PLoS ONE plos plosone PLOS ONE 1932-6203 Public Library of Science San Francisco, CA USA 10.1371/journal.pone.0285878 PONE-D-22-24631 Research Article Research and analysis methodsImmunologic techniquesImmunoassaysEnzyme-linked immunoassays Biology and life sciencesOrganismsVirusesRNA virusesFlavivirusesDengue virus Biology and life sciencesMicrobiologyMedical microbiologyMicrobial pathogensViral pathogensFlavivirusesDengue virus Medicine and health sciencesPathology and laboratory medicinePathogensMicrobial pathogensViral pathogensFlavivirusesDengue virus Biology and life sciencesOrganismsVirusesViral pathogensFlavivirusesDengue virus Medicine and health sciencesMedical conditionsTropical diseasesNeglected tropical diseasesDengue fever Medicine and health sciencesMedical conditionsInfectious diseasesViral diseasesDengue fever Biology and life sciencesPhysiologyImmune physiologyAntibodies Biology and life sciencesImmunologyImmune system proteinsAntibodies Medicine and health sciencesImmunologyImmune system proteinsAntibodies Biology and life sciencesBiochemistryProteinsImmune system proteinsAntibodies Engineering and technologyEquipmentDetectorsBiosensors Biology and life sciencesOrganismsVirusesRNA virusesFlavivirusesZika virus Biology and life sciencesMicrobiologyMedical microbiologyMicrobial pathogensViral pathogensFlavivirusesZika virus Medicine and health sciencesPathology and laboratory medicinePathogensMicrobial pathogensViral pathogensFlavivirusesZika virus Biology and life sciencesOrganismsVirusesViral pathogensFlavivirusesZika virus Biology and life sciencesAnatomyBody fluidsBloodBlood plasma Medicine and health sciencesAnatomyBody fluidsBloodBlood plasma Biology and life sciencesPhysiologyBody fluidsBloodBlood plasma Physical sciencesMaterials scienceMaterialsOligomersDimers Physical sciencesChemistryPolymer chemistryDimers A nonstructural protein 1 capture enzyme-linked immunosorbent assay specific for dengue viruses NS1 capture ELISA for Dengue viruses https://orcid.org/0000-0003-4401-1265 Lim Pei-Yin Conceptualization Data curation Formal analysis Investigation Methodology Supervision Validation Writing – original draft Writing – review & editing 1 ¤a Ramapraba Appanna Conceptualization Data curation Formal analysis Methodology Writing – review & editing 1 ¤b Loy Thomas Data curation Formal analysis Methodology Writing – review & editing 1 ¤c Rouers Angeline Data curation Formal analysis Methodology Writing – review & editing 1 ¤c Thein Tun-Linn Data curation Formal analysis Methodology Writing – review & editing 2 Leo Yee-Sin Data curation Methodology Writing – review & editing 2 Burton Dennis R. Resources Writing – review & editing 3 Fink Katja Conceptualization Funding acquisition Supervision Writing – review & editing 1 ¤d Wang Cheng-I Supervision Writing – review & editing 1 * Singapore Immunology Network, Agency for Science, Technology and Research, Singapore, Singapore National Centre for Infectious Diseases, Singapore, Singapore Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, United States of America Henry Kevin A. Editor National Research Council Canada, CANADA

The authors have declared that no competing interests exist.

Current address: Hilleman Laboratories Pte Ltd, Singapore, Singapore

Current address: Department of Medicine, Surgery & Dentistry, University of Salerno, Baronissi (Salerno), Italy

Current address: A*STAR Infectious Disease Labs, Agency for Science, Technology and Research, Singapore, Singapore

Current address: ImmunoScape Pte Ltd, Singapore, Singapore

 * E-mail: Wang_ChengI@immunol.a-star.edu.sg 18 5 2023 2023 18 5 e0285878 4 9 2022 3 5 2023 2023 Lim et al This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Dengue non-structural protein (NS1) is an important diagnostic marker during the acute phase of infection. Because NS1 is partially conserved across the flaviviruses, a highly specific DENV NS-1 diagnostic test is needed to differentiate dengue infection from Zika virus (ZIKV) infection. In this study, we characterized three newly isolated antibodies against NS1 (A2, D6 and D8) from a dengue-infected patient and a previously published human anti-NS1 antibody (Den3). All four antibodies recognized multimeric forms of NS1 from different serotypes. A2 bound to NS1 from DENV-1, -2, and -3, D6 bound to NS1 from DENV-1, -2, and -4, and D8 and Den3 interacted with NS1 from all four dengue serotypes. Using a competition ELISA, we found that A2 and D6 bound to overlapping epitopes on NS1 whereas D8 recognized an epitope distinct from A2 and D6. In addition, we developed a capture ELISA that specifically detected NS1 from dengue viruses, but not ZIKV, using Den3 as the capture antibody and D8 as the detecting antibody. This assay detected NS1 from all the tested dengue virus strains and dengue-infected patients. In conclusion, we established a dengue-specific capture ELISA using human antibodies against NS1. This assay has the potential to be developed as a point-of-care diagnostic tool.

 SIgN Core grant Fink Katja This work was supported by SIgN Core grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability All relevant data are within the paper and its Supporting Information files. Introduction

Dengue is a global health problem affecting 129 countries, with 3.9 billion people at risk of infections. In 2019, 5.2 million cases of dengue infection were reported to WHO [1]. Infection is caused by the dengue virus (DENV), a mosquito-borne flavivirus consisting of four serotypes (DENV-1 to -4). Infection causes a spectrum of diseases, ranging from asymptomatic, flu-like symptoms to fatal complications involving increased vascular permeability that could result in fluid loss and organ dysfunction. Other mosquito-borne flaviviruses that are closely related to DENV and can cause serious disease include Zika virus (ZIKV), Yellow fever virus, West Nile virus, and Japanese encephalitis virus.

Dengue non-structural protein-1 (NS1) is a 48–50 kDa protein that is 40–70% conserved across the flaviviruses [2]. NS1 is translated from the viral RNA as part of a polyprotein, cleaved into monomers, glycosylated, and then dimerized. In mammalian cells, the dimeric form of NS1 is further processed and then secreted from infected cells as soluble hexamers and potentially other multimers [3]. During the acute phase of infection, NS1 is an important diagnostic marker, especially for the use at point-of-care, because it can be detected in the blood (reviewed by [4]). NS1 has also been shown to play important roles during virus pathogenesis. Severe dengue illness has been associated with high levels of circulating NS1 in the plasma of dengue infected patients [5, 6]. In vitro and animal studies suggest that DENV NS1 causes plasma leakage through disrupting the tight junction or inducing glycocalyx degradations (reviewed by [7]). In addition, antibodies against NS1 have been shown to cross react with host proteins through molecular mimicry, leading to tissue damage, thrombocytopenia, and coagulopathy (reviewed by [8]).

Enzyme-linked immunoassays to detect the presence of dengue NS1 in the blood are commercially available as dengue diagnostic tests and the performance of these assays has been tested by various groups. Depending on the study, the reported sensitivity of these test kits ranges from ~30% to 90%, depending on the serotype of infection [9–12]. In addition, the emergence of ZIKV, another flavivirus, in regions endemic for DENV infection, such as South America and South East Asia [13], has made the detection of DENV NS1 a challenge. Due to the sequence similarity of NS1 from ZIKV to all four serotypes of dengue viruses, the primary concern of using these diagnostic assays is an inability to differentiate between ZIKV and DENV infections. A highly specific dengue diagnostic test is therefore needed.

The goal of this study is to develop an NS1 capture enzyme-linked immunoassay that can positively detect all four dengue serotypes whilst providing a negative signal in the presence of Zika virus.

 Methods and materials Ethics statement

All studies involving human subjects have been reviewed by Institutional Review Board of Singapore National Healthcare Group Ethical Domain. Written informed consent was obtained from all participating human subjects and the data was fully anonymized.

 Sorting of NS1-specific B cells from DENV-positive patient

The study was approved by the Institutional Review Board of Singapore National Healthcare Group Ethical Domain (DSRB Ref: B/05/013). Written informed consent was obtained from the patient. Whole blood from a DENV-positive patient at convalescence phase (3–4 week after fever onset) was collected into CPT tubes (Becton Dickinson). The patient was confirmed DENV positive by DENV-specific RT-PCR, Panbio Dengue IgG Indirect ELISA and Panbio Dengue IgM capture ELISA (Inverness Medical, Queensland, Australia). Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by CPT tube (Becton Dickinson) purification and stored at -80°C until used. Thawed PBMC were labeled with antibodies against CD3-BV450 (BD Horizon, Clone#: UCHT1), CD19-BV605 (Pharmingen, Clone#: SJ25C1), CD27-PE (Pharmingen, Clone#: M-T271), CD45-V500 (BD Horizon, Clone#: H130), IgD-PECy7 (Biolegend, Clone#: IA6-2), and fluorescently labelled recombinant DENV2-NS1 antigen and DENV4-NS1 antigen (The Native Antigen Company). Recombinant NS1 was labeled in house with Alexa Fluor 488 (Alexa Fluor 488 Antibody labeling kit, ThermoFisher Scientific) and Alexa Fluor 647 (Alexa Fluor 647 Antibody Labeling kit, ThermoFisher Scientific), respectively following the manufacturer’s instruction. PBMCs were resuspended in sorting buffer (PBS, 2% FBS, 2 mM EDTA) after incubation with antibodies and antigen. Memory B cells that stained positive for DENV-2-NS1 and DENV-4-NS1 were sorted into 96-well PCR plates containing 10 μl/well of 10 mM Tris-HCL with 40 U/μl RNase inhibitor (Promega), placed on dry ice immediately, and then stored at -80°C.

 Ig cloning, expression, purification, and sequencing

Methods to clone and express the antibodies have been described previously [14]. Briefly, the human IgG heavy and light chains were amplified from mRNA of single B cells using One-step RT-PCR (Qiagen), the resulting RT-PCR products were used for nested PCR with primers including restriction sites. The PCR products were cloned into the pTT5 expression vector (National Research Council of Canada). The plasmids expressing the heavy and light chain (IgG1 format) were co-transfected into HEK293-6E cells using 293fectinTM (Thermo Fisher Scientific) as per manufacturer’s instructions with the following modifications. The transfected cells were cultured in FreeStyle F17 Expression Medium (ThermoFisher Scientific) supplemented with 0.5% (w/v) Tryptone N1. The supernatant was harvested 5 days post-transfection, and the antibodies were purified using Protein G beads (Merck Millipore).

The PCR products of the heavy and light chain variable regions from single B cells were sequenced by Sanger sequencing (1st Base, Singapore). The quality of the sequences was checked using CodonCode Aligner software, and the sequences were trimmed and further analyzed using the IMGT database (http://www.imgt.org/).

 Production and purification of Den3 antibody

Den3 antibodies were expressed in Chinese hamster ovary cells in glutamine-free custom formulated Glasgow minimum essential medium (MediaTech Cellgro) and purified using Protein A beads (Merck Millipore) as previously described [15, 16].

 Selection of NS1-specific antibodies by indirect ELISA

Recombinant NS1 proteins from DENV-1-Nauru/Western Pacific/1974 (Accession: M23027.1), DENV-2-Thailand/16681/84 (U87411.1), DENV-3-Sri Lanka D3/H/IMTSSA-SRI/2000/1266 (AY099336.1), DENV-4-Dominica/814669/1981 (AF326573.1), ZIKV-Suriname (AZS35340.1), and ZIKV-Uganda MR766 (AWF93629.1) produced in HEK293 cells were purchased from The Native Antigen Company (United Kingdom). Nunc Maxisorp plates (ThermoFisher Scientific) were coated with recombinant NS1 (50 μL/well of 2 μg/mL) in coating buffer (0.1M NaHCO3) overnight at 4°C. Wells were washed three times with PBST (PBS supplemented with 0.05% Tween) and blocked with PBS supplemented with 5% skim milk (Sigma-Aldrich) for 1 h at RT. After washing the wells once with PBST, purified antibodies (50 μL of 1μg/mL) was added into the wells for 2 h at RT. Wells were washed three times with PBST, and 50 μL/well of peroxidase-conjugated anti-human IgG (1/5000 dilution, Sigma Aldrich) or HRP-conjugated anti-rabbit IgG (H+L) (1/5000 dilution, Promega) was added for 2 h at RT. After the wells were washed 3X with PBST, 3,3’,5,5’-Tetramethylbenzidine (TMB) Liquid Substrate system for ELISA (50 μL/well, Sigma) was added for ~ 5minutes, and then 1M HCL was added. The OD of the wells were determined at 450nm and 650nm (reference OD).

 Selection of antibody pairs for NS1-capture ELISA

Nunc Maxisorp plates (ThermoFisher Scientific) were coated with purified antibodies at 1 μg/mL in coating buffer overnight at 4°C. Wells were washed three times with PBST and blocked with PBS supplemented with 5% skim milk (Sigma-Aldrich). After 1 h at RT, skim milk solution was removed and recombinant NS1 (50 μL of 2μg/mL) was added into the wells for 2 h at RT. Wells were washed three times with PBST, and 50 μL/well of peroxidase-conjugated purified antibodies (1/200 dilution, Sigma Aldrich) was added for 2h at RT. After the wells were washed 3X with PBST, TMB Liquid Substrate system for ELISA (50 μL/well, Sigma) was added for ~ 5minutes, and then 1M HCL was added. The OD of the wells were determined at 450nm and 650nm (reference OD). Purified antibodies were conjugated with peroxidase using the EZ-Link plus activated peroxidase kit (ThermoFisher Scientific) as per manufacturer’s instructions.

 Evaluation of the performance of the NS1-capture ELISA

NS1-capture ELISA was performed as described above using Den3 [16] as the capture antibodies and D8-HRP as the detecting antibodies. To determine if the NS1-capture ELISA could detect NS1 from different dengue strains/isolated, supernatant from Vero cells infected with different dengue viruses was added into the wells coated with Den3 antibodies. To determine the limit of detection of the assay, various concentrations of purified recombinant NS1 prepared in human plasma from dengue-negative patient were added into the Den3 coated wells and then the NS1-capture ELISA was performed as described above using D8-HRP as the detecting antibodies. The limit of detection (LOD) of the assay was defined as the concentration of NS1 with an OD value that was equal to the 2-fold the average OD of wells containing no NS1.

To detect NS1 from dengue-infected patient samples, NS1-capture ELISA was performed as described above, except that human plasma was added into the wells coated with antibodies, instead of purified recombinant NS1. Clinical specimen was considered positive for NS1 when its OD reading was greater than 2 times the OD of the negative plasma control in each plate [17].

 SDS-PAGE and Western blot analysis

Recombinant NS1 (final concentration at 0.03 μg/μL for DENV-1, -3, and -4, and 0.01 μg/μL for DENV-2) were prepared in PBS and 4X Laemmli Sample Buffer (Bio-Rad). The samples were divided into 2 tubes: (1) untreated and (2) incubated at 90°C for 10 min. The proteins were separated on an Any kD™ Mini-PROTEAN® TGX Stain-Free™ Protein Gels (Bio-Rad) and then transferred onto a Hybond PVDF membrane (0.45μm, Amersham). The membrane was incubated in PBS supplemented with 5% (w/v) skim milk overnight at 4°C, then purified antibodies at 1μg/mL for 2 h at RT. The membranes were washed 3 times with PBST, 5 minutes each, then peroxidase-conjugated anti-human IgG (1/10,000 dilution, Sigma Aldrich) was added for 1 h at RT. The membranes were washed three times with PBST, chemiluminescent substrates (WesternBright Sirius chemiluminescent Detection kit, Advansta) were added and then developed using BioRad ChemiDoc system. For SDS-PAGE, the gel was stained with InstantBlueTM (Expedeon).

 Biolayer light interferometry (BLI) analysis

Analysis of binding of antibodies to recombinant NS1 was performed using an Octet RED96 instrument (ForteBio; Pall Life Sciences) as described previously [18]. Purified antibodies A2, D6, D8, or Den3 (10μg/mL) were loaded onto anti-human IgG Fc Capture (AHC) Biosensors (ForteBio) for 200 sec. The biosensor tips were washed with binding buffer [PBS supplemented with 0.1% Tween and 0.1% (w/v) BSA] for 200 sec, then immersed into different concentrations of recombinant NS1 (0, 1.562, 3.125, 6.25, 12.5, 25, 50, 100 nM) for 200 sec (association), followed by a subsequent immersion in PBS for 600 sec (dissociation). Octet® Data Acquisition software was used for data acquisition, and Octet® Data Analysis software was used for affinity calculations. Briefly, the background signal for each antibody was the signal from the control sensors that were loaded with the specific antibody and incubated with 0 nM of recombinant NS1. Background signal was subtracted from all other results (biosensors loaded with antibodies, incubated with recombinant NS1 at 1.562, 3.125, 6.25, 12.5, 25, 50, 100 nM), the Y-axis was aligned to the association step (199.8 second), and then the association and dissociation constants were analyzed using the 1:1 model with global fitting. New biosensor tips were used for all experiments.

 Preparation of culture supernatant containing NS1 secreted from infected cells

Culture supernatants containing NS1 were prepared in Vero cells that were maintained in RPMI (HycloneTM) supplemented with 5% heat-inactivated Fetal bovine serum (HI-FBS, Gibco) at 37°C, 5%CO2. The virus strains used in this study included DENV-1-WestPac (accession no: U88535.1) [19], DENV-1-08K3126 [14], DENV-2-TSV01 (AY037116.1) [20], DENV-2-D2Y98P derived from an infectious clone (JF327392.1) [21], DENV-3-VN32/96 (EU482459), DENV-3-CHD94-089, DENV-4-2641Y08 (HQ875339.1), DENV-4-TVP360 (GU289913.1) [22], and ZIKV(PLCal) (KX694532.2). Monolayers of Vero cells in T75 flasks were inoculated with 105 focus forming units of virus for 1 h at 37°C, 5%CO2. Viral inoculum was removed, fresh RPMI supplemented with 5% HI-FBS was added, and culture was incubated at 37°C, 5%CO2. Culture supernatant was harvested when >50% cytopathic effect was observed, clarified by centrifugation at 2,000xg for 5 minutes, concentrated using a 30kDa Amicon® Ultra-15 centrifugal filter unit (MerkMillipore), and stored at -80°C.

 Patient cohort for ELISA validation

All patients were recruited following IRB approval from National Healthcare Group domain specific Review Board (NHG DSRB Ref: 2015/0528 and NHG DSRB Ref: 2016/00982). The details of these patients have been previously described [23]. Patients were enrolled into the study after obtaining their consent, and all data was fully anonymized.

 Results Isolation and characterization of DENV NS1-specific antibodies

To isolate antibodies that recognized NS1 from multiple serotypes, we isolated PBMC from a DENV positive patient, incubated the PBMC with antibodies and recombinant NS1, and then sorted memory B cells (CD19+CD27+IgD─) recognizing NS1 of the DENV-2 alone or DENV-2 and DENV-4 from a convalescent DENV-2 patient at one cell per well. The gating strategy of the memory B cells was shown in S1 Fig. The variable regions of the heavy and light chains from these cells were reverse transcribed from the mRNA and amplified by PCR. The PCR products were cloned into the expression vectors. Following the transient expression and purification, the antibodies were tested for their ability to bind a multimeric form of recombinant NS1 from all four dengue serotypes using an indirect ELISA. Through our screening process, we identified three antibodies A2, D6, and D8 that bound to NS1 from multiple dengue serotypes. Specifically, A2 interacted with NS1 from DENV-1, -2, and -3, D6 interacted with NS1 from DENV-1, -2, and -4, and D8 interacted with NS1 from all four dengue serotypes. The positive control Den3, a dengue virus NS1-specific human IgG1 antibody [16], bound to all four dengue serotypes. Finally, the negative control, 3C, a DENV Envelope-specific human IgG1 antibody [24], did not interact with any of the NS1 proteins (Fig 1A).

10.1371/journal.pone.0285878.g001 Human antibodies against NS1 recognize multimeric forms of DENV NS1.

(A) Indirect ELISA demonstrating the specificity of the antibodies. Wells were coated with NS1 from various dengue viruses, blocked, and then purified antibodies were added. The bound antibodies were detected by the addition of HRP-conjugated anti-human IgG followed by TMB substrate. Average OD values ± SEM from 2 experiments, performed in duplicates are shown. (B) Immunoblot analysis. Purified NS1 from various dengue viruses were untreated or incubated at 90°C for 10 minutes, separated on an SDS-PAGE, and then transferred onto a PVDF membrane. The membranes were incubated with the purified antibodies, followed by a HRP-conjugated anti-human IgG and then chemiluminescent substrate. As a loading control, the SDS-PAGE was stained with InstantBlueTM.

To test whether the monomer or dimer forms of NS1 was recognized by these antibodies, we performed an immunoblot analysis using untreated NS1 and NS1 that was heated at 90°C for 10 minutes. Consistent with previously published data [25], treatment of NS1 at 90°C dissociated dimers into monomers. All four antibodies recognized the dimeric form of NS1 from various dengue serotypes. Specifically, A2 and D8 bound to dimeric forms of NS1 from DENV-2 and DENV-3, and D6 to DENV-2 and DENV-4. D6 also bound to monomeric form of NS1 from DENV-2. The loss of detection of the DENV-1 NS1 by A2 and D6, and the loss of detection of the DENV-1 NS1 and DENV-4 NS1 by D8 by immunoblot analysis were possibly due to the lower affinity of the antibodies towards these serotypes, or the requirement of non-denatured protein for binding. Consistent with results from indirect ELISA, the positive control Den3 bound to NS1 dimer from all four DENV serotypes (Fig 1B).

Finally, BLI analysis was performed to examine the Ab binding kinetics and affinity for the commercially available, presumably hexameric, recombinant NS1. Briefly, antibodies were immobilized onto the biosensors, the antibody-loaded biosensors were immersed into solutions containing various concentrations of recombinant NS1 to allow for NS1 to bind to the biosensors, and then NS1-bound biosensors were immersed in buffer to allow for NS1 to dissociate from the antibodies. The association and dissociation constants were calculated using the 1:1 model with global fitting using all concentrations of NS1, and the mean KD values from 2 independent experiments were shown in Fig 2. The serotype-preference of NS1 binding to the immobilized antibodies was similar to that observed from the indirect ELISA. A2 bound to NS1 from DENV-2 and DENV-3 with higher affinities, and DENV-1 and DENV-4 with lower affinities. D6 bound to NS1 from DENV-1, -2, and -4, and D8 and Den3 bound to NS1 from all four serotypes, albeit with different affinity (Fig 2). A slight upward drifting during the dissociation phase was observed for antibodies with high affinities towards the NS1 (KD <10−12 M). The reason for this upward drifting could not be ascertained experimentally. It could theoretically involve a conformational change of Ab-NS1 complex during the dissociation phase. The germline analysis of A2, D6 and D8 were shown in Table 1 and the amino acid and nucleotide sequences of the light chain and heavy chain variable domains of the antibodies are shown in S1 Table. All three antibodies were isolated from the same patient and were derived from different VH and VL genes: IGHV3-30 and IGLV1-51 for A2, IGHV3-9 and IGLV3-27 for D6, and IGHV4-39 and IGLV3-19 for D8 (Table 1). In conclusion, we have identified three antibodies that bind to multimeric forms of NS1 from multiple DENV serotypes.

10.1371/journal.pone.0285878.g002 Analysis of association and dissociation of NS1 from various serotypes of DENV to immobilized purified IgG by BLI.

Purified antibodies were immobilized on the biosensors, the antibodies loaded biosensors were incubated in several concentrations (0, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100nM) of recombinant NS1, and then the biosensors loaded with NS1 bound to antibodies were immersed in buffer to allow for the dissociation of the NS1 from the antibodies. The graph shows raw (black line) and fitted (color lines) binding of 50 nM of NS1 from the different serotypes to immobilize purified IgG. The KD, Kon and Kdis values were calculated using 1:1 model, global fitting analysis using Octet® Data Analysis software, and the mean and standard deviation of two independent experiments were shown.

 10.1371/journal.pone.0285878.t001 Characteristics of the NS1 antibodies.

VJ assignments, CDR3 sequences, % identity.

Heavy Chain Light Chain
Ab V-gene J-gene D-gene CDR3 % Identity V-gene J-gene CDR3 % Identity
A2 IGHV3-30*04 or IGHV3-30-3*03 IGHJ6*02 IGHD5-12*01 ARDRSDSGYDSYYYYYGMDV 92.5 IGLV1-51*01 IGLJ1*01 GSWDNSLSAYV 94.9
D6 IGHV3-9*01 IGHJ4*02 IGHD3-22*01 AKISNGYRPDN 96.6 IGLV3-27*01 IGLJ2*01, or IGLJ3*01 YCAADNNVV 96.8
D8 IGHV4-39*01 IGHJ4*02 IGHD5-18*01, IGHD5-5*01 ARLGIQLWFAIDY 96.0 IGLV3-19*01 IGLJ3*02 YSRDSSGNLWV 95.4
Development of a dengue-specific NS1-capture ELISA

To develop an ELISA that captures NS1 from all dengue serotypes, we tested different combinations of antibodies as capturing and detecting antibody. We combined A2 and D6 as coating antibodies or detecting antibodies in our assays to ensure that we could detect NS1 from all four DENV serotypes (Fig 3). As shown in Fig 3A and 3B, only recombinant DENV-2 NS1 was detected when the following combination of antibodies were used: (1) A2 and D6 as capture antibodies and D8 or Den3 as detecting antibodies (Fig 3A), and (2) D8 as capture antibodies and A2 and D6 or Den3 as detecting antibodies (Fig 3B). To test an alternative approach allowing detection of all four DENV serotypes, we coated the wells with Den3 and used A2/D6, D8, or a polyclonal antibodies against ZIKV NS1 as detecting antibodies. Both A2/D6 and D8 detected recombinant NS1 from all four serotypes but not NS1 from ZIKV. D8 was more sensitive in detecting NS1 from all four DENV serotypes compared to A2/D6 (Fig 3C). To confirm the presence of ZIKV NS1 that was used in the capture ELISAs, we directly coated the NS1 from all the viruses and used polyclonal antibodies against ZIKV NS1 for detection (Fig 3D). In conclusion, we established a dengue-specific NS1 capture ELISA consisting of Den3 as capture antibody and D8 as detecting antibody that detects recombinant NS1 for all dengue serotypes, but not ZIKV NS1.

10.1371/journal.pone.0285878.g003 NS1 capture ELISAs for the detection of NS1 from all dengue serotypes using different combinations of coating and detecting antibodies.

Wells were coated with (A) A2 and D6; (B) D8; or (C) Den3, and then purified recombinant NS1 from various viruses were added. The bound NS1 were detected by the addition of various HRP-conjugated antibodies. (D) Purified NS1 used for the sandwich ELISAs were coated directly on the wells, Den3 or PAb against ZIKV were added, and the bound antibodies were detected after the addition of HRP-conjugated anti-human or HRP-conjugated anti-rabbit. Representative results are shown and the values shown are OD± standard deviation from duplicates.

 Competition ELISA suggests an overlapping epitope for A2 and D6

Based on results from the NS1 capture ELISA (Fig 3), we hypothesized that A2 and D6 bind to overlapping epitopes on dengue NS1. To test this hypothesis and to further characterize the newly identified antibodies, we performed a competition ELISA using unlabeled and HRP-labeled antibodies. Wells were coated with DENV-2-NS1, unlabeled antibodies were added to allow for binding, washed, and then HRP-labeled antibodies were added. In the presence of unlabeled A2, only 12% of A2-HRP was detected to bind to the DENV-2-NS1 and unlabeled D6 and D8 had minimal effect on binding of A2-HRP. Unlabeled A2 and D6 inhibited HRP-D6 from binding to NS1, suggesting that A2 and D6 bound to overlapping on DENV-2-NS1, although competition was only observed in one direction. Finally, only unlabeled D8 prevented binding of HRP-D8, suggesting that D8 bound to an NS1 epitope that is distinct from the A2 and D6 epitope (Fig 4).

10.1371/journal.pone.0285878.g004 Competition indirect ELISA.

Wells were coated with DENV-2 NS1, blocked, and incubated with unlabeled A2, D6, or D8. The unlabeled antibodies were washed and then A2-HRP, D6-HRP or D8-HRP were added. The bound HRP-conjugated antibodies were detected after the addition of TMB substrate. Representative results from 2 experiments are shown and the values shown are OD± standard deviation from duplicates.

 Evaluation of the performance of the NS1 capture ELISA

To ensure that our NS1-capture ELISA that consisted of Den3 as capture antibodies and D8-HRP as detecting antibody could detect NS1 from multiple dengue serotypes and strains, we prepared NS1 by infecting Vero cells with two strains of dengue viruses from each serotype. As shown in Fig 5A, we detected NS1 from all DENV strains tested, but not ZIKV. We then determined the limit of detection of the assay using recombinant NS1 prepared in human plasma. The detection limit of the assay was defined as the concentration of NS1 with an OD value that was equal to 2-fold the mean OD value of the negative control wells. As shown in Fig 5B, the detection limits were 60 ng/mL for DENV-1, 5 ng/mL for DENV-2, 17 ng/mL for DENV-3 NS1, and 58 ng/mL for DENV-4 NS1. These results suggest that the assay was most sensitive in detecting NS1 from DENV-2, followed by NS1 from DENV-3 and DENV-4. The assay was least sensitive in detecting NS1 from DENV-1.

10.1371/journal.pone.0285878.g005 Detection of NS1 by NS1-capture ELISA that consisted of Den3 as capture antibody and D8-HRP as detecting antibody.

Assay was performed using (A) supernatant from virus infected cells, (B) human plasma spiked with purified NS1 from different dengue serotypes, and (C) human plasma from patients infected with dengue viruses from ≤7 days from fever onset or >7 days from fever onset. (A) The mean OD values ± standard deviation from duplicates are shown. (B) The dotted line represents the limit of detection (LOD) of the assay that was derived from 2-fold the mean OD values of the wells containing no NS1. (C) Individual dots represent samples from different patients and the dots on and above the dotted lines are considered positive for NS1.

Finally, we examined if the ELISA could detect NS1 from dengue-infected patients during the acute phase of infection, ie. Less than or equal to 7 days after fever onset. These patients were confirmed NS1 positive using the SD. BIOLINE Dengue Duo kit that could detect NS1 antigen and IgG/IgM against NS1. Of the 60 samples tested, we detected NS1 in 9.5% (2/21) of the DENV-1 samples, 68.8% (22/32) of DENV-2 samples, 100% (6/6) of the DENV-3 samples, and 100% (2/2) of the DENV-4 samples (2/2) using the developed NS-1 capture ELISA. NS1 was not detected in samples collected from the same patient cohort at greater than 7 days after fever onset demonstrating that the assay did not lead to false positive results (Fig 5C). In conclusion, the ELISA detected NS1 from dengue-infected patients.

 Discussion

In this study, we characterized three newly isolated (A2, D6, and D8) and one previously published (Den3) human antibodies against NS1 and developed a dengue NS1 capture ELISA using these antibodies. To develop a DENV-specific NS1 capture ELISA, it is important that all antibodies fulfilled the following criteria: (1) recognize NS1 from multiple dengue serotypes; (2) bind to multimeric forms of NS1; and (3) have high affinity towards NS1. We cloned antibody genes from B cells that bound to NS1 from DENV-2 and/or DENV-4 to ensure that the selected antibodies recognized NS1 from more than one dengue serotypes. Of the three isolated antibodies, both A2 and D6 recognized three out of the four serotypes, whereas D8 recognized NS1 from all four dengue serotypes, albeit at different affinity (Figs 1 and 2). Glycosylated NS1 is secreted by DENV-infected mammalian cells as hexamers [3] and can be detected in the blood of DENV-infected patients [26]. We showed by immunoblot and BLI that all our antibodies recognized multimeric forms of NS1. Finally, antibodies with high affinity towards NS1 allow better and tighter binding of the antigen to the antibodies. Of all the antibodies used in this study, Den3 has the highest affinity to NS1 from all four serotypes.

We predicted that both A2 and D6 bind to the wing and D8 binds to the β-ladder of the NS1 dimer based on serotype sequence comparison and our results (ELISA, Western blot, and BLI analysis). Based on our results, D6 interacted well to NS1 from DENV-1, -2 and -4 but not DENV-3, suggesting that D6 binds to epitopes that are conserved among DENV-1, -2, and -4. We therefore narrowed down potential binding sites for D6 to amino acids 61–71, 132–138, and 183–190, and all these amino acids are located at the wing domain of NS1 [27]. Using similar rationale, we speculated that the potential binding sites for A2 included amino acids 154–171, 252–263, and 310–323, and the potential binding sites for D8 are located at amino acids 110–120 and 154–171 within the wing domain and amino acids 252–263 and 310–323 of the β-ladder domain (Fig 6). We showed by competition ELISA that A2 and D6 recognized overlapping epitopes or epitopes that are close in proximity. We, therefore, propose that A2 binds to amino acids 154–171 (wing domain) whereas D8 binds to amino acid 252–263 or 310–323 (β-ladder domain). Future studies including mutagenesis and solving the structure of the antibody-NS1 complex are needed to test our hypothesis.

10.1371/journal.pone.0285878.g006 <p>(A) Alignment of NS1 amino acid sequences from DENV and ZIKV viruses. Blue boxes are potential epitopes recognized by D6 and red boxes are potential epitopes recognized by A2 and D8. (B) DENV-2 NS1 dimer structure (PDB: 4O6B) prepared using UCSF Chimera [<xref ref-type="bibr" rid="pone.0285878.ref028">28</xref>]. NS1 dimer with one subunit in grey and another subunit in black. Potential epitopes recognized by D6 are colored in blue and the potential epitopes recognized by A2 and D8 are colored in red.</p> </caption> <graphic mimetype="image" position="float" xlink:href="info:doi/10.1371/journal.pone.0285878.g006" xlink:type="simple"/> </fig> <p>The NS1-capture ELISA developed in this study specifically detected NS1 from DENV, and not ZIKV. This assay will need to be tested for the possibility to recognize NS1 from other closely related flaviviruses, such as JEV and WNV, to confirm that the assay is DENV-specific. Several commercial tests to detect for DENV NS1 in the serum are available. Lima and coworkers showed that the Platelia<sup>TM</sup> Dengue NS1 Ag ELISA did not cross-react with ZIKV NS1 using clinical samples from arbovirus suspected cases [<xref ref-type="bibr" rid="pone.0285878.ref029">29</xref>]. In a different study using supernatant from virus-infected cell culture, Tan and coworkers found that SD BIOLINE Dengue NS1 Ag rapid test and Panbio Dengue Early Rapid test could only detect ZIKV NS1 when the virus titers were above 10<sup>6</sup> PFU/mL [<xref ref-type="bibr" rid="pone.0285878.ref030">30</xref>]. It is important to note that the amount of ZIKV NS1 in the virus supernatant tested in their experiments was not known. We therefore do not know the sensitivity of these assays towards ZIKV NS1. One of the limitations of our study is that we did not have clinical samples from ZIKV infected patients to test the cross-reactivity of our assay to ZIKV NS1. To overcome this limitation, we have shown that our capture ELISA did not detect up to 2 μg/mL of purified recombinant ZIKV NS<xref ref-type="supplementary-material" rid="pone.0285878.s001">1</xref> (<xref ref-type="fig" rid="pone.0285878.g003">Fig 3</xref>) and concentrated supernatant from ZIKV infected cell culture (<xref ref-type="fig" rid="pone.0285878.g005">Fig 5A</xref>).</p> <p>Although our NS1 capture ELISA detected NS1 from all the dengue serotypes and strains tested in this study, we were only detecting NS1 in 9.5% of the DENV-1 patient serum and 68.8% of the DENV-2 patient serum. One explanation could be that the patient serum contained NS1 below the limit of detection of our assay at the time of sample collection. It is important to note that these patients were previously confirmed NS1 positive by the detection of NS1 antigen and/or IgG/IgM against NS1 using SD BIOLINE Dengue Duo kit [<xref ref-type="bibr" rid="pone.0285878.ref023">23</xref>]. The presence of antibodies against NS1 in the serum from previous infection could interfere with our assay. Alternatively, it is possible that our assay was not sensitive enough and further optimization of the assay is required to increase the limit of detection of the assay. The level of NS1 in the plasma of patients ranges from several ng/mL to μg/mL [<xref ref-type="bibr" rid="pone.0285878.ref026">26</xref>]. The limit of detection of our NS1 capture assay was 5 ng/mL for DENV-2 NS1, suggesting that our assay is sensitive enough to detect NS1 from DENV-2 infected patients. The limit of detection of the commercially available tests is unknown. However, similar to the commercially available tests [<xref ref-type="bibr" rid="pone.0285878.ref009">9</xref>–<xref ref-type="bibr" rid="pone.0285878.ref012">12</xref>], our assay is less sensitive in detecting NS1 from other dengue serotypes, specifically DENV-1. This result is consistent with the lower K<sub>D</sub> value of D8 towards DENV-1 NS<xref ref-type="supplementary-material" rid="pone.0285878.s001">1</xref> (<xref ref-type="fig" rid="pone.0285878.g002">Fig 2</xref>). It is therefore important that further optimization is performed to increase the limit of detection of the assay towards other dengue serotypes. Several approaches could be used to improve the sensitivity of the assay towards other dengue serotypes. For example, a biotinylated D8 antibody could be used as a detection antibody, followed by horse-radish peroxidase conjugated streptavidin. This method has been shown to significantly increase the sensitivity of an ELISA. Other parameters that we could evaluate include different blocking buffers, the concentrations of the coating antibodies and detecting antibodies, incubation times, incubation temperature, and different substrates.</p> <p>In conclusion, we characterized three newly isolated antibodies against NS1 (A2, D6, and D8) and a previously published antibody (Den3). Using these antibodies, we have established a dengue-specific capture ELISA.</p> </sec> </sec> <sec id="sec020" sec-type="supplementary-material"> <title>Supporting information B cell sorting strategy for patient samples.

Peripheral blood mononuclear cells were incubated with a cocktail of antibodies, and fluorescently labelled recombinant DENV2- and DENV4-NS1. Memory B cells that were positive for DENV2-NS1 or DENV2-NS1 and DENV4-NS1 were sorted into 96-well PCR plates.

(TIF)

 Nucleotide and amino acid sequences of the CDR of heavy and light chains of A2, D6, and D8.

(DOCX)

 Original western blots and SDS-PAGE images.

These blots and gel images were cropped, compiled, and labeled to generate Fig 1B.

(PDF)

We thank Ying-Xiu Toh and Sumathy Velumani for technical support and SIgN flow cytometry for sorting the B cells. Molecular graphics was performed with UCSF Chimera developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311.

 References Organization WH. Dengue and severe dengue 2021. Available from: https://www.who.int/news-room/fact-sheets/detail/dengue-and-severe-dengue. Xu X, Vaughan K, Weiskopf D, Grifoni A, Diamond MS, Sette A, et al. Identifying Candidate Targets of Immune Responses in Zika Virus Based on Homology to Epitopes in Other Flavivirus Species. PLoS Curr. 2016;8. doi: 10.1371/currents.outbreaks.9aa2e1fb61b0f632f58a098773008c4b 28018746 Flamand M, Megret F, Mathieu M, Lepault J, Rey FA, Deubel V. Dengue virus type 1 nonstructural glycoprotein NS1 is secreted from mammalian cells as a soluble hexamer in a glycosylation-dependent fashion. J Virol. 1999;73(7):6104–10. doi: 10.1128/JVI.73.7.6104-6110.1999 10364366 Muller DA, Young PR. The flavivirus NS1 protein: molecular and structural biology, immunology, role in pathogenesis and application as a diagnostic biomarker. Antiviral Res. 2013;98(2):192–208. doi: 10.1016/j.antiviral.2013.03.008 23523765 de la Cruz-Hernandez SI, Flores-Aguilar H, Gonzalez-Mateos S, Lopez-Martinez I, Alpuche-Aranda C, Ludert JE, et al. Determination of viremia and concentration of circulating nonstructural protein 1 in patients infected with dengue virus in Mexico. Am J Trop Med Hyg. 2013;88(3):446–54. doi: 10.4269/ajtmh.12-0023 23339203 Libraty DH, Young PR, Pickering D, Endy TP, Kalayanarooj S, Green S, et al. High circulating levels of the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever. J Infect Dis. 2002;186(8):1165–8. doi: 10.1086/343813 12355369 Chen HR, Lai YC, Yeh TM. Dengue virus non-structural protein 1: a pathogenic factor, therapeutic target, and vaccine candidate. Journal of biomedical science. 2018;25(1):58. doi: 10.1186/s12929-018-0462-0 30037331 Reyes-Sandoval A, Ludert JE. The Dual Role of the Antibody Response Against the Flavivirus Non-structural Protein 1 (NS1) in Protection and Immuno-Pathogenesis. Frontiers in immunology. 2019;10:1651. doi: 10.3389/fimmu.2019.01651 31379848 Kikuti M, Cruz JS, Rodrigues MS, Tavares AS, Paploski IAD, Silva MMO, et al. Accuracy of the SD BIOLINE Dengue Duo for rapid point-of-care diagnosis of dengue. PLoS One. 2019;14(3):e0213301. doi: 10.1371/journal.pone.0213301 30840708 Santoso MS, Yohan B, Denis D, Hayati RF, Haryanto S, Trianty L, et al. Diagnostic accuracy of 5 different brands of dengue virus non-structural protein 1 (NS1) antigen rapid diagnostic tests (RDT) in Indonesia. Diagn Microbiol Infect Dis. 2020;98(2):115116. doi: 10.1016/j.diagmicrobio.2020.115116 32679344 Lima Mda R, Nogueira RM, Bispo de Filippis AM, dos Santos FB. Comparison of two generations of the Panbio dengue NS1 capture enzyme-linked immunosorbent assay. Clin Vaccine Immunol. 2011;18(6):1031–3. doi: 10.1128/CVI.00024-11 21525305 Chong ZL, Sekaran SD, Soe HJ, Peramalah D, Rampal S, Ng CW. Diagnostic accuracy and utility of three dengue diagnostic tests for the diagnosis of acute dengue infection in Malaysia. BMC Infect Dis. 2020;20(1):210. doi: 10.1186/s12879-020-4911-5 32164538 Zika epidemiology update [press release]. https://www.who.int/emergencies/diseases/zika/zika-epidemiology-update-july-2019.pdf?ua=12019. Appanna R, Kg S, Xu MH, Toh YX, Velumani S, Carbajo D, et al. Plasmablasts During Acute Dengue Infection Represent a Small Subset of a Broader Virus-specific Memory B Cell Pool. EBioMedicine. 2016;12:178–88. doi: 10.1016/j.ebiom.2016.09.003 27628668 Hessell AJ, Hangartner L, Hunter M, Havenith CE, Beurskens FJ, Bakker JM, et al. Fc receptor but not complement binding is important in antibody protection against HIV. Nature. 2007;449(7158):101–4. doi: 10.1038/nature06106 17805298 Moldt B, Rakasz EG, Schultz N, Chan-Hui PY, Swiderek K, Weisgrau KL, et al. Highly potent HIV-specific antibody neutralization in vitro translates into effective protection against mucosal SHIV challenge in vivo. Proc Natl Acad Sci U S A. 2012;109(46):18921–5. doi: 10.1073/pnas.1214785109 23100539 Prommool T, Sethanant P, Phaenthaisong N, Tangthawornchaikul N, Songjaeng A, Avirutnan P, et al. High performance dengue virus antigen-based serotyping-NS1-ELISA (plus): A simple alternative approach to identify dengue virus serotypes in acute dengue specimens. PLoS Negl Trop Dis. 2021;15(2):e0009065. doi: 10.1371/journal.pntd.0009065 33635874 Noy-Porat T, Alcalay R, Mechaly A, Peretz E, Makdasi E, Rosenfeld R, et al. Characterization of antibody-antigen interactions using biolayer interferometry. STAR Protoc. 2021;2(4):100836. doi: 10.1016/j.xpro.2021.100836 34568849 Repik PM, Dalrymple JM, Brandt WE, McCown JM, Russell PK. RNA fingerprinting as a method for distinguishing dengue 1 virus strains. Am J Trop Med Hyg. 1983;32(3):577–89. doi: 10.4269/ajtmh.1983.32.577 6859403 Johansson M, Brooks AJ, Jans DA, Vasudevan SG. A small region of the dengue virus-encoded RNA-dependent RNA polymerase, NS5, confers interaction with both the nuclear transport receptor importin-beta and the viral helicase, NS3. J Gen Virol. 2001;82(Pt 4):735–45. doi: 10.1099/0022-1317-82-4-735 11257177 Grant D, Tan GK, Qing M, Ng JK, Yip A, Zou G, et al. A single amino acid in nonstructural protein NS4B confers virulence to dengue virus in AG129 mice through enhancement of viral RNA synthesis. J Virol. 2011;85(15):7775–87. doi: 10.1128/JVI.00665-11 21632767 Eckels KH, Dubois DR, Putnak R, Vaughn DW, Innis BL, Henchal EA, et al. Modification of dengue virus strains by passage in primary dog kidney cells: preparation of candidate vaccines and immunization of monkeys. Am J Trop Med Hyg. 2003;69(6 Suppl):12–6. doi: 10.4269/ajtmh.2003.69.12 14740950 Rouers A, Chng MHY, Lee B, Rajapakse MP, Kaur K, Toh YX, et al. Immune cell phenotypes associated with disease severity and long-term neutralizing antibody titers after natural dengue virus infection. Cell Rep Med. 2021;2(5):100278. doi: 10.1016/j.xcrm.2021.100278 34095880 Xu M, Zuest R, Velumani S, Tukijan F, Toh YX, Appanna R, et al. A potent neutralizing antibody with therapeutic potential against all four serotypes of dengue virus. NPJ Vaccines. 2017;2:2. doi: 10.1038/s41541-016-0003-3 29263863 Pryor MJ, Wright PJ. The effects of site-directed mutagenesis on the dimerization and secretion of the NS1 protein specified by dengue virus. Virology. 1993;194(2):769–80. doi: 10.1006/viro.1993.1318 8389081 Alcon S, Talarmin A, Debruyne M, Falconar A, Deubel V, Flamand M. Enzyme-linked immunosorbent assay specific to Dengue virus type 1 nonstructural protein NS1 reveals circulation of the antigen in the blood during the acute phase of disease in patients experiencing primary or secondary infections. J Clin Microbiol. 2002;40(2):376–81. doi: 10.1128/JCM.40.02.376-381.2002 11825945 Akey DL, Brown WC, Dutta S, Konwerski J, Jose J, Jurkiw TJ, et al. Flavivirus NS1 structures reveal surfaces for associations with membranes and the immune system. Science. 2014;343(6173):881–5. doi: 10.1126/science.1247749 24505133 Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al. UCSF Chimera—a visualization system for exploratory research and analysis. Journal of computational chemistry. 2004;25(13):1605–12. doi: 10.1002/jcc.20084 15264254 Lima M, Chouin-Carneiro T, Azeredo E, Barbosa LS, Souza TMA, Silva J, et al. The inability of a dengue NS1 ELISA to detect Zika infections. Sci Rep. 2019;9(1):18596. doi: 10.1038/s41598-019-55160-4 31819129 Tan LK, Wong WY, Yang HT, Huber RG, Bond PJ, Ng LC, et al. Flavivirus Cross-Reactivity to Dengue Nonstructural Protein 1 Antigen Detection Assays. Diagnostics (Basel). 2019;10(1). doi: 10.3390/diagnostics10010011 31878299 10.1371/journal.pone.0285878.r001 Decision Letter 0 Henry Kevin A. Academic Editor 2023 Kevin A. Henry This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 0

5 Oct 2022

PONE-D-22-24631A nonstructural protein 1 capture enzyme-linked immunosorbent assay specific for dengue virusesPLOS ONE

Dear Dr. Lim,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Both reviewers identified several areas for improvement including further explanation of key experiments and missing methodological information. Please carefully address their comments, especially where they have asked for explanations of potentially discrepant results.

Please submit your revised manuscript by November 17, 2022. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Kevin A. Henry

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.

If you are reporting a retrospective study of medical records or archived samples, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information

Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”).

For additional information about PLOS ONE ethical requirements for human subjects research, please refer to http://journals.plos.org/plosone/s/submission-guidelines#loc-human-subjects-research.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript: 

"This work was supported by SIgN Core grant"

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. 

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: 

"The authors receive no specific funding for this work."

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

4. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. 

  

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

5. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

6. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors developed and characterized three new human monoclonal antibodies that bind to DENV NS1 but not to ZIKV NS1. Using the three new antibodies and a previously described antibody that binds to NS1 from each of the four DENV serotypes the authors devised ELISAs and found a combination of antibodies that were able to detect recombinant NS1 from all four serotypes as well as NS1 in viral supernatants from the four serotypes but did not show signal for ZIKV. Finally, the authors tested the ability of their ELISA using patient samples.

1. In the introduction the authors state that Yellow fever virus, West Nile virus, and Japanese encephalitis virus are also closely related to DENV. Is it important to ensure no cross reactivity with these viruses?

2. Why does the signal continue to go higher during the dissociation phage of the BLI data? Does this make sense? Please offer some explanation about the BLI curves

3. Please explain why A2 has KD of 4.4 nM to DENV-4 NS1 but does not bind to DENV-4 in the indirect ELISA

4. In Figure 5B the developed ELISA shows the best detection of the recombinant DENV 2 NS1. In addition, the D8 antibody was isolated from a convalescent DENV-2 patient. However, 5C shows that about 1/3 of the DENV-2 patient samples are not detected by the author’s ELISA. How could the ELISA be improved to better detect DENV-2 in patient samples as well as to detect DENV-1 in patient samples.

5. It is very unclear how the authors narrowed down the areas of the potential epitopes. Please explain in more detail.

Reviewer #2: This paper describes the isolation of anti-Dengue NS1 antibodies from a Dengue patient, using B-cell sorting. The purpose was to find new reagents to use in Dengue diagnostic ELISA kits which are specific for Dengue NS1 with no cross-reactivity to other flaviviruses such as ZIKA. The authors screened for antibodies which would bind to all four Dengue serotypes, and isolated one pan-specific antibody (D8). When paired with a published pan-specific antibody (Den3) a sandwich ELISA to detect Dengue NS1 was developed. The authors also speculated on the epitope of the D8 and Den3 antibodies, as well as other isolated antibodies which bound one or more Dengue serotypes, by performed competition ELISAs.

General comments:

The manuscript is well written and organised.

It would be good to include more detail on the isolation of the antibodies, and include some results (eg show the flow cytometry gating strategy, and a typical flow cytometry result of a positive hit). How many B-cells were screened, and what was the positive hit rate?

Since the purpose of the study was to find alternative reagents for diagnostic NS1 detection ELISA, it would be good to see a side-by-side comparison of the developed ELISA with an existing commercial kit, especially for limit of detection with recombinant Dengue and ZIKA NS1.

Specific comments:

Line 68: How many patients?

Line 81: Describe sorting/gating conditions

Line 86: Reference 14 is cited for the detailed method for cloning and expressing the antibodies. Please check that the correct reference has been cited, as Ref14 does not contain this methodology

Line 87: Primer sequences? Or are they described in the correct reference 14?

Line 129: This is the first time Den3 antibody has been mentioned (other than the abstract). In the abstract it is referred to as previously published. Please cite the publication here – it is later cited in the results. Also how did you obtain this antibody – from the authors of Ref21? Or expressed from a published sequence – if so, where did the sequence come from? Ref 21 uses the Den3 antibody, but doesn’t give any information about its source – is there a better reference?

Line 159: The method suggests different concentrations of NS1 were included in the analysis, but the results only show one concentration (50nM). Please clarify if multiple concentrations were used, and if so, were these included in the analysis to give more accurate results?

Line 160: Describe evaluation for BLI analysis (fit to a 1:1 binding model?)

Line 288: The word ‘and’ should be ‘an’

Lines 304-311: The samples were confirmed positive using the Bioline Dengue kit. For the samples that were not detected by the newly developed assay, do you think these are false negatives in the new assay or false positives in the Bioline assay? As the new assay was the least sensitive for DENV1, this could explain the low detection of DENV1 samples; however the detection of DENV2 was also fairly low when the assay has the highest sensitivity for DENV2

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

 10.1371/journal.pone.0285878.r002 Author response to Decision Letter 0 Submission Version 1

17 Nov 2022

The response to reviewers and editor comments is included in the rebuttal letter. Please refer to the rebuttal letter as it contains graphs to support the explanation.

Reviewer #1: The authors developed and characterized three new human monoclonal antibodies that bind to DENV NS1 but not to ZIKV NS1. Using the three new antibodies and a previously described antibody that binds to NS1 from each of the four DENV serotypes the authors devised ELISAs and found a combination of antibodies that were able to detect recombinant NS1 from all four serotypes as well as NS1 in viral supernatants from the four serotypes but did not show signal for ZIKV. Finally, the authors tested the ability of their ELISA using patient samples.

1. In the introduction the authors state that Yellow fever virus, West Nile virus, and Japanese encephalitis virus are also closely related to DENV. Is it important to ensure no cross reactivity with these viruses?

Response: Yellow fever virus, West Nile virus, Japanese encephalitis, Dengue viruses, and Zika viruses are considered closely related because they are all members of the Flaviviridae family. These viruses are classified based on their biological properties such as virus morphology, genome structures or antigenic properties. There is no concern about cross reactivity of DENV NS1 with Yellow fever virus, West Nile virus, and Japanese encephalitis virus in the field because the sequence homology of DENV NS1 with these viruses are less than 50% (Xu et. al., 2016).

2. Why does the signal continue to go higher during the dissociation phage of the BLI data? Does this make sense? Please offer some explanation about the BLI curves

Response: In Biolayer interferometry (BLI), the mAbs were immobilized onto a biosensor tip, binding of the antigen (NS1) to the mAb led to an increase of optical thickness, resulting in a wavelength shift. This technique is sensitive and may have a slight deviation due to background, explaining why a slight increase can be observed during the reading for the dissociation step. Fig 2 (in our initially submitted manuscript) represents raw data (not fitted), while the analysis to determine the KD are based on fitted data. For clarity, we have updated Fig 2 in the revised manuscript to show the fitted data, instead of the raw data.

3. Please explain why A2 has KD of 4.4 nM to DENV-4 NS1 but does not bind to DENV-4 in the indirect ELISA

Response: A2 has a KD of 4.4nM to DENV-4 NS1 as measured by BLI but no detectable A2 binding to DENV-4 NS1 was observed by indirect ELISA. This is because indirect ELISA is in general a less sensitive method as compared to BLI due to the differences in technology and means of measuring interactions between antibody and antigen.

BLI is a very sensitive technique with a system limit of detection for KD measurements between 1mM-1pM and the sensitivity of the indirect ELISA depends upon the ELISA. In addition, BLI involves immobilizing the antibodies through its Fc domain on the biosensor tip, and then allowing soluble NS1 to bind in solution. Binding of antigen to the immobilized antibodies causes an increase in optical thickness at the biosensor tip, resulting in a wavelength shift proportional to the extent of binding.

Indirect ELISA, however, involves immobilizing soluble NS1 onto the surface, allowing antibodies to bind to the immobilized NS1, and then the bound antibodies were detected using an HRP-conjugated secondary antibodies and a colorimetric substrate. As the NS1 could be immobilized onto the surface via different orientations, some of these epitopes may not be accessible to the antibodies. This could also contribute to one of the reasons why ELISA may be less sensitive as compared to BLI.

4. In Figure 5B the developed ELISA shows the best detection of the recombinant DENV 2 NS1. In addition, the D8 antibody was isolated from a convalescent DENV-2 patient. However, 5C shows that about 1/3 of the DENV-2 patient samples are not detected by the author’s ELISA. How could the ELISA be improved to better detect DENV-2 in patient samples as well as to detect DENV-1 in patient samples.

Response: There are several ways to improve the sensitivity of the assay. One way is to use a biotinylated-D8 antibody as a detection antibody, followed by horse-radish peroxidase conjugated streptavidin. This method has been shown to significantly increase the sensitivity of an assay. In addition, we could optimize the concentrations of the coating antibodies, the biotinylated-D8 antibody and horse-radish peroxidase conjugated streptavidin. Other parameters that we could examine includes incubation times, incubation temperature, and different TMB substrates.

5. It is very unclear how the authors narrowed down the areas of the potential epitopes. Please explain in more detail.

Response: We have revised the discussion section to further describe how we have speculated the potential epitopes recognized by the antibodies.

Reviewer #2: This paper describes the isolation of anti-Dengue NS1 antibodies from a Dengue patient, using B-cell sorting. The purpose was to find new reagents to use in Dengue diagnostic ELISA kits which are specific for Dengue NS1 with no cross-reactivity to other flaviviruses such as ZIKA. The authors screened for antibodies which would bind to all four Dengue serotypes, and isolated one pan-specific antibody (D8). When paired with a published pan-specific antibody (Den3) a sandwich ELISA to detect Dengue NS1 was developed. The authors also speculated on the epitope of the D8 and Den3 antibodies, as well as other isolated antibodies which bound one or more Dengue serotypes, by performed competition ELISAs.

General comments:

The manuscript is well written and organised.

It would be good to include more detail on the isolation of the antibodies, and include some results (eg show the flow cytometry gating strategy, and a typical flow cytometry result of a positive hit). How many B-cells were screened, and what was the positive hit rate?

Since the purpose of the study was to find alternative reagents for diagnostic NS1 detection ELISA, it would be good to see a side-by-side comparison of the developed ELISA with an existing commercial kit, especially for limit of detection with recombinant Dengue and ZIKA NS1.

Specific comments:

Line 68: How many patients?

Response: We have updated the method and material section to indicate that the antibodies were isolated from one patient.

Line 81: Describe sorting/gating conditions

Response: We have added the sorting/gating strategy as S1 Fig and added the description of the strategy in the results section.

Line 86: Reference 14 is cited for the detailed method for cloning and expressing the antibodies. Please check that the correct reference has been cited, as Ref14 does not contain this methodology

Response: We have checked and confirmed that the correct reference (Appanna et. al., 2016; originally Ref14) section 2.3 described the detailed method for cloning and expressing the antibodies.

Line 87: Primer sequences? Or are they described in the correct reference 14?

Response: We have confirmed that the primer sequences are also listed in table S1 in the publication Appanna et. al., 2016 (originally Reference 14).

Line 129: This is the first time Den3 antibody has been mentioned (other than the abstract). In the abstract it is referred to as previously published. Please cite the publication here – it is later cited in the results. Also how did you obtain this antibody – from the authors of Ref21? Or expressed from a published sequence – if so, where did the sequence come from? Ref 21 uses the Den3 antibody, but doesn’t give any information about its source – is there a better reference?

Response: We have added the reference as per reviewer’s suggestions (currently Line 147). Cells expressing Den3 was obtained from one of the coauthors, Dennis Burton and this antibody was first published in Ref21. We have added a section in the Methods and Materials to describe the production and purification of Den3.

Line 159: The method suggests different concentrations of NS1 were included in the analysis, but the results only show one concentration (50nM). Please clarify if multiple concentrations were used, and if so, were these included in the analysis to give more accurate results?

Response: Several concentrations of NS1 were used in the assay: 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 nM. In general, graph from one concentration is demonstrated in most publications. We have chosen to present only the graph for 50nM as a representation but the KD values shown in the table were calculated by taking all the concentrations into account (global fitting analysis).

Thus, the value presented in the table on the right of Fig 2 include the different concentrations for the calculation in order to deliver more accurate results.

We have added additional information in the Method and Material, the legend for Figure 2, and the Results sections to clarify the analysis.

Line 160: Describe evaluation for BLI analysis (fit to a 1:1 binding model?)

Response: We have added the evaluation of BLI analysis in the Methods and Materials section. We used the Octet® Data Analysis software for affinity calculations as detailed in Noy-Porat et. al., 2021. In short, we used the condition without NS1 antigen (0 nM) as background reference to subtract to the data. Y axis was aligned to the baseline (between 190 and 199.8 sec which was consider the most stable). The association and dissociation steps were analysed using 1:1 model with global fitting.

Line 288: The word ‘and’ should be ‘an’

Response: We have edited the “and” to “an”.

Lines 304-311: The samples were confirmed positive using the Bioline Dengue kit. For the samples that were not detected by the newly developed assay, do you think these are false negatives in the new assay or false positives in the Bioline assay? As the new assay was the least sensitive for DENV1, this could explain the low detection of DENV1 samples; however the detection of DENV2 was also fairly low when the assay has the highest sensitivity for DENV2

Response: We are unable to conclude whether or not the samples that were positive by Bioline Dengue kit but not the newly developed assay were false negatives in the assay or false positive in the Bioline assay for the following reasons: (1) the newly developed assay could be optimized further to increase sensitivity; and (2) SD Bioline Dengue kit detects both NS1 antigen and IgG/IgM against NS1, whereas the newly developed assay only measures NS1 antigen; therefore, SD Bioling Dengue kit could detect more positive samples. We have revised the results section to clarify that Bioline Dengue kit could detect both NS1 antigen and antibodies against NS1.

 10.1371/journal.pone.0285878.r003 Decision Letter 1 Henry Kevin A. Academic Editor 2023 Kevin A. Henry This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 1

4 Dec 2022

PONE-D-22-24631R1A nonstructural protein 1 capture enzyme-linked immunosorbent assay specific for dengue virusesPLOS ONE

Dear Dr. Lim,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Both reviewers appreciated the changes and revisions made in light of their initial comments, but felt that a small number of issues still remained to be addressed prior to publication. See their detailed comments.

Please submit your revised manuscript by Jan 18 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Kevin A. Henry

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: 1. Previously I had made the comment: “In the introduction the authors state that Yellow fever virus, West Nile virus, and Japanese encephalitis virus are also closely related to DENV. Is it important to ensure no cross reactivity with these viruses?” In their response to my comment, the authors stated that “There is no concern about cross reactivity of DENV NS1 with Yellow fever virus, West Nile virus, and Japanese encephalitis virus in the field because the sequence homology of DENV NS1 with these viruses are less than 50% (Xu et. al., 2016)”. However, according to table 2 of that reference, homology to JEV ranges from 51.1% to 53.4%, homology to WNV ranges from 50.3% to 55.4%, and homology to ZIKV ranges from 53.4% to 55.1%. Only YFV has less than 50% homology to the four DENV serotypes.

In order to have the conclusions of this paper be justified by the data, please include a statement indicating that further testing for binding to NS1 from the related WNV, YFV, and JEV will need to be conducted to definitively show the specificity of the developed assay.

2. In Figure 5B the developed ELISA shows the best detection of the recombinant DENV 2 NS1 with a limit of detection of 5 ng/mL. However, 5C shows that about 30% of the DENV-2 patient samples are not detected by the author’s ELISA. Was this expected? Please add a couple of sentences to the discussion section of the manuscript about these results which can include next steps to potentially improve the assay.

3. Please check the size of boxes in figure 6. There is one box that includes one of the ZIKV sequences when they probably all are meant to only include the four DENV sequences.

Reviewer #2: In general, the authors have responded to both reviewer’s comments adequately. However, the following requires further clarification or changes to the manuscript:

Responses to Reviewer 1’s comments:

2. In response to Reviewer 1’s comment about the BLI results, the authors have changed the figure to show the fitted data rather than the raw data. This just hides the issue that the reviewer was concerned about (ie the upward drifting dissociation phase). The graphs should show both the raw data and the fitted curves (eg in solid and dotted lines for each concentration). The upward drift is likely caused by non-specific binding of the analyte to the reference sensor. Although the data has subtracted the 0nM data during the analysis, was there also a reference sensor subtraction for each concentration of analysis (ie binding of the NS1 to a sensor without antibody)?

4. The authors have responded to Reviewer 1’s comments with suggestions on how to optimise the ELISA to improve the sensitivity. Have the authors tried these relatively simple measures and can they be incorporated into the manuscript? If this is not possible, then some comment in the discussion should be included to state that improved sensitivity is required since the ELISA did not detect some of the positive patient samples, and discuss these methods.

Responses to Reviewer 2:

The authors have not commented on Reviewer 2’s suggestion to include a side-by-side comparison with an existing commercial kit. If the purpose is to find reagents to develop a superior assay then this comparison should be included. A comparison of the limit of detection would be ideal.

Line 122: Since Dr Dennis Burton is a co-author, you do not need to acknowledge that you got the cells from Dr Burton. Instead, change the wording to: ‘Chinese hamster ovary cells expressing Den3 antibodies (ref 16) were expressed in glutamine-free…………’

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

 10.1371/journal.pone.0285878.r004 Author response to Decision Letter 1 Submission Version 2

21 Apr 2023

Reviewer #1: 1. Previously I had made the comment: “In the introduction the authors state that Yellow fever virus, West Nile virus, and Japanese encephalitis virus are also closely related to DENV. Is it important to ensure no cross reactivity with these viruses?” In their response to my comment, the authors stated that “There is no concern about cross reactivity of DENV NS1 with Yellow fever virus, West Nile virus, and Japanese encephalitis virus in the field because the sequence homology of DENV NS1 with these viruses are less than 50% (Xu et. al., 2016)”. However, according to table 2 of that reference, homology to JEV ranges from 51.1% to 53.4%, homology to WNV ranges from 50.3% to 55.4%, and homology to ZIKV ranges from 53.4% to 55.1%. Only YFV has less than 50% homology to the four DENV serotypes.

In order to have the conclusions of this paper be justified by the data, please include a statement indicating that further testing for binding to NS1 from the related WNV, YFV, and JEV will need to be conducted to definitively show the specificity of the developed assay.

Response: We have added the statement as suggested by the reviewer (p17).

2. In Figure 5B the developed ELISA shows the best detection of the recombinant DENV 2 NS1 with a limit of detection of 5 ng/mL. However, 5C shows that about 30% of the DENV-2 patient samples are not detected by the author’s ELISA. Was this expected? Please add a couple of sentences to the discussion section of the manuscript about these results which can include next steps to potentially improve the assay.

Response: We have included discussion of these results in the discussion section (p18). As described in the discussion, the patients were previously confirmed NS1 positive using SD BIOLINE Dengue Duo kit that detects Dengue NS1 antigen and/or antibodies in the serum. It is possible that the 30% DENV-2 patient samples contained NS1 below the limit of detection of our assay. Alternatively, these patient samples may contain antibodies against NS1 from previous infection that interfere with our assay.

3. Please check the size of boxes in figure 6. There is one box that includes one of the ZIKV sequences when they probably all are meant to only include the four DENV sequences.

Response: We have amended the Figure to include only the DENV sequences.

Reviewer #2: In general, the authors have responded to both reviewer’s comments adequately. However, the following requires further clarification or changes to the manuscript:

Responses to Reviewer 1’s comments:

2. In response to Reviewer 1’s comment about the BLI results, the authors have changed the figure to show the fitted data rather than the raw data. This just hides the issue that the reviewer was concerned about (ie the upward drifting dissociation phase). The graphs should show both the raw data and the fitted curves (eg in solid and dotted lines for each concentration). The upward drift is likely caused by non-specific binding of the analyte to the reference sensor. Although the data has subtracted the 0nM data during the analysis, was there also a reference sensor subtraction for each concentration of analysis (ie binding of the NS1 to a sensor without antibody)?

Response: We only used the 0nM condition for subtraction of background. We understand the reviewer’s concern and have added both raw data and fitted data in the graphs.

We are unable to explain the upward drifting during the dissociation phase. However, we would like to add the following points that argue against non-specific binding of the analyte to the reference sensor for the following reasons: (1) This upward drifting was only observed in antibodies with high affinity to NS1. Specifically, DENV2 and DENV4 NS1 for A2, DENV2 NS1 for D6, and DENV2 and DENV4 for D8; (2) The experiment has been repeated twice and consistent results were observed; (3) the dissociation phase occurred in a buffer with no antigen; and (4) new biosensor tips were used for all experiments and the tips were not regenerated. We have added a few sentences to describe the observation (p11).

4. The authors have responded to Reviewer 1’s comments with suggestions on how to optimise the ELISA to improve the sensitivity. Have the authors tried these relatively simple measures and can they be incorporated into the manuscript? If this is not possible, then some comment in the discussion should be included to state that improved sensitivity is required since the ELISA did not detect some of the positive patient samples, and discuss these methods.

Response: We have added some comments in the discussion (p18).

Responses to Reviewer 2:

The authors have not commented on Reviewer 2’s suggestion to include a side-by-side comparison with an existing commercial kit. If the purpose is to find reagents to develop a superior assay then this comparison should be included. A comparison of the limit of detection would be ideal.

Response: We acknowledge the reviewer’s suggestion in performing a side-by-side comparison with an existing commercial kit. We would like to emphasize that the goal of this paper is to develop an assay that specifically detect NS1 from dengue viruses and not detect NS1 from Zika virus, and limited studies were performed to examine the specificity of the commercial kits. In addition, our assay needs further optimization before a benchmarking with commercial kits would be conducted. Finally, it is challenging to perform a side-by-side comparison for the concentration of NS1 with an existing commercial kit because commercial kits, at least those we are aware of, are not quantitative tests. The read out of a commercial kit is an OD ratio and there is no standard curve where one could interpolate the amount of NS1 from the standard curve.

Line 122: Since Dr Dennis Burton is a co-author, you do not need to acknowledge that you got the cells from Dr Burton. Instead, change the wording to: ‘Chinese hamster ovary cells expressing Den3 antibodies (ref 16) were expressed in glutamine-free…………’

Response: We have updated the text accordingly.

 10.1371/journal.pone.0285878.r005 Decision Letter 2 Henry Kevin A. Academic Editor 2023 Kevin A. Henry This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 2

4 May 2023

A nonstructural protein 1 capture enzyme-linked immunosorbent assay specific for dengue viruses

PONE-D-22-24631R2

Dear Dr. Wang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Kevin A. Henry

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

 10.1371/journal.pone.0285878.r006 Acceptance letter Henry Kevin A. Academic Editor 2023 Kevin A. Henry This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

9 May 2023

PONE-D-22-24631R2

A nonstructural protein 1 capture enzyme-linked immunosorbent assay specific for dengue viruses

Dear Dr. Wang:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Kevin A. Henry

Academic Editor

PLOS ONE