Infectious diseases including chronic infections such as hepatitis B virus (HBV) remain leading causes of death worldwide. Influenza (Flu) and other respiratory infections kill ~half a million people globally each year, and our vaccines for many infections remain poorly effective, especially in vulnerable populations. Most fundamental insights into the mechanisms of immune responses to pathogens have come from animal models. Interrogating human immune system function has typically been limited to descriptive analyses or confirmation of observations from animal studies. This gap in knowledge limits our ability to use rational manipulation of specific immune pathways to effectively combat chronic infections and improve vaccines. The ongoing revolution in cancer immunotherapy – in which blockade of immune checkpoints such as PD-1 is used to stimulate cancer immunity – has not yet been exploited for human chronic infections and vaccines. Here we will test how PD-1 pathway blockade alters immunity in humans to chronic infections or vaccination, providing an opportunity to dissect the mechanistic role of this pathway in human innate and adaptive immunity. The studies proposed are highly innovative and have potential to reveal groundbreaking new fundamental knowledge of human immunity, because a) the PD-1 pathway is central to T and B cell responses to pathogens; and b) there is almost no information in humans about how blocking PD-1 affects cellular or humoral immunity to chronic viral infection or vaccines. Thus, the central goal of this Program is to employ specific immune perturbation through PD-1 blockade during persistent HBV infection and Flu vaccination, and use this intervention to define the innate and adaptive immune mechanisms controlled by PD-1 for improved therapeutic and preventative treatments for human infections. This Program will develop a broad platform with which to interrogate other existing or emerging immunotherapies and human immunity to infection. Thus, our U19 Program is organized around the following central hypothesis: “We hypothesize that perturbing specific immunoregulatory pathways (e.g., PD-1) during human persisting viral infection and vaccination will provide insights into fundamental immune mechanisms involved in pathogen immunity and lead to novel opportunities for therapeutics and vaccines.” We will capitalize on this revolution in human immune system perturbation – moved to the clinic initially for cancer – to understand basic mechanisms of immune responses to persisting infections and vaccines. We propose 2 Projects to: 1) define how PD-1 blockade in humans re-invigorates HBV-specific CD8 T cell and bystander immune responses in HBV+ patients; and 2) test how PD-1 blockade impacts vaccine-induced CD4 T cell, B cell and antibody responses. The Projects are supported by administrative and scientific Cores to enable clinical sample acquisition, antibody evaluation and TCR, BCR and single-cell RNA sequencing.

Our Center consists of four Cores and two Projects:

John Wherry, Core Co-Lead

The purpose of CORE A is to provide administrative support to the Program Leaders, to the Principal Investigators of the Projects and Cores, and to the scientific personnel of the Projects and Cores of the Pathogen Response on Immunotherapy Study Group (PRIG). CORE A will be located in Dr. Chung’s laboratory/office complex at the Gastrointestinal Division of Massachusetts General Hospital. The main goals of CORE A are to manage and oversee the scientific efforts of the Projects and to coordinate scientific and administrative interactions. Thus, the specific tasks of CORE A will be: (a) to facilitate interactions between Program Investigators, Scientific Advisors and administrative personnel, (b) to plan and coordinate the External Scientific Advisory Group (ESAG) meeting as well as meetings between Program scientists, (c) to organize and coordinate travel for scientific advisors, visiting scientists and Program Investigators, (d) to assist in the organization of the enduring seminar series and annual External Scientific Advisory Group Meeting and Scientific Symposium, (e) to facilitate resource sharing including assisting in the speedy execution of material transfer agreements (MTA) and (f) to assist the Program Directors and Principal Investigators in preparation of progress reports, financial reports and manuscripts for publication. CORE A will also be responsible for the resolution of any potential conflicts.

KIM, ARTHUR Y, Core Lead
Ramin Herati, Core Co-Lead

This U19 aims to determine how PD-1 blockade affects human immune responses induced by chronic viruses and vaccines. The Clinical Core of the proposed MGH-UPenn Pathogen Response on Immunotherapy Study Group (PRIG) will be central to the scientific efforts of this consortium. A unique and expansive sample repository has been created over the last 18 years and well- characterized for clinical, immune, and genetic data. This repository will be maintained and crucially expanded to include longitudinal samples from individuals undergoing immunotherapies via clinical infrastructures at both MGH and UPenn. This concerted effort will maintain a high throughput of sample availability to catalyze scientific efforts across the consortium, including tracking samples to investigators in Projects 1 and 2 and Cores C and D.

Frederick Bushman, Core Co-Lead

A major goal of this U19 is to determine how PD-1 blockade affects human antibody responses against different viral antigens. Core C will fully interrogate serum antibody responses against a range of different viruses from donors undergoing PD-1 therapy in Projects 1 and 2. The Core will standardize assays, complete assays, and provide computational support for the analyses of complex serological datasets. We will offer standardized ELISA-based services that quantify antibodies specific for 5 viruses that are relevant for Projects 1 and 2. We will also assess quality of antibody responses through ELISAs that measure relative affinities and isotype diversity. It is possible that PD-1 blockade affects antiviral antibody responses in more subtle or global ways. To address this, we will complete serological screens to examine how PD-1 blockade affects antibody responses to a large fraction of known human viruses. For this, we will use a new array-based assay, termed ‘Viroscan’, that measures antibody binding to >400,000 non-redundant viral peptides. We will also offer services that intensively analyze antibody responses against a single virus (influenza). All humans are exposed to influenza virus in childhood and all adult encounters with influenza involve both the recall of memory B cells and the stimulation of de novo B cell responses. Our influenza virus antigenic analyses will allow us to determine if PD-1 blockade differentially regulates de novo versus memory antibody responses. Together, these standardized services will allow for the complete antigenic characterization of serum samples for Projects 1 and 2.

Catherine Wu, Core Co-Lead

The overall objective of the U19 is to discover the role of the PD-1 pathway in human immunity by studying the effects of therapeutic PD-1 blockade on immune responses to chronic viruses (Project 1) and preventive vaccines (Project 2). To monitor adaptive immune responses in patients, we need to track the complex repertoire of T and B cells since clonotypes may behave differently over time and in response to infection and therapy. While there are several methods for tracking antigen-specific lymphocytes, large volumes of blood are needed for monitoring multiple antigens, biopsies have limited material for standard pipelines, and clonotypes with distinct antigen receptors are not distinguished. Standard bulk sequencing of TCRs and BCRs can provide quantitative clonotype frequencies; however, its utility is limited because the target antigens are not known for each TCR/BCR and the activation states of each clonotype cannot be determined. Core D provides an innovative service for tracking antigen-specific T and B cells in patient blood and tissues, allowing Projects 1 and 2 to quantify the frequency, antigen specificity and activation states of lymphocytes. Aim 1 provides an approach to match TCRs and BCRs to antigens by sequencing antigen receptors in patient T and B cells stimulated or tagged with viral or vaccine antigens. Aim 2 uses the more cost-effective bulk TCR- and BCR-seq to quantify the frequencies of clonotypes in serial samples of patients treated with anti-PD-1 therapy, but takes advantage of the results in Aim 1 to link antigen receptors from the repertoire with specific viral or vaccine antigens. Aim 3 uses leading-edge droplet-based RNA-seq to sequence paired antigen receptor chains (TCRα & β; IgH & IgK/L) along with thousands of mRNAs in single lymphocytes, linking functional pathways with antigen receptors. The resulting dataset from the three Aims will be integrated to provide the frequencies, antigen specificities and activation states of T and B cells, allowing us to test the hypothesis that anti-PD-1 therapy differentially impacts the proliferation and activation of each lymphocyte subset depending on its antigen specificity and pre-therapy differentiation state. In short, Core D enables the monitoring of antigen- specific lymphocytes at unprecedented resolution, and will help dissect changes in immune protection against chronic infections (including HBV, HCV, CMV, and EBV), and influenza vaccination as result of anti-PD-1 therapy. The establishment and refinement of these approaches and creation of a Core D infrastructure will enable interrogation of immune mechanisms and provide insights into development and durability of immunological memory in the context of checkpoint blockade or any other immunotherapies.

Raymond T Chung and Paul Klenerman, Project Co-Leads

Chronic viral infections remain major threats to global health, with pathogens such as hepatitis B virus (HBV) and hepatitis C virus (HCV), responsible for millions of deaths annually. The recent development of curative antiviral therapy for HCV has been a major breakthrough. However, therapies capable of producing at least functional cure for chronic HBV are urgently needed. A major impediment to cure of HBV is a functionally impaired immune response characterized by markers of exhaustion. The PD-1:PD-L1/2 inhibitory receptor pathway regulates many key aspects of cellular immunity, including T cell exhaustion in chronic viral infection and cancer. Blockade of this pathway has produced dramatic effects in the treatment of advanced cancer and unleashed an immunotherapeutic revolution. However, little is known about the human in vivo effect of PD-1 blockade on the response to chronic infections marked by exhaustion and the mechanisms by which these responses are invigorated. Building on our U19 CCHI Consortium’s important work defining the mechanisms of immune exhaustion in chronic HCV and assessing its reversal upon termination of chronic antigen stimulation, we will now comprehensively investigate how direct and specific blockade of PD-1, as a key mediator of antigen- mediated immune exhaustion, affects the layers of molecular regulation of the antiviral immune response. The overall hypothesis of this project is that blocking PD-1 in humans will alter the magnitude, quality, regulation and composition of pre-existing antiviral CD8 T cell responses, leading to more effective viral control, and will modulate other aspects of cellular immunity, including atypical T cell and macrophage responses. We will test this hypothesis through the following aims. Specifically, in Aim 1 we will test how PD-1 therapy alters pre- existing virus-specific CD8 T cell responses targeting persisting viruses in the blood. We will utilize large PBMC donations from leukapheresis for a comprehensive and in-depth analysis of changes in the phenotype, function, clonal composition, transcriptional state, and epigenetic regulation of HBV-, but also CMV-, EBV-, HBV- and influenza-specific CD8 T cells, as well as of unconventional MAIT T cells. In Aim 2 we will test how PD-1 therapy alters HBV-specific CD8 T cell responses in the liver of patients with chronic hepatitis B. We will complement our analyses from aim 1 with parallel data directly from the site of infection through liver fine needle aspirates. Finally, in Aim 3 we will define the recovery of macrophages through PD-1 blockade in persons with chronic HBV infection. These data will extend our analysis on the effects of PD-1 blockade to other components of the antiviral immune response, by studying the response of macrophages as critical modulators of intrahepatic innate immunity. Collectively, we expect these data to dramatically enhance our understanding of the mechanism of αPD-1 mediated immune recovery that can be utilized not only for the design of pathogen- specific immunotherapy, but also to enable further improvements in the efficacy of immunotherapy in general.

WHERRY, E. JOHN, Project Lead

Infectious diseases contribute three of the top ten causes of death worldwide. Moreover, billions of people remain infected with persisting pathogens such as hepatitis B virus, malaria and HIV, and ~half a million people die annually from influenza (Flu) and other respiratory infections. Much of our knowledge of how the human immune system responds to pathogens or vaccination remains observational rather than direct perturbation of specific molecules. Here, we will exploit the revolution in immunotherapy of cancer to directly interrogate how key immune regulatory pathways impact T cell, B cell and antibody (Ab) responses to human vaccination toward developing a foundation of empirical evidence that informs how to use checkpoint blockade and targeting of other specific immune pathways to treat or prevent infectious disease. T cell and B cell activation and differentiation are regulated in part by the PD-1:PD-L1/2 inhibitory receptor pathway, which also influences T cell exhaustion. These functions are targeted by a new class of biologic drugs that block PD-1 signals to treat cancer. Yet, the role of the PD-1 pathway in human immune responses to vaccination remains unclear. In particular, cells in germinal centers (GC), including follicular helper T cells (TFH), follicular regulatory T cells (TFR), and GC and memory B cells (mBc), have high expression of PD-1 and/or its ligands, but the contribution of the PD-1 pathway to GC responses and humoral, TFH and B cell memory is poorly understood. Further, despite widespread use of anti-PD-1 (αPD-1) therapy in recent years, there is essentially no information on how this treatment impacts TFH, TFR, B cell or Ab responses or whether patients on checkpoint blockade should or should not be vaccinated. Thus, the overarching hypothesis of this Project is that blocking PD-1 in humans will impact the magnitude and/or quality of the TFH, TFR, B cell and humoral response to Flu vaccination through an underlying effect on GC biology. We will: AIM 1: DETERMINE HOW αPD-1 IMPACTS THE FUNCTIONAL DIVERSITY, MAGNITUDE, TCR REPERTOIRE, DURABILITY AND TRANSCRIPTIONAL PROGRAM OF VACCINE-INDUCED CD4 T CELL RESPONSES. AIM 2: DETERMINE HOW αPD-1 IMPACTS THE MAGNITUDE, BCR REPERTOIRE, DURABILITY AND TRANSCRIPTIONAL PROGRAM OF VACCINE-INDUCED B CELL RESPONSES AND QUANTITY/QUALITY OF VACCINE-INDUCED AND OTHER ANTIVIRAL ANTIBODY RESPONSES. This work will inform how to better vaccinate vulnerable populations (e.g. cancer patients; the elderly), and will identify mechanisms by which the PD-1 pathway controls human immunity, providing a platform for future studies of immune perturbation in humans. By its nature Project 2 is highly interactive with Project 1 and all Cores. Core B will generate samples and this Project will use Core C and D services. Projects 1 and 2 will synergize and interact extensively to understand PD-1-based immune perturbation in humans.