Mapping of B-cell epitopic sites and delineation of functional domains on the hemagglutinin-neuraminidase protein of peste des petits ruminants virus.
A recombinant baculovirus expressing membrane bound form of hemagglutinin-neuraminidase (HN) protein of peste des petits ruminants virus (PPRV) was employed to generate monoclonal antibodies (mAbs) against PPRV-HN protein. Four different mAbs were employed for mapping of regions on HN carrying B-cell epitopes using deletion mutants of PPRV-HN and RPV-H proteins expressed in Escherichia coli as well as PPRV-HN deletion proteins expressed transiently in mammalian cells. The immuno-reactivity pattern indicated that all mAbs bind to two discontinuous regions of amino acid sequence 263-368 and 538-609 and hence the epitopes identified are conformation-dependent. The binding regions for three mAbs were shown to be immunodominant employing competitive ELISA with vaccinated sheep sera. Delineation of functional domains on PPRV-HN was carried out by assessing the ability of these mAbs to inhibit neuramindase activity and hemagglutination activity. Two mAbs inhibited NA activity by more than 63% with substrate N-acetyl neuraminolactose, while with Fetuin one mAb showed inhibition of NA activity (95%). Of the three antigenic sites identified based on competitive inhibition assay, site 2 could be antigenically separated into 2a and 2b based on inhibition properties. All the four mAbs are virus neutralizing and recognized PPRV-HN in immunofluorescence assay.
Renukaradhya
G J
GJ
Project Directorate on Animal Disease Monitoring and Surveillance (PD-ADMAS), Hebbal, Bangalore 560024, India.
Sinnathamby
G
G
Seth
Shaguna
S
Rajasekhar
M
M
Shaila
M S
MS
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology; D002478:Cells, Cultured; D002645:Chickens; D002522:Chlorocebus aethiops; D018604:Epitope Mapping; D018985:Epitopes, B-Lymphocyte / Q000276:immunology*; D017353:Gene Deletion; D015707:HN Protein / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D006385:Hemagglutination Inhibition Tests; D006386:Hemagglutination Tests; D006825:Hybridomas; D016056:Immunodominant Epitopes; D018107:Peste-des-petits-ruminants virus / Q000276:immunology*; D011487:Protein Conformation; D018411:Spodoptera; D014709:Vero Cells
D000911:Antibodies, Monoclonal; D018985:Epitopes, B-Lymphocyte; D015707:HN Protein; D016056:Immunodominant Epitopes