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- Borca, Manuel
- Gladue, Douglas
- Zhu, James
- Arzt, Jonathan
- Medina, Gisselle
- Silva, Ediane
- 2023
- 2022
- Publications
- Animal Health (103)

Research Project: Intervention Strategies to Control and Eradicate Foreign Animal Diseases of Swine **Location:
                            Foreign Animal Disease Research** 2023 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: Determine immune mechanisms mediating early protection and its application in blocking infection and preventing transmission. During FY 2023, experiments performed at Plum Island Animal Disease Center (PIADC) were 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 the Classical Swine Fever Virus (CSFV) candidate vaccine, FlagT4G, was alternatively performed in overseas laboratories through collaborative agreements.
In collaboration with Centre de Recerca en Sanitat Animal (CRESA) Barcelona, Spain, studies were conducted to complete the characterization of the early protective immune response induced by the vaccine candidate FlagT4G. Assessment of the induced innate and adaptative immune response were performed in early vaccinated animals (by day 5 post vaccination). These animals were fully protected against the challenge with the highly virulent field strain Margarita. The absence of a virus specific antibody (detected by enzyme-linked immunoassay (ELISA) and virus neutralization assay) as well as the cellular response (detected by interferon gamma ELISPOT) indicates that early protection is the result of cytokine-mediated innate response (mainly interferon alpha) which was analyzed by detecting serum levels of 15 different lymphokines. These results demonstrated for the first time the role of the innate immune mechanisms in the induction of early protection by live attenuated classical swine fever vaccines.
Sub-Objective 1.B: Discover effective CSF vaccine platforms specifically designed for disease control and eradication. In addition, the ARS CSF vaccine candidate FlagT4G was evaluated as an oral vaccine. Initial experiments were performed administering FlagT4G orally to pigs that were later challenged with the virulent Margarita field strain. Results demonstrated that orally administered FlagT4G induced a strong immune response that resulted in solid protection against the challenge. This result opened the possibility of using FlagT4G as an oral vaccine to control the disease in wild suids.
A serologic differentiating infected from vaccinated animals (DIVA) test was developed to differentiate animals vaccinated with FlagT4G from animals infected with field CSFV isolates. The developed DIVA test is a direct ELISA using a solid phase synthetic peptides representing the T4 epitope (a specific epitope in the virus structural protein E2) which was intentionally deleted during the development of the FlagT4G. The test was bench validated, published, patented and is being licensed to a commercial partner which will validate it under field conditions. 
Sub-Objective 2.A: Identify novel virus-host genetic determinants of virulence by systematic screening of almost all previously uncharacterized virus genes. We continued advances in the systematic study of uncharacterized African Swine Fever Virus (ASFV) genes that were initially selected by functional genomics criteria. Seven virus genes (EP296R, A151R, QP509L, E66L, B117L, H240R and O174L) were analyzed for their function and interaction in swine macrophages and 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 later evaluated by swine inoculation. Five genes (EP296R, QP509L, E66L, B117L, and O174L) were not associated with disease production. The other two remaining genes, A151R, and H240R, were shown to be involved in the process of virulence in swine since their individual deletions partially attenuated ASFV Georgia virulence. The significance of this discovery is that A151R, and H240R are among 15 virus genes (all but four discovered at PIADC) which deletion attenuates the virus causing the current Eurasian pandemic of ASF. Importantly, animals surviving the inoculation with recombinant viruses individually lacking the A151R, and H240R genes were completely protected against the challenge of the virulent parental ASFV Georgia isolate. Therefore, these discoveries may be useful for increasing the safety profile of previously developed ASFV vaccine candidates.     
Sub-Objective 2.B: Determine host mechanisms of ASF virus immune protection. We also made progress in the identification of host immune mechanisms mediating protection in vaccinated animals against the infection with ASFV field isolates. We developed a novel methodology that allows the detection of antibodies neutralizing ASFV infectious particles. The new methodology, based in the use of fluorescent cell characterization, allows the objective quantification of circulating neutralizing antibodies (NA). We have also conducted a systematic assessment of the presence ASFV NA in pigs vaccinated with a variety of recombinant live attenuated vaccines and their association to protection against challenge with virulent wild type virus. More than one hundred animals were assessed, and results demonstrated a high association between presence of NA and protection. This is the first time that a direct association of NA and protection is established, making NA an attractive parameter to be used as a potential indicator of protection in vaccinated animals.
Sub-Objective 2.D: Discover effective ASF vaccine platforms specifically designed for disease control and eradication. We previously reported the rational development of ASFV-G-delta177L, a vaccine candidate recombinant strain, which protects swine against challenges with the epidemiologically significant isolate Georgia. Working in collaboration with our partner in Vietnam, Navetco, we also showed that ASFV-G-delta177L protects pigs from the challenge performed with a Vietnamese field isolate as efficiently as it does against the parental Georgia isolate. In addition, we showed that ASFV-G-delta177L effectively induced protection in pigs of a heterogenous genetic background (Vietnamese breeds). Also, in collaboration with Navetco, we demonstrated that ASFV-G-delta177L is a safe and effective vaccine. We completed the corroboration of the lack of residual virulence of ASFV-G-delta177L, a long-term experiment. Pigs inoculated with 1,000 times the vaccine dose and observed for 6 months showed complete absence of residual virulence of the ASFV-G-delta177L. Presence of residual virulence producing chronic forms of the disease is one of the more significant characteristics of the live attenuated ASFV vaccine candidates.
In addition, ASFV-G-delta177L was tested in its ability to induce protection when administered oro-nasally. Animals inoculated by that route were efficiently protected against the challenge with the field virulent isolate. This result opened the possibility of using ASFV-G-delta177L as an oral vaccine to immunize wild swine.
Based on the results on safety and efficacy obtained during our collaboration with Navetco, the Department of Animal Health (DAH) of Vietnam gave Navetco the approval to commercially produce ASFV-G-delta177L. The DHA designed a protocol to test ASFV-G-delta177L under field conditions. Approximately 50,000 doses have been administered so far without experiencing safety problems and reaching 94% of the vaccinated animal’s high levels of anti-ASFV antibodies in serum. ASFV-G-delta177L is the first commercial ASF vaccine ever approved for use under field conditions. **Accomplishments**
                                

1. A safe and effective oral vaccine. Classical Swine Fever (CSF) remains a major disease concern affecting thousands of pigs in endemic areas around the world and representing a threat to the U.S. industry. The disease is difficult to control especially since wild pigs become infected and serve as reservoirs. ARS scientists in Orient Point, New York, previously developed a CSF vaccine (CSF-FlagT4G) that is safe and fully protects pigs as early as 3 days post vaccination. This vaccine also allows the differentiation of infected and vaccinated animals (DIVA). During FY 2023 we showed that the vaccine FlagT4G, when given orally, induces strong virus-specific cellular and antibody responses that result in complete protection against challenge with a highly virulent field strain. In addition, the CSF-FlagT4G vaccine prevented replication of the challenge virus (sterile immunity). The results open the possibility of using FlagT4G as an oral vaccine in wild swine. Furthermore, a companion diagnostic, enzyme-linked immunoassay (ELISA) test for the vaccine was developed, allowing the differentiation between the antibody response elicited by FlagT4G from that elicited in animals infected with field strains. This DIVA ELISA test efficiently differentiated vaccinated from infected animals with 100% accuracy. The oral vaccine and its companion DIVA test represent major contributions to CSFV control and eradication programs. 2. African Swine Fever Virus vaccine, ASFV-G-delta177L, could be used as an oral vaccine. African Swine Fever (ASF) is currently the most dreaded disease affecting pig production in the world. The disease is currently pandemic, covering a large geographical area in Asia, Europe, and the Caribbean. ASF has different transmission cycles that complicate the epidemiological management of the disease. The most important of those cycles involve the infection of wild swine which act as reservoirs and transmitters of the disease. Until now there has not been a safe and effective vaccine that can be delivered to wildlife. ARS scientists in Orient Point, New York, demonstrated that pigs orally vaccinated with the ARS-developed vaccine (ASFV-G-delta 177L) developed a significant virus-specific antibody response and were completely protected against the challenge with a highly virulent field isolate (Georgia). Furthermore, animals developed a “sterile” protection, meaning that no replication of the challenge virus was detected after the challenge. These results open the possibility of using ASFV-G-delta 177L as an oral vaccine which is a critical tool for its use in programs for the vaccination of wild swine. 3. Commercial African Swine Fever vaccine, ASFV-G-delta177L, is being used in the field. Research for the development of African Swine Fever (ASF) vaccines has been taking place for more than 50 years but, until recently, no vaccine was available to control ASF. A live attenuated vaccine candidate developed with ARS scientists in Orient, New York, ASFV-G-delta 177L, is the first commercial ASF vaccine ever approved by Vietnam’s Department of Animal Health (DAH) to be used under controlled field conditions. More than 50,000 doses have been deployed without registering any negative post vaccination reactions. Over 94% of the vaccinated animals developed a significant virus specific antibody response. In addition, groups of animals vaccinated in the field were challenged under laboratory conditions showing 100% efficacy. This vaccine has been recently approved by DAH of Vietnam to be commercialized in Vietnam becoming the first ASF vaccine commercially available. 4. Launched the African swine fever virus genomic center. The first phase of the center of excellence, a community resource that aligns African swine fever (ASF) research among the World Organization of Animal Health reference labs for African swine fever, and African swine fever researchers worldwide was launched. This center although owned by the African swine fever virus research community was launched by efforts led by ARS scientists in Manhattan, Kansas, and Orient Point, New York. Phase one included protein information, aligning reference sequences with all ASFV worldwide laboratories, and providing an easy to view resource of all ASFV proteins. **Review Publications**
                            *Bohórquez, J.A., Wang, M., Diaz, I., Alberch, M., Perez-Simo, M., Rosell, R., Gladue, D.P., Borca, M.V., Llilianne, G. 2022. The FlagT4G vaccine confers a strong and regulated innate immunity that correlates with early virological protection against classical swine fever. Viruses. 14(9). https://doi.org/10.3390/v14091954.*
                                *Hyeon, J., Tseren-Ochir, E., Lee, D., Gladue, D.P., Borca, M.V., Risatti, G.R. 2023. Whole genome sequencing and phylogenetic analysis of African swine fever virus detected from a backyard pig in Mongolia, 2019. Viruses. 10. https://doi.org/10.3389/fvets.2023.1094052.*
                                *Vuono, E., Ramirez Medina, E., Silva, E.B., Berggren, K., Rai, A., Espinoza, N.N., Gladue, D.P., Borca, M.V. 2023. Classical swine fever virus structural glycoprotein E2 interacts with host protein ACADM during the virus infectious cycle. Viruses. 15(5). https://doi.org/10.3390/v15051036.*
                                *Velazquez Salinas, L., Ramirez Medina, E., Rai, A., Pruitt, S.E., Vuono, E., Espinoza, N.N., Gay, C.G., Witte, S.B., Gladue, D.P., Borca, M.V. 2023. Confirming the absence of parental African swine fever virus as a potential contaminant of recombinant live attenuated ASF vaccines. Biologicals. 3;83:101685. https://doi.org/10.1016/j.biologicals.2023.101685.*
                                *Vuono, E., Ramirez Medina, E., Silva, E.B., Rai, A., Pruitt, S.E., Espinoza, N.N., Valladares, A., Velazquez Salinas, L., Gladue, D.P., Borca, M.V. 2022. Deletion of H108R reduces virulence of the Georgia strain of African swine fever virus with surviving animals being protected against virulent challenge. Journal of Virology. https://doi.org/10.1128/jvi.00545-22.*
                                *Ramirez Medina, E., O'Donnell, V., Silva, E.B., Espinoza, N.N., Velazquez Salinas, L., Gladue, D.P., Borca, M.V. 2022. Experimental infection of domestic pigs with an African swine fever virus field strain isolated in 2021 from the Dominican Republic. Viruses.  https://doi.org/10.3390/v14051090.*
                                *Ramirez Medina, E., Vuono, E., Pruitt, S., Rai, A., Espinoza, N.N., Spinard III, E.J., Valadares, A., Silva, E.B., Velazquez Salinas, L., Borca, M.V., Gladue, D.P. 2022. Deletion of an African swine fever ATP-dependent RNA hel-icase QP509L from the highly virulent Georgia 2010 strain does not affect replication or virulence. Viruses. 14(11). https://doi.org/10.3390/v14112548.*
                                *Spinard III, E.J., Azzinaro, P.A., Rai, A., Espinoza, N.N., Ramirez Medina, E., Borca, M.V., Gladue, D.P. 2022. Structural predictions of the complete ASFV-G proteome. Microbiology Resource Announcements. 11(12):e0088122. https://doi.org/10.1128/mra.00881-22.*
                                *Attreed, S.E., Silva, C.M., Abbott, S.T., Ramirez Medina, E., Espinoza, N.N., Borca, M.V., Gladue, D.P., Diaz San Segundo, F.C. 2022. A highly effective African swine fever virus vaccine elicits a memory T cell response in vaccinated swine. Pathogens. 11(12). Article 1438. https://doi.org/10.3390/pathogens11121438.*
                                *Borca, M.V., Ramirez Medina, E., Silva, E.B., Ayushi, R., Espinoza, N.N., Velazquez Salinas, L., Gladue, D.P. 2023. ASF vaccine candidate ASFV-G-deltaI177l does not exhibit residual virulence in long-term clinical studies. Pathogens. 12(6). https://doi.org/10.3390/pathogens12060805.*


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