Fine mapping of bovine herpesvirus-1 (BHV-1) glycoprotein D (gD) neutralizing epitopes by type-specific monoclonal antibodies and sequence comparison with BHV-5 gD.
Overlapping fragments of the bovine herpesvirus-1 (BHV-1) glycoprotein (gD) ORF were expressed as trpE-gD fusion proteins in Escherichia coli to map linear neutralizing epitopes defined by BHV-1-specific MAbs. The MAbs 3402 and R54 reacted with the expressed fragments on Western blots that located the epitopes between the amino acids 52-126 and 165-216, respectively, of gD. Bovine covalescent sera with high neutralizing antibody titers against BHV-1 reacted with these bacterially expressed proteins containing both of the epitopes. Alignment of these sequences from BHV-1 with the corresponding region of the BHV-5 gD ORF sequences (reported here) identified several amino acid mismatches. Since the MAbs 3402 and R54 neutralize the BHV-1 and not BHV-5, it was presumed that these were important amino acids in defining the epitope. To further localize the neutralizing epitopes, synthetic peptides corresponding to these regions in the BHV-1 gD ORF were tested for their capacity to block monoclonal antibody neutralization of BHV-1 infectivity. The peptides encompassing amino acids 92-106 (3402 epitope) and amino acids 202-213 (R54 epitope) of the BHV-1 gD competed with BHV-1 for the binding by MAbs 3402 and R54, respectively, in a dose-dependent manner. Antisera produced in rabbits to these peptides conjugated to a carrier reacted strongly with a 30-kDa protein by Western blotting and had neutralizing antibody titers against BHV-1.
Abdelmagid
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OY
Department of Pathology and Microbiology, College of Veterinary Medicine, Kansas State University, Manhattan 66506.
Minocha
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HC
Collins
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JK
Chowdhury
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SI
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002417:Cattle; D002478:Cells, Cultured; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D007242:Herpesvirus 1, Bovine / Q000276:immunology*; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D016366:Open Reading Frames; D011993:Recombinant Fusion Proteins / Q000276:immunology; D017386:Sequence Homology, Amino Acid; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C039447:glycoprotein D, Human herpesvirus 1; C091563:glycoprotein D, bovine herpesvirus 5