to-day program activities contractual agreements and dispute resolution, consistent with the overall strategy of the Program Project. Core C is housed at the Comprehensive Transplant Center, Northwestern University, Feinberg School of Medicine. Core C provides the following resources to investigators:

Facilitate communication and scientific interaction among investigators involved in each project and core
Manage financial operations, document preparation, grant management, publication and presentation assistance, travel arrangement and overall operations management
Provide coordination for statistical and bioinformatics support

Faculty

Lihui Zhao, PhD, Core Leader

CMV P01 Symposium: Cytomegalovirus Latency and Reactivation

Please join us for the 3rd Annual CMV P01 Symposium: Cytomegalovirus Latency and Reactivation

Presentations from our project core leaders

Date: Tuesday, September 25, 2018

Time: 12:00p.m. to 5:30 p.m.

Location: Prentice Harris Family Atrium – 250 E. Superior, 3rd Floor

Registration is closed.

Recent News

Thank you to all who participated in our third annual CMV P01 Symposium for Cytomegalovirus Latency and Reactivation on Tuesday, September 25, 2018.

Congratulations to Dr. Michael Abecassis and colleagues for the recently awarded P01 grant issued by the National Institute of Health (NIH) titled, Integrating Mechanistic Insights from Diverse Models to Prevent Reactivation of CMV following Transplantation.