Veterinary Microbiology and Preventive Medicine
Non Technical Summary
Bovine respiratory syncytial virus (RSV) is a significant viral pathogens of young cows that is a key component of the respiratory disease complex and often leads to secondary bacterial pneumonia. Prefusion F has recently shown to be highly efficacious in barrier housed RSV challenged cows. However, the difficulty in generating prefusion F along with the cost of its production are a hurdle for adoption to the farm. RSV immunity also tends to wane quickly and given the complications of field or pen raised cattle and their stressors and other circulating diseases, and aprotein vaccine may not prove highly efficacious in the real world. Here, we will test a novel mRNA vaccine system we have developed that substantially lowers the price point for production animals and may lead to more thermal stable transcripts compatible with vaccinating on the farm. The use of an alternative delivery system rather than lipid nanoparticles will also lower the vaccine costs. We expect to demonstrate efficacy of the vaccine platform using mice at first as proof of principle before switching to a full cow vaccination and challenge system in year 2. Our overall goal is to test a novel mRNA system for inducing immunological protection from bovine RSV infection. We hypothesize that a prefusion F mRNA delivered continuously by vaccine implant will lead to prolonged and robust cellular and antibody immunity. Here, we will optimize our vaccine further and then test for potential correlates of protection to examine for in eventually challenged cows.
Animal Health Component
Research Effort Categories
Goals / Objectives
The development of a novel mRNA platform that is cost efficient and thermostable will open the door for vaccinating production animals with this technology. Here, we will develop the platform for a bovine RSV F vaccine as a proof of priniciple for development of vaccines against this pathogen but also as a platform technology for other vaccines as well. Goal 1 will be development of the technology and Goal 2 will be testing for efficacy in vivo.
Objective 1. Express and characterize the BRSV F protein using our mRNA technology.We already have identified a good mRNA expression cassette using TPAV 5' and 3' flanking UTRs reviewed above. We have also already on hand wild-type bovine F and prefusion F commercially synthesized as plasmids using overlapping oligos and have now placed these into our TPAV expression system. While COVID vaccines explored prefusion versus post-fusion versus other formats for stabilization during their development, it was found that mRNA-generating mRNA using mainly wild-type RNA (along with codon optimization) retains the transmembrane domain was the most efficacy in animal studies before testing in humans. However, RSV may not be that simple as the F protein is much more metastable than coronaviral spike proteins leading to spontaneous cleavage into the less favorable post-fusion conformation. Thus, the optimal conformation (pre or post-fusion) needs to be determined for RSV F in an mRNA vaccine format. We would not just jump into using the post-fusion because if the mRNA is more like wild-type viral RNA, we are more distanced from the patented pre-fusion F formats. We just will not know how well the two compare until we test them. The COVID spike protein retained the transmembrane domain, which increases its safety since the protein is highly toxic to cells, and having it make by mRNA vaccines in the body and then having it circulate into the blood or tissues would be rather harmful. F transcripts without the transmembrane domain may express more extracellularly and evoke a more robust B cell response or, like spike, retaining on the cell surface will be more than enough exposure to antigen-presenting cells and B cells to mount a vigorous functional antibody response. Again, we will not know this until we test it.Objective 1a:Test the pre and post-fusion conformations of mRNA expressing wild-type or prefusion conformations. We will test our constructs by transfecting A549 lung epithelial cells with mRNA ranging from 0.1 to 5mg of mRNA. The conformation of the F will be determined by western blotting using either AM14/D25 antibodies (prefusion) or motavizumab (both). Since we know that our mRNA vaccine antigen continues to express for up to at least eight days post-transfection, we will test a series of time points after transfection from day 0-d12 for changes in protein conformation during expression. This time trial will be important to know only know how long our mRNA expresses but also in what conformation and for how long.Objective 1b:Construct F mRNA for prefusion or wild-type without a transmembrane domain.Since we may get better B cells and T cell responses from an F that secretes out of cells, we need to develop this version of our mRNA vaccines for testing. Specifically, we will delete the transmembrane domain from our constructs by PCR cloning the outer domain and replace it within our expression cassettes. Similar qualification as 1a will then be done.Objective 2. Explore optimal ways of increasing target mRNA in EVs and explore their thermostability after lyophilization as bolus or polyanhydride rods.Objective 2a:EVs using our prefusion F mRNA (likely any other format of mRNA will follow suit) will be transfected by DEAE-Dextran and glycerol shock using PCR generated transcripts under a T7 promoter. Our T7 polymerase stable cell lines will then begin to make our TPAV/F mRNA transcripts and incorporate those into EVs. We will qualify the EVs by nanosight for concentration per ml after collection 3 days post-transfection and ultracentrifugation, image them by scanning EM for conformation of structure, and quantify our mRNA transcripts by qRT-PCR against an F calibrator. We will also collect total RNA from all EVs to ensure that most EVs carry our mRNA and not cellular. We will also try two additional things to further increase both EVs and our mRNA within those EVs. (1) We will heat shock our cells post-transfection (40 degrees C) to stress the cells, which is known to enhance EV production. We will then repeat our analysis of EV and their contents as previously stated. We will also explore for mRNA enrichment per EV by incorporating an RNA motif that binds to a known EV RNA packaging/binding protein such as Argonaute (Breakefield et al. 2020) by site mutagenesis into the 5'UTR leader sequence. We will then re-evaluate the number of EVs and mRNA incorporation.Objective 2b:Explore the amount of EV packaged mRNA in rods needed for continuous release versus just the use of DEAE-Dextran carrier.Lyophilized EVs in our rods will help protect the mRNA but also serve as the lipid for delivery. However, the polyanhydride rod that excludes water and oxygen may be enough to protect the mRNA from degradation. The use of DEAE-dextran as a carrier which is capable of transfecting cells with mRNA in cell culture may be enough for transfection in vivo after implant. To test for stability overtime and which format is optimal for continuous release of mRNA, we will test a range (100mg-1mg) of mRNA concentrations either in EVs or pressed just into rods containing DEAE-dextran. Release kinetics will be determined over two weeks by incubating known rod weights in PBS or on top of cells. mRNA concentration and integrity will be assessed by qRT-PCR and MOPS RNA gels and transfection determined by cellular staining for RSV F (we routinely do such staining assays for human RSV). The method that leads to the longest release, most intact mRNA, and highest/longest transfection will be moved forward to animal trials after we know the optimal format of mRNA (see objective 3).Objective 2c: For both rod and EV only mRNA vaccines, understanding thermostability will be important. We will hold some of our vaccine formats at -80C, -20C, 4C, room temp, and 37 degrees for at least six months (sampling every month) and compare for any degradation of mRNA over time or storage condition. These data will be important for future vaccine storage as we gear up for bulk vaccine manufacture for cow vaccines in year 2 and ensure that such vaccines could be used on the farm where vaccine storage is not always optimal.Objective 3.Evaluate thevaccine's in vivoimmunogenicity and efficacy in mice before commencing challenge studies in cows in year 2.Mice (all lab animals, really) are a terrible model for RSV infection, human or bovine. However, mice can be used as a proof of principle that neutralizing antibodies and cellular immunity can be obtained by an RSV vaccine. These correlates are the most critical factors for whether an RSV vaccine will succeed or fail. We are not jumping straight into cows as purchasing, housing, challenges, and reagents along with the costs for initial optimization of our vaccine would be cost-prohibitive under the $20,000 of this year one seed funding. We will directly compare the performance of a licensed bRSV vaccines (likely Boehringer or Zoetis) to our mRNA format in mice in year one and then either choose to apply for year two funding in cows for publication unless USDA or industry funding comes through beforehand for a larger trial. Without the costs of mRNA optimization and joining an ongoing RSV vaccine trial, we could have enough animals at $20,000 in Yr2.?