Bioengineered bacteria-derived outer membrane vesicles as a versatile antigen display platform for tumor vaccination via Plug-and-Display technology

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DAPI was obtained from Life Technologies (Shanghai, China); anti-HA-Tag (catalog no. ab236632, dilution: 1:1000), anti-firefly luciferase monoclonal antibody (catalog no. ab16466, dilution: 1:10,000), and HRP-conjugated goat anti-mouse IgG (catalog no. ab205719, dilution: 1:5000) were purchased from Abcam (UK). Horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (catalog no. A16096, dilution: 1:5000) was bought from Invitrogen (USA). FITC-anti-mouse CD3 (catalog no. 100204, dilution: 1:50), APC-anti-mouse CD8α (catalog no. 100712, dilution: 1:80), PE/Cy7-anti-mouse IFNγ (catalog no. 505826, dilution: 1:20), PE-anti-mouse IFNγ (catalog no. 505808, dilution: 1:80), FITC-anti-mouse CD11c (catalog no. 117306, dilution: 1:200), PE/Cy7-anti-mouse CD80 (catalog no. 104734, dilution: 1:40), APC-anti-mouse CD86 (catalog no. 105012, dilution: 1:80), PE/Cy7-anti-mouse H-2Kb bound to SIINFEKL (PE/Cy7-anti-mouse MHCI-OVA) (catalog no. 114616, dilution: 1:40), PE-anti-mouse H-2Kb bound to SIINFEKL (PE-anti-mouse MHCI-OVA) (catalog no. 114608, dilution: 1:80), PE-anti-mouse Foxp3 (catalog no. 126404, dilution: 1:20), PE-anti-mouse CD44 (catalog no. 103008, dilution: 1:80), PE/Cy7-anti-mouse CD62L (catalog no. 104418, dilution: 1:80), PE/Cy7-anti-mouse F4/80 (catalog no. 123114, dilution: 1:80), APC-anti-mouse Gr1 (catalog no. 108412, dilution: 1:80), APC-anti-mouse CD11b (catalog no. 101212, dilution: 1:80), PE/Cy7-anti-mouse CD8 (catalog no. 100722, dilution: 1:80), FITC-anti-mouse CD4 (catalog no. 100406, dilution: 1:200), PE-anti-mouse CD11b (catalog no. 101208, dilution: 1:80) and FITC-anti-mouse Ly6G (catalog no. 127606, dilution: 1:200) were purchased from BioLegend (USA). FITC-anti-mouse CD8 (catalog no. 2002714, dilution: 1:100) and PE-anti-mouse CD4 (catalog no. 2013481, dilution: 1:160) were purchased from Invitrogen (USA). T-Select H-2Kb OVA Tetramer-SIINFEKL-PE (catalog no. TS-5001-1C, dilution: 1:5) was purchased from MBL Beijing Biotech Co., Ltd. The tumor necrosis factor α (TNF-α) mouse uncoated ELISA kit (catalog no. 88-7324-86), IL-6 mouse uncoated ELISA kit (catalog no. 88-7064-86), and IL-1β mouse uncoated ELISA kit (catalog no. 88-7013-86) were purchased from eBioscience (USA). The mouse IFNγ precoated ELISPOT kit (catalog no. 2210005) was purchased from Dakewe Biotech Co., Ltd (Shenzhen, China). OVA257-264 (SIINFEKL), OVA323-339 (ISQAVHAAHAEINEAGR), TRP2180-188 (SVYDFFVWL), SpT-OTI (VPTIVMVDAYKRYKGGSIINFEKL), SnT-OTII (GKLGDIEFIKVNKGYGGISQAVHAAHAEINEAGR), SnT-TRP2 (GKLGDIEFIKVNKGYGGSVYDFFVWL), SpT-OVA-Cy5.5 (VPTIVMVDAYKRYKGGSIINFEKL-Cy5.5), Adpgk (CGIPVHLELASMTNMELMSSIVHQQVFPT), and SpT-Adpgk (VPTIVMVDAYKRYKGGCGIPVHLELASMTNMELMSSIVHQQVFPT) were synthesized by Top Peptide (Shanghai, China) via Fmoc solid-phase peptide synthesis. Confocal microscopy Petri dishes (Hangzhou Xinyou Biotechnology Co., Ltd., China) were purchased from HuaLiDe Technology Co., Ltd. Hochest 33342 (catalog no. C0030) was purchased from Solarbio Life Science (Beijing, China). According to the quality reports provided by the manufacturer, the purity of all peptides was over 95%.

Animals and cells

Female C57BL/6 mice (6–8-week-old) were purchased from Vital River Laboratory Animal Technology Co. Ltd (Beijing, China). Mice were housed in a room with a temperature of 20–22 °C and a humidity of 30–70%. Feed and water were available ad libitum. Artificial light was provided in a 12-h light/12-h dark cycle. This study complied with relevant ethical regulations for animal testing and research, all animal protocols were approved by the Institutional Animal Care and Use Committee of the National Center for Nanoscience and Technology. The murine melanoma (B16-F10), murine colon cancer (MC38), murine pancreatic cancer (Pan 02), and DC2.4 cell lines were obtained from the ATCC (Manassas, USA). B16-OVA cells were generously provided by P. Wang Hao at the National Center for Nanoscience and Technology. B16-F10, MC38, Pan 02, and B16-OVA cells were cultured in DMEM containing 10% fetal bovine serum, 100 U mL−1 penicillin G sodium, 100 µg mL−1 streptomycin (Pen/Strep), and 20 µM β-mercaptoethanol (β-ME). DC2.4 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U mL−1 penicillin G sodium, and 100 µg mL−1 streptomycin (Pen/Strep). All cell lines were tested mycoplasma-free. The cells were incubated at 37 °C in a humidified environment with 5% CO2. All cell culture medium and FBS were purchased from Wisent (Canada).

Plasmid construction, bacterial strain, and growth

The genes encoding ClyA-OVA-3HA, Luciferase, ClyA-Luciferase, and ClyA-None were cloned into pET28a (Genewiz, Suzhou, China). ClyA-Catcher was cloned into pETDuet-1. The E. coli OMVs production strain Rosetta (DE3) (Tiangen Biotech Beijing Co., Ltd, China, wild-type LPS) which had been transformed with the expression plasmids pET28a-ClyA-OVA-3HA, pET28a-ClyA-Luciferase or pETDuet-1-ClyA-Catcher was grown at 37 °C in LB medium, with shaking at 180 rpm, until the OD600 was 0.6. isopropyl β-D-1-thiogalactopyranoside (IPTG, 0.1 mM) was added to further induce protein expression at 16 °C, and incubation was continued for 14 h with shaking at 160 rpm. Kanamycin (50 ug/μL) or ampicillin (50 μg/mL) was added when appropriate.

OMVs purification and characterization

Briefly, E. coli were cultured as described above, then removed by centrifugation at 5000×g for 10 min at 4 °C. The resulting supernatant (200 mL) was filtered through a 0.45-µm EPS filter (Millipore), then concentrated to 50 mL using a 50-K ultrafiltration tube. The concentrated solution was further filtered with a 0.22-µm EPS membrane (Millipore). OMVs were collected from the filtrate by ultracentrifugation at 150,000×g for 3 h at 4 °C. The collected OMVs were washed with PBS using centrifugation at 150,000×g for 2 h at 4 °C, then finally resuspended in 400 μL PBS and stored at −20 °C until use. The total protein concentration of OMVs preparations was evaluated using the bicinchoninic acid assay, the results of which were defined as the OMVs WT concentration. The size and morphology of the OMVs were characterized using dynamic light scattering (DLS) (Zetasizer Nano ZS90, Malvern, UK) and transmission electron microscopy (TEM) (Tecnai G2 F20 U-TWIN, FEI, USA). The LPS content in OMV was detected by ELISA (CEB526Ge, Cloud-Clone Corp., Wuhan, China) and LAL assay (L00350C, GenScript, Nanjing, China).

Western blot analysis

Total bacterial protein was extracted using the Bacterial Protein Extraction Kit (Beijing Puyihua Science and Technology Co., Ltd., China, catalog no. C600596). Briefly, bacteria were collected and washed by centrifugation at 5000×g for 10 min at 4 °C. The bacteria pellets were resuspended in 400 μL 1 × cell lysis buffer containing 4 μL phenylmethanesulfonyl fluoride (PMSF; Solarbio, China) and 80 μl lysozyme per 1 ml bacterial culture. The suspension was incubated at 37 °C for 30 min. In order to fully lyse the bacteria, the mixture was further incubated on a rocking platform for 10 min. In total, 20 μl DNaseI/RNase was added to the mixture, which was then incubated with rocking for another 10 min at 37 °C. The insoluble debris was removed by centrifugation at 3000×g for 30 min at 4 °C. The supernatant was collected for further use. For the protein extraction of OMVs, the OMVs were resuspended in RIPA buffer (Solarbio, China) containing 1 mM PMSF and lysed for 15 min. After centrifugation at 13,000×g for 15 min, the supernatant was collected. Protein samples were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels (10% polyacrylamide), and then transferred onto a polyvinylidene fluoride membrane. The membranes were blocked in 10% nonfat milk and subsequently incubated with an anti-firefly luciferase monoclonal antibody or anti-HA-Tag antibody for 2 h at room temperature. After washing three times using Tris-buffered saline containing 0.5% Tween 20 (Solarbio, China), the membrane was incubated with HRP-conjugated goat anti-rabbit IgG or HRP-conjugated goat anti-mouse IgG for 1 h at room temperature. Immunoreactive proteins were visualized using Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, USA).

In vitro BMDC maturation, cellular uptake experiments, and cross-presentation assays

Bone marrow cells were flushed from the femurs and tibias of C57BL/6 mice and cultured in RPMI-1640 supplemented with 10% FBS, 100 U mL−1 penicillin G sodium, 100 µg mL−1 streptomycin, 1% HEPES, 0.05 mM β-ME, 20 ng/mL IL-4 and 20 ng/mL GM-CSF after the red blood cells had been lysed. The cultures were initiated by placing ~1–1.5 × 106 bone marrow cells per well into six-well plates. Half the medium was replaced every 2 days. On day 6, non-adherent cells were collected for further investigation. To assess their maturation state, murine BMDCs were cultured with PBS, peptide antigen (50 μg/mL), or a mixture of peptide antigen and the different OMVs formulations (50 μg/mL) in a 1.5-mL tube for 24 h. At the completion of the incubation, the cells were collected for further staining with FITC-anti-mouse CD11c, PE/Cy7-anti-mouse CD80, or APC-anti-mouse CD86 to evaluate BMDC maturation. For the cross-presentation assay, BMDCs, DC2.4, or Pan 02 were incubated with OVA (or SpT-OVA) alone or a mixture of OVA (or SpT-OVA) and different OMVs formulations at 37 °C for 3–24 h. The OVA257-264 presentation by MHCI on the cell surface was then detected using a PE/Cy7-anti-mouse H-2Kb bound to SIINFEKL antibody, and the fluorescence was detected by flow cytometry (BD Accuri C6, BD Biosciences, USA) or laser scanning confocal microscopy (LSCM, Zeiss LSM710, Germany). To assess cellular uptake, SpT-OVA-Cy5.5 or a mixture of SpT-OVA-Cy5.5 and different OMVs formulations (50 µg mL−1) was incubated with murine BMDCs on glass-bottom culture dishes at 37 °C for 6 or 12 h, then examined using LSCM.

Cytokine assay

Bone marrow-derived dendritic cells (BMDCs) from C57BL/6 mice were cultured at 1 × 106 cells/well in 200 µL complete media mentioned before transferring them to 96-well, round-bottomed plates with OVA257–264 alone or a mixture of OVA257–264 and different OMVs formulations at 37 °C for 3–24 h. The supernatant was harvested at 3, 6, 12, and 24 h. TNF-α, IL-1β, and IL-6 were analyzed with ELISA kits according to the manufacturer’s protocols.

Cytotoxicity assay

BMDCs were seeded into 1.5-mL tubes at a density of 1 × 105 cells/tube in the presence of different concentrations of OMVs. After treatment for 24 h, the cells were collected and stained with Annexin V-APC and 7-AAD at room temperature for 30 min. Then cells were washed three times with PBS before analysis by flow cytometry.

Luciferase detection and ex vivo imaging

Fluorescein potassium was added to the bacteria (after they had been treated with IPTG) and their secreted OMVs (addition of ATP before detection). The samples were prepared for bioluminescence and measured using a Maestro in vivo spectrum imaging system (IVIS; Cambridge Research & Instrumentation, Woburn, MA, USA).

Prior to ex vivo imaging, saline, SpT-OVA-Cy5.5, or a mixture of SpT-OVA-Cy5.5 and different OMVs were subcutaneously injected into the tail base of mice for the evaluation of their biodistribution. At various time points thereafter, the inguinal lymph nodes and major organs, including the heart, liver, spleen, lung, and kidney, were collected for ex vivo fluorescence examination using the Maestro system. Subsequently, the inguinal lymph nodes were flash-frozen and cryosectioned (10-μm sections). The sections were stained with a DAPI nuclear stain and examined by LSCM.

Immunization and tumor therapy experiments

C57BL/6 mice (6–8 weeks old; n ≥ 4 for each group) were injected intravenously with B16-OVA or B16-F10 melanoma cells (2 × 105). Animals used for evaluating the anti-tumor effects of single-tumor antigen-displayed OMVs were immunized by subcutaneous injection into the tail base using different OMVs formulations (50 µg per antigen peptide; 50 µg OMVs) or controls on days 3, 6, and 11 after the administration of the tumor cells. For the anti-tumor effects of dual-tumor antigens displayed OMVs, the mice were immunized twice with the different formulations on days 3 and 7 after the administration of the tumor cells. On day 17, the mice were euthanized and the lungs were collected, briefly rinsed with PBS, and fixed with Fekete’s buffer (70 mL 75% alcohol, 10 mL formalin, and 5 mL glacial acetic acid). After 48 h fixation, the lungs were photographed and the pulmonary tumor nodules were counted.

Splenocytes were isolated from the spleen of immunized mice for intracellular IFNγ staining and IFNγ ELISPOT analysis. Briefly, the peptide antigen was co-cultured with the splenocytes overnight. Splenocytes treated with ionomycin (BioGems, USA, Catalog no. 5608212) were used as positive controls. Monensin (BioGems, USA, Catalog no. 2237803) was added to the cells 5 h before staining. The cells were collected for intracellular staining following the protocol provided by the manufacturer (Intracellular Flow Cytometry Staining Protocol provided by BioLegend). The surface markers CD3 and CD8 were stained prior to fixation, and the cells were fixed by a commercially available fixation buffer (BioLegend, USA, Catalog no. 420801) and permeabilized prior to IFNγ staining. The cells were then washed with intracellular staining perm wash buffer (BioLegend, USA, Catalog no. 421002) and resuspended in cell staining buffer for flow cytometry analysis (BD Accuri C6, BD Biosciences, USA). For IFNγ ELISPOT assays, all operations were carried out in accordance with the protocol provided by the manufacturer. Briefly, splenocytes were seeded at 1 × 105 cells per well in a 96-well plate coated with a mouse anti-IFNγ antibody and incubated for 20 h with peptide antigen or ionomycin (positive control). The secreted and captured IFNγ was subsequently detected using a biotinylated antibody specific for IFNγ and alkaline-phosphatase conjugated to streptavidin. After the addition of the substrate solution, a brown precipitate formed and appeared as spots at the sites of cytokine production. Automated spot quantification was performed by Dakewe Biotech Co., Ltd.

A subcutaneous colon cancer model was constructed for evaluating the therapeutic effect of nanovaccine in solid tumors. In total, 1 × 106 MC38 cells were inoculated into the right-back of mice on days 0. The mice were immunized with different formulations subcutaneously (Saline, 50 µg Poly (I:C) + 50 µg SpT-Adpgk, 50 µg SpT-Adpgk + 50 µg CN OMVs, 50 µg CC-SpT-Adpgk OMVs) on days 3, 6, and 11 (n = 10/group). Tumor volume was recorded every other day and calculated by the following equation: tumor volume = length × width2 × 0.5. Mice were sacrificed and tumors were collected on days 29. Tumors were then digested into a single-cell suspension for evaluating the infiltration of immune cells (n = 4). The survival for each mouse was recorded for plotting the survival curve in another set of subcutaneous MC38 tumor model. The survival experiment was terminated when tumor volume reached 1500 mm3 (n = 10).

Immune memory

Female C57BL/6 mice (6–8 weeks, n = 5) were vaccinated on days 0, 3, and 8. Peripheral blood and splenocytes was collected on day 60 to analyze antigen-specific CD8+ T cells (tetramer+ T cells and IFNγ+ cytotoxic T-lymphocyte cells) and memory T cells (Tnaive (CD3+CD8+CD44CD62L+), Tcm (CD3+CD8+CD44+CD62L+), Tem (CD3+CD8+CD44+CD62L)) by flow cytometry analysis.

Splenocytes were further used for the specific killing assays. Splenocytes were cultured with antigen (OVA257–264) overnight and then cultured with B16-OVA cells and MC38 cells at the ratio of 10:1 for 24 h. Non-adherent cells were removed, and adherent cells were washed with PBS. CCK assay was used to calculate the percent of specific killing.

For prophylactic tumor challenge studies, vaccinated mice (n = 10) were challenged on day 60 by intravenous injection of 2 × 105 B16-OVA cells per mouse. On day 80, the mice were euthanized and the lungs were collected, briefly rinsed with PBS, and fixed with Fekete’s buffer. After 48 h fixation, the lungs were photographed, and the pulmonary tumor nodules were counted.

Immune memory in tumor rechallenge model

Female C57BL/6 mice were injected intravenously with 2 × 105 B16-OVA cells on day 0. Mice were immunized by subcutaneous injection with CC-SpT-Adpgk OMVs on days 3, 6, and 11. Survived mice were further subcutaneously inoculated with 1 × 106 B16-OVA cells or 1 × 106 B16-F10 cells per mouse on the right flank on days 60 (n = 6–8). Then, tumor volume was recorded every other day and calculated by the same equation mentioned before.

Immunohistochemical analysis of T-lymphocyte infiltration into lungs

The lungs were harvested when treatments were terminated, fixed in paraformaldehyde, and paraffin-embedded. Lung sections (7 μm) were deparaffinized and rehydrated. The sections were treated with 3% H2O2 at room temperature for 10 min to eliminate the activity of endogenous peroxidase. After antigen retrieval in 10 mM citrate buffer (pH 6.0) at 95 °C for 15 min, the sections were blocked with 5% goat serum/PBS for 1 h and incubated with anti-CD8 (Abcam, UK, catalog no. ab93278, dilution: 1:100) at 4 °C, overnight, followed by incubation with goat anti-rabbit IgG biotinylated antibody (Biorbyt, UK, catalog no. orb153693, dilution: 1:100) at room temperature for 1 h and then HRP-conjugated streptavidin at 37 °C for 30 min. DAB was utilized for color development. Sections were counterstained with hematoxylin. Images were obtained using an Olympus BX 51 microscope (Olympus).

Statistical and reproducibility

Data are presented as the mean ± SD. At least three independent experiments were performed for each in vitro study (Fig. 1a, c-d; 3b-g, i; Supplementary Fig. 1, 2a–c, 9, 10a–c, 11a–c, 12, 14a-b, 15b, 17c-d, 22a-b). Statistical analysis was carried out using SPSS version 19.0. Data were analyzed by a two-tailed unpaired t test for comparison of two groups. Kaplan–Meier curves were analyzed using two-sided log-rank tests. Origin Pro 8.5.1, GraphPad Prism 5, BD Accuri C6 Software, FlowJo V10, and ImageJ v1.8.0 were used to analyze the acquired data.

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