Project and Core Descriptions
Project 1: PI: Jeffrey Ravetch, Rockefeller University
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 proinflammatory, 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 FcγRmediated 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.
Project 2: PIs: Michel Nussenzweig, MD, PhD; Charles Rice, PhD
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). 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. The immune memory to these pathogens is interesting because it can be potentially harmful (e.g. antibody dependent enhancement of infection with DENV). We hypothesize that the memory B cells elicited by DENV and ZIKV in selected individuals are distinct and characterized by the expression of genes that are either general or pathogenspecific 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. 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. Memory transcriptomes of DENV and ZIKV 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 enhance infection (DENV).
Project 3: PI: Taia Wang, MD, PhD, Stanford University
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 receptorbearing cells. We have shown that progression to severe dengue disease is promoted by the presence of antidengue 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 CYDTDV 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 postvaccination/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.
