Source: KANSAS STATE UNIV submitted to NRP
PARTNERSHIP: SINGLE-CYCLE REPLICON-BASED AFRICAN SWINE FEVER VIRUS SUBUNIT VACCINE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1018996
Grant No.
2019-67015-29835
Cumulative Award Amt.
$647,000.00
Proposal No.
2018-06751
Multistate No.
(N/A)
Project Start Date
Jul 1, 2019
Project End Date
Jun 30, 2023
Grant Year
2019
Program Code
[A1221]- Animal Health and Production and Animal Products: Animal Health and Disease
Recipient Organization
KANSAS STATE UNIV
(N/A)
MANHATTAN,KS 66506
Performing Department
Diagnostic Medicine/Pathobiol
Non Technical Summary
The African Swine Fever Virus (ASFV) is a complex high-consequence transboundary animal disease pathogen that poses real danger to the swine industry and it has been identified as a High Priority National Food Security threat by USDA, DHS, and the National Pork Board. The trend in global outbreaks, including ongoing spread in eastern Europe, Russia, China, and Vietnam, shows that ASFV is increasingly becoming a global threat to the swine industry. The U.S.A is a major pork exporter and is thus a major source of food, revenue, and employment. Notably, the U.S.A has a large uncontrolled population of feral pigs in more than forty states that can become a reservoir as it happened in wild boars in Europe and Russia. There is no vaccine or treatment available for controlling this disease and therefore, development of an effective and safe vaccine is necessary for use in case there is an outbreak in North America.Protection against the ASFV can be stimulated with a vaccine since pigs that recover from infection with attenuated isolates are protected against closely related isolates that cause disease. Due to safety concerns, development of a vaccine based on carefully selected virus proteins, as opposed to an attenuated live virus vaccine, is more attractive for use in U.S.A since the virus is not present in North America. However, suitable vaccine candidate proteins and an effective way for immunizing pigs are yet to be identified. Candidate vaccines based on a few virus proteins have failed to stimulate acceptable protection. We envisaged that development of an effective vaccine will require identification and validation of multiple suitable candidate proteins that will induce significant protection in majority of the vaccinated pigs.We have developed rationally designed ASFV vaccine candidates and the goal of the current project is to determine whether the candidates can protect commercial pigs and wild boars against the virus. We will also develop a highly sensitive diagnostic tool that will allow differentiation of infected from vaccinated animals. The study will be conducted by immunizing commercial pigs or wild boars with the candidate vaccines. Naïve animals will serve as negative controls. The animals will be challenged with a lethal dose of the virus to evaluate protection. Development of an effective vaccine is critical for the containment of an ASFV outbreak and safeguarding of the U.S.A pig industry. In addition, such a vaccine will play a critical role in controlling global spread of the virus and reduce outbreaks in endemic regions.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31135991090100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3599 - Swine, general/other;

Field Of Science
1090 - Immunology;
Goals / Objectives
African swine fever (ASF) is an important foreign animal disease that originated from sub-Saharan Africa and it is currently spreading in eastern Europe, Russia, China and Vietnam. It poses a threat to the swine industry in the U.S.A and other countries. Currently, there is no vaccine or treatment available. The purpose of this research is to test the ability of experimental vaccine candidates to protect domestic pigs and wildboars against ASF virus (ASFV). The experimental vaccines are generated using modified adenovirus vectors. We will also develop a highly sensitive Lateral Flow Diagnostic Device (LFD) as a compartibleDIVA component.The specific Project Objectives are;1)Determine whether parenteral immunization of pigs with the prototype replicon-based ASFV vaccine will confer protection upon oral challenge. Commercial pigs will be immunized intramuscularly with the experimental vaccine, immunogenicity and tolerability will be evaluated, and protective efficacy will be determined following oral challenge;2)Determine whether oral immunization of pigs and wild boars with the prototype replicon-based ASFV vaccine will confer protection upon oral challenge. Pigs and wild boars will be immunized orally with a bait containing the experimental vaccine, immunogenicity and tolerability will be evaluated, and protective efficacy will be determined following oral challenge; and3)Test whether a novel LFD will differentiate ASFV-infected from vaccinated pigs and wild boars. We will validate specificity and sensitivity of the LFD using laboratory and field sera from ASFV-infected pigs and wild boars. In addition, we will test whether or not, the LFD can differentiate ASFV-infected from vaccinated pigs and wild boars.
Project Methods
Study 1Experimental Design and Procedures: Master seeds of the recombinant prototype adenovirus-based ASFV vaccine candidate replicons will be used to generate bulk vaccine virus required to conduct efficacy studies. The virus will be purified, concentrated, titrated, and quality control tested to confirm protein expression and authenticity. We will also confirm whether the progeny recombinant virus will still be replication-incompetent in non-complementing cells. In addition, we will generate affinity purified recombinant proteins needed for in vitro immune readouts.Immunization of pigs: Two groups of age-matched [35-45 lbs.] ASFV seronegative and PCR negative commercial piglets (n=15) will be used in this study. Each piglet in the treatment group will be inoculated intramuscularly (IM) with the recombinant prototype adenovirus-based ASFV vaccine candidate replicons (1 x 1011 viral particles of each construct) formulated using TXO adjuvant [Zoetis]. The immunogen formulation is based on our previous studies and guidelines from published data. Negative control piglets (n=15) will similarly be inoculated, but with an equivalent dose of adenovirus-Luciferase virus. The piglets will be boosted one month post-priming using cognate immunizing dose as above and then challenged orally with 104 TCID50 ASFV Georgia 2007/1.Readouts post-immunization and post-challenge: Antibody and T cell responses, post-prime, post-boost, and post-challenge will be evaluated biweekly by ELISA and by IFN-γ EliSpot. Following challenge, clinical signs will be monitored daily by a Veterinarian and recorded and time to death or euthanasia will be the terminal readout. In addition, viremia in blood and nasal swabs will be determined by quantitative PCR. Peripheral blood mononuclear cells and splenocytes [responders] and autologous skin fibroblasts [targets] from each pig will be used to evaluate antigen-specific CTL responses to identify the actual antigens that elicit significant CTL responses that correlate with protection post-challenge. The significance of the differences in immune readouts, clinical scores, viremia, and survival between the treatment and the control groups will be analyzed using Analysis of Variance followed by determining Fishers Least Significant Difference if distributional assumptions for ANOVA are met or a Kruskal-Wallis test (non-parametric equivalent of ANOVA) if ANOVA is not appropriate. A significance level of P<0.05 will be used for all analyses.Immunogen safety and tolerability: The pigswill be monitored daily (rectal temperature, appetite/activity, inoculation site swelling/granuloma) for the first five days post-priming and post-boost to document local and systemic adverse effects caused by the immunogen. Effect of the inoculum on the tissue at the injection site will also be evaluated at the end of the study.Study 2Experimental Design and Procedures: The ability of the replicon ASFV vaccine prototype to confer protection following oral bait immunization will be evaluated in age-matched [35-45 lbs.] ASFV seronegative-PCR negative commercial piglets [n=15] and wild boars [n=15]. Each animal in the treatment group will be primed and boosted, four weeks apart, with a proprietary oral bait containing the experimental ASFV vaccine [1 x 1011 viral particles/each construct]. Negative control piglets [n=15] and wild boars [n=15] will similarly be immunized and boosted, but with an equivalent dose of the Ad-Luciferase virus. The animals will be challenged orally, one month post-boost, with with 104 TCID50 ASFV Georgia 2007/1.Readouts post-immunization and post-challenge: ASFV antigen-specific immune responses post-prime, post-boost, and post-challenge will be evaluated as above. In addition, IFN-γ T-cell and CTL responses in oral lymph nodes/spleenwill be evaluated at study termination. Clinical signs, viremia, and time to death after challenge will be evaluated as above. The safety and tolerability of the immunogen post-priming and post-boost, will be monitored as above. The significance of the differences in immune readouts, clinical scores, viremia, and survival post-challenge between the treatment and the control groups will be analyzed by ANOVA as above and a significance level of P<0.05 will be used for all analyses.Study 3Development of KP1712-based LFD: Baculovirus-expressed KP1712 diagnostic antigen will be affinity purified and protein purity will be validated by PAGE. Antigen authenticity will be confirmed by Western Blotting using the ASFV convalescent serum. The recombinant KP1712 protein will be the target antigen and colloidal gold-labeled anti-porcine IgG/IgM antibody will serve as the conjugate, whereas porcine IgG/IgM antibodies will be the positive control. We are working with DCN Diagnostics Company to generate a prototype LFD. We will optimize the KP1712 LFD and validate performance to generate a fully functional prototype.Validation plan for KP1712 LFD diagnostic tool: The utility of the LFD prototype as a sensitive and specific diagnostic tool will first be tested using body fluids from our previous ASFV subunit vaccine immunogenicity and efficacy studies. The positive control will be ASFV convalescent serum and other validated positive sera, whereas sera from sham immunized pigs will serve as the negative control. We will include the p72 LFD [Ingezim-INGENASA, Spain] in the study as the reference validated diagnostic tool. We have hundreds of sera and saliva samples collected from commercial pigs pre-immunization, post-priming, and post-boost. We also have samples collected daily from treatment and negative control pigs following oral challenge with ASFV Georgia 2007/1 [genotype II virus]. These samples will be used to determine the earliest time point, post-challenge, when the KP1712 and the p72 LFDs can detect ASFV-specific antibodies. We have access to a cohort (n=3000) of laboratory and field sera samples from pigs, wild boars, and warthogs infected with ASFV isolates from around the world. We also have access to sera samples from pigs infected with classical swine fever virus [OIE Reference Lab for ASFV at Universidad Complutense De Madrid] and from naïve commercial pigs. These samples will be used to validate sensitivity and specificity of the KP1712 LFD compared to the p72 LFD. The OIE approved ELISA will be used to generate reference data. Selected samples of the sera from pigs, wild boars, and warthogs infected with different ASFV genotypes will be used to confirm sensitivity and specificity of the KP1712 and p72 LFDs by probing Western blots of whole virus lysate or by immunocytometric analysis of ASFV-infected cells. Following immunization of pigs and wild boars with the replicon-based ASFV vaccine [Studies 1 and 2], we will test whether or not, the KP1712 LFD can reliably and reproducibly differentiate vaccinated suid sera from ASFV-infected suid sera. Sensitivity and specificity of the KP1712 LFD will be calculated using the reference data generated using OIE ELISA as previously described.

Progress 07/01/19 to 06/26/23

Outputs
Target Audience:1. NIFA Project Directors and CRWAD attendees during the 2023 virtual meeting held in Chicago, January 20-24, 2023. 2. One abstract and one poster presentation reached scientiffic community and other stake holders. 3. Publication (reached scientiffic community and other stakeholders). Zajac, M.D., J. Yao, R. Kumar, N. Sangewar, S. Lokhandwala, H. Sang, K. Mallen, J. McCall, L. Burton, D., Kumar, E. Heitmann, T. Burnum, S. D. Waghela, K. Almes, J. D. Trujillo, J. Richt, T. Kim, W. Mwangi. 2023. Immunization of pigs with replication-incompetent Adenovirus-vectored African Swine Fever Virus multiantigens induced humoral immune responses but no protection following contact challenge. Front. Vet. Sci. Volume 10 - 2023. https://doi.org/10.3389/fvets.2023.1208275. Changes/Problems:1. Only one study was conducted in wildboars due to constraints caused by COVID-19. 2. Generation of single-cycle (Adenovirus-6) vectored ASFV antigens was not successful due to instability of virus progenies. One construct that was successfully generated was immunogenic in pigs but it caused diarrhea. This problem was solved by developing an attenuated Replication-Competent Adenovirus-5, which has generated promising results in a pilot study. 3. Constraints caused by COVID-19 [personnel and reagent shortage] coupled with technical hitches hindered development of the proposed Lateral Flow Device. This will be pursued in a different project. What opportunities for training and professional development has the project provided?Two PhD students, four undergraduate and and six DVM students received hands-on training. Specifically, the students received hands-on training in generation of adenovirus-vectored ASFV antigen expression constructs, animal bleeding, processing blood for sera and peripheral blood mononuclear cells, determining viremia, histology, immunohistochemistry, flow cytometry, ELISA/EliSpot, working in ABSL-2 and ABSL-3 [with select agent]. In addition, a Research Associate received hands-on training. How have the results been disseminated to communities of interest?1. NIFA Project Directors and CRWAD attendees during the 2023 virtual meeting held in Chicago, January 20-24, 2023. 2. One abstract and one poster presentation reached scientiffic community and other stake holders. 3. Publications: i) Zajac, M.D., J. Yao, R. Kumar, N. Sangewar, S. Lokhandwala, H. Sang, K. Mallen, J. McCall, L. Burton, D., Kumar, E. Heitmann, T. Burnum, S. D. Waghela, K. Almes, J. D. Trujillo, J. Richt, T. Kim, W. Mwangi. 2023. Immunization of pigs with replication-incompetent Adenovirus-vectored African Swine Fever Virus multiantigens induced humoral immune responses but no protection following contact challenge. Front. Vet. Sci. Volume 10 - 2023. https://doi.org/10.3389/fvets.2023.1208275. ii) Zajac, M.D., J. Yao, R. Kumar, H. Sang, N. Sangewar, S. D. Waghela, T. Kim, and W. Mwangi. 2023. Granzyme B and IFN-γ responses to putative CTL epitopes by lymphocytes from pigs immunized with adenovirus-vectored prototype multi-antigen ASFV vaccine (in-preparation). iii) Kumar, R., Zajac, M.D., H. Sang, E. Heitmann, D., Kumar, J. McCall, J. D. Trujillo, J. Richt, T. Kim, W. Mwangi. 2023. Immunization of pigs with Replication-Competent live-vectored African Swine Fever Virus antigen cocktail conferred protection against challenge with ASFV (Georgia 2007/1) (in-preparation). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1 was completed as reported previously and the results published. Objective 2: i) Results from a study conducted in wildboars that were immunized by IM injection and then challenged by exposure to ASFV contact spreaders was published. ii) A manuscript is in-preparation to report results from an efficacy dose-escalation study that was conducted in domestic pigs which received IM or intranasal immunization and then challenged by exposure to ASFV spreaders. iii) Developed attenuated Replication-Competent Adenovirus vaccine vector and used it to generate a prototype subunit vaccine, designated RC-Ad5-ASFV. Immunization of domestic pigs with this formulation, but not RC-Ad5-GFP, conferred protection to 5/6 following challenge with ASFV (Georgia 2007/1). A manuscript to report the results is in-preparation. iv) Screening of lymphocytes from protected pigs using the in-house generated monoclonal antibodies against porcine granzyme B and the putative epitopes generated using the ASFV motifs that bind strongly to 75 SLA alleles resulted in identification of novel CTL epitopes. A manuscript to report the results is in-preparation.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Zajac, M.D., J. Yao, R. Kumar, N. Sangewar, S. Lokhandwala, H. Sang, K. Mallen, J. McCall, L. Burton, D., Kumar, E. Heitmann, T. Burnum, S. D. Waghela, K. Almes, J. D. Trujillo, J. Richt, T. Kim, W. Mwangi. 2023. Immunization of pigs with replication-incompetent Adenovirus-vectored African Swine Fever Virus multiantigens induced humoral immune responses but no protection following contact challenge. Front. Vet. Sci. Volume 10 - 2023. https://doi.org/10.3389/fvets.2023.1208275.
  • Type: Journal Articles Status: Other Year Published: 2023 Citation: Zajac, M.D., R. Kumar, H. Sang, T. Kim, and W. Mwangi. 2023. Granzyme B and IFN-gamma responses to putative CTL epitopes by lymphocytes from pigs immunized with adenovirus-vectored prototype multi-antigen ASFV vaccine.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Mwangi, W., R. Kumar, M. Zajac, H. Sang, S. Adetunji, J. Trujillo, J. Richt, J. Manuel-Vizcaino, and T. PARTNERSHIP: Single-cycle replicon-based African Swine Fever virus subunit vaccine. CRWAD 2023. Abstract # 77005.
  • Type: Journal Articles Status: Other Year Published: 2023 Citation: Kumar, R., Zajac, M.D., H. Sang, E. Heitmann, D., Kumar, J. McCall, J. D. Trujillo, J. Richt, T. Kim, W. Mwangi. 2023. Immunization of pigs with Replication-Competent live-vectored African Swine Fever Virus antigen cocktail conferred protection against challenge with ASFV (Georgia 2007/1)


Progress 07/01/21 to 06/30/22

Outputs
Target Audience:NIFA Project Directors and CRWAD attendees during the 2021 virtual meeting held in December 3-7 . Changes/Problems:1. This project was negatively impacted by the COVID-19 pandemic and this slowed down progress due to disruption of supply chains and labor shortage. A one year no cost extension was granted and we expect to execute the project as proposed. 2. The single cycle adenovirus-6 (SC-Ad-6) replicon approach that we pursued as vaccine vector encountered technical hitches that we were unable to resolve due to instability of the recombinant viruses generated using ASFV genes. We pursued an alternative approach by generating adenovirus-5 based replicons and the resulting recombinant viruses expressing ASFV genes are stable and scalable. What opportunities for training and professional development has the project provided?Four undergraduate, five graduate, and six DVM students received hands-on training. Specifically, the students received hands-on training in generation of adenovirus-vectored ASFV antigen expression constructs, animal bleeding, processing blood for sera and peripheral blood mononuclear cells, determining viremia, histology, immunohistochemistry, flow cytometry, ELISA/EliSpot, working in ABSL-2 and ABSL-3 [with select agent]. In addition, a Research Associate and Research Assistant Professor received hands-on training. How have the results been disseminated to communities of interest?1. Zajac, M.D., N. Sangewar, S. Lokhandwala, J. Bray, R.P. Bishop, S. D. Waghela, and W. Mwangi*. Adenovirus-vectored African Swine Fever Virus p220 polyprotein induces robust antibody, IFN-gamma, and CTL responses in pigs. Front Vet Sci . 2022 May 31;9:921481. doi: 10.3389/fvets.2022.921481. eCollection 2022. 2. Mwangi, W., J. Yao, S. Lokhandwala, N. Sangewar, H.C. Sang, M. Zajac, J. McCall, R. Kumar, L. Burton, T. Kim, S. Waghela, J. Bray, and J.M. Sanchez-Vizcaino. PARTNERSHIP: Single-cycle replicon-based African Swine Fever virus subunit vaccine. CRWAD 2021 ID: V-P081. 3. Mwangi, W., S. Lokhandwala, N. Sangewar, J. Bray, J. Yao, H. C. Sang, and S. Waghela. Efficacy of prototype live-vectored African swine fever virus vaccines. CRWAD 2021 ID: V-P080. What do you plan to do during the next reporting period to accomplish the goals?1.Complete analysis and publication of data from the just concluded immunogen dose escalation pig study. 2. Utilize T cells isolated from the pigs immunized with the 10^10 immunogen dose to identify the actual ASFV antigens that induce Granzyme B+ T cells. The identified antigens will be used for future development of a second generation subunit vaccine. 3. Evaluate safety, immunogenicity, and protective efficacy of the 10^10 immunogen dose in wildboars [to be conducted by the Co-PI Dr. Viscaino in Madrid, Spain]. 4. Generate adenovirus replicons expressing the remaining ASFV antigens for whole proteome coverage and then conduct the final pig study to determine safety, immunogenicity, and protective efficacy of adenovirus replicon-based whole proteome immunogen formulation using the 10^10 dose.

Impacts
What was accomplished under these goals? Objective 1 was completed as reported previously. Objective 2: Nineteen (19) adenovirus replicons encoding multicistronic expression cassettes of rationally selected ASFV (Georgia 2007/1 isolate) vaccine candidate antigens were generated, tested for protein expression and validated using ASFV convalescent serum. The recombinant adenovirus replicons were scaled up, quality control tested, and a cocktail of the viruses were used to conduct a preliminary dose escalation [10^9; 10^10; and 10^11] study in commercial piglets to evaluate safety, immunogenicity, and protective efficacy. A negative control group received adenovirus replicon expressing GFP. Half of the immunogen was delivered by intranasal spray and the rest was injected IM. Following priming and two boosts, the piglets were challenged by contact with spreaders injected with wildtype ASFV (Georgia 2007/1). This in vivo study has just been completed and the preliminary data show that the 10^10 dose was better than the other two doses as judged by longevity of survival post-challenge and assessment by a pathologist. It was noted that the piglets immunized with the highest dose had diarrhea throughout the study period, suggesting that the intrasal immunization using this dose is not suitable. Preliminary assays show that the immunogen can elicit ASFV-specific cytotoxic T lymphocytes as judged by Granzyme B Intracellular Staining.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Zajac, M.D., N. Sangewar, S. Lokhandwala, J. Bray, R.P. Bishop, S. D. Waghela, and W. Mwangi*. Adenovirus-vectored African Swine Fever Virus p220 polyprotein induces robust antibody, IFN-gamma, and CTL responses in pigs. Front Vet Sci . 2022 May 31;9:921481. doi: 10.3389/fvets.2022.921481. eCollection 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Mwangi, W., J. Yao, S. Lokhandwala, N. Sangewar, H.C. Sang, M. Zajac, J. McCall, R. Kumar, L. Burton, T. Kim, S. Waghela, J. Bray, and J.M. Sanchez-Vizcaino. PARTNERSHIP: Single-cycle replicon-based African Swine Fever virus subunit vaccine. CRWAD 2021 ID: V-P081.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Mwangi, W., S. Lokhandwala, N. Sangewar, J. Bray, J. Yao, H. C. Sang, and S. Waghela. Efficacy of prototype live-vectored African swine fever virus vaccines. CRWAD 2021 ID: V-P080.


Progress 07/01/20 to 06/30/21

Outputs
Target Audience:1. NIFA Project Directors and CRWAD attendees during the 2020 virtual meeting held in December 5-8 . 2. Attendees of Special 'Symposium on African Swine Fever' sponsored by CRWAD and the North American PRRS Symposium. 3. One invited Keynote speaking, two abstracts, and one poster reached stakeholders [live and recorded content]. Changes/Problems:1. This project was negatively impacted by the COVID-19 pandemic and this slowed down progress.In addition, costs of materials and supplies have more than doubled. 2. Our first vaccine vector option based on the proposed single cycle adenovirus-6 (SC-Ad-6) replicon approach encountered technical hitches whereby, assembled recombinant viruses encoding ASFV multicistronic antigen expression cassettes were not stable and failed to amplify post-virus assembly. Alternative approaches were pursued and we have just managed to generate stable virus this month. This antigen delivery platform is superior to the replication-incompetent adenovirus (Ad-5) used in the just concluded pig study. We expect that SC-Ad-6 replicons will induce better immune responses in the next study. What opportunities for training and professional development has the project provided?Three undergraduate, four graduate, and six DVM students received hands-on training. Specifically, the students received hands-on training in generation of adenovirus-vectored ASFV antigen expression constructs, animal bleeding, processing blood for sera and peripheral blood mononuclear cells, determining viremia, histology, immunohistochemistry, flow cytometry, ELISA/EliSpot, working in ABSL-2 and ABSL-3 with select agent. In addition, a Research Associate and Research Assistant Professor received hands-on training. How have the results been disseminated to communities of interest?Abstracts: 1) Mwangi, W., S. Lokhandwala, J. Yao, H.C. Sang, N. Sangewar, and J.M. Sanchez-Vizcaino. PARTNERSHIP: Single-cycle replicon-based African Swine Fever virus subunit vaccine. CRWAD 2020 ID: 132. 2) Mwangi, W., S. Lokhandwala, N. Sangewar, J. Bray, J. Yao, H. C. Sang, S. Waghela, M. Prospects for an African Swine Fever Virus Subunit Vaccine. Special Symposium on African Swine Fever. CRWAD 2020. Oral Presentation: Invited Keynote speaker: Special 'Symposium on African Swine Fever' sponsored by CRWAD and the North American PRRS Symposium', Dec. 2020. Publications; 1) Huldah, S., G. Miller, Q. Manzil, S. Lokhandwala, N. Sangewar, S. D. Waghela, and W. Mwangi*. 2020. Progress towards development of Efficacious and Safe African Swine Fever Virus Prototype Vaccines. Front Vet Sci. 2020 Feb 21;7:84. doi: 10.3389/fvets.2020.00084. eCollection 2020. PMID: 32154279. 2) Cadenas-Fernández, E., J. M. Sánchez-Vizcaíno, A. Kosowska, B. Rivera, A. Rodríguez-Bertos, J. Yao, J. Bray, S. Lokhandwala, W. Mwangi, and J. A. Barasona. 2020. Adenovirus-vectored African swine fever virus antigen cocktail is not protective against virulent Arm07 isolate in Eurasian wild boar. Pathogens J. Feb 28;9(3):171. doi: 10.3390/pathogens9030171. PMID: 32121082. What do you plan to do during the next reporting period to accomplish the goals?1.Complete analysis and publication of data from the just concluded pig study. Tissues and cells from the pig that survived challenge will be used to define possible protective immune responses. 2. Cells isolated from the above mentioned hyperimmunized pigs will be used to screen the peptide library mentioned above to identify cytotoxic T lymphocyte epitopes for design of the next generation prototype subunit vaccine. 3. A group of pigs will be immunized with attenuated ASFV and cells from survivors, post-challenge, will be used to identify protective candidate antigens by screening target cells infected with the recombinant adenoviruses expressing ASFV proteome. The identified antigens will be used to generate multi-antigen prototype vaccine candidate that will then be evaluated for its potential to induce protective immunity following intramuscular or mucosal immunization. 4. Generate and test prototype Lateral Flow Diagnostic devise using the novel diagnostic antigen candidates mentioned above. This will be accomplished in collaboration with a commercial partner.

Impacts
What was accomplished under these goals? Objective 1: Forty two (42) multicistronic expression cassettes covering the whole ASFV (Georgia 2007/1) proteome were generated, tested for protein expression and validated using ASFV convalescent serum. The cassettes were used to generate recombinant adenoviruses and a cocktail of the viruses formulated with and without different adjuvants were tested in pigs to evaluate safety, immunogenicity, and protective efficacy. Following challenge by contact with spreaders injected with ASFV (Georgia 2007/1), only one pig in the group immunized with the virus cocktail without adjuvant survived until study termination 30 days post-challenge. This pig was able to clear challenge virus and overcome classical ASFV clinical symptoms. The animal study was completed in May 2021. Data is being processed and analyzed and a manuscript to report the outcomes is being generated. Tissues and cells collected from the pigs are being used to screen a putative CD8 T cell epitope peptide library based on >75 well characterized SLA-I binding prediction aligorithm. Three separate pigs were hyperimmunized five times with the adenovirus cocktail to generate ASFV-specific T cells. Following termination, peripheral blood mononuclear cells were isolated from maximum amount of blood that could be recovered. Cells were also isolated from the whole spleen, lymph nodes and kidneys. Objective 2: nothing to report. This is awating identification and validation of protective antigens in objective 1 to allow generation of a prototype vaccine candidate that will be evaluated for its ability to induce mucosal immunity. Objective 3: Novel diagnostic candidate antigens were identified by screening the whole ASFV proteome using convalescent serum. Six chimeric genes were generated and tested for protein expression using mammalian cells. The recombinant antigens were validated using ASFV convalescent serum. Futher modifications were done to improve protein yields [invention disclosure has been submitted]. The modified chimeric antigens are highly conserved among diverse ASFV genotypes and the recombinant antigens will be used to develop a prototype Lateral Flow-based diagnostic device for use at point of care. We have attracted a commercial partner interested in further product development.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Mwangi, W., S. Lokhandwala, J. Yao, H.C. Sang, N. Sangewar, and J.M. Sanchez-Vizcaino. PARTNERSHIP: Single-cycle replicon-based African Swine Fever virus subunit vaccine. CRWAD 2020 ID: 132.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Mwangi, W., S. Lokhandwala, N. Sangewar, J. Bray, J. Yao, H. C. Sang, S. Waghela, M. Prospects for an African Swine Fever Virus Subunit Vaccine. Special Symposium on African Swine Fever. CRWAD 2020.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Huldah, S., G. Miller, Q. Manzil, S. Lokhandwala, N. Sangewar, S. D. Waghela, and W. Mwangi*. 2020. Progress towards development of Efficacious and Safe African Swine Fever Virus Prototype Vaccines. Front Vet Sci. 2020 Feb 21;7:84. doi: 10.3389/fvets.2020.00084. eCollection 2020. PMID: 32154279.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Cadenas-Fern�ndez, E., J. M. S�nchez-Vizca�no, A. Kosowska, B. Rivera, A. Rodr�guez-Bertos, J. Yao, J. Bray, S. Lokhandwala, W. Mwangi, and J. A. Barasona. 2020. Adenovirus-vectored African swine fever virus antigen cocktail is not protective against virulent Arm07 isolate in Eurasian wild boar. Pathogens J. Feb 28;9(3):171. doi: 10.3390/pathogens9030171. PMID: 32121082.


Progress 07/01/19 to 06/30/20

Outputs
Target Audience:1. NIFA Project Director and CRWAD attendees during the 2019 meeting held in Chicago on November 4th. A submitted abstract was accepted, and a poster was also presented. 2. Potential commercial partners: Zoetis, CEVA, HUVEPHARMA, ELANCO, MDx [South Korea], and NAVETCO [Vietnam]. Changes/Problems:1. Following efforts to scale up the proposed novel diagnostic antigen [preliminary data had shown that this antigen is strongly recognized by convalescent serum] that we had proposed to use for development of a DIVA lateral flow device, protein yields were low for product development. This problem was addressed by identifying new diagnostic antigen candidates by screening the whole ASFV [2007/1] proteome using ASFV convalescent serum. We identified new strongly recognized antigens and the genes encoding these antigens were used to generate optimized mammalian expression constructs. The original construct was also modified to optimize protein expression. In addition, we acquired a new transient protein expression plaftform [Expi293 system] that has resulted in significantly improved protein yields. 2. We have successfully generated constructs encoding ASFV multicistronic antigen expression cassettes using single-cycle adenovirus backbone vectors and showed that the encoded antigens are well expressed. We used the constructs to rescue recombinant single-cycle adenoviruses. However, after multiple attempts with different constructs, the rescued viruses failed to amplify. Trouble shooting revealed that the multicistronic expression cassettes are not stable in the rescued virus. To address this problem, we have developed an alternative replicon virus vector based on Bovine Parainfluenza 3 Virus genotype C [BPI3Vc]. The novel vector has been used to generate stable recombinant viruses that are expressing the ASFV multicistronic cassettes. We are using this vector to generate ASFV antigen expression replicons which will be used to formulate a prototype vaccine that will be evaluated in domestic pigs and wild boars. What opportunities for training and professional development has the project provided?Several students received training during the reporting period.Specifically, two PhD students [Neha Sangewar, Huldah Sang] received hands-on training in this project. Five undergraduate students [Bailey Fritz, Jason Leftwich, Kayci Sperry, Kylynn Mallen Leeanna Burton (now an MS student in this reserach program)] also received hands-on training. Some of these folks will also be involved in the pending efficacy studies. The project trained Research Associate [Jianxiu Yao] and she was involved in the execution of the studies. How have the results been disseminated to communities of interest?1. Abstract: Mwangi, W*., S. Lokhandwala, J. Yao, H.C. Sang, N. Sangewar, and J.M. Sanchez-Vizcaino. 2019. PARTNERSHIP: Single-cycle replicon-based African Swine Fever virus subunit vaccine. CRWAD ID: P182. 2. Poster: W. Mwangi*, S. Lokhandwala, J. Yao, H. Sang, N. Sangewar, and J. Sanchez-Vizcaino. PARTNERSHIP: Single-cycle replicon-based African Swine Fever Virus subunit vaccine. CRWAD. Chicago, IL, November 4th, 2019. 3. Seminar presentations to potential commercial partners: Zoetis, CEVA, HUVEPHARMA, ELANCO, MDx [South Korea], and NAVETCO [Vietnam]. What do you plan to do during the next reporting period to accomplish the goals?1. Complete generation of ASFV multi-cistronic expression replicons, formulate prototype vaccine and determine whether intramuscular immunization of commercial pigs will elicit ASFV antigen-specific immune responses. We will challenge the pigs to evalute protective efficacy. We will also define immune responses that correlate with protection and use this knowledge to identify protective antigens. Definition of protective antigens will enable formulation of the next generation prototype vaccine that will be tested in wild boars. 2. Complete generation of novel DIVA lateral flow diagnostic device, conduct validation tests using samples from experimentary/naturally infected domestic pigs and wild boars [more than 3000 samples are available]. Work with commercial partner to deploy the device. 3. Publish/report data from the above two research focus areas.

Impacts
What was accomplished under these goals? In Aim 1, constructs encoding ASFV multicistronic antigen expression cassettes, the basic components needed for prototype vaccine formulation, were generated using single-cycle adenovirus backbone vectors, sequenced, and selected subclones were shown to express the encoded antigens. Authenticity of the expressed antigens was validated using ASFV convalescent serum. 2. The constructs mentioned above were used to rescue recombinant single-cycle adenoviruses. 3. Generated six new optimized protein expression mammalian plasmid expression constructs for generation of sufficient amounts of diagnostic antigens needed for development of a novel DIVA lateral flow device [LFD]. We have partnered with a commercial company [MDx, S. Korea] to develop and deploy the LFD.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Mwangi, W*., S. Lokhandwala, J. Yao, H.C. Sang, N. Sangewar, and J.M. Sanchez-Vizcaino. 2019. PARTNERSHIP: Single-cycle replicon-based African Swine Fever virus subunit vaccine. CRWAD ID: P182.