Progress 09/15/24 to 09/14/25
Outputs
Target Audience:The target audience includes the swine industry, food-animal veterinarians, the animal-health vaccine industry, and vaccinology experts. The results obtained during this reporting period were also presented at the 2025 CRWAD and the 2024 NA PRRS/ICSVD meetings. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The fellowship project provided opportunities to attend scientific meetings including 2024 NA PRRS/ICSVD and 2025 CRWAD, which supported PD's professional development. How have the results been disseminated to communities of interest?1) peer-reviewed publication at ACS infectious disease; 2) Oral presentations at2024 NA PRRS/ICSVD and 2025 CRWAD scientific meetings What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will focus on completing Objective 2: In vivo immunogenicity evaluation of candidate ASF mRNA vaccines in pigs, building directly on the success of the first objective completed this year. Using the engineered antigens identified from mouse studies--membrane-bound P72 (MB-P72) and the optimized multi-T-cell-epitope antigen (P30-IRES/P2A-MTE)--we will formulate lipid nanoparticle (LNP)-encapsulated mRNA vaccines for evaluation in swine, the natural host of African swine fever virus (ASFV). Specifically, three groups of 4-week-old piglets will be immunized intramuscularly at weeks 0 and 3 with: MB-P72 mRNA vaccine, P30-IRES/P2A-MTE mRNA vaccine, or a combination cocktail containing both antigens. A placebo group will serve as a negative control. Serum, peripheral blood mononuclear cells (PBMC), nasal swabs, and saliva will be collected weekly to assess systemic and mucosal immune responses. Two weeks after the booster dose, pigs will be euthanized for collection of spleen and draining lymph nodes. Antibody and cellular responses will be evaluated by ELISA, neutralization assay, and IFN-γ/TNF-α ELISpot. Germinal-center formation in lymphoid tissues will be analyzed by immunohistochemistry and confocal microscopy. Data will be statistically analyzed using ANOVA to compare antigen formulations and time points. If challenges arise--such as suboptimal immune responses or unexpected toxicity--we will adjust vaccine dosages, optimize the LNP formulation, or incorporate trimerization or chaperone motifs to stabilize P72 expression, as outlined in our contingency plan. Optimization of antigen dosage and mRNA delivery efficiency will also be explored to ensure reproducibility and scalability. By completing these tasks, we expect to identify one or more mRNA vaccine candidates capable of inducing strong and balanced B-cell and T-cell immunity in pigs. This will mark a critical step toward developing a safe and effective ASF mRNA vaccine, advancing preparedness for potential ASF outbreaks and supporting the health and productivity of the U.S. swine industry.
Impacts
What was accomplished under these goals?
African swine fever (ASF) is a devastating viral disease that kills nearly all infected pigs and causes major economic losses to the pork industry worldwide. Currently, there are no safe and effective vaccines. This project aims to develop a new type of vaccine using mRNA technology that can train the immune system to produce both strong antibody and T cell responses against the African swine fever virus (ASFV). To achieve this, we designed and tested new versions of ASFV antigens--viral proteins that can trigger protective immunity. During this reporting period, we completed Objective 1, which focused on rational design, protein engineering, and validation of candidate antigens in mice using mRNA vaccine technology. Specifically, we engineered two ASFV structural proteins, P72 and penton, into improved forms that can fold and assemble correctly when produced by cells using mRNA. These engineered proteins were tested in both mice and pigs. The results, now published in ACS Infectious Diseases (Cui et al., 2025), showed that the modified "membrane-bound" forms of P72 and penton produced hundreds of times stronger antibody and T cell responses than the native unmodified versions. The engineered proteins folded properly into their natural three-dimensional structures without needing viral helper proteins, overcoming a long-standing technical barrier in ASF vaccine development. This work led to measurable advances in knowledge and capability: Change in knowledge: We identified a simple protein-engineering approach that allows complex viral antigens to be expressed and folded correctly using mRNA technology. Change in action: The new antigen designs are now being applied to develop prototype mRNA vaccines for testing in pigs (Objective 2). Change in condition (potential impact): If successful, this approach could enable the creation of a safe, scalable, and effective ASF vaccine, protecting millions of pigs and supporting the stability of the global pork supply chain. The accomplishment matters because it demonstrates a breakthrough in vaccine antigen design for a major livestock disease where traditional vaccine strategies have failed. The method developed here can also be adapted for other animal or human viral vaccines, expanding its potential benefits to both agriculture and public health.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2025
Citation:
Cui, J.; Yuan, F.; Qin, J.; Jeon, J. H.; Yun, D. S.; Wang, T.; Xu, R.; Cao, H.; Tungate, A. A.; Netherton, C. L.; Chen, J., Membrane Expression Enhances Folding, Multimeric Structure Formation, and Immunogenicity of Viral Capsid Proteins. ACS Infectious Diseases 2025.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
Cui, J.; Yuan, F.; Qin, J.; Jeon, J. H.; Yun, D. S.; Wang, T.; Xu, R.; Cao, H.; Tungate, A. A.; Netherton, C. L.; Chen, J., Membrane Expression Enhances Folding, Multimeric Structure Formation, and Immunogenicity of Viral Capsid Proteins. Oral presentation at the annual meeting for CRWAD, Chicago, 2025
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