Identification and validation of specific B-cell epitopes of hantaviruses associated to hemorrhagic fever and renal syndrome

Abstract

Background: Orthohantavirus 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 findings: In 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/significance: In 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.

Fig 1. Kinetic of IgG serum title against rSHNP and predicted peptides after experimental immunization with rSHNP.

BALB/c mice (n = 3) were immunized with rSHNP emulsified with CFA at day 0 and rSHNP emulsified with IFA at day 14 and 28. IgG serum titer was evaluated by specific ELISA at days 21 and 35 (first and second bleeding). The IgG serum title was calculated interpolating the value of optical density unit of three times the pre-immune serum (3xPIS) on a linear regression graphic of serum dilution, using the mean optical density of each diluted sera in duplicate.

Fig 2. Evaluation of cross reactivity against rSHNP, rJQNP, SHNP(G72-D110), SHNP(P251-D264) and SHNP(I266-A283) elicited by immunization with rSHNP and rJHNP.

Bars represent the optical densities of mice immunized with rSHNP (red bars, n = 3) and immunized with rJHNP (blue bars, n = 3) and non-immunized mice (green bars, n = 6). Traced line indicates the value of Cut-off, defined as 3 times the mean of non-immune optical densities. Data is presented as floating bars with lines indicate minimum, mean and maximum values. Values higher than cut-off were considered responder to antigen and compared by test-T against other groups. * represents difference between immunized groups and non-immunized mice, while + represents statistical differences between immunized groups.

Fig 3. Predicted SHNP homology model.

(A) SHNP representation as cartoon with predicted linear peptides spatial localization represented by colors where: red—SHNP_(G72-D110); green—SHNP_(P251-D264); yellow—SHNP_(I266-A283). (B) SHNP electrostatic potential surface representing the positive (blue) and negative (red) charges of predicted protein. Peptides sticks representations of SHNP_(G72-D110) (C), SHNP_(P251-D264) (D), SHNP_(I266-A283) (E) and their respective locations and electrostatic characteristics on the protein surface; SHNP_(G72-D110) is in a more electronegative region whereas SHNP_(P251-D264) and SHNP_(I266-A283) are found in regions with more neutral features.