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2019 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, identifying the immune mechanism mediating effective homologous and heterologous protection against virus infection, researching potential antigenic vaccine markers to differentiate infected from vaccinated animals (DIVA), and identifying host cell factors that contribute to ASFV growth in cell culture conditions to inform the development of a cell line for ASFV vaccine production.
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.
Sub-Objective 2.iii: identifying the immune mechanism mediating effective homologous and heterologous protection against virus infection.
Sub-Objective 2.iv: researching potential antigenic vaccine markers to differentiate infected from vaccinated animals (DIVA).
Sub-Objective 2.v: identifying host cell factors that contribute to ASFV growth in cell culture conditions to inform the development of a cell line for ASFV vaccine production.
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 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 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. This research will lead to the identification of genes which may be modified or deleted to create attenuated virus strains for use in vaccine development. 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. Research will be focused in the identification of potential antigenic vaccine markers to DIVA. Studies will be focused in a systematic identification of highly immunogenic virus antigens to be used as target in the development of DIVA compatible vaccines. Efforts will also include the development of a stable cell line capable of supporting ASFV growth for use in commercial vaccine production. Host cell factors contributing to ASFV growth will be analyzed studying patterns of gene expression in susceptible versus non-susceptible cell line. 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. Efforts will bedevoted to identification of host immune mechanism mediating effective protection against the challenge with homologous and heterologous viruses.
Progress Report
During FY 2019 animal facilities were not available until January 2019. Animal experiments focusing on the development of African swine fever vaccine was defined as a priority, and the evaluation of CSFV candidate vaccine FlagT4G was delayed until early FY 2020. Importantly, in collaboration with CReSA, Barcelona, Spain we developed and optimized, at the experimental level, a DIVA test based in a direct ELISA test using synthetic peptides. Results distinguished FlagT4G vaccinated from C-strain or CSFV infected animals. CReSA is preparing a document to file the corresponding patent covering this invention.
We reported last year the development a novel approach to identify and characterize regions of CSFV major structural glycoprotein E2 that specifically interact with swine host proteins. This information allowed us to develope 14 recombinant CSFV mutants that have abolished their ability to interact with a specific swine host protein. As a result, three novel determinants of virulence have been mapped in the virus major structural protein E2. Studies on E2-host proteins interactions led to the identification of the precise area in E2 interacting with each of three host proteins. Recombinant viruses having disrupted each of these interactions have been shown to have a decreased virulence in swine indicating that each of these protein-protein interactions are critical in defining virus virulence. These novel attenuated CSFV may be the bases of novel candidates for the development of live attenuated vaccines.
In the area of ASFV, we previously reported the development of a series of attenuated vaccine candidates harboring additional gene deletions based on our previously developed vaccine strain ASFV-G-delta 9GL/delta UK, in order to strengthen its genetic stability and safeness. Those viruses were tested in swine and results demonstrated that, although there was a gain in safety, there was a significant decrease in their immunogenicity. An important conclusion we arrived at with these results is that one particular virus gene may play a different function depending the ASFV strain considered, important information for the development of ASFV attenuated strains by genetic manipulation. Also, we used a transcriptomic approach to analyze host and virus whole genome gene expression changes after virus infection. The results suggest that infected pigs could be sickened and killed by so-called “cytokine storm” due to over-production of a group of pro-inflammatory cytokines, an important issue that have been under discussion for several years. In addition, approximately 20 previously uncharacterized virus genes were selected by functional genomics criteria. All of them were characterized in their transcriptional activity in cell cultures of swine macrophages by next generation mRNA sequencing kinetics experiments. Their potential interaction with host proteins during the replication cycle was studied using the yeast to hybrid methodology. Five (I177L, MGF360-16, MGF110-1L, X69R and E120R) of the 20 virus genes analyzed in vitro were selected to perform in vivo studies. Recombinant ASFV having individually deleted (by genetic manipulation) each of the gene were developed. The replication ability was compared with the parental virus in swine macrophage cell cultures. The effect of these genes in virulence was evaluated by using each of the recombinant virus in pathogenesis studies in swine. All but ASFV lacking I177L were virulent, although at different levels, when compared with the parental virus. Animals infected with ASFV recombinant virus lacking I177L gene (ASFV-G-delta I177L) remained clinically normal indicating I177L play a critical role in ASFV virulence in the domestic swine. Importantly, the potential effect of ASFV-G-delta I177L as a vaccine candidate was evaluated. Animals infected with ASFV-G-delta I177L developed a significant anti-ASFV response and remained protected (clinically normal) when challenged with the parental virulent virus, indicating ASFV-G-delta I177L is a potential candidate for the development of a live attenuated vaccine strain. I177L is the fifth virus gene that has been ever described as affecting ASFV virulence and the third one identified as a potential tool to develop a live attenuated vaccine against the epidemiologically significant isolate Georgia. In addition, we have made progress towards the addition of DIVA markers to our ASFV vaccine candidates, a critical tool to use a vaccine in a control/eradication program under different epidemiological circumstances. Different virus genes were identified as DIVA candidates using a peptide microarray methodology screening all ASFV proteins. Based in that information several vaccine strains harboring DIVA markers are under development. In addition, we continued our efforts to establish a cell line able to support ASFV replication as a possible substrate for vaccine production. Many cell lines obtained from different laboratories around the world were tested at PIADC. Preliminary results have demonstrated that is possible to grow our vaccine candidates in a particular cell line after a short process of adaptation. The produced virus stock needs to be genetically and antigenically characterized to confirm that the adaptation did not significantly affect its protective efficacy. In addition, our effort to develop an ASFV experimental subunit vaccine, using raccoon pox viruses as vaccine vector, demonstrated that a recombinant virus co-expressing 6 different ASFV proteins (rRPV6), although immunogenic when tested in swine, did not induce protection against challenge with virulent ASFV. We are currently modifying rRPV6 including 6 additional ASFV proteins in order to expand its immunogenicity.
Accomplishments
1. Development of companion ELISA DIVA test for marked vaccine candidate FlagT4G. There is a need to develop a test to differentiate vaccinated from infected animals (DIVA) as a companion tool for current vaccine strategies. Researchers from USDA, ARS, Orient Point, New York have developed a companion ELISA DIVA test for our live attenuated DIVA marked vaccine candidate for African Swine Fever Virus called, FlagT4G virus. This development is a key complement to the FlagT4G vaccine, which was recently nominated in a Federal Registration call seeking for a commercial partner to transfer FlagT4G virus to industry.
2. Discovery of three novel virus genetic determinant of virulence for Classical Swine Fever Virus. Three novel virus genetic determinants of virulence have been discovered associated with major Classical Swine Fever Virus (CSFV) structural protein E2 by USDA, ARS researchers from Orient Point, New York in 2019. The discovery of genetic determinants of virulence and their further manipulation are key issues in the rational development of live vaccine strain candidate against CSFV.
3. Development of ASFV-G-delta I177L vaccine candidate for African Swine Fever Virus. African Swine Fever (ASF) is a devastating and highly lethal disease of pigs for which there are no commercial vaccines. The use of live attenuated strains is so far the only reliable methodology to protect pigs against the infection with virulent virus strains. The rationally development of attenuated virus strains by genetic manipulation, removing specific genes, is an effective methodology. The base of that methodology resides in the identification of the virus genes which, by removal, attenuates virulence. Researchers from USDA, ARS, Orient Point, New York have discovered that ASFV gene I177L is critical for virus virulence and its removal from ASFV strain Georgia genome completely attenuates virulence. The developed virus, ASFV-G-delta I177L, induced effective protection against the challenge with virulent parental virus ASFV Georgia.
Review Publications
Gladue, D.P., Largo, E., Holinka-Patterson, L.G., Ramirez-Medina, E., Vuono, E.A., Berggren, K., Risatti, G.R., Nieva, J.L., Borca, M.V. 2018. Classical swine fever virus p7 protein interacts with host protein CAMLG and regulates calcium permeability at the endoplasmic reticulum. Virus Research. 10(9):E460. https://doi.org/10.3390/v10090460.
Borca, M.V., Berggren, K., Ramirez, E., Vuono, E., Gladue, D.P. 2018. CRISPR/Cas gene editing of a large DNA virus: African Swine Fever Virus. Bio-protocol. 8(16). https://doi.org/10.21769/BioProtoc.2978.
Borca, M.V., Holinka-Patterson, L.G., Ramirez-Medina, E., Vuono, E., Berggren, K., Gladue, D.P. 2018. Identification of structural glycoprotein E2 domain critical to mediate Classical Swine Fever Virus replication in SK6 cells. Virology. 526:38-44. https://doi.org/10.1016/j.virol.2018.10.004.
Vuono, E., Ramirez-Medina, E., Holinka-Patterson, L.G., Baker-Branstetter, R., Borca, M.V., Gladue, D.P. 2019. Interaction of structural glycoprotein E2 of Classical Swine Fever Virus with protein phosphatase 1 catalytic subunit beta (PP1ß). Virology. Viruses 2019, 11(4), 307. https://doi.org/10.3390/v11040307.
Gladue, D.P., Largo, E., De La Arada, I., Aguilella, V., Alcaraz, A., Arrondo, J., Holinka-Patterson, L.G., Brocchi, E., Ramirez-Medina, E., Vuono, E., Berggren, K., Carrillo, C., Borca, M.V. 2018. Molecular characterization of the viroporin function of foot-and-mouth disease virus non-structureal protein 2B. Journal of Virology. https://doi.org/10.1128/JVI.01360-18.
Elias, E.H., McVey, D.S., Peters, D.C., Derner, J.D., Pelzel-McCluskey, A., Schrader, T.S., Rodriguez, L.L. 2018. Contributions of hydrology to Vesicular Stomatitis Virus emergence in the western United States. Ecosystems. 22:416-433. https://doi.org/10.1007/s10021-018-0278-5.
