Significant progress in basic immunology research over the last three decades has resulted in numerous medical advances and dissected the general mechanisms by which the human immune system responds to foreign antigens. However, a much more substantial understanding of the coordinated molecular mechanisms involved in eliciting immunity will be required, as each viral pathogen poses unique challenges to the immune system and the elicited immune responses are characterized by substantial heterogeneity that impacts disease susceptibility and pathogenesis. Indeed, it is expected that B-cell responses against diverse viral pathogens are uniquely evolved during infection to shape the functional activity of IgG antibodies. Studies from viral infectious diseases have shown that antiviral IgG antibodies have the capacity to mediate a wide spectrum of opposing functions: (i) protective functions, including neutralization, viral opsonization, and clearance of infected cells and (ii) pathogenic activities, which enhance viral infectivity, disease susceptibility and severity; a phenomenon termed as antibody-mediated enhancement (ADE) of disease. ADE mechanisms have been previously suggested to account for susceptibility to dengue disease, as epidemiological data support that prior flavivirus infection is the major risk factor for dengue disease, implicating the presence of cross-reactive, non-neutralizing IgG antibodies to this process. Understanding the heterogeneity of IgG responses elicited upon infection or vaccination with diverse viral antigens is therefore critical for characterizing the immunological mechanisms that drive human immunity and determine the protective vs. pathogenic activity of IgG antibodies. Our Center will feature three Projects directed by Drs. Ravetch (Project 1: Fc domain effector activity in dengue disease), Nussenzweig and Rice (Project 2: Understanding B cell memory in response to diverse virus infections), and Wang (Project 3: Immunity to dengue viruses), supported by a scientific core (Core A: Transgenic mouse core) and the administrative core (Core B). Through a series of collaborative studies between the three Projects, our Center aims to study human antiviral immune responses during infection and vaccination and characterize the immune mechanisms that regulate the function of IgG antibodies in humans. More specifically, we aim to characterize the heterogeneity of IgG responses elicited upon vaccination or infection with diverse viral pathogens, including HBV and flaviviruses, like Zika and dengue. Additionally, we will dissect the ADE mechanisms by which IgG antibodies mediate disease-enhancing activities and contribute to dengue disease susceptibility and pathogenesis. These studies will provide novel insights into the mechanisms that drive protective immunity and modulate antibody function, having a broader impact on the development of vaccination strategies against infectious pathogens.
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Our U19 Center consists of two Cores and three Projects:
ADMINISTRATIVE CORE
RAVETCH, JEFFREY VICTOR, Core Lead
The Administrative Core will provide overall management of the administrative functions of the CCHI. This includes proving clear and regular communication channels between the CCHI participants, the NIH, and the External Scientific Advisory Group, reporting research findings annually and through publications, allocating research resources between the projects and the core, and ensuring compliance on animal and human subjects usage. The goals of the Administrative Core are 1) to provide scientific leadership and management for the Center; 2) maintain regulatory and fiscal compliance and administrative oversight; and 3) coordinate communications and research dissemination. The result of the execution of these goals will be a Center with synergized research projects, streamlined and accurate reporting, and vast public dissemination. Deliverables from the Administrative Core will include quarterly seminars and annual Distinguished Investigator lectures, a CCHI-specific website with both public and private access, annual progress reports to the NIAID following NIH policy and guidelines, multiple publications resulting from CCHI research, and data input to the ImmPort system.
TRANSGENIC MOUSE CORE
BOURNAZOS, STYLIANOS, Core Lead
The ability of an IgG antibody to mediate biological effects in vivo, such as protection from viral infection, results from its bispecific nature. The Fab region of an antibody recognizes epitopes on the viral spike and may interfere with virus binding to target cells, while the Fc domain mediates diverse immunomodulatory activities through specific interactions with Fcγ receptors (FcγRs) expressed by effector leukocytes. Studies on several infectious pathogens have shown that Fc-FcγR interactions have the capacity to mediate a wide spectrum of opposing functions – from viral opsonization and clearance of infected cells (protective) to enhanced viral infection and disease severity (pathogenic). Understanding the precise mechanisms by which IgG antibodies mediate protective or pathogenic functions necessitates the use of well-defined in vivo experimental systems that reflect the unique complexity of effector leukocytes that participate in an immune response. Indeed, although in vitro assays using cell lines or primary effector cells represent well-established approaches for characterizing the function of IgG antibodies, such assays provide limited information on the precise molecular mechanisms that drive human immunity during infection or upon vaccination. We will provide a Core that will maintain, characterize, and distribute a series of genetically-engineered humanized mouse strains to the Center investigators in support of the proposed studies on the characterization of the pathogenic activity of anti-flavivirus antibodies that confer susceptibility to dengue disease, as well as on the evaluation of the protective antiviral function of neutralizing antibodies against hepatitis B viruses. Through specific deletions of key immune signaling pathways or reconstitution with human primary cells that represent the natural viral host cell type, these engineered mouse strains support the infection and replication of flaviviruses and hepatitis viruses in vivo, thereby facilitating the study of viral disease pathogenesis, as well as the characterization of the molecular mechanisms that contribute to the IgG antibody activity during viral infection.
FC DOMAIN EFFECTOR ACTIVITY IN DENGUE DISEASE
RAVETCH, JEFFREY VICTOR, Project Lead
Flaviviruses, such as dengue, Zika, and West Nile have a significant impact on public health with tremendous socioeconomic consequences for a large fraction of the world’s population. A feature common to all flaviviruses is the clear distinction between infection and disease. For example, only a small fraction of dengue-infected individuals develops dengue disease, which is characterized by a diverse spectrum of clinical symptoms of variable severity. A large body of epidemiological data suggests that prior flavivirus infection represents the major risk factor for dengue disease susceptibility. Indeed, susceptibility to severe dengue disease is associated with the titers of cross-reactive, non-neutralizing IgG antibodies that are elicited during primary infection with other flaviviruses. The established mechanistic model by which IgG antibodies contribute to disease susceptibility is based upon the in vitro observation that these antibodies mediate infection of leukocytes through increased uptake of virus-IgG complexes via specific interactions of their Fc domains with Fcγ receptors (FcγRs); a phenomenon termed antibody-dependent enhancement (ADE) of infection. Although this model can sufficiently explain susceptibility to dengue disease, it is likely that complex host susceptibility factors exist that contribute to disease pathogenesis and determine severity among symptomatic dengue patients. Consistent with this hypothesis, our recent analysis of the Fc domain structure of IgG antibodies derived from dengue patients with variable disease severity revealed that specific Fc domain characteristics that confer increased affinity for pro- inflammatory, activating FcγRs, are enriched in patients with severe disease and evidence for specific clinical manifestations, including thrombocytopenia and vascular leakage. These antibodies exacerbate disease severity by inducing platelet depletion via FcγR-mediated mechanisms, suggesting that previously-uncharacterized ADE mechanisms contribute to disease pathology. Understanding the mechanisms that mediate dengue ADE is essential for predicting the susceptibility to severe dengue disease in high-risk patient groups and developing approaches to prevent or reduce disease-associated clinical manifestations. In the proposed studies, we will analyze the IgG responses from cohorts of dengue-infected patients with variable disease severity to identify the specific IgG features that are associated with dengue disease severity and clinical manifestations. Follow-up mechanistic studies in mouse models of dengue disease using strains fully humanized for all classes of FcγRs will be performed to determine the role of specific human FcγRs in dengue disease and characterize the precise FcγR pathways that contribute to disease pathogenesis. Lastly, we will characterize IgG responses elicited upon influenza vaccination of individuals with differential susceptibility to severe flavivirus infection to determine whether changes in the Fc domain structure represent immune determinants for predicting disease susceptibility. Our studies will provide novel insights into the mechanisms by which pathogenic IgG antibodies mediate dengue disease and have a broader impact on our understanding of the pathogenesis of other flaviviruses, like Zika.
UNDERSTANDING B CELL MEMORY IN RESPONSE TO DIVERSE VIRUS INFECTIONS
NUSSENZWEIG, MICHEL C, Project Lead
Memory B cells are mediators of immunological memory and vaccine responses. Memory B cells are a heterogeneous group of cells, that can be distinguished based on phenotypic markers, function, location, the antibody isotype expressed, and the degree of antibody gene somatic mutation. Our understanding of memory B cell biology is based primarily on sophisticated experiments using genetically modified mice and model antigens. In contrast, we know much less about the human memory B cells, in particular about those that develop in response to a specific pathogen and during natural infection. To date most studies have focused on sequencing the antibodies from the memory B cells, while many basic aspects of their biology could not be studied in part due to the difficulty at identifying pathogen-specific cells and their relative paucity. Advances in single-cell technologies are starting to make these investigations possible. In Project 2, the Rice and Nussenzweig laboratories propose to work together to investigate and compare the B cell memory that develops in response to important human pathogens: the dengue and Zika flaviviruses (DENV and ZIKV) and the hepatitis B virus (HBV). These viruses are responsible for considerable morbidity and mortality. More than 40% of the world population lives in areas at risk for infection by DENV and ZIKV flaviviruses and about 250 million people are living with HBV infection worldwide, despite the existence of an efficacious HBV vaccine. The immune memory to these pathogens is interesting because it can be either protective (e.g. HBV vaccination) or potentially harmful (e.g. antibody dependent enhancement of infection with DENV). We hypothesize that the memory B cells elicited by DENV, ZIKV and HBV in selected individuals are distinct and characterized by the expression of genes that are either general or pathogen-specific and that are linked to features of the antibodies that they express. To test this hypothesis, we will combine antigen-specific B cell purification by cell sorting with single cell transcriptomics analysis. Our approach will take advantage of large cohorts of human samples obtained through ongoing collaborations with investigators in DENV and ZIKV endemic areas of Brazil and Mexico, as well as of HBV samples from naturally infected or vaccinated individuals obtained at the Rockefeller University Hospital. Samples from individuals with high DENV and ZIKV neutralizing activity from Brazil and Mexico will be identified and single memory B cells specific for DENV will be subjected to single cell RNA-seq to characterize their transcriptome and to obtain paired antibody heavy and light chain sequences (Aim 1). Similarly, we will analyze single memory B cells elicited by HBV infection or vaccination (Aim 2), and the memory transcriptomes of DENV, ZIKV and HBV will be compared to each other and to those of naïve B cells. In Aims 3 and 4 we will clone and characterize the antibodies from the same cells, and link this information to the transcriptome and to the antibodies’ ability to either protect (HBV) or enhance infection (DENV).
IMMUNITY TO DENGUE VIRUSES
WANG, TAIA, Project Lead
Dengue viruses are mosquito-borne flaviviruses of immense public health impact that cause a spectrum of disease in humans ranging from mild to fatal. Progression to severe dengue disease is promoted by the presence of non-neutralizing anti-dengue IgGs that modulate virus and cytokine production in Fc receptor- bearing cells. We have shown that progression to severe dengue disease is promoted by the presence of anti- dengue antibodies with abundant afucosylated Fc glycoforms, a modification that enhances affinity of the Fc for a specific activating Fc receptor, FcγRIIIa. Thus, our data point to a role for FcγRIIIa in the pathogenesis of dengue disease. In this proposal we will study samples from Phase III trials of a live, attenuated tetravalent dengue virus vaccine, CYD-TDV (Dengvaxia, Sanofi Pasteur), to define mechanism involved in human immunity to dengue viruses. Recent analyses of data from the Phase III trials of CYD-TDV showed that risk for disease was increased in some study cohorts by vaccination. This finding highlights the importance of understanding how antibody responses to dengue vaccination are regulated and molecular mechanisms by which antibodies can enhance dengue infections. Aims in this proposal will: a) define regulators of Fc fucosylation on antibodies elicited by CYD-TDV vaccination or by natural dengue infection in humans; b) define associations between post- vaccination/pre-infection anti-dengue antibody repertoires and susceptibility to dengue disease during a 5-year follow-up period after vaccination; c) define mechanisms by which FcγRIIIa impacts dengue infections and disease pathogenesis. Collectively, these aims will advance our fundamental understanding of mechanisms regulating human immunity to dengue viruses and guide the design of safe, effective dengue virus vaccines.
