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2020 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 FY2020 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 US. Therefore, the evaluation of Classical Swine Fever Virus (CSFV) candidate vaccine FlagT4G was delayed. In addition, COVID-19 pandemic halted all laboratory activities at PIADC in Q2 and Q3 of FY2020.
Despite these delays, we were able to show that rationally designed live attenuated FlagT4G vaccine candidate was genetically stable. Reversion to virulence studies, which is an official OIE protocol designed to assess CSFV qualification, demonstrated that this vaccine candidate remains genetically and antigenically stable throughout a successive passage in groups of highly susceptible pigs.
The previously reported FlagT4G accompanying diagnostic DIVA (differentiating infected from vaccinated animals) test, developed in collaboration with CReSA (Barcelona, Spain) will be submitted for patenting by CReSA after ARS OTT approval. This DIVA test efficiently allows the serological differentiation of animals that has been vaccinated with FlagT4G from animals vaccinated or infected with any other strain of CSFV.
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. Four CSFV mutants each containing specific mutations that abolished interaction with a specific swine host protein were created and tested in animals. As a result, one novel determinant of virulence was mapped in the virus major structural protein E2 that interacted with host protein SERTAD1. The recombinant virus lacking the region of E2 that interacted with SERTAD1 was shown to have a decreased virulence in swine indicating that this protein-protein interaction is critical in defining virus virulence. This novel attenuated CSFV may be the basis of novel candidates for the development of live attenuated vaccines.
We continued advances in the systematic study of uncharacterized African swine fever virus genes that were initially selected by functional genomics criteria. Five 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 will be evaluated in pathogenesis studies in swine in Q4 of FY2020, provided that the COVID 19 pandemic situation allows access to the animal facility.
In FY2019, we reported the rational development of ASFV-G-delta I177L, a vaccine candidate recombinant strain, which induces a significant anti-ASFV response and protects swine against challenge with the epidemiologically significant isolate Georgia.
In FY2020, we extended our characterization of ASFV-G-delta I177L as vaccine candidate, showing that this vaccine candidate (i) is effective as vaccine even when used at very low doses (ii) is completely attenuated in swine even when administered at high doses, (iii) produce sterile immunity when used at the right doses and (iv) does not shed from vaccinated animals. These characteristics make ASFV-G-delta I177L the most promising experimental vaccine strain reported so far to protect swine against ASFV Georgia. A patent covering ASFV-G- delta I177L vaccine virus was filed. Several companies are in the process of licensing ASFV-G- delta I177L patent.
It is important to mention that as a result of the work we have done characterizing ASFV-G-delta 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 advance development of ASFV-G-delta I177L as potential commercial vaccine. Similarly, recombinant vaccine viruses previously developed in our laboratory, strains G-delta 9GL/delta UK and G-delta 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-delta I177L vaccine candidate can be administrated by 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 need to be solved to translate the production of these viruses to a commercial partner is the use of stable cell lines as substrate to produce vaccine virus. In FY2020 we have continued our effort to solve this problem using two different technical approaches: (i) discover a stable cell line that can efficiently support ASFV replication, and (ii) the adaptation of our vaccine candidates to grow in a stable cell line. More than 40 cell lines were tested obtained from research laboratories and commercial sources worldwide without, so far, finding one that efficiently support virus growth. This effort will continue during FY2021. In addition, we initiated the process of adaptation of our vaccine strains, G-delta 9GL/delta UK, G-delta MGF, and ASFV-G-delta I177L, to different established cell lines. Progress have been made in some of them and stocks of adapted vaccine viruses have been produced and genetically characterized. Additionally, preliminary results demonstrated that G-delta 9GL/delta UK virus could be adapted to grow in a stable cell line. This adapted G-delta delta9GL/ UK strain presented distinctive genetic changes related to the parental virus but still was able to protect domestic swine. This strain along with the adapted version of the others vaccine candidates will be further tested in more detail in their ability to induce protection against the virulent challenge in FY21. If results are successful, these actions will enormously facilitate the process of adoption of our vaccine candidates by commercial partners.
An important result obtained during the systematic evaluation of established cell lines supporting ASFV replication was the discovery that MA-104, a monkey derived cell line readily available from ATCC, was highly susceptible to being infected with ASF field isolates. We demonstrated that MA-104 cells were readily infected by all field ASFV isolates belonging to more than 10 different genotypes, and the sensibility of detection was just below that of the primary swine macrophage cultures. This discovery is of paramount importance for ASFV diagnostic since enable regional diagnostic laboratories that are usually unable to produce primary swine macrophage cultures and can now perform detection of ASFV infectious particles. A patent covering the use of MA-104 cells for ASFV diagnostic was filed along with the respective publication. Unfortunately, MA-104 do not yield high titers of virus, therefore we need to continue work to develop an appropriate cell line for vaccine virus production.
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. Several recombinants harboring DIVA markers were developed using G-delta 9GL/delta UK and G-delta MGF strains as template. Four recombinant DIVA marked virus strains were produced and genetically characterized. So far, all four viruses failed to induce protection at the same level that its corresponding parental virus. Several additional recombinant viruses considering different DIVA genes are being developed and planned to test during FY2021.
We continued our efforts to develop an ASFV experimental subunit vaccine using raccoon pox viruses as a vaccine vector. We extended our previous data that showed a recombinant virus co-expressing 6 different ASFV proteins, named rRPV6, by developing a novel construct expressing now 14 different ASFV proteins, named rRPV14. This construct, when tested in swine, did not induce protection against challenge with virulent ASFV. We are currently creating a second raccoon pox virus, harboring another 10-12 different ASFV proteins, named rRPV12, that seeks to expand the spectrum of ASFV proteins to be offered as subunit experimental vaccine. We plan to test an immunizing protocol contemplating a vaccine formulation including both, rRPV14 and rRPV12.
Accomplishments
1. Development of effective attenuated African Swine Fever Virus Vaccine -ASFV-G-deltaI177L. 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-delta I177L which possesses a remarkable therapeutic index, at levels exceeding all other ASV vaccine candidates. For the first time we have achieved sterile protection, absence of vaccine shedding, and a high safety profile, where high doses of the vaccine did not induce any clinical signs in pigs. As result, ASFV-G-delta I177L is the most promising vaccine candidate reported so far, capable of inducing protection, even at very low doses against the highly virulent ASFV strain Georgia. A patent covering the development ASFV-G-delta I177L was filed and several international commercial partners have initiated the process of licensing ASFV-G-delta I177L.
2. Discovery of stablished cell line to detect African Swine Fever Virus infectious field isolates. African Swine Fever Virus (ASFV) field isolates only replicate in primary cultures of swine macrophages, which is complicated and time consuming to prepare and requires a heard of healthy donor pigs. These factors make swine macrophage cultures inaccessible for most of the diagnostic laboratories trying to diagnose infection in suspect field samples. ARS scientists from Greenport, New York, discovered a cell line of monkey origin, Ma-104, was highly susceptible to infection with field isolates of ASFV. Infection was easily detected by two simple laboratory techniques. Ma-104 cells can be readily infected by all ASFV isolates tested and the sensitivity of detection was just below that of the gold standard, primary swine macrophage cultures, and above the sensitivity of conventional real-time PCR methods. This discovery is of paramount importance for ASFV diagnostics as it will enable diagnostic laboratories worldwide to perform detection of ASFV infectious particles, using a readily available, easy to grow cell line. A patent covering the use of Ma-104 cells for ASFV diagnostic was filed by ARS OTT.
Review Publications
Ramirez-Medina, E., Vuono, E., O'Donnell, V., Holinka, L.G., Silva, E., Rai, A., Pruitt, S., Carrillo, C., Gladue, D.P., Borca, M.V. 2019. The differential effect of the deletion of African swine fever virus virulence-associated genes in the induction of attenuation of the highly virulent Georgia strain. Virology. https://doi.org/10.3390/v11070599.
Velazquez-Salinas, L., Verdugo-Rodriguez, A., Rodriguez, L.L., Borca, M.V. 2019. The role of interleukin 6 during viral infections. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2019.01057.
Vuonno, E., Ramirez-Media, E., Berggren, K., Rai, A., Pruitt, S., Silva, E., Velazquez-Salinas, L., Gladue, D.P., Borca, M.V. 2020. Swine host protein coiled-coil domain containing 115 (CCDC115) interacts with classical swine fever virus structural glycoprotein E2 during virus. Viruses. https://doi.org/10.3390/v12040388.
Vuono, E., Ramirez-Medina, E., Azzinaro, P.A., Berggren, K., Rai, A., Pruitt, S., Silva, E., Velazquez-Salinas, L., Borca, M.V., Gladue, D.P. 2020. SERTA domain containing protein 1 (SERTAD1) interacts with classical swine fever virus structural glycoprotein E2 which is involved in virus virulence in swine. Journal of Virology. https://doi.org/10.3390/v12040421.
