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United States Department of Agriculture

Agricultural Research Service

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Location: Foreign Animal Disease Research

2013 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.

3.Progress Report:

The main objective of this research project was to determine if native and/or modified forms of Classical Swine Fever Virus (CSFV) proteins present in the surface of the virion were able to induce a rapid and protective immune response against CSFV. Two approaches were conducted;.
1)an assessment of the capability each CSFV envelope proteins E0, E1, and E2 for eliciting a protective immune response against the disease, and.
2)introduction of modifications into CSFV envelope proteins to increase their capability of inducing an effective early protection against the virus. All three proteins (E0, E1, and E2) were produced using a baculovirus/insect cell system to obtain high protein yields. Proteins were purified, tested for purity, combined with adjuvant , and used to immunize 30-40 lbs pigs. Animals were inoculated via intra-muscular route (IM) receiving boosters at 28, 42, and 56 days post primo inoculation. All pigs were challenged intra-nasally (IN) 1 week after the last boost. Using this immunization scheme only those animals that were immunized with E0 or E2 elicited high antibody titers and survived the challenge. E1 protein did not elicit a detectable antibody response nor did it elicit a protective immunity in pigs against CSFV. All animals succumbed to challenge. To further understand the protective efficacy of E0 and E2 proteins groups of 4 week-old swine were inoculated with 1 or 2 doses of each protein. These animals were then exposed to CSFV IN at 21 days after single or last inoculation. All the animals survived the infection, suggesting that lesser doses are sufficient to induce protection. Similarly animals receiving three doses of the proteins and challenged 1 week after last inoculation were protected against CSFV, suggesting that strong response can be induced shortly after 3 inoculations of both proteins. After the protective efficacy of wild-type E0 and E2 proteins was established, modifications were introduced to these proteins aimed to enhance the immune response against CSFV. E0 and E2 proteins were fused to flagellin; or to single chain anti-class II antibody sequences. Production and purification of modified proteins had to be adjusted from original procedures to obtain high yields of modified proteins. Proteins were then used to assess their capability to induce an antibody response in pigs. Unexpectedly, inoculation of these proteins into swine did not elicit a measurable antibody response suggesting that as formulated E0 and E2 will not induce an efficient immune response in pigs against CSFV. No technologies were transferred in FY 2013. No publications were produced during FY 2013. This collaborative research project, which was established to support a National Pork Board grant, terminated in March 2013.

Last Modified: 4/20/2014
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