2012 Annual Report
1a.Objectives (from AD-416):
This research project seeks to evaluate native and modified forms of Classical Swine Fever (CSF) envelope proteins for their capacity to induce rapid protective immune responses against CSF virus. Specific objectives included the creation of recombinant baculoviruses to synthesize native and modified forms of CSFV strain Brescia E0, E1 and E2 glycoproteins. These baculovirus will be used to express massive amounts of these proteins in order to assess their immunogenicity in swine.
1b.Approach (from AD-416):
The assessment of each of the CSF virus structural proteins E0, E1 and E2 in the elicitation of a protective immune response in swine against CSF infection will be conducted. Genetic constructs will be designed and tested to determine levels of immunogenicity. Subsequent studies will be conducted to determine the levels of glycosylation needed to produce enhanced immune response.
We will synthesize several forms of His-tagged CSFV E0, E1, or E2 envelope proteins from insect cells (Spodoptera frugiperda derived, Sf9) infected with recombinant baculoviruses. To assess the immunogenicity and antigenicity of these expressed proteins, forty lbs swine will be serially immunized with purified proteins followed by an intranasal challenge with 105 TCID50 of highly virulent strain Brescia (BICv) 7 days after the last inoculation. Antigenicity will be determined by measuring antibody response of immunized animals using commercially available ELISA tests and by virus neutralization. The ability of modified forms of CSFV envelope proteins to induce an antibody response will be compared relative to the antibody response induced by non-modified forms of the proteins. Immunogenicity will be determined by assessing swine survival after challenge with virulent CSFV s BICv relative to non-immunized control animals.
Wild type and mutant genes of the three CSFV envelope proteins (E0, E1, and E2) will be synthesized. Recombinant baculoviruses will be produced and expressed and the resulting proteins purified. We will design and perform the antigenicity and immunogenicity studies in conjunction with the protein production.
As a result of this research, CSFV proteins or combination of CSFV proteins that confer a robust immunity against CSFV will be identified. By using different CSFV proteins, both in their native as well as in their modified forms, their antigenicity and immunogenicity may be improved. These proteins will be safe and efficient against CSFV.
In FY 2012, the individual effect of each of CSFV structural proteins Erns, E1, and E2 on the induction of humoral response and protection against the infection was analyzed. Native version of each of these proteins was produced in baculovirus. Infected insect cells were used to analyze the expression of CSFV envelope proteins by Western blot. The fractions collected during purification were analyzed by Western blot. In vivo challenge studies were conducted using thirty to forty lbs pigs that were vaccinated intramuscularly with purified proteins followed by an intranasal challenge with highly virulent strain Brescia (BICv) 7 days after the last inoculation. In each experiment, a control group was mock vaccinated and received the challenge as previously described. Samples were collected and the viral loads analyzied by real time RT-PCR. Serum samples were collected throughout the experiment and the presence of CSFV Erns and E2 antibodies were assessed using commercial ELISA test kits. Neutralizing antibody response was assessed using virus neutralization assays. Results demonstrated that as expected, E2 is able to induce a neutralizing antibody response as well as to protect swine against the virulent challenge. Interestingly, purified Erns was also efficient in raising a neutralizing antibody response and protection against the challenge.
Modifications to the Erns and E2 structure were made. Work with E1 was discontinued due to its complete inability to induce protection in its native form. Cells of the innate immune system recognize conserved pathogen-associated molecular patterns (PAMPs) through Toll-like receptors (TLRs). Flagellin is recognized by TLR5 that signals through TLR5/TLR4, likely activating the interferon pathway and is therefore a potent immune activator that rapidly induces expression of proinflammatory cytokines, chemokines, and costimulatory molecules. Flagellin gene was kindly provided by collaborators from INIA, Spain and was synthetically fused to the His-tagged Erns and E2. We used a recombinant single chain antibody attempting to target Erns and E2 to antigen presenting cells in the immunized animals. Recombinant single chain antibody (scFV) directed an invariant epitope of the porcine MHC II DR, derived from monoclonal antibody (mAb) 1F12, was expressed as a fusion product along with Erns and E2 Analysis of protein expression in Western blot using a commercial anti-HIS antibody was conducted confirming the expression of the fusion proteins scFV Erns and scFVE2 .
Publications for FY 12 include:
Gavrilov BK, Rogers K, Fernandez-Sainz IJ, Holinka LG, Borca MV, Risatti GR. (2011). Effects of glycosylation on antigenicity and immunogenicity of classical swine fever virus envelope proteins. Virology 420(2):135-45.