SAFE AND COST-EFFECTIVE NEXT GENERATION VACCINES AGAINST INFECTIOUS VIRUSES USING ESTABLISHED IMMORTAL AVIAN CELL LINES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 1009381
• Project Start Date: Mar 17, 2016
• Project End Date: Sep 30, 2020
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF ARKANSAS
(N/A)
FAYETTEVILLE,AR 72703

Performing Department
Poultry Sciences

Non Technical Summary
Avian viral diseases, such as avian influenza, Newcastle disease, infectious bronchitis, pneumovirus, Marek's disease, or laryngotracheitis cause economic losses in poultry industries by decrease productivities. Effective detection and prevention of avian viruses are of the utmost importance to maintain the US leadership in world poultry product markets. Development of more rapidly produced, safer,and more effective vaccinesare required to minimize the loss in poultry production. This project for optimal vaccine production will address a stable and safer baseline for protection of avian infectious viral pathogens. By doing so, the improvement in sustainability of US poultry may be realized by the much safer control of long-term threat of virus spread (as either an epidemic or even pandemic).

Animal Health Component

30%

Research Effort Categories

Basic

40%

Applied

30%

Developmental

30%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	4030	1101	60%
311	4030	1040	20%
311	3299	3100	20%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3299 - Poultry, general/other; 4030 - Viruses;

Field Of Science
1040 - Molecular biology; 1101 - Virology; 3100 - Management;

Keywords

vaccine

immortal cell lines

poultry viruses

Goals / Objectives
The goal of this proposal is to improve animal health by developing an optimal systme for the producionofinfectiousvaccine viruseofgenetically engineered-, non-reversible and live attenuated vaccines using established immortal avian cell lines. The specific objectives are to:1. Attenuate virulent and vaccine virus strains using immortal avian cell lines.2. Develop a new reverse genetics system retaining the complete full length sequence of viral RNA genome of vaccine strain using a novel artificial bacterial chromosome (BAC) vector and immortal avian cell line substrates to produce vaccine candidates.3. Compare commercial vaccines with the newly generated vaccines in this project in terms of immunogenic activity and protective efficacy.4. Produce a large quantity of optimally infectious vaccine strains for the use of vaccination in commercial setting.

Project Methods
Propagate and attenuate virulent and vaccine strains ofinfectious viruseusing immortal avian cell lines. Immortalized chicken cell lines will be utilized as continuous, stable cell substrates for the production of attenuated vaccinevirus strains. Virulent and vaccinevirus strains will be inoculated inimmortal aviancells in addition to primary chicken embryo tissue cultured cells as control cells. Media containing viruses will be collected at appropriate time points post infection. Collected viruses will be re-infected to freshly prepared cells for the attenuation. Attenuation will be performed for more than 100 passages. Virus titers will be determined by plaque assay and reverse-transcription (RT)-PCR at attenuation passages of 2, 4, 6, and every 10th passage. To identify the possible genetic variations possibly occurring during attenuation, the full-length viral genome will be sequenced at every 10th passage using 96 well format virus genome sequencing system.Develop a new reverse genetics system retaining the complete full length sequence of viral genomes. To construct reverse genetics system, commercially available genome cloning kit will be used to put amplified smallvirus gene segments into BAC vector. BAC clone containingvirus genomewill be subjected to site-directed mutagenesis to produce various mutant strains that may be fully attenuated and that can propagate more efficiently in the cell cultures. BAC clone will be transfected into cultured cells and virus titer and cytopathic effects will be determined at the second passage after transfection.Evaluate vaccine efficacy fornewly produced vaccines. Attenuated and genetically engineered vaccine candidates will be used for the vaccination to 1 day old chicks and, if needed, the vaccination will be boosted 2 - 3 weeks later. Blood samples will be collected from each vaccinated chickens at various time points and virus protective efficacy and antibody titers will be determined by virus neutralization assays using the in-vitro culture infection system by treating collected serums. IBV stocks highly neutralizing virulent strain will be subjected to an in-vivo challenge study. Vaccinated birds will be infected by virulent IBV viruses and the data including clinical signs, gross pathology, immunohistochemistry, weight gain, virus detection, and reversion to virulence will be collected.Produce a large quantity of optimally infectious vaccine viruses. To produce large volumes of high titer of the best vaccine for flock vaccination, we will use our immortal cell lines and the vaccine strain that is deemed the most infectious and protective from our above studies. As a result, we expect to produce a large quantity and high titer of a new vaccine for the application to larger population of chickens.

Progress 03/17/16 to 09/30/20

Outputs
Target Audience:Poultry Production; Animal Vaccine Industry Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A postdoctoral researcher was involved in this project. Two graduate student had performed this research. One student completed graduate program and moved to next career position (postdoctoral researcher). How have the results been disseminated to communities of interest?Results were presented at international conference meeting (The International Poultry Scientific Forum) and at the invited seminars. Results was published in journal, Poultry Science. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? During the project years, IBV attenuation in avian cells (Specific Aim 1) and BAC cloning for IBV (Specific Aim 2) have been conducted. Vero (African green monkey kidney), TT-1, CEK-im, CEL-im, and Caco2 (human epithelial colorectal adenocarcinoma as a negative control) cells were used in this project. As results, Vero, TT-1, CEK-im, and CEL-im cells were permissive by the infection of all three IBV strains compared with a negative control cell (CaCo2). Of cells tested, avian cell lines including TT-1, CEK-im, and CEL-im cells showed higher virus titers that mammalian Vero cells. During viral attenuation, virus titers were not increased as passaging. Additionally, primary cells and embryo itself were used for IBV attenuation and IBV attenuation in embryos and primary cells were confirmed by testing up to 14th passages. IBV titers were determined by reverse-transcription (RT-PCR) and the very low titers were detected from all substrates tested at 14th passage. Finally, IBV was attenuated using Leghorn male hepatoma (LMH) cell line and virus propagation were confirmed by testing up to 10th passages. IBV titers were determined by RT-PCR and moderate titers were detected (Specific Aim 1). For Specific Aim 2 (Develop a new reverse genetics system retaining the complete full length sequence of RNA genome of IBV vaccine strain), small segments at kilo-base pair level for genes (1a, 1b, 3a, 3b, S, sM, M, N, 5a, and 5b) of ARK-DPI strains were collected by PCR methods. Small fragments were cloned into plasmid vectors for further recombination to make longer fragments. Certain longer fragments were produced cloned into vector until the termination of this project (Specific Aim 2).

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Yang Y, Latorre JD, Khatri B, Kwon YM, Kong BW, Teague KD, Graham LE, Wolfenden AD, Mahaffey BD, Baxter M, Hernandez-Velasco X, Merino-Guzman R, Hargis BM, Tellez G. Characterization and evaluation of lactic acid bacteria candidates for intestinal epithelial permeability and Salmonella Typhimurium colonization in neonatal turkey poults. Poult Sci. 2018 Feb 1;97(2):515-521.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2020 Citation: Mandal A, Mandal R, Yang Y, Khatri B, Kong B, and Kwon Y. 2020. In vitro characterization of chicken gut bacterial isolates for probiotic potentials. Poultry Science. Accepted. https://doi.org/10.1016/j.psj.2020.11.025
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Khatri B, Hayden A, Smartt A, Seo D, Anthony N, Kong BW. Arkansas Progressor and Regressor chicken lines: important models for tumor biomarker study. International Poultry Scientific Forum. Poultry Sci. 2016; 95(E-Suppl. 1): 260 (Abst. #P197).

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Poultry Production; Animal Vaccine Industry Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A postdoctoral researcher was involved in this project. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?IBV will be attenuated during longer passages with LMH cell lines. A new graduate student will join this project.

Impacts
What was accomplished under these goals? We attenuatedIBV using Leghorn male hepatoma (LMH) cell line and virus propagation were confirmed by testing up to 10th passages. IBV titers were determined by reverse-transcription (RT-PCR) and moderatetiters were detected (Specific Aim 1). Construction of BAC vector based- reverse genetics system for IBV ARK-DPI strain was continued and 1a, 1b, and 3a framents were cloned into vector (Specific Aim 2).

Publications

• Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Mandal A, Mandal R, Yang Y, Khatri B, Kong B, and Kwon Y. 2019. In vitro characterization of chicken gut bacterial isolates for probiotic potentials. Poultry Science.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Poultry Production; Animal Vaccine Industry Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Agraduate student hadperformed this research, completed graduate program and move to next career position (post-doctoral researcher). How have the results been disseminated to communities of interest?Results was published in journal, Poultry Science and a manucript was submitted to Journal of Applied Poultry Reserch. What do you plan to do during the next reporting period to accomplish the goals?We will work on developing recombinant vaccine against poultry infectious viruses during next reporting period.

Impacts
What was accomplished under these goals? We continuedto optimize the attenuation procedures of IBV using primary cells and embryo itself in addition to established cell lines. IBV attenuation in embryos and primary cells were confirmed by testing up to 14thpassages. IBV titers weredetermined by reverse-transcription (RT-PCR) and the very low titers were detected from all substrates tested (Specific Aim 1). Construction of BAC vector based- reverse genetics system for IBV ARK-DPI strain was continued and 1a, 1b, and 3a framents were cloned into vector (Specific Aim 2). During the year reported, a new viral vaccine project was initiated and the accomplishments on the new project will be reported the following year.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2018 Citation: Vuong C, Barros T, Latorre J, Kong B, Moore R, Sellers H, Garritty C, Hargis B. 2018. Observed effects and occurrence of secondary infections in broilers after recovery from acute inclusion body hepatitis. Journal of Applied Poultry Research. Submitted.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Yang Y, Latorre J, Khatri B, Kwon Y, Kong B, Teague K, Graham L, Wolfenden A, Mahaffey B, Baxter M, Hern�ndez-Velasco X, Merino-Guzman R, Hargis B, and Tellez G. 2018. Characterization and evaluation of lactic acid bacteria candidates for reduction of intestinal epithelial permeability and Salmonella Typhimurium colonization in neonatal turkey poults. Poultry Science. 97(2):515-521. doi: 10.3382/ps/pex311

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Poultry Production; Animal Vaccine Industry Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student has performed this research. How have the results been disseminated to communities of interest?Results were presented at the invited seminars. What do you plan to do during the next reporting period to accomplish the goals?We will continue to optimize the attenuation procedures of IBV in avian immortal cell lines. Construction of reverse genetics system for IBV ARK-DPI strain will be continued. Another poultry infectious virus will be tested for permissiveness to poultryimmortal cell lines.

Impacts
What was accomplished under these goals? In thesecond year of this project, BAC cloning for IBV (Specific Aim 2) were mainly performed. We continued tocollect small segments at kilo-base pair level for genes (1a, 1b, 3a, 3b, S, sM, M, N, 5a, and 5b) of ARK-DPI strains by PCR methods. Small fragments are being cloned into plasmid vectors for further recombination to make longer fragments.

Publications

Progress 03/17/16 to 09/30/16

Outputs
Target Audience:Poultry Production; Animal Vaccine Industry Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One postdoctoral researcher and a graduate student have performed this research. How have the results been disseminated to communities of interest?Results were presented at international conference meeting (The International Poultry Scientific Forum). What do you plan to do during the next reporting period to accomplish the goals?We will continue to optimize the attenuation procedures of IBV in avian immortal cell lines. Construction of reverse genetics system for IBV ARK-DPI strain will be continued.

Impacts
What was accomplished under these goals? In the first yearof this project, IBV attenuation in avian cells (Specific Aim 1) and BAC cloning for IBV (Specific Aim 2) were initiated. Vero (African green monkey kidney), TT-1, CEK-im, CEL-im, and Caco2 (human epithelial colorectal adenocarcinoma as a negative control) cells were used in this project. As results, Vero, TT-1, CEK-im, and CEL-im cells were permissive by the infection of all three IBV strains compared with a negative control cell (CaCo2). Ofcells tested, avian cell lines including TT-1, CEK-im, and CEL-im cells showed higher virus titers that mammalian Vero cells.During viralattenuation, virus titers were not increased as passaging. For Specific Aim 2 (Develop a new reverse genetics system retaining the complete full length sequence of RNA genome of IBV vaccine strain), we are collecting small segments at kilo-base pair level for genes (1a, 1b, 3a, 3b, S, sM, M, N, 5a, and 5b) of ARK-DPI strains by PCR methods.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Khatri B, Hayden A, Smartt A, Seo D, Anthony N, Kong BW. Arkansas Progressor and Regressor chicken lines: important models for tumor biomarker study. International Poultry Scientific Forum. Poultry Sci. 2016; 95(E-Suppl. 1): 260 (Abst. #P197).