Mapping of functional epitopes of Japanese encephalitis virus using monoclonal antibodies.

Epitopes involved in the important functions, hemagglutination (HA) and neutralization (NT), were mapped on Japanese encephalitis (JE) virus proteins by using monoclonal antibodies (MAbs). Fourteen MAbs raised against Nakayama-Yoken strain of JE virus characterized by hemagglutination inhibition (HI) and plaque reduction neutralization test (PRNT) were used to map the epitopes on the JE proteins by Western blot analysis in which non-reducing conditions were used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). With these MAbs, at least 8 functional epitopes were demonstrated comprising (i) epitopes recognized by 5 MAbs which gave strong HI but weak NT activities and were mapped on the envelope (E) 53 kDa protein; (ii) epitopes recognized by 2 MAbs which showed weak HI but strong NT activities and were mapped also on the E protein; (iii) epitopes recognized by 2 MAbs which possessed weak HI but no NT activities and were mapped on the E protein; (iv) an epitope recognized by 1 MAb which gave weak NT and no HI activities and was mapped on the nonstructural protein 5 (NS5); (v) an epitope recognized by 1 MAb which showed activities similar to (i) but was mapped on both E and NS5; (vi) an epitope recognized by 1 MAb which had high activities to both HI and NT and was mapped on E and NS5; (vii and viii) epitopes recognized by 1 MAb which also gave low HI but high NT, and strong HI as well as strong NT activities respectively, but their location could not be demonstrated by SDS-PAGE under non-reducing condition.(ABSTRACT TRUNCATED AT 250 WORDS)

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

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.