Design of recombinant baculoviruses
For the generation of recombinant baculoviruses was used the expression vector pFastbacTM1 of Bac-to-Bac® baculovirus expression system (Thermo Fisher, USA, cat. no.10359–016). Under the promoter of polyhedrin (polh),, the genetic sequence of the first 330 bp of the N-terminal region of the polyhedrin was cloned and the genetic sequence of the GFP was ligated into its C-terminal in an open reading frame to generate a fusion protein called PH(1–110)GFP . The polh promoter and the polyhedrin sequence were taken from Autographa californica multiple nucleopolyhedrovirus virus (AcMNPV). For the generation of the PH-WT-GFP chimeric polyhedra, the pFastbacTM Dual expression vector (Thermo Fisher, USA, cat.No.10712024) was used, the WT polyhedrin was cloned under the p10 promoter and the PH(1–110)GFP under polh promoter. The baculoviruses were amplified, purified and titrated by following the recommendations and protocols provided by the supplier (Thermo Fisher, USA).
Cell line and recombinant baculovirus
To propagate the recombinant baculoviruses and titrate them we used the Spodoptera frugiperda cell line, Sf9 (ATCC®, USA, cat. no. CRL–1711). Cells were maintained in Grace’s medium (Thermo Fisher, USA, cat. no. 11300–027) supplemented with 10% inactivated fetal bovine serum (FBS) (Biowest, France, cat. no. S1650–500), lactoalbumin (Sigma-Aldrich, USA, cat. no. 19010), yeastolate (Thermo Fisher, USA, cat. no. 292805), antibiotic-antimycotic (Thermo Fisher, USA, cat. no. 15240–062) and 0.1% pluronic acid F- 68 (Sigma-Aldrich, USA, cat. no. P1300) at 27 °C under agitation, as previously described .
Production and purification of PH(1–110)GFP particles
SF9 cells (2x106 cel/ml) were infected using a multiplicity of infection (moi) of 10 with the recombinant baculoviruses, the cells were maintained at 27 °C under agitation at 100 RPM, 72 hours post infection (hpi) the cultures were centrifuged at 4200 g for 15 min to recover the viruses and obtain the cell pellet. The pellets were resuspended in phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) and were sonicated with 5 cycles of 20 seconds per pulse with 30% amplitude (Qsonica 700, USA). Between each cycle were maintained on ice for 5 minutes. After the last cycle, the PH(1–110)GFP particles were washed 5 times with PBS, between each wash the samples were centrifuged at 14,000 g. Finally, they were resuspended in PBS. In addition, chimeric polyhedra were generated by infecting SF9 cells with baculovirus with the WT polyhedrin and recombinant polyhedrin PH(1–110)GFP.
Separation of PH(1–110)GFP particles by sucrose gradients
The PH(1–110)GFP particles were separated in a discontinuous gradient of sucrose. To form the discontinuous gradient, 3 different sucrose concentrations were used, 40%, 50% and 60% (w/v) in distilled water, ultracentrifugation was performed at 17,738 g (SW 40 ti rotor, Beckman Coulter, USA) for 10 min at 4 °C. The PH(1–110)GFP particles of the different gradients were recovered and 3 washes were carried out with PBS, centrifuging the samples at 14,000 g after each wash.
The total protein of the lysates and the particles of the different gradients was determined using the PierceTM BCA Protein Assay Kit (Thermo Fisher, USA, cat. no. 23225) based on bicinchoninic acid (BCA) for colorimetric detection.
Cell confocal microscopy
SF9 cells infected with recombinant baculoviruses, 72 hpi were washed with PBS and incubated for 5 minutes with DAPI (4’, 6-diamino–2-phenylindole) to mark the nucleus (Thermo Fisher, USA, cat. no. D3571) at a 1:1000 dilution and fixed in slide glass (76x26 mm) with DAKO Fluorescent Mounting Medium (Agilent, USA, cat. no. S3023) . The GFP of the PH(1–110)GFP particles was excited at 473 and DAPI was excited at 405 nm. Fluorescence emission was collected at 510 nm for GFP and 420 nm for DAPI. All images were taken with a Fluoview FV10i confocal microscope (Olympus®, Japan), using the 60 × NA 1.35 oil immersion objective (UPLSAPO60XO). The images were analyzed with the software, FV10ASW.
PH(1–110)GFP particles confocal microscopy and 3D reconstruction
The purified PH(1–110)GFP particles were fixed with DAKO Fluorescent Mounting Medium in glass slides (76x26 mm). To obtain the images, we used a wide-field inverted IX81 Olympus® microscope with 60 × 1.42 NA oil immersion objective, to MT–20 illumination system and EMCCD camera iXon–897 (Andor Technology South Windsor, CT, USA). The used excitation and emission filters were 470 and 520 nm/40 bandpass respectively. The images were analyzed using ImageJ software. Imaris software was used for the 3D recostruction of confocal images (Additional file 1).
Transmission electron microscopy (TEM)
SF9 cells infected with recombinant baculovirus PH(1–110)GFP were centrifuged, the pellet was washed with cacodylate buffer (0.08 M, pH 7.4) and fixed with 0.6% glutaraldehyde and 0.4% paraformaldehyde in cacodylate buffer for 10 min. Post-fixation was made with 1% osmium tetroxide in cacodylate buffer. The cells were included in an epoxy resin and cuts of 90 nm thickness were made. Then the samples were contrasted with uranyl acetate 1% for 10 min and with lead citrate for 2.5 min. The JEOL JEM 12,000 EXII microscope at 80 kV (Jeol USA, USA) was used to observe the samples.
Scanning electron microscopy (SEM)
Briefly, the particles were purified and fixed with 2.5% glutaraldehyde in phosphate buffer (0.1 M, pH 7.4). Post-fixation was performed with 1% osmium tetroxide in phosphate buffer. The samples were dehydrated with alcohol gradients and dried to critical point and coated with gold for observation. Finally, the JEOL JSM 5410LV microscope (Jeol USA, USA) was used to observe the samples.
We peformed Fluorescence Recovery After Photobleaching (FRAP) using a Zeiss LSM 780 scanning confocal microscope (Axio observer.Z1/7) with an objective Plan-Apochromat 63 × / 1.40 oil DIC M27 (Carl Zeiss, Germany). The photobleaching protocol consisted in exposing the circular region-of-interest (ROI) to 488 nm Ar + laser at 100% of relative intensity in each PH(1–110)GFP particles. The photobleaching lasted for approximately 1–2 s, and the fluorescence intensity images after photobleaching were collected at intervals of 4 min during 2 h, resolution using a pinhole of 40.96 μm. Detection wavelength was at 510 nm. Laser intensity settings of 1% were sufficient to illuminate the fluorescent label without causing significant photobleaching. The images were analyzed with ZEN 2012 software (blue edition, Carl Zeiss, Germany) and the final images were edited with ImageJ 1.52n (NIH, USA). We compared the recovery of fluorescence between the PH(1–110)GFP particles (n = 14) and the chimeric particles PH-WT-GFP (n = 10) at different times. For this experiment, the PH(1–110)GFP particles and the PH-WT-GFP particles were prepared in the same way as for confocal microscopy.
Fresh PH(1–110)GFP particles or PH(1–110)GFP particles recovered from the different gradients of sucrose and GFP (Merck Millipore, USA, cat. no. 14–392) were mixed with 5 × Laemmli buffer (50 mM Tris-HCL, 3% SDS, 1% β-mercaptoethanol, 20% glycerol, 0.7% bromophenol blue, pH 6.8). The proteins were separated by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at 85 V for 2 h and stained using Coomassie brilliant blue R–250.
For western blot analysis, proteins contained in the SDS-PAGE were transferred to a nitrocellulose membrane (Merck Millipore, USA, cat. no HATF00010) at 100 V for 1 h in wet chamber using transfer buffer (48 mM Tris base, 39 mM glycine, 0.037% SDS, 20% methanol). Membrane was blocked with 5% fat-free milk in Tris-buffered saline (TBS, 50 mM Tris-Cl, pH 7.6, 150 mM NaCl) over night (ON). The anti-GFP antibody utilized in these studies was produced in mice in our laboratory. The antibody was used at a 1:2000 dilution in TBS-T (0.05% Tween) and 0.5% fat-free milk. Membranes were incubated with anti-GFP antibody for 3 hours with agitation at room temperature (RT). The secondary antibody was horseradish peroxidase-coupled (HRP) anti-mouse IgG (Sigma-Aldrich, USA, cat. no. A9044) was used at dilution 1:5000 in TBS-T and 0.5% fat-free milk. The secondary antibody was incubated 1 h in agitation at RT. The membranes were analyzed with a C-Digit Blot scanner (LI-COR, USA) and the signal generated by the SuperSignal® West Femto substrate (Thermo Fisher, USA, cat. no. 34095) was taken using the Image Studio software.
Purity and conservation of PH(1–110)GFP particls
The purity of fresh PH(1–110)GFP particles (Additional file 2) was evaluated by run electrophoretic assays using the Agilent Bioanalyzer 2100 (Agilent Technologies, USA) equipped with the Protein 230 assay kit according to the manufacturer’s recommended protocol. The electropherograms and gel-like images results were analyzed with Agilent 2100 expert software (Agilent technologies, USA).
Nanoparticle tracking analysis (NTA)
The NanoSight instrument (Malvern Panalytical, UK) was used to determine the size of the polyhedrin particles produced by the recombinant baculoviruses. The PH(1–110)GFP particles resuspended in sterile water were injected in a volume of 1 ml into the sample chamber. Five readings were made for each sample processed to obtain the average particle sizes. The NanoSight software (Malvern Panalytical, UK) tracked the Brownian motion in real-time to determine the center of the PH(1–110)GFP particles and determine the diffusion coefficient of each particle. Finally, the software based on the Stokes-Einstein equation calculated the size of the particles [40, 41].
For the care, feeding and management of the animals, we followed the guidelines established by the Official Mexican Standard NOM–062-ZOO–1999, by the Institutional Subcommittee for the Care and Use of Experimental Animals (SICUAE) of the Faculty of Veterinary Medicine and Zootechnics (Protocol number DC–2017/2–3) and by the Internal Committee for the Care and Use of Laboratory Animals (CICUAL) of the Institute of Cellular Physiology (Protocol number LVD102 (66)–16), both committees attached to the National Autonomous University of Mexico (UNAM).
Female BALB/c mice 6–8 weeks of age were used for all experiments. The immunization route was intramuscular (i.m.). All treatment groups received the dose of antigen on days 0 and 14, this was decided after the dose response assay (Additional file 3). Blood samples were collected from day 0 until the end of each study at 2-week intervals. Samples were centrifuged, and the sera were stored at –70 °C until analysis by Enzyme Linked Immunosorbent Assay (ELISA). In each study, particular points are described.
Dose response assay
Five groups (n = 5) were subjected to the following treatments: Group 1: PH(1–110)GFP 25 μg (one dose); Group 2: PH(1–110)GFP 25 μg (two doses); Group 3: PH(1–110)GFP 100 μg (one dose); Group 4: PH(1–110)GFP 100 μg (two doses); and Group 5: PBS (control group) (Additional file 3). Blood samples were taken at 2-week intervals for 6 months.
PH(1–110)GFP particles vs aluminum hydroxide (Alum)
From the dose response assay the treatment with PH(1–110)GFP 25μg two doses for subsequent experiments was selected. The following groups (n = 5) were evaluated: Group 1: GFP 25 μg; Group 2: GFP 25 μg + Alum; Group 3: PH(1–110)GFP 25 μg; Group 4: PH(1–110)GFP 25 μg + Alum; and Group 5: PBS + Alum (control group). The dilution used for Alum was 1:1. Blood samples were collected at 2-week intervals. With the serum samples obtained, the immune response was measured over time and antibody titers were evaluated at weeks 4 (Figure 4B), 14 and 24 (Additional file 4). To evaluate the Th1 and Th2 responses, total IgG, IgG1, IgG2a, and IgG2b were measured and the IgG2a/IgG1 ratio was calculated (Figure 5).
Long-lived antibody responses
In week 24th week of the experiment PH(1–110)GFP particles vs aluminum hydroxide (Alum), all groups received a boost with 5 μg of free GFP without adjuvant. Serum samples were obtained at day 4, 7, 14 and 21 post-immunization (Additional file 5).
Stocks of PH(1–110)GFP particles were stored at different conditions: 1) Room Temperature Dehydrated (RTD); 2) Room Temperature (RT); 3) 4 °C; 4) –20 °C; and 5) –70 °C. After 1, 3, 6 and 12 months of maintaining the particles in the different conditions, stock of each condition was taken and 6 mice group (n = 5) including a control group (PBS) were immunized. The RTD particles were dehydrated using a vacufugeTM concentrator 5301 (Eppendorf, Germany, cat. no. 5301) at a centrifugal force of 240 g at 30 °C for 30 min and were resuspended in PBS before being injected. In this experiment, no adjuvant was used. Blood sampling was performed for two months at 2-week intervals.
Immune response with PH(1–110)GFP particles of different sizes
With the particles purified by discontinuous gradient of sucrose, the following groups of mice were immunized (n = 5): Group 1: PH(1–110)GFP particles gradient 40%; Group 2: PH(1–110)GFP particles gradient 50%; Group 3: PH(1–110)GFP particles gradient 60%; Group 4: PH(1–110)GFP particles gradient > 60%; Group 5: PH(1–110)GFP particles gradients mixture; and Group 6: PBS. All treatments were conducted without adjuvant. Blood samples were collected for 10 weeks every 14 days.
Immunization for proliferation assay
For this assay, 3 groups of mice were immunized (n = 5): Group 1: PH(1–110)GFP particles 25 μg: Groups 2: PH(1–110)GFP particles 25 μg + Adjuvant; and Group 3: PBS + Adjuvant. In this experiment the complete Freund’s adjuvant (CFA) (Sigma-Aldrich, USA, cat. no. F5881) and incomplete Freund’s adjuvant (IFA) (Sigma-Aldrich, USA, cat. no. F5506) were used. We decided to use the CFA and IFA in this experiment because, unlike Alum, broader stimulation of the cellular response has been previously observed [32, 42]. Blood samples were taken for 6 weeks at 14-day intervals (Additional file 6A).
Mice were euthanized at week 6 post-immunization. Splenocytes were isolated from 3 animals from each treatment group by spleen perfusion with RPMI 1640 medium (Thermo Fisher, USA, cat. no. 31800022). Cells were treated and resuspended in RPMI 1640 supplemented medium and incubated with 5-(and–6) -Carboxyfluorescein Diacetate, Succinimidyl Ester (CFSE) (Thermo Fisher, USA, cat. no. C1157) as previously described . Cells were stimulated with concanavalin A (ConA) (3 μg mL–1) (data not shown), GFP (10 μg mL–1), PH(1–110)GFP (10 μg mL–1) or Albumin (10 μg mL–1, as a non-related antigen), and finally incubated in flat-bottomed microtiter plates (5 × 105 cells/well), for 5 days at 37 °C in a 5% CO2 humidified atmosphere.
Flow cytometry analysis
Cell proliferation was evaluated using standard flow cytometry protocols. [43, 44] After 5 days cells were harvested and stained with Phycoerythrin Cyanin 5.1 (PE-Cy ™ 5)-conjugated anti-CD3 (BD Biosciences, USA, cat. no. 553065). T lymphocytes proliferation was determined by measuring the progressive loss of CFSE fluorescence within daughter cells in each cell division. Results were expressed as a percentage of proliferation (Additional file 6B). The cells were analyzed on the Attune® Acoustic Focusing Cytometer (blue/red system) using the Attune® Cytometric Software (Thermo Fisher, USA). At least 10,000 events were collected. The final analysis of the data was performed using FlowJo 7.6.2 software (FlowJo LLC, USA).
Enzyme-linked immunosorbent assay (ELISA)
To determine the presence of GFP-specific antibodies in immunized mouse sera, samples were analyzed by ELISA. ELISA analysis was carried out using microtiter plates (Corning, USA, cat. no 3590) coated overnight with 50 μL of GFP at a concentration of 1 μg mL–1 in 0.1 M sodium bicarbonate buffer (pH 9.2). Microplates were washed 5 times with 200 μL of PBS containing 0.2% Triton X–100 and blocked with PBS-Triton + 5% fat-free milk for 1 h at 37 °C. Then, 50 μL of the sera diluted 1:100 in PBS-Triton-fat-free milk (for the experiment of PH(1–110)GFP particles of different sizes a dilution 1:400 was used) were added and plates were incubated 1 h at 37 °C. After washing as described above, 50 μL of anti-mouse IgG diluted 1:5000 (Sigma-Aldrich, USA, cat. no. A9044) or anti-mouse IgG1 diluted 1:3000 (Thermo Fisher, USA, cat. no. 04–6120) or anti-mouse IgG2a diluted 1:3000 (Abcam, UK, cat. no. ab98698) or anti-mouse IgG2b diluted 1:3000 (Thermo Fisher, USA, cat. no. 610320) (all HRP-conjugated) were added and plates were incubated 1 h at 37 °C. Plates were washed 5 times as described, 50 μL of the 3,3’,5,5’-Tetramethylbenzidine (TMB) substratum was added to each well (Sigma-Aldrich, USA, cat. no. 00–2023) and microplates were incubated at RT for 20 min. 50 μL of 0.16 M sulfuric acid solution was added to each well to stop the reaction. The OD reading at 450 nm was registered using Multiskan FC 3.1 microplate reader (Thermo Fisher, USA). For the titration of antibodies, sera were tested by performing serial 2-fold dilutions from 1:50 to 1:102400.
All statistical analyses were performing using GraphPad Prism 7 software (GraphPad software, USA). Results were expressed as the means ± SD. All experiments were repeated at least once with comparable results. Data were analyzed by two-way ANOVA with a Tukey or Dunnett post-tests to correct for multiple comparison test. In the FRAP experiment to obtain the percentage of fluorescence recovery, the initial post-bleaching value (10 min) was subtracted from the last value obtained (140 min). To calculate FRAP differences was used in an unpaired, two-tailed Student’s t-test. To determine the cutoff in the titration of antibodies, the previously described methodology was used . In the lymphoproliferation assay to obtain the absolute percentage of proliferation the PBS+Alum group value was subtracted from the other groups. A p-value <0.05 was considered statistically significant. * p <0.05; ** p <0.01; *** p <0.001 and ns = not significant.