FcγRIIIA-Activating Antibodies in Dengue Virus Infection Reveals a Distinct, Transient Cross-Reactive Profile   Claudio Soto-Garita1, Tatiana Murillo1, Hartmut Hengel2, Eugenia Corrales-Aguilar3, 1*   1Virology Section, Research Centre for Tropical Diseases and Faculty of Microbiology, University of Costa Rica, San José, Costa Rica, 2Institute of Virology, University Medical Center, Faculty of Medicine, University of Freiburg; Freiburg, Germany, Freiburg, Germany, 3University of Costa Rica, San José, Costa Rica   Submitted to Journal:   Frontiers in Immunology   Specialty Section:   Viral Immunology   Article type:   Brief Research Report Article   Manuscript ID:   1662138   Received on:   08 Jul 2025   Revised on:   25 Aug 2025   Journal website link:   www.frontiersin.org In review http://www.frontiersin.org/           Scope Statement Dengue virus has four types and is spread mainly by mosquitoes, especially Aedes aegypti. When someone gets infected, their immune system makes two types of antibodies: one that targets the specific virus they got and another that reacts to the other types. These antibodies can both help fight the virus and, in some cases, actually make the disease worse. This study focused on a part of the immune system that isn’t usually examined closely — a receptor called FcγRIIIA, which helps immune cells recognize and respond to antibodies. We used special cells to test how this receptor reacted to blood samples from people who had been infected with dengue, both recently and in the past. We found that a certain type of antibody activity—mainly from antibodies that react to multiple dengue types—increases shortly after the illness but fades within two years. This type of antibody activity doesn’t match up exactly with how well the antibodies can block the virus or how they might help it infect cells more easily. Overall, this study offers new insight into how antibodies behave during and after dengue infection. Understanding this better could help in developing vaccines or treatments that avoid triggering harmful immune responses.       Conflict of interest statement   The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest       Credit Author Statement   Claudio Soto-Garita: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. Eugenia Corrales-Aguilar: Conceptualization, Formal Analysis, Funding acquisition, Methodology, Project administration, Supervision, Writing – original draft, Writing – review & editing. Hartmut Hengel: Conceptualization, Formal Analysis, Investigation, Resources, Writing – review & editing. Tatiana Murillo: Data curation, Formal Analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing.       Keywords   denv, humoral response, Antibody-dependent enhancement (ADE), FcγRIIIA, CD16A       Abstract Word count: 246   Dengue viruses (DENVs), members of the Flavivirus genus, comprise four antigenically distinct serotypes (DENV-1 to DENV-4) transmitted primarily by Aedes aegypti. Clinical outcomes of DENV infection range from mild to severe, with the host antiviral immune response playing a pivotal role in disease progression. Antibody responses to DENV include serotype-specific (homotypic) and cross-reactive (heterotypic) antibodies, both of which can mediate protective immunity or contribute to immunopathogenesis through antibody-dependent enhancement (ADE). The balance between these outcomes is influenced by multiple host and viral factors. Although antibody effector mechanisms rely on Fc-gamma receptor (FcγR) interactions, these are often overlooked in the assessment of antibody function. In particular, FcγRIIIA has been implicated in both protective and pathogenic roles during viral infection. To investigate its contribution, we employed FcγRIIIA-CD3ζ reporter cells to evaluate receptor activation by polyclonal sera from individuals with acute and past DENV infections. Neutralization capacity and enhancement potential were also analyzed. The FcγRIIIA activation assay revealed a distinct humoral profile, primarily mediated by cross- reactive antibodies, which differed from neutralization and enhancement patterns. This profile increased during the post-acute phase of infection but waned within two years. These findings highlight the dynamic nature of antibody responses, where the same antibody populations may contribute to cross-protection or immunopotentiation depending on the context. Overall, this study underscores the importance of FcγR-mediated effector functions in shaping DENV immunity and pathogenesis. The FcγRIIIA activation assay provides a valuable tool to characterize functional antibody responses, informing future efforts in vaccine and therapeutic development.       Funding information   Funding for the project (except for publication fees) was obtained from the University of Costa Rica (Grant numbers B7360, B7331, and ED- 3257)       Funding statement   The author(s) declare that financial support was received for the research and/or publication of this article. In review       Ethics statements   Studies involving animal subjects Generated Statement: No animal studies are presented in this manuscript.       Studies involving human subjects Generated Statement: The studies involving humans were approved by Scientific Ethical Committee from the Vicerrectory of Research of the University of Costa Rica. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.       Inclusion of identifiable human data Generated Statement: No potentially identifiable images or data are presented in this study.       Data availability statement Generated Statement: The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.           Generative AI disclosure   No Generative AI was used in the preparation of this manuscript.   In review Brief Research Report 1 FcγRIIIA-Activating Antibodies in Dengue Virus Infection Reveals a 2 Distinct, Transient Cross-Reactive Profile 3 Soto-Garita, Claudio1; Murillo, Tatiana1; Hengel, Hartmut2; Corrales-Aguilar, Eugenia1* 4 1Virology Section, Research Centre for Tropical Diseases and Faculty of Microbiology, University of Costa 5 Rica 6 2Institute of Virology, University Medical Center, Faculty of Medicine, University of Freiburg; Freiburg, 7 Germany 8 *Correspondence: 9 Eugenia Corrales-Aguilar 10 eugenia.corrales@ucr.ac.cr 11 Number of words: 4818 12 Number of figures: 2 13 Number of tables: 1 14 Keywords: DENV, Humoral response, Antibody-dependent enhancement (ADE), FcγRIIIA, 15 CD16a 16 Abstract 17 Dengue viruses belong to the genus Flavivirus and consist of a serocomplex of four serotypes (DENV-18 1, DENV-2, DENV-3, and DENV-4). As arthropod-borne viruses (arboviruses), their transmission is 19 mediated primarily by the vector Aedes aegypti. Antiviral immune response is one of the most crucial 20 factors influencing the progression from uncomplicated to severe dengue virus (DENV) infection. Two 21 types of antibody responses are elicited during a DENV infection: one specific to the infecting serotype 22 (serotype-specific or homotypic response) and another that cross-reacts with other serotypes (cross-23 reactive or heterotypic response). Both responses play roles in the protection against and in the 24 induction of immunopathogenesis of DENV disease. In the case of the humoral immune response, the 25 balance between protective and pathogenic effects mediated by antibodies (antibody-dependent 26 enhancement, ADE) is highly dynamic and influenced by multiple factors. Although many downstream 27 effector mechanisms depend on antibody recognition by Fc-gamma receptors (FcγRs) present on 28 immune effector cells, this interaction is traditionally not considered when evaluating antibody 29 properties. Specifically, FcγRIIIA has been implicated in both protection and immunopathogenesis of 30 virus infection. To assess its role within the humoral immune response to DENV, we took advantage 31 of FcγRIIIA-CD3ζ reporter cells and tested receptor activation by polyclonal sera from individuals 32 with past and acute DENV infections. In addition, the neutralizing capacity and the potential 33 enhancement of infection were analyzed. The FcγRIIIA activation assay revealed a humoral profile 34 distinct from neutralization and immunopotentiation, primarily mediated by cross-reactive antibodies. 35 Notably, this profile increases during the post-acute period but disappears within two years after 36 infection. Because these two types of antibodies are found during both the cross-protective and disease-37 In review 2 This is a provisional file, not the final typeset article enhancing (immunopotentiation) phases, their exact function in each situation is still not clearly 38 understood. The results of this study provide a valuable measurement of the effector function of anti-39 DENV antibodies, contributing to the understanding of their role in both protective and disease 40 enhancing courses of DENV infection. 41 1 Introduction 42 Dengue virus (DENV) is an arthropod-borne virus (arbovirus) transmitted by Aedes aegypti and Aedes 43 albopictus, being the former the most important vector. DENV is assigned to the family Flaviviridae and the 44 genus Orthoflavivirus and poses a major global health burden in tropical and subtropical regions. The World 45 Health Organization (WHO) estimates that between 100 to 400 million infections occur yearly and that half the 46 world population is at risk of infection (1). Costa Rica is considered a hyperendemic country for dengue, with 47 co-circulation of all four DENV serotypes and recurring outbreaks that continue to pose major public health 48 concerns (2). DENV infection is characterized by an incubation period of 4 to 10 days after the mosquito bites 49 and produces a spectrum of clinical manifestations. Although many of the infections are asymptomatic, it can 50 produce a self-limited but debilitating clinical presentation characterized by high fever, headache, retroorbital 51 pain, myalgia, arthralgia, nausea, vomiting, lymphadenopathy and rash. The major risk of DENV infection is 52 for those patients who develop dengue-hemorrhagic fever (DHF) which can be death threatening (3). DHF has 53 three phases: febrile, critical and recovery. In the critical phase the increase in capillary permeability leads to 54 plasma leakage and hypovolemic shock with multiorgan failure, metabolic acidosis, disseminated intravascular 55 coagulation and hemorrhage (4). Some critical patients can develop hepatitis, encephalitis, myocarditis, and 56 severe hemorrhage without plasma leakage. In these cases, intravenous rehydration treatment can reduce 57 mortality from 20% to 1% (3). 58 Four DENV serotypes (1, 2, 3, 4) exist, sharing between 60%-70% of their coding sequence (5). DENV 59 pathogenicity in the human host can be partially explained by differences in viral virulence due to genotype and 60 serotype (6). For instance, the Asian genotype of DENV-2 produces a more severe disease than the American 61 genotype (7, 8). Host factors are also implicated in the severity of the disease, including the humoral immune 62 response. The immune response against DENV differs between serotypes, a serotypic-specific or homotypic 63 response is produced against the infecting serotype while a cross-reacting or heterotypic response is generated 64 against other serotypes (9). A heterotypic immune response provides protection for an estimated period of six 65 months to three years while a homotypic immune response should give a lifetime protection (10). However, 66 once a cross-reacting immune response cannot protect the host anymore, it can contribute to the 67 immunopathogenesis of the disease by exacerbating inflammation through a cytokine storm or 68 immunopotentiation (ADE) (11). The overproduction of cytokines produces endothelial cell damage increasing 69 vascular permeability and plasma leakage characteristic of DHF (12). Complement activation and the production 70 of a temporal autoimmune response may also occur (13, 14). 71 Both the cellular and humoral heterotypic immune response may induce immunopathogenesis. Cross-reacting 72 cytotoxic T cells are ineffective at controlling the infection and increase the production of cytokines (15). 73 Antibody-dependent enhancement (ADE) of infection occurs when IgG antibodies bind the viral particles but 74 are uncapable to neutralize them and instead, form immune complexes that bind to the Fc receptors (FcR) on 75 immune cells, favoring viral infection of these cells followed by uncontrolled immune cell activation (16). 76 Antibody specificity determines the risk of developing ADE. Antibodies targeting the I- and II- domain of the 77 envelope (E) viral glycoprotein are highly serotype cross-reactive and associated with ADE (11, 17). The 78 tridimensional disposition of the epitopes and antibody concentration also has an impact on the development of 79 ADE (18). Linear epitopes and neutralizing antibodies at low concentrations can favor ADE (19, 20). Thus, 80 serotype-specific, and cross-reactive antibodies may produce ADE depending on their concentration (18). 81 Fcγ receptors (FcγRs) belong to the immunoglobulin superfamily and are expressed on the surface of various 82 immune cells, including monocytes, macrophages, neutrophils, and NK cells. The three main classes—FcγRI 83 (CD64), FcγRII (CD32), and FcγRIII (CD16)—differ in structure, cellular distribution, affinity for IgG 84 subclasses, and the signaling pathways they activate (21). In the context of DENV infection, FcγRs play a dual 85 In review 3 role: they can mediate protective immune clearance or contribute to ADE, depending on the antibody 86 characteristics and the receptor involved. Notably, FcγRIIIa (CD16a), expressed primarily on NK cells and some 87 myeloid populations, has been implicated in both beneficial effector functions such as antibody dependent cell-88 mediated cytoxicity (ADCC) and potentially in facilitating ADE under certain conditions (22, 23). Despite its 89 relevance, the dynamics of FcγRIIIa activation during acute dengue infection remain poorly understood. In this 90 study, we aim to characterize the FcγRIIIa-activating antibody profile in individuals with acute dengue infection, 91 evaluate how it relates to other antibody effector functions and compare it to the profile found in convalescent 92 patients. 93 2 Materials and Methods 94 2.1 Serum Samples 95 96 Two sets of serum samples were analyzed. A first set consisted of seven anonymous convalescent serum samples 97 (S) collected in Golfito and Puntarenas, which represented DENV hyperendemic regions in Costa Rica, for a 98 previous sero-epidemiological study during 2005-2006 (24). The second set of samples were collected from 99 seven acute dengue adult patients with follow-up serial sample collections (table 1). All sera were collected 100 from non-severe dengue cases. Previous exposure to DENV infection was assessed with IgG detection in the 101 acute sample with a commercial ELISA. Individuals with IgG antibodies against DENV during acute infection 102 were categorized as non-primary infection (NP) and those where antibodies were not detected were classified 103 as primary infection (P). Ethical approval for the use of human samples was given to the project B7360 in the 104 resolution VI-3178-2017 by the Scientific Ethical Committee from the Vice rectory of Research of the 105 University of Costa Rica. 106 2.2 Anti-DENV IgG and IgM detection 107 To detect IgG and IgM antibodies against DENV, two highly sensitive commercial ELISA kits were used (26): 108 the Human Dengue IgG ELISA Test Kit (Diagnostic Automation, Cortez Diagnostics Inc., CA, USA) with 109 94.7% sensitivity and 97.4% specificity, and the Human Dengue IgM ELISA Test Kit (Diagnostic Automation, 110 Cortez Diagnostics Inc., CA, USA) with 97.8% sensitivity and 93.5% specificity. Both assays were performed 111 following the manufacturer's protocol. Optical density (OD) values were measured after a 25-minute reading at 112 450 nm and 630 nm using the Epoch spectrophotometer (BioTek, Vermont, USA). 113 114 2.3 Molecular detection and serotyping of DENV 115 116 Viral RNA was extracted from 200 μl of serum or urine using the MagNA Pure LC RNA Isolation Kit I (Roche, 117 Basel, Switzerland) according to the manufacturer's instructions, using the MagNA Pure LC 2.0 extraction 118 system (Roche, Basel, Switzerland). Detection and confirmation of DENV, ZIKV, and CHIKV were conducted 119 on RNA samples using real-time reverse transcription PCR (RT-PCR) with Modular Diagnostic Kits for 120 Dengue, Zika, and Chikungunya viruses, along with Multiplex RNA Master Mix on the LightCycler II (Roche, 121 Basel, Switzerland), following the manufacturer protocol. Dengue serotyping was carried out following the 122 protocol described by Lanciotti et al., using specific serotype controls (25). 123 124 2.4 Viral strains and cell lines 125 Dengue virus prototype strains all grown in the C6/36 cell line (ATCC® CRL-1660™ RRID:CVCLZ230), 126 donated by the Pedro Kourí Institute in Cuba, were used in the K562 (ATCC® CCL-243™ RRID:CVCL0004) 127 immune enhancement and FcγRIIIA–CD3ζ activation assays (26). The strains were DENV-1 Angola (12 128 passages), DENV-2 Jamaica (19 passages), DENV-3 Nicaragua (13 passages) and DENV-4 Dominica (16 129 passages). 130 For neutralization assays, chimeric viruses (ChimeriVax – DENV1, DENV2, DENV3 and DENV4) produced 131 by Sanofi Pasteur and grown in Vero cells (ATCC® CCL-81™ RRID:CVCL0059) were used (27). These 132 In review 4 This is a provisional file, not the final typeset article viruses are based on the yellow fever 17D vaccine backbone and express only the prM and E genes of each 133 DENV serotype, thereby assessing the neutralizing activity of antibodies directed against the major structural 134 antigens involved in viral entry. Using this approach restricts the readout to neutralization-relevant epitopes, 135 thereby minimizing contributions from other viral proteins. These viruses were donated by Sanofi Pasteur 136 through the CDC Arbovirus Reference Collection under a material transfer agreement (MTA). 137 138 2.5 Reporter cell BW:FcγRIII-ζ assay 139 140 The assay used to evaluate individual antibody-dependent activation of FcγRIII (CD16) involved co-culturing 141 antigen-bearing cells with BW5147 reporter cells that stably express chimeric FcγRIII-ζ chain receptors. These 142 receptors trigger mouse IL-2 production upon receptor crosslinking by immune-complexed IgG, provided the 143 opsonizing IgG is recognized by specific FcγR (26). This assay was standardized before in Corrales-Aguilar et 144 al (26). Briefly, to assess antibody-dependent activation of BW:FcγRIII-ζ reporter transfectants, Vero cells 145 where infected with 0.1 multiplicity of infection (MOI) of each DENV serotype for a 72-hour period, then virus 146 was inactivated by UV-light. After inactivation, mock-infected and virus-infected cells were incubated with 147 serial two-fold dilutions of human sera in D-MEM (Sigma-Aldrich, MO, USA). containing 10% (v/v) FCS 148 (Thermo Fisher Scientific, MA, EE.UU.) for 30 minutes at 37 °C in a 5% CO₂ atmosphere. Non-bound IgG was 149 removed by washing the cells three times with D-MEM containing 10% (v/v) FCS before co-culturing them 150 with 100 000 BW:FcγRIII-ζ reporter cells per well for 16 to 24 hours at 37 °C in a 5% CO₂ atmosphere in RPMI 151 medium (Thermo Fisher Scientific, MA, EE.UU.) supplemented with 10% (v/v) FCS. Unless otherwise noted, 152 experiments were conducted in triplicate with a MOI of 0.1. After the 16 to 24-hour co-cultivation, supernatants 153 were diluted 1:2 in ELISA sample buffer (PBS with 10% [v/v] FCS and 0.1% [v/v] Tween-20). Mouse IL-2 154 levels were then measured by ELISA using the capture antibody JES6-1A12 and the biotinylated detection 155 antibody JES6-5H4 (BD Pharmingen™, Erembodegem, Belgium. RRID:AB2067783 and RRID:AB2621654 156 respectively) following the manufacturer instructions. The cutoff point for result interpretation was calculated 157 by adding the mean of mIL-2 production in virus-free (or mock) cells and three standard deviations. Values 158 above this cutoff point were considered positive. The magnitude of IL-2 production was interpreted as an 159 indicator of the strength of receptor engagement by IgG–virus immune complexes. Higher IL-2 values reflect 160 more efficient crosslinking of FcγRIIIA (26, 28). 161 162 2.6 Focus reduction neutralization test (FRNT) 163 164 For the FRNT assay, ChimeriVax strains (YFV-DENV1, 2, 3, and 4), validated for viral neutralization studies 165 (29), were used. A focus-reduction microneutralization assay (FRNT) was performed in flat-bottom 96-well 166 plates (30). Serial two-fold dilutions of sera, starting at 1:40, were incubated for 1 hour at 37 °C with viral stocks, 167 adjusted to yield 30–200 foci per well in at least four wells. The mixture was then inoculated (50 μL/well) into 168 confluent Vero cell monolayers and incubated for an additional hour to allow viral adsorption. The adsorption 169 medium was replaced by 100 μL of 1.5% carboxymethylcellulose overlay medium to restrict infection. DENV-170 1, DENV-2, and DENV-3 were incubated at 37 °C for 48 hours, while DENV-4 was incubated for 24 hours. 171 Post-incubation, the overlay medium was removed, wells were washed with PBS (Thermo Fisher Scientific, 172 MA, EE.UU.) and fixed with 100 μL of cold methanol per well. Plates were stored at −20 °C for at least 24 173 hours. For focus visualization, immunostaining was performed using an anti-flavivirus group monoclonal 174 antibody 4G2 (GeneTex, CA, USA. RRID:AB3074294) (1:600 dilution) followed by a secondary anti-mouse 175 IgG antibody conjugated with peroxidase (1:600 dilution). The signal was developed using 3-amino-9-176 ethylcarbazole (AEC) substrate, incubated for 30 minutes at room temperature in darkness. Foci were imaged 177 using a stereoscope and manually counted with ImageJ software (RRID:SCR_003070). FRNT50 was 178 determined in Prism 10 (GraphPad, San Diego, CA, USA) by nonlinear regression, identifying the dilution that 179 reduced foci by 50% (FRNT50). High FRNT50 values indicate stronger neutralizing capacity against the tested 180 DENV serotype. 181 182 2.7 Antibody dependent enhancement test (ADE) 183 184 In review 5 This study used the semi-adherent K562 cell line, which constitutively expresses FcγRIIa (31), based on a 185 monolayer methodology (32). Plates were coated with fibronectin and 30 000 cells per well were added. Serial 186 dilutions of test sera were mixed with DENV serotypes at a MOI of 0.5 (DENV4) to 0.1 (other serotypes) and 187 incubated at 37°C for 24 (DENV-4) to 48 (other serotypes) hours. Post-incubation, cells were fixed, 188 immunostained with the 4G2 antibody and secondary anti-mouse peroxidase-conjugated antibodies and stained 189 with AEC to visualize infected cells as described before. Infected cells, identified by a precipitated brown color, 190 were observed under light microscopy, and the number of infected cells per 40X field was quantified using 191 ImageJ software. The percentage of infection for all serial dilutions was plotted, and the level of 192 immunopotentiation was determined based on the width of the curve. Samples that exhibited broad curves 193 against more than one DENV serotype were considered to have a high level of immunopotentiation as defined 194 in other studies (18). The magnitude of enhancement was interpreted based on the breadth and height of the 195 curve: narrow, low curves were considered low enhancement, whereas broad curves with high percentages of 196 infection across multiple dilutions indicated strong enhancement potential. 197 198 2.8 Statistical analysis 199 All assays were performed in triplicate unless otherwise indicated. Data are shown as individual values or as 200 mean ± standard deviation (SD). For the FcγRIIIA activation assay, the cutoff for a positive response was 201 defined as the mean IL-2 production of mock-infected cells plus three standard deviations. Neutralization titers 202 (FRNT50) were determined by nonlinear regression analysis using GraphPad Prism 10 (GraphPad Software, 203 San Diego, CA, USA). No formal hypothesis testing was performed due to the small sample size; instead, results 204 are presented descriptively to illustrate individual antibody profiles over time. 205 206 3 Results 207 3.1 Serological and Functional Characterization of samples 208 Serum samples were classified into two main groups based on clinical and serological criteria: past infections 209 and acute infections. The past infection group (S) consisted of asymptomatic individuals with serological 210 evidence of prior DENV exposure, while the acute infection group included laboratory-confirmed cases of active 211 dengue virus infection. Acute-phase samples were further subdivided into primary (P) and non-primary 212 infections (NP), based on the presence or absence of anti-DENV IgG within the first seven days following 213 symptom onset. The detection of IgG at this early stage was used as a proxy to distinguish primary infections 214 from those that were likely secondary or beyond. Due to limitations in discriminating between secondary and 215 tertiary or quaternary responses, all early IgG-positive acute cases were conservatively grouped as non-primary 216 (NP) infections. 217 218 The samples from past infections presented highly diverse profiles depending on IgG antibody concentration 219 measured as OD values. Samples with anti-DENV IgG optical density (OD) values below 0.500 displayed a 220 monotypic neutralization profile, showing serotype-specific activity restricted to either DENV-3 (Figure 1, S1) 221 or DENV-2 (Figure 1, S5).These specimens exhibited minimal ADE activity, revealed by the short breath of 222 the curves against the four serotypes, and failed to induce significant activation of the FcγRIIIA–CD3ζ receptor, 223 suggesting limited effector function in this group. 224 225 In contrast, samples with intermediate anti-DENV ELISA OD values (0.5–1.0) exhibited broader serotype 226 recognition, neutralizing two (Figure 1, S3 and S7) or three (Figure 1, S4) DENV serotypes. Moderate ADE 227 activity was observed across these samples. Notably, FcγRIIIA–CD3ζ activation was detected exclusively in 228 S4. Interestingly, despite having the highest neutralizing titer against DENV-3, the strongest receptor activation 229 in S4 was induced by DENV-1, highlighting a potential uncoupling between neutralization capacity and Fc-230 mediated effector activation. 231 232 Only two samples exhibited high anti-DENV IgG OD values (>1.0). Both (Figure 1, S2 and S6) neutralized 233 three serotypes and displayed the highest levels of ADE and FcγRIIIA–CD3ζ activation among all specimens 234 In review 6 This is a provisional file, not the final typeset article analyzed from the past infection cohort. S2 showed peak FcγRIIIA activation in response to DENV-4, with 235 neutralization strongest against DENV-1. In contrast, in S6 the strongest receptor activation occurred in response 236 to DENV-1, while the highest neutralization titer targeted DENV-3. These findings underscore the complex 237 relationships among antibody specificity, enhancement potential, and Fc-mediated effector functions following 238 natural DENV exposure. 239 240 3.2 Longitudinal Analysis of Serum Samples from Acute DENV Infections 241 242 In the longitudinal study, the values for all antibody characterization assays for each patient were plotted across 243 all collected samples (T1-T4) (Figure 2). In primary DENV infections (Figure 2, P1 and P2), the immune 244 response followed classical kinetics, marked by the induction of anti-DENV IgG and a progressive increase in 245 functional activity. Neutralization peaked at T3 timepoint, with strong titers against the infecting serotype 246 (DENV-3). ADE activity rose during the T2 timepoints, with moderate levels persisting in the T3 subacute 247 phase. FcγRIIIA–CD3ζ activation was largely absent, except for a minimal, above-threshold response to DENV-248 4 in P2 at T3. 249 250 In non-primary infections (NP), antibody dynamics were more heterogeneous. In all cases, the infecting serotype 251 was DENV-1. Patient NP2 showed detectable IgG during the acute phase, but without measurable neutralizing 252 activity. By T2, neutralization peaked against DENV-2, and ADE activity increased notably. A low but 253 detectable FcγRIIIA–CD3ζ activation signal was recorded in T3 against DENV-4 (Figure 2, NP2). For NP3, 254 all measured antibody activities—including neutralization, ADE, and FcγRIIIA–CD3ζ activation—peaked at 255 T2 and declined by T3 timepoint. Neutralizing responses were strongest against DENV-2 across all timepoints, 256 followed by DENV-1, suggesting that DENV-2 was likely the priming serotype. FcγRIIIA activation in this case 257 was restricted to DENV-4, which peaked at T2 and decrease by T3 (Figure 2, NP3). 258 259 NP5 was the only case with a fourth sample collected nearly five years post-infection (Figure 2, NP5). The 260 acute-phase sample (T1) showed the highest IgG OD value among all evaluated samples, along with the 261 strongest neutralizing response against DENV-2, followed by DENV-1, supporting DENV-2 as the primary 262 infecting serotype. FcγRIIIA–CD3ζ activation was significant for DENV-1, DENV-2, and DENV-3, with 263 DENV-1 showing the highest signal from T1. An elevated enhancing activity is seen for all serotypes in all time 264 points. It should be noted that in this case the acute sample had a broad neutralizing activity, recognizing all four 265 serotypes. Although antibody function remained relatively high through T3, all profiles declined markedly by 266 T4. 267 268 Patient NP4 showed persistently high IgG OD values and a broad neutralization activity across all timepoints, 269 being the strongest against DENV-2, suggesting this serotype as the primary exposure. ADE activity increased 270 over time, and FcγRIIIA–CD3ζ activation was pronounced against DENV-1 from the acute phase through T3. 271 A secondary, though significant, activation signal was also observed for DENV-4 (Figure 2, NP4). In the case 272 of NP1, the neutralization profile also pointed to DENV-2 as the primary infecting serotype. Enhancing activity 273 was initially low but increased by T2. FcγRIIIA–CD3ζ activation was undetectable in the acute phase, but 274 increased significantly in the subacute sample, particularly in response to DENV-4, DENV-3, and DENV-2. 275 276 Collectively, these findings highlight the dynamic and individualized nature of DENV-specific antibody 277 responses following natural infection. Primary infections showed a more predictable trajectory of rising 278 neutralization and ADE activity, with minimal detection of FcγRIIIA activation. In contrast, non-primary 279 infections were characterized by broader serotype recognition, variable neutralization targets, and a more 280 prominent engagement of FcγRIIIA-mediated triggering. Notably, patients NP4 and NP5—who exhibited the 281 broadest neutralization profiles—were also the only individuals with the detectable FcγRIIIA activation against 282 the infecting serotype (DENV-1) with peaks at relatively late time-points (T3) after symptom onset. 283 284 4 Discussion 285 In this pilot study, we determined for the first time distinct antibody effector functions and profiles by ELISA, 286 FRNT, ADE test and FcγRIIIA activation assay across different immunological contexts of DENV 1-4 infection. 287 In review 7 Notably, in most cases, FcγRIIIA–CD3ζ activation did not consistently correlate with neutralization profiles, 288 one explanation may be that the epitopes driving neutralization differ from those responsible for Fc-mediated 289 functions (28). Neutralization is typically mediated by antibodies targeting structurally critical regions on the 290 virion, such as quaternary epitopes recognizing multiple envelope (E) protein subunits or serotype-specific sites 291 on the E protein domain III (33). By contrast, robust FcγRIIIA activation often arises from highly cross-reactive 292 IgG antibodies against conserved epitopes that confer little DENV neutralization. Notably, many human anti-293 DENV antibodies dominantly target the precursor membrane (prM) protein and the conserved fusion-loop of E 294 domain II; these antibodies are broadly cross-reactive among serotypes yet poorly neutralizing, even at high 295 concentrations, and can still efficiently opsonize infected cells and virions, triggering FcγRIIIA (17, 33). Indeed, 296 the FcγRIIIA activation assay of this study utilized DENV-infected Vero cells that display both E and uncleaved 297 prM on their surface, providing abundant targets for Fc binding in comparison to neutralization assay (34). In 298 summary, the antigenic determinants of neutralization versus FcγRIIIA-mediated effector function only partially 299 overlap, leading to an uncoupling dissection of these profiles in many samples. 300 Past infection data likely reflect a range of diverse time points post DENV infection (Figure 1). Samples with 301 broader serotype reactivity and enhanced Fc-mediated function are consistent with non-primary infections or 302 specimens taken within two years after exposure, when cross-reactive antibodies remain elevated (35). 303 Longitudinal analysis of acute cases provides a clearer view on the kinetics of the humoral response and its 304 associated effector functions. Individuals with secondary or multiple infections exhibited notably stronger 305 FcγRIIIA–CD3ζ activation compared to primary cases. This increased activity reflects not only higher antibody 306 titers but also qualitative differences in the IgG response, possibly due to subclass distribution and Fcγ N297 307 glycosylation pattern as demonstrated in COVID-19 patients (36-38). DENV infection predominantly induces 308 IgG1 and IgG3, both capable of engaging FcγRIIIA. IgG3 is short-lived and more potently neutralizing, while 309 IgG1 is longer-lasting and subject to glycan modification (22). It has been described that afucosylation of IgG1 310 is more prominent in dengue secondary infections, and that elevated levels of afucosylated anti-E IgG1 are 311 present early on severe dengue (22). Afucosylation significantly enhances FcγRIIIA binding (22) which may 312 explain the difference observed between P and NP individuals. 313 314 Analysis of past infection samples revealed a consistent association between the breadth of serotype recognition 315 by neutralization and the magnitude of FcγRIIIA-mediated effector activity. In acute infections, broadly 316 neutralizing sera, typically from those with non-primary infection, tended to activate FcγRIIIA across multiple 317 serotypes more robustly than narrow, type-specific sera. A broader neutralization profile implies a more 318 extensive distribution of IgG bound to diverse epitopes on the virion surface or the infected cells membrane, 319 thereby increasing the valency, defined as the multivalent engagement of antibodies with multiple epitopes, and 320 the density of immune complexes (22). This configuration enhances the odds of cross-linking of FcγRIIIA on 321 effector cells, a prerequisite for efficient receptor signaling (39). This finding is consistent with the concept that 322 a minimum concentration and opsonization density of IgG must be achieved to overcome the activation 323 threshold of FcγRIIIA. Prior studies of dengue immunity have noted that intermediate antibody levels can 324 exacerbate infection (via ADE), but sufficiently high antibody levels confer protection (40, 41). Analogously, 325 only the samples with high IgG binding levels were potent in FcγRIIIA triggering, whereas those with modest 326 titers did not (23, 42). Thus, a higher abundance and breadth of antibodies likely ensures that FcγRIIIA is 327 engaged in antiviral effector functions rather than in enhancing pathways. 328 329 The longitudinal FcγRIIIA activation profiles observed in individuals NP5 and NP4 provide valuable insight 330 into the dynamics of Fc-mediated antibody responses during acute dengue infection. In both cases, a marked 331 FcγRIIIA/CD16 activation signal was detected in response to the infecting serotype (DENV-1) during the acute 332 phase, indicating the presence of FcγRIIIA-activating IgG early in infection. Interestingly, both individuals 333 exhibited a transitory decline in activation at the T2 timepoint, followed by a peak in T3. This transient reduction 334 may reflect in vivo engagement of FcγRIIIA-expressing effector cells, such as natural killer (NK) cells or 335 monocytes, by IgG-virus immune complexes, leading to ADCC or phagocytosis and temporary clearance of 336 activating antibodies in immune complexes (22, 23, 42, 43). The increased CD16 activation signal observed in 337 T3 may be due to clonal expansion against the epitopes recognized in the acute infection (44). Notably, while 338 both individuals shared similar FcγRIIIA activation kinetics against the infecting serotype, they differed in their 339 In review 8 This is a provisional file, not the final typeset article ADE profile: NP5 displayed high ADE in acute sample, while NP4 did not. This immune assessment enabled 340 the distinction between FcγRIIIA-activating antibody profiles with low enhancing potential and those with 341 strong enhancing activity. 342 343 Our study focused exclusively on FcγRIIIA activation profile, which does not capture the full range of FcγR-344 mediated effector mechanisms. Furthermore, FcγR polymorphisms such as FcγRIIA-H131R and FcγRIIIA-345 V158F, which affect the affinity of Fcγ receptors for IgG subclasses, have been associated with increased 346 susceptibility and protection against severe dengue, respectively (45, 46). Therefore, a broader approach 347 incorporating additional FcγRs, and their key polymorphic variants, along with FcγR reporter cell assay settings 348 selective for certain ligands including soluble multimeric immune complexes and C reactive Protein isoforms 349 (47, 48), should be undertaken to evaluate the full effector potential of dengue-specific antibodies and to identify 350 thresholds that help define the spectrum of clinical outcomes from DENV infection. The hyperendemic setting 351 in Costa Rica, where multiple flaviviruses co-circulate, highlights the need for a broader viral panel to better 352 interpret antibody profiles. This would allow for the inclusion of both severe and non-severe patients. (2). 353 Increasing the number of patients, outcomes of DENV-disease, and timepoints during the early acute and 354 convalescent phases would provide a more detailed understanding of how FcγR activation evolves. This would 355 also help to clarify its complex role in the dual nature of the humoral response in dengue infection. 356 357 Recent studies highlight the dual impact of FcγRIIIA interactions: afucosylated IgG1 enhancing FcγRIIIA 358 binding has been linked to severe dengue (23, 42, 49, 50), dengue immune complexes can activate NK cells and 359 suppress ADE (51), and stronger FcγRIIIA-driven effector functions, including NK activation, associate with 360 protection from symptomatic infection (22). While NK cell–based assays are highly informative to evaluate the 361 protective role of CD16-activating antibodies, our reporter system allows the measurement of the broader 362 fraction of antibodies capable of engaging FcγRIIIA, including those that may also contribute to 363 immunopathogenic outcomes, since FcγRIIIA expression is not restricted to NK cells but includes monocytes 364 implicated in infection and inflammation (52). This distinction provides a complementary view, revealing 365 potentially different functional profiles of dengue antibodies. Additionally, Kao et al recently revealed that CD8 366 T cells, which typically do not express Fcγ receptors, can specifically induce the activating FcγRIIIa receptor in 367 response to viral infections like COVID-19 and dengue (53). While FcγRIIIa expression closely follows the 368 immune response timeline, its activation alone does not trigger CD8 T cell function; however, it synergizes with 369 T cell receptor (TCR) stimulation to enhance activation (53). These findings uncover a novel costimulatory role 370 for FcγRIIIa, showing how virus-induced antibodies can modulate CD8 T cell responses. By providing a scalable 371 and reproducible way to measure FcγRIIIA engagement beyond natural killer and CD8 T cell functions, our 372 assay offers a novel framework to characterize the balance between protective and pathogenic antibody 373 responses. 374 375 Taken together, our data shows that neutralization and FcγRIIIA-mediated antibody functions against Dengue 376 viruses are often uncoupled which has already been observed with other viral infections before (28). 377 Furthermore, the different epitopes involved in each process may lead to distinct antibody functional profiles. 378 Cross-reactive antibodies (e.g., anti-prM, fusion-loop) may not neutralize dengue virus effectively but still 379 trigger immune effector mechanisms via Fc receptors. To better understand how antibody effector mechanisms 380 and Fc-mediated immunity influence dengue outcomes, different FcγRs and their polymorphisms, distinct 381 immune complex forms, more patients and defined timepoints of sampling should be studied. Our foremost 382 rationale for using these tests will be to evaluate the functional quality of antibodies, especially cross-reactive 383 ones, during different phases of dengue infection (acute and post-acute). This may help elucidate their dual role 384 in both protection and immunopathogenesis, improving our understanding of disease progression and immune 385 responses, and potentially guiding vaccine development by distinguishing between protective and pathogenic 386 antibody profiles. 387 388 4 Conflict of Interest 389 In review 9 The authors declare that the research was conducted in the absence of any commercial or financial 390 relationships that could be construed as a potential conflict of interest. 391 5 Author Contributions 392 CSG: Methodology, Investigation, Data curation, Formal analysis, Validation, Visualization, Writing-393 Original draft, Writing-Review & Editing 394 TM: Methodology, Investigation, Data curation, Formal analysis, Validation, Visualization, Writing-395 Original draft, Writing-Review & Editing 396 HH: Investigation, Conceptualization, Resources, Formal analysis, Writing-Review & Editing 397 ECA: Funding acquisition, Conceptualization, Formal analysis, Project administration, Supervision, 398 Writing-Original draft, Writing-Review & Editing 399 6 Funding 400 Funding for the project (except for publication fees) was obtained from the University of Costa Rica 401 (Grant numbers B7360, B7331, and ED-3257) 402 7 Acknowledgments 403 The authors wish to thank the serum donors for their invaluable contribution to this study. 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Proc Natl Acad Sci U S A. 546 2025;122(27):e2509016122. 547 548 549 550 551 552 553 554 555 In review 13 12 Table and Figure Legends 556 Table 1. List of samples from individuals with a confirmed acute infection and with sequential sample collection 557 at different time points (T1-T4), including the number of days post-symptom onset for sample collection. NP: 558 non-primary infection; P: primary infection. 559 560 ID Infecting DENV Serotype Days post-symptoms onset Sample 1 Sample 2 Sample 3 Sample 4 (T1) (T2) (T3) (T4) P1 DENV-3 3 14 82 - P2 DENV-3 2 13 81 - NP1 DENV-1 2 17 - - NP2 DENV-1 4 21 - - NP3 DENV-1 4 33 127 - NP4 DENV-1 6 36 116 - NP5 DENV-1 2 11 48 1719 561 562 Figure 1. Anti-DENV antibody profile of seven participants from a serosurvey. The ELISA OD values for IgG 563 detection are indicated below each participant code. NT: Neutralization profile; ADE: Antibody-dependent 564 enhancement profile; BW:CD16: FcγRIIIA activation profile. For FcγRIIIA activation, data points represent the 565 mean of three independent experiments ± standard deviation (SD), and the cutoff for a positive response (dotted 566 line) was defined as the mean IL-2 production of mock-infected cells plus three standard deviations. 567 Neutralization titers (FRNT50) were determined by nonlinear regression analysis. Negative controls included 568 mock-infected cells (for FcγRIIIA assay) and seronegative human sera (for ELISA, ADE, and FRNT assays). 569 Figure 2. Anti-DENV antibody profile of seven dengue patients with sequential samples collected at different 570 time points (T1–T4) post-symptom onset. Infecting serotype is indicated below each patient. ELISA: anti-DENV 571 IgG and IgM profile; NT: Neutralization profile; ADE: Antibody-dependent enhancement profile; BW:CD16: 572 FcγRIIIA activation profile. For FcγRIIIA activation, data points represent the mean of three independent 573 experiments ± standard deviation (SD), and the cutoff for a positive response (dotted line) was defined as the 574 mean IL-2 production of mock-infected cells plus three standard deviations. Neutralization titers (FRNT50) 575 were determined by nonlinear regression analysis. Negative controls included mock-infected cells (for FcγRIIIA 576 assay) and seronegative human sera (for ELISA, ADE, and FRNT assays). 577 578 579 580 In review Figure 1.JPEG In review Figure 2.JPEG In review