
==== Front
J Extracell Vesicles
J Extracell Vesicles
10.1002/(ISSN)2001-3078
JEV2
Journal of Extracellular Vesicles
2001-3078
John Wiley and Sons Inc. Hoboken

10.1002/jev2.12514
JEV212514
Research Article
Research Article
Enhancing protective immunity against bacterial infection via coating nano‐Rehmannia glutinosa polysaccharide with outer membrane vesicles
HUANG et al.
Huang Yee https://orcid.org/0000-0003-2281-9589
1
Sun Jiaying 1 2
Cui Xuemei 1
Li Xuefeng 1
Hu Zizhe 1
Ji Quanan 1
Bao Guolian 1 baoguolian@163.com

Liu Yan 1 ly-liuyan@163.com

1 Institute of Animal Husbandry and Veterinary Science Zhejiang Academy of Agricultural Sciences Hangzhou Zhejiang China
2 College of Life Sciences China Jiliang University Hangzhou Zhejiang China
* Correspondence
Guolian Bao and Yan Liu, Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang, China.
Email: baoguolian@163.com and ly-liuyan@163.com

24 9 2024
9 2024
13 9 10.1002/jev2.v13.9 e1251412 10 2023
28 8 2024
© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

Abstract

With the coming of the post‐antibiotic era, there is an increasingly urgent need for safe and efficient antibacterial vaccines. Bacterial outer membrane vesicles (OMVs) have received increased attention recently as a potential subunit vaccine. OMVs are non‐replicative and contain the principle immunogenic bacterial antigen, which circumvents the safety concerns of live‐attenuated vaccines. Here, we developed a novel nano‐vaccine by coating OMVs onto PEGylated nano‐Rehmannia glutinosa polysaccharide (pRL) in a structure consisting of concentric circles, resulting in a more stable vaccine with improved immunogenicity. The immunological function of the pRL‐OMV formulation was evaluated in vivo and in vitro, and the underlying mechanism was studied though transcriptomic analysis. The pRL‐OMV formulation significantly increased dendritic cell (DC) proliferation and cytokine secretion. Efficient phagocytosis of the formulation by DCs was accompanied by DC maturation. Further, the formulation demonstrated superior lymph node targeting, contributing to a potent mixed cellular response and bacterial‐specific antibody response against Bordetella bronchiseptica infection. Specifically, transcriptomic analysis revealed that the immune protection function correlated with T‐cell receptor signalling and Th1/Th2/Th17 differentiation, among other markers of enhanced immunological activity. These findings have implications for the future application of OMV‐coated nano‐carriers in antimicrobial immunotherapy.

Bordetella bronchiseptica
dendritic cells
outer membrane vesicle
subunit vaccine
transcriptomic analysis
China Agriculture Research System of MOF and MARA“Pioneer” and “Leading Goose” R&D Program of ZhejiangKey Research and Development Program of Zhejiang Province 10.13039/100022963 2019C02052 2021C02007 National Natural Science Foundation of China 10.13039/501100001809 32002323 32102714 source-schema-version-number2.0
cover-dateSeptember 2024
details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_JATSPMC version:6.4.8 mode:remove_FC converted:24.09.2024
Huang, Y. , Sun, J. , Cui, X. , Li, X. , Hu, Z. , Ji, Q. , Bao, G. , & Liu, Y. (2024). Enhancing protective immunity against bacterial infection via coating nano‐Rehmannia glutinosa polysaccharide with outer membrane vesicles. Journal of Extracellular Vesicles, 13 , e12514. 10.1002/jev2.12514
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pmc1 INTRODUCTION

Pulmonary infections are one of the leading contributors to global disease burden in humans and livestock. Despite advances in controlling a substantial proportion of zoonotic diseases, there is a need for development and approval of safe and effective vaccines against many common respiratory pathogens, including those caused by bacteria (Adams et al., 2011; Ali et al., 2023; Wu et al., 2022) such as Bordetella bronchiseptica (Bb). Bb is a Gram‐negative bacterium that colonizes the respiratory tract of mammals including mice, rabbits, dogs and pigs. In humans, Bb can cause pneumonia in infants and immunologically compromised individuals (Zhang et al., 2019). Currently, antimicrobials are the last medical resource to treat the disease. However, a dramatic increase in bacterial resistance to antimicrobials has created an urgent need for studies exploring safe and efficient human vaccines.

Recently, outer membrane vesicles (OMVs) have received increased attention as a potential subunit acellular vaccine candidate against pathogenic bacteria (Li et al., 2020). OMVs are non‐replicative, spherical, proteolipid nanoparticles that are naturally released by Gram‐negative bacteria throughout growth (Ribeiro de Freitas et al., 2022; Schetters et al., 2019). They are primarily comprised of a range of immunogenic antigens of the parental bacteria either on the surface or encapsulated in the interior, which are required to stimulate both innate and adaptive immunity (Weyant et al., 2023). OMVs are replication‐defective and therefore addresses the major safety concerns in applications of traditional attenuated bacterial vaccines (Chen et al., 2020). Moreover, the nanosize of the OMV (10–300 nm) facilitates their uptake by antigen presenting cells (APCs), such as dendritic cells (DCs). Activation and maturation of DCs in turn stimulate naïve T cells and induce B cell activity for antibody production, capacities critical for protective immunization (Micoli & MacLennan, 2020). Several studies have been carried out to exploit the potential of OMVs as an antibacterial vaccine. Among them, the current state‐of‐the‐art of OMV‐based vaccine is Group B meningococcal vaccine Bexsero®, which is clinically approved and successful in limiting the incidence and mortality of meningitis in New Zealand (Petousis‐Harris et al., 2017). Despite this, some disadvantages are associated with free OMVs, such as compromised vesicle stability and heterogeneous size (Jiang et al., 2019; Li et al., 2020; Wu et al., 2019).

Since 2011, cell membrane coating technology has been widely used in vaccine research and development in the field of biomedicine (Fang et al., 2018; Gan et al., 2020). Coating OMVs onto synthetic nanoparticles will preserve the complex biological characteristics of bacteria and mimics the physiologic process of antigen presentation to the immune system (Gao et al., 2015). Simultaneously, the physiochemical properties of the synthetic nanoparticles can be finely tuned on demand. Therefore, nanoparticle facilitated OMV vaccines have the potential to maximize the immunization effect of free OMVs. Inspired by this technology, we combined Bb‐derived OMVs with PEGylated nano‐Rehmannia glutinosa polysaccharide (pRL) to synthesize a novel nano vaccine and explored its ability to induce antigen‐specific immune responses in vivo.

R. glutinosa polysaccharide (RGP) is a naturally produced polysaccharide derived from the Chinese herbal medicine R. glutinosa. In our previous study we discovered that, as an adjuvant, pRL is capable of effectively inducing humoral and cellular immunity against Bb (Huang et al., 2021). In order to develop a safe and highly efficient vaccine, we applied the pRL to our current OMV‐based subunit vaccine formulation. In vitro, application of pRL‐OMV significantly promoted dendritic cell (DC) proliferation and activation. In vivo, pRL‐OMV demonstrated lymph node (LN)‐targeting antigen presentation, and effectively activated DCs within LNs, which consequently contributed to efficient induction of potent antigen‐specific immune responses in the vaccinated animals. Transcriptomic analysis revealed that the immunological function of pRL‐OMV was related to several key immunological pathways, including T‐cell receptor signalling pathway and differentiation of Th1, Th2, and Th17 cells.

Collectively, our findings suggested that the use of pRL coupled with Bb‐derived OMV could function as a promising subunit vaccine towards effective treatment of respiratory bacterial infections.

2 MATERIALS AND METHODS

2.1 Materials

B. bronchiseptica (Bb) strain FX used in this study was isolated from a rabbit farm in Zhejiang Province in China and maintained in our laboratory. Tryptone soya broth (TSB) was purchased from Thermo Fisher (Waltham, MA, USA). R. glutinosa polysaccharide (RGP, with a purity of 98% verified by HPLC, MW: 3.57 × 104) was purchased from Ciyuan Biotechnology Co. Ltd (Shanxi, China). Soybean phospholipid (C42H80NO8P, MW: 758.06, 98% purity) and DSPE‐PEG2000 (C172H336N2Na2O62P2, MW:3532.41) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Cholesterol (C27H46O, MW: 386.65) was purchased from Sigma Aldrich (St. Louis, MO, USA). RPMI‐1640 medium supplemented with benzylpenicillin 100 IU mL−1, streptomycin 100 IU mL−1 and 10% fetal bovine serum, all of which were purchased from Gibco (New York, USA). CCK‐8 cell viability assay was purchased from Abcam (Cambridge, UK). ConA and Lipopolysaccharide powder (LPS, from Escherichia coli O111:B4) was obtained from Sigma‐Aldrich (St. Louis, Missouri, USA). HRP‐conjugated Goat anti‐Mouse IgG was purchased from Abcam (Cambridge, UK). FITC‐conjugated anti‐mouse MHC Class II, FITC‐conjugated anti‐mouse CD80, PE‐conjugated anti‐mouse CD86, APC‐conjugated anti‐mouse CD11c, FITC‐conjugated anti‐mouse CD4, PE‐conjugated anti‐mouse CD8a, APC‐conjugated anti‐mouse CD3 and PE/Cy7‐conjugated anti‐mouse CD19 antibodies were all obtained from Multi Sciences (Hangzhou, Zhejiang, China). ELISA kits for IL‐6, IL‐12, IL‐1β and TNF‐α were all purchased from Solarbio (Beijing, China).

2.2 Synthesis of nano‐R. glutinosa polysaccharide coated with outer membrane vesicles

Nano‐R. glutinosa polysaccharide (pRL) was prepared as previously reported (Huang et al., 2019). Briefly, soybean phospholipid, cholesterol, and DSPE‐PEG2000 (molar ratio = 1000:125:48 respectively) were dissolved in a 1:1 by volume solution of chloroform and methanol. The organic solvent was removed via vacuum evaporation at 56°C until dry. The resulting dry film was hydrated with RGP solution. After rotary evaporation, the resulting mixture was homogenized in an ultrasonic cell disintegrator (JY92‐II DN, Xinzhi Bio‐technology and Science Inc., Zhejiang, China) to obtain a homogeneous solution. The solution was successively filtered first using a 0.45‐µm and then a 0.22‐µm millipore membrane. pRL without PEGylation (RL) was made using the same method as above, while omitting the DSPE‐PEG2000. A blank carrier without RGP (BL) was also generated, using the original method while omitting RGP.

Outer membrane vesicles (OMVs) were obtained from B. bronchiseptica (Bb) strain FX. Bacteria were cultured in TSB (2 mL/tube) at 37°C and on a standard shaker (220 rpm) for 10 h. The resulting cultures were then refreshed with TSB enriched with 64 µg/mL cefalexin at a 1:100 dilution. It was continued to culture at 37°C for 18 h until the exponential growth phase was reached. Cultures were centrifuged (Beckman Coulter Optima LE‐80 K, Type 70 Ti rotor, California, USA) at 10,000 × g for 20 min at 4°C. The cell free filtrates were concentrated using 100‐kDa cutoff centrifugal filter units (Millipore, Massachusetts, USA). OMVs were pelleted by ultracentrifugation 100,000 × g, 4°C for 2 h, and resuspended in phosphate‐buffered saline (PBS) or 2 mM Tris–HCl (pH 7.5). The stock solution of OMV was stored at 4°C after filtration by 0.22 µm filter.

The OMV solution was diluted as need and then mixed with pRL, RL, BL or RGP at equal volume. The mixture was extruded ten times with an Avanti Mini‐Extruder extrusion device (Avanti Polar Lipids, Alabaster, AL, USA), and the unloaded OMV was separated by centrifugation (10,000 × g, 20 min, 4°C). As for Alum‐OMV, OMV solution was added to the Alum (10 mg/mL, Thermo Fisher, Massachusetts, USA) were mixed and stirred on the magnetic mixer at room temperature for 3 h. The OMV protein content on the nanoparticles was determined by Bradford assay (Solarbio, Beijing, China).

In some experiment, DiI‐labelled OMVs were used to mix with pRL, RL, BL and RGP, respectively. OMVs suspension (300 µg/mL) mixed with DiI dye (2 µL, 1 mg/mL) and incubated at 37°C for 30 min. Excess dye was removed by washing OMVs three times with PBS using a 10 kDa MWCO filtration column (Millipore).

The particle size and zeta potential of pRL‐OMV were measured by the nanoparticle tracking analyser (NTA, Particle Metrix, Germany). The morphology of pRL‐OMV was determined via transmission electron microscopy (TEM, Model H‐7650, Hitachi, High Technologies Co., Ltd., Tokyo, Japan). Samples were negatively stained for TEM with 1% solution of phosphotungstic acid.

2.3 Cell viability assay

DC2.4 cells were obtained from the Chinese Academy of Science Cell Bank (Shanghai, China) and cultured in RPMI‐1640 containing 10% foetal bovine serum (FBS), 100 µg/mL penicillin, and 100 µg/mL streptomycin in an incubator at 37°C with 5% CO2.

Following initial incubation, DCs were seeded in a 96‐well plate at a density of 5×103 cells/well and incubated at 37°C, 5% CO2 for 24 h. Subsequently, the complete medium containing predetermined concentrations of either pRL‐OMV, RL‐OMV, BL‐OMV, RGP‐OMV or OMV was added. The final concentration of RGP was 6.25 and 100 µg/mL, and the final concentration of OMV was 0.625 and 10 µg/mL. LPS (10 µg/mL) was used as a positive control, while culture medium treatment was used as a blank control (BC). The culture was continued for an additional 24 h. Then, CCK‐8 reagent was added to each well and cells were further incubated for 2 h. The absorbance was measured at 450 nm.

2.4 Co‐localization of nano‐vaccine with DCs

DC2.4 cells were cultured as described in Section 2.3. DiI‐labelled pRL‐OMV, RL‐OMV, BL‐OMV, RGP‐OMV and OMV were co‐cultured with DC2.4 for 4 h. The final concentration of RGP was 100 µg/mL, and the final concentration of OMV was 10 µg/mL. The cells were then washed with PBS to remove the DiI‐labelled OMV formulations. The cells were stained with DAPI and Lyso‐Tracker Yellow dye in room temperature for 30 min, and the remaining dye was washed off. The cells were observed under a confocal laser scanning microscope (Zeiss, Germany) to determine the cellular uptake and subcellular distribution. Quantitative information about the cellular uptake of OMVs and OMV formulations was obtained by performing flow cytometry (FACSCanto™, BD Biosciences, Franklin Lakes, NJ, USA) to evaluate the cellular uptake of DiI‐labelled OMVs formulations after incubating for 4 h.

2.5 DCs maturation assay

Bone marrow‐derived dendritic cells (BMDCs) were flushed from the femurs and tibias of BALB/c mice and cultured in RPMI‐1640 supplemented with 10% FBS, 100 µg/mL benzylpenicillin, 100 µg/mL streptomycin, 20 ng/mL murine IL‐4 (PeproTech, New Jersey, USA) and 20 ng/mL murine GM‐CSF (PeproTech, New Jersey, USA) after the red blood cells were lysed. The cultures were initiated by placing 1 × 106 cells/well into 6‐well plates. Half of the media was replaced every 2 days with fresh, supplemented RPMI‐1640. On day 6, non‐adherent cells were collected for further investigation.

To assess the effect of different OMV formulations on DC maturation, murine BMDCs were cultured in the presence of distinct OMV formulations for 24 h. The final concentration of RGP was 100 µg/mL, and the final concentration of OMV was 10 µg/mL. At the completion of the incubation, the cells were collected for further staining with FITC anti‐mouse CD11c, PE‐anti‐mouse CD86, APC‐anti‐mouse CD80 or PerCP anti‐mouse MHC II to evaluate BMDC maturation. Flow data were acquired on a flow cytometer (BD FACSCanto) and analysed by Flow Jo software (Version 10.8.1).

After incubation for 24 h under the same incubation conditions as above, the BMDCs were harvested and fixed. The morphological changes with and without pRL‐OMV treatment were observed under a scanning electron microscope (Hitachi SU8200, Japan).

2.6 Animal immunization

Female BALB/c mice (6‐weeks‐old) were obtained from the Institute of Medicine, Zhejiang province and housed in ventilated cages under 12‐h dark and 12‐h light cycle. Standard rodent chow and water were available ad libitum. All animal experiments were performed in accordance with the protocols approved by the Animal Welfare and Ethics Committee of Zhejiang Academy of Agricultural Sciences (Ethics protocol no. 1935).

The mice were randomly separated into seven groups (n = 6) and injected subcutaneously with 0.2 mL of different OMV formulations: pRL‐OMV (2.5 mg/mL of RGP and 5 µg of OMV), RL‐OMV (2.5 mg/mL of RGP and 5 µg of OMV), BL‐OMV (5 µg of OMV), RGP‐OMV (2.5 mg/mL of RGP and 5 µg of OMV), Alum‐OMV (5 µg of OMV), OMV (5 µg of OMV) and PBS (Blank control, BC). The first immunization was administered on day 0, followed by a booster immunization on day 14. The body weight was monitored during immunization. Serum was obtained for biochemical detection, antibody detection and cytokine detection at predetermined time points and kept at −80°C. Upon the experimental endpoint, animals were euthanized, spleens and lungs were aseptically dissected for subsequent detection of immunological responsivity. Blood samples were collected into EDTA microcentrifuge tubes for haematology analysis.

2.7 Anti‐Bb IgG titters analysis

The antibody response mounted against OMV or Bb whole‐cell lysates in serum was analysed by TMB‐based colorimetric ELISA (Huang et al., 2022). OMV (10 µg/mL) and the whole‐cell lysate from Bb bacteria (10 µg/mL) were dissolved in 0.05 M carbonate buffer (pH = 9.6), respectively. Then, 96‐well plates were coated with the OMV or lysate protein solution (100 µL/well) and allowed to incubate at 37°C for 2 h and then kept in 4°C overnight, after which they were washed with PBST three times. Then the plates were blocked with 5% non‐fat milk in PBS for 2 h, followed by the addition of diluted serum samples and HRP‐conjugated goat anti‐mouse IgG (1:10,000), IgG1 (1:5000), and IgG2a (1:5000) (Abcam, Cambridge, UK). Next, ELISA TMB (Multi Sciences, Hangzhou, Zhejiang, China) was added, and after incubation at 37°C in the dark for 15 min, the reaction was stopped with 100 µL/well of stop reagent (0.5 M H2SO4). The absorbance was measured at 450 nm on a microplate reader (ThermoFisher Scientific, Waltham, MA, USA).

2.8 Ex vivo imaging

For ex vivo imaging, PBS, OVA‐Cy5.5 (Xi'an ruixi Biological Technology Co., Ltd., China), OMV‐OVA‐Cy5.5 or pRL‐OMV‐OVA‐Cy5.5 were subcutaneously injected into the tail base of mice for the evaluation of their biodistribution. The inguinal lymph nodes (LNs) and major organs including the heart, liver, spleen, lung, and kidney were collected 24 h later for ex vivo fluorescence examination using the IVIS Lumina III (PerkinElmer, Waltham, MA, USA). Subsequently, the inguinal LNs were flash‐frozen and cryo‐sectioned (10‐µm sections). The sections were stained with DAPI and examined by confocal laser scanning microscopy (CLSM, LSM980, Carl Zeiss, Oberkochen, German).

2.9 Maturation of DCs in popliteal lymph nodes

Female BALB/c mice were immunized as described in 2.6. Popliteal LNs were harvested aseptically and processed into single‐cell suspensions. Cells were stained with a mixture of anti‐mouse antibodies (FITC‐CD11c, PE‐MHC I, PerCP‐MHC II, APC‐CD80 and PE CY7‐CD86, Multi Sciences, Hangzhou, Zhejiang, China). Surface expression of MHC I, MHC II, CD80, and CD86 on DCs was determined using BD FACS‐Verse (BD Biosciences, San Diego, CA, USA) and analysed by Flow Jo (version 10.8.1).

2.10 Spleen and lung analysis

Spleens and lungs were obtained aseptically from inoculated mice (n = 4/group) and individual cells were isolated using a cell strainer and a sterile plunger from a 5‐mL syringe. RBCs were lysed by mixing RBC lysis buffer with cell suspension (v:v = 2:1). After washed twice with PBS, an equivalent of 5 × 106 cells were plated into 96‐well cell culture plates and stimulated in vitro for 72 h with Con A (Concanavalin A, 2.5 µg/well), LPS (Lipopolysaccharide, 2 µg/well), and OMVs (1 µg/well). The CCK‐8 (Cell counting kit‐8) method was used to measure cell proliferation.

Individual splenocytes and lung cells were obtained by a similar method from challenged mice. The samples were stained with FITC‐conjugated anti‐mouse CD4 (0.5 mg/mL, 0.125 µg/test), PE‐conjugated anti‐mouse CD8a (0.2 mg/mL, 0.125 µg/test), and APC‐conjugated anti‐mouse CD3 (0.2 mg/mL, 0.25 µg/test) for 30 min at 4°C. Flow data were acquired on a flow cytometer (BD Biosciences, San Diego, CA, USA) and analysed by Flow Jo software (version 10.8.1).

2.11 Cytokine expression in restimulated splenocytes and serum

As described in 2.10, splenocytes from mice (n = 6/group) were harvested on day 42. The single‐cell suspension obtained from the spleen was stimulated for 3 days with OMV (1 µg/well) to induce cytokine production. Then the supernatant was collected, and the concentration of T‐helper‐related cytokines, interleukin‐4 (IL‐4), IL‐6, IL‐17 and IFN‐γ, was determined using the corresponding ELISA kits according to manufacturer guidelines.

The presence of Th1‐ (IFN‐γ and IL‐12p70), Th2‐ (IL‐4 and IL‐5) and Th17‐ (IL‐17 and TNF‐α) associated cytokines in the serum samples was determined using mouse ELISA kits (Multi Sciences, Hangzhou, Zhejiang, China) in accordance with the manufacturer's instructions.

2.12 Complement‐mediated bactericidal assay

The bactericidal activity of the serum collected from mice 42 days after vaccination was tested in vitro. Briefly, Bb was grown on TSA medium and diluted to 1 × 103 colony‐forming unit (CFU)/mL in PBS. Both immune and naïve sera were heat‐inactivated at 56°C for 30 min prior to use. Guinea pig complement was selected for its absence of bactericidal activity against the test strains (Figure S1). The serum samples were serially diluted in a 96‐well plate (from 1:2 to 1:512), and 40 µL of diluted test serum was mixed with 20 µL of guinea pig complement and 20 µL of the bacterial suspension. The suspensions of bacteria in PBS without guinea pig complement, or without serum, were used as the two controls. Next, the 96‐well plates were sealed and incubated on a shaker incubator (100 rpm) at 37°C for 1 h. Subsequently, 50 µL of each mixture was plated on TSA medium (Solarbio, Beijing, China), incubated at 37°C for 24 h, and the colonies were counted on a colony counter (Scan 1200, Interscience, Saint Nom, France). The data were expressed as a bactericidal rate, calculated as a percentage based on the following formula: Bactericidal rate%=coloniesnaive sera−coloniesimmune sera/coloniesnaive sera×100

2.13 Challenge assay

To confirm the protective effect of pRL‐OMV against bacterial infection, both immunized and naive mice (n = 6/group) were challenged with Bb (FX strain) (6 × 106 CFU/mice) on day 28 through intravenous injection. Then, the mice were sacrificed on the 7th day post‐challenge. The lungs were homogenized for colonization assay in 2 mL TBS using a bullet blender (JXFSTPRP‐CL, Jingxin, Shanghai, China) at 60 Hz for 3 min. Serial dilutions (1:10, 1:100, and 1:1000) of each lung homogenate were plated on TSA incubated in 37°C, and cell counts were performed using a colony counter (XY‐C100, Shandong, China) to determine the number of Bb inside the representative lung samples with 3 replications of each sample.

2.14 Transcriptome sequencing

2.14.1 RNA extraction and library construction

Splenocytes from mice immunized with pRL‐OMV were used for transcriptome sequencing. Splenocytes from mice injected with PBS were used as the control group. The collected samples were snap frozen in liquid nitrogen and the total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. The PE150 sequencing mode was selected and paired‐end sequencing was performed using the Illumina Novaseq™ 6000 (LC‐BioTechnology CO., Ltd., Hangzhou, China) according to the standard procedure.

2.14.2 RNA sequencing and DEG analysis

LC‐Biotechnology Co., Ltd. (Zhejiang, China) performed the transcriptome sequencing. CleanData was aligned to the Rattus norvegicus genome using HISAT2 software, and then mRNA expression levels were analysed by StringTie and ballgown software packages. The differences in mRNA expression levels between the two groups were analysed by the R package. The screening conditions for significantly different genes were p‐value < 0.05 and |log2FoldChange| ≥ 1.

2.14.3 Gene functional annotation

Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed by topGO and clusterProfiler, respectively, and a corrected p‐value < 0.05 was used as the screening condition for significant functional enrichment.

2.15 Statistical analysis

All values are expressed as mean ± standard error (SE). A two‐tailed Student's t‐test was performed for comparisons between two independent groups. A one‐way analysis of variance (ANOVA) followed by a post hoc Tukey test was performed to compare more than two independent groups. All statistical analyses were performed using GraphPad Prism (version 9, GraphPad software, Boston, MA, USA). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 indicated significant differences.

3 RESULTS

3.1 Characterization of pRL‐OMV

In this study, pRL was functionalized with OMV for improved subunit vaccine performance. OMVs were derived from the supernatant of cultured Bb and fused onto the surface of pRL. The average size of OMV and pRL, as determined by NTA, was 114 ± 2.14 and 109.8 ± 0.82 nm, respectively. After pRL was combined with OMV, its size increased to 122.4 ± 3.23 nm (Figure 1a). The zeta potential of pRL, OMV, and pRL‐OMV, measured by NTA, were −24.47 ± 0.95, −22.95 ± 1.34, and −36.94 ± 1.31 mV, respectively (Figure 1b). During the 5 weeks of storage at 4°C, the size of the OMVs increased to 176 nm, approximately a 55% increase. Under the same conditions, the size of the pRL‐OMV formulation increased by less than 9% (from 122.4 to 130.7 nm). The size of pRL increased by 19% (from 109.8 to 130.7 nm). The morphology of fresh prepared pRL‐OMV and OMV were compared with those stored at 4°C for 1 year by TEM (Figure S2). After 1‐year storge, the size of the single OMV were more than doubled, and the spherical surface was no longer as smooth as the fresh one. While there was a slight accumulation of pRL‐OMV, and the particle size of pRL‐OMV showed a slight increase. These results suggested that the presence of a pRL core within the OMV improved morphological stability under conditions of prolonged storage.

FIGURE 1 Characterization of synthesized pRL‐OMV. (a) Size distribution of pRL, OMV and pRL‐OMV analysed by nanoparticle tracking analyser. (b) Zeta potentials of pRL, OMV and pRL‐OMV. (c) The average size of pRL with or without OMV coating compared to that of free OMV over 5 weeks of storage. (d) Representative TEM images of pRL, OMV and pRL‐OMV.

The morphology of pRL, OMV and pRL‐OMV formulations were observed using TEM. The pRL alone appeared as a spherical particle with uniform size. The OMV possessed a bilayer structure, and the pRL‐OMV complex demonstrated a uniform structure consisting of concentric circles (Figure 1d). The protein components of pRL‐OMV and OMV were analysed by SDS‐PAGE (Figure S3), by which the membrane coating was further confirmed. The result showed that the protein composition of OMV was preserved after coating with pRL.

3.2 Cellular proliferation, uptake, and cytokine secretion properties of pRL‐OMV in vitro

DCs are the principal antigen‐presenting cells (APCs) and are pivotal cells for initiation and regulation of an adaptive immune responses, which is required to pathogen clearance and long lasting immune protection (Moran et al., 2018). In this study, DC2.4 cells were stimulated with pRL‐OMV, RL‐OMV, BL‐OMV, RGP‐OMV, OMV or LPS for 24 h. The effect of different OMV formulations on DC2.4 proliferation was evaluated by CCK‐8 assay. The OD450nm was used as the index for cell proliferation. The results demonstrated that pRL‐OMV induced significantly greater cell proliferation than other formulations delivered at equivalent concentrations. When the three highest concentrations were applied (25 + 2.5, 50 + 5, and 100 + 10 µg/mL), the OD value of pRL‐OMV group was more than 1.5 times that of the OMV and LPS groups (Figure 2a). When comparing pRL‐OMV with RL‐OMV, it was found that PEGylation on the RL significantly enhanced the proliferation effect (Figure 2a).

FIGURE 2 pRL‐OMV increased cell viability and cytokine production of DCs. (a) DC2.4 cells treated with increasing concentrations of different OMV formulations for 24 h. Cell viability was measured by the CCK‐8 method. (b) TNF‐ α, IL‐1β, IL‐12p70 and IL‐6 production of DC2.4 were evaluated after co‐culture with different OMV formulations for 24 h. (c) Confocal images of OMV uptake by DC2.4 cells treated with different DiI‐labelled OMV formulations for 4 h. The nucleus and lysosomes were stained with DAPI (blue) and Lysotracker (yellow), respectively. Scale bar, 20 µm. (d) The cellular uptake of DiI‐OMV was quantified by flow cytometry. (e) The quantitative data of the flow cytometry results. Data were expressed in the form of mean ± SE (n = 6). ns: not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Cytokine secretion plays a pivotal role in the development of the immune response, and certain cytokines are also markers of DC activation. The concentration of several cytokines in the supernatant of DC2.4 treated with different OMV formulations was examined, including TNF‐α, IL‐1β, IL‐6 and IL‐12p70. Based on the CCK‐8 test, the concentration used was 50 µg/mL RGP and 5 µg/mL OMV, under which pRL‐OMV induced highest proliferation level of DC. These findings demonstrated that the pro‐inflammatory cytokine secretion triggered by pRL‐OMV was greater than that of any other formulation, suggesting that treatment with pRL‐OMV induced the highest degree of DC activation (Figure 2b).

Nano‐vaccines are generally designed to be effectively taken up by APCs in order to stimulate host immune responses, rather than to release antigen into the extracellular environment (Nevagi et al., 2019). Here, the efficiency of antigen uptake by DCs was evaluated after treatment with one of the aforementioned formulations: pRL‐OMV, RL‐OMV, BL‐OMV, RGP‐OMV or OMV. OMV was labelled with a fluorescent dye (DiI), and then its cellular uptake into DCs was evaluated by confocal microscopy and flow cytometry. Confocal imagery revealed that pRL‐OMV mainly aggregated within the DC lysosome, whereas OMV alone localized only partially to the lysosome, and the remainder was scattered in the DC cytoplasm (Figure 2c). The images demonstrated that pRL‐OMV, unlike OMV, could be effectively phagocytosed by DCs. Supporting this visual evaluation, the phagocytosis ratio of pRL‐OMV was significantly higher than that of the other groups subjected to the same incubation conditions (Figure 2d,e). A potential explanation for these findings is that a stable structure could be helpful in promoting uptake by APCs (Wu et al., 2019). It is also likely that PEGylation improved uptake efficiency because of its high hydration capacity, which is also consistent with our previous research (Huang et al., 2021).

3.3 BMDC maturation induced by pRL‐OMV

DCs are the crucial APC for priming naïve T lymphocytes, and therefore form a bridge between the innate and adaptive immune response. As a sentinel‐like immune cell, DCs constantly take up and present antigen to T cells. During the process of antigen presentation DCs become activated, a phenomenon that is characterized by a rapid increase in surface expression of co‐stimulatory molecules (Walsh & Mills, 2013). As OMVs can serve as an antigen capable of eliciting DC responses, we next evaluated the immunological effects of different OMV formulations on the maturation of bone marrow‐derived dendritic cells (BMDCs). Flow cytometry revealed that pRL‐OMV significantly upregulated the surface expression of MHC II, CD86 and CD80 in BMDCs (Figure 3a–d). Additionally, scanning electron microscopy (SEM) was employed to identify surface morphological changes of BMDCs under the different treatments. Under the SEM, pRL‐OMV treated BMDCs had more elongated and denser synapses compared to the other treatments (Figure 3e), which showed a good effect on the maturation of DCs.

FIGURE 3 pRL‐OMV induced DC maturation in vitro. (a–c) Expression levels of (a) MHC II, (b) CD86 and (c) CD80 in BMDCs treated with pRL‐OMV, RL‐OMV, BL‐OMV, RGP‐OMV, OMV, LPS and medium for 24 h. (d) Quantitative data from the results of flow cytometry. Data are expressed in the form of mean ± SE (n = 4). ns, not significant; *p < 0.05. (e) Surface topographical changes were documented by SEM after pRL‐OMV treatment. Scale bar, 5 µm.

3.4 Bb‐specific antibody responses were induced by pRL‐OMV

Further investigation was performed to evaluate the ability of the nano‐vaccine to induce antigen‐specific immunity in vivo. Following the experimental timeline (Figure 4a), mice were vaccinated with 100 µL vaccine (1 µg OMV and 0.5 mg RGP in different vaccine formulations) on day 0 and given a single equivalent booster dose on day 14. Anti‐OMV and anti‐Bb specific IgG in serum were detected by indirect ELISA. Additionally, OMV‐ and Bb‐specific IgG1 and IgG2a were further evaluated as specific indicators of Th2‐ and Th1‐biased immune responses, respectively.

FIGURE 4 pRL‐OMV induced a strong antigen‐specific IgG immune response and a potent Th1/Th2/Th17 mixed cellular response. (a) Vaccination schedule of BALB/c mice (n = 6) subcutaneously immunized twice using different OMV formulations. Serum was collected for antibody detection via ELISA 2, 4 and 6 weeks after the second vaccination. OMV‐specific IgG (b), IgG1 (c), IgG2a (d) and Bb‐specific IgG (e), IgG1 (f), and IgG2a (g) were measured. (h) Levels of Th1 (IFN‐γ and IL‐12p70), Th2 (IL‐4 and IL‐5) and Th17 (IL‐17 and TNF‐α) cytokines in the serum of immunized mice were analysed. (i) Vaccination and sampling schedule of mice receiving immunization and booster of different OMV formulations. Four weeks after the second vaccination, splenocytes and blood were collected. (j) The proliferation rate of ConA/LPS/OMV‐stimulated lymphocytes from the spleen of immunized mice. (k) Concentration of various cytokines detected in the splenocyte culture supernatant. (l) Changes in circulating immune cells in each group 6 weeks post‐immunization. Data are expressed in the form of mean ± SE (n = 6). ns: not significant; *p < 0.05, **p < 0.01, ***p < 0.001.

As shown in Figure 4b–d, during the 6 weeks post‐immunization the concentration of anti‐OMV IgG, IgG1 and IgG2a gradually increased, demonstrating the OMV formulations elicited a strong antibody response in vivo. The concentration of anti‐OMV IgG and IgG2a in the serum of mice receiving pRL‐OMV was significantly higher than that in other groups, except for Alum‐OMV (Figure 4b,‐d). As for anti‐OMV IgG1, pRL‐OMV elicited a significantly greater response than most other groups, except RL‐OMV and Alum‐OMV (Figure 4c). ELISA also revealed that over time, mice immunized with pRL‐OMV developed robust IgG response to Bb. Six weeks post‐vaccination, Bb‐IgG and IgG1 responses elicited by the pRL‐OMV group were significantly increased compared to other groups, whereas at 2–4 weeks post‐immunization, the differences were not significant (Figure 4e,f). A similar delayed trend was observed with Bb‐IgG2a; the pRL‐OMV group developed a significantly greater Bb‐IgG2a response than Alum‐OMV and OMV at 6 weeks post‐vaccination, while the differences between them at the first two time points were not significant (Figure 4g).

Besides, the safety profiles of pRL‐OMV were also evaluated during the immunization. The body weight of mice immunized with pRL‐OMV increased gradually before and after immunization, and the difference was not significantly compared with the blank control (BC) group (Figure S4). Serum biochemical tests were done 5 days post immunization, and the results showed that there were no significant differences between pRL‐OMV and BC group (Figure S5).

The robust antibody response in vivo is indicative of an enhanced bacteria‐specific B cell response, which has been shown to contribute to protective immunity (Huang et al., 2021; Sahu et al., 2020). Safety is a necessary and important attribute of vaccines. Taken together, these findings provided strong support for the conclusion that pRL combined with OMV could effectively induced a robust Bb‐specific antibody response in vivo with a safety profile.

3.5 Th1/Th2/Th17 cytokine production and cell proliferation were increased by pRL‐OMV in vivo

Up to this point, we had established that vaccination with the pRL‐OMV formulation was effective at inducing B cell‐mediated antibody responses in vivo. Next, to determine the effect of pRL‐OMV immunization on T‐cell responses, levels of Th1 (IFN‐γ and IL‐12p70), Th2 (IL‐4 and IL‐5) and Th17 (IL‐17 and TNF‐α) cytokines in the serum of immunized mice were analysed. Four weeks post‐vaccination, mice immunized with different OMV formulations demonstrated increased cytokine production. Indeed, the results from analysis of IgG1 and IgG2a production already indicated that pRL‐OMV could effectively induce Th1 and Th2 immune responses. The resultant secretion of Th1‐ and Th2‐associated cytokines supported these previous findings. IFN‐γ and IL‐12p70, indicators of the Th1 cellular response (Wang et al., 2014), were observed to be significantly higher following vaccination with pRL‐OMV compared to that of the Alum‐OMV and OMV control groups (Figure 4h). Similar results were discovered in IL‐4 and IL‐5 production (Figure 4h), which are collectively indicative of the Th2 response (Piao et al., 2023). Finally, it was also found that pRL‐OMV significantly increased the production of IL‐17 and TNF‐α (Figure 4h), which are classically associated with a Th17 response (Yero et al., 2023).

The spleen is home to B and T lymphocytes, which are mainly involved in humoral and cellular immunity. Given this, splenic lymphocyte proliferation can indirectly demonstrate an organism's immune status. In this study, on the 42nd day splenic lymphocytes from immunized and non‐immunized mice were isolated in suspension and stimulated with ConA, LPS and OMV to identify the effects of immunization upon lymphocyte production (Figure 4i). Mice immunized with pRL‐OMV possessed a significantly higher lymphocyte proliferation rate compared to other groups (Figure 4j). As Bb is a respiratory pathogen, the lung lymphocyte differentiation was evaluated though flow cytometry. The data showed that after immunization, the ratio of CD4/CD8 of pRL‐OMV was significantly higher than that of RL‐OMV, Alum‐OMV and BC group (Figure S6).

In addition, the presence of IFN‐γ, IL‐4, IL‐6 and IL‐17 was detected in the supernatant of OMV‐stimulated splenocytes isolated from different test groups. Splenocytes separated from mice immunized with pRL‐OMV demonstrated a significantly and globally increased cytokine secretion, which was collectively consistent with the changes in serum cytokines from the same group (Figure 4k). Results from routine blood tests taken at the same time point confirmed that pRL‐OMV immunized mice produced a significantly higher concentration of circulating white blood cells (WBC), lymphocytes (SCC), neutrophils (LCC) and monocytes (MCC) (Figure 4l).

Taken together, pRL‐OMV effectively increased lymphocytic proliferation, which is necessary for initiating both humoral and cellular immunity. Importantly, pRL‐OMV could effectively stimulate a potent Th1/Th2/Th17 mixed cellular response, which is one of the major features of a protective immune response against bacterial infection (Ali et al., 2023; Raeven et al., 2020).

3.6 Targeting and activation of lymph nodes by pRL‐OMV in vivo

Targeting DCs in the lymph nodes (LNs) can effectively enhance the antigen‐specific immune response induced by a vaccine (Jiang et al., 2017). Therefore, we next evaluated the ability of pRL‐OMV to target the LNs and activate LN‐associated DCs. OMV formulations were first fluorescently labelled with Cy5.5, and then the labelled vaccine was injected subcutaneously into mice. Forty‐eight hours later, organs including the heart, liver, spleen, kidney, and inguinal LNs were collected and imaged. Simultaneously, the inguinal LNs were sectioned, stained, and imaged via confocal laser scanning microscopy (CLSM).

By 48 h post‐vaccination, there was a clear global accumulation of fluorescence in the inguinal LNs of pRL‐OMV‐injected mice (Figure 5a). Simultaneously, pRL‐OMV showed reduced the accumulation of fluorescence in the liver compared to OMV‐OVA‐Cy5.5 (Figure 5a). Upon sectioning and imaging via CSLM, it was confirmed that OVA‐Cy5.5 labelled pRL‐OMV (pRL‐OMV‐OVA‐Cy5.5) demonstrated stronger fluorescence in LNs than any other group examined (Figure 5b). Efficient accumulation of OMVs into the LNs can likely be attributed to the small size of the pRL‐OMV formulation, as well as to their natural immunogenicity (Cheng, Zhao et al., 2021).

FIGURE 5 pRL‐OMV increased the enrichment of antigen in inguinal lymph nodes and activated lymph node‐associated DCs. (a) Lymph node accumulation of OMVs labelled with OVA‐Cy5.5 in vivo (n = 3). The inguinal draining lymph nodes, along with several organs, were collected 24 h after subcutaneous injection with the indicated OMV formulations to examine the accumulation of Cy5.5 fluorescence. (b) After ex vivo imaging, the inguinal lymph nodes were collected, frozen, and sectioned and stained for fluorescent imaging via CSLM. Cell nuclei are shown stained in blue (DAPI). Scale bar, 50 µm. (c) Expression of MHC I, MHC II, CD80 and CD86 on DCs in inguinal lymph nodes 7d following the second immunization (n = 4). LNs were collected and processed into a single‐cell suspension. Following this, cells were analysed by flow cytometry. (d) Quantitative data derived from the flow cytometry results. Data are expressed in the form of mean ± SE (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001.

Efficient delivery of vaccines into the lymph nodes is only the first step of inducing a potent immune response. The vaccine must also be efficiently taken up by, and strongly activate, key APCs. Therefore, we further examined the activation of DCs within the LNs.

As shown in Figure 5c and d, pRL‐OMV elicited a significant increase in the proportion of CD80+, CD86+, MHC II+ and MHC I+ DCs in the LNs compared to that of the RL‐OMV and Alum‐OMV groups, confirming that pRL‐OMV was capable of inducing a strong adaptive immune response.

3.7 Evaluation of the ability of pRL‐OMV to prevent Bb bacterial infection in vivo

Assessment of protective immunity is considered essential for the development of an efficient subunit vaccine (Sadeghi et al., 2020). The infection prevention capacity of OMV formulations was therefore evaluated through tests of in vivo immunity following Bb challenge (Figure 6a). The bactericidal activity of serum from pRL‐OMV immunized mice was significantly greater than that of the alum‐OMV and OMV groups, indicating that pRL improved the anti‐infective properties of vaccination by OMVs with or without alum (Figure 6b). Indeed, the serum from animals immunized with the pRL‐OMV formulation outpaced the bactericidal rate even of RL‐OMV, indicating that PEGylation could contribute to an overall better adjuvant (Figure 6b).

FIGURE 6 Immunization with pRL‐OMV prevented pulmonary Bb colonization in vivo. (a) Vaccination and sampling schedule of mice receiving the previously established standard immunization schedule of different OMV formulations. Six weeks after the last vaccination, the serum of immunized and non‐challenged mice (n = 4) was collected for in vitro bactericidal assay against plated Bb colonies (refer to Methods 2.12 for rate formula). In a separate cohort of mice (n = 5), two weeks after the second vaccination, mice were subjected to a complement‐mediated Bb challenge (6.0 × 106 CFU/mouse), and the lungs were collected 7 days later for analysis of in vivo bactericidal activity. (b) Bactericidal activity of the serum collected from immunized mice. (c) The number of Bb CFUs/mL colonized within the lungs of immunized and naïve mice. Data are expressed in the form of mean ± SE. *p < 0.05, **p < 0.01, ***p < 0.001.

Colonization of bacteria within host tissues and organs facilitates the evaluation of the protective effect of vaccines (Fathi et al., 2023; Halder et al., 2023). Accordingly, the population density of bacteria invading the lungs of immunized mice following Bb challenge was analysed. As expected, the lungs of mice in the BC group were heavily colonized after Bb challenge (Figure 6c). In contrast, the lowest pulmonary colonization was detected in pRL‐OMV immunized mice, followed by the RL‐OMV group (Figure 6c). As an additional measure of immune protection, splenic lymphocyte differentiation after Bb challenge was evaluated by flow cytometry. The ratio of CD4+/CD8+ elicited from the pRL‐OMV group was significantly higher than that of the alum‐OMV and all other OMV groups (Figure S7). CD19+ splenic lymphocytes demonstrated a similar trend, with the pRL‐OMV group producing significantly greater differentiation than RL‐OMV, alum‐OMV and other OMV groups (Figure S7). Taken together, these findings suggest that immunization with pRL‐OMV induced a strong protective immune response against Bb infection.

3.8 Several immunity‐related pathways were enriched in pRL‐OMV immunized animals

To explore the underlying mechanism of how pRL‐OMV affected the murine immune system, transcriptome sequencing was conducted. A total of 3,072 differentially expressed genes (DEGs) were identified, and the clustering results of DEGs are shown in Figure 7a. A volcano plot of the DEGs expressed in two directionalities (i.e., upregulated or downregulated) is pictured in Figure 7b; this plot intuitively reflects the distribution and statistical significance of the identified DEGs. Compared with the BC group, 1318 genes were upregulated and 1,754 genes were downregulated in pRL‐OMV group (Figure 7b). Furthermore, KEGG pathway enrichment analysis was performed to obtain information about the functional annotation of DEGs. As shown in Figure 7c, DEGs were highly correlated with pathways involved in T cell receptor signalling, Th1 and Th2 cell differentiation, Th17 cell differentiation, the intestinal immune network for IgA production, primary immunodeficiency and hematopoietic cell lineage, all pathways known to be involved in immune regulation.

FIGURE 7 The immune protection function of pRL‐OMV was associated with changes in the expression of genes responsible for regulating various immune‐related pathways. (a) Clustered heatmap of differentially expressed genes expressed in splenocytes from mice (n = 3). Mice were immunized twice at a 2‐week interval with pRL‐OMV and PBS. The conditions for screening significantly different genes were p‐value < 0.05 and |log2FoldChange|≥ 1. Red represents increased expression, whereas blue represents decreased expression. (b) Volcano plot of DEGs. Red dots indicate genes that were significantly upregulated, while the blue dots represent genes that were significantly downregulated. Values on the x‐axis and the y‐axis are log2 fold change (log2FC) differences in the gene expression and ‐log10‐corrected p values (‐log10(pValue)), respectively. (c) Scatterplot of KEGG pathways enrichment analysis. The x‐axis represents the value of the gene ration, while the y‐axis represents the enrichment term of the pathway. The size of the dot represents the number of DEGs, and the dot colour represents an adjusted p value. (d) Bar plot of GO functional enrichment analysis. GO terms with a corrected p value < 0.05 were considered significantly enriched by DEGs. The x‐axis represents the enrichment term of the pathway, while the y‐axis represents the percentage of genes enriched.

To further explore the potential relationship between pRL‐OMV vaccination and these differential proteins, bioinformatics analysis was also performed (Figure 7d). GO annotation classification and GO enrichment analysis revealed that the biological process of these differential genes was associated with signal transduction, the immune system process, the response to bacterium, and B cell receptor signalling pathways, among others. The cellular association of the DEGs correlated with the membrane, cytoplasm, nucleus, and plasma membrane. The molecular function was mainly related to protein binding, metal ion binding and transferase activity. Among these DEGs, the expression levels of Slc4a1, Gfap, Gypa, Spta1, Pklr, Cyp26b1, Cnga1, Scd4 and Chrna6 were verified by qRT‐PCR (Figure S8, Table S1). These genes were confirmed to be upregulated or downregulated, consistent with the RNA sequencing data.

4 DISCUSSION

Bacterial infection is one of the leading causes of death globally (Antimicrobial Resistance, 2022; Xuan et al., 2023). The ever‐increasing number and global distribution of pathogens resistant to antimicrobial drugs has caused a major global health challenge (Wang et al., 2020). Antibacterial vaccines are becoming increasingly important for addressing the challenges in bacterial infections because they are capable of killing germs without using antibiotics. Prophylactic use of antibacterial vaccines, for example, could dramatically decrease rates of infection and illness caused by microbial pathogens by inducing humoral and/or cellular immunity. Importantly, novel nano‐platforms have recently provided new toolsets to design ground‐breaking strategies against bacterial infections, which is accelerating antibacterial vaccine development (Baker et al., 2018; Lahiri et al., 2022).

OMVs are outer membrane spherical buds with a diameter range of 30–200 nm that are ubiquitously generated by Gram negative bacteria (Richter et al., 2021). OMVs have gained tremendous interest in biomedical science due to their inherent immunogenicity and ability to deliver their cargo both locally and systematically to recipient cells (Xie et al., 2022; Xie et al., 2023). However, OMVs suffer from heterogeneity in size resulting in variable stability upon delivery. To address this and make uniformly sized and stable OMVs, here we generated a biomimetic nano‐system by coating OMVs on the surface of R. glutinosa polysaccharide nanocarriers (pRL) though repeated extrusion. The mechanical force provided by the extrusion is believed to disrupt the membrane structure and enable it to reform around the nanoparticulate cores. According to Zhang's report, OMV‐coated gold nanoparticles showed enhanced stability and generated stronger and durable antibody responses (Gao et al., 2015).

Nanoparticles coated with OMVs could also potentially achieve a high targeting efficiency. Qin and colleagues coated OMVs onto copper sulphide nanoparticles for systemic photothermal‐immunotherapeutic synergy. The resulting biomimetic nanoparticle exhibited good tumour targeting capacity, and elicited strong immunogenic cell death of tumour cells by promoting DC maturation and repolarizing M2‐like tumour‐associated macrophages into an M1‐like phenotype (Qin et al., 2022). Taken together, OMV‐coated nanocarriers could improve the immune effect of free OMVs by improving their stability and targeting efficiency. However, the type of nanoparticle chosen could affect the degree to which OMV properties are improved. Consequently, selection of the optimal nanoparticle core is vital.

According to our previous study, PEGylated nano‐R. glutinosa polysaccharide (pRL) improved the immune profile of nano‐R. glutionosa (Huang et al., 2021) and acted as a promising adjuvant. In this study, pRL was chosen as the nanoparticulate core to be coated by OMVs in an attempt to develop an ideal nano subunit anti‐bacterial vaccine. Strong vaccine‐based protection against infection and transmission requires induction of adaptive immunity, which involves antigen presentation to APCs, processing by APCs, and APC activation (Guo et al., 2021; Yan et al., 2020). DCs as a key type of APCs, internalize antigen from their surroundings and present it through MHC I/II, leading to either naïve CD8+ or CD4+ T cell proliferation and activation (Dacoba et al., 2017). DCs thus form a functional bridge between the innate and adaptive immune system.

PEGylation and cell membrane coating are both considered active targeting strategies. PEGylation is a bottom‐up approach that cannot meet the requirements of large batch production, whereas cell membrane coating, a top‐down approach, is a relatively facile technique (Dash et al., 2020). In this study, we designed a pRL‐OMV formulation consisting of a concentric circle structure which demonstrated superior stability to free OMVs (Figure 1). And also, it showed a good safety profile in vivo (Figures S4 and S5).

Further, the ability to activate APCs was studied both in vitro and in vivo. A potent vaccine must efficiently release and deliver antigen to APCs and then undergo effective APC‐directed processing. This process is accompanied by the maturation of APCs, a key example being dendritic cells (DCs), which is critical for eliciting a robust antigen‐specific immune response (Li et al., 2020). The findings of this study demonstrate that pRL‐OMV could promote DC proliferation and cytokine secretion, which is prerequisite for the potent evolution of adaptive immune responses (Zhong et al., 2019). After injection, pRL‐OMV was effectively localized to the draining lymph nodes (LNs) and induced DC maturation. These findings were substantiated by significantly increased accumulation of fluorescently labelled antigen in inguinal LNs and by increased expression of CD80, CD86, major histocompatibility complex I (MHC I) and MHC II (Figure 5). Consistent with the in vivo data, the in vitro data also showed that pRL‐OMV was effectively phagocytosed by DCs, processed in lysosomes, and then subsequently induced high expression of surface molecules such as CD80, CD86 and MHC II (Figure 3).

Lymphocyte proliferation is the first step in the immune response to create effector lymphocytes (Levin et al., 2020). T lymphocytes are mainly responsible for the cell‐mediated immune response, while B lymphocytes are responsible for humoral (antibody‐directed) immune response. Immunization of mice with the pRL‐OMV formulation significantly promoted splenic lymphocyte proliferation (Figure 4j) and increased CD4/CD8 ratio of the lung lymphocyte (Figure S6).

As mentioned before, a potent mixed Th1/Th2/Th17 immune response is one of the major features of protective immune response against bacterial infection (Ali et al., 2023; Raeven et al., 2020). We further detected Th1, Th2 and Th17 cytokines production. According to the results, pRL‐OMV immunization significantly increased the cytokine release of splenic lymphocytes, which was further consistent with the concentration of Th1, Th2 and Th17 cytokines in the serum of immunized mice (Figure 4h). In addition, the production of IL‐4 and IL‐17 in the splenic lymphocytes of pRL‐OMV group were significantly higher than those in Alum‐OMV group. Whereas the levels of IFN‐γ and IL‐6 in the two groups showed no significant difference (Figure 4k). It indicated that pRL was significantly superior to alum adjuvant in inducing Th2 and Th17 immune responses.

A dramatic increase in long‐lasting OMV‐ and Bb‐specific antibodies were also detected in pRL‐OMV group, which provided the most direct evidence that pRL‐OMV could effectively induce a long‐lasting bacterial‐specific antibody response (Figure 4). These findings also suggest that combining pRL with OMVs made OMV a better antibacterial subunit vaccine overall.

The anti‐Bb infection function of the subunit vaccine was further confirmed by the results of bactericidal assay following an in vivo Bb challenge. After Bb challenge, pRL‐OMV was proven to be effective against Bb infection (Figure 6). The bactericidal activity of serum from pRL‐OMV immunized mice was higher than other groups. These results provide strong evidence that pRL‐OMV could effectively induce Th1/Th2/Th17 mixed cellular responses and bacterial‐specific antibody responses against Bb infection.

RNA‐sequence based transcriptome analysis (RNA‐seq) allows a deeper understanding of complicated physiological pathways, including immune responses (Cui et al., 2019). Here, transcriptome sequencing was performed to investigate potential mechanisms underlying the immunological function of OMV‐based subunit vaccines (Figure 7). Thousands of DEGs were identified between the pRL‐OMV‐immunized group and blank control (BC) group. GO enrichment analysis revealed that pRL‐OMV significantly activated the GO terms associated with various immunological responses including signal transduction, the immune system process, the response to bacterium, and B cell receptor signalling pathways. KEGG enrichment analysis showed the DEGs were highly related to T cell receptor signalling pathways, Th1 and Th2 cell differentiation, Th17 cell differentiation, the intestinal immune network for IgA production, primary immunodeficiency and hematopoietic cell lineage (Figure 7). Vaccines against intracellular pathogens are usually designed to act through the Th1 pathway, whereas vaccines for extracellular organisms are aimed to predominantly stimulate the Th2 pathway (Nevagi et al., 2019). Activated Th1 cells produce cytokines, such as interferon‐γ (IFN‐γ), which are mainly responsible for the stimulation of cell‐mediated immunity. Th2 cells interact with B cells, which further differentiate into plasma cells and memory cells (Cai et al., 2021). Plasma cells in turn secrete antibodies designed against the invading antigen, whereas memory cells are responsible for inducing a faster and stronger immune protective response against future attacks. Th17 cells are key mediators of the host defence against airway infection by respiratory pathogens (Zhang et al., 2023). Host production of IL‐17 is essential for protection against several respiratory mucosal pathogens. The results of transcriptome analysis were consistent with the in vivo experimental results described above, and s that the pRL‐OMV formulation has potential for development as a potent subunit vaccine against bacteria.

5 CONCLUSION

In this study, a novel antibacterial strategy was explored by coating OMV onto pRL nano‐carriers. The pRL‐OMV formulation was constructed as a nano subunit vaccine to fight against Bb bacterial infection. The prophylactic effects of the subcutaneously‐delivered pRL‐OMV vaccine were investigated and substantiated. pRL‐OMV showed LN‐targeted delivery and effectively activated DCs, which was further supported by an increase in DC‐associated cytokine production and enhanced DC‐mediated phagocytosis. pRL‐OMV effectively promoted DC maturation, resulting in the induction of potent Th1/Th2/Th17 mixed cellular responses and bacterial‐specific antibody responses against Bb infection. The results of transcriptomic analysis demonstrated that the immune protection role pRL‐OMV played correlated with genetic regulation of T cell receptor signalling pathways, Th1 and Th2 cell differentiation, and Th17 cell differentiation. Altogether, our results provide implications for the prospective application of OMV‐coated nano‐carriers in antimicrobial immunotherapy.

AUTHOR CONTRIBUTIONS

Yee Huang: Conceptualization (equal); data curation (lead); investigation (lead); methodology (equal); writing—original draft (lead); funding acquisition (equal). Jiaying Sun: Investigation (equal); methodology (equal). Xuemei Cui: Investigation (equal); writing—review and editing (equal). Xuefeng Li: Investigation (equal); formal analysis (equal). Zizhe Hu: Investigation (equal). Quanan Ji: Methodology (equal). Yan Liu: Conceptualization (equal); investigation (equal); supervision (equal); writing—review and editing (equal); funding acquisition (lead). Guolian Bao: Data curation (equal); conceptualization (equal); methodology (equal); resources (equal); funding acquisition (equal); supervision (equal); writing—review and editing (equal).

CONFLICT OF INTEREST STATEMENT

The authors declares no conflicts of interest.

Supporting information

Supporting information

ACKNOWLEDGEMENTS

This project was supported by ‘Pioneer’ and ‘Leading Goose’ R&D Program of Zhejiang (grant number 2023C02047), National Natural Science Foundation of China (grant number 32002323, 32102714), China Agriculture Research System of MOF and MARA (grant number CARS‐43‐C‐2), Key Research and Development Program of Zhejiang Province (No. 2021C02007 and No. 2019C02052). We are grateful to all other staff in the Institute of Animal Husbandry and Veterinary Science for their assistance in this study. We also thank Xin Ma from Shiyanjia Lab (www.shiyanjia.com) for the TEM work, and LetPub (www.letpub.com) for linguistic assistance and pre‐submission expert review.
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