Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108
Performing Department
Veterinary Population Medicine
Non Technical Summary
COVID-19 vaccine based on mRNA of the virus has been hugely successful. We believe that mRNA vaccine against PRRSV will also be successful in protecting pigs against this virus.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
To develop an mRNA vaccine against PRRSV and evaluate it in vivo in pigs for safety and efficacy.
Project Methods
Preparation of virus and selection of target genes: A fully sequenced, low passaged new variant PRRSV (strain 1-4-4) will be used to infect Marc-145 cells and/or immortalized porcine alveolar macrophages (iPAM). RNA will be extracted with EZ1&2 Virus Mini Kit v2.0 (Qiagen). Three ORF regions (ORF5=606 nucleotide length (nt), ORF 6=522 nt, ORF 7=387 nt), which are shown to be associated with high humoral immunity in and laboratory and by others (Dortmans et al., 2019), will be selected for cloning. Each ORF will be amplified separately and then purified using high Fusion High-Fidelity PCR Kit (Thermo Scientific™, Cat.F553) and ORF-speci?c primer pairs that have Kozak motif (5-GCCGCCGCC-3) for facilitating the translation of mRNA in eukaryotic cells. The amplified product will be subjected to Sanger sequencing to confirm the presence of target region.Cloning of the targeted genes: Each ORF region will be cloned individually in their suitable plasmid vectors. ORF5 will be cloned in pcDNA™3.1/V5-His TOPO™ TA Expression Kit (Thermo Fisher) using the TOPO ligation principle as described previously (Barfoed et al., 2004). ORF6 and ORF7 will be cloned in pVAX1 (Thermo Fisher) using the T4 DNA ligase kit. The ligation products (1 µL) will be used to transform in 100µL of competent E. coli DH5α (Thermo Fisher, Cat#18265017 using the heat-shock method followed by plating on LB agar plates containing appropriate antibiotics for selection. After incubation at 37oC for 24 hours, positive colonies will be picked followed by extraction of plasmid DNA. ORF insertion will be confirmed by Colony PCR by using REDExtract-N-Amp™ PCR ReadyMix™ (Sigma, Cat# R4775) followed by Sanger sequencing.3. Synthesis of mRNA from the cloned genes: Linearized plasmid DNA will be obtained by using BamHI restriction enzyme. The cloned products will be used for synthesis of mRNA using HiScribe™ T7 ARCA mRNA Kit (New England Biolabs, NEB #E2065S) after which capping and poly(A) tailing process will be achieved. Synthesis of mRNA will be followed by purification using a spin column-based method (Monarch RNA Cleanup Kits, NEB #T2030)). Purified mRNA will be integrated in lipid nanoparticles using ionized lipids (SM-102) for delivery (Kim et al., 2021; Hou et al., 2021). Dr. James Marti at the Minnesota Nano Center in the College of Science and Engineering has agreed to help us in this regard.4. In vivo testing of final products: The final products will be tested in piglets (see Table below). Weaned piglets at 3-weeks of age (n=48) will be purchased from a PRRS-negative farm, divided in eight groups of six each and housed in individual rooms in the Veterinary Isolation Facility. The vaccines will be injected at 3 weeks of age and the challenge with PRRSV (1-4-4) will be at 5 weeks of age. The pigs will be monitored daily for the appearance of clinical signs of PRRS. All pigs will be euthanized at 7 weeks of age (2 weeks after the challenge). Blood of all pigs will be subjected to rRT-PCR to detect the presence of PRRSV. Serum samples will be subjected to virus neutralization test to determine the levels of PRRSV neutralizing antibodies in the treatment groups. We will assess T lymphocytes response in the blood to vaccine and PRRSV challenge by measuring the expression of CD154 (CD40L) and the cytokines TNF-α and IFN-γ in CD4+ T lymphocytes using multicolor flow cytometry. T Cells will be stimulated ex vivo with either inactivated purified purified virus or recombinant viral proteins to quantify the magnitude and antigen specific T cell response to vaccine and/or challenge virus. Antigen specificity will be compared to nonspecific proteins (KLH/MOPC) and unstimulated PBMCs. Positive control for the assay will be developed using Concavalin A stimulated PBMCs from the same animal. The statistical strength of differences between treatment groups and infection groups will be evaluated by a two-way ANOVA. Samples of lungs will be stored in buffered formalin and will be used at a later date for histopathological examination when we receive additional funding from AFRI-NIFA-USDA.