
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00727-2
10.1016/j.psj.2024.104148
104148
IMMUNOLOGY, HEALTH AND DISEASE
Exploiting membrane vesicles derived from avian pathogenic Escherichia coli as a cross-protective subunit vaccine candidate against avian colibacillosis
Zhu Dongyu *†
Zhang Yuting *
Wang Zhongxing *†
Dai Jianjun †
Zhuge Xiangkai zhugexk@ntu.edu.cn
*1
⁎ Department of Nutrition and Food Hygiene, School of Public Health, Nantong University, Nantong, Jiangsu 226019, China
† MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China.
1 Corresponding author: zhugexk@ntu.edu.cn
02 8 2024
10 2024
02 8 2024
103 10 10414814 4 2024
26 7 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Avian pathogenic Escherichia coli (APEC) is a notable pathogen that frequently leads to avian colibacillosis, posing a substantial risk to both the poultry industry and public health. The commercial vaccines against avian colibacillosis are primarily inactivated vaccines, but their effectiveness is limited to specific serotypes. Recent advances have highlighted bacterial membrane vesicles (MV) as a promising candidate in vaccine research. How to produce bacterial MVs vaccines on a large scale is a significant challenge for the industrialization of MVs. The msbB gene encodes an acyltransferase and has been implicated in altering the acylation pattern of lipid A, leading to a decrease in lipid A content in lipopolysaccharides (LPS). Here, we evaluated the immunoprotective efficacy of MVs derived from the LPS low-expressed APEC strain FY26ΔmsbB, which was an APEC mutant strain with a deletion of the msbB gene. The nitrogen cavitation technique was employed to extract APEC MVs, with results indicating a significant increase in MVs yield compared to that obtained under natural culture. The immunization effectiveness was assessed, revealing that FY26ΔmsbB MVs elicited an antibody response of laying hens and facilitated bacterial clearance. Protective efficacy studies demonstrated that immunization with FY26ΔmsbB MVs conferred the immune protection in chickens challenged with the wild-type APEC strain FY26. Notably, LPS low-carried MVs recovered from the mutant FY26ΔmsbB also displayed cross-protective capabilities, and effectively safeguarding against infections caused by O1, O7, O45, O78, and O101 serotypes virulent APEC strains. These findings suggest that MVs generated from the LPS low-expressed APEC strain FY26ΔmsbB represent a novel and empirically validated subunit vaccine for the prevention and control of infections by various APEC serotypes.

Key words

Avian pathogenic E. coli
membrane vesicles
nitrogen cavitation
subunit vaccine
cross-protection
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pmcINTRODUCTION

Avian pathogenic Escherichia coli (APEC) is a significant pathogen responsible for severe infections in poultry, leading to avian colibacillosis. The high incidence of avian colibacillosis presents a grave challenge to the global poultry industry, resulting in notable economic losses (Collingwood, et al., 2014; Wang, et al., 2017). The primary approach for prevention and treatment of avian colibacillosis has been the administration of antibiotics. The overuse of antibiotics has led to various complications, including the emergence of drug resistance. Consequently, vaccines might become the effective means for the prevention and control of this disease. Presently, killed E. coli vaccines, including autovaccines, have generally not achieved the anticipated outcomes (Li, et al., 2017). In 2006, the USA registered Poulvac E. coli, a live vaccine derived from an aroA-mutant strain EC34195 of serotype O78. Reports indicate that this vaccine can mitigate colibacillosis-like lesions, decrease total mortality, and enhance average daily weight gain in broilers under farm conditions (Christensen and Nielsen, 2020; La Ragione, et al., 2013). However, its effectiveness is limited to certain serotypes. This limitation emphasizes the urgent need for the development of cross-protective and broad-spectrum vaccines to combat the diverse serotypes of APEC.

Bacterial membrane vesicles (MV), also known as extracellular vesicles (EV), were initially defined as outer-membrane vesicles (OMV). As research progressed, the structure of MVs is not limited to a single outer membrane layer but can also encompass a bilayer structure with inner and outer membranes (Kaparakis-Liaskos and Ferrero, 2015; Jiang, et al., 2022). Thus, they are now uniformly referred to as bacterial membrane vesicles (MV) (Schwechheimer and Kuehn, 2015; Toyofuku, et al., 2019). These nanoscale spherical structures, with diameters ranging from 20 to 250 nanometers, are naturally released nanovesicles during bacterial growth (Palomino, et al., 2021). They carry a variety of biomolecules, including lipopolysaccharides (LPS), enzymes, and peptidoglycans (Schwechheimer and Kuehn, 2015; Hong, et al., 2019). Bacterial MVs play a crucial role in mediating communication both within and between species (Dhital, et al., 2021; Wang, et al., 2023b).

Bacterial MVs, due to their diverse bacterial content, non-replicative nature, and potent antigenic properties, are promising candidates for subunit vaccine development (Ghunaim, et al., 2014; Lee, et al., 2015; Treanor, 2020). MV-based vaccinations confer heterologous immunity, effectively extending protective responses across several serotypes within the same pathogenic species (Gnopo, et al., 2017; Zurita, et al., 2019). Nevertheless, the native secretion and subsequent purification of MVs from bacterial cultures typically result in low yields, posing significant challenges to their scalable commercial production (Liu, et al., 2016). Remarkably, recent advancements in microbiological methods have seen researchers achieve breakthroughs in generating membrane vesicles from P. aeruginosa via nitrogen cavitation method (Wang, et al., 2018). This technique stands to revolutionize the preparation process of vesicles, potentially enhancing their yield and applicability in commercial-scale productions.

Lipopolysaccharides (LPS), are critical component of the outer membrane of gram-negative bacteria, and also referred to as endotoxin. Its composition predominantly includes lipid A, core polysaccharide, and O antigen (Hagelueken, et al., 2015). Notably, lipid A constitutes the endotoxic segment of LPS, playing a crucial role in triggering immune responses. The release of LPS into a host initiates a potent immune reaction mediated by lipid A, characterized by symptoms such as fever, inflammation, and increased vascular permeability (Bishop, et al., 2000). The msbB gene in E. coli encodes a lipid A acyltransferase responsible for adding laurate (C12) to the lipid A component of lipopolysaccharide (LPS). Deletion of the msbB gene leads to a biochemical alteration wherein hexa-acylated lipid A is converted to its penta-acylated form. This molecular transformation results in a reduction of the endotoxicity (Kim, et al., 2009). LPS is a high-quality adjuvant that can enhance the efficacy of vaccines. However, high doses of LPS can induce strong immune responses, potentially causing severe damage to the organism (Vanaja, et al., 2016; Pfalzgraff, et al., 2019). Additionally, the development of subunit vaccines based on bacterial MVs encounters significant hurdles due to the predominance of non-functional antigens (especially lipopolysaccharides) in comparison to the scant presence of immunoprotective antigens (Hu, et al., 2020). Based on these issues, genetic engineering methods, such as the deletion of related genes, removal, addition, or alteration of bacterial outer membrane proteins and other components (van der Pol, et al., 2015), can be employed to reduce the endotoxin content in MVs while fully exposing surface antigenic components, thereby enhancing the immune effect of MVs vaccines.

In this study, we constructed an APEC msbB gene mutant strain (FY26ΔmsbB) with reduced LPS expression. In order to obtain a significantly higher yield compared to natural culture methods, the nitrogen cavitation technique was used to extract APEC MVs. We assessed the immunization effectiveness of these LPS-low MVs by evaluating their cross-protection against infections from heterologous strains, highlighting the broad-spectrum effectiveness of subunit vaccines based on APEC LPS-low MVs in preventing avian colibacillosis.

MATERIALS AND METHODS

Chickens and Housing

Beijing White 904 layer hen is a well-regarded breed in China, known for its high productivity and adaptability in egg production. Three-day-old layer lens were housed in isolators and provided with commercial feed and drinking water. All animal experiments conducted adhered to animal welfare standards and were granted approval by the Ethical Committee for Animal Experiments of Nanjing Agricultural University, Nanjing, China (permit number: NJAU.NO20230915136).

Bacterial Strains and Culture Conditions

The virulent APEC strains FY26 (O2 serotype), Jnd25 (O1 serotype), CE-F-122 (O7 serotype), CE-F-140 (O45 serotype), CVCC1553 (O78 serotype), and E125 (O101 serotype) were used to infect chickens to evaluate the cross-protective immunity of APEC MVs (Ge, et al., 2014; Zhuge, et al., 2019; Zhuge, et al., 2021). In order to acquire an APEC strain capable of producing MVs with low LPS content, the mutant strain FY26ΔmsbB was constructed using the λ-Red homologous recombination system (Datsenko and Wanner, 2000). The wild-type FY26 and its LPS low-expressed strain FY26ΔmsbB were grown on Luria-Bertani (LB) agar or in LB medium at 37°C.

Isolation and Purification of APEC MVs Produced From Bacterial Culture Supernatant

APEC MVs were prepared and purified as previously described (Jiang, et al., 2022). Initially, the bacteria were cultured in 1.0 L of LB medium for 12 h at 37°C with continuous shaking at 180 rpm. After incubation, the culture supernatant was separated by centrifugation at 10,000 × g for 10 min. This supernatant was then filtered twice using a 0.22 μm sterile filter to eliminate any residual bacteria. To collect the crude APEC MVs, ultracentrifugation was performed at 200,000 × g for 2 h at 4°C with a 50.2 Ti rotor (Beckman-Coulter), followed by resuspension in 1.0 mL of TE buffer. For further purification, the MVs were subjected to OptiPrep gradient centrifugation. The MVs were mixed with 45% OptiPrep medium and layered at the bottom of a gradient ranging from 25 to 40% OptiPrep (v/v). This mixture was then ultracentrifuged at 200,000 × g for 16 h at 4°C using an SW41 Ti rotor (Beckman-Coulter). Post-centrifugation, ten fractions were collected sequentially from the gradient's top to bottom. Fractions rich in MVs were combined and subjected to another round of ultracentrifugation at 200,000 × g for 2 h at 4°C with a 70.1 Ti rotor (Beckman-Coulter) to obtain the purified MVs. Finally, the MVs were resuspended in 1.0 mL of TE buffer, and their protein concentration was measured using the BCA Protein Assay Kit (Thermo Scientific).

Preparing APEC MVs Utilizing the Nitrogen Cavitation

MVs produced by FY26 and FY26ΔmsbB were isolated from bacterial culture supernatant or created by nitrogen cavitation, and we have previously reported on the extraction of naturally occurring APEC MVs. Here, we primarily focus on detailing the methodology for preparing MVs utilizing the nitrogen cavitation (Wang, et al., 2018). Briefly, bacteria were grown in 1.0 L LB broth for 12 h, followed by the collection of bacteria through centrifugation (at 8,000 × g for 10 min), and about 3.0×1012 CFU bacteria could be obtained. The bacterial pellet was resuspended in 500 mL of PBS and then subjected to a pre-cooled nitrogen cavitation device, specifically the Cell Disruption Vessel (920 mL vessel, 4635 series, Parr Instrument Company, Moline, IL), for cell disruption. To ensure the homogeneity of the suspension, a micromagnetic stirring bar was placed inside the nitrogen air pressurization device, which was subsequently positioned on a magnetic stirrer (Zhou and Philips, 2017). The device was gradually pressurized until the pressure gauge displayed an approximate reading of 1,500 PSI. Finally, all valves were closed and disconnected (Simpson, 2010). Following 2 repetitions of this process, the high purified MVs were acquired through the utilization of ultracentrifugation and OptiPrep density gradient centrifugation.

Transmission Electron Microscopy and Nanoparticle Tracking Analysis

The size and morphology of the MVs were characterized using TEM. Briefly, a copper grid was utilized to apply 5.0 µL of MVs solution, which was subsequently fixed and stained with a 1% aqueous solution of phosphotungstic acid for a duration of 90s. The samples were further examined using a Hitachi electron microscope (Model HT7800, Hitachi, Tokyo) at an accelerating voltage of 80 kV, and images were captured. The size distributions and concentrations of MVs were quantified using nanoparticle tracking analysis (NTA), as outlined in our previous study (Wang, et al., 2023b). For this purpose, 50 μL aliquots of EV solutions were subjected to NTA employing a ZetaView nanoparticle analyzer (Beijing ECHO Biotech, China).

Determination of LPS Content in APEC and its MVs

Lipopolysaccharides (LPS) were isolated from both the parental FY26 strain and its mutant counterpart utilizing the Non-Phenol LPS Extraction Solution (Catalog: 220508-5, BINGENE, Beijing, China) following the provided protocol. Subsequently, aliquots of 10 μg from each LPS sample were subjected to separation via 12% SDS-PAGE and visualized using the LPS PAGE Silver Staining Kit (Catalog: 220509-10, BINGENE) to examine the LPS profiles. The quantification of LPS extracted from bacteria or MVs was measured by the PyroGene Recombinant Factor C Endotoxin Detection Assay (Lonza, Basel, Switzerland), executed in alignment with the manufacturer's guidance (Liu, et al., 2019). Briefly, 100 µL aliquots of endotoxin standards, diluted LPS samples, or blank controls were allocated to designated microplate wells and subjected to a preliminary incubation phase of 10 min at 37°C. Subsequently, each well received 100 µL of the active reagent, comprising recombinant factor C enzyme, a fluorogenic substrate, and the assay buffer. Fluorescence measurements for each well were performed using a fluorescence microplate reader immediately and following the incubation at 37°C for 1 h, quantifying the optical density at 450 nm (OD450). LPS concentrations were deduced from a standard calibration curve and reported in international units (IU) per milliliter. This protocol was also applied to ascertain the LPS levels in APEC MVs. Replicability was ensured by conducting the procedure in triplicate across 3 separate experimental runs.

Cytotoxicity Assays

The cytotoxic impact of MVs on HD11 macrophage cell lines was evaluated using the Cell Counting Kit-8 (CCK-8; HY-K0301, MedChemExpress) following previously established procedures (Wang, et al., 2023b). In brief, HD11 cells at a density of 5 × 105 cells/mL were seeded into 96-well plates. The cells were then exposed to varying concentrations of MVs (25 or 50 µg/mL), and toxicity was assessed at 0, 1, 2, 4, 8, 16, and 24 h postexposure. After the exposure period, 10 µL of CCK-8 solution was added to each well and incubated for 3 h at 37°C. Following incubation, the optical density at 450 nm (OD450) was recorded to assess cell viability. The CCK-8 solution contains a water-soluble tetrazolium salt (WST-8) that is reduced by cellular dehydrogenases to produce a water-soluble formazan dye. The amount of formazan produced is directly proportional to the number of living cells, as only viable cells possess the active dehydrogenases required for this reaction. By measuring OD450, the amount of formazan can be quantified, providing an indirect measurement of cell viability. A decrease in OD450 indicates increased cytotoxicity, as it reflects a reduction in the number of viable cells.

Immunization and Enzyme-Linked Immunosorbent Assay

For the vaccination study, approximately 1,000 chickens were randomly allocated into vaccination groups of 200 chickens each and one PBS control group for 400 chickens. At about 1 wk of age (d 7), the chickens were received subcutaneous injections in the neck. The control group received 100 µL of PBS, while the vaccination groups received a 50 µg dose of MVs vaccine mixed with aluminum hydroxide adjuvant (1:1 volume ratio). The vaccines were prepared using MVs from natural production of FY26 and FY26ΔmsbB, and FY26ΔmsbB MVs created by nitrogen cavitation. Immunizations were administered on d 7 and d 21. Serum antibody titers were measured by ELISA on d 14 and d 28.

The antibody response was analyzed using an indirect ELISA protocol. In brief, 100 µL of FY26 whole bacteria (at a final protein concentration of 2.0 µg/mL per well) were used to coat 96-well plates (Thermo Scientific, Waltham, MA), followed by overnight incubation at 4°C. The plates were then washed 3 times with PBS containing 0.1% Tween 20 (PBST) and blocked with 1% bovine serum albumin (BSA) for 2 h the next day. Serum samples diluted in a 2-fold series were added to the wells as the primary antibody and incubated at 37°C for 1 h. After washing, the wells were incubated with sheep anti-chicken antibody conjugated with horseradish peroxidase (Bersee, Beijing, China), and the working dilution for the ELISA was 1:5000. Specific IgY antibodies were detected by adding a tetramethylbenzidine substrate, with absorbance readings taken at 450 nm using a microplate reader.

Immune Protection, Bacteria Colonization, and Cytokine Level Tests

Twenty laying hens from each immunized group were challenged intratracheally with wild-type FY26 (1.2 × 108 CFU/chicken, equal to the LD90 for 35-day-old chickens) on d 35 after the second immunization. To assess the protective ability of the vaccine, each group was challenged with the LD90 dose of wild-type FY26. This immune protection test was repeated 3 times. The survival rate of each group was monitored for up to 7 d and calculated using GraphPad Prism software 9.0. Staircase with ticks (Starting at 100%) was used as the model for the survival analysis.

Quantification of bacterial colonization in lungs and liver was performed for 15 chickens from each immunized group through colony counting 24 h after bacterial challenge. Cytokine levels were determined using commercial ELISA kits. Briefly, serum samples were collected from both PBS and MVs-immunized chickens 24 h post-challenge to measure proinflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α using the Chicken IL-1β ELISA Kit (Catalog No. BES0589K, BIOESN, Shanghai, China), Chicken IL-6 ELISA Kit (Catalog No. BES0608K, BIOESN), Chicken IL-8 ELISA Kit (Catalog No. BES0241K, BIOESN), and Chicken TNF-α ELISA Kit (Catalog No. F13235, YOYOBIO, Shanghai, China), following the manufacturer's instructions. The cytokine level for vaccinated chickens prior to challenge acted as the negative control.

Preparation of Pathological Tissue Sections

The chicken lung and liver tissues of all groups were fixed with formalin, embedded in melted solid paraffin, and sectioned after dehydration. The prepared pathological sections were stained with hematoxylin and eosin (HE) and then sealed for observation.

Cross-Protection Ability Testing

The LD90 of different serotype strains against laying hens was determined using various doses (ranging from 5.0 × 105 to 5 × 109 CFU). To determine the cross-protection ability, chickens of each immunized group were challenged with LD90 doses of bacteria. Each virulent strain of each serotype was evaluated separately. For each virulent strain, we challenged 10 chickens from each immunization group with an LD90 dose. This immunization assessment experiment was repeated twice. The survival rate of each group was monitored for up to 7 d.

Statistical Analyses

For analyses involving more than 2 groups, 1-way analysis of variance (ANOVA) was employed. The data are expressed as the mean ± Standard Error (SE) or Standard Error of the Mean (SEM) based on a minimum of 3 independent experiments. Statistical significance was established at P < 0.05, indicating by an asterisk (*). Data were statistically analyzed using GraphPad Prism software 9.0 or SPSS.

RESULTS

Isolation and Characterization of APEC MVs

To assess the effect of msbB gene deletion on bacterial growth, growth curves of the wild-type APEC strain FY26 and its LPS low-expressed strain FY26ΔmsbB were measured. Comparative analysis revealed no significant difference in the growth curves of FY26ΔmsbB relative to FY26 (Figure 1A). MVs from the FY26ΔmsbB strain were isolated via both natural production and nitrogen cavitation handling, respectively. Subsequent MVs purification was achieved using OptiPrep gradient ultracentrifugation. TEM analysis confirmed that APEC MVs produced via 2 distinct methods were spherical in morphology (Figure 1, Figure 1). NTA showed that the size distribution of MVs produced via 2 methods ranged from 50 to 200 nm, with no significant differences observed. However, FY26ΔmsbB MVs yield was notably higher in samples prepared by nitrogen cavitation compared to those obtained through natural production, and the concentration of the total MVs were approximately 1.32× 1014 particles/mL more than 1.15 × 1012 particles/mL for culture collection (Figure 1, Figure 1).Figure 1 Characterization of MVs derived from FY26ΔmsbB. (A) The growth curve of FY26 and its msbB mutant strain FY26ΔmsbB over 24 h. Data are the means of 3 biological replicates ± SD. (B) TEM image of FY26ΔmsbB MVs produced from bacterial culture supernatant and purified by density gradient centrifugation. Scale bars: 200 nm. (C) TEM image of FY26ΔmsbB MVs produced by nitrogen cavitation and purified by density gradient centrifugation. Scale bars: 200 nm. (D) Size distribution of FY26ΔmsbB MVs produced from bacterial culture supernatant determined with NTA. Data are the means of 3 biological replicates ± SD. (E) Size distribution of FY26ΔmsbB MVs produced by nitrogen cavitation determined with NTA. Data are the means of 3 biological replicates ± SD.

Figure 1

Deletion of msbB Gene Reduces its LPS Content in APEC and its MVs

The LPS of wild-type FY26 and the mutant FY26ΔmsbB were extracted, and SDS-PAGE and silver staining was performed to identify silver-stained LPS profiles. As shown in Figure 2A, compared with silver-stained ladder bands of the wild-type FY26, the LPS ladder bands in the mutant FY26ΔmsbB changed and the length of the LPS bands varied after the absence of the msbB gene. The production of lipopolysaccharides was evaluated in both the wild-type FY26 and its mutant FY26ΔmsbB. As shown in Figure 2B, a distinct discrepancy in LPS levels was observed when extracted from an equivalent bacterial load (1.0 × 108 CFU) of each strain, with the mutant FY26ΔmsbB demonstrating a notably reduced LPS yield compared to the wild-type FY26, reaching statistical significance (P < 0.05). LPS was similarly isolated from an identical quantity of MVs (∼ 1.0 × 1012), derived from both the natural production of wild-type FY26 and the mutant FY26ΔmsbB, and in addition to FY26ΔmsbB MVs produced via nitrogen cavitation. The LPS content in MVs from FY26ΔmsbB was significantly lower than that from the wild-type FY26 MVs (Figure 2C) (P < 0.05). Similarly, the lower content of LPS bands were also observed in the mutant FY26ΔmsbB and recovered by nitrogen cavitation relative to that in wild-type FY26 (Figure 2D).Figure 2 Determination of LPS content in APEC and its MVs. (A) The silver staining profiles of LPS from wild type FY26 and the mutant FY26ΔmsbB. (B) LPS level in the wild-type FY26 and its mutant FY26ΔmsbB were quantified using the PyroGene™ Recombinant Factor C Endotoxin Detection Assay. The differential LPS production was statistically evaluated using one-way ANOVA, denoting significance with * for P < 0.05. (C) LPS level in MVs derived from the wild-type FY26 and its mutant FY26ΔmsbB, and MVs recovered by nitrogen cavitation (NC) were quantified. (D) The silver staining LPS content from the MVs naturally produced by wild type FY26 and the mutant FY26ΔmsbB, and recovered by nitrogen cavitation for an identical quantity of MVs (∼ 1.0×1012).

Figure 2

MVs produced by APEC msbB mutant FY26ΔmsbB cause significantly milder cytotoxicity against macrophages

Macrophages play a crucial role in the immune system, acting as a primary defense mechanism against pathogens. In our previous study, we revealed that the cytotoxicity of MVs from the APEC wild-type strain FY26 on host cells is dose-dependents (Wang, et al., 2023b). In this context, our investigation focused on the potential of MVs recovered from LPS low-expressed strain FY26ΔmsbB to induce macrophage injury. Therefore, we selected 2 concentrations of APEC MVs to evaluate the cytotoxicity of MV. We exposed HD11 macrophages to various doses of MVs (25 or 50 µg/mL) for a 24-h period, subsequently assessing the MVs cytotoxic effects on these cells using the CCK-8 at intervals. As shown in the Figure 3A, after 24 h of MVs exposure, the cell viability of macrophages was approximately 46.2% at an FY26 MVs concentration of 25 µg/mL, and approximately 38.6% at an FY26 MVs concentration of 50 µg/mL (Figure 3B). The FY26 MVs at a concentration of 50 µg/mL exhibited stronger cytotoxicity towards macrophages (P < 0.05). As shown in the Figure 3A, after 24 h of MVs exposure, the cell viability of macrophages was approximately 74.2% at an FY26ΔmsbB MVs concentration of 25 µg/mL, and approximately 70.4% at an FY26ΔmsbB MVs concentration of 50 µg/mL. The difference in damage to macrophages between the 2 concentrations of MVs was not significant (P ≥ 0.05). This may be due to the deletion of msbB, which significantly reduces the LPS content in APEC MVs, resulting in less pronounced differences in the cytotoxicity of LPS low-carried MVs on macrophages compared to wild-type FY26 MVs. Conversely, MVs inherently produced by the FY26ΔmsbB mutant or those isolated from bacterial sediments of FY26ΔmsbB through nitrogen cavitation exhibited substantially reduced cytotoxic effects on macrophages (Figure 3, Figure 3).Figure 3 MVs derived from the FY26ΔmsbB mutant demonstrate diminished cytotoxicity towards macrophages. (A) HD11 macrophages were subjected to various doses of MVs from the wild-type FY26 and the mutant FY26ΔmsbB (25 µg/mL), and extracted from the bacterial whole cells of FY26ΔmsbB by employing nitrogen cavitation (NC) across a 24 h period. Viability of cells was evaluated via the CCK-8 kit, with data for each time point adjusted based on a control group that did not receive treatment. Data represent average values ± SEM from 3 distinct experiments. Statistical evaluations were performed using one-way ANOVA, indicating significance with * for P < 0.05. (B) Macrophages received treatments with 50 µg/mL concentration of APEC MVs.

Figure 3

MVs Collected From LPS Low-Expressed APEC Elicit an Antibody Response

To evaluate the antigenicity of LPS low-carried MVs, chickens were immunized with 50 μg doses of MVs derived from FY26 and FY26ΔmsbB on d 7 and d 21. Subsequent to immunization, serum samples were collected, and IgY titers were quantified on d 14, d 28, and d 35. ELISA result revealed that the level of antigen-specific IgY at d 14 was no significant change (Figure 4). Furthermore, a marked increase in IgY titers was observed at d 28 and d 35 relative to d 14 (Figure 4). Notably, chickens immunized with LPS low-carried MVs exhibited equally high antibody IgY titer relative to chicken immunized with wild-type FY26 MVs. Similarly, MVs isolated from FY26ΔmsbB through nitrogen cavitation exhibited high IgY titers (Figure 4). These results validate that MVs derived from both wild-type and mutant strains are capable of eliciting potent immune reactions in chickens postvaccination. The deletion of a particular genetic element, resulting in shortened LPS, preserves the immunostimulatory capability of APEC MVs. This modification markedly reduces the toxicity of APEC MVs, underscoring its viability as a safer vaccine candidate with efficacious immunogenic characteristics.Figure 4 Antibody response induced by wild-type FY26 and FY26ΔmsbB MVs. Chickens were immunized with 50 μg MVs naturally produced by wild type FY26 and the mutant FY26ΔmsbB, and FY26ΔmsbB MVs recovered by nitrogen cavitation (NC) on d 7 and d 21, respectively. At least 30 chickens are immunized in each immunization group, and at d 7 and 21, blood samples are randomly collected from 10 chickens in each group. Antigen-specific IgY titers in serum against FY26 whole cells were determined on d 14, d 28, and d 35, respectively.

Figure 4

The Immunization Efficacy of LPS Low-Carried MVs in Protection From APEC Infections

To determine the immunization protection of APEC MVs, 35 day-old laying hens (equal to immunized chickens at 14 d after the second immunization) were first challenged with wild-type FY26 (ranging from 5.0 × 105 to 5.0 × 109 CFU/chicken). The 90% lethal dose (LD90) for chickens was approximately 1.2 × 108 CFU for wild-type FY26. The laying hens from different immunized groups were challenged with this lethal dose 1.2 × 108 CFU/chicken, and the survival rates were observed over 7 d postinfection. The PBS control group experienced complete mortality within 7 d. However, chickens immunized with MVs collected from supernatant of wild-type FY26 and FY26ΔmsbB cultures exhibited significantly lower mortality rates, and protection rate of 90% in the wild-type FY26 MVs-immunized group and 95% in the FY26ΔmsbB MVs-immunized groups was detected (Figure 5) (P < 0.05). Similarly, the chickens immunized with MVs recovered from bacterial sediments of FY26ΔmsbB by nitrogen cavitation exhibited a high survival during the virulent APEC infection. This result provided strong evidence indicating that FY26ΔmsbB MVs immune protected chickens against wild-type FY26 infection with a lethal dose, highlighting its potential as a subunit vaccine candidate.Figure 5 Protective efficacy of APEC LPS low-carried MVs produced by FY26ΔmsbB against wild-type FY26 infection. Chickens were immunized with 50 μg MVs from wild-type FY26, LPS low-carried MVs naturally derived by FY26ΔmsbB, and FY26ΔmsbB MVs recovered by nitrogen cavitation (NC). The control group was immunized with PBS. After challenge with 1.2×108 CFU of wild-type FY26, the number of surviving layer chickens was monitored for 7 d.

Figure 5

Subsequent analysis of the bacterial loads in the lung and liver of infected chickens, the lungs and liver of chickens that did not receive the MV vaccine exhibited notable colonization following infection. In contrast, there was a significant reduction within the MVs-immunized groups compared to the control group (Figure 6A) (P < 0.05). There was no obvious presence of the bacterial loads observed in in the lungs and liver of chickens administered with MVs derived from wild-type FY26 and FY26ΔmsbB (P ≥ 0.05). Additionally, the levels of pro-inflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α in serum samples of infected chickens for the MV-immunized or PBS control groups have been quantified at 24 h post-bacterial infection. There was a notable decrease in the levels of IL-1β, IL-6, IL-8, and TNF-α compared to control group (Figure 6B) (P < 0.05). Conversely, there is no difference between FY26ΔmsbB and FY26 MVs. These results indicated that MVs with reduced LPS content contributed to the enhancement of protective immunity by diminishing both the bacterial load and the expression levels of pro-inflammatory cytokines, thereby mitigating the inflammatory response elicited by APEC infection.Figure 6 Post-infection bacterial loads and serum pro-inflammatory cytokine levels after immunized with wild-type FY26 and FY26ΔmsbB MVs. Chickens were immunized with 50 μg MVs naturally produced by wild type FY26 and the mutant FY26ΔmsbB, and FY26ΔmsbB MVs recovered by nitrogen cavitation (NC) on d 7 and 21 or with PBS as a control, and infected with 1.2×108 CFU of wild-type FY26 on d 35. Bacterial loads of chicken lungs and liver after 24 h post-infection were determined. (A) Bacterial colonization in lungs and liver for chickens immunized by wild-type FY26 MVs, LPS low-carried MVs naturally derived by FY26ΔmsbB, and FY26ΔmsbB MVs recovered by nitrogen cavitation (NC), respectively. Statistical test: one-way ANOVA, * indicting P < 0.05. (B) Serum levels of IL-1β, IL-6, IL-8, and TNF-α were determined after 24 h post-infection. Statistical test: one-way ANOVA, P < 0.05. Data are the means of 5 biological replicates ± SEM.

Figure 6

Pathological Changes of MVs-Immunized Chickens After Infection

After infection, various tissues (including lungs and liver) of MVs-immunized laying hens post 24 h infection were collected to conduct histopathological sectioning and subsequent analysis. After HE staining, lung and liver tissues from the negative control group infected with wild-type FY26 exhibited severe pathological alterations when examined under light microscopy. The infected lungs tissues displayed significant pathological changes, including pronounced thickening of the alveolar septa, vascular congestion, and infiltration by inflammatory exudates. Conversely, lung specimens from chickens administered with MVs-based immunotherapy presented with preserved architectural integrity, featuring patent and adequately aerated alveoli devoid of cellular infiltration, signifying a marked alleviation of pulmonary damage attributed to the immunization with APEC-derived MVs (Figure 7A). Notably, liver tissues in control chicken group displayed characteristic pathological features including nuclear shrinkage and fragmentation, and the bacteria were observed in the liver tissue (Figure 7B). Strikingly, none of such pathological changes were observed in the groups immunized by APEC MVs at the challenged dose 1.2×108 CFU/chicken. This indicates that high dose of 1.2 × 108 CFU attacks through the APEC natural infection pathway (respiratory tract infection) couldn't cause organ damage in the immunized chickens. This APEC MVs subunit vaccine showed good immune effects at an LD90 infection dose.Figure 7 Pathological changes in tissues from FY26 infected chickens immunized by APEC LPS low-carried MVs. (A) Pathological changes in lung tissues of chickens immunized with (a) negative control PBS, (b) FY26 MVs, (c) LPS low-carried MVs naturally produced by FY26ΔmsbB, and (d) FY26ΔmsbB MVs collected after nitrogen cavitation. The red arrows indicate pronounced thickening of the alveolar septa, while the black arrows indicate architectural integrity, featuring patent and adequately aerated alveoli devoid of cellular infiltration. (B) Pathological changes in liver tissues of chickens immunized with (e) negative control PBS, (f) FY26 MVs, (g) MVs for FY26ΔmsbB natural production, and (h) FY26ΔmsbB MVs collected after nitrogen cavitation. The red arrows indicate the presence of bacteria in the liver tissue, and the black arrows indicate intact hepatocytes without nuclear shrinkage and fragmentation.

Figure 7

Evaluation of Cross-Protective Capability of APEC LPS Low-Carried MVs Subunit Vaccine

To assess the cross-protective efficacy, the O1, O7, O45, O78, and O101 virulent APEC strains were employed to infect different MVs-immunized chickens. The cross-protective potential of MVs derived from wild-type FY26 and FY26ΔmsbB against diverse serotypes bacterial infections was evaluated. Initially, the 35 day-old laying hens were infected with varying doses as specified, and the LD90 of each strain was determined. The LD90 of APEC strains Jnd25 (O1), CE-F-122 (O7), CE-F-140 (O45), CVCC1553 (O78), and E125 (O101) for 35 day-old chickens was approximately 2.3×108 CFU, 8.5 × 107 CFU, 1.6×108 CFU, 2.1 × 108 CFU, 3.1 × 108 CFU, and 4.3 × 108 CFU, respectively. Subsequently, the MVs-immunized groups and negative control group were subjected to infection with different doses of APEC strains (O1, O45, O78, and O101 strains). The mortality of chickens in each group was observed within 7 d. The results revealed that all chickens in the PBS negative-control group succumbed to infection within 7 d, whereas the mortality rate in MVs-immunized groups exhibited a significant reduction (Table 1). Chickens immunized with MVs collected from the supernatant of FY26ΔmsbB cultures showed significantly lower mortality rates compared to chickens immunized by wild-type FY26 MVs (Table 1). Similarly, MVs recovered from bacterial sediments of FY26ΔmsbB by nitrogen cavitation to immunize the chickens held cross-protective capabilities against several virulent APEC strains with different serotypes.Table 1 Cross-protective efficacy of APEC LPS low-carried MVs.

Table 1Infetious strains\Vaccine type	Immune protection (survival rate, %) against APEC infection	
	Jnd25 (O1)	CE-F-122 (O7)	CE-F-140 (O45)	CVCC1553 (O78)	E125 (O101)	
PBS	10%	5%	5%	10%	10%	
FY26 MVs	55%	40%	45%	40%	35%	
FY26ΔmsbB MVs	85%	80%	75%	80%	75%	
FY26ΔmsbB MVs by nitrogen cavitation	85%	85%	85%	75%	85%	
(A) Chickens were immunized with 50 μg MVs from wild-type FY26, LPS low-carried MVs naturally derived by FY26ΔmsbB, and FY26ΔmsbB MVs recovered by nitrogen cavitation. The control group was immunized with PBS. After challenge with various serotypes of APEC strains at LD90 doses, the number of surviving chickens was monitored for 7 d.

DISCUSSION

While bacterial MVs subunit vaccine offers advantages over traditional live attenuated and inactivated vaccines in terms of safety and the induction of host immune responses, concerns regarding the potential harm posed by endotoxins packaged in MVs and challenges related to their production represent significant hurdles to their widespread adoption and use (Mancini, et al., 2020). The emergence of multidrug-resistant bacteria has made the control and prevention of animal diseases increasingly critical (Wang, et al., 2021; Wang, et al., 2023a). The development and application of new broad-spectrum and multivalent subunit vaccine have become one of the best options for controlling the occurrence of bacterial diseases in poultry. Bacterial MVs have gained significant attention as a promising vaccine platform, primarily due to their robust immunogenicity and non-replicative nature (Sartorio, et al., 2021). A notable example is the commercial production of MVs-based vaccines derived from N. meningitidis in several countries (Holst, et al., 2009).

In this study, we employed homologous recombination to delete the msbB gene, which is involved in LPS synthesis. The msbB gene encodes an acyltransferase and has been implicated in altering the acylation pattern of lipid A, leading to a decrease in lipid A content in LPS (Kim, et al., 2009). This reduction in lipid A is hypothesized to mitigate the endotoxic effect and enhance the safety profile of bacterial MVs for vaccine candidate. Furthermore, we performed nitrogen cavitation for the preparation of APEC MVs. Nitrogen cavitation utilizes high-pressure disruption to rapidly lyse bacterial cells (Figure 8). The resulting broken cell membranes form nanovesicles, encapsulating bacterial contents. The yield of APEC MVs obtained via nitrogen cavitation significantly surpasses that of traditional extraction methods. This demonstrated that nitrogen cavitation could enhance the efficiency of APEC MVs extraction, setting a foundation for the large-scale production of bacterial MVs (Wang, et al., 2018). Additionally, TEM analysis indicated that the size and morphology of APEC MVs obtained through nitrogen cavitation are comparable to those acquired via conventional methods. MVs derived from the LPS low-expressed APEC mutant FY26ΔmsbB were subsequently used to immunize chickens. This LPS low-carried MVs subunit vaccine held the capability to induce the production of anti-APEC antibodies in chickens. Our previous study has demonstrated that APEC-derived MVs can be internalized by macrophages, triggering immune responses, and thereby APEC MVs might elicit both humoral and cellular immunity (Wang, et al., 2023b). Moreover, the alterations in lipid A to reduced LPS content on membrane surface could not impede the ability of APEC MVs to induce antibody production in vivo (Pulido, et al., 2020), implying that LPS couldn't be the primary immunogenic component in bacterial MVs to defense against bacterial infections, although its contribution to the immune response is non-negligible.Figure 8 The main method steps for exploiting LPS low-carried MVs derived from APEC mutant strain FY26ΔmsbB as a cross-protective subunit vaccine candidate against avian colibacillosis. The APEC mutant strain FY26ΔmsbB was cultured for 16h, and then the bacterial cells were centrifuged and collected (Operation Step 1). The bacterial cells were treated with nitrogen cavitation to generating vesicles, and the MVs were purified to assess the LPS content and identify the molecular characterization (Operation Step 2). The immunogenicity and toxicity of LPS low-carried MVs was assessed to prepare this APEC MVs subunit vaccine (Operation Step 3). The chicken received subcutaneous injections into the neck with MVs subunit vaccine (Operation Step 4). The MVs-immunized chickens were infected with the O1, O2, O45, O78, and O101 virulent APEC strains, and the immunization efficacy of LPS low-carried MVs in protection from APEC infections was evaluated (Operation Step 5).

Figure 8

The incursion of pathogenic bacteria has been known to stimulate the release of inflammatory mediators (Lu, et al., 2020). This MVs subunit vaccine can offer protective immunity to chickens against bacterial infection. The efficacy of vaccines is a critical metric for assessing vaccine quality. Our findings demonstrated that APEC LPS low-carried MVs vaccine confer protection against lethal bacterial doses, underscoring their potential as effective vaccine candidate for avian colibacillosis. Post-immunization challenge with pathogenic bacteria revealed a significant reduction in bacterial load within the internal organs of chickens immunized with MVs from LPS low-expressed strain FY26ΔmsbB, accompanied by a marked decrease in the inflammatory level. This indicates that LPS low-carried MVs can enhance the host's capacity to eliminate pathogenic bacteria and mitigate the inflammatory response (Pulido, et al., 2020). LPS low-carried MVs vaccine derived from FY26ΔmsbB exhibits enhanced cross-protective capability against various serotypes of APEC compared to MVs vaccine from wild-type FY26. We speculated that this enhanced protection may be attributed to the blockage in lipid A synthesis, leading to a truncation of LPS. This shortening potentially allowed to fully disclosure membrane antigens on the surface of vesicles, thereby amplifying their immunogenicity and conferring robust protection against infection by different serotypes APEC virulent strains.

MVs offered more effective immunoprotection against current isolates and induced durable immunity compared to whole-cell vaccines (Gaillard, et al., 2014). A dual vaccine based on bacterial MVs against Influenza A virus H1N1 and MERS-CoV stimulates antibodies that could effectively protect mice from H1N1pdm09 and MERS-CoV infections (Shehata, et al., 2019), indicating that bacterial MVs can serve as carriers for presenting different viral antigens simultaneously. The recombinant N. meningitidis MVs subunit vaccine prepared by methods such as capsular gene deletion, LPS toxicity reduction, increased MV yield, and overexpression of fHBP v.1, has been considered as an affordable vaccine option to prevent infections by all major serogroups of Meningococcus causing meningitis in sub-Saharan Africa (Koeberling, et al., 2014). Moreover, Acinetobacter baumannii MVs vaccine has been reported to prevent mouse infections by A. baumannii (Pulido, et al., 2020). MVs as effective subunit vaccine have been shown to combat avian salmonellosis caused by Salmonella enteritidis (Li et al., 2020). MVs as candidate vaccines could avoid cross-infections among different APEC strains, among others. These studies indicate that MVs subunit vaccines are an important direction for the development of broad-spectrum bacterial vaccines and an effective means to prevent significant bacterial diseases in poultry under the context of “ban and restrict antibiotics”.

CONCLUSIONS

In summary, this study employs genetic engineering techniques to modify pathogenic E. coli in poultry, reducing the LPS content in membrane vesicles. To enhance the production of MVs from the mutant FY26ΔmsbB, we utilized the nitrogen cavitation to processing bacterial cells. Simultaneously, we conducted animal trials to assess the effectiveness of this FY26ΔmsbB MVs used as a subunit vaccine, and the findings indicated that this MVs subunit vaccine generated by FY26ΔmsbB exhibited a significant capacity for cross-protection against APEC strains of varying serotypes (Figure 8). Nitrogen cavitation allows for the mass production of nanosized bacterial vesicles at low cost, meeting commercial production requirements and suitable for use in large-scale poultry farms. The results obtained from these experiments establish a strong basis for future investigations into the potential use of MVs in cross-protective subunit vaccine development.

DISCLOSURES

The authors declare no conflicts of interest.

ACKNOWLEDGMENTS

This study was supported by the Jiangsu Province Key Research and Development Program (Modern Agriculture) Project (Grant No. BE2022329 ) and National Natural Science Foundation of China (Grant No. 32172855 and 31702252 ). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Data Availability Statement: All data generated or analyzed during this study are included in this published article.
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