Chimeric virus-like particles presenting tumour-associated MUC1 epitope result in high titers of specific IgG antibodies in the presence of squalene oil-in-water adjuvant: towards safe cancer immunotherapy | Journal of Nanobiotechnology

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Construction of nanostructures presenting multiple copies of MUC1 epitope

DNA coding sequence of MUC1 epitope (PDTRPAPGSTAPPAHGVTSA) was synthesized by Gene Art Gene Synthesis (Thermo Fisher Scientific). The nanostructure, presenting multiple copies of MUC1 epitope, was based on VP1 capsid protein of human NoV. The GII.4 NoV 2012 pandemic variant (Hu/GII.4/Sydney /NSW0514/2012/AU) Vp1 DNA coding sequence (1637 bp) was modified by addition of cloning sites for XbaI and MspCI restriction enzymes in second loop of P2 domain of VP1 protein, between 371 and 374 amino acids. The sequence was optimized using L. tarentolae-adapted codon and synthesized by Gene Art Gene Synthesis (Thermo Fisher Scientific). The synthetized gene was ligated into BglII-NotI restriction sites in the pLEXSY_I-blecherry vector (Jena Bioscience) resulting in platform vector: pLEXSY_Iblecherry3_VP1. The synthetic epitope was cloned as a XbaI/MspCI fragment into platform vector pLEXSY_Iblecherry3_VP1. Resulting plasmid pLEXSY_Iblecherry3_VP1_MUC1 was used for protein expression in LEXSY expression system. As a control VP1 gen was cloned into BglII-NotI restriction sites in the pLEXSY_I-blecherry vector resulting in vector: pLEXSY_Iblecherry3_cVP1.

VP1-MUC1 protein expression in L. tarentolae

The inducible LEXSY expression system (Jena Bioscience) was used for NoV VP1-MUC1 protein expression according to the manufacturer’s instructions Briefly, the pLEXSY_Iblecherry3_VP1_MUC1 plasmid was delivered into L. tarentolae cells by electroporation. The electroporated cells were grown in a suspension culture in BHI medium supplemented with bleomycin (100 µg/ml). Subsequently, the recombinant cell line was cultivated in 25 cm2 tissue culture flasks filled with the selective medium containing bleomycin at 26 °C and kept in the dark. The T7 promoter-driven transcription was induced by adding tetracycline at the final concentration of 15 µg/ml.

SDS-PAGE and western blotting

NoV VP1-MUC1, NoV VP1 and WT L. tarentolae cell lysates were loaded on 10–20% precast WedgeWell Gel (Thermo Fisher Scientific) and run at the constant voltage of 165 V. After electrophoresis, semi-dry electrotransfer of proteins onto polyvinylidene difluoride membranes was performed. Membranes were then blocked for 1 h in a 5% semi-skimmed milk solution (5%milk/TBS/0,01%Tween20) and incubated overnight with primary antibodies solution: Armenian hamster anti-MUC1 antibody (MA5-11,202, Thermo Fisher Scientific; 1:100 in 5%milk/TBS/0,01%Tween20) or rabbit anti-N terminal capsid protein of NoV antibodies (ab92976, Abcam; 1:1000 in 5%milk/TBS/0,01%Tween20). The following day, the membranes were washed (TBS/0,01%Tween20) and incubated with solution of Peroxidase-conjugated mouse anti-Armenian hamster antibody (Santa Cruz Biotechnology; 1:4000 in 5%milk/TBS/0,01%Tween20) or AffiniPure goat anti-rabbit antibodies (Jackson Immuno Research; 1:4000 in 5%milk/TBS/0,01%Tween20). A reaction was developed with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific).

MUC1 presenting VLP production and purification

Cell lysis

Tetracycline-induced L. tarentolae cell culture was grown in shake flasks for 72 h, at 26 °C, in agitated culture to the final optical density of 4–5 at OD600. Cells were then centrifuged for 15 min, 8000 rpm, at 4 °C. The cell pellet was resuspended in ice-cold lysis buffer (PBS/0,6%Tween-20) and sonicated (for 40 min, 40% amplitude, 10 s time on, 15 s time off). The lysed cells were centrifuged for 40 min, 8000 rpm, at 4 °C, the cell pellet was discarded, and the lysate was left for 16 h at 4 °C to allow particle formation.

Ultracentrifugation in a non-ionic iodixanol-based medium gradient (OptiPrep gradient)

The lysate containing VP1-MUC1 VLPs was layered on OptiPrep gradient (Sigma-Aldrich) formed in ultra-clear tube (2 ml of 40% (v/v) OptiPrep, 2,5 ml 36% (v/v) OptiPrep, 2,5 ml 33% (v/v) OptiPrep, 2 ml 30% (v/v) OptiPrep in ultra-clear water) and ultracentrifuged at 27,000 rpm for 16 h at 4 °C. Then, 500 µl fractions were collected and analysed. The OptiPrep buffer was replaced with PBS using Amicon® Ultra 100 K centrifugal units (Merck Millipore). The purity of the fractions was evaluated by SDS-PAGE with InstantBlue (Expedeon) Coomassie based staining.

Transmission electron microscopy

Chimeric MUC1 NoV VLPs produced in the protozoan expression system were purified through ultracentrifugation in OptiPrep Gradient and diluted in TM buffer (50 mM Tris HCl, pH 7,4, 10 mM MgCl2). In order to visualize, the chimeric VLPs were absorbed on the carbon coated grids followed by negative staining with 2% uranyl acetate. The particles were observed at 120 kV in the Tecnai Spirit BioTWIN (FEI, USA).

Detection of MUC1 presenting VLPs using ELISA assay

A 96-well ELISA plate (Pierce Streptavidin High Binding Capacity, Clear) was coated with 100 µl/well of biotinylated PDTRPAPGSTAPPAHGVTSA MUC1 peptide (synthesized by JPT Peptide Technologies) adjusted to 10 µg/ml. The coated plate was incubated for 2 h with shaking at room temperature. Then the plate was washed 4 × 5 min with 200 µl/well of wash buffer (Tris buffered saline pH 7,2/0,1%BSA/0,05%Tween20). Next, 100 µl/well of L. tarentolae cell lysates (WT, NoV VP1 and NoV VP1-MUC1 lysates) were used to coat the plate. The plate was incubated for 2 h with shaking in room temperature. The plate was washed as previously. Next, 100 µl/well of Armenian hamster anti-MUC1 antibody (MA5-11,202, Thermo Fisher Scientific; 1:100 in wash buffer) was added and the plate was incubated for 1 h at room temperature, then washed as previously. Then 100 µl/well of Peroxidase-conjugated mouse anti-Armenian hamster antibody (Santa Cruz Biotechnology; 1:2000 in wash buffer) was added and incubated for 1 h at room temperature. The plate was washed as previously and 100 µl/well of HRP-substrate solution was added (1-Step Turbo TMB-ELISA, Thermo Scientific). The plate was incubated in dark until the blue color developed and the reaction was stopped by adding 50 µL of 0,5 M sulfuric acid to each well. Signal intensity was measured at 450 nm using a plate reader (Epoch, Biotek).

Characterization of NoV VP1-MUC1 VLPs by ELISA assay

A 96-well ELISA plate (Greiner Microlon High-Binding, clear) was coated with 100 µl/well of sequential dilutions purified L. tarentolae-derived VLPs (NoV VP1 and NoV VP1-MUC1). WT L. tarentolae cell lysate served as a negative control. The coated plate was incubated overnight at 4 °C. Next, the plate was washed 4 × 5 min with 200 µl/well of washing buffer (PBS/0,05%Tween20) and blocked for 2 h with 250 µl/well of blocking buffer (3%BSA/PBS/0,05%Tween20) at 37 °C. The plate was washed as previously and 100 µl/well of primary rabbit anti-NoV antibodies (Abcam ab92976; in 3%BSA/PBS/0,05%Tween20) and Armenian hamster anti-MUC1 antibody (MA5-11,202, Thermo Fisher Scientific) were added. After incubation the plate was washed as previously, and the appropriate secondary antibody solution (Jackson Immuno Research; in 3%BSA/PBS/0,05%Tween20) was used for detection. Finally, following the last plate-washing step (6 × 5 min with 200 µl/well), 100 µl/well of HRP-substrate solution was added (1-Step Turbo TMB-ELISA, Thermo Scientific), the plate was incubated in dark until the blue color developed, and the reaction was stopped by adding 50 µL of 0,5 M sulfuric acid to each well. Signal intensity at 450 nm was measured using a plate reader (Epoch, Biotek).

Animal immunization

Two groups of 6 BALB/c male mice, 6 weeks old, were immunized subcutaneously with either 15 µg (day 0) or 10 µg of VP1-MUC1 protein (days: 14, 28, 42) and mixed in the 1:1 ratio with squalene-based oil-in-water nanoemulsion adjuvant MF59 (Addavax, InvivoGen). The mice serving as negative controls were immunized with 1:1 PBS-adjuvant mixture. Additionally, a group of 6 BALB/c male mice (6 weeks old) were immunized subcutaneously with (day 0) 10 µg of VP1 protein (days: 14, 28, 42) and mixed in the 1:1 ratio with squalene-based oil-in-water nanoemulsion adjuvant MF59 (Addavax, InvivoGen). All experiments on animals were conducted by an accredited company (Tri-City Central Animal Laboratory Research and Service Center, Medical University of Gdańsk) in accordance with the current guidelines for animal experimentation. The protocols were approved by the Committee on the Ethics of Animal Experiments of the Medical University of Gdańsk (Permit Number: 45/2015). All surgery procedures were performed under isoflurane anaesthesia, and all efforts were taken to minimize animal suffering.

Analysis of the antibody response

Mouse sera were collected on day 56 and pooled. Antibody response was measured by ELISA assay using (i) 10 µg/ml 100 µl/well of mucins from pig gastric mucous (Porcine gastric mucin, PGM, Sigma Aldrich) in PBS (ii) 10 µg/ml 100 µl/well of biotinylated PDTRPAPGSTAPPAHGVTSA MUC1 peptide (synthesized by JPT Peptide Technologies), (iii) 100 µl/well of L. tarentolae cell lysates—WT, (iv) 100 µl/well of L. tarentolae cell lysates—NoV VP1 and (v) 100 µl/well of WT L. tarentolae cell lysates—NoV VP1-MUC1. The coated plate was incubated for 2 h with shaking at room temperature. Then the plate was washed 4 × 5 min with 200 µl/well of wash buffer (Tris buffered saline pH 7,2/0,1%BSA/0,05%Tween20). Next, 100 µl/well of pooled serum 1:100 was added, and the plate was incubated for 1 h at room temperature, then washed as previously. Then 100 µl/well of Peroxidase-conjugated anti-mouse antibody (Jackson Immuno Research; 1:2000 in 3%BSA/PBS/0,05%Tween20) was added and incubated for 1 h at room temperature. The plate was washed as previously and 100 µl/well of HRP-substrate solution was added (1-Step Turbo TMB-ELISA, Thermo Scientific). The plate was incubated in dark until the blue color developed and the reaction was stopped by adding 50 µL of 0,5 M sulfuric acid to each well. Signal intensity was measured at 450 nm using a plate reader (Epoch, Biotek).

End-point titration of NoV VP1-MUC1 mouse sera by ELISA assay

Sera from immunized mice were collected and pooled on day 56 after immunization. A 96-well ELISA plate (Greiner Microlon High-Binding, clear) was coated with 100 µl/well of L. tarentolae cell lysates (WT and NoV VP1-MUC1 lysates). The coated plate was incubated overnight at 4 °C. Next, the plate was washed 4 × 5 min with 200 µl/well of washing buffer (PBS/0,05%Tween20) and blocked for 2 h with 250 µl/well of blocking buffer (3%BSA/PBS/0,05%Tween20) at 37 °C. The plate was washed as previously and serial dilutions of pooled mouse sera (in 3%BSA/PBS/0,05%Tween20) were added to the wells and incubated for 1 h at room temperature. Serial dilutions of Armenian hamster anti-MUC1 antibody (MA5-11,202, Thermo Fisher Scientific; in 3%BSA/PBS/0,05%Tween20) served as a positive control. After incubation the plate was washed as previously, and appropriate secondary antibody solution (Jackson Immuno Research; in 3%BSA/PBS/0,05%Tween20) was used for detection. Finally, following the last plate-washing step (6 × 5 min with 200 µl/well), 100 µl/well of HRP-substrate solution was added (1-Step Turbo TMB-ELISA, Thermo Scientific), the plate was incubated in dark until the blue color developed, and the reaction was stopped by adding 50 µL of 0,5 M sulfuric acid to each well. Signal intensity at 450 nm was measured using a plate reader (Epoch, Biotek).

Cross-reactivity of NoV VP1-MUC1 mouse sera by ELISA assay

A 96-well ELISA plate (Greiner Microlon High-Binding, clear) was coated with 100 µl/well of sequential dilutions of L. tarentolae cell lysates (WT, NoV VP1 and NoV VP1-MUC1 lysates). The coated plate was incubated overnight at 4 °C. Next, the plate was washed 4 × 5 min with 200 µl/well of washing buffer (PBS/0,05%Tween20) and blocked for 2 h with 250 µl/well of blocking buffer (3%BSA/PBS/0,05%Tween20) at 37 °C. The plate was washed as previously and 100 µl/well of NoV VP1-MUC1 serum, NoV VP1 serum or control mouse sera were added and followed by 2 h incubation at room temperature. After incubation the plate was washed as previously, and the appropriate secondary antibody solution (Jackson Immuno Research; in 3%BSA/PBS/0,05%Tween20) was used for detection. Finally, following the last plate-washing step (6 × 5 min with 200 µl/well), 100 µl/well of HRP-substrate solution was added (1-Step Turbo TMB-ELISA, Thermo Scientific), the plate was incubated in dark until the blue color developed, and the reaction was stopped by adding 50 µL of 0,5 M sulfuric acid to each well. Signal intensity at 450 nm was measured using a plate reader (Epoch, Biotek).

Characterization of immune response by ELISA isotyping assay

Ig isotyping ELISA test was performed using Ig Isotyping Mouse Uncoated ELISA Kit (Invitrogen) according to manufacturer protocol. Briefly, a 96-well ELISA plate (Greiner Microlon High-Binding, clear) was coated with 100 µl/well of isotype-specific capture antibody in coating buffer according to the suggested plate layout. The plate was sealed and incubated overnight at 4 °C. Next, the plate was washed 4 × 5 min with 200 µl/well of washing buffer and blocked for 2 h with 250 µl/well of blocking buffer at room temperature. The plate was washed as previously and 100 µl/well of mouse sera were added, as well as negative and positive control included in the kit. Mouse sera used for the analysis: NoV VP1-MUC1, NoV VP1 and PBS + MF59 Adjuvant (dilution used—1:1000). The plate was sealed and incubated at room temperature for 1 h on microplate shaker. Next, the plate was washed as previously and 100 µl/well of detection antibody was added. The plate was sealed and incubated at room temperature for 1 h on microplate shaker. Finally, following the last plate-washing step (6 × 5 min with 200 µl/well), 100 µl/well of substrate solution was added and the plate was incubated in dark until the blue colour developed, and the reaction was stopped by adding 100 µL of stop solution. Signal intensity at 450 nm and 570 nm was measured using a plate reader (Epoch, Biotek).

Statistical analysis

All statistical analyses were performed using Sigmaplot 12.0 software (SYSTAT Software). Statistical differences between the means of the two groups were analysed using t-test. Each experiment was performed in triplicates and the P value p< 0,05 was considered to be statistically significant.

NoV VP1 internal controls

Purified NoV VP1 VLPs produced in L. tarentolae cells (empty epitope presenting platform) and NoV VP1 sera obtained after mouse immunization described previously [36] served as internal controls for all the experiments.

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