Natural immunogenic properties of bioinformatically predicted linear B-cell epitopes of dengue envelope and pre-membrane proteins.

BACKGROUND: The natural antibody responses to B-cell epitopes from dengue structural proteins were assessed using immune sera from people having well-defined past dengue infections with one of the four serotypes. METHOD: Based on an immune-computational analysis previously conducted, nineteen epitopes from the envelope (E) and eight epitopes from pre-membrane (prM), which were more than 50% conserved across all the four DENV serotypes, were selected. Peptides to represent these B-cell epitopes were obtained from commercially available arrays, and were subjected to enzyme linked immunosorbent assay with sera obtained from dengue seropositive healthy volunteers (DENV1 n = 12: DENV2 n = 12: DENV3 n = 12 and DENV4 n = 12), and 10 dengue seronegative healthy volunteers from Sri Lanka. The cut-off value for the positive antibody response was set by taking the mean response of a peptide to the negative sera plus three standard deviations. The peptides (N = 7) showing the broad immune responses were used to generate antibodies in three mice (Balb/c) batches. The mice antisera were then subjected to microneutralization assays against all the four DENV serotypes. An EC50 viral neutralization ≥ 40 times the serum dilution was considered as neutralizing. RESULTS: Five of the E-peptide and two prM peptides were recognised by most individuls exposed to infections with each of the four serotypes, showing a serotype cross-reactive broad antibody response. The mice immune sera against the peptides representing the five E protein epitopes neutralized all the four DENV serotypes. Two of these five epitopes are from the Domain II, whereas one of them includes the whole bc-loop region. CONCLUSION: The antibody responses of highly conserved epitopes across the serotypes, were broadly responsive with sera of all four DENV serotypes collected from individuals infected with only one DENV serotype. Weakly conserved epitopes showed rather specific antibody responses dominated by one or few serotypes.

Natural antibody responses to the capsid protein in sera of Dengue infected patients from Sri Lanka.

This study aims to characterize the antigenicity of the Capsid (C) protein and the human antibody responses to C protein from the four dengue virus (DENV) serotypes. Parker hydrophilicity prediction, Emini surface accessibility prediction and Karplus & Schulz flexibility predictions were used to bioinformatically characterize antigenicity. The human antibody response to C protein was assessed by ELISA using immune sera and an array of overlapping DENV2 C peptides. DENV2 C protein peptides P1 (located on C protein at 2-18 a.a), P11 (79-95 a.a) and P12 (86-101 a.a) were recognized by most individuals exposed to infections with only one of the 4 DENV serotypes as well as people exposed to infections with two serotypes. These conserved peptide epitopes are located on the amino (1-40 a.a) and carboxy (70-100 a.a) terminal regions of C protein, which were predicted to be antigenic using different bioinformatic tools. DENV2 C peptide P6 (39-56 a.a) was recognized by all individuals exposed to DENV2 infections, some individuals exposed to DENV4 infections and none of the individuals exposed to DENV1 or 3 infections. Thus, unlike C peptides P1, P11 and P12, which contain epitopes, recognized by DENV serotype cross-reactive antibodies, DENV2 peptide P6 contains an epitope that is preferentially recognized by antibodies in people exposed to this serotype compared to other serotypes. We discuss our results in the context of the known structure of C protein and recent work on the human B-cell response to DENV infection.