Detoxified synthetic bacterial membrane vesicles as a vaccine platform against bacteria and SARS-CoV-2

Animals

Wild-type mice in the C57BL/6 genetic background (6 weeks old) were purchased from Charles River. The mice were maintained at the Experimental Biomedicine facility at the University of Gothenburg, Sweden. The study was approved by the local Animal Ethics Committee in Gothenburg, Sweden (Dnr 5.8.18–03598/2019), and was carried out according to institutional animal use and care guidelines.

Bacterial strains and cell culture

P. aeruginosa PAO1 (ATCC, Manassas, VA) and E. coli BL21 (DE3) (Thermo Fisher Scientific, Waltham, MA) were cultured in Luria-Bertani broth at 37 °C. MH-S (ATCC), a murine alveolar macrophage cell line, was maintained in RPMI 1640 medium (HyClone, Logan, UT) containing 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. The cells were cultured at 37 °C in an atmosphere of 5% CO2.

Isolation of natural OMV

Bacterial cultures were pelleted at 6,000 × g at 4 °C for 20 min, and the supernatants were applied to 0.45 μm vacuum filters to remove cell debris. The filtrate was concentrated with a Vivaflow 200 module equipped with a 100 kDa cut-off membrane (Sartorius, Goettingen, Germany). The concentrated solution was ultracentrifuged at 150,000 × g at 4 °C for 3 h to pellet the OMV, and the pelleted OMV were resuspended with PBS.

Preparation of SyBV

To collect bacterial outer membranes, the bacterial cultures were first pelleted and resuspended in 20 mM Tris-HCl (pH 8.0) with lysozyme (600 µg/g cells) and 0.1 M EDTA (0.2 mL/g cells). The resulting spheroplasts were sonicated, and total membranes were obtained by centrifuging at 40,000 × g for 1 h at 4 °C (Fig. 1a). The outer membranes were isolated by incubation with 0.5% Sarkosyl (Sigma Aldrich, St. Louis, MO) followed by centrifuging at 40,000 × g for 1 h at 4 °C. The outer membrane pellets were treated with high pH solution (200 mM Na2CO3 (pH 11)) to disrupt the membrane integrity. The open membranes were resuspended in 4 mL of 50% iodixanol (Axis-Shield PoC AS, Oslo, Norway) and applied onto a step gradient (4 mL of 30% iodixanol and 2 mL of 10% iodixanol) in a 14 mL ultracentrifuge tube (Beckman Coulter, Brea, CA). The tube was ultracentrifuged at 100,000 × g for 2 h at 4 °C to collect vesicles between the 10% and 30% iodixanol layer. Finally, the vesicles were mildly sonicated and considered as SyBV.

TEM

OMV and SyBV were analyzed by negative-stain TEM. The vesicles were applied onto glow-discharged formvar carbon-coated 200-mesh copper grids (Electron Microscopy Sciences, Hatfield, PA). After washing with water, the vesicles were fixed with 2.5% glutaraldehyde dissolved in PBS, followed by staining with 2% uranyl acetate for 1.5 min. Negative-stained bacterial vesicles were visualized using a LEO 912AB Omega electron microscope (Carl Zeiss SMT, Oberkochen, Germany) at 120 kV with a Veleta CCD camera (Olympus-SiS, Stuttgart, Germany).

Nanoparticle tracking analysis

OMV and SyBV (10 µg/mL) were measured using ZetaView® PMX 120 (Particle Metrix GmbH, Meerbuch, Germany). Measurements were done in triplicate, and each individual data point was obtained from two stationary layers with five measurements in each layer. The sensitivity of the camera was set to 70 for all measurements. Data were interpreted using ZetaView analysis software version 8.2.30.1 with a minimum size of 10, a maximum size of 1000, and a minimum brightness of 30.

RNA analysis

RNA from bacterial vesicles was purified with the miRCURY™ RNA isolation kit for biofluids (Exiqon, Vedbaek, Denmark) according to the manufacturer’s protocol. One microliter of isolated RNA was examined for its quality, yield, and nucleotide length by capillary electrophoresis using an Agilent RNA 6000 Nanochip on an Agilent 2100 Bioanalyzer® (Agilent Technologies GmbH, Berlin, Germany).

LC-MS/MS analysis

Two biological replicates of OMV and SyBV (30 µg of both) were digested with trypsin using the filter-aided sample preparation (FASP) method and C18 spin columns according to the manufacturer’s instructions [52]. All fractions were dried on a Speedvac and reconstituted in 3% acetonitrile and 0.2% formic acid and examined on an Orbitrap Fusion Tribrid mass spectrometer interfaced with an Easy-nLC 1200 (Thermo Fisher Scientific). Peptides were captured on an Acclaim Pepmap 100 C18 trap column (100 μm × 2 cm, particle size 5 μm; Thermo Fischer Scientific) and separated on an in-house packed C18 analytical column (75 μm × 30 cm, particle size 3 μm) using a gradient from 5.6 to 80% acetonitrile in 0.2% formic acid over 90 min at a flow of 300 nL/min. Precursor ion mass spectra were monitored at 120,000 resolution, and the most intense precursor ions were fragmented using HCD at a collision energy setting of 30. The MS/MS spectra were recorded at 30,000 resolution with a maximum injection time of 110 ms and an isolation window of 1.2 Da. Charge states 2 to 7 were selected for fragmentation, and dynamic exclusion was set to 20 s with 10 ppm tolerance.

Database search

Data were analyzed with Proteome Discoverer version 1.4 (Thermo Fisher Scientific). The database search was performed against the Swissprot P. aeruginosa database. Mascot 2.5.1 (Matrix Science, London, UK) was used as the search engine with a precursor mass tolerance of 5 ppm and fragment mass tolerance of 0.5 Da, and one missed cleavage was recognized, mono-oxidation on methionine was set as a variable modification, and methylthiolation on cysteine was set as a fixed modification. Percolator was used for the validation of the identification results with the strict target false discovery rate of 1%, and proteins were only accepted when identified in all replicates. Gene ontology analysis was performed using the Funrich analysis tool, and principal component analysis and hierarchical cluster analysis were performed with the ClustVis software. The mass spectrometry data has been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD033690.

Immunotoxicity assay

The immunotoxicity of the OMV and SyBV was compared with a cytokine release assay in MH-S cells. The cells were applied onto 24-well plates and two concentrations of OMV or SyBV were added, and pro-inflammatory cytokine release was measured 24 h later. The secretion of TNF-α and IL-6 in the supernatants was quantified by a DuoSet ELISA Development kit (R&D Systems, Minneapolis, MN). For the in vivo safety assay, mice were intraperitoneally injected with the same number of OMV or SyBV (5 × 109) and then sacrificed after 6 h following anesthetization with intraperitoneal injection of xylazine chloride (10 mg/kg; Bayer, Gothenburg, Sweden) and ketamine hydrochloride (100 mg/kg; Pfizer AB, Kent, UK). Rectal temperature was measured with a thermometer (Bioseb, Chaville, France). Blood was acquired by cardiac puncture and peritoneal fluid and BAL fluid were acquired, and after centrifugation the supernatants were maintained at − 80 °C for cytokine analysis.

TLR screening assay

Engineered HEK-293 cell lines expressing different murine TLR were purchased by InvivoGen (Toulouse, France). In case of TLR2, only single receptor was tested without heterodimer formation with TLR1 or TLR6. The receptors were linked with a reporter gene which is a secreted alkaline phosphatase. The activation of the reporter gene was mediated by a NF-ĸB inducible promoter, and then TLR activation results were determined as optical density values after 18 h stimulation of the cells with OMV or SyBV. As a positive control for activation of murine, the following ligands were used: TLR2 (PAM2), TLR3 (Poly I:C), TLR4 (LPS), TLR5 (Flagellin), TLR7 (R848), TLR8 (TL8-506), and TLR9 (ODN 1826).

Cell uptake experiment

OMV or SyBV were stained with DiO (Molecular Probes, Eugene, OR) for 1 h at 37 °C. MH-S cells labelled with Cellmask Deep Red (Thermo Fisher Scientific, Waltham, MA) were incubated with the DiO-labelled vesicles for 6 h. Flow cytometry was analyzed using a BD FACSVerse Flow Cytometer running BD FACSuit Software (BD Biosciences, San Jose, CA) and FlowJo Software (Tree Star Inc., Ashland, OR).

Immunization protocol and induction of pulmonary inflammation

Mice were intraperitoneally injected with 5 × 109P. aeruginosa-derived OMV or SyBV once a week for three weeks. For P. aeruginosa-induced lung inflammation, mice were intranasally challenged with 4 × 108 CFU of P. aeruginosa followed by collection of BAL fluid and lungs after 48 h. For lung histology, lungs were fixed with 4% paraformaldehyde, sectioned at 4 μm, and stained with hematoxylin and eosin. The images were acquired using the EVOS XL Core Imaging System (Life Technologies, Bothell, WA).

Immunization protocol for sepsis

Mice were intraperitoneally immunized with 5 × 109E. coli-derived OMV or SyBV three times every week. For E. coli-induced sepsis, immunized mice were intraperitoneally injected with E. coli (1 × 108 CFU), and survival was monitored every 3 or 12 h for 5 days. Rectal temperature and serum cytokines were measured 3 h after challenge with E. coli. For heat inactivation of SyBV, the vesicles were incubated for 20 min at 100 °C. And then survival rate of mice immunized with heat-inactivated SyBV (5 × 109) was determined for 5 days following challenge with lethal dose of E. coli.

Measurement of antibody titers against bacterial proteins

Serum samples were obtained from mice 3 days after each immunization and assayed for IgG antibodies specific for bacterial proteins by ELISA. Briefly, the mouse serum was diluted 1:500 in 1% BSA/PBS and applied to 96-well plates coated with 200 ng bacterial lysates. After incubation for 2 h, the IgG antibody levels were quantified with a peroxidase-conjugated anti-mouse IgG antibody followed by a luminescent substrate (Thermo Fisher Scientific).

Cytokine release by splenic T-cells

CD4+ T-cells were obtained from mouse spleens following immunization using a cell isolation kit (Miltenyi Biotec, Bergish Gladbach, Germany) according to the manufacturer’s instructions. The CD4+ T-cells (5 × 105) were incubated for 72 h with 1 µg/mL of bacterial proteins, followed by measurement of IFN-γ and IL-4 in the supernatants using a DuoSet ELISA Development kit (R&D Systems).

Flow cytometry analysis

Mouse spleens were removed upon sacrifice after immunization, and single cell suspensions were prepared. Viable cells were blocked for non-specific staining with 2.4G2 (anti-Fc-receptor) and stained with the following antibodies: anti-CD4-APC-H7 (GK1.5), anti-CD19-BUV395 (1D3), anti CD62L-BV605 (MEL-14), anti-CD44-BV786 (IM7), anti-CXCR5-BV605 (2G8), anti-Rorgt-PECF594 (Q31-378), anti-ki67-V450 (B56), and anti-BCL6-AF488 (K112-91) obtained from BD Biosciences (San Jose, CA); anti-TCRb-PeCy7 (H57-597), anti-MHCII-AF700 (M5/114.15.2), anti-PD1-BV711 (29 F.1A12), and anti-Foxp3-AF647 (150D) obtained from Biolegend (San Diego, CA); and anti-GL7-PE (GL-7) obtained from eBioscience (San Diego, CA). To exclude dead cells, Live/dead Aqua (Thermo Fisher Scientific) was used to exclude positive cells from the analysis. Intracellular staining was performed using the FOXP3 transcription factor staining kit (eBioscience) according to the manufacturer’s instructions. Events were collected and analyzed using an LSR-II or LSR Fortessa-X20 (BD Biosciences) and FlowJo software (Treestar Inc., Ashland, Oregon).

Immunohistochemistry

Mouse spleens were obtained upon sacrifice after immunization and fixed in 4% paraformaldehyde and 10% sucrose for 1 h and then transferred to 30% sucrose solution for overnight incubation at 4 °C. The spleens were embedded in TissueTek OCT compound and snap frozen in liquid nitrogen. Frozen Sects. (7–9 μm thick) were fixed in 100% acetone and blocked with 5% normal horse serum in PBS for 15 min. The antibodies used to stain sections were anti-mouse B220-FITC (clone: RA3-6B2; BD Biosciences, San Jose, CA) and GL-7-biotin (clone: GL7; eBioscience) followed by Streptavidin-Alexa Flour 594 (Thermo Fisher Scientific). Microscopy was performed with a confocal Zeiss LSM 700 inverted system and LSM software (Carl Zeiss, Oberkochen, Germany).

Display of spike protein S1 on the bacterial surface

E. coli BL21 (DE3) was used to express recombinant fusion proteins. Plasmid pET-28a(+) contained a fusion of the signal sequence and the first nine amino acids of Lpp, the sequence for five outer membrane-spanning domains of OmpA, the full sequence for the SARS-CoV-2 spike protein S1, and a His tag. For S1 overexpression, transformed bacterial cultures were inoculated at a ratio of 1/50 from overnight cultures and grown in a large volume of medium supplemented with 50 µg/mL kanamycin. When cultures reached an optical density of OD600 = 0.6, protein expression was induced by the addition of 0.1 mM IPTG (Thermo Fisher Scientific).

SDS-PAGE

Bacterial whole-cell lysates and SyBV overexpressing S1 were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The blocked membrane was then incubated with anti-His tag antibody (Thermo Fisher Scientific). After incubation with horseradish peroxidase-conjugated secondary antibody, the immunoreactive bands were detected with a chemiluminescent substrate.

Immunization with vesicles expressing S1

Mice were intraperitoneally injected with 5 × 109 OMV or SyBV overexpressing S1 three times every week. Serum samples were taken from mice 3 days after each immunization to measure the antibody titer against recombinant S1 proteins (Arigo Biolaboratories, Hsinchu City, Taiwan). For splenic cytokines, isolated CD4+ T-cells (5 × 105) were incubated for 72 h with 1 µg/mL of S1 proteins followed by measurement of cytokines in the supernatants using a DuoSet ELISA Development kit (R&D Systems).

Statistical analysis

Data analysis was performed using GraphPad Prism 7. Results are shown as means and standard errors of the mean. Unpaired two-tailed Student’s t-test was performed to compare two groups. One-way ANOVA followed by Tukey’s multiple comparison test was used to evaluate the difference between multiple groups with one independent variable, and two-way ANOVA was applied to compare multiple groups with two independent variables followed by Tukey’s multiple comparison test. Statistical significance for the survival curve was evaluated by the Mantel–Cox log-rank test. P < 0.05 was considered to be statistically significant.

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