Research Project: Countermeasures to Control and Eradicate Foreign Animal Diseases of Swine

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2021 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, and also identifying the molecular viral antigenic determinants that drive heterologous protection. 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. 3: Determine the mechanisms that drive ASF viral evolution, including determine the molecular determinants that drive virus evolution in ASF-historical endemic settings and determine the molecular determinants that are affecting virus evolution in new ASF-endemic settings.

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 2021, animal experiments were mainly focused on the development of African Swine Fever (ASF) vaccines, which was defined as a program priority given the international epidemic situation and increased threat to the U.S. Therefore, the evaluation of Classical Swine Fever Virus (CSFV) candidate vaccine FlagT4G was delayed. In addition, the COVID-19 pandemic affects all laboratory activities at PIADC in Q3 of FY2020 and Q1, Q2, and Q3 in 2021. Despite these delays, we were able to show that our live attenuated FlagT4G vaccine candidate is efficacious when orally administered. Animals receiving a dose of FlagT4G, directly by installation or delivered using baits, were protected against the challenge with a virulent field isolate currently acting in the Caribbean. This result demonstrated that the FlagT4G virus could potentially be used to immunize wild swine, a critical fact in the epidemiological control of the disease. A patent was filed covering the FlagT4G accompanying diagnostic DIVA (differencing infected from vaccinated animals) test, developed in collaboration with the Centre de Recerca en Sanitat Animal (CRESA) Barcelona, Spain. The test was further successfully assessed at CRESA using a large set of sera from infected and vaccinated animals. We continued characterization of the CSFV major structural glycoprotein E2 in order to identify regions that specifically interact with swine host proteins and, in this way, discover novel genetic determinants of virulence. CSFV mutant containing specific mutations that abolished interaction of the major structural protein E2 with a specific swine host protein, Tora1, were created and used to demonstrate that this interaction is critical for virus replication. This information may be the basis of novel countermeasures to control virus replication. We continued advances in the systematic study of uncharacterized African swine fever virus genes that were initially selected by functional genomics criteria. None of these genes have been previously studied; therefore, their function was previously unknown. Eight virus genes were analyzed for their function and interaction in swine macrophages and were selected for pathogenesis studies in pigs. For this purpose, recombinant ASFV single deletions of each of the genes were developed and characterized for their replication ability in swine macrophage cultures. The effect of these genes in virulence was evaluated by swine inoculation. Deletion of six of those genes in the genome of ASFV Georgia isolate showed none of those genes were associated with disease production. The other two remaining genes, A137R and E184L, were shown to be involved in the process of virulence in swine. Individual deletions of E184L and A137L partially and completely attenuates ASFV Georgia virulence, respectively. The significance of this discovery is that A137L and E184L are among the only 8 virus genes (all but two discover at PIADC) whose deletion attenuates the virus causing the current Eurasian pandemic of ASF. Importantly, animals inoculated with recombinant viruses lacking the respective genes (ASFV-G-A137L and ASFV-G-E184L) were completed protected against the challenge of the virulent parental ASFV Georgia isolate. Therefore, these discoveries may originate novel ASFV candidates. In FY2019/20, we reported the rational development of ASFV-G-I177L, a vaccine candidate recombinant strain, which protects swine against challenges with the epidemiologically significant isolate Georgia. This vaccine candidate is effective as a vaccine even when used at very low doses, is completely attenuated in swine even when administered at high doses, and produces sterile immunity when used at the right doses. Making ASFV-G-I177L the most promising experimental vaccine strain reported so far. This year we extend our studies on this vaccine candidate, showing that, besides parenterally, it can be administrated oro-nasally. Animals inoculated by that route were shown to be completely protected against challenge with parental virulent ASFV strain Georgia. The immune response, as well as the efficacy of this vaccine candidate, was similar in animals receiving the vaccine oro-nasally to those intramuscularly inoculated. The significance of this result resides in the potential use of ASFV-G-I177L to vaccinate wild swine populations, a critical issue in the epidemiological management of an ASF endemic area. It is important to mention that as a result of the work we have done characterizing ASFV-G-I177L the virus has been excluded by APHIS from the Select Agent list, which will allow future research and development of the vaccine candidate outside BSL-3 facilities. This will significantly expedite the advanced development of ASFV-G-I177L as a potential commercial vaccine. Similarly, recombinant vaccine viruses previously developed in our laboratory, strains G-9GL/UK and G-MGF, have also been excluded from the Select Agent list and have been transferred to vaccine industry partners for further development. Additionally, we have preliminary results that demonstrated that ASFV-G-I177L vaccine candidates can be administrated by the oronasal route and still induce protection against the challenge with virulent parental virus Georgia strain. All ASFV vaccine candidates developed in our laboratory need to be grown in primary swine macrophage cultures. An important issue that needs to be solved to translate the production of these viruses to a commercial partner is the use of stable cell lines as the substrate to produce vaccine viruses. In FY2020, we have continued our effort to solve this problem using two different technical approaches. This year we have adapted ASFV-G-I177L to grow in a stable epithelial cell line of swine origin (PIPEC). After a few passages, ASFV-G-I177L adapted to grow in PIPEC cells with similar yields to those of growing in primary swine macrophages cultures. The genetic changes induced in the adapted virus (ASFV-G-I177L/LVR) remain stable until passage 20 in PIPEC cells. Importantly, all positive vaccine characteristics of ASFV-G-I177L regarding safety and efficacy inducing protection in swine remained unchanged in ASFV-G-I177L/LVR. This development is of paramount importance to facilitate the process of adoption of our vaccine candidates by commercial partners. We continued making 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. We previously reported that several virus genes were identified as DIVA candidates using an in-house developed peptide microarray methodology. During this year, we developed a recombinant virus by deleting a highly immunogenic ASFV gene, E184L. As described earlier, this virus presents a partial attenuation. Importantly, animals surviving the infection with ASFV-G-E184L present a protective antibody response that, while recognizing all immunogenic ASFV proteins, failed to recognize E184L gene product. Conversely, animals inoculated with different attenuated strains of virus strongly recognize E184L. Therefore, the protein encoded by E184L gene is a perfect candidate to be used as a negative antigenic marker to develop a DIVA test. This result constitutes the first reported experimental proof of a functional antigenic marker in ASFV.

Accomplishments
1. African Swine Fever Virus vaccine candidate ASFV-G-I177L can be orally administered. African Swine Fever (ASF) is a devastating and highly lethal disease of pigs for which there are no commercial vaccines. As a result of genetic manipulation, ARS scientists from Greenport, New York, have developed an attenuated vaccine strain called ASFV-GI177L, which possesses a remarkable therapeutic index at levels exceeding all other ASV vaccine candidates achieving sterile protection, absence of vaccine shedding, and a high safety profile. ASFV-G-I177L is the most promising vaccine candidate reported so far. Now, it is shown that ASFV-G-I177L can be administered by the oro-nasal route, being as efficacious as when parenterally administered. This discovery opens the potential use of ASFV-G-I177L as an oral vaccine. This would permit the vaccination of wild swine, a critical host factor in the complex epidemiological scenario of ASF in endemic areas.

2. Development of effective attenuated African Swine Fever Virus Vaccine ASFV-G-177L that replicates in stable cell line. ASFV recombinant vaccines only replicate in primary cultures of swine macrophages, which is complicated and time-consuming to prepare and requires a herd of healthy donor pigs. This issue is a serious obstacle in manufacturing an ASFV vaccine at the industrial level. ARS scientists from Greenport, New York, have achieved the modification of the vaccine candidate strain ASFV-G-I177L (named ASFV-G-I177L/LVR), which is now able to grow in a stable swine cell line (PIPEC), also developed at PIADC. Importantly, ASFV-G-I177L/LVR has been shown to be as safe and efficacious as ASFV-G-I177L. This development is of paramount importance to the transference of this highly efficacious vaccine candidate to commercial partners. A patent covering the development ASFV-G-I177L was filed, and several international commercial partners have initiated the process of licensing ASFV-G-I177L.

Review Publications
Ramirez-Medina, E., Vuono, E.A., Pruitt, S.E., Rai, A., Silva, E., Zhu, J.J., Velazquez-Salinas, L., Gladue, D.P., Borca, M.V. 2020. X69R is a non-essential gene that when deleted from African swine fever does not affect virulence in swine. Viruses. https://doi.org/10.3390/v12090918.
Gladue, D.P., O'Donnell, V., Ramierez-Medina, E., Rai, A., Pruitt, S.E., Vuono, E., Silva, E., Velazquez-Salinas, L., Borca, M.V. 2020. Deletion of CD2-like (CD2v) and C-type lectin-like (EP153R) genes from African swine fever virus Georgia- 9GL abrogates its effectiveness as an experimental vaccine. Viruses. https://doi.org/10.3390/v12101185.
Ramierez-Medina, E., Vuono, E., Rai, A., Pruitt, S.E., Silva, E., Velazquez-Salinas, L., Zhu, J.J., Gladue, D.P., Borca, M.V. 2020. Evaluation in swine of a recombinant African swine fever virus lacking the MGF-360-1L gene. Viruses. https://doi.org/10.3390/v12101193.
Rai, A., Pruitt, S.E., Ramierez-Medina, E., Vuono, E., Silva, E., Velazquez-Salinas, L., Carrillo, C., Gladue, D.P., Borca, M.V. 2020. Detection and quantification of African swine fever in MA104 cells. Bio-protocol. https://doi.org/10.21769/BioProtoc.3955.
Velazquez-Salinas, L., Zarate, S., Eberl, S., Gladue, D.P., Novella, I., Borca, M.V. 2020. Positive selection of ORF1ab, ORF3a, and ORF8 genes drives the early evolutionary trends of SARS-CoV-2 during the 2020 COVID-19 pandemic. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2020.550674.
Ramirez-Medina, E., Vuono, E., Pruitt, S.E., Rai, A., Espinoza, N.N., Velazquez-Salinas, L., Gladue, D.P., Borca, M.V. 2021. Evaluation of the function of ASFV KP177R gene, encoding for structural protein p22, in the process of virus replication and in swine virulence. Viruses. https://doi.org/10.3390/v13060986.
Borca, M.V., Rai, A., Ramirez-Medina, E., Silva, E.B., Velazquez-Salinas, L., Vuono, E., Espinoza, N.N., Pruitt, S.E., Gladue, D.P. 2021. A cell culture-adapted vaccine virus against the current pandemic African swine fever virus strain. Nature Communications. https://doi.org/10.1128/JVI.00123-21.
Vuono, E., Ramirez-Media, E., Velazquez-Salinas, L., Berggren, K., Rai, A., Pruitt, S.E., Espinoza, N.N., Gladue, D.P., Borca, M.V. 2021. Structural glycoprotein E2 of classical swine fever virus critically interacts with host protein Torsin-1A during the virus infectious cycle. Virology Journal. https://doi.org/10.1128/JVI.00314-21.
Borca, M.V., Ramirez Medina, E., Silva, E., Vuono, E., Rai, A., Pruitt, S.E., Gay, C.G., Espinoza, N.N., Velazquez-Salinas, L., Gladue, D.P. 2021. ASFV-G-I177L is an effective oral nasal vaccine against the Eurasia Strain of Africa swine fever. Viruses. https://doi.org/10.3390/v13050765.
Ramirez-Medina, E., Vuono, E., Rai, A., Pruitt, S.E., Silva, E., Espinoza, N.N., Velazquez-Salinas, L., Zhu, J.J., Borca, M.V., Gladue, D.P. 2021. Development and in vivo evaluation of a recombinant African swine fever strain Georgia with a deletion in the MGF110-1L gene. Viruses. https://doi.org/10.3390/v13020286.
Vuono, E., Ramirez-Medina, E., Rai, A., Pruitt, S.E., Silva, E., Espinoza, N.N., Velazquez-Salinas, L., Zhu, J.J., Borca, M.V., Gladue, D.P. 2020. Evaluation in swine of a recombinant Georgia 2010 African Swine Fever Virus lacking the I8L Gene. Viruses. https://doi.org/10.3390/v13010039.