DNA vaccine targeting betacoronavirus spike protein blocks neuroinvasion and neuroinflammation in swine via dual antiviral-immunomodulatory action

Abstract

Porcine hemagglutinating encephalomyelitis virus (PHEV), a neurotropic betacoronavirus, causes fatal neurological disease in piglets, yet no licensed vaccines exist. Here, we developed a DNA vaccine encoding the receptor-binding domain (RBD) of PHEV spike protein fused to IgG1 Fc, adjuvanted with GEL01 (RBD + GEL01). Immunization in mice and piglets elicited robust neutralizing antibodies (titers up to 1:446 and 1:147, respectively) and Th1-biased cellular immunity. The vaccine restricted viral neuroinvasion, reducing brain viral loads by >90% and confining PHEV to discrete olfactory and cortical regions. Vaccinated animals exhibited preserved motor coordination, cognitive function, and minimal neuropathology. Transcriptomic analysis revealed suppression of proinflammatory mediators (e.g., Cxcl2, Saa3) and enhanced neural repair pathways, highlighting dual virological control and immunomodulatory mechanisms. As the first DNA vaccine against PHEV, the RBD + GEL01 candidate offers scalable protection against neurotropic coronaviruses by dual antiviral-immunomodulatory strategy, underscoring its potential to mitigate economic and zoonotic risks.

Fig. 1. Design of DNA vaccine targeting PHEV spike protein.

a Monomeric structure of PHEV spike protein (PDB: 6nzk.1.A) modeled using SWISS-MODEL and visualized with PyMOL. b Schematic of PHEV trimeric NTD and RBD constructs. c Western blot and SDS-PAGE verification of recombinant proteins.

Fig. 2. Immunogenicity of PHEV DNA vaccine in mice.

a Immunization schedule (prime: day 0; boost: day 21). b Serum IgG titers by ELISA. c Neutralizing antibody titers. d Flow cytometry analysis of splenic CD4⁺ and CD8⁺ T cell frequencies. e, f Serum cytokine levels. Data represent mean ± SD (n = 5/group). Lowercase letters indicate statistically significant differences (P < 0.05, one-way ANOVA with Tukey’s test).

Fig. 3. Protective efficacy of PHEV DNA vaccine in challenged mice.

a Flow chart of PHEV challenge in mice after immunization. b Survival curves. c Body weight changes. d Brain viral loads and titers were determined by qRT-PCR and TCID₅₀ assay, respectively. e IFA detection of PHEV in brain regions. CTX cerebral cortex, HPC hippocampus, STR striatum, OT olfactory tubercle, TL thalamus, HT hypothalamus, OB olfactory bulb, OC olfactory cortex, OP olfactory peduncle, CBL cerebellum, BS brainstem. PHEV-N (green), nucleocapsid protein of PHEV. DAPI (blue), nucleus. f Quantification of PHEV N protein-positive areas. Data represent mean ± SD (n = 3/group; ***P < 0.001 by unpaired two-tailed Student’s t test).

Fig. 4. Distribution of PHEV and associated pathological changes in challenged mice.

a Viral localization in hippocampal (HPC), prefrontal cortical (PFC), olfactory peduncle (OP), and hypothalamic (HT) regions across treatment groups. Quantification data was listed on the left panel. PHEV-N (green), nucleocapsid protein of PHEV. DAPI (blue), nucleus. b H&E-stained brain sections. Asterisks (*) indicates meningitis. Arrows indicate perivascular cuffing.

Fig. 5. Behavioral and transcriptomic analyses of vaccinated mice.

a Elevated plus maze performance. b Forced swim test immobility duration. c Differential gene expression in brain tissue (RBD + GEL01 vs Fc control) involve in immunoregulation (purple), humoral immunity (pink), olfactory and other neural function (yellow), inflammation (blue). d GO enrichment of inflammatory and CNS function pathways (RBD + GEL01 vs Fc control). Data show mean ± SD (n = 5/group; *P < 0.05, **P < 0.01 by unpaired two-tailed Student’s t test).

Fig. 6. Vaccine immunogenicity and protection in piglets.

a Flow chart of PHEV challenge in piglets after immunization. b Body temperature and c weight monitoring post-immunization. d Serum cytokine levels. e Anti-RBD IgG titers. f Neutralizing antibody titers on 35 days post-immunization. g Flow cytometry analysis of splenic CD4⁺ and CD8⁺ T cell frequencies on 35 days post-immunization. h Brain viral loads and titers on 7 days post-challenge were determined by qRT-PCR and TCID₅₀ assay, respectively. i Viral protein detection by Western blot analysis on 7 days post-challenge. j IFA detection of PHEV distribution in brain regions on 7 days post-challenge. k H&E-stained brain sections on 7 days post-challenge. Data represent mean ± SD (**P < 0.01, ***P < 0.001, and ****P < 0.0001 by unpaired two-tailed Student’s t test).