Awarded Pilot Abstracts

Title: Analysis of IgG-FcγR interactions in protection against tuberculosis
Pilot PI: Jeremy Rock, PhD
Abstract: The existence of natural immunity to tuberculosis (TB) is supported by the fact that nine out of 10 individuals infected with Mycobacterium tuberculosis (Mtb) control the disease in a state of clinical latency. While cell mediated immunity is essential for this control, the humoral response has long been assumed to be irrelevant for defense against intracellular pathogens such as Mtb1. However, growing evidence demands a reappraisal of this view. A protective role for B cells and IgG antibodies has been demonstrated for numerous intracellular pathogens such as Salmonella spp2 and Cryptococcus neoformans3. Moreover, recent evidence suggests that sera from vaccinated mice is sufficient to contribute to Mtb control in mouse models4 and that the human humoral response may be distinct in patients with active vs latent TB5. Protection against pathogens is conferred through two functional domains of IgG antibodies: the Fab domain confers antigen specificity, and the Fc domain mediates effector functions through specific interactions with diverse Fcγ receptors (FcγR) expressed on the surface of effector leukocytes. Depending on the FcγR and cell type engaged, IgG-FcγR interactions can be pro-inflammatory, anti-inflammatory, or immunomodulatory. The ability of an IgG molecule to engage the various FcγRs is a dynamic and tightly regulated process that is controlled predominantly by the intrinsic structure and heterogeneity of the Fc domain (IgG subclass and associated glycan). Importantly, mouse model results support a role for FcγRs in promoting TB control6. However, while it is known that humans mount a dynamic and heterogeneous humoral response to Mtb infection7, the existence and characteristics of functionally protective antibodies is poorly understood. There is an urgent need to fill this gap in knowledge because, until we have it, the rational design of an effective B-cell directed TB vaccine will remain beyond reach. Our long-term goal is to define, and ultimately to harness, the mechanisms by which the human humoral response restricts TB disease. Our central hypothesis is that Fc-FcγR interactions represent a critical mechanism for determining outcome of TB exposure by modulating the interplay between the innate and adaptive immune responses against Mtb. This hypothesis is supported by published studies demonstrating that anti-Mtb antibodies modulate the macrophage-Mtb interaction by enhancing immunity via FcγR-mediated phagocytosis8, and that Fc-FcγR interactions contribute to TB control in the mouse6. Our overall objective here is to: 1) establish the humanized FcγR mouse as a novel small animal TB model; and 2) to use this model to define the efficacy of human monoclonal antibodies (mAbs) to potentially protective Mtb antigens.


Title: A genetic dissection of severe influenza and other viral infections in humans
Pilot PI: Qian Zhang
Abstract: We recently identified inborn errors in the type I interferon (IFN) pathway that lead to severe influenza infections in otherwise healthy individuals, including TLR3, IRF7, and IRF9 (unpublished data and [1]). These discoveries highlighted the importance of three steps of the type I IFN responses: the local respiratory epithelial cells respond to intracellular infection by activating TLR3 pathway and producing type I IFN; the plasmacytoid dendritic cells (pDC) capture the viral products from infected cells and produce large amount of IFN by activating IRF7; and the amplification of IFN signaling through the activation of ISGF3 complex (STAT1, STAT2, and IRF9) [2]. Yet, these discoveries poses new questions worth further investigation. Defects in the TLR3 pathway have also been shown to cause herpes simplex encephalitis (HSE), a rare but fatal infection in children [3]. We hypothesize that defects in the TLR3 pathway lead to increased susceptibility to influenza with incomplete penetrance. To test this hypothesis, we propose to test the type I IFN response to influenza A virus (IAV) in cells from patients with TLR3, UNC93B1, TRAF3, TRIF, TBK1, or IRF3 deficiency, as well as search for defects of these genes in patients with severe influenza. Moreover, both of the IRF7 and IRF9 patients experienced MMR infections after vaccination, which resembles the severe vaccine strain measles infections in patients with IFNAR2 or STAT2 deficiencies [1, 4, 5]. Therefore, we hypothesize that type I IFN response is essential to control both influenza and vaccine strain measles infections. To test this hypothesis, we propose to test type I IFN responses to both IAV and measles virus (MeV) vaccine strain in cells from patients with IRF7 or IRF9 deficiency. Taken together, we hypothesize that inborn errors in the type I IFN pathway lead to monogenic, non-Mendelian diseases with susceptibility to a narrow spectrum of viral infections including influenza. Therefore, we propose to extend our search for new genetic defects in this pathway in patients with severe influenza infections.


Title: A genetic dissection of severe influenza in humans
Pilot PI: Michael Ciancanelli, PhD
Abstract: Influenza usually causes relatively mild upper respiratory disease that is cleared with little need for medical intervention1, but infection with either seasonal or pandemic strains can be fatal due to acute respiratory distress syndrome (ARDS). Intriguingly, the >300 known primary immunodeficiencies, including the congenital lack of T cells, B cells, and NK cells, do not underlie severe influenza2. Most cases of severe influenza in otherwise healthy individuals thus remain unexplained. Our lab has shown that single-gene inborn errors of immunity can selectively predispose otherwise healthy children to severe bacterial, fungal and viral diseases3-9. Using whole exome sequencing (WES) methods pioneered in our lab for the investigation of infectious diseases, I identified the first genetic deficiency predisposing humans to severe influenza, autosomal recessive (AR) IRF7 deficiency, in a child with severe pneumonitis due to influenza A virus (IAV)9,10. This study demonstrated the requirement for the production and IRF7-dependent amplification of IFN to limit IAV pathology. In this application, I will continue to test the hypothesis that severe influenza is a heterogeneous collection of rare monogenic defects affecting the immune response of children without known risk factors. WES analysis on a cohort of 80 patients with sporadic severe influenza identified rare heterozygous mutations in TLR3 in two patients and a private mutation in IRF9 in one patient. TLR3 is an endosomal sensor of viral RNA that leads to the production of antiviral cytokines including interferon (IFN). IFN triggers IRF9 to direct the transcription of >400 IFN-stimulated genes (ISGs) limiting virus replication and spread. I propose a biochemical dissection of the IFN pathways, cytokine production and signaling downstream of receptor engagement, in hematopoietic and non-hematopoietic tissues from the three patients. We will assess the effect of the mutations on gene expression; on protein function at the molecular level; and the integrity of the IFN pathway in the context of IAV infection of cells isolated from the patients. Additionally, I will mine the WES data from 80 other patients to find novel mutations to add to this study. These findings would reinforce the centrality of the IFN pathway in anti-IAV immunity and validate in vitro and in vivo work using a human model.


Title: Continuation: Isolation of Zika Virus Specific B Cells for Human Antibody Cloning
Pilot PI: Davide Robbiani, MD, PhD
Abstract: Zika virus (ZIKV) infection typically produces mild symptoms consisting of fever, rash and arthralgia that resolve rapidly, and like many other viral illnesses it is occasionally associated with Guillain-Barré Syndrome. However, when infection occurs during pregnancy, vertical transmission can lead to a broad spectrum of devastating neurodevelopmental and other abnormalities collectively referred to as Congenital Zika Syndrome. Although the data are still incomplete, infants born from mothers infected with ZIKV during pregnancy can suffer an up to 42% risk of clinical abnormal or brain imaging findings. ZIKV belongs to the Flavivirus genus, which includes Yellow Fever (YFV), West Nile (WNV), and the 4 serotypes of Dengue virus (DENV1-4). These positive-stranded RNA viruses are responsible for a considerable burden of morbidity and mortality in the equatorial and subequatorial regions populated by their mosquito vectors. Unlike other flaviviruses, ZIKV can also be transmitted sexually, and on occasion persists for months. Currently there is no prophylaxis or treatment against ZIKV. Although vaccines are under development, there is no guarantee that they will be either safe or effective. Human anti-ZIKV antibodies represent an alternative that will be almost certainly safe and more rapid to develop. The ultimate goal of this proposal is to discover potent ZIKV-specific neutralizing antibodies to be used for passive protection and to identify targets for vaccine design. The objective of the proposed pilot project is to develop tools to enable the isolation of B lymphocytes bearing antibodies that are specific to ZIKV from the peripheral blood of patients recovering from infection. Antibodies will then be cloned from single B cells, produced in vitro, and tested for binding and neutralization against ZIKV. The proposed strategy is similar to the one, which was previously used in the Nussenzweig laboratory to discover potent neutralizing antibodies against HIV, which are now being evaluated in clinical trials. In Year 1 of funding we have developed a method to isolate antibodies binding to the E protein Domain III of ZIKV from memory B cells of individuals from Zika outbreak areas in Brazil. Importantly, most of the antibodies that we discovered with this approach cross-reacted with at least one other flavivirus. Cross-reactive antibodies are not desirable for therapeutic purposes, since they have the potential of causing adverse effects through Antibody-Dependent Enhancement. Therefore, in Year 2 we propose to improve and advance the methodology to help distinguish memory B cells with antibodies that react uniquely to ZIKV from those that also bind to other flaviviruses. In addition to diagnostic applications, this method could accelerate the identification of ZIKV-specific antibodies to be used for passive immunotherapy in populations at risk (e.g. during pregnancy).


Title: "The role of IgG3 in immunity”
Pilot PI: Stylianos Bournazos, PhD
Abstract: IgG3 exhibits a number of structural and functional characteristics unique among the human IgG subclasses, suggesting thereby a key role for this subclass in immunity. Indeed, IgG3 presents the highest affinity for all the classes of human FcγRs, mediating potent Fc effector activity, and is characterized by an exceptionally long hinge domain that comprises several glycan structures. Additionally, a number of genetic variants (Gm allotypes) have been described for IgG3, representing the most genetically diverse IgG subclass. Based on these unique characteristics of IgG3, we aim to evaluate the role of this IgG subclass in immunity and determine the impact of structural and genetic determinants on the functional activity of IgG3. Using a combination of biochemical, genetic, and immunological approaches, the role of the IgG hinge structure as well as the impact of IgG3 Gm allotypes on the antibody function will be evaluated. Furthermore, we will analyze the activity of IgG3 antibody responses elicited upon influenza vaccination to investigate any association between IgG3 and antiviral antibody activity. These studies will provide novel mechanistic insights into the role and activity of IgG3 in immunity, elucidating several aspects related to the function of this IgG subclass.


Title: "Isolation of Zika Virus Specific B Cells for Human Antibody Cloning"
Pilot PI: Davide Robbiani
Abstract: The rapid spread of Zika virus (ZIKV) across the Americas calls for prompt interventions to limit the harm ZIKV is causing to human health (1). The discovery of human monoclonal antibodies capable of neutralizing ZIKV would provide a means to rapidly and effectively attack the virus in settings where the infection causes serious damage. Many uncertainties still surround the biology of ZIKV, including its target tissues, persistence in vivo, and means of transmission (2-5). Nevertheless, a growing body of evidence links ZIKV to severe health conditions. For example, an increased incidence of miscarriages, microcephaly and other developmental defects is observed in fetuses of mothers infected by ZIKV early during pregnancy (6). Moreover, a fraction of ZIKV-infected individuals develop Guillain-Barré syndrome, a life-threatening autoimmune disorder characterized by muscle weakness and paralysis (7). The ultimate goal of this proposal is to discover potent ZIKV-neutralizing antibodies, to be used for passive protection and to identify targets for vaccine design. The objective of the proposed pilot project is to develop tools to enable the isolation of B lymphocytes bearing antibodies specific to ZIKV from the peripheral blood of patients recovering from infection. Antibodies will then be cloned from single memory B cells, produced in vitro, and tested for binding and neutralization against ZIKV. The proposed strategy is similar to the one, which was previously used in the lab to discover potent neutralizing antibodies against HIV (8), which are now being evaluated in clinical trials (9). To accomplish this goal we will develop a flow cytometer-based binding assay for the Envelope (E) protein, ZIKV’s major surface antigen to B lymphocytes from the blood of convalescent individuals. The coding region for the extracellular domain of the E protein will be cloned into an expression plasmid. The E proteins of related flaviviruses (Dengues 1-4 and Yellow Fever) will also be produced and used as control for cross-reactivity. E proteins will be produced in mammalian cells and isolated by column purification, prior to conjugation to distinct fluorophores. Fluorophore-labeled E proteins will then be used to stain peripheral blood mononuclear cells from individuals recovering from ZIKV. Non-exposed individuals will serve as control for staining specificity. Donors are individuals from Northeastern Brazil, a region with high prevalence of Zika disease. Dengue is also highly prevalent in the same region (all four serotypes). Moreover, vaccination with the live attenuated Yellow Fever 17D strain is common in Brazil. Cells will be acquired through an ongoing collaboration with Dr. Albert Ko (Yale University), who is conducting studies in this area. The generation of these tools and optimization of the staining protocols are essential to enable the discovery of neutralizing human monoclonal antibodies against ZIKV to use for passive immunotherapy.

Title: "Effects of chronic viral infection on immune response to Zoster vaccination"
Pilot PI: Oyebisi Jegede, MBBS, PhD
Abstract: Zoster vaccine (Zostavax, Merck) is recommended for the prevention of herpes zoster (HZ) reactivation (shingles; painful blistering rash). It is recommended for individuals > 50 years without an underlying immune deficiency (HIV, malignancies, immunosuppression and transplantation). In non-immunocompromised individuals, zoster vaccine decreases shingles by 51-61%. In the U.S., 99.5% of adults >40 years have been infected with Varicella zoster virus (VZV) and are at risk of herpes zoster virus reactivation and its complications (acute or chronic PHN, osteonecrosis, HZ ophthalmicus with visual impairment, increased risk of blindness and a 4-fold risk of cerebral vasculitis-associated stroke)1,2. Chronic infections with mycobacteria, helminthes and viruses are associated with increased susceptibility to other pathogens and decreased vaccination efficacy3-6. Although chronic Hepatitis C virus (HCV) infection is not considered a clinically immunosuppressed state, it is associated with persistent immune activation and decreased vaccination response7. Zostavax is routinely administered to chronic HCV patients. However, at present, no other study has documented the immune responses elicited by Zoster vaccination in this population. This study aims to identify the innate and adaptive immune signatures elicited by zoster vaccination in chronic HCV as compared to healthy volunteers. Results from this study could identify targets for vaccine optimization for this population and other chronic dysregulated states (HIV, diabetes, older age, cancers and transplantation).

Title: Mechanisms of human Th1- and Th17-dependent immunity to mycobacteria: Insights from patients with novel inborn errors of IL-12Rβ2, IL-23R, T-bet, and RORγT
Pilot PI: Janet Markle, Ph.D., M.P.H.
Abstract: Only a small fraction of children infected by a given microbe will go on to develop clinical disease. The huge inter-individual variability in response to primary infection in childhood – the major enigma in infectious disease research – results at least in part from variability in host genetic determinants of immunity. For two decades, our research group has unraveled the cellular and molecular mechanisms of human antimycobacterial immunity by studying patients with genetic mutations causing susceptibility to mycobacteria but not to other microbes (1). Mendelian susceptibility to mycobacterial disease (MSMD) is a rare disorder predisposing individuals to severe clinical disease upon infection with weakly virulent mycobacteria, including Mycobacterium bovis Bacille Calmette-Guérin (BCG) vaccines (2-4). Eighteen genetic etiologies of MSMD have been reported, involving mutations in nine genes (1, 5), all of which are involved in the production of, or response to, IFN-γ. IL-12Rβ1 deficiency is the most common genetic etiology of MSMD (6, 7), however it is unclear whether disease in these patients results from disrupted signalling of the IL-12Rβ1/IL-12Rβ2 heterodimer, which responds to IL-12, or the IL-12Rβ1/IL-23R heterodimer, which responds to IL-23. IL-12 is a key cytokine for Th1 responses, whereas IL-23 is a key cytokine for Th17 responses, and both cytokines are induced by mycobacterial infection (8). Yet the mechanistic contributions of Th1 vs. Th17 cells to mycobacterial clearance are not known. This project aims to clarify the roles of Th1 and Th17 cells in mycobacterial immunity by studying, in parallel, the hallmark cytokines and transcription factors required for the development of Th1 and Th17 responses. We have identified rare, damaging mutations in T-bet and IL-12Rβ2 (required for Th1) and RORγT and IL-23R (required for Th17) in patients with severe mycobacterial infections. Molecular and cellular investigation of these mutations will clarify the immunological basis of mycobacterial immunity.

Title: The role of human NK cells in HBV infected mice
Pilot PI: Ype de Jong, MD, PhD; Eva Billerbeck, PhD
Abstract: The majority of individuals exposed to hepatitis B virus (HBV) mounts an effective immune response that controls viral replication but ~400 million people worldwide remain chronically viremic, putting them at risk for hepatocellular carcinoma (HCC) and cirrhosis. Current treatments can efficiently suppress HBV viremia but, even after years of therapy, do not result in durable immune control. New strategies need to be developed to enhance immune activation in chronically viremic individuals, with the goal of achieving immune control of viral replication. Classical depletion studies in chimpanzees have shown that both CD4+ and CD8+ T cells are essential for viral control, but the role of natural killer (NK) cells as first responders to acute HBV infection remains poorly defined. With the dwindling support for chimpanzee research and because mice are not susceptible to HBV infection we have recently developed a humanized mouse model that can be doubly engrafted with human liver cells and syngeneic human immune cells (Fig 1). Using a NK cell depletion approach we propose to test if and how NK cells contribute to T cell activation in early HBV infection. We anticipate that a better understanding of how NK cells function in this model will lead to the identification of pathways that may become therapeutic targets to activate the immune system in HBV infected patients.

Fig 1: Creation of doubly engrafted mice. Fresh hepatoblasts obtained from human fetal livers are transplanted in immunodeficient fah-/- mice. After surgery mice are cycled off the protective drug NTBC and will receive human oncostatin-M (hOSM) for one month. Once the hepatoblasts have expanded mice are irradiated and transplanted with syngeneic hematopoietic stem cells (HSCs) from the same donor. Mice will continue to be cycled off NTBC whilst the HSCs develop into human immune cells.

Title: Sialylated IgG Fc glyans modulate affinity maturation through CD23
Pilot PI: Taia Wang, MD, PhD
Abstract: Following exposure to antigen, the antibody response undergoes a maturation process, resulting in high affinity, class switched antibodies capable of efficiently neutralizing pathogenic challenges. This process is driven by antigen in the form of immune complexes (ICs), retained on FDCs in the germinal center and directing affinity maturation and B cell selection. Activation thresholds for B cell selection result, in part, from the ability of the ICs to interact not only with antigen receptors on B cells and APCs, but also with Fc receptors expressed on those cells. Fc receptors are members of a diverse family of cell surface molecules and can transduce either activating, inhibitory or immunomodulatory signals. The specific pathways triggered by ICs are determined by the specific structure the Fc adopts which, in turn, results from both the IgG subclass and glycan composition. The Fc glycan is an N-linked, complex, biantennary glycan, attached at Asn-297 of each IgG heavy chain. Composition of the core Fc glycan can be modified by addition of specific saccharide units (fucose, N-acetylglucosamine, galactose and sialic acid); these modifications are dynamic and act to regulate the biological activity of IgG molecules by modulating Fc structure and, as a consequence, FcR interactions. For example, terminal sialic acid modification of Fc glycans (sialylated Fc) converts IgGs to anti-inflammatory molecules in a variable region-independent manner by switching specificity of the Fc from type 1 (Ig superfamily members) to the type 2 (C-type lectins) Fc receptor DC-SIGN [Pincetic 2014]. We have recently found a second type 2 Fc receptor pathway for sialylted Fc signaling that modulates the selection of B cells during an antibody response. Sialylated Fcs within ICs were found to trigger upregulation of the inhibitory FcgRIIB on B cells via CD23 binding. In turn, this elevates the threshold for the affinity of B cell receptor that is required for cell survival. Thus, sialylated Fcs, previously known to trigger anti-inflammatory signaling through SIGN molecules, have been found to participate in a separate, CD23-dependent pathway that contributes to selection of B cells during antibody responses. We have used vaccination in healthy subjects as a model system for the study of Fc glycan modulations during antibody responses; we now wish to expand our studies to analyze Fc glycan modulations and maturation of antibody responses in patients with rheumatoid arthritis who are known to have dysregulated Fc glycan composition.

Title: Analysis of IgG-FcγR interactions in protection against tuberculosisPilot PI: Jeremy Rock, PhDAbstract: The existence of natural immunity to tuberculosis (TB) is supported by the fact tha