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

Location: Foreign Animal Disease Research

2024 Annual Report

Objectives
Objective 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 emergency response to a disease outbreak, disease control and eradication. Sub-Objective 1.A: Determine immune mechanisms mediating early protection and its application in blocking infection and preventing transmission. Sub-Objective 1.B: Discover effective CSF vaccine platforms specifically designed for disease control and eradication. Objective 2. Develop intervention strategies to control African Swine Fever Virus (ASFV), including identify functional genomics of virus-host determinants of virulence and transmission, determining host mechanisms of ASF immune protection, and discovering effective ASF vaccine platforms specifically designed for emergency response, disease control and eradication, including the identification of antigenic markers that can be deleted from attenuated live ASV vaccine strains to differentiate infected from vaccinated animals to be used in the development of differentiation of infected from vaccinated animals (DIVA) vaccines. Sub-Objective 2.A: Identify novel virus-host genetic determinants of virulence by systematic screening of almost all previously uncharacterized virus genes. Sub-Objective 2.B: Determine host mechanisms of ASF virus immune protection. Sub-Objective 2.C: Identify antigenic markers that can be deleted from attenuated live ASF vaccine strains to differentiate infected from vaccinated animals (DIVA capability). Sub-Objective 2.D: Discover effective ASF vaccine platforms specifically designed for disease control and eradication.

Approach
Sub-Obj. 1.A: Host mechanisms of early protection will be studied by using our live attenuated vaccine (LAV) model (FlagT4Gv) that induces protection within 3 days post inoculation. Studies will analyze virological and immunological factors present in animals that are protected at early times post vaccination. Different immunological markers of cellular and humoral immune response will be evaluated at different times post vaccination and correlated with presence or absence of protection against virulent challenge. Sub-Obj. 1.B: A full evaluation of the of the second-generation marker LAV vaccine FlagT4Gv will be conducted. Activities will focus on completing the assessment of toxicity, immunogenicity, and protective effect of FlagT4G. Serological DIVA tests will be optimized and validated to differentiate infected from vaccinated animals accompanying FlagT4Gv. Sub-Obj. 2.A: The ASFV genome harbors more than 150 genes, most of which have not been characterized. The knowledge obtained from their characterization will provide critical information to understand mechanisms of virus replication, the virus and the host cell interactions, and virulence in the natural hosts. Uncharacterized virus genes will be studied. Full characterization of the selected genes will include their interaction with host proteins, the production of recombinant ASFV lacking the gene or harboring modified versions of it to assess the protein functionality in vitro, and virulence during infection in swine. This research will lead to the identification of genes which may give origin to potential attenuated vaccine candidates. Sub-Obj. 2.B: There is no consensus about the immune mechanism mediating protection in ASF. Identifying those mechanisms may improve the chances of developing more efficacious vaccines. An evaluation to determine the presence of immune mechanisms in both innate and acquired immune response in animals protected against challenge after vaccination with our LAV candidates will be done, and correlate them with the presence or absence of protection. Sub-Obj. 2.C: Experimental LAVs have been developed through genetic manipulation by deleting single virus genes involved in virulence. Depending of the epidemiological scenario it will be important to have those vaccines harbor antigenic markers that confer to them DIVA capabilities. LAVs vaccines harboring antigenic markers will be developed that will enable differentiation between vaccinated animals from those infected with field isolates. Highly antigenic genes will be identified and later deleted from the vaccine candidates to produce vaccine viruses that can be antigenically differentiated from field isolates. Sub-Obj. 2.D LAVs will be transferred to commercial partners for use as potential vaccine candidates. Actions will be focused to increase the safety profile of our LAV strains by using different combinations of additional virulence associated viral genes discovered in Sub-Objective 2.A. In addition, the development of a stable cell line capable of supporting the growth of our LAV candidate viruses will be conducted to afford the possibility of commercially develop an ASFV vaccine.

Progress Report
Sub-objective 1.A.1: Our in-house developed live attenuated marker vaccine, FlagT4Gv can induce early (3 days post immunization) protection after vaccination. Induction of this early status of protection is crucial in vaccines used to control a disease outbreak in disease free areas. Host mechanisms mediating this early protection must involve those of the innate immune response. Host innate mechanisms mediating early (3-5 days post vaccination) protection were analyzed in FlagT4Gv vaccinated animals. Different parameters of cellular and humoral immune response were studied, and it was found that detection of interferon alpha clearly correlated with presence of protection against virulent challenge. Besides providing information of clarifying mechanisms of protection induced by FlagT4Gv, this result also opens the possibility of using interferon alpha as bio therapeutics in classical swine fever virus (CSFV) infection. A commercial partner in Vietnam licensed FlagT4Gv and an agreement was established between ARS and Vietnamese cooperator DABACO to further commercial development. Sub-objective 1.B.2: FlagT4Gv vaccine was designed to contain antigenic markers that would allow the development of a differentiate infected from vaccinated animals (DIVA) test that would differentiate the serological response in animals that have been vaccinated from those infected with CSFV field strains. The availability of DIVA tests makes the corresponding vaccine an invaluable tool to be used in the epidemiological management of disease outbreaks in disease free areas. In collaboration with Centre de Recerca en Sanitat Animal (CRESA) Barcelona, Spain, we developed a peptide based direct enzyme-linked immunoassay (ELISA) DIVA test that allows the differentiation between animals vaccinated with FlagT4Gv and other CSFV field strains. The test was bench validated, patented, licensed and transferred to a commercial partner (DABACO, Vietnam) for its further commercial development. Sub-objective 1.B.3: Usually, the epidemiological scenario of Classic swine fever (CSF) involves domestic and wild pigs. These later constitute a natural reservoir in nature. Immunization of wild pigs is an essential issue in programs of disease eradication in endemic areas. Therefore, administration of the vaccine by the oral route is essential in a CSF vaccine to be used in wild pigs. In collaboration with CRESA (Barcelona, Spain), we have studied the feasibility of administering FlagT4Gv by the oral route and showed that vaccinated pigs developed a strong immune response that fully protects against the virulent challenge. No challenge virus replication or presence of clinical signs associated with CSF were observed in these orally vaccinated animals. The potential use of FlagT4Gv by the oral route opens the potential use of the vaccine in wild pigs making FlagT4Gv an attractive vaccine candidate to be used in both endemic and disease-free areas. Sub-objective 2.A.2., 2.A.3, 2.C.2 and 2.C.3: The study of the function of African swine flu vaccine (ASFV) genes have been a key issue in understanding basic aspects of virus replication and disease production. For years, our laboratory has studied gene function by deleting selected genes from the genome of highly virulent ASFV field strains, developing recombinant viruses. These developed recombinant viruses have been used as tools to study the effect of the deleted gene in vitro and in vivo tests. This approach was critical in the identification of genes involved in virulence and the use of this knowledge for the rational development of live attenuated ASFV vaccine candidates. During 2024 three new previously uncharacterized virus genes were studied, genes O174L, E111R and B134L. Recombinant ASFV were produced, harboring individual deletions of each of these genes, and each of these viruses were evaluated in their characteristics both in vitro and in animals. Results indicated that deletion of any of these three virus genes did not significantly affect ASFV replication in cell cultures or during the experimental infection in pigs. Animals inoculated with the recombinant viruses did not show any alteration of the virulence when compared with the parental virulent field isolate indicating that genes under studied were not involved in the process of the disease pathogenesis. Sub-objective 2.B: Animals infected with attenuated strains of ASFV are usually protected against the challenge with virulent homologous viruses. Immune mechanism involved in protection has not been fully identified and this issue has been the center of controversy for years. In particular, the role of virus neutralizing antibodies in African swine fever (ASF) protection is probably the most controversial immune mechanism under discussion. In 2023, we presented clear evidence that the presence of neutralizing antibodies closely associates with the presence of protection in animals tested at different times post vaccination and vaccinated with different types of live attenuated vaccines. During 2024, we have extended those studies and presented the development a new methodology that allows the accurate detection and quantification of ASFV neutralizing antibodies. The new method, based in quantification of target infected cells by cell cytometry, quickly magnify the levels of neutralizing antibodies in the sera of vaccinated pigs correlating the values obtained with the presence of protection against the challenge of the vaccinated animals with virulent field isolates. Sub-objective 2.D.1.C: One of the biggest challenges in transferring to commercial partners the live attenuated recombinant vaccine viruses developed in our laboratory resides in the need to use primary cultures of swine macrophages as substrate for virus replication. Therefore, we have invested a significant effort in evaluating established cell lines that would efficiently support replication of our vaccine virus. During 2024, we have demonstrated that a particular cell line derived from swine macrophages, IPKM, was suitable to support the efficient replication of two of our vaccine candidates, ASFV-G-DI177L and ASFV-G-D9GL/DUK. Both vaccine viruses grow well in these cells without need of adaptation, showing genomic stability and unaltered patterns of safety and efficacy when compared with the parental virus. This is an important step to facilitate the transfer of these vaccine viruses to pharmaceutical partners. Sub-objective 2.D.2: Live attenuated ASFV strains may have a common problem that is the presence of residual virulence, particularly, in certain types of more susceptible animals. Young animals and females in reproductive activity usually appear as highly susceptible hosts. During 2024, in collaboration with our partner NAVETCO (Vietnam), we evaluated on the possibility of vaccinating piglets at 4 weeks of age. Several experiments were successfully performed demonstrating that the vaccine is safe and efficacious in those young animals. Vaccinated animals did not show any clinical sign associated with the vaccination and all of them were fully protected against the experimental challenge. In addition, several alternative vaccination protocols were designed to vaccinate sows that were dedicated to reproduction. Some of those protocols were selected since they were not affecting the reproductive efficacy of the sows inducing good levels of immunity. Based on these results, the commercial NAVETCO vaccine (which is based on the use of the ARS strain ASFV-G-DI177L) was approved by the Department of Animal Health of Vietnam (DHA) to be administered at 4 weeks of age (previous authorization limited the use of the vaccine to animals between 8-10 weeks of age). In addition, the use of the vaccine in sows involved in reproduction is currently being considered by the DAH. Incorporating young animals, as well as reproductive sows into the vaccination scheme is a very valuable issue to reach a systemic protection status against ASFV infection.

Accomplishments
1. Development of a commercial version of FlagT4Gv and the accompanying DIVA test. A safe and effective differentiate infected from vaccinated animals (DIVA) test compatible Classical Swine Fever (CSF) vaccine is still a concern affecting swine production in endemic countries worldwide and a constant threat to the U.S. ARS scientists in Plum Island, New York, previously developed a safe CSF vaccine (FlagT4Gv) that is safe and fully protects pigs as early as 3 days post vaccination. During 2024 we completed studies which showed that FlagT4G could be administered by the oral route inducing complete protection against challenge with a highly virulent field strain. In addition, during the recently developed diagnostic enzyme-linked immunoassay (ELISA) test, allowing the differentiation between the antibody response induced by FlagT4G from that elicited in animals infected with field strains, was bench validated and patented. An agreement between ARS and the pharmaceutical Vietnamese company DABACO was initiated allowing for the commercial development of the FlagT4G vaccine and the accompanying DIVA test. The availability of a commercial safe and efficacious, DIVA compatible, live attenuated CSF vaccine with potential to be used in wild swine is an important tool for the control of the disease in endemic areas as well as for the proper epidemiological management of a disease outbreak in a disease-free country.

2. Commercial version of the ARS developed African Swine Fever vaccine ASFV-G-DI177L is actively used in the field. The commercial African Swine Fever vaccine ASFV-G-D177L is being heavily used in the field in Vietnam. In 2023, and after more than 50 years or research, the first commercial African Swine Fever virus (ASFV) vaccine started being used in the field. NAVETCO, a Vietnamese pharmaceutical company, in collaboration with the vaccine inventors, ARS scientists at Orient, New York, deployed the vaccine in the field. During 2024, NAVETCO performed safety and efficacy studies of the ASFV-G-D177L involving more than 150,000 vaccinated pigs showing that only 0.06% of the animals presented clinical problems associated with vaccination while over 94% developed a significant virus specific antibody response. To date, more than 600,000 vaccine doses have been delivered in the field showing that the ASFV-G-D177L vaccine is safe and effective.

3. ARS live attenuated vaccine candidates can be produced in an established cell line. ARS live attenuated vaccine candidates can be produced in an established cell line. Since the African Swine Fever (ASF) program was restored in 2011, ARS scientists working at Orient, New York, have created a methodological approach that allowed the development of several live attenuated vaccine candidates. The ARS research group became the most prolific laboratory developing African Swine Fever virus (ASFV) vaccine candidates worldwide. All candidates were experimentally demonstrated to be safe and induce a strong protective immune response that effectively protect vaccinated pigs against infection with virulent field strains. All vaccine candidates were developed and need to be replicated in cell cultures based in the use of swine macrophages. This constitutes a limitation since this type of culture requires the constant use of healthy pigs as cell donors. Therefore, this methodological issue makes the massive production of the virus with commercial purposes a significant problem obstructing the transfer of the vaccine candidates to commercial pharmaceutical partners. ARS scientist have solved this problem by systematically testing available cell lines (which can be maintained and reproduced almost indefinitely) that may support the efficient replication of the vaccine strains. This systematic cell line screening allowed the identification of a particular cell line (named IPKM) that fully supported the replication of at least two of the most efficacious ARS vaccine candidates, ASFV-G-DI177L and ASFV-G-D9GL/DUK. Studies performed demonstrated that these two vaccine strains efficiently replicate in IPKM cells without acquiring any anomalous genomic changes and remain as safe and effective when used as vaccines as their corresponding original virus growth in swine macrophages. This is an important step in vaccine development facilitating the transfer of the ARS candidates to commercial partners.

4. Development of the first DIVA compatible African Swine Fever vaccine ASFV-G-DI177L/DEP402R. All African Swine Fever vaccine (ASFV) live attenuated vaccines developed to date, although safe and effective, lack antigenic markers that would allow the development of differentiate infected from vaccinated animals (DIVA) serological test to differentiate vaccinated animals from those that were infected with field virus strains. During 2024, ARS scientist at Orient, New York, developed several vaccine candidates harboring modifications in their antigenic profile, and created a vaccine candidate, ASFV-G-DI177L/DEP402R, that is DIVA compatible. This new vaccine, based on the modification of the ASFV-G-DI177L strain, harbors an additional gene deletion (EP402R gene) that encodes for a very immunogenic protein. ASFV-G-DI177L/DEP402R has been shown to be as safe and effective as ASFV-G-DI177L and the antibody response elicited in vaccinated animals can be differentiated from the response induced by ASFV field strains. In addition, our laboratory also developed the proof -of- concept of the corresponding DIVA ELISA test. ASFV-G-DI177L/DEP402R is a safe and effective DIVA compatible ASFV vaccine.

5. Creation of the WOAH Collaborative Center for Genome Monitoring of Swine Viral Diseases. During 2024, the World Organization for Animal Health (WOAH) has approved the National Bio and Agro-Defense Facility WOAH Center for Genome Monitoring of Swine Viral Diseases. This newly created Center provides support and training to collaborating WOAH member laboratories and other laboratories, in genomic characterization of viral pathogens causing disease outbreaks in pigs, particularly those caused by African swine fever virus (ASFV), Classical swine fever, Japanese encephalitis and Nipah virus. The collaborative Center will provide protocols and training for sample collection, nucleic acid extraction, next-generation sequencing and sequence analysis. Initially, the center will focus on ASFV. Viral DNA can be sent to these collaborating or other reference WOAH laboratories for next generation sequencing (NGS). The center will perform NGS of the full-length virus genome for those laboratories that lack or have limited sequencing capacity. In addition, the Center will establish reliable bioinformatic pipelines and associated tools to deliver standardized genomic data, annotation and classification for archival and new ASFV isolates. The Center will carry out site visits to determine laboratory capabilities, helping in the identification of needs to process analyze and interpret genomic data.

Review Publications
Vuono, E., Ramirez Medina, E., Pruitt, S.E., Rai, A., Espinoza, N.N., Silva, E.B., Velazquez Salinas, L., Gladue, D.P., Borca, M.V. 2022. Deletion of the ASFV dUTPase gene E165R from the genome of highly virulent African swine fever virus Georgia 2010 does not affect virus replication or virulence in domestic pigs. Viruses. 14(7). Article 1409. https://doi.org/10.3390/v14071409.
O'Donnel, V., Pierce, J., Osipenko, O., Xu, L., Berninger, A., Lakin, S., Barrette, R., Gladue, D.P., Faburay, B. 2024. Rapid detection and quick characterization of African swine fever virus using the Voltrax automated library preparation platform. Viruses. 16(5). Article 731. https://doi.org/10.3390/v16050731.
Ambagala, A., Goonewardene, K., Lamboo, L., Goolia, M., Erdelyan, C., Fisher, M., Handel, K., Lung, O., Blome, S., King, J., Forth, J.H., Calvelage, S., Spinard III, E.J., Gladue, D.P., Masembe, C., Adedeji, A.J., Olubade, T., Maurice, N.A., Ularamu, H.G., Luka, P.D. 2023. Characterization of a novel African swine fever virus p72 genotype II from Nigeria. Viruses. 15(4). Article 915. https://doi.org/10.3390/v15040915.
Spinard III, E.J., Rai, A., Osei-Bonsu, J., O'Donnell, V., Ababio, P., Tawuah-Yingar, D., Baah, D., Ramirez Medina, E., Espinoza, N.N., Valladares, A., Faburay, B., Ambagala, A., Odoom, T., Borca, M.V., Gladue, D.P. 2023. The 2022 outbreaks of African swine fever virus demonstrate the first report of genotype II in Ghana. Viruses. 15(8). Article 1722. https://doi.org/10.3390/v15081722.
Borca, M.V., Rai, A., Espinoza, N.N., Ramirez Medina, E., Spinard III, E.J., Velazquez Salinas, L., Valladares, A., Silva, E.B., Burton, L.J., Meyers, A., Gay, C.G., Gladue, D.P. 2023. African swine fever vaccine candidate ASFV-g-deltaI177l produced in the swine macrophage-derived cell line IPKM remains genetically stable and protective against homologous virulent challenge. Viruses. 5(10). Article 2064. https://doi.org/10.3390/v15102064.
Borca, M.V., Ramirez Medina, E., Espinoza, N.N., Rai, A., Spinard III, E.J., Velazquez Salinas, L., Valladares, A., Silva, E.B., Burton, L.J., Meyers, A., Clark, J.A., Wu, P., Gay, C.G., Gladue, D.P. 2024. Deletion of the EP402R gene from the genome of African swine fever vaccine strain ASFV-G-deltaI177L provides the potential capability of differentiating between infected and vaccinated animals. Viruses. 16(3). Article 376. https://doi.org/10.3390/v16030376.
Wang, L., Madera, R., Li, Y., Gladue, D.P., Borca, M.V., McIntosh, M., Shi, J. 2023. Development of porcine monoclonal antibodies with in vitro neutralizing activity against classical swine fever virus from C-strain E2-specific single B cells. Viruses. 15(4). Article 863. https://doi.org/10.3390/v15040863.
Ramirez Medina, E., Vuono, E., Rai, A., Espinoza, N.N., Valladares, A., Spinard III, E.J., Velazquez Salinas, L., Gladue, D.P., Borca, M.V. 2023. Evaluation of the function of ASFV gene E66L in the process of virus replication and virulence in swine. Viruses. 15(2). Article 566. https://doi.org/10.3390/v15020566.
Silva, E.B., Krug, P., Ramirez Medina, E., Pruitt, S., Rai, A., Espinoza, N.N., Valladares, A., Gladue, D.P., Borca, M.V. 2022. The presence of virus neutralizing antibodies is highly associated with protection against virulent challenge in domestic pigs immunized with ASFV live attenuated vaccine candidates. Pathogens. 11(11). Article 1311. https://doi.org/10.3390/pathogens11111311.
Ramirez Medina, E., Velazquez Salinas, L., Rai, A., Espinoza, N.N., Valladares, A., Silva, E.B., Burton, L.J., Spinard III, E.J., Meyers, A., Risatti, G., Calvelage, S., Blome, S., Gladue, D.P., Borca, M.V. 2023. Evaluation of the deletion of the African swine fever virus gene O174L from the genome of the Georgia isolate. Viruses. 15(10). Article 2134. https://doi.org/10.3390/v15102134.
Spinard III, E.J., O'Donnell, V., Vuono, E., Davis, C., Rai, A., Ramirez Medina, E., Espinoza, N.N., Valladares, A., Borca, M.V., Gladue, D.P. 2023. Full genome sequence for the African swine fever virus outbreak in the Dominican Republic in 1980. Scientific Reports. 13. Article 1024. https://doi.org/10.1038/s41598-022-25987-5.
Spinard III, E.J., Dinhobl, M.W., Tesler, N., Birtley, H., Signore, A., Ambagala, A., Masembe, C., Borca, M.V., Gladue, D.P. 2023. A re-evaluation of African swine fever genotypes based on p72 sequences reveals only 5 distinct p72 groups. Viruses. 15(8). Article 1722. https://doi.org/10.3390/v15081722.
Spinard III, E.J., Wade, A., Unger, H., Nenkam, R., Mayega, F.J., Sreenu, V., Da Silva Filipe, A., Mair, D., Borca, M.V., Gladue, D.P., Mesambe, C. 2024. Near-complete genome sequences of multiple genotype I African swine fever virus isolates from 2016 to 2018 in Cameroon. Microbiology Resource Announcements. 13. Article e00978-23. https://doi.org/10.1128/mra.00978-23.
Ramirez Medina, E., Rai, A., Espinoza, N.N., Spinard III, E.J., Meyers, A., Valladares, A., Velazquez Salinas, L., Gay, C.G., Gladue, D.P., Borca, M.V., Silva, E.B., Burton, L.J., Clark, J.A. 2023. African swine fever vaccine candidate ASFV-G-deltaI177L produced in the swine macrophage-derived cell line IPKM remains genetically stable and protective against homologous virulent challenge. Pathogens. 15(10). Article 2064. https://doi.org/10.3390/v15102064.
Ramirez Medina, E., Vuono, E., Pruitt, S., Rai, A., Espinoza, N.N., Valladares, A., Spinard III, E.J., Silva, E.B., Velazquez Salinas, L., Gladue, D.P., Borca, M.V. 2022. ASFV Gene A151R is involved in the process of virulence in domestic swine. Viruses. 14(8). Article 1834. https://doi.org/10.3390/v14081834.