DNA vaccine in lipid nanoparticles triggers balanced antibody and T-cell responses in pigs with maternal antibodies

Swine influenza A/swine/Minnesota/A01392045/2013 (H1N2) (GenBank accession number: KF715130.1↗), belonging to the H1 1B.2.2.1 clade, was obtained from the National Veterinary Services Laboratories (NVSL, Ames, IA, USA). The virus was propagated in Madin-Darby canine kidney cells (MDCK-ATCC CCL-34) in infection medium (Dulbecco's modified Eagle medium [DMEM, Gibco, #12800-058] supplemented with 0.2% bovine serum albumin fraction V [Sigma, #A8412], 25 mM HEPES [Hyclone, #SH30237.01], 100 U/mL penicillin and 100 µg/mL streptomycin [Gibco, #15140-122], and 1 µg/mL TPCK-treated trypsin [Sigma, #T1426]). After 60 h of incubation, the culture supernatant was collected, clarified by centrifugation at 3,000 × g for 10 min, aliquoted, and stored at −80°C. Virus titer was determined by titration on MDCK cells and expressed as log₁₀ tissue infectious dose 50 (TCID₅₀) per mL.

The HA sequence of the swine IAV H1N2 (GenBank protein_id: AGZ62252.1↗) was codon-optimized for Sus scrofa. To facilitate protein detection, a FLAG-tag sequence (DYKDDDDK) was fused in-frame to the 3′ end of the gene. The gene fragment was synthesized using a commercial DNA synthesis service (GenScript) and cloned into the pCI plasmid backbone (Promega, #E1731) between the NheI and NotI sites. The recombinant plasmid was amplified in E. coli DH5α and purified using the Giga Prep Kit (Zymo Research, #D4204).

The LNP-DNA vaccine containing the HA gene was prepared as previously described (42, 43). The lipids used included DLin-MC3-DMA (MC3, Nanosoft Polymer, #1224606-06-7), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP, Avanti Polar Lipids, #132172-61-3), distearoylphosphatidylcholine (DSPC, Avanti Polar Lipids, #816-94-4), cholesterol (Sigma Aldrich, #57885), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000, Avanti Polar Lipids, #160743-62-4). Each lipid was individually dissolved in absolute ethanol. Subsequently, MC3, DOTAP, DSPC, cholesterol, and DMG-PEG2000 were combined at a molar ratio of 42:10:8:38:2, respectively, to prepare the lipid mixture with a total lipid concentration of 15 mM. The DNA plasmid was diluted in 25 mM sodium acetate pH 4.0. The lipid and DNA solutions were combined using the Mixer-4 chip (PreciGenome, #CHP-MIX-4) under the control of a microfluidic device (NanoGenerator Flex-M, PreciGenome) at a DNA-to-lipid flow rate ratio of 3:1, with a total flow rate of 4 mL per minute. The nitrogen-to-phosphate ratio (mol/mol) was 5.5. The resulting products were dialyzed against Tris-Cl buffer pH 7.4 using Slide-A-Lyzer G2 dialysis cassettes with a 10 kDa molecular weight cutoff (Thermo Fisher Scientific, #87732) for 4 h at room temperature and overnight at 4°C. After dialysis, the LNPs were filtered through a 0.45 µm polyethersulfone membrane filter (Thermo Scientific, #09-740-114). The concentration of encapsulated DNA plasmid was quantified using a Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, #P7589), following the procedure described previously (44). Each dose of the LNP-DNA vaccine contained 500 µg of encapsulated DNA plasmid in a 5 mL volume. The vaccine was administered intramuscularly to pigs on the same day of preparation.

To inactivate the virus, β-propiolactone (BPL, Sigma-Aldrich, #P5648) was added to the virus stock at a final concentration of 0.05% (vol/vol), followed by continuous stirring overnight at 4°C. The next morning, the BPL was hydrolyzed by incubation at 37°C for 2 h. To confirm complete inactivation, the virus was inoculated onto MDCK cells for three successive rounds. The inactivated virus stock was purified by ultracentrifugation on a 20% sucrose cushion in the NTC buffer (100 mM NaCl, 20 mM Tris/Cl pH 7.4, and 5 mM CaCl₂) at 175,000 × g, using a SW32Ti rotor (Beckman Coulter) for 2 h at 4°C. The resulting virus pellet was resuspended in sterile phosphate-buffered saline (PBS, pH 7.4). Virus titers were determined using a hemagglutination (HA) assay. The virus concentration was adjusted in PBS and mixed with the Emulsigen-DL 90 adjuvant (Phibro Animal Health) at a 4:1 (vol/vol) ratio. The vaccine dose for sows contained 512 HA units in a 2 mL volume, while the vaccine for piglets contained 32 HA units, also in a 2 mL volume. In both cases, the vaccine was administered intramuscularly to pigs on the same day of preparation.

Four sows were procured from the University of Nebraska-Lincoln (UNL) Swine Research Farm and housed in the animal biosafety level 2 (ABSL-2) research facility at UNL. These sows were tested negative for antibodies against porcine reproductive and respiratory syndrome virus (PRRSV) and swine IAV using commercial ELISA kits purchased from IDEXX Laboratories (PRRS X3, #99-18070 for PRRSV; and Swine Influenza AB, #99-0000900 for IAV). After 1-week acclimation, two sows were vaccinated intramuscularly with 2 doses of the WIV vaccine 44 and 23 days before the expected parturition. The remaining two sows received no treatment and served as NV controls. Serum samples were collected from the sows every 2 weeks following vaccination to monitor antibody responses. All sows farrowed naturally, and the piglets were allowed to nurse from their own dams.

On day 14 post-farrowing, blood samples were drawn from all piglets and serum was collected to evaluate for the hemagglutination inhibition (HI) antibody titers. On day 21 post-farrowing, the piglets were administered a dose of ceftiofur crystalline-free acid to mitigate the risk of respiratory bacterial infections and were subsequently weaned. During the weaning, 18 piglets nursed by the two WIV-vaccinated sows, which exhibited HI titers between 1:320 and 1:640 at 14 days of age, were selected and assigned to three groups: MDA⁽⁺⁾/LNP, MDA⁽⁺⁾/WIV, and MDA⁽⁺⁾/NV (Table 1). Similarly, 18 piglets nursed by the NV sows were selected and allocated into three groups: MDA⁽⁻⁾/LNP, MDA⁽⁻⁾/WIV, and MDA⁽⁻⁾/NV. Each treatment group within the MDA-positive and MDA-negative categories received the same number of piglets from each respective sow.

On day 28 post-farrowing, day 0 post-vaccination, the MDA⁽⁺⁾/LNP and MDA⁽⁻⁾/LNP groups received an intramuscular injection of the LNP-encapsulated DNA plasmid. Meanwhile, the MDA⁽⁺⁾/WIV and MDA⁽⁻⁾/WIV groups received two intramuscular injections of the WIV vaccine, administered 21 days apart (days 0 and 21 post-vaccination). The remaining groups, MDA⁽⁺⁾/NV and MDA⁽⁻⁾/NV, received no treatment and served as NV controls (Table 1).

On day 36 post-vaccination, the pigs were challenged with 2 × 10⁵ TCID₅₀ of the homologous H1N2 virus, via both intratracheal and intranasal routes, as previously described (43). On day 5 post-challenge, the pigs were humanely euthanized with an overdose of sodium pentobarbital (Fatal-Plus Solution, Vortech Pharmaceutical). Necropsies were performed by a board-certified pathologist, who is one of the co-authors and was intentionally blinded to the animal treatment groups.

Whole blood samples were collected with and without an anticoagulant before vaccination and on days 14, 21, 28, and 35 post-vaccination and day 5 post-challenge, corresponding to day 41 post-vaccination. Serum samples were isolated from blood collected without an anticoagulant to measure antibody responses. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples collected in EDTA tubes by density gradient centrifugation using Lymphoprep density gradient medium (STEMCELL Technologies, #07851) and SepMate−50 tubes (STEMCELL Technologies, #85450). After isolation, cell density was counted, and the cells were cryopreserved in a freezing medium consisting of 40% RPMI-1640 (Gibco, # 61870036), 50% fetal bovine serum (FBS; Sigma-Aldrich, #12,107C), and 10% dimethyl sulfoxide (DMSO; Sigma-Aldrich, #D2650).

Nasal swabs were collected daily post-challenge, using sterile ultrafine polyester flocked swabs (Puritan, #25-3317-U) and immersed in 1 mL of DMEM containing 100 units/mL penicillin and 100 µg/mL streptomycin. The samples were centrifuged at 3,000 × g for 10 min to remove debris, aliquoted into two tubes of approximately 400 µL each, and stored at –80°C until use.

Oral swabs were collected prior to challenge (i.e., day 36 post-vaccination). Additionally, tracheal swabs were collected during necropsy. Sterile 4 × 4 cotton gauze pads (Gauze Care) were used for sample collection. Each pad was placed in a 50 mL conical tube containing 3 mL of cold PBS. Subsequently, the pad was transferred into a 30 mL syringe (Exelint International, #26290), and the plunger was used to compress the pad. The recovered fluid was collected and centrifuged at 3,000 × g for 10 min to remove debris. The clarified supernatant was aliquoted into small volumes and stored at −80°C until use.

Bronchoalveolar lavage fluid (BALF) was collected during necropsy. After the lungs were removed from the chest cavity, the trachea was cut away near the bifurcation. Fifty milliliters of cold PBS was instilled into the lung, followed by gentle massage to distribute the fluid. The lavage fluid was then recovered, centrifuged at 3,000 × g for 10 min, and stored at –80°C until use.

A Bio-Thermo microchip (Merck Animal Health, #362118) was implanted intramuscularly in the neck muscle of the pigs to facilitate body temperature measurement. Body temperature was recorded daily using a Global pocket Reader Plus (Destron Fearing, model GPR+) from 1 day before challenge to day 5 post-challenge.

Gross lung lesions were scored during necropsy. The percentage of lung surface exhibiting consolidation typical of IAV infection was estimated for each lung lobe. The total lung consolidation was then calculated based on the weighted proportions of each lobe relative to the total lung volume (45, 46).

During necropsy, samples from the trachea and the apical, middle, and caudal lung lobes were collected and fixed in 10% buffered formalin for 24 h. The samples were then submitted to the Nebraska Veterinary Diagnostic Center, where they were embedded in paraffin and sectioned at a thickness of 4 µm. Lung sections were stained with hematoxylin and eosin (H&E) for histological evaluation. Lung microscopic lesions were scored based on four parameters proposed by Gauger et al. (47), including necrotizing, suppurative bronchitis and bronchiolitis, peribronchiolar lymphocytic cuffing, and alveolar septal inflammation. An additional parameter, microscopic lung consolidation, was introduced in this study. Each parameter was scored according to the percentage of cross-sectional area affected, using a scale of 0–3, where 0 = normal, 1 = <5%, 1.5 = 5–25%, 2.0 = 26–50%, 2.5 = 51–75%, and 3.0 = >75% affected. The total score (maximum of 15) was calculated for each lung lobe, and the average across the three lobes was reported per animal.

Additionally, sections of the cardiac lung lobe and trachea were subjected to RNA ISH using a probe specific to viral nucleoprotein (NP) transcripts (Advanced Cell Diagnostics, #579001) and the RNAscope 2.5 HD Reagent Kit-BROWN (Advanced Cell Diagnostics, #322300), as previously described (48). The ISH score was assigned based on the abundance of virus-infected cells within a microscopic field at 40× objective magnification, as previously described (49). Scale: 0 = none; 1 = 1–10 cells; 2 = 11–30 cells; 3 = 31–100 cells; and 4 = >100 cells.

Serum samples were first incubated at 56°C for 30 min. The samples were then mixed at a 1:4 (vol/vol) ratio with 20% kaolin and incubated for 20 min at room temperature, followed by centrifugation at 800 × g for 5 min. Subsequently, the supernatants were incubated with an equal volume of 0.5% turkey red blood cells (RBCs, Lampire Biological Laboratories, #7209403) for 20 min at room temperature, followed by centrifugation at 400 × g for 5 min to remove non-specific inhibitors. The treated sera were then serially diluted twofold in PBS in a 96-well V-bottom plate (Greiner Bio-One, #651101), starting at a 1:10 dilution. An equal volume of the H1N2 virus containing 4 HA units was added and incubated for 30 min. Subsequently, 0.5% turkey RBCs were added, and the plates were incubated for 1 hour at room temperature. HI titers were determined as the reciprocal of the highest serum dilution, which completely inhibited hemagglutination.

Antibodies specific to the influenza NP were detected using a blocking ELISA kit (IDEXX Laboratories, Swine Influenza AB, #99-0000900), following the manufacturer's instructions. As a blocking enzyme-linked immunosorbent assay (ELISA), color development is inversely proportional to concentrations of NP-specific antibodies in the test samples. Results were expressed as the ratio of the optical density (OD) of the test sample to the negative control (S/N ratio). Samples with an S/N ratio below 0.6 were considered positive.

An indirect ELISA was developed to assess HA-specific IgG and IgA antibodies in oral swabs. To produce the HA antigen for coating the ELISA plates, HEK-293T cells cultured in a 100 mm tissue culture dish (TPP Techno Plastic Products, #93100) were transfected with 30 µg of the plasmid containing the HA gene of the swine IAV H1N2 virus, the same construct used to formulate the LNP vaccine. At 48 h post-transfection, the cells were harvested and lysed in 1,200 µL of Pierce RIPA buffer (Thermo Fisher Scientific, #89901). The resulting lysate was dialyzed overnight at 4°C against PBS using a Slide-A-Lyzer G2 dialysis cassette, MWCO of 10 kDa (Thermo Fisher Scientific, #87730). The total protein concentration of the lysate was quantified using the bicinchoninic acid protein assay kit (Thermo Fisher Scientific, #23227).

To coat the ELISA plates, the protein lysate was diluted in 0.1 M carbonate/bicarbonate buffer (pH 9.6) to a final concentration of 6.25 µg/mL. A volume of 100 µL of the diluted protein was added to each well of LockWell Maxisorp ELISA plates (Thermo Fisher Scientific, #446469) and incubated overnight at 4°C. The plates were washed with tris-buffered saline, pH 7.4, containing 0.1% Tween 20 (TBS-T20), followed by blocking at room temperature for 1 h using a sample dilution buffer (ChonBlock blocking/sample buffer [Chondrex Inc, #9068] diluted 1:1 with TBS-T20). Oral swab samples were diluted 1:4 in the sample dilution buffer. Then, 100 µL of the diluted samples was added in duplicate to each well of the ELISA plates and incubated for 1 h at room temperature. After five washes with TBS-T20, goat anti-pig IgG (H + L) HRP-conjugated (Bethyl Laboratories, #A100-105P) or goat anti-pig IgA HRP-conjugated (Bio-Rad, #AAI40P) secondary antibodies were added and incubated for 1 h. Both antibodies were diluted at 1:2,000 in the sample dilution buffer. Following five more washes with TBS-T20, the reaction was developed with TMB (3,3′,5,5′-tetramethylbenzidine) substrate (Alpha Diagnostic International, #80093-1000) for 7 min, stopped with a stop solution (Alpha Diagnostic International, #80100), and absorbance was measured at 450 nm using Biotek Synergy LX Plate Reader (BioTek Instruments Inc.).

The IFN-γ ELISpot assay was performed, as previously described (48). Sterile 96-well filter plates with 0.45 µm pore size hydrophobic polyvinylidene fluoride membranes (Millipore Sigma, #MSIPS4W10) were coated with 50 µL/well of anti-porcine IFN-γ antibody (BD Biosciences Pharmingen, clone P2G10, #559961) at a concentration of 10 µg/mL overnight at 4°C. Plates were washed three times with sterile PBS and blocked by incubation with 250 µL/well of complete RPMI (RPMI-1640 supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin) for 2 h at 37°C in a 5% CO₂ incubator.

Cryopreserved PBMCs were thawed, washed, and resuspended in complete RPMI, and 100 µL containing 500,000 cells were added to each well. Six wells were used per PBMC sample. Two wells were stimulated with the H1N2 virus at a concentration of 10⁶ TCID₅₀ per well in 100 µL of complete RPMI. Two wells were stimulated with a cocktail of phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, #P8139, final concentration 10 ng/mL) and ionomycin (Sigma-Aldrich, #I0634, final concentration 1 µg/mL), diluted in 100 µL of complete RPMI, as the positive control. The remaining two wells received 100 µL of complete RPMI and served as unstimulated controls.

After 20 h of incubation at 37°C in a 5% CO₂ incubator, the cells were removed, and plates were washed three times with 250 µL per well of PBS containing 0.05% Tween 20 (PBS-T20). Fifty microliters of biotinylated mouse anti-pig IFN-γ antibody (BD Biosciences Pharmingen, #559958), diluted to a final concentration of 2 µg/mL in PBS-T20, was added to each well, followed by incubation at room temperature for 1 h. After five washes with PBS-T20, plates were incubated with streptavidin-conjugated alkaline phosphatase (Southern Biotech, #7105-04), diluted 1:1,000 in PBS-T20. Following another five washes with PBS-T20, plates were incubated with alkaline phosphatase substrate (Vector Laboratories, #SK-5300) for 15 min. Color development was stopped by washing with water. Spots were analyzed and counted using the CTL Immunospot S5 Analyzer (ImmunoSpot). The number of spots in wells stimulated with H1N2 virus was subtracted from the number of spots in unstimulated wells. Data are presented as the number of IFN-γ-secreting cells (IFN-γ-SC) per 5 × 10⁵ PBMCs.

Virus titers in the nasal swabs and BALF samples were determined by titration in MDCK cells, as previously described (50). The titers were calculated using the Reed and Muench method and expressed as the log₁₀ TCID₅₀ per mL. Samples with undetected infectious virus were assigned a value of 0 log₁₀ for graphical presentation and statistical analysis.

Graphical and statistical analyses were conducted using GraphPad Prism 10 (GraphPad Software Inc.). Prior to statistical testing, HI titers and viral infectious titers were log-transformed using base 2 and base 10, respectively. Continuous single-variable data were analyzed using Brown-Forsythe and Welch's ANOVA, followed by unpaired t-tests with Welch's correction. For continuous two-variable data, including HI titers in piglets post-vaccination and viral titers following challenge, two-way ANOVA was performed, followed by Tukey's multiple comparisons test. Ranked data, including H&E and ISH scores, were analyzed using the Kruskal-Wallis test, followed by uncorrected Dunn's multiple comparisons test.

To produce piglets with and without IAV-specific MDAs, two sows were administered two doses of the WIV vaccine on days 44 and 23 before the expected parturition, while two other sows served as NV controls. The WIV-vaccinated sows developed high HI antibody titers, reaching 1:640 on day 17 before parturition. On the day of weaning, 21 days post-parturition, the HI antibody titers of the two WIV-vaccinated were still relatively high, measuring at 1:160 and 1:320, respectively. The NV control sows had no detectable HI titers throughout the study (Fig. 1A). As a result, piglets nursed by the WIV-vaccinated sows had high HI titers in their serum on day 14 after birth, with the titers ranging from 1:80 to 1:1,280. Conversely, piglets nursed by the NV sows had no detectable HI titers (Fig. 1B).

Eighteen piglets from NV- and 18 piglets from WIV-vaccinated sows were selected for a vaccination study. At the time of vaccination, 28 days of age, all 18 piglets from the NV sows had undetectable HI titers (Fig. 2A). Of these, the six piglets assigned to the MDA^(–)/NV group maintained undetectable HI titers throughout the study. In the MDA^(–)/LNP group, the mean HI titer was 1:80 on day 14 post-vaccination and continued to rise, reaching 1:640 by day 35 post-vaccination. In the MDA⁽⁻⁾/WIV group, the mean HI titer was 1:18 on day 14 post-vaccination, sharply increased to 1:960 on day 28, corresponding to 7 days after the second immunization, and then declined. The mean HI titers in the MDA⁽⁻⁾/LNP group were statistically higher than those in the MDA⁽⁻⁾/WIV group on days 14, 21, and 41 post-vaccination (Table S1).

At the time of vaccination (day 0), the 18 piglets selected from WIV-vaccinated sows had HI titers ranging from 1:160 to 1:640, with the mean HI titers not significantly different among the groups (Fig. 2A). The HI titers of the MDA⁽⁺⁾/NV and MDA⁽⁺⁾/WIV groups steadily declined at a similar rate, reaching mean titers of 1:22 and 1:50, respectively, on day 41 post-vaccination, corresponding to day 5 post-challenge. Interestingly, in the MDA⁽⁺⁾/LNP group, the HI titer initially dropped to a mean of 1:90 by day 21 post-vaccination but gradually increased afterward, reaching a mean titer of 1:143 by day 41 post-vaccination. On days 35 and 41 post-vaccination, the HI titers in the MDA⁽⁺⁾/LNP group were statistically higher than those in the MDA⁽⁺⁾/WIV group (Fig. 2A and Table S1).

Serum antibodies specific to the viral NP were measured using a commercially available ELISA kit, which is widely used for IAV serodiagnosis. On day 0 post-vaccination, NP-specific antibodies were not detected in any of the 18 pigs from the NV control sows (i.e., MDA-negative pigs) (Fig. 2B). Additionally, NP-specific antibodies were not detected at any time in the MDA⁽⁻⁾/LNP group throughout the study. In the MDA⁽⁻⁾/WIV group, NP-specific antibodies were detected on day 28 post-vaccination, 7 days after the second immunization. These results were expected as the NP antigen was included in the WIV but not the LNP-DNA vaccine. On day 0 post-vaccination, NP-specific antibodies were detected in 13 of 18 pigs born to WIV-vaccinated sows, although their S/N values were near the detection threshold (Fig. 2B). In the MDA⁽⁺⁾/NV and MDA⁽⁺⁾/LNP groups, the maternally derived NP-specific antibodies gradually declined over time and became undetectable by the NP-based ELISA by day 14 post-vaccination. Similarly, the NP-specific antibodies in the MDA⁽⁺⁾/WIV group also gradually declined and became undetectable, even though this group received two doses of the WIV which contained the NP antigen, suggesting that MDAs hindered the development of NP-specific antibody responses in these piglets (Fig. 2B).

To access the presence of antigen-specific antibodies at the mucosal surface, oral swabs were collected on day 36 post-vaccination, and HA-specific IgG and IgA antibodies were measured using indirect ELISAs. In MDA-negative pigs, the OD values for IgG and IgA in both the MDA⁽⁻⁾/LNP and MDA⁽⁻⁾/WIV groups were significantly higher than those in the MDA⁽⁻⁾/NV group (Fig. 2C and D). However, in MDA-positive pigs, only the MDA⁽⁺⁾/LNP group showed significantly higher IgG OD values compared to the MDA⁽⁺⁾/NV group. The OD values for IgA, however, were not significantly different among the MDA⁽⁺⁾ groups.

The IFN-γ ELISpot assay was used to evaluate T-cell responses. IFN-γ-SC were virtually undetectable in PBMCs collected from the MDA⁽⁺⁾/NV and MDA⁽⁻⁾/NV groups on days 14 and 28 post-vaccination (Fig. 3; Fig. S1), indicating the absence of maternally transferred virus-specific T cells. The WIV vaccine induced low but detectable T-cell responses in MDA-negative pigs. In the MDA⁽⁻⁾/WIV group, the mean numbers of IFN-γ-SC were 33 and 17 per 5 × 10⁵ PBMCs on days 14 and 28 post-vaccination, respectively. However, due to substantial inter-individual variability, these responses were not statistically different from those observed in the NV groups. In the MDA⁽⁺⁾/WIV group, the corresponding values were 3 and 29 cells per 5 × 10⁵ PBMCs. In contrast, the LNP-DNA vaccine induced stronger T-cell responses in both MDA-negative and MDA-positive pigs. On both days 14 and 28 post-vaccination, the mean numbers of IFN-γ-SC in the MDA⁽⁻⁾/LNP and MDA⁽⁺⁾/LNP groups were statistically higher than those in the NV groups. The means IFN-γ-SC in the MDA⁽⁺⁾/LNP were slightly lower than those in the MDA⁽⁻⁾/LNP group (108 vs 112 on day 14 and 82 vs 130 on day 28), but these differences were not statistically significant. Following challenge infection with the H1N2 virus, the numbers of IFN-γ-SC increased across all treatment groups, with the most pronounced increase observed in the MDA⁽⁺⁾/LNP and MDA⁽⁺⁾/WIV groups, indicating an anamnestic T-cell response.

Collectively, these results demonstrate that a single dose of the LNP-DNA vaccine induces high T-cell responses regardless of MDA status, whereas the WIV vaccine elicits weaker T-cell responses, even in MDA-negative pigs. However, the WIV vaccine effectively primes the immune system, leading to a strong anamnestic T-cell response following challenge.

The IAV strain used for the challenge did not induce a significant febrile response. The body temperature after infection ranged between 39°C and 40°C (Fig. 4), remaining below the 40.5°C threshold, which is considered indicative of fever.

Nasal swabs were collected from all pigs following challenge inoculation with the H1N2 virus to assess viral shedding. In both MDA⁽⁺⁾/NV and MDA⁽⁻⁾/NV groups, infectious virus was detected in nasal swabs on day 1 post-challenge, with mean titers of 10² and 10^2.2 TCID₅₀/mL, respectively (Fig. 5A). Virus titers increased through day 5 post-challenge, reaching mean titers of 10⁵ and 10^4.5 TCID₅₀/mL, with no statistically significant difference observed between the two groups. In MDA-negative pigs, both the LNP-DNA and WIV vaccines effectively prevented viral shedding. Infectious virus was detected in only one swab from the MDA⁽⁻⁾/WIV group on day 1 post-challenge, while infectious virus was not detected in nasal swabs from the MDA⁽⁻⁾/LNP group throughout the 5-day observation period. In MDA-positive pigs, the WIV vaccine failed to prevent viral shedding in nasal secretions. Infectious virus was consistently detected in nasal swabs from the MDA⁽⁺⁾/WIV group throughout the 5-day observation period, with shedding kinetics and magnitudes similar to those of the NV control groups. In the MDA⁽⁺⁾/LNP group, infectious virus was also detected in nasal swabs, with mean titers ranging from 10^0.3 TCID₅₀/mL on day 1 to 10^3.9 TCID₅₀/mL on day 5 post-challenge. However, these titers were significantly lower than those in both the NV control groups and the MDA⁽⁺⁾/WIV group (Fig. 5A and Table S2).

During necropsy, tracheal swabs were collected to evaluate the presence of infectious virus in the lower respiratory tract. Virus titers ranging from 10⁴ to 10⁷ TCID₅₀/mL were detected in the swabs from both NV control groups, with no statistically significant difference between them (Fig. 5B). Infectious virus was not detected in any swabs from the MDA⁽⁻⁾/WIV, MDA⁽⁻⁾/LNP, and MDA⁽⁺⁾/LNP groups. In the MDA⁽⁺⁾/WIV group, five of six swabs contained infectious virus, with titers ranging from 10³ to 10⁶ TCID₅₀/mL. The mean virus titers in this group were significantly lower than those in the MDA⁽⁻⁾/NV control group.

During necropsy, BALF samples were also collected to quantify the presence of the virus in the bronchoalveolar space. Infectious virus was detected in the BALF from both MDA⁽⁻⁾/NV and MDA⁽⁺⁾/NV groups, with a mean titer of 10⁵.⁷⁵ and 10^5.13 TCID₅₀/mL, respectively (Fig. 5C). In contrast, no infectious virus was detected in any samples from the MDA⁽⁻⁾/LNP and MDA⁽⁺⁾/LNP groups. Infectious virus was found in only one sample from each of the MDA⁽⁺⁾/LNP and MDA⁽⁺⁾/WIV groups.

Overall, in MDA-negative pigs, both the LNP-DNA and WIV vaccines effectively prevented infection. In MDA-positive pigs, the WIV vaccine failed to prevent viral shedding in nasal secretions and the tracheal tract but successfully protected the lungs from infection. In contrast, the LNP-DNA vaccine significantly reduced viral shedding in nasal secretions and nearly prevented viral replication in the tracheal tract and lungs.

On day 5 post-challenge with the H1N2 virus, purple-red consolidation typical of swine IAV infection was prominently observed in the lung of pigs in the MDA⁽⁻⁾/NV group, particularly in the apical and cardiac lobes, with an average of 3.4% of the total lung surface affected (Fig. 6A and B). In contrast, lung consolidation was not observed in either MDA⁽⁻⁾/WIV or MDA⁽⁻⁾/LNP groups. The MDA⁽⁺⁾/NV group exhibited less severe lung consolidation than the MDA⁽⁻⁾/NV group (1% vs 3.4%), suggesting that the presence of MDAs at the time of challenge infection may help mitigate macroscopic lung lesions. Mild consolidation was observed in four of six pigs in the MDA⁽⁺⁾/WIV group and in one of six pigs in the MDA⁽⁺⁾/LNP group (Fig. 6B).

Despite differences in macroscopic lesions, lung sections from both NV control groups exhibited a similar degree of microscopic lesions, characterized by moderate peribronchiolar lymphocytic cuffing, interstitial pneumonia, epithelial necrosis, and immune cell infiltration into the alveolar septa, along with areas of suppurative bronchiolitis (Fig. 6C). Minimal microscopic alterations were observed in MDA-negative pigs vaccinated with either the LNP-DNA or WIV vaccine, consistent with the absence of macroscopic lesions. In contrast, severe microscopic changes were observed in the lung sections of pigs in the MDA⁽⁺⁾/WIV group, with microscopic scores not significantly different from those of the NV control groups (Fig. 6D). Interestingly, only minimal microscopic changes were detected in the MDA-positive pigs vaccinated with the LNP-DNA vaccine. Particularly, the microscopic score for the MDA⁽⁺⁾/LNP group was not significantly different from those of the MDA⁽⁻⁾/LNP or MDA⁽⁻⁾/WIV groups but was significantly lower than the scores of the NV control groups (Fig. 6D).

The ISH assay was performed to detect virus-infected cells in the lung and tracheal tissues. Numerous ISH-positive cells were observed in the tracheal and bronchiolar epithelium of both NV control groups (Fig. 6E and G). In MDA-negative pigs, ISH-positive cells were virtually absent in both lung and tracheal sections, regardless of vaccination with either the LNP or WIV vaccines. Similarly, no ISH-positive cells were found in the lung and tracheal tissues of MDA⁽⁺⁾/LNP pigs (Fig. 6F and H). However, ISH-positive cells were clearly detected in the lung and tracheal tissues of MDA⁽⁺⁾/WIV pigs, although the number of ISH-positive cells in these tissues was significantly lower than in the NV control groups.

Overall, in the presence of MDA, LNP formulations appear to provide better protection than WIV in terms of reducing lung lesions and virus-infected cells in the lung and tracheal tissues.