Dominant epitopes of cross-reactive anti-domain III human antibody response change from early to late convalescence of infection with dengue virus.

Cross-neutralizing activity of human antibody response against Dengue virus complex (DENV) changes importantly over time. Domain III (DIII) of the envelope protein of DENV elicits a potently neutralizing and mostly type-specific IgG response. We used sera from 24 individuals from early- or late convalescence of DENV1 infection to investigate the evolution of anti-DIII human IgG with the time lapse since the infection. We evaluated the correlation between the serotype-specific reactivity against recombinant DIII proteins and the neutralization capacity against the four serotypes, and examined its behavior with the time of convalescence. Also, we use a library of 71 alanine mutants of surface-exposed amino acid residues to investigate the dominant epitopes. In early convalescence anti-DIII titers and potency of virus neutralization were positively associated with correlation coefficients from 0.82 to 1.0 for the four serotypes. For late convalescence, a positive correlation (r = 0.69) was found only for DENV1. The dominant epitope of the type-specific response is centered in the FG-loop (G383, E384, and K385) and includes most of the lateral ridge. The dominant epitope of the anti-DIII cross-reactive IgG in secondary infections shifts from the A-strand during early convalescence to a site centered in residues E314-H317 of the AB-loop and I352-E368 of the DI/DIII interface, in late convalescence. An immunoassay based on the detection of IgG anti-DIII response can be implemented for detection of infecting serotype in diagnosis of DENV infection, either primary or secondary. Human dominant epitopes of the cross-reactive circulating antibodies change with time of convalescence.

A time-efficient workflow to purify a library of alanine mutants of surface-exposed residues of the domain III of the envelope protein of dengue virus serotype 1.

Dengue complex is formed by four viral serotypes that cause the disease of the same name. Dengue is the arthropod-borne disease with the highest incidence worldwide. The envelope glycoprotein comprises three structural domains. The domain III (DIII) induces neutralizing antibodies and is involved in the interactions with soluble plasma factors from human host. Recombinant DIII proteins have been used as analytical tools for the characterization of virus-host interactions and have been evaluated as sub-unit vaccines. Here, we report a purification procedure of recombinant DIII protein and seventy-four alanine mutants refolding by size exclusion chromatography that allows obtaining highly homogeneous protein preparations and suitable for efficient purification and folding check. Four positions are identified that significantly affect either the protein expression or folding of recombinant DIIIE1, K310, G304, D330 and P332.