Awarded Pilot Abstracts

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: "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, su