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. 1996 Dec;70(12):8492–8501. doi: 10.1128/jvi.70.12.8492-8501.1996

Immunogenicity of an aphthovirus chimera of the glycoprotein of vesicular stomatitis virus.

P R Grigera 1, M Garcia-Briones 1, O Periolo 1, J L la Torre 1, R R Wagner 1
PMCID: PMC190940  PMID: 8970972

Abstract

An oligodeoxynucleotide coding for amino acids 139 through 149 of antigenic site A (ASA) of the VP1 capsid protein of the foot-and-mouth disease virus C3 serotype (FMDV C3) was inserted into three different in-frame sites of the vesicular stomatitis virus New Jersey serotype (VSV-NJ) glycoprotein (G) gene cDNA present in plasmid pKG97 under control of the bacteriophage T7 polymerase promoter. Transfection of these plasmids into CV1 cells coinfected with the T7 polymerase-expressing vaccinia virus recombinant vTF1-6,2 resulted in expression of chimeric proteins efficiently reactive with both anti-FMDV and anti-VSV G antibodies. However, in vitro translation of transcripts of these VSV-G/FMDV-ASA chimeric plasmids resulted in proteins that were recognized by anti-G serum but not by anti-FMDV serum, indicating a requirement for in vivo conformation to expose the ASA antigenic determinant. Insertion of DNA coding for a dimer of the ASA unidecapeptide between the VSV-NJ G gene region coding for amino acids 160 and 161 gave rise to a chimeric ASA-dimer protein designated GF2d, which reacted twice as strongly with anti-FMDV antibody as did chimeric proteins in which the ASA monomer was inserted in the same position or two other G-gene positions. For even greater expression of chimeric VSV-G/FMDV-ASA proteins, plasmid pGF2d and a deletion mutant p(delta)GF2d (G protein deleted of 324 C-terminal amino acids) were inserted into baculovirus vectors expressing chimeric proteins GF2d-bac and deltaGF2d-bac produced in Sf9 insect cells. Mice vaccinated with three booster injections of 30 microg each of partially purified GF2d-bac protein responded by enzyme-linked immunosorbent assay with FMDV antibody titers of 1,000 units, and those injected with equivalent amounts of deltaGF2d-bac protein showed serum titers of up to 10,000 units. Particularly impressive were FMDV neutralizing antibody titers in serum of mice vaccinated with deltaGF2d-bac protein, which approached those in the sera of mice vaccinated with three 1-microg doses of native FMDV virions. Despite excellent reactivity with native FMDV, the anti-deltaGF2d-bac antibody present in vaccinated mouse serum showed no capacity to bind to sodium dodecyl sulfate (SDS)-denatured FMDV virions and only minimal reactivity with VP1 protein by Western blotting (immunoblotting) after SDS-polyacrylamide gel electrophoresis. It was also shown in a competitive binding assay that a synthetic ASA unidecapeptide, up to concentrations of 200 microg/ml, was quite limited in its ability to inhibit binding of anti-deltaGF2-bac antibody to native FMDV virions. These results suggest that the chimeric VSV-G/FMDV-ASA proteins mimic the capacity of FMDV to raise and react with neutralizing antibodies to a restricted number of ASA conformations present on the surface of native FMDV particles.

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Selected References

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