To assess the potential contribution of DENV-reactive IgA to a functional anti-DENV humoral immune response, we synthesized two pairs of previously described DENV-reactive monoclonal antibodies with either an IgG or an IgA Fc domain ( Figure 1A ). Both mAbs selected for this analysis were previously determined to bind the fusion loop of the DENV E protein and to react with all 4 DENV serotypes (32). However, VDB33 was initially identified as an IgG clone, while VDB50 was discovered as an IgA clone ( Table 1 ). This cross-conversion strategy was chosen so as to determine if the native F_c configuration of a given antibody influenced its functionality as either an IgG or IgA protein product.

The DENV-binding capacity of the IgG and IgA versions of VDB33 and VDB50 was initially assessed with a DENV virion-capture ELISA. For this analysis, DENV-3 was chosen as the prototypic DENV serotype as previous work demonstrated that the IgG versions of both VDB33 and VDB50 exhibited significant DENV-3 reactivity (32). Consistent with previously published reports, both VDB33 and VDB50 exhibited potent DENV-3 binding activity with VDB33 demonstrating ~200 fold higher affinity for DENV-3 than VDB50 ( Figure 1B and Table 2 ). However, the DENV-binding capacity of the two mAbs was not impacted by their conversion to either an IgG or IgA format ( Figure 1B and Table 2 ). Furthermore, this cross-conversion of VDB33 and VDB50 to either an IgG or IgA format minimally impacted the DENV-3 neutralization activity of the clones when assessed using a flow cytometry-based neutralization assay ( Figure 1C and Table 2 ). These results indicate that both IgG and IgA isotype antibodies are equally capable of binding and neutralizing DENV, reaffirming that antibody epitope/paratope interactions occur independently of an antibody’s F_c domain.

Having demonstrated that the antigen binding and neutralization capacity of DENV-reactive monoclonal antibodies is negligibly impacted by the isotype of the construct, we endeavored to determine if the infection-enhancing capability of these antibodies was impacted by their isotype conversion. To this end, we utilized a K562-based ADE assay, wherein antibody/DENV immune complexes were pre-formed and added to the F_c-receptor expressing K562 cell line to assess the ability of defined antibody complexes to enhance DENV infection.

The IgG versions of both VDB33 and VDB50 exhibited potent infection-enhancing activity in the K562 ADE assay, with both antibodies capable of facilitating DENV infection/enhancement in a dose-dependent fashion ( Figures 2A–D ). Consistent with their relative EC₅₀/IC₅₀ values, VDB33-IgG exhibited notably higher ADE activity than VDB50-IgG, but with the peak of ADE activity occurring at a similar antibody concentration. However, no infection enhancement was observed when the same assay was performed with either VDB33-IgA or VDB50-IgA ( Figures 2A–D ). This was despite the fact that these IgA isotype antibodies exhibit nearly identical virus binding and neutralization activity as their IgG counterparts, underlining the obligate role of an antibody’s F_c domain in determining the ADE potential of an antibody.

In light of the inability of VDB33-IgA and VDB50-IgA to facilitate ADE of DENV-3, we next endeavored to determine how DENV-reactive IgG and IgA behave in a competitive setting. IgG and IgA antibodies are never found in isolation in a dengue immune individual, so determining how these antibodies function in a complex/poly-immune setting is critical for understanding their potential contribution to functional anti-DENV immunity.

To this end, we utilized the same K562 ADE assay as previously described, but used a fractional IgG/IgA replacement strategy wherein the total amount of antibody remained the same across the different titration schemes but the ratio of IgG to IgA was varied from 100:0 to 0:100. The fractional addition of DENV-reactive IgA significantly reduced the ADE activity observed in cultures containing either VDB33-IgG or VDB50-IgG ( Figure 3 ). While both VDB33-IgA and VDB50-IgA were capable of antagonizing IgG-mediated ADE of DENV-3, the highly avid yet non-enhancing VDB33-IgA antibody was capable of dramatically blunting IgG-mediated ADE even when used at low fractional concentrations. Of note, the addition of DENV-reactive IgA to these ADE assays does not appear to shift the antibody dilution at which maximal ADE activity is observed for any of the cultures. Rather, the addition of DENV-reactive IgA reduces the magnitude of infection achieved at any given antibody dilution. These results are consistent with IgA actively antagonizing IgG mediated ADE by competing with DENV-reactive IgG for the same viral epitopes.

A limitation of the analysis presented thus far is that all the monoclonal antibodies used in this analysis have the same antigen specificity; namely, the fusion loop of the DENV E protein. Therefore, it is unclear what impact–if any–DENV-reactive IgA would have in the presence of a polyclonal IgG repertoire of divergent DENV antigen specificity. Therefore, we endeavored to determine how the presence of either VDB33-IgA or VDB50-IgA impacts the infection-enhancing potential of polyclonal/DENV-immune serum.

Plasma from DENV-immune donors was screened to identify samples with both high DENV-3 reactive IgG titers by ELISA as well as DENV-3 enhancing activity in the K562 ADE assay. Samples from four subjects were selected for additional analysis based on these criteria ( Figure 4A , Supplementary Figures 3 , 4 ). VDB33-IgA or VDB50-IgA were then titrated into cultures containing this enhancing DENV-immune plasma to determine if IgA isotype monoclonal antibodies could antagonize polyclonal enhancement of DENV-3 infection.

Consistent with what was observed with IgG monoclonal antibodies, the addition of VDB33-IgA or VDB50-IgA significantly suppressed ADE-mediated K562 infection with DENV-3 ( Figures 4B, C ). The additional of DENV-reactive IgA in these assays suppressed ADE-mediated infection by 75%-90% in a dose-dependent fashion, a result consistent with the concept that IgG antibodies targeting the fusion loop of the DENV E protein are particularly amenable to facilitating ADE activity and are abundant in DENV-immune serum (34, 35). Notably, an equal amount of isotype control human serum IgA had no impact on the ADE activity, suggesting that the observed suppression via VDB33- and VDB50-IgA is a function of antigen binding rather than F_c -mediated ( Supplementary Figure 5 ).These data also indicate that even modest concentrations of DENV-reactive IgA can significantly antagonize polyclonal IgG-mediated enhancement of DENV infection, signifying that the presence of DENV E reactive IgA (especially fusion loop reactive IgA) has the potential to significantly modulate DENV infection and associated immunopathogenesis.

DENV-3 (strain CH53489) propagated in Vero cells were utilized for ELISA, FlowNT50, and ADE assays. Virus for ELISA was purified by ultracentrifugation through a 30% sucrose solution and the virus pellet was resuspended in PBS.

Cell lines: Human K562 cells were maintained in IMDM supplemented with 10% FBS, penicillin, and streptomycin. U937-DC-SIGN cells were maintained in RPMI supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin.

The variable regions from the heavy and light chains were codon optimized, synthesized in vitro and subcloned into a pcDNA3.4 vector containing the human IgG1 or IgA1 Fc region by a commercial partner (Genscript). Transfection grade plasmids were purified by maxiprep and transfected into a 293-6E expression system. Cells were grown in serum-free FreeStyle 293 Expression Medium (Thermo Fisher), and the cell supernatants collected on day 6 for antibody purification. Following centrifugation and filtration, the cell culture supernatant was loaded onto an affinity purification column, washed, eluted, and buffer exchanged to the final formulation buffer (PBS). Antibody lot purity was assessed by SDS-PAGE, and the final concentration determined by 280 nm absorption. The clonotype information for all monoclonal antibodies generated as part of this study is listed in Table 1 . Dengue IgG antibody positive plasma was purchased from SeraCare. Donor ID and batch numbers are shown in Supplementary Table 1 . Purified polyclonal IgA from human serum was purchased from a commercial source (Sigma, I4036-1mg).

Monoclonal antibody and plasma DENV-reactivity was assessed using a 4G2 DENV capture ELISA protocol. In short, 96 well NUNC MaxSorb flat-bottom plates were coated with 2 μg/ml flavivirus group-reactive mouse monoclonal antibody 4G2 (Envigo Bioproducts, Inc.) diluted in borate saline buffer. Plates were washed and blocked with 0.25% BSA + 1% Normal Goat Serum in PBS after overnight incubation. DENV-3 (strain CH53489) diluted in blocking buffer was captured for 2 hr, followed by extensive washing with PBS + 0.1% Tween 20. Serially diluted monoclonal antibody or plasma samples were incubated for 1 hr at RT on the captured virus, and DENV-specific antibody binding quantified using anti-human IgG HRP (Sigma-Aldrich, SAB3701362), anti-human IgA HRP (Biolegend, 411,002), or anti-human IgM HRP (SeraCare, 5220-0328). Secondary antibody binding was quantified using the TMB Microwell Peroxidase Substrate System (KPL, cat. #50-76-00) and Synergy HT plate reader (BioTek, Winooski, VT). Monoclonal antibody binding data were analyzed by nonlinear regression (One site total binding) to determine EC50 titers in GraphPad Prism 8 (GraphPad Software, La Jolla, CA). End-point titers of DENV-reactive plasma samples were determined as the reciprocal of the final dilution at which the optical density (OD) was greater than 2× of a control flavivirus naïve serum.

Neutralizing titers of monoclonal antibodies and heat-inactivated plasma were assessed using a flow cytometry-based neutralization assay in U937 cells expressing DC-SIGN as previously described (51, 52). Four-fold dilutions of antibody or sera were mixed with an equal volume of virus diluted to a concentration to achieve 10%–15% infection of U937-DC-SIGN cells in the absence of antibody. The antibody/virus mixture was incubated for 1 h at 37°C, after which an equal volume of medium (RPMI-1640 supplemented with 10% FBS, 1% penicillin/streptomycin, 1% l-glutamine (200 mM) containing 5 × 10⁴ U937-DC-SIGN cells was added to each well and incubated 18–20 hr overnight in a 37°C, 5% CO2, humidified incubator. Following overnight incubation, the cells were fixed with IC Fixation Buffer (Invitrogen, 00-82222-49), permeabilized using IC Permeabilization Buffer (Invitrogen, 00-8333-56) and immunostained with flavivirus group-reactive mouse monoclonal antibody 4G2 (Envigo Bioproducts, Inc.), and secondary polyclonal goat anti-mouse IgG PE-conjugated antibody (#550589, BD Biosciences). The percentage of infected cells were quantified on a BD Accuri C6 Plus flow cytometer (BD Biosciences). Data were analyzed by nonlinear regression to determine 50% neutralization titers in GraphPad Prism 8 (GraphPad Software, La Jolla, CA).

In vitro antibody-dependent enhancement (ADE) of DENV-3 infection was quantified as previously described (32, 53). Four-fold serial dilutions of antibody or heat-inactivated sera were incubated with virus (in sufficient amounts to infect 10%–15% of U937-DC-SIGN cells) at a 1:1 ratio for 1 h at 37°C. This mixture was then added to a 96-well plate containing 5 × 10⁴ K562 cells per well in duplicate. Cells were cultured for 18–20 hr overnight in a 37°C, 5% CO2, humidified incubator. Processing and quantification continued as outlined in the FlowNT50 methods.

All statistical analysis was performed using GraphPad Prism 8 Software (GraphPad Software, La Jolla, CA). A P-value < 0.05 was considered significant.