Development and characterization of serotype-specific monoclonal antibodies against the dengue virus-4 (DENV-4) non-structural protein (NS1)

Gelanew and Hunsperger Virology Journal (2018) 15:30 DOI 10.1186/s12985-018-0925-7 METHODOLOGY Open Access Development and characterization of serotype-specific monoclonal antibodies against the dengue virus-4 (DENV-4) non-structural protein (NS1) Tesfaye Gelanew and Elizabeth Hunsperger* Abstract Background: Dengue, caused by one of the four serologically distinct dengue viruses (DENV-1 to − 4), is a mosquito-borne disease of serious global health significance. Reliable and cost-effective diagnostic tests, along with effective vaccines and vector-control strategies, are highly required to reduce dengue morbidity and mortality. Evaluation studies revealed that many commercially available NS1 antigen (Ag) tests have limited sensitivity to DENV-4 serotype compared to the other three serotypes. These studies indicated the need for development of new NS1 Ag detection test with improved sensitivity to DENV-4. An NS1 capture enzyme linked immunoassay (ELISA) specific to DENV-4 may improve the detection of DENV-4 cases worldwide. In addition, a serotype-specific NS1 Ag test identifies both DENV and the infecting serotype. Methods: In this study, we used a small-ubiquitin-like modifier (SUMO*) cloning vector to express a SUMO*-DENV-4 rNS1 fusion protein to develop NS1 DENV-4 specific monoclonal antibodies (MAbs). These newly developed MAbs were then optimized for use in an anti-NS1 DENV-4 capture ELISA. The serotype specificity and sensitivity of this ELISA was evaluated using (i) supernatants from DENV (1–4)-infected Vero cell cultures, (ii) rNS1s from all the four DENV (1–4) and, (iii) rNS1s of related flaviviruses (yellow fever virus; YFV and West Nile virus; WNV). Results: From the evaluation studies of the newly developed MAbs, we identified three DENV-4 specific anti-NS1 MAbs: 3H7A9, 8A6F2 and 6D4B10. Two of these MAbs were optimal for use in a DENV-4 serotype-specific NS1 capture ELISA: MAb 8A6F2 as the capture antibody and 6D4B10 as a detection antibody. Conclusion: This ELISA was sensitive and specific to DENV-4 with no cross-reactivity to other three DENV (1–3) serotypes and other heterologous flaviviruses. Taken together these data indicated that our MAbs are useful reagents for the development of DENV-4 immunodiagnostic tests. Keywords: Dengue virus 4, Non-structural protein 1, NS1, Diagnostic, flavivirus, Monoclonal antibody Background Dengue is a serious disease of public importance with increasing worldwide spread. It is caused by an infection with any one of the four antigenically distinct dengue virus serotypes (DENV1–4). Current diagnosis of an acute DENV infection primarily relies on reverse transcription polymerase chain reaction (RT-PCR), a highly * Correspondence: Dengue Branch, Division of Vector-Borne Diseases, National Center for Enteric and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention (CDC), San Juan, PR, USA sophisticated test. To improve dengue case detection in dengue endemic countries, a highly sensitive, specific and simple rapid assay is needed to provide early support for patients and to accurately differentiate dengue from other acute febrile illnesses. DENV non-structural protein 1 (NS1) is a unique diagnostic marker for early detection of DENV compared to serological tests (i.e., anti-DENV IgM) because it is detected in the serum of DENV-infected patients as early as one day post onset of symptoms (DPO) to 18 DPO at concentration up to 50 μg/ml and it is a confirmatory test [1]. Whereas anti- © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gelanew and Hunsperger Virology Journal (2018) 15:30 DENV IgM is not present until five days after illness onset, can cross react with other flaviviruses thus not considered a confirmatory test especially in regions where multiple flaviviruses co-circulate. NS1 is highly conserved glycoprotein and possesses both group-specific and serotype-specific epitopes hence it can discern DENV from other flaviviruses and has the potential to differentiate between DENV serotypes [2]. Rapid tests such as the NS1 enzyme-linked immunosorbent assay (ELISA) are commercially available for DENV with relatively good sensitivity and specificity [3]. Recently several studies have critically evaluated the performance of the current commercial NS1 ELISA kits by DENV serotype. Results from these studies showed that these tests had lower sensitivity for the detection of DENV-4. Additionally, these commercial tests had decreased sensitivity in detecting secondary dengue infections, common in dengue endemic countries [3–9]. Commercially available NS1 antigen (Ag) tests manufactured by BioRad (Platelia) and Panbio (Dengue Early) both have low sensitivity to DENV-4 infections [8, 10– 12]. Previous evaluations of Dengue Early test showed a sensitivity as low as 19% for DENV4 [6, 8, 13–15]. Although the overall sensitivity of Platelia test was good however when comparing the sensitivities between all DENV serotypes, the BioRad test had the lowest sensitivity for DENV-4 (58.3%) [10]. Our rationale to develop a DENV4 ELISA was due to the limited sensitivity of NS1 tests for detection DENV4 infections. Recent evaluation study using retrospective samples from South America showed lower sensitivity of seven commercially available NS1 Ag tests for DENV-4 compared to the other three serotypes [7]. Also, metaanalysis for DENV detection in Asia showed the lowest sensitivity of the commercial NS1 Ag tests was against DENV-4 detection [16]. Further, studies conducted in Brazil showed the lower sensitivity of Platelia NS1 Ag ELISA (BioRad) for DENV4 detection [13, 14]. Collectively, these data suggest the need for development of new NS1 Ag detection test that incorporates monoclonal antibodies (MAbs) with higher sensitivity to DENV-4. In order to improve the performance of NS1 Ag detection tests with higher sensitivity to DENV-4, it is important to understand why the current NS1 Ag detection tests failed to detect DENV4 infections efficiently. Almost all the present commercial NS1 Ag detection tests are based on cross-reactive anti-NS1 MAbs to all four DENV serotypes due to common epitopes. Epitope mapping studies have identified epitopes LX1 (113YSWKTWG-119) and LD2 (24-VHTWTEOYK-32) that are common to all four DENV serotype [2, 17, 18]. Several studies showed the absence of significant amino acid sequence variation in the epitopes of serotype-crossreactive M (...truncated)