Source: UNIVERSITY OF ILLINOIS submitted to NRP
TOWARD A CELL-FREE MAREK'S DISEASE VACCINE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1032080
Grant No.
2024-67015-42412
Cumulative Award Amt.
$650,000.00
Proposal No.
2023-07978
Multistate No.
(N/A)
Project Start Date
Jul 1, 2024
Project End Date
Jun 30, 2027
Grant Year
2024
Program Code
[A1221]- Animal Health and Production and Animal Products: Animal Health and Disease
Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801
Performing Department
(N/A)
Non Technical Summary
Over 103 million metric tons of chicken meat was produced in the world in 2023, with 20% produced in the USA, ranking poultry as the second most-produced meat in the world. Americans also consume the most chicken meat in the world, despite having a smaller population than other countries like China or India. The cost of meat and egg production has increased over the years partly due to incidences of diseases such as Marek's disease. Marek's disease is caused by a herpes virus called Marek's disease virus (MDV) and causes cancer and other ailments within the first few weeks after hatching. This ultimately leads to decreased growth of the chickens, susceptibility to other diseases, condemnation of the meat at the processing plant, and death, contributing to the overall cost of production. Most licensed vaccines against MD are administered as cell-associated preparations, also increasing the costs of production since these vaccines require storage and transportation in liquid nitrogen and require a significant amount of primary cell culture for their production. Therefore, a cell-free Marek's disease vaccine that can be prepared as a stable, lyophilized preparation would greatly benefit the poultry industry, especially in countries or rural settings that cannot afford liquid nitrogen storage for current MD vaccines. A major obstacle in designing new cell-free vaccines is the lack of information on viral genes important for this essential step in the virus life cycle. We have identified specific virus genes that are essential for the transmission of MDV and the production of a cell-free virus termed the conserved herpesvirus protein kinase (CHPK). We have identified modifications of this protein kinase during infection in chickens that are not present during replication in cell culture, the technique used to produce Marek's disease vaccines. Our goal here is to better understand these specific modifications of the protein kinase that we can use to produce cell-free Marek's disease vaccines. The long-term impact of this work is the development of novel vaccines that can be used to provide better protection for chickens without the added cost of maintaining a cold chain for administering the vaccine to poultry producers.
Animal Health Component
20%
Research Effort Categories
Basic
70%
Applied
20%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113299110180%
3113299104020%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3299 - Poultry, general/other;

Field Of Science
1040 - Molecular biology; 1101 - Virology;
Goals / Objectives
We hypothesize the conserved herpesvirus protein kinase (CHPK) of Marek's disease virus (MDV) is a master regulator of cell-free virus (CFV) production. Accumulating data has suggested specific MDV proteins are not expressed or are "inactive" during cell culture propagation; thereby, MDV is unable to complete infectious CFV assembly. These same viral genes are abundantly expressed or "active" in skin cells. This facilitates CFV production and transmission in chickens. Our goal here is to identify these specific viral proteins targeted by CHPK and modify those to be expressed or active in cell culture to produce CFV. Our hypothesis will be examined using three Specific Aims.Specific Aim 1: Determine post-translational modifications (PTMs) on CHPK during semi- and fully productive replication.We do not know the present state of "activation" of CHPK in cell culture and skin cells representing semi- and fully productive replication, respectively. Based on the differential phosphorylation of viral proteins in skin cells during vCHPKwt (fully productive) and vΔCHPK (semi-productive) replication, we hypothesize CHPK is "modified" in skin cells to be "active". We have four objectives to achieve this specific aim.Analyze PTMs on CHPK during fully productive replication in vivo.Analyze PTMs on CHPK during semi-productive replication in cell culture.Test specific PTMs of CHPK during replication in cell culture.Test specific PTMs on pCHPK for in vivo replication and transmission.Specific Aim 2: Determine PTMs on viral proteins during semi- and fully productive replication.Using LC/MS-MS-based proteomics, we detected 20 viral proteins phosphorylated in MDV-infected skin cells, while these same protein peptides were detected in vΔCHPK-infected skin cells but were not phosphorylated. Here, we will examine the modifications of viral proteins during semi-productive replication in cell culture and compare these results to fully productive replication in skin cells. We hypothesize that specific viral proteins, such as pUL47 and pICP27, will be differentially phosphorylated when examined during semi- (cell culture) and fully (skin cells) productive replication. We have four objectives to achieve this specific aim.Analyze PTMs on MDV proteins during fully productive replication.Analyze PTMs on MDV proteins during semi-productive replication in cell culture.Test specific viral protein PTMs in cell culture.Test specific PTMs for in vivo replication and transmission.Specific Aim 3: Determine PTMs on cellular proteins during semi- and fully productive replication.We do not know the present state of activation of CHPK in cell culture and skin cells representing semi- and fully productive replication, respectively; however, Specific Aim 1 is focused on identifying PTMs on pCHPK, while Specific Aim 2 is directed at validating our current information on viral protein PTMs. Based on the differential phosphorylation of cellular proteins in skin cells during vCHPKwt (fully productive) and vΔCHPK (semi-productive) replication, we hypothesize specific cellular proteins or pathways are not present or "active" in cell culture. In contrast, these cellular proteins or pathways are expressed and "active" during fully productive MDV replication in skin cells. Alternatively, it is possible CHPK "activity" is inhibited by cellular proteins or pathway in cell culture cells that is not present in skin cells. We would predict the former to be most likely and this Specific Aim will address these contrasting hypotheses with two objectives.Analyze cellular PTMs during semi- and fully productive replication.Test specific cellular PTMs during replication in cell culture.
Project Methods
Methods to be used include MS-based proteomics studies, generation of mutant MDV, testing for CFV infectivity, and experimental/natural infection studies.We utilized LC/MS-MS-based proteomics with the UIUC Proteomics Core Facility and will continue with the methodology recently published. For all groups, a total of three independent samples will be used. Triplicate analyses are the standard for label-free quantitative proteomics work with good coverage of the proteome without extensive pre-fractionation.To determine the importance of viral proteins for CFV production, we will use our experimental/natural infection model with recombinant viruses. This is a straightforward system for our laboratory. We use two-step Red recombination of bacterial artificial chromosome (BAC) clones containing the complete MDV genome to efficiently modify specific genes. These BAC clones are then reconstituted into infectious viruses, titrated, and then inoculated into chickens to experimentally infect the chickens. These experimentally infected birds are housed with naïve contact chickens that can be infected, through the natural route, to test whether a respective recombinant virus can horizontally transmit. Fluorescently tagged MDV are used to track the virus from chicken to chicken.Using MDV generated in this project, we will test whether specific modifications in the MDV proteins affect their functions in cell culture. Using a refined protocol for CFV extraction in our laboratory, infectivity assays will be used to determine whether modifications led to infectious CFV.

Progress 07/01/24 to 06/30/25

Outputs
Target Audience:The primary target audiences for this reporting period are herpes virologists and the poultry industry, particularly vaccine companies interested in developing Marek's disease vaccines, as well as breeders seeking genetic resistance to MD. Changes/Problems:We recently upgraded our ABL2 poultry housing units, which has prevented us from performing some important chicken experiments. The units are now operational, but this has delayed some work by about six months. What opportunities for training and professional development has the project provided?Two Graduate Research Assistants and two Undergraduate Students were provided mentoring and training during this reporting year in Dr. Jarosinski's laboratory. How have the results been disseminated to communities of interest?Results of this project were presented locally at the University of Illinois PATH Graduate Student EXPO during this reporting period. Some data was presented nationally at the Conference of Research Workers in Animal Disease (CRWAD) in January 2024 by a graduate student. One paper was recently submitted for publication, but it has not been reviewed during this reporting period. What do you plan to do during the next reporting period to accomplish the goals?During this reporting period (07/01/2025-6/30/2026), multiple experiments are planned to address the three Specific Aims. Specific Aim 1: Determine post-translational modifications (PTMs) on CHPK during semi- and fully productive replication. Analyze PTMs on CHPK during fully productive replication in vivo. We plan to conduct animal experiments related to this objective in early 2026. Analyze PTMs on CHPK during semi-productive replication in cell culture. We will be analyzing the PTMs on CHPK late 2025 and early 2026. Test specific PTMs of CHPK during replication in cell culture. We will be generating amino acid substitutions in CHPKI in the coming year. We will determine whether these amino acids are important for the function and localization of CHPK in cells using immunofluorescence assays (IFAs). Test specific PTMs on CHPK for in vivo replication and transmission. We do not have the data on specific PTMs on CHPK during in vivo replication, thus far, but should have some targets to modify by the end of the next reporting period. Specific Aim 2: Determine PTMs on viral proteins during semi- and fully productive replication. Analyze PTMs on MDV proteins during fully productive replication. We have plans to perform animal experiments related to this objective in early 2026 Analyze PTMs on MDV proteins during semi-productive replication in cell culture. We will be analyzing the PTMs on CHPK late 2025 and early 2026. Test specific viral protein PTMs in cell culture. We will be testing how the modification of specific PTMs on ICP27 and UL47 will affect their function in cell culture. Test specific PTMs for in vivo replication and transmission. We have identified putative NES and NLS regions of ICP27 and will generate mutant viruses in the next reporting period. In vivo experiments are expected to be performed at the end of the next reporting period. Specific Aim 3: Determine PTMs on cellular proteins during semi- and fully productive replication. Analyze cellular PTMs during semi- and fully productive replication. We will complete the analysis of cellular PTMs in the next reporting period. Test specific cellular PTMs during replication in cell culture. Once we have specific cellular proteins to target, experiments are planned to examine these PTMs in the next reporting period.

Impacts
What was accomplished under these goals? During this reporting period (07/01/2024-6/30/2025), some goals were accomplished or are in progress. Specific Aim 1: Determine post-translational modifications (PTMs) on CHPK during semi- and fully productive replication. Analyze PTMs on CHPK during fully productive replication in vivo. We have not yet performed the animal experiment for this work. Analyze PTMs on CHPK during semi-productive replication in cell culture. We have completed the MS-based proteomics and are in the process of examining the specific PTMs on CHPK. Test specific PTMs of CHPK during replication in cell culture. We are currently generating specific mutations to CHPK, including the following, in the context of transient expression via transfection and immunofluorescence assays (IFAs). Mutation of putative nuclear export sequences (NESs) Mutation of putative nuclear localization sequences (NLSs) Mutation of the putative autophosphorylation site (S82) on CHPK. Currently, we have identified the putative NES and NLS regions of CHPK and are making specific amino acid substitutions to characterize these sequences further. Test specific PTMs on CHPK for in vivo replication and transmission. We do not have data on specific PTMs on CHPK during in vivo replication to date. Specific Aim 2: Determine PTMs on viral proteins during semi- and fully productive replication. Analyze PTMs on MDV proteins during fully productive replication. We have not yet performed the animal experiment for this work. Analyze PTMs on MDV proteins during semi-productive replication in cell culture. We have completed the MS-based proteomics and are in the process of examining the specific PTMs on specific viral proteins. Test specific viral protein PTMs in cell culture. Based on some preliminary data, we have started to modify specific PTMs on ICP27 and UL47, but have not performed function experiments thus far. Test specific PTMs for in vivo replication and transmission. We have identified putative NES and NLS regions of UL47 and have generated mutant viruses. In vivo experiments were completed this month, and we found that UL47 is required for transmission, as previously shown, and that the UL47 NES is also necessary for transmission. These data suggest localization of UL47 is crucial for UL47 function in vivo. Specific Aim 3: Determine PTMs on cellular proteins during semi- and fully productive replication. Analyze cellular PTMs during semi- and fully productive replication. We have consulted with a company to run the analysis on the cellular PTMs we currently have. Test specific cellular PTMs during replication in cell culture. We have not performed any studies related to this sub-aim at this time.

Publications