VARIANT-RESISTANT KILLED WHOLE CELL GENOME REDUCED BACTERIAL VACCINE FOR PORCINE CORONAVIRUSES

Project Methods
?MethodsGoal 1. Engineer a Killed Whole Cell (KWC) Genome-Reduced Bacterial (GRB) PEDV vaccine with enhanced immunogenicity.We will test multiple versions of PEDV vaccines directly in the small animal mouse model, vaccines targeting both the PEDV Spike protein Fusion Peptide (FP) and vaccines targeting the Spike Protein stalk region. For the FP we will make vaccines with: 5-mer FP with AQQASSS(x3) linkers, 5-mer FP with AQQASSS(x3) linkers with the rSIP immunomodulator, 5-mer FP with AQQASSS(x3) linkers with the non-cognate universal T-cell antigen PADRE, and 1-mer FP (original published vaccine) as control. However, we are continuing our immunogenicity optimization engineering program for our candidate HIV vaccines and will incorporate any significant improvements we identify in the PEDV vaccine effort. For stalk vaccines, we will design and produce at least 21 different alternative designs, with alternative amounts of stalk sequence and different immunomodulators. Prior to DNA synthesis, we will evaluate predicted structures using AlphaFold to confirm that we are likely to produce a vaccine antigen with the desired tertiary structure. For each candidate construct, we will test antigen expression by quantitative immunoblot, and by flow cytometry to also confirm surface expression. We will use Twist Biosciences for DNA synthesis.We will test approximately 10 different candidate vaccines in a mouse small animal model. We will use 6 w old quasi-outbred Het3 mice (Jax). We will vaccinate mice via the IM route. We will also vaccinate mice intranasally (formalin-fixed bacteria in 200ul 1XPBS + 1 ug of cholera toxin B subunit (CTB)).Prior to immunizations, we will obtain prebleeds at weeks -4 and -2, sample 2 weeks after immunization, with boosts at 3 and 6 weeks with sampling, and final collection 2 weeks after the last dose, with euthanasia accompanied by exsanguination, harvest of spleens for isolation of spleen mononuclear cells for cellular immunology assays, and necropsy. Fecal samples will be collected weekly, with preparation of extracts for assessment of mucosal anti-FP antibody production. Sampling will occur before and immediately after immunizations, and at the conclusion of the experiments. At each blood sampling we will also obtain vaginal washes from female mice, by instilling and aspirating 50 ul of PBS using a micropipette, to enable evaluation of genital tract mucosal Abs.We will evaluate humoral immunity with antibody capture ELISAs. In addition to the anti-mouse IgG secondary Ab, we will also assay for IgM and IgA (sera and fecal extracts). We will evaluate mucosal immune responses in fecal extracts. We will resuspend fecal pellets, vortex until homogenized, clarify by centrifugation, and store supernatants at -80C. We will conduct ELISA assays as described above for the sera. We will also conduct neutralization assays. To evaluate CMI induction against FP and stalk, we will compare T-cell phenotypes of spleen cells obtained at necropsy in the unvaccinated versus vaccinated mouse samples, and chemokine receptor expression, using antibodies CCR7, CCR4 and CXCR3 and CLA. We will also evaluate T-cell cytokine release by intracellular cytokine assay and Cytokine Bead Array. To determine whether vaccination can elicit a T-cell response to FP or stalk, we will measure the frequency of CTL precursors specific for FP or stalk synthetic peptide by ELIspot. If we fail to detect Ag-specific T-cells, we will increase sensitivity by adding a prior stimulation step (Ag pulse), then test in an IFN-γ ELISpot assay.Goal 2. Evaluate immunogenicity and protective efficacy of enhanced immunogenicity KWC GRB PEDV vaccine in post-weaning piglets.We will test effects of the vaccine on a total of 56 post-weaning, specific pathogen free (SPF) pigs of 3-4 weeks of age, purchased from a PEDV-free farm (Oak Hill Genetics, Ewing IL). We will divide the pigs randomly into seven groups of eight pigs each. We will vaccinate the pigs IM or intranasally with the best individual or combined vaccine identified in Aim 1. We will also vaccinate the pigs with a commercial killed PEDV vaccine (Zoetis-PED) as a positive control. We will challenge the pigs with infectious PEDV, and determine daily clinical scores, and viral RNA load in feces, sera, and tissues.At necropsy, we will evaluate and score the gross and histologic lesions as previously described. We will quantitate virus excreted in feces daily by quantitative RT-PCR. We will evaluate immune responses by ELISA for humoral immune responses. We will assess PEDV immunity by determining the frequency of antigen specific T cells producing protective cytokines such as IFN-γ and TNF-α, neutralizing antibody responses as measured by serum virus neutralization (SVN) assay, and the ability of T cells to proliferate after exposure to vaccine antigen. We will determine anti-PEDV neutralizing antibodies using a SVN with PEDV strain CO/13 infection of Vero cells, and we will deter-mine the IgA responses in fecal samples and serum using a previously described IgA-specific ELISA.We will measure antigen-specific T cell frequencies by exposing PBMCs to vaccine antigen for 18h with Brefeldin A added for the final 5 h. We will isolate CD3+ T cells by fluorescence-activated cell sorting (FACS) using anti-CD3 Abs, staining for cell surface markers CD4 or CD8α with directly conjugated antibodies (Innova Biosciences). After surface staining, we will permeabilize cells with Leucoperm (AbD Serotec), staining for intracellular cytokines IFN-γ, TNF-α or IL-2 using directly-conjugated antibodies (Innova Biosciences). We will determine frequencies of cells producing both IFN-γ and IL-2 and polyfunctional CD8+ T cells producing both IFN-γ and TNF-α in the stimulated CD3+ T cell populations. We will determine the ability of T cell to proliferate in response to vaccine Ag in vitro by labeling PBMCs from the vaccinated pigs with carboxyfluorescein succinimidyl ester (CFSE) after 5 day, using a phytohemagglutinin (PHA) (positive control) or medium only (mock/negative control). We will determine frequencies of "low- CFSE" (defined as percent of T cells that have lost any amount of CFSE labeling) cells after 5 day, using flow cytometry.Goal 3. Determine the protective efficacy of KWC GRB PEDV vaccine with enhanced immunogenicity in piglets born to vaccinated pregnant sows.We will use the best vaccine candidate determined in Goal 2 to vaccinate pregnant sows to induce lactogenic immunity for transfer from sows to newborn piglets to protect against PEDV infection, A total of 28 pregnant gilts will be divided into 4 groups, 7 per group. For the infectious PEDV challenge, we will select 21 piglets born to the 7 gilts in each group, selected approximately equally from each gilt. We will vaccinate pregnant gilts either IM or IN. We will include a group of gilts vaccinated with commercial PEDV vaccine (Zoetis-PED) as a positive control. We will challenge the piglets 4 days post-farrowing (DPF) with infectious PEDV to assess the protective efficacy of the vaccine. We will use essentially the same methods as described for Aim 2 for pathologic examination, viral RNA load quantification by qRT-PCR, neutralizing immune response evaluation, and evaluation of cell-mediated immunity.We will also evaluate humoral and cell-mediated immune responses at different times post-immunization. Immune responses in pregnant gilts will also be correlated with the protection efficacy in newborn piglets. We will induce farrowing in all gilts on the same day. We will collect colostrum samples on the day of farrowing. We will euthanize extra piglets such that each experimental group will have 3 piglets/sow. We will challenge the piglets orally with a heterologous PEDV strain, IA2 126 at 4 DPF at a dose of 104.5 TCID50/piglet. The piglets will be monitored daily for clinical scores, and necropsied at 7 DPC.