Intranasal delivery of inactivated PRRSV loaded cationic nanoparticles coupled with enterotoxin subunit B induces PRRSV-specific immune responses in pigs

This study was conducted to evaluate the induction of systemic and mucosal immune responses and protective efficacy following the intranasal administration of inactivated porcine reproductive and respiratory syndrome virus (PRRSV) loaded in polylactic acid (PLA) nanoparticles coupled with heat-labile enterotoxin subunit B (LTB) and dimethyldioctadecylammonium bromide (DDA). Here, 42- to 3-week-old PRRSV-free pigs were randomly allocated into 7 groups of 6 pigs each. Two groups represented the negative (nonvaccinated pigs/nonchallenged pigs, NoVacNoChal) and challenge (nonvaccinated/challenged, NoVacChal) controls. The pigs in the other 5 groups, namely, PLA nanoparticles/challenged (blank NPs), LTB-DDA coupled with PLA nanoparticles/challenged (adjuvant-blank NPs), PLA nanoparticles-encapsulating inactivated PRRSV/challenged (KNPs), LTB-DDA coupled with PLA nanoparticles loaded with inactivated PRRSV/challenged pigs (adjuvant-KNPs) and inactivated PRRSV/challenged pigs (inactivated PRRSV), were intranasally vaccinated with previously described vaccines at 0, 7 and 14 days post-vaccination (DPV). Serum and nasal swab samples were collected weekly and assayed by ELISA to detect the presence of IgG and IgA, respectively. Viral neutralizing titer (VNT) in sera, IFN-γ-producing cells and IL-10 secretion in stimulated peripheral blood mononuclear cells (PBMCs) were also measured. The pigs were intranasally challenged with PRRSV-2 at 28 DPV and necropsied at 35 DPV, and then macro- and microscopic lung lesions were evaluated. The results demonstrated that following vaccination, adjuvant-KNP-vaccinated pigs had significantly higher levels of IFN-γ-producing cells, VNT and IgG in sera, and IgA in nasal swab samples and significantly lower IL-10 levels than the other vaccinated groups. Following challenge, the adjuvant-KNP-vaccinated pigs had significantly lower PRRSV RNA and macro- and microscopic lung lesions than the other vaccinated groups. In conclusion, the results of the study demonstrated that adjuvant-KNPs are effective in eliciting immune responses against PRRSV and protecting against PRRSV infections over KNPs and inactivated PRRSV and can be used as an adjuvant for intranasal PRRSV vaccines.

www.nature.com/scientificreports/ PRRSV propagation and inactivation. PRRSV type 2 (S1/17 MA2-2 0117 ORF5US) were isolated from swine farms in Thailand experiencing PRRS virus outbreaks. The viruses were propagated in MARC-145 cells at 0.01 multiplicity of infection (MOI). MARC-145 cells were harvested when exhibiting 80% cytopathic effect (CPE) and supernatant was collected by centrifugation at 2500 rpm under 4 °C for 15 min. The PRRSV-containing supernatant was then overlaid onto 20% sucrose and ultracentrifuged at 35,000 rounds per minute (rpm) for 3 h to pellet the virus at 4 °C. Viral pellets were then resuspended in sterile PBS and inactivated with UV radiation dose of 1000 mJ/cm 2 for 10 min, using a UV generator (Hoenle UV Technology, Munich, Germany). The inactivated virus was stored at − 80 °C until used.
PRRSV complete inactivation analysis. To determine whether the virus was totally inactivated, MARC-145 cells were inoculated with 1 mL of each inactivated viral suspension. The cells were cultured for one week at 37 °C in a humidified environment with 5% CO 2 . MARC-145 cells were inoculated with 1 mL of noninactivated virus and mock medium as positive and negative controls, respectively. CPE was observed to identify infected cells.
Total PRRSV protein determination. The total PRRSV protein content was determined using a bicinchoninic acid (BCA) assay kit (Sigma-Aldrich, MO, USA) by the help of series of bovine serum albumin (BSA) standard prepared in phosphate-buffered saline (PBS). The inactivated PRRSV was kept at − 80 °C until use.

Formulation. Preparation of polylactic acid (PLA) nanoparticles loaded with inactivated PRRSV. PLA na-
noparticles loaded with inactivated PRRSV (KNPs) were prepared according to our previous study using the double emulsion evaporation technique 27 . Briefly, inactivated virus pellets equivalent to 5 mg total viral protein content were added to 5% Resomer 202H poly-(D,L-lactide) or PLA (MW 10,000-18,000 gmol −1 ) (Evonik Nutrition & Care GmbH, Essen, Germany) in dichloromethane (DCM) solution (EMSURE, Merk, Germany). The mixture was sonicated at 30% amplitude for 1 min using a SON-1 VCX750 probe sonicator (Sonics & Materials, Inc., CT, USA) to form a primary emulsion. The emulsion was then added to 5% polyvinyl alcohol solution (Sigma-Aldrich, MO, USA) and sonicated at 30% amplitude for 2 min to generate a water-in-oil-in-water (w/o/w) double emulsion. The double emulsion was stirred overnight at ambient temperature to obtain nanoparticles encapsulated with inactivated PRRSV. The nanoparticles were washed three times with deionized water, ultracentrifuged (Beckman Coulter Inc., CA, USA) at 35,000 rpm at 4 °C for 30 min, freeze dried using a lyophilizer (Lyophilization system, Inc., NY, USA) and stored at 4 °C. For the preparation of LTB-DDA coupled with PLA nanoparticles loaded with inactivated PRRSV (adjuvant-KNPs), an aliquot of inactivated virus pellets was emulsified with the mixture of PLA and DDA in DCM under the same conditions as previously described 27 . After that, ten milligrams of the freeze-dried nanoparticles were resuspended in 1 mL of PBS (pH 7.4) containing 2 μg of LTB (Sigma-Aldrich, MO, USA) and mixed for 10 min before use. PBS was used instead of inactivated virus pellets as a blank in the preparation of PLA nanoparticles (blank NPs) and LTB-DDA coupled with PLA nanoparticles (adjuvant-blank NPs). Both groups were served as nanoparticle controls.
Characterization of the nanoparticles. Size, size distribution and zeta potential measurement. The particle size, size distribution and zeta potential of the freeze-dried nanoparticles were determined using a Zetasizer Nano ZS (Malvern, UK). Ten milligrams of freeze-dried nanoparticles were resuspended in 1 mL of deionized water and subjected to size analysis. All measurements were performed in triplicate at 25 °C.
Scanning electron microscope. The morphology of the freeze-dried nanoparticles was visualized using scanning electron microscopy (JEOL Ltd., Tokyo, Japan). The nanoparticles were mounted on adhesive tape and coated with gold-platinum under a vacuum using an ion coater (Blazers, Liechtenstein). The coated samples were examined under a microscope at 10 kV (JSM-6610) HV/LV with EDX and at a magnification of 15,000 ×.
Protein entrapment efficiency (EE) of nanoparticles. Ten milligrams of freeze-dried nanoparticles were degraded in 1 mL of 0.1 N NaOH for 1 h at ambient temperature with a constant stirring at 500 rpm. The mixture was ultracentrifuged at 35,000 rpm for 10 min. After that, the supernatant was collected to determine the total viral protein content using a BCA protein assay kit. The % EE was calculated by dividing the amount of encapsulated protein by the total amount of added protein as the following equation: The series of BSA samples with different concentrations in 0.1 N NaOH served as the reference standards.
Western blot analysis of PRRSV protein in nanoparticles. Freeze-dried nanoparticles were degraded for 24 h at room temperature in 1 mL of 0.1 N NaOH with continuous stirring at 500 rpm. The suspension was ultracentrifuged for 10 min at 35,000 rpm. The supernatant was then separated and quantified for the total viral protein content using a BCA assay kit. The sample was prepared in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (Bio-Rad, CN, USA) with equal amounts of viral protein and boiled for 5 min before loading onto a 15% acrylamide gel. SDS-PAGE was used to separate viral proteins according to the manufacturer's recommendations (Bio-Rad, CN, USA). Plus Protein WesternC Blotting Standards (Bio-Rad,

%EE =
The amount of entrapped protein in nanoparticles The total amount of added protein × 100. www.nature.com/scientificreports/ CN, USA) were used as molecular-weight size markers. Viral proteins were transferred onto a nitrocellulose membrane for western blot detection using a Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad, CN, USA). The membranes were incubated for 1 h at room temperature in blocking buffer (5% skim milk in Tris-buffered saline and Tween 20; TBST). The viral proteins were detected using antibody in serum generated from a pig vaccinated with PRRS MLV, which was diluted in blocking buffer overnight at 4 °C. The membranes were washed twice with TBST and incubated with HRP-coupled secondary antibody in blocking buffer for 1 h at room temperature. The antigen-antibody complexes were examined using chemiluminescence substrate (SuperSignal West Pico, MA, USA).
In vitro cell culture studies. 3 Animal studies. Experimental design. Forty-two, castrated male, crossbred (LRxLWxD) pigs of approximately 3 weeks of age were procured from a PRRSV negative herd. All pigs were tested for PRRSV by serological testing and PCR prior to shipment. Upon arrival, pigs were randomly allocated into 7 treatments groups of 6 pigs each.  (7) inactivated PRRSV/challenged pigs (Inactivated PRRSV) were intranasally vaccinated for 3 consecutive weeks (500 µL/nostril) at 0, 7 and 14 days post vaccination (DPV). The amount of PRRSV protein antigens in each vaccine dose was 1 mg. At 28 DPV, pigs in challenged groups were inoculated intranasally with PRRSV-2 (S1/17 MA2-2 0117) at 10 6 TCID 50 (1 mL/nostril). Pigs were euthanized at 7 days post challenge (DPC), 35 DPV. Macro-and microscopic lung lesions were examined. The animal studies were performed as previously described 35 .
All animal procedures were carried out in accordance with the requirements of the Guide for the Care and Use of Laboratory Animals of the National Research Council of Thailand according to protocols that were reviewed and authorized through the Chulalongkorn University Animal Care and Use Committee (protocol number 1731047). The animal study is reported in compliance with ARRIVE guidelines 36 .
Collection of blood, nasal swab samples and lung for analyses. Blood and nasal swab samples were collected on 0, 14, 28 and 35 DPV. Sera were separated and assayed for the presence of antibody using virus neutralization assay (VNA) and immunoglobulin (Ig) G enzyme-linked immunosorbent assay (ELISA). Nasal swab samples were obtained by deeply inserting swabs of cotton wool into nasal cavity and placed in sterile PBS. Swab samples were centrifuged and clarified supernatants were assayed for the presence of IgA antibody using ELISA. Lung tissues were collected at necropsy. Lung lysates were prepared and assayed for the presence of PRRSV RNA and antibody using PCR and VNA, respectively.
Preparation of lung lysates. Lung lysates were prepared with a slight modification according to a previously described protocol 37 . In brief, 5 g of lung tissue were minced into pieces and finely mashed using a mortar and pestle. Tissues were clarified by centrifugation at 2,500 rpm under 4 °C for 15 min. Supernatant was then collected, passed through 0.2 μm membrane filter and subjected for further determination of virus neutralizing titer (VNT).

Determination of virus neutralizing titer.
To measure VNT in sera and lung lysates, VNA was performed. In brief, sera were heated inactivated at 56 °C for 30 min. To conduct VNA, sera were serially two-fold diluted and incubated with an equal volume of PRRSV (10 2 TCID 50 ). Following a 2 h incubation at 37 °C, 100 µL of the samples/virus mixes were transferred into a 96-well plate containing a confluence monolayer of MARC-145 cells.
Cells were incubated at 37 °C for 48 h in a CO 2 incubator prior evaluation of CPE through microscope. VNT in sera and lung lysates were determined by the highest dilution that inhibited CPE. Neutralization titers were expressed as the geometric mean titer (GMT) of the reciprocal of highest serum dilution that completely inhibit virus infection (no CPE).
Analysis of PRRSV-specific antibodies. The level of IgG in sera and IgA in nasal swab samples were determined using ELISA. Briefly, 96-well ELISA plates (Nunc MaxiSorp flat-bottom, Thermo Fisher Scientific, CPH, Denmark) were coated with whole PRRSV (10 µg/mL) in carbonate buffer (pH 9.6) and incubated overnight at 4 °C. ELISA plates were washed and treated with blocking buffer containing 1% BSA and 0.1% Tween 20 in PBS for 2 h at ambient temperature. Sera or nasal swabs samples were added and incubated for 2 h at ambient temperature. Plates were then washed with blocking buffer and incubated with either anti-pig IgA-horseradish peroxidase (HRP) or anti-pig IgG-HRP (Bio-Rad Laboratories, CA, USA) for 3 h at ambient temperature to detect IgG and IgA in serum and nasal swab samples, respectively. Following a wash, 3,3' ,5,5'-tetramethylbenzidine (TMB) (Sigma-Aldrich, MO, USA) was added. The reaction was stopped and the optical density was measured at 450 nm using an ELISA plate reader (AccuReader, Taipei, Taiwan). Data were analyzed as OD values which were subtracted from those of control wells.
Determination of lymphocytes producing IFN-γ. The percentage of PRRSV-specific lymphocytes producing IFN-γ after stimulation with homologous virus was evaluated using a flow cytometric technique. Briefly, 1 × 10 6 PBMCs were seeded into a 96-well plate containing a mock suspension, PMA (25 ng/ml)/ionomycin (1 μM Fig. S1). The percentage of lymphocytes producing IFN-γ was analyzed based on lymphocyte gating, as determined by a forward scatter versus side scatter graph after the acquisition of at least 30,000 events.
Quantification of PRRSV RNA copy number. PRRSV RNA in the lungs was evaluated by quantitative PRC (qPCR) after PRRSV challenge. Briefly, total RNA was extracted from lung samples (25 mg wet weight/pig) using a NucleoSpin RNA Virus tissue extraction kit (Macherey-Nagel, Germany) in accordance with the manufacturer's protocol. The RNA quality was measured using a NanoDrop spectrophotometer (Colibri Spectrometer, Titerterk-Berthold, Germany) and converted to cDNA. The cDNA was used for qPCR. Primers specific for the ORF5 gene in PRRSV type 2 and the forward and reverse primers used to amplify viral RNA were 5′-GAA GAG AAA CCC GGA GAA GC-3′ and 5′-CGT AGG CAA ACT AAA TTC CACAG-3′, respectively. The copy number of the viral RNA was then quantified according to a previously published method 39 with minor modifications. The reaction was carried out in a QuantStudio 3 Real-time PCR machine (Thermo-Fisher, USA). The copy numbers of the viral RNA estimated by a QuantStudio 3 Real-time PCR machine were normalized by dividing the weight of the lung and reported as the copy numbers of the viral RNA/mg in the lung.
Pathological examination. All pigs were necropsied at 7 DPC, PRRSV-induced pneumonia lung lesions were evaluated according to a previously described method 40 . For the macroscopic lung lesion score, the lungs were scored to estimate the percentage of the lung affected by pneumonia. Each lung lobe was assigned a number to reflect the approximate percentage of the volume of the entire lung, and the percentage volume of each lobe was added to the entire lung score (ranging from 0 to 100% of the affected lung). For the microscopic lung, lung sections were stained for hematoxylin and eosin (H&E) as described previously 41 and then examined by a blinded observer and given an estimated score according to the severity of interstitial pneumonia. Lung sections were scored according to the severity of interstitial pneumonia as follows; 0 = no microscopic lesions; 1 = mild interstitial pneumonia; 2 = moderate multifocal interstitial pneumonia; www.nature.com/scientificreports/ 3 = moderate diffuse interstitial pneumonia; 4 = severe interstitial pneumonia. The mean values of microscopic score of each group were calculated.
Statistical analyses. The data from repeated measurements were expressed as the mean ± SD or SEM and analyzed using analysis of variance (ANOVA) followed by Turkey's HSD post hoc test by SPSS version 19 (SPSS Inc., IL, USA). The statistical significance was assessed as p < 0.05.

Results
Formulation. Characterization of polylactic acid nanoparticle-encapsulated inactivated PRRSV. The average size of PLA nanoparticles loaded with inactivated PRRSV (KNPs) was 312 nm, and they had a PDI of 0.20, zeta potential of − 12.80 mV and %EE of 67.01 (Fig. 1A).  www.nature.com/scientificreports/ reduced to 53.67 (Fig. 1B). The SEM images indicated that both nanoparticle systems were spherical in shape with a smooth surface (Fig. 1C,D). The western blot results showed that the PRRSV protein remained intact in both nanoparticle systems (Fig. 1E,F).       www.nature.com/scientificreports/ Antibody responses as measured by ELISA. Antibody response was not detected in the negative group (NoVac-NoChal) throughout the study. At 28 DPV, the Adjuvant-KNPs and KNPs group had detectable levels of IgG in sera and the levels were significantly higher compared with the other groups. The IgG levels in sera of these 2 groups continues to increase at 35 DPV (p < 0.05, Fig. 6C) and again the level was significantly highest in the Adjuvant-KNPs group.

In vitro cell
In nasal swab samples, the detectable IgA levels were observed in the Adjuvant-KNPs, KNPs and inactivated PRRSV groups as early as 28 DPV and the levels were significantly higher than in that of other groups (p < 0.05, Fig. 6D). Both IgG and IgA level of the Adjuvant-KNPs and KNPs groups continued to increase and the levels were significantly higher compared with the other groups at 35 DPV.
Quantification of PRRSV RNA in lungs. PRRSV RNA was not detectable in lungs of the negative control group (NoVacNoChal). The viral RNA copy number of the KNPs, Adjuvant-KNPs, and inactivated PRRSV groups were significantly lower compared with the other groups at 7 DPC. No significant differences in viral RNA copy number among the control nanoparticle groups (Blank NPs and Adjuvant-Blank NPs) and NoVacChal groups. Pigs in the Adjuvant-KNPs showed significantly lower viral RNA copy number than in other groups (p < 0.05, Fig. 7).
Pathological examination. Macroscopic lung lesion scores. Macroscopic lung lesions were characterized by multifocal, mottled tan areas with irregular and indistinct borders. The macroscopic lung lesion scores are summarized in Table 1. No macroscopic lung lesions were observed in the negative control group (NoVac-NoChal). The nonvaccinated, challenged group (NoVacChal) and the control nanoparticle groups (blank NPs and adjuvant-blank NPs) had significantly higher macroscopic lung lesion scores than the other groups, and differences were not observed between them. Among the three vaccinated groups, the adjuvant-KNP group Figure 7. PRRSV RNA copy numbers in lung homogenate samples. Copy numbers were analyzed using qRT-PCR. The data indicate the average RNA copy number per milligram of lung ± SEM (n = 6). Different lowercase letters indicate significant differences between groups (p < 0.05). Table 1. Levels of PRRSV RNA in lungs, and macro-and microscopic lung lesions, lung lesions of intranasally vaccinated pigs following challenged with PRRSV-2 at 7 days post challenge. Values are present in mean ± SEM. The difference of lowercase letters exhibited significantly differences between groups (p < 0.05) in each column. www.nature.com/scientificreports/ had significantly lower PRRSV-induced pneumonic macroscopic lung scores than the other vaccinated groups (p < 0.05, Fig. 8A,C).

Descriptions PRRSV RNA (10 3 copies/mg) Macroscopic lung scores Microscopic lung scores
Microscopic lung lesion scores. Microscopic lung lesions associated with PRRSV infection were characterized by thickened alveolar septa with increased numbers of interstitial macrophages and lymphocytes and by type II pneumocyte hyperplasia. The pigs in the negative group had the lowest microscopic lung lesion scores. The microscopic lung lesion scores were consistent with the macroscopic lung lesion scores ( Table 1). The nonvaccinated, challenged group (NoVacChal) and the control nanoparticle groups (blank NPs and adjuvant-blank NPs) had the highest microscopic lung lesion scores. Although there was no difference between them, the scores were relatively higher than those of the KNP and inactivated PRRSV groups. The adjuvant-KNP group had a significantly lower microscopic lung lesion score than the other groups (p < 0.05) (Fig. 8B,C).

Discussion
This study was conducted to investigate the induction of systemic and mucosal immune responses and the protective efficacy following the intranasal administration of inactivated PRRSV loaded in PLA-based nanoparticles coupled with LTB and DDA (adjuvant-KNPs). It was demonstrated that pigs intranasally administered adjuvant-KNPs had a strong positive influence on both systemic and mucosal immune responses, as observed by increased VNT and IgG levels in sera and increased IgA levels in the nasal swab samples. A significantly induced cell-mediated immune response in the adjuvant-KNP group was indicated by the increase in IFN-γ-producing lymphocytes and lower level of IL-10 secreted from PBMCs compared to that in the other vaccinated groups. The lowest lung lesion scores and PRRSV RNA copies in the lungs following PRRSV-2 challenge indicated the protective efficacy of the adjuvant-KNP group. The study suggested that when administered intranasally, PLAbased nanoparticles coupled with LTB and DDA are crucial adjuvants to improve the efficacy of inactivated PRRSV in eliciting immune responses and providing protective efficacy. The findings presented herein suggest the development of intranasal vaccines that can be used for livestock. One of the obstacles for the success of intranasal vaccines is the transport of antigens across M cells to be taken up by immune cells. Our previous in vitro studies indicated that when a cationic lipid DDA is incorporated into the matrix of PLA, it creates positively charged PLA nanoparticles 27 . The positive charge on the surface of nanoparticles is an important key to inducing the initial contact between sialic acid and the heparan sulfate proteoglycan receptor on the surfaces of porcine alveolar macrophages, which facilitates the cellular uptake of antigens loaded within the DDA-PLA nanoparticles via the endocytosis pathway 27,42 . Moreover, the transport of antigens across cultured M cells is also improved following the combination of LTB, a common mucosal adjuvant, with www.nature.com/scientificreports/ DDA-PLA nanoparticles 27 . LTB is known to bind to the monosialotetrahexosylganglioside (GM1) receptor. The receptor has easy accessibility to M cell areas and therefore can facilitate the transport of vaccine nanoparticles across M cells to underlying immune cells 42,43 . Thus, the improved induction of the immune response observed in pigs vaccinated with LTB-DDA coupled with PLA nanoparticles loaded with inactivated PRRSV could be contributed by increased antigen transportation into M cells and immune cells by LTB coupled with DDA-PLA nanoparticles 27 . Compared with LTB-DDA-coupled PLA nanoparticles, PLA nanoparticles alone (without DDA and LTB) possess a net negative charge on their surface. It is generally accepted that negatively charged nanoparticles are less internalized by cells than positively charged nanoparticles 44 , which might explain why the pigs in the PLA nanoparticles loaded with the inactivated PRRSV group (KNPs) had weaker immune responses than the pigs in the LTB-DDA coupled with PLA nanoparticles loaded with inactivated PRRSV group. Inactivated virus is poorly immunogenic; therefore, unsurprisingly, pigs intranasally vaccinated with inactivated PRRSV alone displayed poor immune induction. Inactivated PRRSV vaccines induce no detectable level of immune response when intramuscularly administered to PRRSV-free pigs. However, a rapidly increased immune response was observed following intramuscular vaccination with live virus in naïve pigs previously vaccinated with inactivated PRRSV vaccine 45 . The mechanisms underlying this behavior have not been clarified, although they could be associated with memory cells generated following the immune response. Although intramuscular injection in naïve pigs does not lead to detectable levels of immunity, pigs have already been infected by foreign antigens, such as PRRSV. Although the immune response might have been initiated, the level may be below the sensitivity of current assays, such as ELISA. In the present study, similar evidence was observed. Inactivated virus alone when administered intranasally vaccinated induced no detectable level of immune response. However, a significant increase in the immune response was observed following challenge with live virus compared to that in pigs challenged only with inactivated PRRSV. These results suggested that inactivated PRRSV was present and could potentially induce a low level of immune response. To induce a higher immune response, adjuvants are needed. In this study, PLA nanoparticles coupled with DDA and LTB were selected. Encapsulating inactivated PRRSV in PLA nanoparticles is beneficial to protect the antigen from proteolytic degradation, prolong their bioavailability and maintain slow and sustained antigen release, especially in coupling with DDA 27 . After the uptake of nanoparticles in APCs, the nanoparticles can induce the production of various innate cytokines that regulate humoral and cellular immunity 46 . All of these properties facilitated the induction of better immune responses of inactivated PRRSV when loaded in LTB-DDA coupled with PLA nanoparticles. The findings found in the present study are in accordance with earlier studies that reported an increased uptake of inactivated PRRSV in porcine alveolar macrophages with the assistance of PLGA nanoparticles 25,47 . The intranasal vaccine administration of these PLGA nanoparticles resulted in the induction of significantly higher immune responses compared with the intranasal administration of inactivated virus alone, which elicited no response.
Humoral immunity is crucial for controlling PRRSV infection. Neutralizing antibodies contributed mostly to viral clearance by neutralizing viral infection, thereby inhibiting viral replication in the lung and controlling the systemic spread of PRRS virus 48,49 . We found that adjuvant-KNP-vaccinated pigs elicited the highest humoral immunity in both systemic and local responses against homologous PRRSV, as indicated by higher levels of VNT in both the sera and lung lysate samples, IgG in sera, and IgA in nasal swab samples at 14-35 DPV compared to that in the other groups (p < 0.05). The significantly high VNT from the serum of pigs vaccinated with adjuvant-KNPs also correlated with the high level of serum IgG from the same group, implying that adjuvant-KNPs have a strong positive influence on humoral responses in systemic and mucosal sites. This was likely due to the great uptake of adjuvant-KNPs in immune cells facilitated by DDA and LTB 27 . Previous studies reported that inactivated PRRSV vaccines elicit a predominantly humoral immune response, as indicated by increased levels of specific PRRSV antibodies 50 and VNT, and such immunity was augmented by using nanoparticles 25 . Therefore, it is possible to induce high levels of VNT and elicit an effective memory response against PRRSV by using our vaccine.
For the lung lysate samples, we observed high VNT in the adjuvant-KNP-, KNP-and inactivated PRRSVvaccinated pigs. The adjuvant-KNP group had the highest VNT, and the inactivated PRRSV group had the lowest VNT. In agreement with our studies, commercially available inactivated PRRSV vaccines induced very low VNT 51,52 , and inactivated PRRSV encapsulated in nanoparticles elicited higher VNT in both serum and lung samples than unencapsulated inactivated PRRS virus 52 . Therefore, it is possible that LTB and DDA could play a role in the production of higher neutralizing antibody titers.
Strong cell-mediated immunity is an essential key for protection against PRRSV infection 53 . Significantly increased IFN-γ producing cells, a key moderator of cell-mediated immunity, in the LTB-DDA coupled with PLA nanoparticles loaded with inactivated PRRSV group could indicate a strong stimulation toward a Th1 response, as indicated by the increased number of IFN-γ producing lymphocytes after vaccination. Consistent with our results, previous studies showed that DDA is capable of producing high levels of IFN-γ in CD4 + CD8 − lymphocytes 54,55 . Additionally, LTB could potentially induce the production of IFN-γ in both CD4 + CD8 − and CD4 − CD8 + lymphocytes 56 . Mechanisms involving LTB and DDA and lymphocyte stimulation are not well understood. However, it may involve the ability of LTB to facilitate transcytosis of antigens loaded in nanoparticles across M cells to underlying APCs 57 and the adjuvant property of DDA. In addition, DDA could be involved in cross-presentation via major histocompatibility complex (MHC) class I molecules by mediating nanoparticles taken up by APCs 50 and inducing lysosomal escape due to its positively charged nature 58 .
IL-10 is an anti-inflammatory cytokine that maintains the balance of the immune response, allowing effective pathogen clearance with minimal host damage. The expression of IL-10 has been associated with decreases in cell-mediated immune responses against PRRSV by downregulating the expression of proinflammatory cytokines such as IFN-γ 59 . In this study, the induction of IL-10 levels was low in all vaccinated groups. Following challenge, we observed an increase in IL-10 levels in all vaccinated groups except the adjuvant-KNP group, where the IL-10 www.nature.com/scientificreports/ level showed no significant difference from the negative control group. It has been proposed that IL-10 induction by PRRSV can result in the ineffective induction of an IFN-γ-specific response. The low induction of IL-10 in the adjuvant-KNP group both during vaccination and after challenge indicated that the LTB-DDA coupled with PLA nanoparticle system did not induce a significant IL-10 response to mediate immune dysfunction, as demonstrated by the high number of lymphocytes producing IFN-γ. This finding suggested that LTB-DDA coupled with PLA nanoparticles could be a good candidate for improving the efficacy of inactivated PRRSV vaccines.
For lung pathogens such as PRRSV, preventing initial infection at local sites could contribute to the success of vaccines. IgA in the respiratory tract is mostly responsible for PRRSV clearance at the nasal mucosa, thus helping control primary infection and limiting shedding and lung infection 20,21 . We observed significant differences in IgA levels in nasal swab samples of pigs in the adjuvant-KNP, KNP and inactivated PRRSV groups compared to the other groups. The highest IgA level was found in the adjuvant-KNP group, and the lowest IgA level was detected in the inactivated PRRSV group. This increased IgA level in the nasal swab samples (Fig. 6D) could also be related to the reduction in the PRRSV RNA copy number in the lungs of the challenged pigs (Fig. 7). These data suggested that induction of strong local mucosal immunity is the most important method of clearing detectable replicating challenge PRRSV.
Following challenge, all vaccination groups (KNP, adjuvant-KNP, and inactivated PRRSV groups) were partially protected against PRRSV challenge, as evaluated by the reduction in PRRSV RNA in the lungs and lung lesion scores at 7 DPC. The greatest potential in reducing the copy number of PRRSV RNA in the lungs and lowering lung lesion scores was observed in the adjuvant-KNP group.
The pathological examination of lungs in both macroscopic and microscopic images was typical of those associated with PRRSV infection. Consequently, pathological examination is a critical process for evaluating the efficacy of PRRSV vaccines. The pathology of the lungs was examined at 35 DPV (7 DPC). A previous study indicated that the most extensive and severe lesions were observed at Day 7 after challenge with PRRSV and that resolved lesions were observed at 21 DPC 60 . Our study revealed that the macroscopic lung lesion scores were consistent with the microscopic lung lesion scores and showed similar trends (Fig. 8A,B). The lowest macroscopic lung lesion scores and microscopic lung lesion scores were observed in the adjuvant-KNP group, suggesting the efficacy of this vaccine in protecting against PRRSV infection in the lungs (Fig. 8C). The results suggested that adjuvant-KNPs showed the greatest ability to induce CMI and HRI at both systemic and local sites and provided protective efficacy against homologous PRRSV challenge.
It is noteworthy that heterologous protection against different PRRSV isolates is less reliable, varying from partial to none. Further studies evaluating the protective efficacy of the intranasal PRRSV vaccine against heterologous strains of PRRSV are needed to demonstrate whether intranasal vaccination can broaden the protection. In the present study, mucosal immunity (IgA) in the nasal cavity might be a key issue that plays crucial roles in protecting the mucous membranes against colonization and invasion by PRRSV and preventing the development of potentially damaging immune responses to PRRSV if they reach the pig body.
In summary, this is the first study to illustrate the efficacy of LTB-DDA coupled with PLA nanoparticles loaded with inactivated PRRSV for inducing humoral and cell-mediated anti-PRRSV immune responses, which better improved immune responses and protected PRRSV infection than PLA nanoparticles loaded with inactivated PRRSV and free inactivated PRRSV. Further studies are needed to evaluate the efficacy of LTB-DDA coupled with PLA nanoparticles loaded with inactivated PRRSV compared with commercially available PRRSV vaccines and to investigate anti-PRRSV cross-protective immunity in vaccinated pigs. Our study successfully produced a novel promising vaccine against PRRS administered by nasal vaccination. The novel vaccine may have the potential to control PRRSV outbreaks and reduce economic losses of swine farms.