Dengue is a major global health burden caused by infection with one of the four dengue virus (DENV) serotypes, which belong to the Flaviviridae family. Most DENV infections are asymptomatic; however, a subset of patients develops severe disease characterized by hemorrhage, plasma leakage, thrombocytopenia, and, in extreme cases, shock and death. To facilitate early recognition and better clinical management, the World Health Organization (WHO) introduced a revised dengue severity classification system in 2009, categorizing infections into Dengue without warning signs (DOS), Dengue with warning signs (DWS), and Severe Dengue (SD) (WHO, 2009).

DENV is an enveloped, positive-sense RNA virus with a ∼10.9 kb genome encoding three structural proteins—capsid (C), envelope (E), and membrane (M)—and seven non-structural proteins—NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. The mature virion comprises the capsid protein enclosing the viral genome and the M and E proteins embedded in the lipid envelope ((Rani et al., 2024; Rodenhuis-Zybert et al., 2010). Among these, the envelope (E) protein is the principal target of neutralizing antibody responses, forming the basis for most dengue vaccine strategies. One of the leading hypotheses explaining dengue pathogenesis is antibody-dependent enhancement (ADE), wherein secondary infection with a heterologous DENV serotype exacerbates disease severity. Cross-reactive but subneutralizing antibodies generated from a previous infection can bind to the new infecting serotype and promote viral entry into Fcγ receptor-bearing cells, enhancing viral replication (Katzelnick et al., 2017, Shukla et al., 2020).

Among the non-structural proteins, NS1 is of particular interest. It exists as a monomer intracellularly, dimerizes in the endoplasmic reticulum (ER), and is secreted as a hexamer. NS1 is the only non-structural protein secreted into the bloodstream, making it a valuable diagnostic marker during the acute phase of infection ((Muller and Young, 2013). Within host cells, NS1 plays an essential role in viral replication (Gutsche et al., 2011). Structurally, NS1 is a 352-amino-acid glycoprotein comprising three domains: the N-terminal β-roll domain (1–29 aa), the wing domain (30–180 aa), and the β-ladder domain (181–352 aa). High-resolution crystallographic analyses have shown that NS1 is a multi-domain protein comprising: an N-terminal β-roll domain (involved in dimerization and membrane association), a wing domain (implicated in immune modulation and host interactions), and a C-terminal β-ladder domain (critical for oligomerization, secretion, and pathogenic functions) (Akey et al., 2014, Edeling et al., 2014).

Recent studies have revealed that NS1 directly contributes to dengue pathogenesis. Secreted NS1 can disrupt the endothelial glycocalyx, inducing vascular hyperpermeability by upregulating sialidases, heparanases, and cathepsin L (Puerta-Guardo et al., 2016). It can also activate Toll-like receptor 4 (TLR4), leading to proinflammatory cytokine secretion and increased endothelial permeability (Chao et al., 2019). Additionally, NS1 interacts with complement components, facilitating immune evasion by inhibiting complement-mediated clearance (Conde et al., 2016a).

While antibody responses in dengue infection have traditionally focused on structural proteins such as the E protein, not all E-specific antibodies are protective. Some epitopes induce non-neutralizing antibodies capable of mediating ADE, meaning that E-specific responses do not consistently correlate with viral load or disease severity (Katzelnick et al., 2017, Pathak et al., 2024). In contrast, NS1 represents a unique target for immune responses since it is not a component of the mature virion, thereby eliminating the risk of ADE. NS1 is abundantly secreted during the acute phase and directly contributes to endothelial dysfunction and vascular leakage, key features of severe dengue. Antibodies against NS1 may confer protection by neutralizing its pathogenic effects, as shown in several in vitro and mouse studies ((Lai et al., 2017; Sanchez-Vargas et al., 2024).

However, the role of NS1 antibodies remains controversial. Some studies report that NS1-specific antibodies cross-react with host proteins, including platelet and coagulation components such as plasminogen and thrombin, potentially leading to endothelial cell apoptosis and contributing to vascular damage (Cheng et al., 2009, Sun et al., 2007). Thus, the exact contribution of NS1 antibodies to disease protection versus pathogenesis is still unclear.

Despite several animal studies demonstrating the protective potential of NS1-targeted immunity, human data on NS1-specific antibody responses and their correlation with disease severity remain limited. The present study aims to delineate domain-specific NS1 antibody profiles in pediatric dengue patients and to examine their association with clinical outcomes. Identifying such immunological correlates of protection or pathogenesis may provide predictive biomarkers for disease progression and inform the development of safer, more effective dengue vaccines.