24 INTRODUCTION Dengue fever is a major public health issue faced in many countries, especially in tropical and subtropical areas of the world (1). More than 390 million cases of dengue virus (DENV) infection have been estimated to occur annually (2). Dengvaxia® (CYD-TDV) is the first dengue vaccine that has been approved and made available in some countries. However, uneven stimulation of the immune response to four dengue virus serotypes (DENV-1, -2, -3, or -4) limits the application of this vaccine (3). Infection with any of the four serotypes results in symptoms ranging from mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Secondary infection with a different serotype is considered as a major risk factor of severe dengue disease that is caused due to antibody-dependent enhancement (ADE) (4,5). DENV is a positive-sense single-stranded RNA virus that belongs to the genus Flavivirus in the family Flaviviridae. DENV genome encodes three structural proteins, including capsid [C], pre-membrane [prM], and envelope [E], and seven nonstructural proteins, including NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. NS1 is expressed in the host cytosol as a monomer, and present at the cell membrane as a dimer, and as a hexamer in the secreted form (6). NS1 is known to be immunogenic for the induction of humoral immune response to DENV infection (7). High levels of secreted NS1 (sNS1) have been detected in the sera of patients in the early stage of DENV infection (8), and it is produced at high levels during the secondary infections (9). The autoimmune efects induced by anti- NS1 antibodies (Abs) contribute to the pathogenesis of severe DF (10,11). Several studies have demonstrated that anti-NS1 Abs in the sera of patients with DF or mouse anti-NS1 monoclonal antibodies (MAbs) exhibit cross-reactivity with platelets and endothelial cells, thereby resulting in platelet dysfunction and endothelial cell damage (12,13). Moreover, in patients with severe DF, anti-NS1 Abs are suggested to be the causative agents of the hemostatic imbalance that may contribute to the hemorrhagic status of patients with DHF/DSS. Nonetheless, the activity of anti-NS1 Abs that confer protection against DENV infection has been previously reported using a mouse model (14,15). Since most of the reported anti-NS1 Abs were based on mouse or human polyclonal Abs, in this study, we characterized nine anti- NS1 human monoclonal antibodies (HuMAbs) isolated from patients infected with DENV and investigated their cross-reactivity against human plasminogen, thrombin, and human microvascular endothelial cells (HMEC-1). MATERIALS AND METHODS Viruses and cells: DENV-1 (Mochizuki strain), DENV-2 (New Guinea C or NGC strain), DENV-3 (H87 Original Article Characterization of Human Anti-Dengue NS1 Monoclonal Antibodies Derived from Patients Infected with DENV-2 Siriporn Kowaboot 1 , Wilarat Puangmanee 1 , Surachet Benjathummarak 2 , Khwanchit Boonha 2 , Urai Chaisri 3 , Pongrama Ramasoota 1,2 , and Pannamthip Pitaksajjakul 1,2 * 1 Department of Social and Environmental Medicine, 2 Center of Excellence for Antibody Research, and 3 Department of Tropical Pathology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand ABSTRACT: Mouse antibodies specifc to dengue NS1 have been widely investigated for their cross- reactivity with several human biomolecules. This is the frst study demonstrating the cross-reactivity of human monoclonal antibodies (HuMAbs) specifc to dengue NS1 isolated from patients infected with dengue virus serotype-2 (DENV-2). Nine anti-NS1 HuMAbs, which were mainly derived from patients in convalescent-phase after secondary infection of DENV-2, were characterized. Their cross-reactivity with plasminogen, thrombin, and endothelial cells was investigated, following which plasmin-formation assays were performed. All anti-NS1 HuMAbs exhibited cross-reactivity with human plasminogen (Plg), but not with thrombin or endothelial cells. Moreover, all HuMAbs exhibiting cross-reactivity with Plg converted Plg to plasmin in the plasmin-formation assay. These results suggest the implications and drawbacks of using anti-NS1 antibodies in immunotherapy. Received January 4, 2021. Accepted May 12, 2021. J-STAGE Advance Publication May 31, 2021. DOI: 10.7883/yoken.JJID.2020.1071 *Corresponding author: Mailing address: Faculty of Tropical Medicine, Mahidol University, 420/6 Ratchawithi Road, Ratchadewee, Bangkok 10400, Thailand. Tel: +66 0 2306 9100-10 ext. 1564, Fax: +66 0 2306 9179, E-mail: pannamthip.pit@mahidol.ac.th Jpn. J. Infect. Dis., 75, 24-30, 2022