To localize the specific mAbs binding sites/epitopes, peptide mapping array experiments were performed (Physique S2)

To localize the specific mAbs binding sites/epitopes, peptide mapping array experiments were performed (Physique S2). after reaction with mAb 4H2, which also did not cross react with ZIKV. The selection and characterization of the epitope, specificity of anti-NS1 mAbs, may contribute to the development of diagnostic tools able to differentiate DENV and ZIKV infections. Keywords: dengue computer virus, NS1, Zika computer virus, mAbs, antibody acknowledgement, amino acid sequences 1. Introduction Dengue fever is an important mosquito-borne and the most prevalent and costly arbovirus affecting humans, caused by one of the four serotypes of dengue computer virus (DENV 1C4) [1]. In the last decade, a large number of dengue epidemics have occurred, HS-10296 hydrochloride which resulted in enormous economic and human loss in parts of Asia and South America [2,3]. Considering Brazil only, more than three million cases of confirmed dengue infections occurred between 2015 and 2017, with 70 cases per 100,000 inhabitants [4]. The DENV genome is composed of a single positive-sense RNA that encodes a Rabbit polyclonal to Tumstatin single viral polyprotein that is further processed by viral and host proteases into three structural proteins (C, prM/M, and E) and HS-10296 hydrochloride seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). NS1 is the first nonstructural protein to be translated and is essential to computer virus replication [5]. It is a conserved N-linked glycoprotein with a variable molecular mass of 46C55 kDa, which depends on its glycosylation status [6]. The NS1 protein can be found as a dimer associated with vesicular compartments within the cell, where it plays an important role as an essential cofactor in the computer virus replication process [7]. Alternatively, NS1 can be secreted into the extracellular space as a hexameric lipoprotein particle [8] that interacts with several plasma proteins [9,10]. The recent introduction of the Zika computer virus (ZIKV) to the American continent represented a regional and worldwide public health challenge [11]. The close evolutionary relationship between DENV and ZIKV is usually reflected by the high sequence conservation of both structural and non-structural proteins [12]. In this aspect, the identification of monoclonal antibodies (mAbs) able to react specifically with DENV or cross-react with ZIKV proteins is a relevant feature for the validation of the diagnostic tools based on the NS1 protein. In pioneering work by Falconar et al. [8], the immunogenic regions of DENV2 NS1 employing mAbs were extensively analyzed. Recently, certain studies have been using new methods to predict the binding epitopes of proteins to specific antibodies [13,14]. This approach was also applied to identify binding epitopes of DENV NS1 protein serotypes [15,16,17]. Also, the crystal structure of the DENV2 NS1 protein (PDB code: 4O6B) has been solved in both dimeric and hexameric configurations [6], which provides a useful guideline for the selection of potential epitopes for therapy and vaccine strategies. In the present study, recombinant DENV2 NS1 was used to immunize mice and generate murine mAbs. Four mAbs were isolated, purified, characterized and tested for reactivity with native NS1 produced by all DENV serotypes in Vero-infected cells and also for cross-reactivity with ZIKV NS1. 2. Results 2.1. Isolation and Characterization of NS1-Specific DENV mAbs Fusion of popliteal lymph node cells, from mice immunized with DENV2 rNS1, with a non-Ig-secreting or synthesizing collection derived from a cell collection produced by fusing a BALB/c mouse spleen cell and the mouse myeloma P3X63Ag8 (SP2/O-Ag14) mouse myeloma cells, generated 25 secretory hybridomas. Among them, four hybridomas were selected by enzyme-linked immunosorbent assay (ELISA) and sub cloned by limiting dilution and named as 4F6, 4H2, 4H1BC, and 2H5. The clones were expanded, supernatants collected and mAbs purified for further characterization. Accordingly, mAbs 4F6 and 4H2 were characterized as IgG2a (immunoglobulin G), and 2H5 and 4H1BC as IgG1. The affinity constants were comparable (10?8 M) as well as their reactivity with and limits of detection of NS1 (Table 1). Table 1 Characteristics of the monoclonal antibodies (mAbs) against dengue computer virus (DENV) nonstructural protein 1 (NS1). < 0.01; ** < 0.1; * < 0.5). 2.2. Detection of Native DENV2 NS1 and Epitope Mapping After selection, mAbs were tested by immunofluorescence assays using fixed DENV2-infected Vero cells. All four mAbs acknowledged the native viral NS1 expressed in infected cells, as shown in Physique 2. To localize the specific mAbs binding HS-10296 hydrochloride sites/epitopes, peptide mapping array experiments were performed (Physique S2). The results showed that mAb 4F6 reacted with the peptide corresponding to the sequence 25VHTWTEQYKFQPES38 of NS1 (Table 1), which is located in an external loop of the protein 3D structure (Physique 3). The 4H2 mAb acknowledged the peptide corresponding to the sequence 127ELHNQTFLIDGPETAEC143 of NS1 (Table 1), which is located in beta-sheets in an external region of the protein 3D structure (Physique 4). The other two mAbs (2H5 and 4H1BC) showed.