Source: LARAD INC. submitted to NRP
DIFFERENTIATION BETWEEN MAREK`S DISEASE VIRUS INFECTED AND HERPESVIRUS OF TURKEYS VECTORED VACCINATED POULTRY USING ELISA.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
1031027
Grant No.
2023-40000-40697
Cumulative Award Amt.
$598,800.00
Proposal No.
2023-03976
Multistate No.
(N/A)
Project Start Date
Sep 1, 2023
Project End Date
Aug 31, 2025
Grant Year
2023
Program Code
[8.3]- Animal Production & Protection
Recipient Organization
LARAD INC.
132 E LIBERTY ST
WOOSTER,OH 446914346
Performing Department
(N/A)
Non Technical Summary
In this Phase II projectwe propose to develop a marketable technology to improve animal health and well-being. Marek's Disease (MD) is caused by Marek's Disease Virus (MDV), a highly contagious, ubiquitous herpesvirus infection of poultry that can cause tumors and immune suppression as well as decreased growth and egg production. There are vaccines for MD that include the Herpesvirus of Turkeys (HVT). HVT is also used as the backbone in vectored vaccines for other poultry diseases. The three serotypes of avian alpha herpesviruses are MDV-1, MDV-2 and MDV-3 (HVT). The close antigenic relatedness of these viruses makes it difficult to easily identify MDV infected birds from HVT vaccinated birds. To date, no rapid diagnostic tests exist for differentiating between MDV infected and HVT and HVT-vectored vaccinated birds. We are proposing to refine the differentiating ELISA that we developed during our Phase I project so it can be offered as a commercial ELISA kit to specifically detect antibodies to HVT. This ELISA will allow for better HVT vaccine management and control of MDV plus a variety of diseases covered by the HVT-vectored vaccines.
Animal Health Component
50%
Research Effort Categories
Basic
25%
Applied
50%
Developmental
25%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113299110150%
3114030110150%
Knowledge Area
311 - Animal Diseases;

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

Field Of Science
1101 - Virology;
Goals / Objectives
In this project, we plan to create baculovirus expressed truncated gB-MBP fusion proteins and purify them for use as ELISA antigens to specifically detect antibodies to HVT in chicken sera. The R&D studies proposed for our Phase 1 SBIR project demonstrated that whole gB-MBP antigen could differentiate HVT specific antibodies from antibodies specific for MDV-1 and MDV-2.Construct HVT gB-MBPrecombinantbaculovirus transfer plasmids containing three truncated gB proteins and confirm the nucleotide sequence of the inserts.Transfect the gB-MBP genes into baculovirus and generate seed stocks of the r-baculoviruses.Produce and purify the truncated gB-MBP proteins through affinity chromatography and test the antigenicity of the three truncated gB-MBP proteins in an ELISA using chicken antisera specific for serotype 1 MDV, serotype 2 MDV and HVT.Optimize the ELISA protocol by evaluating ideal antigen concentration, incubation times, and reagents to create a commercially viable HVT antibody specific ELISA kit and de-risk the USDA licensing process for our strategic partner.
Project Methods
To develop an ELISA specific for antibodies to HVT, we propose to clone and express three regions of the gB protien of this virus. PCR and Gibson cloning will be used to produce transfer vectors containig the truncated HVT gB genes. The baculovirus expression system will be used to express the three gB protein fragments as fusions to the maltose-binding protein (MBP). The MBP region will be used to purify the expression products via affinity chromatography. Purified truncated gB fusion proteins will be used to coat a variety of ELISA plate platforms.To optimize the ELISA protocol we will evaluate ideal antigen concentrations, incubation times, and reagents to obtain the best signal to backgroud ratio possible.

Progress 09/01/23 to 08/31/24

Outputs
Target Audience:Scientists and business professionals employed at international vaccine and diagnostics companies were the target audience during this period. Changes/Problems:We are having issues with expression of the gB1-MBP and gB3-MBP fusion proteins. It is possible that baculovirus defective interfering particles are affecting expression. We are infecting Sf9 cells with a lower MOI to reduce the likelihood of these particles. Other issues may be that expression isoccurring but the fusion proteins may not be folding correctly or there may be an issue with our purification procedure or we may need to try to excise the MBP tag from the protein. We will be working on all of these issues in the next granting period. What opportunities for training and professional development has the project provided?This project has provided training and professional development for the Larad technical staff and project director in the area of molecular expression of proteins in the baculovirus system. The knowledge gained from conducting this project is being applied to other Larad products that are still in development. How have the results been disseminated to communities of interest?For proprietary reasons and for future patenting of the intellectual property, none of the results contained in this report have been publicly disclosed. We have a confidentiality agreement with our diagnostic company partner, and have disclosed some but not all of these results to that company. What do you plan to do during the next reporting period to accomplish the goals? Objective 1. Construct HVT gB-MBPrecombinantbaculovirus transfer plasmids containing truncated gB proteins and confirm the nucleotide sequence of the inserts. Due to the lack of response in ELISA, we will produce truncated gB-MBP proteins that encompass different regions of the gB protein. Objective 2. Transfect the truncated gB-MBP genes into baculovirus and generate seed stocks of the r-baculoviruses. We will be continuing to work on improving expression of our current truncated gB fusions and also will be making stocks of the new truncated gB antigens that will be generated in further work on Objective 1. Objective 3. Produce and purify the truncated gB-MBP proteins through affinity chromatography and test the antigenicity of the three truncated gB-MBP proteins in an ELISA using chicken antisera specific for serotype 1 MDV, serotype 2 MDV and HVT. We are going to look into different purification methods such as adding a His tag as well as an MBP tag which would allow us to try purification with a Ni agarose column Objective 4. Optimize the ELISA protocol by evaluating ideal antigen concentration, incubation times, and reagents to create a commercially viable HVT antibody specific ELISA kit and de-risk the future USDA-CVB submission for our strategic partner. We will continue to work on optimizing our ELISA with our current full length gB-MBP antigen as well as any truncated antigens we produce that give a signal.

Impacts
What was accomplished under these goals? Objective 1: Construct HVT gB-MBP recombinant baculovirus transfer plasmids containing truncated gB proteins and confirm the nucleotide sequence of the inserts. We produced three truncated gB-MBP plasmids, each consisting of a different portion of gB and an MBP tag. Objective 2: Transfect the gB-MBP genes into baculovirus and generate seed stocks of the r-baculoviruses. All three of the truncated gB plasmids were transfected into baculovirus and seed stocks generated. Objective 3: Produce and purify the truncated gB-MBP proteins through affinity chromatography and test the antigenicity of the three truncated gB-MBP proteins in an ELISA using chicken antisera specific for serotype 1 MDV, serotype 2 MDV and HVT. None of the truncated gB antigens gave a significant signal above the background level when tested in ELISA with MDV-1, MDV-2 or HVT antisera. Only the gB2-MBP antigen gave a positive result in an MBP tag ELISA. We are not sure if the expression level of gB1-MBP and gB3-MBP antigens is low or if they are not folding correctly. Objective 4: Optimize the ELISA protocol by evaluating ideal antigen concentration, incubation times, and reagents to create a commercially viable HVT antibody specific ELISA kit and de-risk the future USDA-CVB submission for our strategic partner. Currently the full-length gB-MBP fusion has performed the best in the ELISA. We are continuing to work on the expressing higher quantities of truncated gB-MBP antigens.

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