Source: SOUTH DAKOTA STATE UNIVERSITY submitted to NRP
NOVEL PORCINE EPIDEMIC DIARRHEA VIRUS VACCINE PIPELINE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1008929
Grant No.
2016-67016-24949
Cumulative Award Amt.
$149,987.00
Proposal No.
2015-07006
Multistate No.
(N/A)
Project Start Date
Feb 15, 2016
Project End Date
Aug 14, 2019
Grant Year
2016
Program Code
[A1221]- Animal Health and Production and Animal Products: Animal Health and Disease
Recipient Organization
SOUTH DAKOTA STATE UNIVERSITY
PO BOX 2275A
BROOKINGS,SD 57007
Performing Department
Biology & Microbiology
Non Technical Summary
Recent emergence of extremely contagious Porcine Epidemic Diarrhea virus (PEDV) with 80-100% mortality in neonate pigscalls for the development of an effective prophylactic vaccine. PED has been such an important disease that it brought attentionto the NIFA Animal Health and Disease priority area (A1221). In this seed grant proposal, we propose to develop an effectivenovel vaccine candidate against PEDV infection.We will produce a ferritin nanoparticle immunogen coated with PEDV's spike (S) glycoprotein to induce neutralizing antibodies.We will formulate this novel nanoparticle into a Newcastle virus (NDV) vector with mucosal tropism derived from an attenuatedvaccine strain LaSota. The NDV vector is effective in inducing protective immunity at both the mucosal and systemic levels andit has been extensively used to express various antigens of important viral diseases including the full-length spike glycoproteinof SARS coronavirus. Resultant vector vaccines have shown safe and protective efficacies in different animal models, and NDVvector replication in eggs is amenable to large scale and rapid production.We hypothesize that the nanoparticle vaccine delivered by NDV vector mimic native viruses, which in turn elicit high titerneutralizing antibody responses and provide protection against PEDV infection in pigs. We propose to employ a pregnant sowmodel to test this hypothesis. Sow model is of clinical relevance because successful vaccination of sows is likely to play animportant role in the control and eradication of PED epidemics in piglets.
Animal Health Component
30%
Research Effort Categories
Basic
20%
Applied
30%
Developmental
50%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31135991101100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3599 - Swine, general/other;

Field Of Science
1101 - Virology;
Goals / Objectives
It is our hypothesis that NDV-delivered nanoparticles presenting multiple copies of PEDV S protein mimic native viruses, whichin turn elicit high titer neutralizing antibody responses and provide protection against PEDV infection. Two specific objectives areproposed below to address this central hypothesis:1. To determine in vitro functional properties and characterize immunogenicity of NDV-delivered S-ferritin NPs displaying thespike (S) glycoproteins of PEDV in mice.2. To evaluate the ability of NDV-delivered PEDV nanoparticle vaccine in inducing protective antibody response in pregnantsows to prevent clinical infection of piglets against PEDV.
Project Methods
We will test the central hypothesis that NDV-delivered PEDV S-ferritin NP vaccine can generate a strong neutralizing antibodyresponse in pregnant sows that will protect piglets against a lethal PEDV virus challenge. We will first generate NDV expressingPEDV S-ferritin NP and determine its functional properties prior to conducting the immunogenicity experiment in mice (Objective1). We will then evaluate its protective efficacy in piglets through immunization of pregnant sows. Cell-mediated immunity (CMI)may be involved in conferring protection against PEDV. We will plan an independent study of the CMI elicited by PEDV NPvaccination after the successful completion of this seed project.

Progress 02/15/16 to 08/14/19

Outputs
Target Audience:Swine industry. Our research has the potential to offer the swine industry an effective control and prevention strategy for Porcine Epidemic Diarrhea (PED), which is caused by a coronavirus. Changes/Problems:There have been no changes in the objectives of this project. Due to the initial challenge to generate a stable NDV vector under our experimental conditions, we switched our expression platform for delivering PEDV Spike protein ferritin nanoparticle to the baculovirus expression system, which is widely used for human and veterinary vaccine production. The ferritin nanoparticle strategy for PEDV is the same as what has been described in the original application. The mice study further validated the utility of baculovirus expression platform for this project. The candidate vaccine will be tested in a piglet experiment. The pig experiment has been significantly postponed due to a long wait for available biosafety level 2 plus animal facilities. Therefore, we have requested a second, non-cost extension of six months to allow the completion of the proposed pig experiment. What opportunities for training and professional development has the project provided?Jieshi Yu, PhD student, Biology and Microbiology conducted the work to develop the PEDV nanoparticle vaccine, and performed pig experiment to evaluate the vaccine's efficacy. Caleb Kragenbring, undergraduate student, Microbiology and Biotechnology participated in baculovirus expression work. Katherine Meyer, undergraduate student, Biology participated in generation of baculovirus expression constructs. Ella Lee, undergraduate student, Microbiology and Biotechnology participated in pig experiment. All the above listed students had participated in preparing the following conference presentation: Yu J, Sreenivasan C, Uprety T, Huang C, Gao R, Kragenbring C, Meyer K, Lee EJ, Dilberger-Lawson S, Singrey A, Diel DG, Nelson E, Li F, Wang D. 2019. Enhancement of PEDV replication and diarrhea by immunization with a Baculovirus-expressed recombinant spike glycoprotein. The 100th Annual Conference of Research Workers in Animal Diseases (CRWAD). November 2-5. Chicago, IL. Jieshi Yu was the presenter. He was also a presenter/author of a few other conference presentations and submitted manuscript: 1. Yu J, Liu R, Zhou B, Sheng Z, Li F, and Wang D. 2019. Development of Swine Influenza D Virus reverse genetics system and identification of nucleoprotein substitutions primarily determining in vitro viral replication fitness. 38th Annual ASV Meeting - July 20-24. Minneapolis, MN. 2. Yu J, Li F, Wang D. 2018. Influenza D Virus diverges from Its related Influenza C Virus in the recognition of 9-O-acetylated N-acetyl- or 9-O-glycolyl-neuraminic acid-containing glycan receptors. 2018 Annual meeting of American Society of Virology. July 14-18. College Park, MD. 3. Yu J, Sreenivasan C, Diel D, Nelson E, Li F, Wang D. 2018. Generation and immunogenicity analysis of baculovirus-delivered ferritin-driven porcine epidemic diarrhea virus spike nanoparticles. The 99th Annual Conference of Research Workers in Animal Diseases (CRWAD). December 1-4. Chicago, IL. 4. Yu J, Sreenivasan C, Li F, Wang D. 2020. Baculovirus-delivered ferritin-driven spike nanoparticles generate high-titers antibodies in mice that potently neutralize porcine epidemic diarrhea virus. Vaccine. (submitted). How have the results been disseminated to communities of interest?During the 2018 CRWAD meeting we presented our mice study showing the utility of ferritin-derived PEDV spike nanoparticle in stimulation of potent PEDV-neutralizing antibodies. Our data generated significant interest among fellow researchers due to the novel approach and produced high-titer neutralizing antibodies against PEDV. During the 2019 CRWAD meeting, we delivered an oral presentation entitled "Enhancement of PEDV Replication and Diarrhea By Immunization with a Baculovirus-Expressed Recombinant Spike Glycoprotein". Our presentation was well received, highlighting a critical need to evaluate the adverse effect that a PEDV subunit vaccine may stimulate in vaccinated piglets. 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: To determine in vitro functional properties and characterize immunogenicity of NDV-delivered S-ferritin NPs displaying the spike (S) glycoproteins of PEDV in mice. (100% Accomplished) Following the challenge to produce NDV-delivered S-ferritin nanoparticles, we have switched to the baculovirus expression platform, which has been a robust delivery platform for human and animal subunit vaccines. After development of baculovirus-based ferritin-driven PEDV S nanoparticle constructs, we successfully detected the expression of PEDV S nanoparticles through Western blot assay. Our mice study data showed that mice receiving intramuscular injection of ferritin-derived PEDV spike nanoparticles developed much higher titers of neutralizing antibodies against PEDV (titers ≥ 320) than those receiving the inactivated whole PEDV antigen (titers ≈ 80). One-way ANOVA analysis indicated the titers of the PEDV S nanoparticle group are significantly higher than that of the inactivated whole PEDV (P<0.05). No PEDV antibodies were detected in mice receiving either PBS or Ferrtin protein. The successful completion of Objective 1 supports further evaluation of PEDV nanoparticle vaccine efficacy in piglets, which is Objective 2 of our project. Objective 2: To evaluate the ability of NDV-delivered PEDV nanoparticle vaccine in inducing protective antibody response in pregnant sows to prevent clinical infection of piglets against PEDV. (100% Accomplished) Baculovirus-expressed PEDV Spike protein vaccine was tested for protective efficacy against PEDV challenge in PEDV antibody-free piglets at South Dakota State University's Animal Research Facility. In addition to the baculovirus-expressed PEDV Spike nanoparticle vaccine, the traditionally inactivated PEDV vaccine was also included in our vaccine study. Mock vaccination groups with or without PEDV challenge served as controls. Immunization of weaned piglets resulted in a neutralizing antibody response similar to that observed in mice as measured in fluorescent focus neutralization (FFN) assay. Interestingly, following the PEDV challenge, weaned piglets in the immunization group had a higher level of fecal viral RNA shedding and developed more severe diarrhea than the piglets receiving the baculovirus-derived control vaccine expressing only ferritin or the inactivated PEDV vaccine. In addition, moderate protective efficacy was observed in piglets receiving the inactivated PEDV vaccine. These data suggest that despite the stimulation of high-level neutralizing antibodies, vaccination with the baculovirus-expressed recombinant spike glycoprotein can lead to enhancement of PEDV replication and disease. This study provides a novel in vivo model for examining in detail the mechanisms of the vaccine-associated enhancement of PEDV infection and highlights the importance to avoid the production of deleterious immune responses in PEDV vaccine design. In summary, we have completed the objectives of this USDA/NIFA seed proposal. Despite the fact that the outcome was not expected, we strongly believe that vaccination of piglets with the baculovirus-expressed PEDV subunit vaccine exacerbating clinical diarrhea and disease should offer novel insights to future design of a safe and effective nanoparticle vaccine against porcine epidemic diarrhea.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Wang Z, Huang B, Thomas M, Sreenivasan CC, Sheng Z, Yu J, Hause BM, Wang D, Francis DH, Kaushik RS, Li F. 2018. Detailed mapping of the linear B Cell epitopes of the hemagglutinin (HA) protein of swine influenza virus. Virology 522:131-137.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Wan Y, Kang G, Sreenivasan C, Daharsh L, Zhang J, Fan W, Wang D, Moriyama H, Li F, Li Q. 2018. A DNA vaccine expressing Consensus Hemagglutinin-Esterase Fusion Protein protected Guinea Pigs from infection by two lineages of Influenza D Virus. J Virol 92: e00110-18.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Thomas M, Pierson M, Uprety T, Zhu L, Ran Z, Sreenivasan CC, Wang D, Hause B, Francis DH, Li F, Kaushik RS. 2018. Comparison of porcine airway and intestinal epithelial cell lines for the susceptibility and expression of pattern recognition receptors upon Influenza virus infection. Viruses 10:312
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Sheng Z, Liu R, Yu J, Ran Z, Newkirk SJ, An W, Li F, Wang D. 2018. Identification and characterization of viral defective RNA genomes in influenza B virus. J Gen Virol 99:475-488.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang Z, Yu J, Thomas M, Sreenivasan CC, Hause BM, Wang D, Francis DH, Kaushik RS, Li F. 2019. Pre-exposure with influenza A virus A/WSN/1933(H1N1) resulted in viral shedding reduction from pigs challenged with either swine H1N1 or H3N2 virus. Vet Microbiol. Jan;228:26-31. doi: 10.1016/j.vetmic.2018.11.008. Epub 2018 Nov 16.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Sreenivasan CC, Thomas M, Antony L, Wormstadt T, Hildreth M.B, Wang D, Hause B, Francis DH, Li F, Kaushik RS. 2019. Development and characterization of swine primary respiratory epithelial cells and their susceptibility to infection by four influenza virus types. Virology. 528:152163.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Sreenivasan CC, Thomas M, Kaushik RS, Wang D, Li F. 2019. Influenza A in bovine species: A narrative literature review. Viruses. 11:561.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Yu J, Liu R, Zhou B, Chou T-W, Ghedin E, Sheng Z, Gao R, Zhai S-L, Wang D, Li F. 2019. Development and characterization of a reverse-genetics system for influenza D virus. J Virol. 93:e01186-19. https://doi.org/10.1128/JVI.01186-19.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Sreenivasan CC, Jandhyala SS, Luo S, Hause BM, Thomas M, Knudsen DEB, Leslie-Steen P, Clement T, Reedy SE, Chambers TM, Christopher-Hennings J, Nelson E, Wang D, Kaushik RS, Li F. 2018. Phylogenetic analysis and characterization of a sporadic isolate of Equine Influenza A H3N8 from an unvaccinated horse in 2015. Viruses 10:31.


Progress 02/15/18 to 02/14/19

Outputs
Target Audience:Swine industry. Our research has the potential in offering swine industry an effective control and prevention strategy for Porcine Epidemic Diarrhea (PED), which is caused by a coronavirus. ? Changes/Problems:There have been no changes in the objectives of this project. Due to the initial challenge to generate a stable NDV vector under our experimental conditions, we switched our expression platform for delivering PEDV Spike protein ferritin nanoparticle to the baculovirus expression system, which is widely used for human and veterinary vaccine production. The ferritin nanoparticle strategy for PEDV is the same as what has been described in the original application. The mice study further validated the utility of baculovirus expression platform for this project. The candidate vaccine will be tested in a piglet experiment. The pig experiment has been significantly postponed due to a long wait for available biosafety level 2 plus animal facilities. Therefore, we have requested a second, non-cost extension of six months to allow the completion of the proposed pig experiment. What opportunities for training and professional development has the project provided?Jieshi Yu, PhD student, conducted the work to develop the PEDV nanoparticle vaccine, and apply it for NDV vector production using the baculovirus expression platform This graduate student is working under the supervision of the PI. With funding from the project, the PD, co-PD Dr. Li, and Jieshi Yu participated in 2018 CRWAD meeting (December 2018. Chicago, IL). How have the results been disseminated to communities of interest?During the 2018 CRWAD meeting we presented our mice study showing the utility of ferritin-derived PEDV spike nanoparticle in stimulation of potent PEDV-neutralizing antibodies. Our data generated significant interest among fellow researchers due to the novel approach and produced high-titer neutralizing antibodies against PEDV. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: To determine in vitro functional properties and characterize immunogenicity of NDV-delivered S-ferritin NPs displaying the spike (S) glycoproteins of PEDV in mice. We have completed Objective 1 of this seed project. Objective 2: To evaluate the ability of NDV-delivered PEDV nanoparticle vaccine in inducing protective antibody response in pregnant sows to prevent clinical infection of piglets against PEDV. In next reporting period we will focus on a piglet experiment to evaluate the ability of the PEDV ferritin-derived S nanoparticle vaccine to protect piglets from PED infection.

Impacts
What was accomplished under these goals? Objective 1: To determine in vitro functional properties and characterize immunogenicity of NDV-delivered S-ferritin NPs displaying the spike (S) glycoproteins of PEDV in mice. (100% Accomplished) Following the challenge to produce NDV-delivered S-ferritin nanoparticles, we have switched to the baculovirus expression platform, which has been a robust delivery platform for human and animal subunit vaccines. After the development of baculovirus-based ferritin-driven PEDV S nanoparticle constructs, we have successfully detected the expression of PEDV S nanoparticles through Western blot assay. Our mice study data showed that mice receiving intramuscular injection of ferritin-derived PEDV spike nanoparticles developed much higher titers of neutralizing antibodies against PEDV (titers ≥ 320) than those receiving the inactivated whole PEDV antigen (titers ≈ 80). One-way ANOVA analysis indicated the titers of the PEDV S nanoparticle group are significantly higher than that of the inactivated whole PEDV (P<0.05). No PEDV antibodies were detected in mice receiving either PBS or Ferrtin protein. The successful completion of Objective 1 supports further evaluation of PEDV nanoparticle vaccine efficacy in piglets, which is Objective 2 of our project. Objective 2: To evaluate the ability of NDV-delivered PEDV nanoparticle vaccine in inducing protective antibody response in pregnant sows to prevent clinical infection of piglets against PEDV. (30% Accomplished) We have generated ferritin-derived PEDV spike nanoparticles from the baculovirus expression platform, which will be tested for protection of PEDV infection of piglets. We have cultured the PEDV-Colorado strain for this virus challenge experiment. We were unable to complete this project because we could not reserve an appropriate animal facility for PEDV vaccine efficacy study that needs a specific BSL3-like containment condition. We are currently working with the SDSU animal facility to conduct a pig challenge experiment in early 2019. In addition, we are working with SDSU's Office of Research and Sponsored programs to apply for a second, no-cost-extension which needs to be approved by USDA/NIFA.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Sheng Z, Liu R, Yu J, Ran Z, Newkirk SJ, An W, Li F, Wang D. 2018. Identification and characterization of viral defective RNA genomes in influenza B virus. J Gen Virol 99:475-488.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Sreenivasan CC, Jandhyala SS, Luo S, Hause BM, Thomas M, Knudsen DEB, Leslie-Steen P, Clement T, Reedy SE, Chambers TM, Christopher-Hennings J, Nelson E, Wang D, Kaushik RS, Li F. 2018. Phylogenetic Analysis and Characterization of a Sporadic Isolate of Equine Influenza A H3N8 from an Unvaccinated Horse in 2015. Viruses 10.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Thomas M, Pierson M, Uprety T, Zhu L, Ran Z, Sreenivasan CC, Wang D, Hause B, Francis DH, Li F, Kaushik RS. 2018. Comparison of Porcine Airway and Intestinal Epithelial Cell Lines for the Susceptibility and Expression of Pattern Recognition Receptors upon Influenza Virus Infection. Viruses 10.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Wan Y, Kang G, Sreenivasan C, Daharsh L, Zhang J, Fan W, Wang D, Moriyama H, Li F, Li Q. 2018. A DNA Vaccine Expressing Consensus Hemagglutinin-Esterase Fusion Protein Protected Guinea Pigs from Infection by Two Lineages of Influenza D Virus. J Virol 92.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Wang Z, Huang B, Thomas M, Sreenivasan CC, Sheng Z, Yu J, Hause BM, Wang D, Francis DH, Kaushik RS, Li F. 2018. Detailed mapping of the linear B Cell epitopes of the hemagglutinin (HA) protein of swine influenza virus. Virology 522:131-137.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Rongyuan Gao, Dan Wang, and Feng Li. 2018. Trimer Interface Binding Human Monoclonal Antibody Recognizes Diverse Hemagglutinin (HA) Subtypes of Influenza A Viruses. 2018 Annual meeting of American Society of Virology. July 14-18. College Park, Maryland.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Jieshi Yu, Feng Li, and Dan Wang. 2018. Influenza D Virus Diverges from Its Related Influenza C Virus in the Recognition of 9-O-Acetylated N-Acetyl- or 9-O-Glycolyl-Neuraminic Acid-Containing Glycan Receptors. 2018 Annual meeting of American Society of Virology. July 14-18. College Park, Maryland.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Jieshi Yu, Chithra Sreenivasan, Diego Diel, Eric Nelson, Feng Li, and Dan Wang. 2018. Generation and immunogenicity analysis of baculovirus-delivered ferritin-driven porcine epidemic diarrhea virus spike nanoparticles. The 99th Annual Conference of Research Workers in Animal Diseases (CRWAD). December 1-4. Chicago, Illinois.
  • Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Jieshi Yu, Chithra Sreenivasan, Feng Li, and Dan Wang. Baculovirus-delivered ferritin-driven spike nanoparticles generate high-titers antibodies in mice that potently neutralize porcine epidemic diarrhea virus. Vaccine.


Progress 02/15/17 to 02/14/18

Outputs
Target Audience:Animal health industry; swine producers, and fellow researchers. Changes/Problems:We are currently requesting a 1-year no-cost extension for our USDA/NIFA seed project. The requested extension will enable us complete the objectives of our project, publish our data, and apply for a USDA/NIFA standard grant. What opportunities for training and professional development has the project provided?Three graduate students and one undergraduate student are working in this project. Jieshi Yu, PhD student, spent 6 months on the project in this year, working on virus culture, viral vector production, expression construct design. Other scholarly activities: Seventeenth Annual Symposium in Virology at the Nebraska Center for Virology. October 20, 2017. He has published a manuscript at mSphere as the first author. mSphere 2:e00254-17. 2017 Aug 9;2(4). He has also generated preliminary data for two grant proposals that were funded in last year: 1) Center grant "South Dakota Center for Biologics Research & Development", 07/01/2017-06/30/2022, sponsored by South Dakota Governor's Office of Economic Development; 2) NIH R21 grant "ZIKA assembly inhibitors", 07/01/2017--6/30/2019, sponsored by NIH/NIAID. Zhao Wang, PhD student, spent 2 months on the project in this year, working on plasmid purification and cloning. Other scholarly activities: He has submitted three first-author manuscripts that are currently under review or revision. Sunayana shyam Jandhyala, MS student, spent 6 months on the project in this year, working on structural modeling of PEDV spike protein. Other scholarly activities: Seventeenth Annual Symposium in Virology at the Nebraska Center for Virology. Caleb Kragenbring, undergraduate student, spent 12 months on the project in this year, working on the baculoviral vector expression of PEDV spike protein and spike-ferritin nanoparticles. Other scholarly activities: Poster presentation: Baculovirus Expression and Immunological Detection of the Spike Protein of Porcine Epidemic Diarrhea Virus. November 28, 2017. Poster presentation at "Fall 17 Day of Scholars" of Department of Biology and Microbiology, South Dakota State University. How have the results been disseminated to communities of interest?A poster presentation entitled" Baculovirus Expression of the PEDV Spike Protein nanoparticle vaccines" was presented at South Dakota State University's Scholar day on November 19, 2017. A manuscript entitled "Comparative epidemiology of porcine circovirus type 3 in pigs with different clinical presentations" was published in Virology Journal. The manuscript has been highlighted in AASV newsletter (https://www.aasv.org/news/story.php?id=10380), which provides updated coverage in the swine industry. What do you plan to do during the next reporting period to accomplish the goals?? Objective 1: To determine in vitro functional properties and characterize immunogenicity of NDV-delivered S-ferritin NPs displaying the spike (S) glycoproteins of PEDV in mice. We will complete mice experiments to determine the immunogenicity of the PEDV S-ferritin nanoparticles in mice. Objective 2: To evaluate the ability of NDV-delivered PEDV nanoparticle vaccine in inducing protective antibody response in pregnant sows to prevent clinical infection of piglets against PEDV. We will produce both baculovirus and NDV vectors expressing PEDV nanoparticles and complete a pig experiment to evaluate the efficacy of the PEDV S-ferritin nanoparticle vaccine for its ability in protecting piglets by vaccination of pregnant sows against PEDV.

Impacts
What was accomplished under these goals? Objective 1: To determine in vitro functional properties and characterize immunogenicity of NDV-delivered S-ferritin NPs displaying the spike (S) glycoproteins of PEDV in mice (60% accomplished). We have developed baculovirus vector-based expression systems for both PEDV's spike protein and spike-ferritin fusion protein. These constructs have been confirmed by PCR coupled Sanger sequencing method. Protein expressions were further verified in Western-blot assay. We have introduced these constructs into insect Sf9 cells and are currently in the process of generating a sufficient amount of purified proteins. These nanoparticle antigens will be evaluated in mice experiments for their ability in stimulating protective antibody responses against PEDV Colorado 2013 strain. Objective 2: To evaluate the ability of NDV-delivered PEDV nanoparticle vaccine in inducing protective antibody response in pregnant sows to prevent clinical infection of piglets against PEDV (40% accomplished). We have replicated PEDV-Colorado strain in the lab, which will be used in our virus challenge experiment in pigs. Over the past year, we developed a neutralization antibody assay that enables us to evaluate vaccine-mediated protection in pigs. We have produced NDV vectors in our laboratory and are collaborating with our USDA collaborator Dr. Qingzhong Yu to produce recombinant NDV vectors expressing both PEDV's spike protein and spike-ferritin nanoparticles. Dr. Yu is a CoPD of this project. Considering genetic and antigenic diversity of PEDVs, we have employed a computational biology approach to model the virus-receptor interaction complex. Predicted structure will enable investigating antigenic sites of PEDV and addressing how genetic polymorphisms at these sites affect viral tropism and antigenic drift. Antibodies produced in our mice and pig experiment will further evaluate the modeled structure.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Zhai SL, Zhang H, Chen SN, Zhou X, Lin T, Liu R, Lv DH, Wen XH, Wei WK, Wang D, Li F. Influenza D Virus in Animal Species in Guangdong Province, Southern China. Emerg Infect Dis. 2017 Aug; 23(8):1392-1396. doi: 10.3201/eid2308.170059.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Yu J, Hika B, Liu R, Sheng Z, Hause BM, Li F, Wang D. 2017. The hemagglutinin-esterase fusion glycoprotein is a primary determinant of the exceptional thermal and acid stability of influenza D virus. mSphere 2:e00254-17. 2017 Aug 9;2(4).
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Zhai SL, Lin T, Chen YW, Liu R, Lv DH, Wen XH, Zhu XL, Wang D, Li F, Wei WK. First complete genome sequence of canine bocavirus 2 in mainland China. New Microbes New Infect. 2017 Apr 13;18:47-49. doi: 10.1016/j.nmni.2017.04.002. eCollection 2017 Jul.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Wang D, Ma W. Visualization of IAV Genomes at the Single-Cell Level. Trends Microbiol. 2017 Oct; 25(10):781-782. doi: 10.1016/j.tim.2017.08.003. Epub 2017 Aug 23.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Shao-Lun Zhai, Xia Zhou, He Zhang, Ben M. Hause, Tao Lin, Runxia Liu, Qin-Ling Chen, Wen-Kang Wei, Dian-Hong Lv, Xiao-Hui Wen, Feng Li and Wang D. Comparative epidemiology of porcine circovirus type 3 in pigs with different clinical presentations; Virology Journal; 13 November 2017 14:222


Progress 02/15/16 to 02/14/17

Outputs
Target Audience:Animal health industry; swine producers, and fellow researchers.Our efforts have resulted better understanding about PEDV and other porcine corona viruses that are threatening swine health. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students were involved in this project in the past year. Mr. Jieshi Yu is a first-year PhD student who arrived at SDSU late August, 2016, and Ms. Runxia Liu is a PhD student. Both graduate students are working under the supervision of the PD and co-PDs. This project provided 4-5 months of graduate research assistantship for each of them, allowing for significantly increased one-to-one mentor and mentee interactions. With support of the fund, the PD and co-PD Dr. Li participated in the USDA-NIFA Animal Health and Well-Being Project Director's (PD) meeting and CRWAD meeting (December 2016. Chicago, IL) and visited Dr. Qingzhong Yu's laboratory (December 2016. Athens, GA) to get training in NDV vector-based vaccine techniques. How have the results been disseminated to communities of interest? A manuscript entitled "Occurrence and sequence analysis of porcine deltacoronaviruses in southern China" has been published in Virology Journal. The manuscript has been highlighted in American Association of Swine Vaterinarians (AASV) newsletter (AASV e-Letter, August 10th, 2016), which provides updated coverage to the swine industry. Link: :https://www.aasv.org/news/story.php?id=9156 The research narratives have been included by the "Department of Biology & Microbiology Research, South Dakota State University" that was published last year as hard copies and posted on our university website as well. Our research activities have drawn attention from current and prospective undergraduate students. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: To determine in vitro functional properties and characterize immunogenicity of NDV-delivered S-ferritin NPs displaying the spike (S) glycoproteins of PEDV in mice. Upon functional validation and generation of NDV vector-based nanoparticles expressing PEDV spike-ferritin protein, we will determine the immunogenicity of the PEDV S-ferritin nanoparticles in mice. Objective 2: To evaluate the ability of NDV-delivered PEDV nanoparticle vaccine in inducing protective antibody response in pregnant sows to prevent clinical infection of piglets against PEDV. We will plan and conduct a pig experiment to evaluate the efficacy of the PEDV S-ferritin nanoparticle vaccine for its ability to protect piglets by vaccination of pregnant sows against PEDV.

Impacts
What was accomplished under these goals? Objective 1: To determine in vitro functional properties and characterize immunogenicity of NDV-delivered S-ferritin NPs displaying the spike (S) glycoproteins of PEDV in mice (50% accomplished) We have generated codon-optimized PEDV's spike gene expression constructs and fused them into our ferritin nanoparticle scaffold plasmid. We are currently characterizing the expression and antigenicity of these nanoparticle constructs in vitro prior to producing NDV vector expressing the PEDV spike-ferritin nanoparticles. In addition, we have cultured a PEDVColorado strain for virus challenge experiments. Objective 2: To evaluate the ability of NDV-delivered PEDV nanoparticle vaccine in inducing protective antibody response in pregnant sows to prevent clinical infection of piglets against PEDV (20% accomplished) We have formed a strong team with necessary swine vaccine trial experience. Two graduate students (Mr. Jieshi Yu and Mr. Hunter Nedland), one undergraduate student (Mr. Jared Wollman) and a visiting scholar (Dr. Shaolun Zhai) have attained relevant animal handling skills during the last year with the support from South Dakota and AES funds. They will be added to the personnel of the IACUC (15-116A) and participate in the animal experiments. The immunogenecity of three forms of PEDV nanoparticle vaccines will be evaluated in mice beginning August 2017. A pig PEDV nanoparticle vaccine trial will follow. Overall, 40% of the stated objectives have been completed. Upon the successful completion of constructs characterization and identification of suitable nanoparticle vaccine candidates, we will move forward to in vivo work to test the immunogenicity and protective efficacy of these nanoparticle vaccines.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Zhai SL, Wei WK, Li XP, Wen XH, Zhou X, Zhang H, Lv DH, Li F, Wang D; Occurrence and Sequence Analysis of Porcine Deltacoronaviruses in Southern China; Virol J. 2016 Aug 5;13:136. doi: 10.1186/s12985-016-0591-6. PMID: 27496131 DOI: 10.1186/s12985-016-0591-6
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Zhai SL, Wei WK, Li XP, Wen XH, Zhou X, Zhang H, Lv DH, Li F, Wang D. Occurrence and evolutionary characterization of porcine Deltacoronaviruses in southern China. Global Veterinary Microbiology and Veterinary Medicine Summit. October 17-18, 2016 Chicago, USA. Abstract accepted by J Veterinar Sci Techno. DOI: 10.4172/2157-7579.C1.019