Source: LARAD INC. submitted to
DIFFERENTIATING BETWEEN MAREK’S DISEASE VIRUS INFECTED AND HERPESVIRUS OF TURKEYS VECTORED VACCINATED POULTRY.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
1028492
Grant No.
2022-33530-37153
Cumulative Award Amt.
$135,484.00
Proposal No.
2022-00898
Multistate No.
(N/A)
Project Start Date
Jul 1, 2022
Project End Date
Feb 28, 2023
Grant Year
2022
Program Code
[8.3]- Animal Production & Protection
Project Director
Jackwood, D. J.
Recipient Organization
LARAD INC.
132 E LIBERTY ST
WOOSTER,OH 446914346
Performing Department
(N/A)
Non Technical Summary
Poultry companies use ELISA to determine if their flocks have been adequately vaccinated against specific poultry pathogens. These assays are a valuable tool in the control of avian diseases. Herpesvirus of turkeys (HVT) has been used as a vector to create recombinant vaccines (rHVT) that protect against Marek's disease virus (MDV) and other poultry diseases. These rHVT vaccines are widely used in modern poultry operations yet no ELISA kit has been developed to differentiate between MDV infected and HVT vectored vaccinated birds. Such an ELISA would address research priority 3, improving animal health and well-being, by allowing producers to determine if a flock has been successfully vaccinated leading to better disease management and control. The cross reactivity between HVT and pathogenic (MDV-1) and vaccine strains (MDV-2) presents a challenge. We propose to approach this problem by using individual purified protein antigens from HVT. We will target the glycoproteins from HVT since these surface proteins have the most sequence diversity between HVT and MDV. Glycoprotein genes will be cloned separately into a vector containing the maltose binding protein (MBP) gene to facilitate purification. The MBP fusion proteins will be expressed in the baculovirus system and then affinity purified. Purified antigens will be checked for their utility as antigens in the ELISA with monospecific HVT antisera. Those that produce sufficient signals with HVT antisera will then be retested using monospecific MDV-1 and MDV-2 antisera. The ideal antigen candidate will be the MBP-glycoprotein that gives a strong signal with only the HVT antisera.
Animal Health Component
30%
Research Effort Categories
Basic
70%
Applied
30%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31132991101100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3299 - Poultry, general/other;

Field Of Science
1101 - Virology;
Goals / Objectives
There is a need for an antibody test that can be used to differentiate the antibody response to Herpesvirus of Turkeys (HVT) and the HVT-vectored vaccines from antibodies produced to the pathogenic and vaccine strains of Marek's Disease Virus (MDV) (MDV-1 and MDV-2 respectively).Currently is is notpossible to differentiate between vaccinated and infected animals or to determine if a flock has been successfully vaccinated using one of the HVT-vectored vaccines. We propose to use purified glycoproteins from HVT as ELISA antigens to create a differentiating assay that will distinguish the antibody response to HVT from MDV-1, and MDV-2. Such an assay will give poultry producers a tool to determine if their flock has been adequately vaccinated using the HVT-vectored vaccines.
Project Methods
To create HVT antigens which can be easily purified, we will make a fusion of an HVT glycoprotein gene to the maltose binding protein gene in a baculovirus transfer vector. Gibson assembly will be used to create a plasmid containing a HVT glycoprotein-MBP fusion for each protein using primer pairs containing the appropriate overlapping sequence regions.The pHVT gX-MBP fusion plasmid constructs will be used to transfect the fusion protein gene into the baculovirus genome. The transfection procedure is conducted using Sf9 cells and a commercial transfection kit.The gX-MBP ELISA antigens will be produced in serum-free Sf9 cells. Following the appropriate incubation period (usually 72 hrs), the protein antigens will be harvested and purified over dextrin sepharose in an MBPTrap HP column. Each purified HVT glycoprotein fusion will be used to coat ELISA plates and tested for their ability to bind antibodies specific for HVT. The glycoprotein fusionswill also be tested for any binding to antibodies made to other Marek's disease viruses (MDV-1 and MDV-2)

Progress 07/01/22 to 02/28/23

Outputs
Target Audience:The target audience is poultry producers that use HVT vectored vaccines to protect their flocks from not only Marek's disease but also diseases like infectious bursal disease and Newcastle disease. These producers have a need for an ELISA test that can verify their flocks have been adequately vaccinated. Vaccine companies that make these HVT vectored vaccines are also a target audience. They would use the HVT ELISA to help promote and provide efficacy data on their vaccines. Diagnostic companies will use the products from this project to produce and sell the HVT specific ELISA to vaccine companies and poultry producers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Some results and conclusions of this project were disseminated to scientists and business managers at an international diagnostic company. The goal was to develop an interest and working relationship with this company to help commercialize this technology. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The HVT genes coding for glycoproteins gB, gC, gD, gH. gE, gI, gK and gL were cloned separately into a baculovirus expression system and expressed as a fusion with the maltose binding protein (MBP). The MBP component of each gX HVT protein was used with affinity chromatography to purify the antigens. Expression ranged from <1ug/ml to 50ug/ml. Each of the gX-MBP fusions were coated onto ELISA plates at a range of concentrations that were dependent on the quantity of protein expressed. In some cases, very little antigen was available. Antisera specific for HVT (serotype 3) demonstrated that in the ELISA the gB, gC, gD and gH proteins reacted with this homologous HVT serum. The gE, gI, gK and gL constructs produced only small amounts of antigen which may have contributed to their poor reaction to the HVT specific antiserum. To determine if the gB, gC, gD and gH antigens react specifically to the HVT antibodies, these HVT-MBP fusion antigens were tested using heterologous antisera specific for either serotype 1 or serotype 2 Marek's disease. A range of antigen concentrations (0.0039 ug/ml up to 0.5 ug/ml) was tested against the antisera diluted 1:500. Of the four HVT antigens tested with the homologous sera, only the gB-MBP antigen was discriminating between the homologous serotype 3 antiserum and heterologous serotype 1 and 2 antisera. The best serotype 3 specificity was observed at a gB concentration of 0.25ug/ml. At that antigen concentration, the gB signal with its homologous serotype 3 antisera was about 3X higher than the signal with the serotype 2 antisera and 4-5X higher than the signal with serotype 1 antisera. It was concluded that the gB-MBP antigen has the potential to be developed into a commercial ELISA kit that is specific for serotype 3 Alpha herpesvirus (HVT) antibodies.

Publications