Non Technical Summary
Classical Swine Fever (CSF) is a highly contagious viral disease of pigs that is present in several countries in central and South America and can soon reach the U.S. unless preventative measures are taken in endemic regions and novel diagnostic methods for detection are developed. The rationale for this proposal is that the current CSF vaccine used in countries with CSF is outdated and poses significant limitations. It is a live-attenduated virus vaccine, which at present cannot be used in disease-free countries such as the U.S. Moreover, it fails to differentiate between infected and vaccinated animals (DIVA), a crucial feature of novel vaccine candidates. The major goals of this project are to develop (i) an efficacious DIVA-compatible CSF vaccine that produces rapid and strong protection against CSF disease in pigs, and (ii) develop novel diagnostic methods for differentiating wild-type infected animals from vaccinated animals. The two Specific Aims are to: (i) develop CSF virus-envelope protein 2 (E2) expressing vaccine vectors and assess them for immune responses and protection against CSF disease in pigs; and (ii) establish diagnostic assays that differentiate wild-type CSF-infected pigs from vaccinated animals using modern antibody-based detection methods. Based on our past experiences and preliminary results, we expect to deliver efficacious and safe CSF vaccine candidates which are able to prevent and control CSF disease in pigs. This study will also establish a novel diagnostic method for CSFV. The results of this research hold immense potential for the swine industry worldwide. By preventing the spread and controlling the devastating CSF disease in swine globally, it could potentially save billions of dollars, making it a crucial investment for the industry.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3599	1101	50%
807	3599	1101	50%

Knowledge Area
807 - Disaster Preparedness, Mitigation, Response, and Recovery; 311 - Animal Diseases;

Subject Of Investigation
3599 - Swine, general/other;

Field Of Science
1101 - Virology;

Goals / Objectives
The overarching goals of this proposal are to develop and establish novel virus-vectored vaccine candidates and diagnostics for CSFV that are DIVA compatible.The Specific Aims are to:Develop virus-vectored vaccine candidates for CSFV using recombinant vesicular stomatitis virus (rVSV) and recombinant orf virus (rORFV) as vector platforms; andEstablish DIVA diagnostics for the CSFV subunit vaccines including indirect and blocking ELISAs.The proposed work includes experimental studies in pigs that will evaluate (i) the immunogenicity; and (ii) the efficacy of the rVSV- and rORFV-vectored vaccine candidates, individually and in combination, for CSFV. Clinical samples from CSFV-vaccinated and -challenged pigs will be used for the establishment of DIVA diagnostic assays.

Project Methods
We propose to develop safe and efficacious virus-vectored subunit vaccine platforms for CSFV (Aim 1) and novel DIVA-compatible diagnostic assays to detect CSF disease rapidly (Aim 2). Two types of recombinant virus vectors, rVSV, and rORFVs, that are non-pathogenic to pigs will be used to generate CSFV vaccine candidates.Aim 1. Develop virus-vectored vaccine candidates for CSFV using recombinant VSV and recombinant ORFV as vector platforms.To generate the rVSV vaccine candidate expressing the CSFV envelope proteins (Erns, E1, E2), the respective genes will be cloned into the VSV vector in place of VSV's G gene (glycoprotein) and expressed as a tri-cistronic cassette. The inclusion of the picornavirus 2A peptide in the sequence will enable the processing of the polyprotein into individual proteins. The vaccine construct and required VSV accessory elements will be transfected into suitable permissive cell lines for multiplication and vaccine use.Viral vectors without CSFV genes will be used as vector controls for vaccination.To generate rORFV-CSF's E vaccine candidate, a similar approach to the above will be used to insert the Erns, E1, and E2 genes into theORFV121locus of the ORFV genome. Required regulatory sequences will also be incorporated for functional expression. Viral vectors without CSFV genes will be used as vector controls for vaccination.Based on our preliminary results, generating two types of recombinant viral vaccine candidates offers advantages for optimizing/evaluating them for enhanced antiviral humoral and T-cell responses, which might offer good protection against CSFV in pigs.The efficacy of the rVSV- and rORFV-vectored vaccine candidates for CSFV will be evaluated individually and in combination in pigs.Aim 2. Establish DIVA diagnostics for the CSFV subunit vaccines.The currently used live-attenuated vaccine for CSFV produces antibodies against its E and NS proteins, and it is not DIVA compatible for diagnostics.Therefore, we propose to develop diagnostic assays that differentiate wild-type infected from vaccinated animals (DIVA). For this purpose, sera produced from Aim 1 and wild-type CSFV-infected pigs will be used. Recombinant CSFV Erns, E1, E2, and NS3 proteins will be expressed in baculovirus andE. coli and purified to raise monoclonal antibodies (mAbs). An indirect and blocking ELISA (Enzyme-linked immunosorbent assay, ELISA) will be developed using the purified E antigens and the NS3 antigen as wild-type infection marker.Animals naturally infected or exposed to CSFV will produce antibodies to the structural E proteins (Erns, E1, E2) and to the non-structural protein NS3. Vaccinated animals will produce antibodies to the structural E proteins (Erns, E1, E2) but not to the non-structural protein NS3. Using this approach, we anticipate being able to differentiate infected from vaccinated animals.