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2017 Annual Report
Objectives
1. Develop intervention strategies to control and eradicate Classical Swine Fever (CSF), including determining immune mechanisms mediating early protection and its application in blocking infection and preventing transmission, and discovering effective CSF vaccine platforms specifically designed for disease control and eradication. Immune mechanisms mediating early protection and its application in blocking infection and preventing transmission will be developed. Studies designed to develop effective CSF vaccine platforms specifically designed for disease control and eradication will be completed.
Sub-Objective 1.i: Determine immune mechanisms mediating early protection and its application in blocking infection and preventing transmission.
Sub-Objective 1.ii: Discover effective CSF vaccine platforms specifically designed for disease control and eradication.
2. Develop intervention strategies to control African Swine Fever (ASF) including identify functional genomics of virus-host determinants of virulence and transmission, determining host mechanisms of ASF immune protection, determining host mechanisms of ASF disease tolerance in wild suids. Additional efforts include the identification of effective ASF vaccine platforms specifically designed for disease control and eradication.
Sub-Objective 2.i: Identify novel virus-host genetic determinants of virulence by systematic screening of almost all previously uncharacterized virus genes.
Sub-Objective 2.ii: Discover effective ASF vaccine platforms specifically designed for disease control and eradication.
Approach
The development of intervention strategies to control Classical Swine Fever will based on research of live attenuated vaccines (LAV). Research will be aimed at determining the virological and immunological factors present in animals that are protected at early times post vaccination, emphasizing on expression profiles of pro-inflammatory chemical mediators (PCMs) produced the first few days after vaccination. The potential therapeutic effect of any PCMs identified will then be assessed. An evaluation of the second generation marker live attenuated vaccine (LAV) FlagT4Gv vaccine will be conducted focusing on toxicity, immunogenicity, protective effect and genetic stability. Efforts will be devoted to develop and optimize serological DIVA (to differentiate infected from vaccinated animals) tests to accompany the FlagT4G strain. Additional vaccine candidates and companion DIVA tests will also be assessed.
To develop strategies to control African Swine Fever Virus (ASFV) studies will be conducted to provide information about the mechanisms of viral replication, virus-host interaction and virulence in the natural host. This information will be used to identify genes that determine the viral virulence that could targeted for deletion of mutation in order to yield attenuated viral strains with potential as vaccine candidates. Identification of candidate target genes will be determined through in silico analysis and/or interaction with host proteins. Full characterization of selected genes will include their interaction with host proteins, production of recombinant ASFV to assess the protein functionality in vitro and virulence during infection in swine. It is anticipated that this research will lead to the identification of genes which may be modified or deleted to created attenuated virus strains for use in vaccine development. In addition to testing attenuated strains containing single gene deletions, strains containing two or more gene deletions/modifications will be produced and assessed to evaluate their ability to protect against homologous and heterologous virulent strains. Efforts will also include the development of a stable cell line capable of supporting ASFV growth for use in commercial vaccine production. As contingency to the LAV approach, experimental subunit vaccines will be tested for their ability to protect against homologous virulent ASFV. The vaccine antigens will be delivered using, a modified vaccinia Ankara virus (MVA) vector co-expressing the ASFV recombinant proteins. These vectors will be assessed in their efficiency of expressing the ASFV recombinant proteins and their immunogenicity and efficacy in protecting swine against challenge.
Progress Report
During FY 2017 work continued on the development of intervention strategies to control and eradicate Classical Swine Fever (CSF) and African Swine Fever (ASF). In the area of CSF, we started the study of the mechanisms mediating early protection after the administration of live attenuated CSFV vaccines. Using an animal model developed in our laboratory based in the use of in house developed vaccine strain FlagT4G, we determined the target organs for vaccine virus replication after its parenteral administration. Importantly, we determined that immunity provided by FlagT4G completely abrogates replication of the virus used to challenge animals as early as 3 days after vaccination. Using the same animal model we initiated the determination of the profiles of locally and systemically produced pro-inflammatory chemical mediators during early times after vaccination. Initial results discovered a significant role of alpha-IFN in early protection induced by vaccination. The transitory unavailability of animal facilities at PIADC impeded to perform any evaluation of the marker LAV FlagT4Gv vaccine strain in swine. New progress has been performed in the discovery of genetic determinants of virulence. We discover and characterize a specific area in major virus structural glycoprotein E2 that mediates critical interaction with the host cell membrane. Genetic modification of this area allowed the development a novel vaccine strain candidate.
In the ASFV area we developed a unique attenuated vaccine strain by specifically deleting two virus genes that are involved in virus virulence. The double gene deletion strength the genetic stability and safeness of this novel virus strain, named ASFVdelta9GL/deltaUK. Under experimental conditions ASFVdelta9GL/deltaUK showed to be safer and more immunogenic than our previous single gene deleted vaccine strains inducing an earlier and stronger protection efficacy against the infection. In addition, we were able to identify virus genes which are candidates to be important in virus virulence. Ten different previously uncharacterized virus genes were selected by functional genetics and further analyzed in their ability to interact with host genes by yeast two-hybrid methodology. Additional analysis of their function includes the development of recombinant viruses harboring deletions or modified forms of the genes under study. In addition, several swine cell lines are being evaluated in their susceptibility to support ASFV replication as possible substrate for vaccine production. Although, so far none of these cell lines fully supported virus replication we are adapting virus strains to grow in some of these cell lines. Virus adaptation is closed monitored in terms of avoiding major virus genome modifications, which can seriously affect virus ability as immunogen. We started to explore the development of ASFV experimental subunit vaccines using poxviruses as vaccine vector. Modify Vaccine Ankara (MVA) and Raccoon pox (RP) viruses were selected as vectors. A preliminary study was performed to optimize the use of promoters and genomic signals to achieve by the first time high levels of co-expression of several ASFV proteins in these pox vectors. Based in those results recombinant MVA and RP viruses harboring 4-6 ASFV proteins were designed and developed.
Accomplishments
1. New generation vaccine to control Classical Swine Fever. Classical Swine Fever Virus (CSFV) is a deadly disease effecting swine. An outbreak in the U.S. would cause an estimated $8 billion in economic loss. ARS scientists in Orient, New York have developed a new highly effective vaccine which provides protection as early as three days after vaccination and allows for the differentiation between infected and vaccinated animals, a critical feature for outbreak control and eradication efforts. This vaccine is an ideal addition to the USDA APHIS National Veterinary Stockpile to safeguard the pork industry against economic loss from this devastating foreign animal disease.
2. New vaccine to fight deadly African disease in pigs. African Swine Fever Virus (ASFV) is a deadly disease effecting swine, causing near 100% mortality, trade restrictions and significant economic losses globally. As its name indcicated, the disease occurs in Africa. However, since 2007 disease appeared in the Caucasus (Republic of Georgia) and subsequently spread into Russia and the Ukraine and is now present in multiple countries of Eastern Europe including Poland and most recently the Czech Republic, posing an imminent threat to the European and global swine industries. Currently there are no vaccines to protect swine against ASF. ARS scientists in Orient, New York, have developed a vaccine which can protect swine as early as 2 weeks post vaccination. This is the first experimental vaccine inducing early protection against ASF in swine. This vaccine can be used globally to protect the swine from this deadly disease and safeguard the pork industry against economic loss caused by the increasing incursion of this devastating disease.
Review Publications
Holinka-Patterson, L.G., O'Donnell, V., Risatti, G., Azzinaro, P.A., Arzt, J., Stenfeldt, C., Velazquez-Salinas, L., Gladue, D.P., Borca, M.V. 2017. Early protection events in swine immunized with an experimental live attenuated classical swine fever marker vaccine, FlagT4G. PLoS One. doi: 10.1371/journal.pone.0177433.
Borca, M.V., O'Donnell, V., Holinka-Patterson, L.G., Sanford, B., Azzinaro, P.A., Risatti, G., Gladue, D.P. 2017. Development of a fluorescent ASFV strain that retains the ability to cause disease in swine. Nature Scientific Reports. 7:46747. doi: 10.1038/srep46747.
O'Donnell, V., Risatti, G.R., Holinka-Patterson, L.G., Krug, P.W., Carlson, J., Velazquez-Salinas, L., Azzinaro, P., Gladue, D.P., Borca, M.V. 2017. Simultaneous deletion of the 9GL and UK genes from the African swine fever virus Georgia 2007 isolate results in virus attenuation and may be a potential virus vaccine strain. Journal of Virology. 91(1):e01760-16.
Carlson, J., O'Donnell, V., Alsfonso, M., Velazquez Salinas, L., Holinka-Patterson, L.G., Krug, P.W., Gladue, D.P., Higgs, S., Borca, M.V. 2016. Association of the host immune response with protection using a live attenuated african swine fever virus model. Viruses. 8(10):291. doi: 10.3390/v8100291.
Holinka-Patterson, L.G., Largo, E., Gladue, D.P., O'Donnell, V.K., Risatti, G.R., Nieva, J.L., Borca, M.V. 2016. Alteration of a second putative fusion peptide of structural glycoprotein E2 of Classical Swine Fever Virus alters virus replication and virulence in swine. Journal of Virology. 90:10299-10308. doi: 10.1128/JVI.01530-16.
Borca, M.V., O'Donnell, V., Holinka-Patterson, L.G., Rai, D., Sanford, B., Alfano, M., Carlson, J., Azzinaro, P., Alonso, C., Gladue, D.P. 2016. The Ep152R ORF of African Swine Fever Virus strain Georgia encodes for an essential gene that interacts with host protein BAG6. Virus Research. 223:181-189. doi: 10.1016/j.virusres.2016.07.013.
