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

Abstract

An oligodeoxynucleotide coding for amino acids 139 through 149 of antigenic site A (ASA) of the VP 1 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⌬GF2d (G protein deleted of 324 C-terminal amino acids) were inserted into baculovirus vectors expressing chimeric proteins GF2d-bac and ⌬GF2d-bac produced in Sf9 insect cells. Mice vaccinated with three booster injections of 30 g 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 ⌬GF2d-bac protein showed serum titers of up to 10,000 units. Particularly impressive were FMDV neutralizing antibody titers in serum of mice vaccinated with ⌬GF2d-bac protein, which approached those in the sera of mice vaccinated with three 1-g doses of native FMDV virions. Despite excellent reactivity with native FMDV, the anti-⌬GF2d-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 VP 1 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 g/ml, was quite limited in its ability to inhibit binding of anti-⌬GF2-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.

Single dilution Avidity-Blocking ELISA as an alternative to the Bovine Viral Diarrhea Virus neutralization test

“…For the Avidity-Blocking ELISA, plates were washed twice with PBS (300 l/well), subsequently washed with PBS-6M Urea (Promega, USA) for 15 min at room temperature and followed by two regular-PBS washing steps. Blocking Detector Antibody (50 l/well, diluted 1:2000) was then added to the plates and incubated for 1 h at 37 • C. Non-specific adsorption was controlled by pre-treating (for 30 min at room temperature) all samples, Blocking Detector Antibody and conjugate with a custom-prepared pre-adsorption solution containing bovine and mouse BVDVnegative (naïve) sera (1 l of undiluted serum for each sample), culture medium supernatant (TNMFH medium, 5 l per sample) from an heterologous Baculovirus infection (titer 1 × 10 7 PFU/ml) (Grigera et al, 1996) and Baculovirus-mock infected SF 21 cells. Following five washing steps with PBS, BtE2-specific antibodies were detected with HRP-labeled anti-guinea pig conjugate diluted 1:1500 and incubated for 1 h at 37 • C. The colorimetric reaction was revealed with chromogen/substrate mixture ABTS/H 2 O 2 [ABTS: 2,2 -azino-bis (3-ethylbenzthiazoline-6-sulphonic acid)] at room temperature, protected from light exposure.…”