Lipid Nanoparticle-Encapsulated DNA Vaccine Encoding African Swine Fever Virus p54 Antigen Elicits Robust Immune Responses in Pigs

Authors: The N. Nguyen, Danh C. Lai, Sarah Sillman, Erika Petro-Turnquist, Eric A. Weaver, Hiep L. X. Vu

Affiliation: Department of Veterinary Medicine, University of Nebraska-Lincoln; PreciGenome Institute, San Jose, CA

Fig 1. Morphology and transfection efficiency of the LNP-DNA. (A) Representative TEM micrograph of the LNP-DNA. Scale bar = 100 nm. (B & C) HEK-293T cells in a 24-well plate were transfected with 500 ng of the LNP-encapsulated (LNP) or unencapsulated (UnCap) DNA plasmid encoding the p54 gene. For comparative purposes, the cells were transfected with an equal amount of DNA plasmid complexed with polyethyleneimine (PEI). At 48 hpt, p54 expression was assessed by IFA (B) or by immunoblotting (C) using an anti-Flag antibody. In Panel B, cells expressing p54 antigen are shown in green.

Background

This study by Nguyen and colleagues advances the field of veterinary vaccinology, specifically targeting African swine fever virus (ASFV), a highly contagious pathogen responsible for catastrophic losses in global swine populations. ASFV’s complex structure and antigenic diversity have hindered vaccine development, with prior efforts focusing on subunit vaccines targeting immunodominant proteins like p54, a structural antigen critical for viral entry and immune evasion. The paper explores a lipid nanoparticle (LNP)-encapsulated DNA vaccine platform—a strategy validated during the COVID-19 pandemic—to enhance antigen delivery and immunogenicity. By encoding the p54 antigen within a DNA plasmid encapsulated in LNPs, the study aims to overcome limitations of traditional ASFV vaccines, such as poor cellular uptake and transient immunity.

Materials and Methodology

The p54 gene (E183L) was codon-optimized for swine cells, cloned into a pCI plasmid backbone, and administered either unencapsulated or encapsulated in LNPs using PreciGenome’s Flex-M Nanoparticle Synthesis System. The LNP formulation utilized a lipid mixture (MC3, DOTAP, DSPC, cholesterol, DMG-PEG2000) at a nitrogen-to-phosphate ratio of 5.5, optimized for porcine cell transfection. LNPs were characterized for size (90–120 nm), polydispersity (<0.2), and encapsulation efficiency (>95%).

Pigs were divided into three experimental groups which each received two intramuscular doses. One group received unencapsulated 500 µg plasmid DNA, another 500 µg LNP encapsulated plasmid DNA, and the last PBS as a negative control. Sera was collected every two weeks post-vaccination. Immune responses were assessed via immunofluorescent assay, flow cytometry, ELISA (anti-p54 IgG) and IFN-γ ELISpot.

Results and Key Findings

The study elicited a robust humoral and cellular immune response in LNP vaccinated pigs. Peptide-based ELISAs showed that encapsulated DNA displayed strong antibody reactivity versus unencapsulated DNA, which in contrast was almost undetectable. Additionally, while increased antigen-specific CD8+ T-cell responses were detected via ELISpot for both DNA vaccines, only the LNP-DNA group had statistically significant differences from the negative control.

Fig 2. Linear B-cell epitopes within p54 protein. Peptide-based ELISAs were developed using an overlapping peptide library encompassing the entire p54 protein. Serum samples collected at 53 days post-vaccination were tested on the peptide ELISAs. (A) The intensity of IgG antibody binding to each of the sequential overlapping peptides. The y-axis represents the signal-to-noise (S/N) ratio. Asterisks indicate peptides for which pigs vaccinated with the LNP-DNA vaccine exhibit significantly higher S/N ratios than nonvaccinated (NV) pigs. * * p < 0.01, * ** p < 0.001, * ** * p < 0.0001. (B) The number of pigs within each treatment group (n = 5) that exhibited IgG antibodies specific to the peptides.

Conclusion

Nguyen and colleagues showed strong evidence for the feasibility of DNA-LNP vaccines for ASFV. A dual immunization induced a robust immune response, providing a promising solution to the logistical issues of livestock vaccination. More importantly, LNP encapuslation directly improved antibody protection versus administration of free DNA. This highlights the benefits of LNPs as efficient delivery vehicles while also offering a viable route for further research. LNP vaccines have already been proven with mRNA-LNP technology, with a key benefit being rapid scalability. This ensures research on the DNA-LNP vaccine with larger test groups will be viable as long as the LNPs involved retain their critical quality attributes (CQAs).

The use of PreciGenome’s Flex-M system highlights its utility in veterinary nanoparticle synthesis. Controlled fluid flow and scalable production are both crucial for repeatable results with LNP administered drugs. While further trials are needed to assess field efficacy, this platform offers a promising template for next-generation ASFV vaccines, with potential applications for other swine pathogens like porcine epidemic diarrhea virus.

For more details on this innovative research, you can access the full paper at:

https://www.sciencedirect.com/science/article/pii/S0378113525001439