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Maternal Anti-Dengue IgG Fucosylation Predicts Susceptibility to Dengue Disease in Infants

SUMMARY Infant mortality from dengue disease is a devastating global health burden that could be minimized with the ability to identify susceptibility for severe disease prior to infection. Although most primary infant dengue infections are asymptomatic, maternally derived anti-dengue immunoglobulin G (IgGs) present during infection can trigger progression to severe disease through antibody-dependent enhancement mechanisms. Importantly, specific characteristics of maternal IgGs that herald progression to severe infant dengue are unknown. Here, we define ≥10% afucosylation of maternal anti-dengue IgGs as a risk factor for susceptibility of infants to symptomatic dengue infections. Mechanistic experiments show that afucosylation of anti-dengue IgGs promotes FcγRMIa signaling during infection, in turn enhancing dengue virus replication in FcγRIIIa+ monocytes. These studies identify a post-translational modification of anti-dengue IgGs that correlates with risk for symptomatic infant dengue infections and define a mechanism by which afucosylated antibodies and FcγRMIa enhance dengue infections.


Correspondence taiawang@stanford.edu
In Brief Thulin et al. show that reduced fucosylation (afucosylation) of maternally derived anti-dengue IgGs is associated with symptomatic dengue infections in infants. Afucosylation of dengue immune complexes promotes FcgRIIIa signaling in monocytes, in turn enhancing infection through a post-entry pathway that is dependent on the calcineurin signaling network.

INTRODUCTION
Antibody-mediated inflammatory responses are critical in immunity against infectious organisms. These responses can promote pathogen clearance but can also exacerbate symptoms during infections (Bournazos et al., 2017). Antibody-mediated inflammation is triggered when pathogens or infected cells are bound by immunoglobulin G (IgG) antibodies, forming immune complexes that signal through Fc gamma receptors (FcgRs) on effector cells. The outcome of effector cell responses depends on the balance of activating to inhibitory (A/I) FcgR signaling that arises from interactions with Fc domains within immune complexes. Activating FcgRs contain immunoreceptor tyrosine-based activation motifs (ITAMs), while the inhibitory FcgR balances this activity through immunoreceptor tyrosine-based inhibitory motif (ITIM) signaling. One factor that modulates the ratio of A/I FcgR signaling is the glycosylation state of the IgG Fc domains within immune complexes. For example, sialylation of the Fc promotes anti-inflammatory effector responses, whereas absence of core fucosylation, afucosylation, of the Fc is pro-in-flammatory due to increasing affinity of the Fc for the activating FcgRIIIa, found on natural killer (NK) cells, as well as on subsets of macrophages and monocytes (Anthony et al., 2011;Bournazos et al., 2017;Rafiq et al., 2013). The second major determinant of A/I FcgR signaling by immune complexes is the distribution of IgG subclasses within the complex, with IgG1 being the dominant subclass promoting pro-inflammatory responses and IgG2 signaling through the inhibitory FcgR (Pincetic et al., 2014).
Dengue virus infections are unusual in that non-neutralizing anti-dengue virus IgGs can play a central role in triggering progression to the severe forms of disease through antibodydependent enhancement (ADE) mechanisms (Anderson et al., 2014;Burke et al., 1988;Chau et al., 2009;Guzmá n et al., 1990;Halstead et al., 1970;Katzelnick et al., 2017;Libraty et al., 2009;Sangkawibha et al., 1984;Wang et al., 2017). ADE can occur in the presence of reactive, non-neutralizing IgGs, as are found in secondary heterologous dengue infections or in primary infections in infants of dengue-immune mothers due to acquisition of anti-dengue virus IgGs during gestation (Halstead et al., 1970;Kliks et al., 1988;Simmons et al., 2007). These antibodies are thought to promote disease by forming immune complexes with the virus that modulate infection in FcgRbearing cells, primarily monocytes and macrophages, leading to higher viral titers and altered cytokine production during infection (Aye et al., 2014;Durbin et al., 2008;Kou et al., 2008;Thein et al., 1997). Still, the vast majority of dengue infections that occur in the presence of non-neutralizing IgGs are asymptomatic, and specific features of antibodies that enhance one's basal susceptibility for dengue disease are unknown. Importantly, mortality rates in severe dengue can exceed 20% when patients are not hospitalized, but can be reduced to <1% with in-patient care (Anderson et al., 2014;Gordon et al., 2013). Therefore, the identification of serologic markers for increased baseline susceptibility to dengue disease could dramatically reduce mortality associated with these viruses by enabling early hospitalization of those at highest risk for disease progression.
Because maternal IgGs are transferred to the fetus during gestation, we hypothesized that the repertoire of maternal antidengue virus IgG Fc domains might impact basal susceptibility of infants to disease during dengue virus infections. To address this, we performed a global analysis of maternal anti-dengue IgG Fc domain features from a prospective cohort of mothers and infants in a dengue-endemic region of the Philippines in order to define characteristics that predicted progression to significant infant dengue disease. We found that maternal anti-dengue IgGs enriched for afucosylated Fc glycoforms predicted clinically significant disease in their infants during primary dengue infections. Because afucosylation enhances affinity of the Fc for FcgRIIIa, this suggested a role for this receptor in the pathogenesis of dengue disease. We therefore performed studies to dissect the mechanism by which FcgRIIIa and afucosylated IgGs impact dengue virus infections. Our findings identify a pathway by which dengue replication can be enhanced through a FcgRIIIa signaling mechanism requiring the calcineurin signaling network. These studies identify a mechanistic basis for differential susceptibility to dengue disease based on the presence of afucosylated anti-dengue IgGs and suggest novel strategies for the treatment of dengue virus infections.

Maternal Anti-Dengue Fucosylation Predicts
Susceptibility to Clinically Significant Disease during Infant Dengue Infections Maternal IgG levels in the serum of newborns generally match or exceed those in maternal serum (1,012 mg/mL IgG), and the halflife of maternal IgGs in infants is >40 days (Watanaveeradej et al., 2003). Thus, when a mother is seropositive for dengue viruses, maternal anti-dengue IgGs have the potential to modulate infant dengue infections for over 1 year after birth (Alkan Ozdemir et al., 2016;Nicoara et al., 1999;Simmons et al., 2007;Thompson et al., 2016).
To define specific maternal anti-dengue IgG features that herald risk for infant dengue disease, we performed a global analysis of anti-dengue envelope (E) IgGs in mothers of infants with symptomatic (febrile) or asymptomatic primary dengue infections. To do this, we studied samples from a prospective cohort in San Pablo, Laguna, Philippines, a dengue-endemic region.
Given the role of FcgRs in ADE of dengue virus infections, we first asked whether any maternal anti-E Fc glycosylation patterns could predict susceptibility to infant dengue disease. Characterization of anti-E IgG Fc glycoforms showed that mothers of infants who had clinically significant (symptomatic) primary dengue infections had significantly increased levels of afucosylated anti-E Fc glycoforms compared with mothers of infants who had asymptomatic primary infections ( Figure 1A). Indeed, R10% afucosylated Fc glycans was predictive of elevated risk for symptomatic infant dengue infections; this threshold predicted symptomatic infections in infants with a positive predictive value of 88%. Specificity of this marker was 93%, indicating that it nearly always predicted symptomatic dengue infections in infants. Sensitivity was lower, 46%, as many, but not all, symptomatic infections could be predicted using this serologic marker. Even omitting the sample with the highest level of afucosylation in the symptomatic group, the 10% threshold remained predictive with unchanged specificity (93%) and slightly reduced sensitivity (43%). Interestingly, this specific threshold of afucosylation was also identified in a prior study of hospitalized dengue patients; this prior study found that anti-E IgG with R10% afucosylated Fc glycans was predictive of the most severe disease outcomes during acute dengue infections that occurred in the presence of reactive, non-neutralizing IgGs (Wang et al., 2017). Aside from Fc fucosylation, other Fc glycoforms did not vary between mothers of infants with symptomatic and asymptomatic primary dengue infections ( Figure 1B).
Besides Fc glycosylation, the other features that can impact the A/I ratio of Fc-FcgR interactions were also measured. Extensive characterization of the maternal anti-E IgG response showed that symptomatic infant dengue infections could not be predicted by the IgG1/IgG2 ratio of anti-E subclasses, a measurement that accounts for the most abundant IgG subclasses, with IgG1 having increased affinity for activating FcgRs over IgG2 ( Figure 1C). The distribution of all maternal anti-E IgG subclasses, the titer of anti-E IgGs, and the binding avidity of anti-E IgGs ( Figures 1D-1F) were also not different between groups. In addition, there was no correlation between either symptomatic infant dengue infections or the levels of maternal anti-E afucosylation with infant sex, age at infection, days of fever during symptomatic infections, or serotype of the infecting dengue virus strain (Table S1). Because anti-NS1 IgGs can modulate the severity of dengue infections, we further assessed whether the maternal titer of anti-NS1 IgGs predicted symptomatic dengue infections in their infants and found no correlation ( Figure S1A) (Hertz et al., 2017;Jayathilaka et al., 2018). In addition, there was no significant difference in the abundance of anti-NS1 or total IgG Fc fucosylation from mothers of infants with symptomatic or asymptomatic primary dengue infections ( Figure S1B). The presence of R10% afucosylated glycans on anti-E IgG was the only factor identified in our characterizations that predicted risk for dengue disease in infants. Because Fc afucosylation increases affinity for FcgRIIIa, this finding pointed toward a role for FcgRIIIa in antibody-enhanced dengue infections.

FcgRIIIa Signaling Is Driven by the Fucosylation Level in Dengue Immune Complexes and Enhances Dengue Virus Replication in Human U937 Monocytes
To study how FcgRIIIa signaling by dengue immune complexes might be impacted by variable levels of anti-E fucosylation, we used the maternal IgGs to generate dengue immune complexes and defined their signaling capacity in a FcgRIIIa reporter assay. This showed a linear correlation between endogenously produced levels of anti-E afucosylation and FcgRIIIa-mediated signaling ( Figure 2A). Next, we generated dengue virus immune complexes using pooled maternal IgGs with R10% or <10% afucosylation of anti-E IgG; immune complexes with R10% afucosylation drove significantly higher FcgRIIIa signaling (Figure 2B). To assess whether anti-E domain specificity might modulate FcgRIIIa signaling activity, we expressed monoclonal antibodies (mAbs) that bound to distinct domains of the E protein with fucosylated or afucosylated Fc glycans. All mAbs triggered significantly enhanced FcgRIIIa signaling when expressed with afucosylated Fc glycoforms regardless of their anti-E binding specificity ( Figure 2C; Figure S2; Table S2).
Although FcgRIIIa is present on macrophage and pro-inflammatory monocyte subsets that are associated with dengue disease, FcgRIIIa is not expressed on human monocytic or other granulocytic lineage cell lines that are often used to study ADE of dengue virus infections, such as THP-1, K562, or U937 cells (Fleit et al., 1982;Tridandapani et al., 2002;van de Winkel and Anderson, 1991). These cell lines express the high-affinity activating FcgR, FcgRI (except for K562 cells), along with the low-affinity FcgRs, activating FcgRIIa and inhibitory FcgRIIb. Further, primary cells and in vivo mouse models are not robust systems for studying the role of FcgRIIIa in dengue infections because FcgRIIIa is unstable on primary monocytes/macrophages that are cultured ex vivo (Ong et al., 2019;Wang lab, unpublished data), and mouse models that are humanized for FcgR expression, and thus express FcgRIIIa, are immunocompetent, rendering them incompatible with current dengue infection models (Smith et al., 2012;Zellweger and Shresta, 2014). Thus, to dissect the role of FcgRIIIa in ADE of dengue infections, we generated human U937 monocytes expressing different combinations of the activating low-affinity FcgRs, FcgRIIa and , but a role for FcgRIIIa has not been defined. First, U937 monocytes expressing different combinations of FcgRIIa and FcgRIIIa were infected with immune complexes made from dengue virus and five different maternal IgGs from the San Pablo cohort. Single-cycle infection was enhanced by up to 300% in monocytes expressing FcgRIIIa (FcgRIIa + Fc-gRIIIa + ) compared with those expressing only FcgRIIa (FcgRIIa + FcgRIIIa À ) ( Figure 3A). Next, we determined whether FcgRIIIa ITAM signaling was required for FcgRIIIa-mediated ADE, using cells expressing signaling-competent (FcgRIIa + Fc-gRIIIa + ) or signaling-null [FcgRIIa + FcgRIIIa (ITAMÀ) ] versions of FcgRIIIa ( Figure S3A) (Blá zquez-Moreno et al., 2017). Interestingly, the FcgRIIIa-mediated enhancement of infection required ITAM signaling ( Figure 3A). To assess whether anti-E domain specificity might contribute to the involvement of FcgRIIIa in ADE, we tested the panel of afucosylated anti-E mAbs for their ability to modulate dengue infections. In all cases, anti-E mAbs mediated significantly increased infection in FcgRIIa + FcgRIIIa + cells over U937 monocytes expressing only FcgRIIa. As observed with the maternal IgG, FcgRIIIa-mediated ADE required FcgRIIIa signaling in all cases ( Figure 3B). Overall, these studies showed that increasing the cellular A/I FcgR signaling ratio via FcgRIIIa ITAM signaling promoted ADE of dengue virus infections.
We next asked whether modulating cellular A/I FcgR signaling in parallel with dengue infection might impact ADE. Crosslinking individual FcgRs in FcgRIIa + FcgRIIIa + U937 cells showed that increased signaling by the activating, ITAM-containing FcgRIIa or FcgRIIIa enhanced cell infectibility, whereas signaling through the inhibitory, ITIM-containing FcgRIIb reduced infection ( , 1997). To verify this, we treated cells with the calcium ionophore, ionomycin, prior to infection. This treatment increased ADE of infection in a dose-dependent manner ( Figure 3E).
Having found that FcgRIIIa ITAM signaling could increase dengue infections, we next sought to determine whether FcgRIIIa might have a separate role in mediating attachment of dengue immune complexes. To investigate this, we used wellcharacterized blocking mAbs against FcgRI, FcgRIIa, or FcgRIIIa to prevent immune complex attachment to cells prior to infection (Boruchov et al., 2005;Fleit et al., 1982;Mandelboim et al., 1999;Tamm and Schmidt, 1996;Zeller and Sullivan, 1993). In FcgRIIa + FcgRIIIa + U937 cells, blocking either FcgRI or FcgRIIa during immune complex attachment prevented $50% infection, whereas blocking FcgRIIIa alone with the blocking mAb, 3G8, did not inhibit infection ( Figure 4A) (Boruchov et al., 2005;Fleit et al., 1982;Mandelboim et al., 1999;Tamm and Schmidt, 1996;Zeller and Sullivan, 1993). Combining blocking mAbs for FcgRI and FcgRIIa inhibited infection by $80%, and inclusion of the FcgRIIIa blocking mAb did not further diminish infection. In freshly isolated primary monocytes, blocking attachment of immune complexes to FcgRIIa prior to entry prevented nearly all infection, whereas blocking FcgRI, FcgRIIb, or FcgRIIIa did not reduce infection ( Figure 4B). These results are consistent with studies showing that FcgRIIa on monocytes mediates dengue immune complex attachment and entry (Boonnak et al., 2013;Kou et al., 2008;Littaua et al., 1990) and suggested that the FcgRIIIa-mediated enhancement is due to modulation of a post-attachment step of infection.

Inhibitors of the Calcineurin Signaling Network Block Dengue Virus Replication
Engagement and crosslinking of the activating FcgRs trigger phosphorylation of ITAM domains by Src family kinases and intracellular calcium flux (Bournazos and Ravetch, 2015;Hippen et al., 1997). Calcium flux, in turn, activates calcineurin, a serine/ threonine phosphatase that recognizes a host of substrates, scaffolds, and regulator proteins through specific docking motifs (Roy and Cyert, 2009;Roy et al., 2007). To dissect how enhanced A/I ratios contribute to ADE of dengue infections, we treated cells with inhibitors of the ITAM signaling pathway prior to infection.
First, FcgRIIa + FcgRIIIa + monocytes were treated with PRT, an inhibitor of Syk tyrosine kinase activity, required for initiation of ITAM signaling (Lowell, 2011;Reilly et al., 2011). This inhibited dengue infection ( Figure 5A), supporting the requirement for ITAM signaling in ADE of dengue virus infection.
Next, we tested the hypothesis that ADE of dengue infection is enhanced by calcium flux via the calcineurin signaling network. The small-molecule inhibitor INCA-6 (Bretz et al., 2013) and the peptide inhibitor 11R-VIVIT (VIVIT) (Aramburu et al., 1999) act selectively to prevent the association of calcineurin with substrates that bind via the calcineurin PxIxIT docking motif (Roy et al., 2007). Both inhibitors prevented dengue virus infection in U937 monocytes (Figures 5B and 5C). Inhibitors were used at concentrations that did not impact cell viability ( Figure S4). To assess the specificity of this observation for dengue infections, we studied the impact of the VIVIT inhibitor on infection of U937 cells by influenza A viruses. Although dengue serotypes 2 and 3 were robustly inhibited by VIVIT, influenza A viruses (H1N1 A/Netherlands/602/2019 and H3N2 A/HK/1/1968 strains) were not affected by this inhibitor of the calcineurin signaling network ( Figure 5D). We next treated cells with the VIVIT inhibitor during or at different time points after dengue infection to characterize the kinetics of the required calcineurin network activity. Treatment of cells up to 6 h after entry significantly reduced infection, consistent with disruption of a post-entry stage of infection ( Figure 5E). Interestingly, FK506 and cyclosporin A, two clinically used drugs that block calcineurin through mechanisms distinct from INCA-6 and VIVIT (Ho et al., 1996), did not prevent dengue infections ( Figure S5). These studies show that calcineurin-PxIxIT site interactions are required for maximal dengue virus infections, suggesting new clinical strategies for treatment of dengue infections.

IgG Fucosylation Is Stable over Time, Suggesting Utility as a Predictive Marker for Dengue Disease Risk
To be a useful marker of dengue disease susceptibility, the abundance of Fc afucosylation would have to be stable over time and withstand immune perturbation. To assess stability of this IgG modification, we performed a longitudinal study of IgGs from healthy adults in a North American cohort who were challenged with influenza A virus (Memoli et al., 2015). We characterized IgG afucosylation prior to and over a period of 60 days after influenza

DISCUSSION
Here, we show that elevated afucosylated Fc glycans (R10%) on maternal anti-E IgGs is one risk factor for susceptibility of infants to disease during primary dengue virus infections. Mechanistic studies performed to identify how afucosylated anti-E IgGs can impact dengue infections support a model whereby the presence of afucosylated dengue immune complexes triggers a FcgRIIIa-modulated infection characterized by increased ITAM signaling. Enhanced ITAM signaling, in turn, promoted dengue infections in U937 monocytes through an intrinsic ADE mechanism ( Figure 5F) Figure 5G). This mechanism suggests novel treatment strategies for dengue infections that target this pathway. An important topic for future studies on the pathogenesis of dengue disease is the cellular activities associated with afucosylated IgGs during dengue infection that were not studied here. We have used percent infection as the readout of activity conferred by anti-dengue Fc afucosylation and of FcgRIIIa signaling in ADE of dengue virus infections, yet FcgRIIIa signaling activity likely impacts disease pathogenesis through multiple mechanisms, including modulation of cytokine production by FcgRIIIa-expressing cells.
Because mortality rates in severe dengue cases can be nearly eliminated with intensive clinical monitoring (Anderson et al., 2014;Gordon et al., 2013), identifying markers for susceptibility to dengue disease could dramatically improve the clinical management of dengue patients. Here we show that maternal IgG Fc fucosylation could be used in the cohort studied to identify a significant subset of susceptible infants. Prospective cohort studies are now needed to examine testing of antibody fucosylation to guide the treatment of primary infant dengue infections and acute dengue infections in non-infant populations.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

Lead Contact
The lead contact for this study is Taia T. Wang (taiawang@stanford.edu).

Materials Availability
This study did not generate new unique reagents. Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact.

Data and Code Availability
This study did not generate any unique datasets or code. An unpaired Student's t test was utilized to calculate p value: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Table  See attached file

EXPERIMENTAL MODEL AND SUBJECT DETAILS
Clinical cohorts and samples: Samples from mothers of infants who had primary dengue infections were from a study that was approved by the institutional review boards of the Research Institute for Tropical Medicine, Philippines, and the University of Massachusetts Medical School. Mothers and their healthy infants were recruited and enrolled after providing written informed consent (ClinicalTrials.gov NCT00377754) (Libraty et al., 2009). Symptomatic dengue was defined as febrile illness with a positive result by dengue serotype-specific reverse-transcription PCR in acute phase sera (Lanciotti et al., 1992). Clinically inapparent dengue infection (asymptomatic infection) was determined using a hemagglutination inhibition assay (HAI assay); a R four-fold increase in HAI against one or more dengue serotypes in the absence of febrile illness confirmed asymptomatic infection (Libraty et al., 2009). The outcome assessed was clinical phenotype during dengue virus infection (symptomatic infection versus asymptomatic infection) (Table S1). Samples from the controlled influenza A virus challenge study have been previously described (Memoli et al., 2015). The study (ClinicalTrials.gov NCT01646138) was approved by the National Institute of Allergy and Infectious Diseases institutional review board and underwent an ethics review by the NIH Department of Bioethics prior to being conducted. The study was conducted in accordance with the provisions of the Declaration of Helsinki and Good Clinical Practice guidelines.

METHOD DETAILS
IgG Fc glycan and IgG subclass analysis: Methods for relative quantification of Fc Glycans and IgG subclasses have been previously described Wang et al., 2017). Briefly, IgGs were isolated from serum by protein G purification. Antigen-specific IgGs were isolated on NHS agarose resin (ThermoFisher; 26196) coupled to the protein of interest. Mixed envelope proteins from the four DENV serotypes were used (ProSpec; DEN-021, DEN-022, DEN-023, DEN-024). Following tryptic digestion of purified IgG bound to antigen-coated beads, nanoLC-MS/MS analysis for characterization of glycosylation sites was performed on an Ulti-Mate3000 nanoLC (Dionex) coupled with a hybrid triple quadrupole linear ion trap mass spectrometer, the 4000 Q Trap (SCIEX).  (U937) was generated that express all combinations of these activating FcγRs. A) Expression of FcγRIIa, FcγRIIIa, and/or FcγRIIb on U937 cell lines was confirmed by FACS. B) Cross-linking of ITAM-containing FcγRIIa or FcγRIIIa induced signaling in cell lines that express the respective receptor as indicated by tyrosine phosphorylation. Signaling by FcγRIIIa was observed in the FcγRIIa + FcγRIIIa + cell line but not the FcγRIIIa ITAM-deficient FcγRIIa + FcγRIIIa (ITAM-) line or in FcγRIIa + FcγRIIIa -(wildtype) cells. All cell lines express FcγRI and FcγRIIb, with or without FcγRIIa and/or FcγRIIIa C) Broadly reactive anti-E mAb 235 was expressed as a human IgG1 (mAb 235), afucosylated Fc (afucFc) mAb235, mAb 235 Fc null variant N297A. The DENV3 specific mAb 5J7 was also expressed as a human IgG1. Binding of mAbs was assessed by ELISA on dengue 2infected cells. D) FACS gating strategy for dengue-infected cells. Infection in single, live cells was assessed by staining of envelope protein using mAb 4G2 at 24 hours unless otherwise specified. E) DENV2 infection in wild-type U937 cells increased over time when infection occurred in the presence of mAb 235 expressed as a wild-type human IgG1. Infection did not increase when mAb 235 was expressed as an Fc-null variant, N297A or when immune complexes were made with deactivated virus (iDENV). F) The degree of enhanced infectability depended on the concentration of mAb 235. Data are representative of a minimum of three experiments. Error bars represent the standard error of three technical replicates.
Replicates were performed at all points; where error bars are not present, the standard error was smaller than the width of the symbol marking the data point.       Figure 1 and Experimental Model and Subject Details Methods. Maternal Subject ID shown with the mothers age as well as their infants sex, age at infection, clinical outcome of primary infant infections (symptomatic=sympt., asymptomatic=asympt., days of fever during primary dengue infections and the dengue serotype of the infecting virus. For asymptomatic infections, the dates of sample collection between which seroconversion was observed are indicated.  Swanstrom, J.A., et al., 2016 ) 752-2 C8 C8 Dimer EDE1 DENV1-4 Swanstrom, J.A., et al., 2016;de Alwis, R et al., 2012) 5J7 5J7 E protein herringbone Quarternary epitope DENV3 (de Alwis, R et al., 2012;Fibriansah, G., et al., 2015a;Fibriansah, G., et al., 2015b)