Western blot analysis of antigens specifically recognized by natural immune responses of patients with Japanese encephalitis infections.

Abstract

Specific recognition of antigenic proteins of Japanese encephalitis virus (JEV) by JE patients was investigated by using non-reducing and reducing Western immunoblot analysis. Under non-reducing conditions, the profile of JEV proteins recognized comprised E (52 kDa), NS1 (45 and 41 kDa), NS3 (66.2 kDa) and NS5 (103 and 97.4 kDa). When recognition patterns of sera from JE and dengue patients were compared, only slight differences between JE and dengue sera were found (under non-reducing conditions), involving only the 66.2 kDa protein: to this protein, JE sera exhibited greater reactivity, but not in greater frequency, than did dengue sera. In contrast, cerebrospinal fluid (CSF) from JE patients showed more differences from JE sera: CSF antibody lacked recognition of the 41 kDa protein and had lower frequencies, as well as less reactivities to several other proteins. These results suggested that restricted populations of lymphocytes were localized in the central nervous system of JE patients. The effect of reducing agent (2 beta-mercaptoethanol) on the recognition patterns of those groups of sera was also analysed: the reducing agent affected all the proteins mentioned above, however, the effects were not uniform. It is proposed that JE and dengue sera may recognize different epitopes on some or all of these proteins. Such differences cannot be detected by Western immunoblot analysis, but it would be feasible to test this hypothesis using epitope mapping with synthetic peptides in a multi-pin ELISA. Analysis in this fine detail is essential for designing improved JE vaccines.