Viruses, plasmids, cells, adjuvant, and animals
The PHEV CC14 strain (GenBank: MF083115.1) was isolated and maintained in our laboratory. The plasmid pFUSE-hIgG1-Fc1 was generously provided by Prof. Yaowei Huang (South China Agricultural University, China). Human embryonic kidney (HEK) 293T cells and Neuro-2a cells were cultured in Dulbecco’s modified eagle medium (Meilunbio, Dalian, China) supplemented with 10% fetal bovine serum (Biological Industries, Beit HaEmek, Israel), at 37 °C under 5% CO2. The adjuvant (MontanideTM GEL01 PR, GEL01) was purchased from Shanghai Seppic Company. Previous studies have confirmed its safety profile, with no reported toxicity or adverse effects in animals14. Three-week-old specific-pathogen-free (SPF) BALB/c mice were obtained from Changsheng Biotechnology Co., Ltd. (Liaoning, China), and 10-day-old piglets were sourced from Jilin Tai Shuo Agriculture Co., LTD (Jilin, China).
Ethics statement
All animal experiments involving mice and suckling piglets were conducted in strict accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals, as approved by the State Council of the People’s Republic of China and the Institutional Animal Care and Use Committee of Jilin University (number of permit: KT202003232 and KT202402234). All procedures were designed to minimize animal suffering. Animals were housed in specific pathogen-free facility with ad libitum access to food and water under 22 ± 1 °C, 50% humidity.
Construction of recombinant vaccine and protein purification
The N-terminal domain (NTD) and receptor binding domain (RBD) of PHEV were identified using the NCBI Conserved Domain Database (https://www.ncbi.nlm.nih.gov/guide/domains-structures/). Gene-specific PCR primers were designed using SnapGene software. Total RNA was extracted from PHEV using the RNA Extraction Kit (TransGen Biotech, Beijing, China) and reverse-transcribed into cDNA. The NTD and RBD genes were amplified by PCR using PrimeSTAR Max DNA Polymerase (Takara Bio, Kusatsu, Japan). The amplified fragments were subsequently cloned into two expression vectors: pFUSE-hIgG1-Fc1 for mammalian expression and pET-32a for prokaryotic expression. Recombinant protein expression was verified by Western blot analysis and SDS-PAGE. Plasmid DNA was purified using the EndoFree Maxi Plasmid Kit (TIANGEN, Beijing, China) for subsequent animal immunization studies.
Animal immunization and health monitoring
For mouse immunization studies, 55 BALB/c mice were randomly allocated into 11 experimental groups (n = 5 per group): Naïve (unvaccinated control), Sterile PBS (negative control), Fc plasmid (vector control), 0.25 mg NTD, 0.5 mg NTD, 0.25 mg NTD + GEL01, 0.5 mg NTD + GEL01, 0.25 mg RBD, 0.5 mg RBD, 0.25 mg RBD + GEL01, 0.5 mg RBD + GEL01. For piglet studies, 20 animals were randomly assigned to 4 groups (n = 5 per group): Normal (unvaccinated), Sterile PBS, Fc plasmid, 5 mg RBD + GEL01. All animals received two intramuscular immunizations. Following vaccination, subjects were monitored daily for 35 days, with particular attention to general appearance and behavior, neurological responsiveness, body weight and rectal temperature. Following experimental endpoint, mice were anesthetized via intraperitoneal injection of pentobarbital sodium (50 mg/kg) combined with inhaled isoflurane (4% induction, 1.5–2% maintenance in 100% oxygen). Cervical dislocation was performed under deep anesthesia. Piglets were euthanized under deep anesthesia by intravenous injection of potassium chloride (1–2 mEq/kg) followed by bilateral thoracotomy as a secondary physical method.
Cytokine ELSIA analysis
Following primary immunization, blood samples were collected via the posterior orbital venous plexus (in mice) or the precaval vein (in piglets) at three time points (days 0, 14, and 35 post-immunization). Whole blood samples were maintained at 4 °C overnight, then centrifuged at 2000 × g for 10 min at 4 °C to isolate serum. Serum concentrations of IFN-γ, TNF-α, IL-2, and IL-4 were quantified using commercial mouse-specific ELISA kits (Jianglai Bio, Shanghai, China) according to the manufacturer’s protocols. Optical density measurements were performed using an ELx808 Absorbance Microplate Reader (BioTek Instruments, USA), with standard curves generated for each cytokine to determine sample concentrations.
Neutralizing antibody detection
On day 35 post-primary immunization, serum samples from all vaccination groups were heat-inactivated at 56 °C for 30 min. Serial two-fold dilutions of each serum sample were prepared in serum-free DMEM culture medium. The diluted serum samples were then mixed with 100 TCID50 of PHEV and incubated at 37 °C for 1 h to allow antibody-virus interaction. For the neutralization assay, Neuro-2a cells seeded in 96-well plates were inoculated with the serum-virus mixtures. Appropriate virus control (positive) and cell control (negative) groups were included in each assay. Cytopathic effects (CPE) were monitored daily under microscopy to determine the neutralizing antibody titer, defined as the highest serum dilution that completely inhibited viral CPE.
ELISA antibody detection
The ELISA plates were coated with a 1:1 mixture of purified RBD or NTD proteins (1 μg/mL each) in antigen coating buffer (Solarbio, Beijing, China) and incubated at 4 °C overnight. Following coating, plates were washed three times with PBST (PBS containing 0.05% Tween-20) and blocked with 5% skim milk in PBS for 1 h at room temperature. Serum samples collected at days 0, 14, and 35 from both mice and piglets were diluted 1:100 in PBS and added to the coated wells (100 μL/well). After 1 h incubation at 37 °C, plates were washed three times with PBST and incubated with HRP-conjugated anti-mouse/anti-pig IgG (Beyotime, Shanghai, China) for 1 h at 37 °C. Following additional washing steps, the reaction was developed using TMB substrate (Beyotime, Shanghai, China) and absorbance was measured at 450 nm using an ELx808 microplate reader (BioTek, USA).
Flow cytometry analysis
On day 35 post-primary immunization, mice and piglets were euthanized, and spleens were aseptically collected. Single-cell suspensions were prepared by mechanically dissociating splenic tissue through a 70 μm cell strainer, followed by washing with PBS. Erythrocytes were lysed by incubating splenocytes with ACK lysis buffer on ice for 5 min, followed by centrifugation at 500 × g for 5 min at 4 °C. Cells were then washed twice with sterile PBS and resuspended in FACS buffer (PBS containing 1% FBS). For immunophenotyping, splenocytes were stained with PE-labeled anti-CD8 and FITC-labeled anti-CD4 (BD Biosciences, NJ, USA). Cells were incubated with antibodies for 30 min at 4 °C in the dark. After washing, stained cells were analyzed using a BD FACSAria II flow cytometer (BD Biosciences), and data were processed with FlowJo software (v10.8.1; Tree Star, Ashland, OR, USA).
Histopathological and immunofluorescence analysis
Thirty-five days following primary immunization, mice and piglets were challenged intranasally with 104.8 TCID50 of PHEV. At 7 days post-challenge, animals were humanely euthanized and brain tissues were collected for Western blot, quantitative RT-PCR, and histopathological and immunofluorescence analysis. For histological examination, brain tissues were fixed in 10% neutral buffered formalin for 48 h, processed through graded alcohols, and embedded in paraffin. Serial sections (3 μm) were stained with hematoxylin and eosin (H&E) for histopathological evaluation. For immunofluorescence, antigen retrieval was performed using citrate buffer (pH 6.0, Servicebio, China) at 95 °C for 15 min. Sections were incubated with primary antibodies overnight at 4 °C, followed by Alexa Fluor-conjugated secondary antibodies and DAPI counterstain (Beyotime, China). Images were acquired using a DMi8 fluorescence microscope (Leica, Germany).
Neurobehavioral assessment
Following immunization and viral challenge, mice underwent comprehensive behavioral testing at designated intervals (days 3–7 post-challenge) to evaluate motor function, depressive-like behavior, and anxiety-related responses. Three standardized behavioral paradigms including Footprint Test, Forced Swim Test (FST), and Elevated Plus Maze (EPM) were employed. For Footprint Test, forepaws and hindpaws were coated with non-toxic red and blue dyes, respectively, and the mice traversed a 100-cm long × 10-cm wide corridor lined with absorbent paper. Gait parameters were quantified from sequential footprints, i.e., stride length (distance between consecutive paw prints), base width (lateral distance between ipsilateral paws), and stride overlap (distance between forepaw and hindpaw placements). Three complete stride cycles were analyzed per animal using ImageJ software. For FST, mice were placed in cylindrical glass tanks (30 cm diameter ×50 cm height) where the water maintained at 23–25 °C. Six-minute test sessions were video-recorded under controlled lighting. The immobility time (defined as passive floating with minimal limb movement) was scored during the final 4 min by blinded observers. For EPM test, the elevated plus maze comprised two open arms and two enclosed arms, each measuring 50 cm in length, positioned 50 cm above the ground. Mice were individually placed at the central intersection of the maze and permitted to explore freely. The duration spent in both the open and enclosed arms was recorded over a period of 6 minutes. The maze configuration comprised two open arms (50 × 10 cm) without walls, two enclosed arms (50 × 10 × 40 cm) with opaque walls, and central platform (10 × 10 cm) 50 cm above floor level. Mice were placed facing an open arm and allowed free exploration for 6 min. Behavioral parameters recorded by time spent in open vs. enclosed arms.
Statistical analysis
All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA). Continuous variables were expressed as mean ± standard deviation (SD). Statistical significance was considered at *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
