Location: Foreign Animal Disease Research
Project Number: 3022-32000-064-008-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2018
End Date: Jul 31, 2023
Objective:
The overall objective of this project is the development of Foot-and-Mouth Disease Virus (FMDV) live attenuated vaccine candidates. Specific mutations will be introduced in the viral leader coding region, called Lpro, to limit its ability to counteract the innate immune system without significantly affecting virus growth in cell culture.
Specific objectives include:
1. Identify and mutate the key residues that specifically affect FMDV Lpro deUbiquitinase (DUB) activity without impairing the Lpro function on the viral polyprotein.
2. Create candidate vaccine viruses with reduced DUB activity by introducing mutations into identified key residues using the currently available FMDV infectious clone.
3. Assess the potential application of FMDV-DUB mutants in vaccine development in swine, the natural host.
Approach:
1. Identify and mutate the key residues that affect FMDV Lpro DUB activity: By using molecular modeling, we will determine which are the putative residues on Lpro that physically interact with Ubiquitin. We will then construct these mutants in plasmids that allow for expression of recombinant Lpro in bacteria. Bacterially expressed Lpro mutants will be evaluated for their ability of removing Ubiquitin (deUbiquitinase –DUB-) from commercially available synthetic peptides or from proteins synthetized in cell free systems.
2. Construction of viruses with mutations that affect Lpro DUB activity. Mutations identified in Objective 1 will be introduced in the FMDV infectious clone to assay for viability and function. Viable mutants will be characterized for their kinetics of growth, the DUB effect on host proteins, and their ability to induce innate immune responses, in comparison to wild type FMDV derived under the same conditions. Attenuated mutants will be selected for further evaluation of virulence in animals.
3. Preliminary evaluation of FMDV-DUB mutants as live attenuated vaccine candidates in swine. The virulence of one attenuated DUB deficient recombinant FMDV will be evaluated in pigs. Groups of animals will be inoculated with different amounts of DUB mutant and one group will be inoculated with wild type virus as a control. After inoculation animals will be evaluated for the appearance of clinical signs, viremia, virus shedding and induction of systemic immune responses. At 21 days post infection, animals inoculated with the mutant virus that did not get sick, will be challenged with virulent FMDV to assess for efficacy and potency.
