Characterization of an Escherichia coli-derived triple-type chimeric vaccine against human papillomavirus types 39, 68 and 70

Ethics statement

The experimental protocols were approved by the Xiamen University Laboratory Animal Management Ethics Committee. All manipulations were strictly conducted in compliance with animal ethics guidelines and approved protocols.

Strains and vector construction

N-terminally truncated HPV39 (P24838), HPV68 (AAZ39498.1) and HPV70 (P50793) L1 genes were cloned into the pTO-T7 expression vector by Gibson assembly, and plasmids were transferred into the ER2566 E. coli strain for L1 protein expression.

Protein purification and particle assembly

ER2566 E. coli strains containing HPV39, −68, −70 WT or the chimeric L1 plasmid were cultured in LB liquid medium at 37 °C until reaching an OD600 of 0.6. Protein expression was then induced by the addition of isopropyl-β-D-thiogalactoside (IPTG, final concentration of 5 μM) and samples were further cultured at 24 °C for 10 h. Cells were then harvested by centrifugation (7000 × g, 10 min), resuspended in lysis buffer (50 mM Tris-Base [pH 7.2], 10 mM EDTA and 0.3 M NaCl) and lysed by ultrasonication. The ultrasonication output power was set at 45% of 500 W, and the ultrasonication time was 15 min per gram of E. coli, with a frequency of 2 s pulsed and 4 s paused. HPV L1 proteins were released from the cells and treated with 20 mM DTT for 12 h at room temperature. Then the supernatant was purified by chromatography using SP or XS sepharose (GE Healthcare). For the preparation of VLPs, the purified L1 protein was then dialyzed into a neutral buffer (10 mM pB6.5, 0.5 M NaCl) without DTT to allow VLP self-assembly and purified by Superdex 200.

Sequence alignment and phylogenic tree construction

HPV39 (GenBank: P24838), HPV68 (AAZ39498.1), HPV70 (P50793), HPV6 (7EW5_A), HPV30 (YP_009508159.1), HPV34 (NP_041812.1), HPV51 (ACV88631.1), HPV58 (P26535.1), HPV66 (ABO76865.1), HPV67 (ALT54969.1), HPV69 (AHV83654.1), HPV73 (ALJ32570.1), HPV82 (ALJ32344.1), HPV85 (YP_009362313.1), HPV97 (ABO27083.1), and HPV11, −16, −18, −26, −31, −33, −35, −45, −52, −53, −56, −5951 L1 genes were from NCBI. L1 protein sequence alignment was performed using BLAST, and evolutionary tree building was performed by the Neighbor-joining method in MEGA X software (https://www.megasoftware.net).

SDS-PAGE and western blotting

SDS-PAGE samples were diluted with an equal volume of sample buffer (125 mM Tris-HCl, pH 6.8, 4% (w/v) SDS, 20% (w/v) glycerol, 200 mM dithiothreitol, and 0.002% (w/v) bromophenol blue) to a final protein concentration of 0.3–0.5 mg/mL, heated at 80 °C for 10 min, and then loaded into the wells of a 10% separating gel. The gels were subjected to standard laboratory methods for gel electrophoresis (running at voltage of 80 V at concentration phase for 15 min, and then increase to 120 V at separation phase for additional 60 min in a BioRad MINI-PROTEAN Tetra system [BioRad Laboratories, CA, USA]) and stained with Coomassie Brilliant Blue R-250 (Bio-Rad) for 30 min at room temperature.

For western blotting, gels were transferred to nitrocellulose membranes, blocked with 5% skimmed milk for 1 h, and then incubated with HPV broad-spectrum linear monoclonal antibody 4B3 (1:1000 dilution) for 1 h at room temperature. Membranes were washed, and then incubated with goat anti-mouse alkaline phosphatase-conjugated antibodies (Abcam; Cambridge, UK; 1:5000 dilution) for 1 h. NBT/BCIP (Pierce Biotechnology; Rockford, IL) reagent was used to develop the color (5 min). All blots or gels derive from the same experiment and that they were processed in parallel. And source data are provided in Supplementary Fig. 4 and in the source data file.

Transmission electron microscopy (TEM)

Samples were diluted to 0.15 mg/mL with HPV L1 storage buffer. Each sample (10 μL) was pipetted onto carbon-coated copper grids for 10 min, absorbing any residual liquid before adding dropwise 2% phosphotungstic acid (pH 6.4) and incubating the reaction for 5 min. An FEI Tecnai T12 TEM with an accelerating voltage of 120 kV was used to observe and measure HPV VLP morphology.

High-performance size-exclusion chromatography (HPSEC)

The homogeneity of WT and chimeric VLPs was assessed using an Agilent 1200 high-performance liquid chromatography system, with a pre-installed TSK G5000 pwxl 7.5 mm × 300 mm column (TOSOH, Tokyo, Japan). The system flow rate was 0.5 mL/min, and the detection wavelength was set to 280 nm.

Analytical ultra-centrifugation (AUC)

Sedimentation analysis was carried out on a Beckman XL-A analytical ultracentrifuge at 20 °C. 10 mM phosphate buffer pH 6.5 with 0.5 M NaCl was used as the reference solution. WT and chimeric VLPs were diluted to 0.5 mg/mL with reference solution, and centrifuged at 3951 × g using an An-60 Ti rotor (Beckman Coulter; Fullerton, CA). The sedimentation coefficient was determined using Sedfit software kindly provided by Dr. P. Schuck at the National Institutes of Health (Bethesda, MD).

Differential scanning calorimetry (DSC)

The thermal stability of WT and chimeric VLPs was measured using MicroCal VP-Capillary differential scanning calorimetry (DSC) (GE Healthcare, MicroCal Products Group, Northampton, MA). Samples were diluted to 0.5 mg/mL with 10 mM phosphate buffer, pH 6.5, with 0.5 M NaCl. The temperature range was set as 10 °C to 90 °C, with a scanning rate of 100 °C/h. MicroCal Origin 8.0 software (Origin-Lab Corp., Northampton, MA) was used to analyze the DSC curve and the melting temperatures (Tm) of the samples.

Indirect enzyme-linked immunosorbent assay (ELISA)

The wells of 96-well microplates were coated with WT or chimeric VLPs (300 ng per well) for 2 h at 37 °C and then blocked with 200 μL blocking solution for 2 h at 37 °C. The wells were then incubated with 100 μL of twofold serially diluted mAbs at a starting concentration of 1 μg/mL for 45 min at 37 °C. The wells were then washed and incubated with 100 μL horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (diluted 1:5000 in HS-PBS, Abcam; Cambridge, UK), followed by 50 μL of 3, 3’, 5, 5’-tetramethylbenzidine liquid substrate (Sigma-Aldrich, St Louis, MO) per well for 10 min at 37 °C and the reaction was quenched with the addition of 50 µL 2 M H2SO4. An automated ELISA reader (TECAN, Männedorf, Switzerland) was used to detect the absorbance at 450 nm (reference, 620 nm). GraphPad Prism (GraphPad Software, San Diego, CA) were used to calculate the median effective concentration (EC50, ng/mL), which is the concentration of antibody that binds to 50% of the antigen.

Cryo-electron microscopy (Cryo-EM) and three-dimensional (3D) reconstruction

The WT and chimeric VLPs (~2.0 mg/mL) were vitrified on Quantifoil holey carbon grids using an automated Thermo Fisher Vitrobot IV, and the cryo-EM images were collected using the Thermo Fisher TF30 electron microscope equipped with Thermo Fisher Falcon II and Falcon III cameras. The acceleration voltage was set to 300 kV and the magnification was set to 93,000×, resulting in a pixel size of 1.120 Å. Data were collected automatically by using Thermo Fisher EPU software. Icosahedral 3D reconstruction were performed by using AUTO3DEM49 or cisTEM50 software. The resolutions of the final 3D density maps were estimated based on the gold-standard FSC curve with a cut-off of 0.14357. Visualization of the density maps were performed with software ChimeraX58.

Murine monoclonal antibodies (mAbs)

HPV WT and chimeric VLPs, formulated with Freund’s complete or incomplete adjuvant (20 μg/dose), were subcutaneously injected into BALB/c mice, with three injections delivered in 2-week intervals. The first injection used Freund’s incomplete adjuvant whereas the last two injections used Freund’s complete adjuvant. Cell fusion experiments were performed 2 weeks after a boost immunization. Fused hybridomas were isolated through hypoxanthine-aminopterin-thymidine medium (Sigma, Atlanta, GA) selection, and supernatants were screened by a indirect ELISA and PBNA for reactivity. Positive cells were cloned by limiting dilution at least three times until a single cell clone was attained.

The screened monoclonal hybridoma cells were injected into the peritoneal cavities of pristane-primed BALB/c mice. Ascitic fluid was collected after 9–12 days, and added with an equal volume of saturated ammonium sulfate solution, incubated at 4 °C for 30 min, centrifuged at 13,000 × g for 10 min, and the pellet was resuspended with 0.2 M Na2HPO4, and finally purified by protein A affinity chromatography. The resultant purified anti-HPV39 mAbs, anti-HPV68 mAbs and anti-HPV70 mAbs were diluted to 1.0 mg/mL in PBS and stored at −20 °C.

HPV pseudoviruses preparation

The pvitro (for HPV 68 and 70) or pshell (for HPV39)-L1-L2 co-expression vector and the pcDNA3.1-EGFP expression vector used in the experiment were kindly provided by Dr. J. T. Schiller59. HPV39, -68 and -70 pseudoviruses (PsVs) were produced in HEK293FT cells, as follows. First, HEK293FT adherent cells were prepared with high activity and passaged one day before plasmid transfection, and when the cell density reached to 80–90%, then half of the medium was changed to ES serum-free dulbecco’s modified eagle medium (DMEM), the cell culture was incubated at 37 °C for 30 min. Second, the transfection system with 0.15 M NaCl buffer were prepared, 1 mL of the transfection system contains 10 μg of co-expressing plasmid carrying codon-optimized HPV L1-L2, 10 μg of pcDNA3.1-EGFP plasmid, and 65 μg of PEI (Yeasen Biotechnology (Shanghai) Co. Ltd.). The system was mixed and incubated at room temperature for 15 min, protected from light throughout. Third, the plasmid and PEI mixture were slowly added to the cells, 1 mL per plate, incubated at 37 °C for 4 h, then half of the medium was changed to ES serum-containing DMEM, and 0.5 mL ES serum was added to each cells plate, cultivated at 37 °C for 72 h. Fourth, the medium was aspirated and the cells were carefully washed with 2 mL DPBS, then resuspended with 4 mL DPBS and collected by centrifugation at 1,300 × g for 3 min at room temperature. Fifth, the cells were then lysed in 150 μL cell lysis buffer comprising 0.5% Brij58 (Sigma-Aldrich), 0.2% Benzonase (Merck Millipore; Darmstadt, Germany), 0.2% Plasmid-Safe ATP-Dependent DNase (Epicenter Biotechnologies, Madison, WI) and DPBS-Mg solution for 24 h at 37 °C. Finally, the above cell lysate was ice-bathed for 15 min, and 5 M NaCl of 0.19 times the volume of the lysate was added, ice-bathed for 15 min too after mixing, and then centrifuged at 1,300 × g for 10 min at 4 °C, the obtained supernatant was the pseudovirus used in neutralization assay.

The measured of tissue culture infectious dose (TCID50)

The tissue culture infectious dose (TCID50) of the supernatant was then measured to determine the titers of the PsVs, calculated according to the classical Reed–Muench method60. First, HEK293FT cells in seeded into the wells of a 96-well plate at a density of 1.5 × 104 cells/well and cultured for 4–6 h at 37 °C, and then a 10-fold gradient dilution was performed on the pseudovirus, with a total of 7 gradients. Second, the pseudovirus were added to the cells plate at the ratio of 100 μL/well, 8 wells of each gradient were repeated, cultured at 37 °C for 72 h. Third, the expression of EGFP in each well of each gradient were recorded by an inverted fluorescence microscope, and the proportional distance was calculated according to the Reed-Muench method: proportional distance = (infection rate at dilution next above 50%-50%) / (infection rate next above 50% - infection rate next below 50%); and the logarithm TCID50 can be obtained by multiplying the logarithm of the dilution coefficient the proportional distance, and plus the highest dilution of the virus with an infection rate higher than 50%. So the TCID50 value can be obtained too, and PsVs were diluted to 3 ×105 TCID50/μL for PBNA assay.

Pseudovirus-based neutralization assay (PBNA)

HEK293FT adherent cells were prepared with high activity and passaged one day before PBNA assay, and when the cell density reached to 80–90%, seeded into the wells of a 96-well plate at a density of 1.5 × 104 cells/well and cultured for 4–6 h at 37 °C. Antibody sera were first diluted according to different dilution (2.5–1000) with ES serum-containing DMEM, and then subjected to 2-fold serial dilutions, for a total of 12 gradients, and PsVs were diluted with ES serum-containing DMEM to 3 × 105 TCID50/μL. Sera and PsVs were then mixed at a ratio of 1:1 (60 μL each), and incubated at 37 °C for 1 h. Equal volumes (60 μL) PsV diluent and culture medium mixture was used as the negative control. Cells were then incubated with 100 μL of PsV-serum mixture for 72 h at 37 °C. The number of green fluorescent spots in each cell well was read with Elispot, and the PsV infection of the cell wells with the spots number lower than half of the average value of the sum of the negative control wells were considered to inhibited by serum neutralizing antibodies. Then the neutralization titers of the antibodies were calculated as the log10 of the highest sera dilution with a percentile of infection inhibition higher than 50%.

Animals, immunizations and serological analysis

To initially assess the immunogenicity of chimeric VLPs, special pathogen-free (SPF) BALB/c female mice (n = 5) were immunized intraperitoneally three times at an interval of 2 weeks (week 0, 2 and 4) with WT or chimeric VLPs diluted in aluminum adjuvant prepared at three different doses (5, 1 or 0.2 μg per dose). In the mixed WT VLP group, the dose of each VLP was equal to that of the same VLP in the single-type groups. Serum samples were collected at week 8, and the neutralizing titers were analyzed by PBNA.

For ED50 analysis, SPF BALB/c female mice (n = 8) were vaccinated via an intraperitoneal injection of a single dose (0.300, 0.100, 0.033 or 0.011 μg) of WT or chimeric VLPs diluted in aluminum adjuvant. In the mixed WT VLP group, the dose of each VLP was equal to that of the same VLP in the single-type groups. Serum samples were taken 5 weeks after immunization, and the ED50, (i.e., the immune dose that causes 50% seroconversion in the mice), were analyzed based on a dose-response curve using the Reed–Muench model60.

Statistical methods

SPSS statistics (International Business Machines Corporation, New York, USA) was used for statistical analysis of data sets. All data were analyzed by one-way analysis of variance (ANOVA) and are presented as the mean ± standard deviation (SD). P values < 0.05 was considered statistically significant.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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