Rationally designed viral capsid improves immune response against PCV2 and protects against evolving new strains.
(You may click on the images below for an enlargement.)
Immune Refocused PCV2 Vaccine
Discovery of protective and non-protective epitopes:
Large pools (A) and smaller pools (B) of synthetic peptides representing potential neutralizing and non-neutralizing epitopes were used in a competition assay with sera from infected animals.
Peptides that reduce neutralization as shown by the bar extending downward identify neutralizing epitopes. Peptides that increase neutralization as shown by the bars extending upward identify non-neutralizing epitopes.
Epitope mapping on capsid structure:
Amino acid residues participating in epitopes are mapped to the capsid monomer (A and B, 3R0R.pdb) and the whole virus particle (C and D, 6E2R.pdb).
Immune refocusing mutations were introduced into two epitopes using a amino acid substitution strategy to modify the antigenicity of the epitopes.
In addition, sequences encoding a marker peptide were cloned at the 5’ end of the capsid gene to distinguish between immunized and infected animals.
Experimental Design:
An infectious plasmid clone containing the genome of a PCV2b strain was re-engineered to contain two IRT mutation sites and a N-terminal DIVA marker peptide (in red). The marker peptide allows differentiation between immunized and infected animals.
The Clone was used to derive virus which was used to infect the animals. Animals were later challenged with a PCV2d strain to assess cross-protection.
Virus Neutralization Responses:
Sera taken 14 and 28 days after immunization of pigs with the IRT vaccine (rPCV2-MLV), a commercial vaccine, or mock vaccine were tested for neutralization of PCV2a, 2b, and 2d viruses.
The asterisks (*) denote significant improvement from the mock vaccine sera.
The ampersand (@) denotes improvements of the IRT vaccine over the commercial vaccine.
Antibody response to the mutated epitopes:
Surface plasmon resonance shows reduced antibody recognition to Epitopes A and B (sites of IRT mutations) in animals immunized with the IRT vaccine, rPCV2-Vac.
Antibody responses to Immunogenic marker:
ELISA data shows that animals immunized with the IRT vaccine
generate antibodies to the incorporated marker peptide
from Neospora canium SRS2 protein.
Sera from the IRT-rPCV2 recognizes the marker peptide.
rPCV2-Vac reduces tissue lesions in challenged pigs:
a = significant improvement over unvaccinated. b = significant improvement over commercial vaccine.
(p <0.05, Mann-Whitney U test).
