BackgroundOrthohantavirus infection is a neglected global health problem affecting approximately 200,000 people/year, spread by rodent hosts and associated to fatal human diseases, such as hemorrhagic fever with renal syndrome (HFRS) and orthohantavirus cardiopulmonary syndrome (HCPS). Circulation of HFRS-associated orthohantaviruses, such as Seoul, Gou, Amur, Dobrava and Hantaan, are supposed to be restricted to Eurasian countries even though their hosts can be a worldwide distribution. Few confirmed HFRS orthohantavirus infections in humans have been reported in American countries, but due to lower medical awareness of the symptoms of this zoonosis, it could be associated to viral underreporting or to misdiagnosis with several tropical hemorrhagic diseases. Serological evidence of orthohantavirus infections, using enzyme-linked immunosorbent assay for the presence of immunoglobulin M and G against recombinant nucleoprotein protein, remains as an essential assay for viral surveillance. In this study, we aimed to identify in silico immunogenic B-cell linear epitopes present on orthohantavirus nucleoprotein that are exclusive to HFRS-related species.Methodology/Principal findingsIn silico analysis were performed using Seoul orthohantavirus nucleoprotein (SHNP) sequence as a model. Linear B-cell-epitopes on SHNP and its immunogenicity were predicted by BepiPred-2.0 and Vaxijen algorithms, respectively. The conservancy of predicted epitopes was compared with the most clinically relevant HFRS or HCPS-associated orthohantavirus, aiming to identify specific sequences from HFRS-orthohantavirus. Peptide validation was carried out by ELISA using Balb/c mice sera immunized with purified recombinant rSHNP. Peptides cross-reactivity against HCPS orthohantavirus were evaluated using immunized sera from mice injected with recombinant Juquitiba orthohantavirus nucleoprotein (rJHNP).Conclusion/SignificanceIn silico analysis revealed nine potential immunogenic linear B-cell epitopes from SHNP; among them, SHNP(G72-D110) and SHNP(P251-D264) showed a high degree of sequence conservation among HFRS-related orthohantavirus and were experimentally validated against rSHNP-IMS and negatively validated against rJHNP-IMS. Taken together, we identified and validated two potential antigenic B-cell epitopes on SHNP, which were conserved among HFRS-associated orthohantavirus and could be applied to the development of novel immunodiagnostic tools for orthohantavirus surveillance.

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Zika virus became a major public health problem in early 2015, when cases of Guillain–Barré syndrome and microcephaly were associated with viral infection. Currently, ZIKV is endemic in all tropical areas of the world, and the chance for future Zika epidemics remains very real and accurate diagnosis is crucial. The aim of this work was to select specific ssDNA aptamers that bind to the entire Zika virus and can be used to compose specific diagnostics, without cross-reactivity with other flaviviruses. Zika virus was cultivated in Vero cells and used as a target for aptamer selection. Aptamers specific for the ZIKV were selected using whole-virus SELEX, with counterselection for other flavivirus. Secondary and tertiary structures were evaluated and the molecular anchoring between the aptamers and target were simulated by the HDOCK server. Aptamer interaction was evaluated by ELISA/ELASA and the dissociation constant (Kd) was calculated by thermophoresis. Four ZIKV-specific aptamers were selected. The best two were further characterized and proved to be specific for ZIKV. Aptamers are capable of binding specifically to the ZIKV and differentiate from Dengue virus. The aptamers selected in this work can be used as capture agents in the composition of diagnostic tests to specifically detect ZIKV infection.

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The successful introduction of antibody-based immunotherapies into the arsenal of treatments for cancer patients has been reinvigorated by antibody engineering technology. New antitumor strategies include enhancement of T-cell responses provided by monoclonal antibody activation of costimulatory molecules present on T-cell surface. OX40, a member of the TNF receptor superfamily (TNRFS4), is a key T-cell costimulator and a promising cancer immunotherapy target. Currently, several pharmaceutical companies have invested in clinical studies using mAb anti-OX40 for cancer and/or autoimmune treatment. In this context, in silico antibodies engineering emerges as a promising approach to develop novel biopharmaceuticals with improved specificity and affinity. Objective: To develop an in silico strategy for novel biopharmaceutical development, using OX40 mAb as a model. Methodology: Heavy and light chain amino acid sequences of anti-OX40 antibody were obtained from a patent prospection in the Integrity database (patent number: WO2018/178074). The human-scFv (single-chain variable fragment) model was constructed by comparative molecular modeling through the Modeller software. Predicted model quality was evaluated using Molprobity and Verify3D servers. ScFv model was subjected to molecular docking against the OX40 structure (PDB: 2HEV) on Cluspro server. The best complex according to Cluspro parameters was submitted to Robetta Alanine Scanning server to identify hotspots. Point mutations were defined using DUET server and performed on specific amino acids in order to increase the interaction and stability of the complex. Thereafter, a new molecular docking with mutated scFv's was performed in order to compare the results before and after the mutations. Results: Anti-OX40 antibody was constructed by comparative modeling using as model PDB archive 6EHY that presented 84% amino acids sequence homology. An initial docking was performed. The scFv+OX40 complex was submitted to alanine scanning server. Seven important hotspots for the complex stability were identified in the scFv CDR's (complementarity determining region). Besides these, four possible mutation points in the CDR's were also identified. The choice of amino acids substitute was performed on the DUET server. Three substitutions were inferred as possibly being able to increase the complex stability (SER>ASP, SER>MET, ASP>LEU). This step originated seven variants of scFv. After a new docking with all possible mutants scFv's, it was possible to observe an increase in predicted complex interaction in 4 of 7 models tested, according to the parameters defined by the Cluspro server (members and weighted score). In the best result, with only one amino acid mutation, it was observed an increase in the Cluspro score from 124 to 249 members and, from-309,9 to-355,1 (Weighted Score) when compared to native molecule. Conclusion: The proposed workflow resulted in improved predicted antibodies that showed increased in silico stability and better interaction with its correlated antige...