Epitope detection in the envelope of intracellular naked orthopox viruses and identification of encoding genes.
Monoclonal antibodies (MAbs) were generated against vaccinia virus, cowpox virus KR2 Brighton, monkeypox virus Copenhagen, or ectromelia virus. Pairwise epitope specificity studies by competition ELISAs identified 23 distinct antigenic sites in 19 different orthopox virus strains. Six epitopes were completely independent of each other, and 17 closely related antigenic sites formed three separate epitope complexes. As shown by immunogold electron microscopy (ELMI), all MAbs reacted with epitopes in the envelope of intracellular naked virus, 16 MAbs recognized proteins of 32, 30, 16 or 14 kDa in Western blotting (WB), and 9 MAbs neutralized virus infectivity. In rabbitpox virus (RPV) 18 epitopes were detected. A lambda gt11 expression library of RPV DNA was screened with the corresponding 18 MAbs. Fourteen recombinant bacteriophage clones (ph) were isolated. Cross-hybridizations of phage and RPV DNA demonstrated a reaction with the HindIII A, HindIII D, or HindIII H fragments, respectively. DNA of ph3D was related to the A25L gene, which corresponds to the A-type inclusion body gene of cowpox virus. Two phage clones contained sequences of the 14-kDa fusion protein gene (A27L gene). Ph1A contained nearly the entire 14-kDa gene encoding 4 neutralizing (neutr) and 2 nonneutr epitopes. Ph5, expressing only half of this gene product, encoded 1 nonneutr epitope. The fusion protein of vaccinia virus MVA was isolated by immune-affinity chromatography with a neutr. catching MAb. The protein formed hollow rods (ELMI) and the 6 antigenic sites that were present were identical to those expressed by Escherichia coli infected with ph1A. WB detection with a polyclonal hyperimmune serum detected protein bands of 54, 32, 30, 16, and 14 kDa. The catching MAb bound only to a 16-kDa band. The purified fusion protein induced neutralizing antibodies in mice and rabbits.
Czerny
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Institute of Medical Microbiology, Infectious and Epidemic Diseases, Veterinary Faculty, Ludwig-Maximillans-University, Munich, Germany.
Johann
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Hölzle
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Meyer
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000096:biosynthesis / Q000276:immunology; D000956:Antigens, Viral / Q000235:genetics*; D001667:Binding, Competitive; D002478:Cells, Cultured; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000235:genetics*; D005814:Genes, Viral / Q000276:immunology*; D008954:Models, Biological; D009500:Neutralization Tests; D018155:Orthopoxvirus / Q000235:genetics* / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D013045:Species Specificity; D014616:Vaccinia virus / Q000737:chemistry; D014760:Viral Fusion Proteins / Q000276:immunology / Q000302:isolation & purification / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D000939:Epitopes; D011994:Recombinant Proteins; D014760:Viral Fusion Proteins