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

Location: Foreign Animal Disease Research

2022 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
During FY 2022, still animal experiments performed at PIADC 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 and alternatively performed in overseas laboratories through collaborative agreements. In addition, the COVID-19 pandemic still affected all laboratory activities at PIADC in Q4 of FY2021 and Q1 in 2022. Collaborative studies with Centre de Recerca en Sanitat Animal (CRESA) Barcelona, Spain were conducted to characterize the early (5 days post vaccination) protective immune response in FlagT4G vaccinated animals. Evaluation of the presence of element of the innate and adaptative immune response were performed in vaccinated animals which resulted sterile protected against the challenge with highly virulent field strain Margarita. Absence of virus specific antibody (detected by ELISA and virus neutralization assay) as cellular response (detected by gIFN ELISPOT) indicate that early protection is entirely produced by a regulated cytokine mediated innate response (mainly alpha IFN) which was analyzed by detecting serum levels of 15 different lymphokines. These results demonstrated by the first time the role of the innate immune mechanisms in the induction of early protection induced by live attenuated CSF vaccines. 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. Six virus genes (E184L, A859L, A104R, H108R, MGF110-5L-6L and A165R) 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 three of those genes (A859L, MGF110-5L-6L and A165R) in the genome of ASFV Georgia isolate showed none of those genes were associated with disease production. The other three remaining genes, E184L, A104R, and H108R), were shown to be involved in the process of virulence in swine since their individual deletions partially attenuates ASFV Georgia virulence. The significance of this discovery is that E184L, A104R, and H108R are among the only 12 virus genes (all but four discovered at PIADC) whose deletion attenuates the virus causing the current Eurasian pandemic of ASF. Importantly, animals surviving the inoculation with recombinant viruses individually lacking the E184L, and H108R genes were completed 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. We previously 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 the most promising vaccine candidate reported so far. This year we extend our studies on this vaccine candidate. Working in collaboration with our MTRA partner in Vietnam, we showed that ASFV-G-I177L protects pigs from the challenge performed with a Vietnamese field isolate as efficiently as it does against the parental Georgia isolate. This result indicates the presence of effective coverage against field strain that have been evolved genetically and antigenically in the field for over 15 years. In addition, in these studies it was shown that ASFV-G-I177L effectively induce protection in pigs of an heterogenous genetic background (Vietnamese breeds). Also, in collaboration, we performed a complete set of experiments evaluating the safety of the ASFV-G-I177L vaccine. Results demonstrated that ASFV-G-I177L is genetically stable remaining attenuated in Reversion to Virulence experiments. Also, the virus shedding, and the absence of local and systemic toxicity was evaluated. The results obtained indicate that ASFV-G-I177L safe and effective vaccine and has led to commercial production and regulatory approval in Vietnam. It is important to mention that because 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 commercial vaccine. Similarly, recombinant vaccine strain ASFV-G-9GL/UK has also been excluded from the Select Agent list and have been transferred to vaccine industry partners for further development. 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. This year we expanded that knowledge by testing all ASFV proteins encoded in the genome of the ASFV strain Georgia in an eukaryotic expression system. The immunogenicity of all ASFV proteins were tested by their reactivity with sera from pigs vaccinated with our vaccine candidates and were protected against the challenge. The information obtained allowed us to identify a novel DIVA candidate, the MGF110-5L-6L gene. Sera from animals immunized with vaccine candidates ASFV-dMGF, ASFV-dI177L and ASFV-d9GL/dUK all strongly recognized MGF110-5L-6L protein expressed in eukaryotic cells. We developed a recombinant virus by deleting this gene in the genome of the ASFV-G-dI177L vaccine candidate. Therefore, the protein encoded by MGF110-5L-6L gene is a good candidate to be used as a negative antigenic marker to develop a DIVA test. This result constitutes the second reported experimental proof of a functional antigenic marker in ASFV. It is important to remark that our laboratory has developed genetic DIVA test for all the four vaccine candidates that has been transferred to pharmaceutical companies, ASFV-dMGF, ASFV-d9GL/dUK, ASFV-dI177L and ASFV-dI177L/dLVR. The genetic DIVA tests are quantitative real time PCR that specifically detect the presence of parental ASFV Georgia isolate. The sensitivity of the test is similar to the commercial real time PCR based in the detection of p72 gene. In addition, our laboratory was the first in characterize the virulence and transmissibility of the ASFV virus isolated in the Dominican Republic (ASFV DR21 isolate). The ASFV DR21 isolate was genetically homologous to the ASFV Georgia isolate. However, results demonstrated the DR21 isolate, when inoculated by oronasal route or by contact, presented a reduced virulence and transmissibility when inoculated in domestic pigs when compared to that of the ASFV Georgia isolate. Approximately 50% of animals oronasally or inoculated by contact survived infection with the DR21 virus and, importantly, all of them developed a strong virus specific response. These results may be important in the epidemiological management of infected animals in the Dominican Republic.

Accomplishments
1. African Swine Fever Virus vaccine. African Swine Fever Virus vaccine candidate ASFV-G-dI177L have been the first ASFV vaccine approved for commercial use in Vietnam. 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-G-dI177L, 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-dI177L is the most promising vaccine candidate reported so far. In partnership, through a MTRA, in Vietnam ARS scientist have developed master stocks of ASFV-GI177L and successfully tested their protective efficacy against local virus field strains in pigs of European and Asian genetic background. In addition, our pertner has shown that ASFV-G-I177L is genetically stable, remains attenuated and lacks local and general toxicity when inoculated in domestic pigs. Based in those results our partner has received the certificate of Marketing Authorization from Department of Animal Health of Vietnam government 6/3/2022. Making of ASFV-GI177L the first ASFV vaccine approved for commercial use.

2. Phenotypic and genetic characterization of the first ASFV. Phenotypic and genetic characterization of the first ASFV field isolate detected in the Western hemisphere in more than 40 years. Our laboratory was the first and only in characterizing the genotype, virulence and transmissibility of the ASFV virus isolated in the Dominican Republic (ASFV DR21 isolate) during the first outbreak of the disease occurred in the western hemisphere since 1980. Although the ASFV DR21 isolate genome was homologous to the ASFV Georgia isolate, our results demonstrated the DR21 isolate presented a reduced virulence and transmissibility, compared with the Georgia isolate when inoculated in domestic pigs by the natural route, surviving approximately 50% of the inoculated animals. These results are of critical importance in the epidemiological management of the disease in Hispaniola Island and in the prevention of the potential spreading of the disease to the USA.

3. Systematic identification of the role of ASFV genes in in virus virulence. We have developed recombinant viruses that have deletions of individual genes in order to assess their role in virulence. There have been approximately 40 reported single gene deletions in ASFV, with the majority of these single deletions being performed in our laboratory at PIADC. In this 2021 period alone we have tested 6 different virus genes using recombinant ASFV viruses lacking a single viral gene deletion. Testing of these individual gene deletions has led us to the identification of new determinants of virulence and potential DIVA markers (antigenic markers that can differentiate between vaccinated and previously infected animals), a necessary tool for any successful vaccine eradication program. This information will be used to further develop next-generation vaccines for ASF and is critical to the field of ASFV research to provides the basis for any rationally designed vaccine.

Review Publications
Canter, J.A., Aponte, T., Ramirez Medina, E., Pruitt, S.E., Neilan, J., Gladue, D.P., Borca, M.V., Zhu, J.J. 2022. Determining hyperimmune serum neutralizing effect on African Swine Fever Virus and serum enhancing effect on extracellular virion infectivity in adherent pig PBMC by flow cytometry. Scientific Reports. https://doi.org/10.3390/v14061249.
Ramirez Medina, E., Vuono, E., Pruitt, S.E., Rai, A., Valladares, A., Espinoza, N.N., Velazquez Salinas, L., Gladue, D.P., Borca, M.V. 2021. Evaluation of the deletion of ASFV MGF110-5L-6L on swine virulence and its potential use as a DIVA vaccine marker gene. Viruses. https://doi.org/10.3390/v13020286.
Ramirez Medina, E., Vuono, E., Rai, A., Pruitt, S.E., Espinoza, N.N., Velazquez Salinas, L., Pina-Pedrero, S., Zhu, J.J., Rodriguez, F., Borca, M.V., Gladue, D.P. 2021. Deletion of E184L, a putative DIVA target from the pandemic strain of African Swine Fever Virus, produces a reduction in virulence and protection against virulent challenge. Viruses. https://doi.org/10.1128/JVI.01419-21.
Bohórquez, J., Defaus, S., Pérez-Simó, M., Alberch, M., Gladue, D.P., Borca, M.V., Andreu, D., Ganges, L. 2021. Development of a dendrimeric peptide-based approach for the differentiation of animals vaccinated with FlagT4G against classical swine fever from infected pigs. Viruses. https://doi.org/10.3390/v13101980.
Tran, X., Le Thi Thu, P., Nguyen Quang, H., Do Thanh, T., Nguyen Van, D., Gay, C.G., Borca, M.V., Gladue, D.P. 2021. African swine fever virus vaccine candidate ASFV-G-I177L efficiently protects European and native pig breeds against circulating Vietnamese field strain. Transboundary and Emerging Diseases. https://doi.org//10.1111/tbed.14329.
Xuan, T., Le Thi Thu, P., Nguyen Quang, H., Do Thanh, T., Nguyen Van, D., Pham Hào, Q., Quách Võ, N., Gay, C.G., Gladue, D.P., Borca, M.V. 2022. Evaluation of the safety profile of the ASFV vaccine candidate ASFV-G-I177L. Viruses. https://doi.org/10.3390/v14050896.
Jaime, L., Lex, M., Gladue, D.P., Borca, M.V., Jorge, O. 2021. Optimization in the expression of ASFV proteins for the development of subunit vaccines using poxviruses as delivery vectors. Scientific Reports. https://doi.org/10.1038/s41598-021-02949-x.
Velazquez Salinas, L., Ramirez Medina, E., Rai, A., Pruitt, S.E., Vuono, E.A., Espinoza, N.N., Gladue, D.P., Borca, M.V. 2021. Development real-time PCR assays to genetically differentiate vaccinated pigs from pigs infected with the Eurasian strain of African swine fever virus. Frontiers in Veterinary Science. https://doi.org/10.3389/fvets.2021.768869.