Location: Foreign Animal Disease Research
Project Number: 8064-32000-060-17-I
Project Type: Interagency Reimbursable Agreement
Start Date: Aug 13, 2018
End Date: Sep 30, 2020
The objective of this research project is to rationally develop an antigenically marker vaccine strain of African Swine Fever Virus harboring the antibody immune response that also includes the capability to differentiate vaccinated from infected animals (DIVA). Specific objectives include: 1. Identify immunogenic ASFV as putative antigenic marker targets. Non-essential ASFV genes encoding for highly immunogenic viral proteins will be identified. These viral proteins must be strongly recognized by the host antibody response during the process of virus infection. 2. Develop recombinant ASF viruses lacking the proteins under study. Recombinant ASFV lacking each of the proteins previously identified will be produced using as virus template ASFV strains already attenuated by genetic manipulation in our laboratory. These strains already have been successfully tested as vaccine candidates preventing disease caused by the epidemiologically relevant strain Georgia 2007. Therefore, we will attempt to produce viable vaccine candidates able to protect against Georgia 2007 virulent strains that harbor a potential negative antigenic marker. 3. Evaluation of protective efficacy and immunogenic functionality of the antigenically marker viruses. Selected vaccine virus candidates will be evaluated in their ability to still induce protection against virulent challenge and to induce a differential immune response compared with that induced by virus strains harboring a wild type antigenic phenotype.
1. The identification of antigenic ASFV proteins will be conducted by peptide scanning using immune microarray. The predicted full length amino acid sequence of all ASFV open reading frames (ORFs) will be commercially synthesized and placed in immune microarrays. Initially, ORFs already know to be essential for virus growth will be excluded of the screen since those genes cannot be deleted from a replicating ASFV. ASFV proteins encoding for immunodominating epitopes will be identified in immune microarray using a pool of sera of pigs infected with attenuated strains of different ASFV isolates. The subsequent evaluation of immunogenicity of selected proteins will be assessed, cloned, tagged and purified. Reactivity to each of the selected proteins will be assessed against selected sera. Those proteins producing the earliest, strongest and longer antibody responses will be selected for further work. 2. Develop recombinant ASF viruses lacking the proteins under study. Previously identified proteins will be produced using as virus template ASFV strains already attenuated by genetic manipulation. These strains already have been successfully tested as vaccine candidates preventing disease caused by the epidemiologically relevant strain Georgia 2007. A recombinant plasmid will be designed and constructed targeting each of the selected ASFV ORF and commercially synthesized. A recombinant virus will then be developed utilizing a specific gene deletion. Two different vaccine strains will be used as parental viruses, Georgia delta9GL/deltaUK and BA71 CReSA-deltaCD2/PI. Both viruses have been showed to efficiently induce protection against the challenge with ASFV Georgia 07 strain. The resulting recombinant viruses will then be purified and genetically characterized. The growth characteristics will be evaluated and their ability to grow in swine macrophages will be evaluated and compared with that of the parental viruses. Only recombinant viruses showing acceptable replication rates will be selected for the following step. 3. An evaluation of the protective efficacy and immunogenic functionality of resulting antigenically marked virus candidates will be conducted to assess their ability to induce protection against virulent challenge and to induce a differential immune response compared with that induced by virus strains harboring a wild type antigenic phenotype. In vivo challenge study will be performed using ASFV Georgia07 strain. Those recombinant viruses still being able to mediate protection will be consider as vaccine candidates with DIVA capability. An assessment of the functionality of the negative antigenic marker will also be conducted.for their reactivity to the corresponding protein encoded for the deleted ORF. Proteins cloned, expressed and purified as denoted in objective 1 will be used as detecting antigens in direct ELISA to assess the presence/absence of antibody response in the vaccinated/challenged animals. Recombinant viruses able to elicit solid differential antibody response to their negative antigenic marker, early times post vaccination/challenge will be selected as live attenuated vaccine candidates.