Viruses
Three IAVs isolated from swine were selected for the preparation of the influenza vaccine: A/swine/Brazil/025 − 15/2015 1 A.3.3.2 (H1N1pdm; NCBI GenBank Accession HA = MH559931 and NA = MH559933; BRMSA 1710), A/swine/Brazil/223-15-1/2015 1B.2.4 (H1N2; NCBI GenBank Accession HA = MH560035 and NA = MH560037; BRMSA 1698) and A/swine/Brazil/028-15-8/2015 (H3N2; NCBI GenBank Accession HA = MH559963 and NA = MH559965; BRMSA 1697). H1N1 and H1N2 viral samples were propagated in SPF (Specific Pathogen-Free) embryonated chicken eggs, and H3N2 virus was inoculated into Madin-Darby Canine Kidney (MDCK; BCRJ) cells, according to Zhang and Gauger [29]. To confirm the IAV presence, the cell supernatant and chorioallantoic fluid harvested from eggs were tested by hemagglutination assay [30] and by RT-qPCR [31].
Virus concentration
Approximately 1 L of each virus was individually concentrated by tangential ultrafiltration using a flow pump coupled to a cassette containing a dialysis membrane consisting of polyethersulfone (PES) with a cut-off of 100 kDa (Vivaflow 200 System, Sartorius, Germany). Then, 50 mL of each viral concentrate was ultracentrifuged at 100,000 x g for 4 h at 4 °C (Optima LE 80 K, Beckman Coulter, USA). The supernatants were discharged, and the pellets of each virus were diluted to a final volume of 5 mL in TNE buffer (10 mM Tris, 100 mM NaCl and 1 mM EDTA, pH 7.4). The concentrated viruses were titrated by hemagglutination assay [30], and the hemagglutinin content was determined by SDS-PAGE (NuPAGE™ Bis-Tris, Thermo Fisher, USA) using acrylamide gel plate (4–12%) and silver staining according to the manufacturer’s recommendations. The HA concentration was calculated from the intensity of the bands (obtained with the ImageJ software) using an equation obtained from a standard calibration curve of albumin.
Virosome preparation and characterization
The multivalent virosome was prepared according to de Jonge, Leenhouts [32] with modifications. Briefly, the same volume of each virus was mixed (1:1:1) and diluted in a 200 mM solution of 1,2-dicaproyl-sn-glycero-3-phosphocholine (DCPC, Avanti Polar Lipids, USA). After gentle homogenization, the pooled viruses were further diluted 1:2 (v/v) in the TNE buffer, and the final mixture was kept in an ice bath for 30 min to ensure the dissolution of the viral envelopes. Afterward, the mixture was ultracentrifuged at 100,000 x g for 30 min at 4 °C to remove the viral nucleocapsids. The supernatant was extensively dialyzed in a cellulose dialysis tube (cut-off 10 kDa, SpectraPor, USA) against TNE buffer for 48 h at 4 °C to remove the surfactant (DCPC), which led to the self-assembling of the virosome vesicles. Subsequently, the physical-chemical characteristics of the dialyzed virosome formulation were determined, measuring the particle size and zeta potential by dynamic laser scattering and laser Doppler electrophoresis (Zetasizer, Malvern). The HA antigen content from H1N1, H1N2, and H3N2 was determined by SDS-PAGE.
Electron microscopy
Transmission electron microscopy (TEM) was used to examine the morphology and ultrastructure of the virosomes using the microscope JEOL JEM-1011 (Jeol, Japan). The samples were used pure or at a 50% dilution in ultrapure water. Before analysis, 3 µL of virosome suspension was deposited into a copper grid covered with Formvar®. The copper grids were fixed and after they had completely dried, they were contrasted with 1% Osmium Tetroxide vapor. Thus, 40 µL of Osmium Tetroxide were placed at the bottom of a petri dish containing the copper grids for 40 min. Virosomes were digitized using an UltraScan® camera connected to Digital Micrograph 3.6.5® computer software (Gatan, USA). To eliminate any doubt about what were virosomes and what were artifacts from the contrast with 1% Osmium Tetroxide, a negative control was created during the analysis that used only ultrapure water and Osmium Tetroxide [33].
Assessment of the infectivity and cytotoxicity of influenza virosomes
In order to evaluate the infectivity of virosome, it was inoculated into embryonated chicken eggs and incubated at 37 °C for 4 days. Virosome was also inoculated into MDCK cells and monitored for 7 days.
For the in vitro cytotoxicity assays, immortalized macrophage lines (RAW 264.7 cells, BCRJ-0212) were cultured in complete Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen) modified to contain 4 mM GlutaMAX (Gibco), 4500 mg/L glucose, 1 mM sodium pyruvate (Sigma-Aldrich), and 1500 mg/L sodium bicarbonate (Sigma-Aldrich) with 10% of fetal bovine serum (FBS) (Sigma-Aldrich). RAW 264.7 cells were cultured and maintained at 37 °C and 5% CO2. After the formation of the cell monolayer, the adherent cells were detached by scraping. Initially, RAW 264.7 cells (2 × 105 cells/well) were seeded in 96-well plates, cultured for 24 h for adhesion and then treated with different dilutions of the virosome formulation (1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128 and 1:256, v/v) for 24, 48 and 72 h. This procedure was repeated for different passages of the cell culture until the desired sample size (n = 8) was reached. The control cells received the same volume of a simple liposome (prepared with phospholipids – Lipoid® S100, Lipoid). The cell viability was measured by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich) assay. At the end of each incubation time, the medium was removed, and the cells were washed twice with DPBS (Sigma-Aldrich) and incubated for 3 h with 5 mg/mL MTT solution at 37 °C. After incubation, the precipitated formazan crystals were dissolved in 200 µL of dimethyl sulfoxide (DMSO, Sigma-Aldrich). Optical densities (OD) were measured at 540 nm using a Multiskan™ FC Microplate Photometer (Thermo Fisher Scientific). The absorbance values recorded for untreated cells (negative control) represent 100% of cell viability and were used as a reference to calculate the percentage of cell viability in the presence of each sample concentration. Complementary cytotoxicity analysis was performed using the enzyme terminal deoxynucleotidyl transferase (TdT) and the propidium iodide (PI) staining kit (APO-DIRECT™, BD Biosciences). RAW 264.7 cells were plated and treated with the virosome dilutions (1:32 and 1:64) as described above. The assay was done according to the manufacturer’s guidelines at 24, 48 and 72 h after exposure to the virosomes [33]. The staining protocol consisted of cell incubation with TdT-FITC enzyme and staining with propidium iodide. After 24 and 48 h of exposure, cells were analyzed using a flow cytometer (Accuri C6 Plus, Becton-Dickinson, USA), and the percentage of intact cell membranes per group was determined. The percentage of live cells was calculated from the fluorescence readings defined according to the kit instructions.
Immunization of mice with multivalent influenza virosomes
The protocols and the use of animals for this research complied with the Animal Use Ethics Committee of Embrapa Swine and Poultry (protocol number 001/2016). C57BL/6 mice (female, 6–8 weeks old) were reared under SPF conditions and divided into 3 groups as follows: non-vaccinated control (NV, n = 20), intranasal vaccinated (5 µL/nostril or 10 µL/animal, IN, n = 20), intramuscular vaccinated (100 µL/animal, IM, n = 20). An additional group, non-vaccinated liposome control (n = 20), served as a control for virosomes. The G4 group did not show any difference in the analyzes performed when compared to the animals in the NV group (data not shown). A mucoadhesive adjuvant (carboxymethyl cellulose – CMC) was added to the formulation (0.125%, m/v) for intranasal administration, and Emulsigen-D® (MVP Adjuvants, USA) for intramuscular administration (20%, v/v) [34]. The experimental protocol consisted of the administration of two doses of the vaccine 2 weeks apart (days 1 and 15). At 21 days (day 36) and eight months later (day 255) after the second vaccine dose, ten animals/group from all three groups were euthanized using intraperitoneal injection of sodium pentobarbital (80 µg/g body weight).
Biochemical determinations
For blood collection, mice were anesthetized with intraperitoneal ketamine-xylazin (ketamine 60 µg/g body weight, and xylazin 10 µg/g body weight). Blood samples were drawn by retro-orbital bleeding on days 0 (before vaccination), 3 and 17 (two days after each immunization). In order to assess the possible toxicity of the vaccine, quantification of biochemical markers from the serum samples was performed, evaluating the hepatic (AST and ALT) and renal (urea and creatinine) functions. These assays were performed with colorimetric kits, according to the manufacturer’s instructions (Labtest, Brazil).
Morphologic assessment
For histopathology analysis, liver, kidney and lung tissue samples were collected at necropsy and fixed with 4% buffered paraformaldehyde, dehydrated in a graded series of ethanol, paraffin-embedded and sectioned at 4 μm. This material was stained with hematoxylin-eosin (H&E). Furthermore, to assess the in vivo cytotoxicity of the virosome, staining for apoptosis was performed using the In Situ Cell Death Detection kit (Roche, Germany), according to the manufacturer’s instructions. Nuclei were counterstained with 3,3-diaminobenzidine (Sigma-Aldrich, USA). The TUNEL assay is employed to identify and quantify apoptotic nuclei by an in situ reaction involving TdT-mediated dUTP-X nick end labeling. TUNEL-positive nuclei were quantified using light microscopy under magnification of 400x. The degree of TUNEL expression was calculated in 25 distinct fields (corresponding to a total area of 0.08 mm2). Results were expressed as cells/mm2.
Bronchoalveolar lavage
Bronchoalveolar lavage fluid (BALF) from mice was obtained at necropsy (days 36 and 255), through a tracheostomy procedure in a biosafety cabinet. BALF was collected by flushing the lungs four times with 0.2 mL sterile physiological saline (0.9% NaCl) via the tracheal cannula. After BALF collection, a protease inhibitor cocktail was added to a final concentration of 1x and also phenylmethylsulfonyl fluoride (PMSF) to a final concentration of 1 mM. One portion of the BALF samples was stored at -80 °C for ELISA assay. ELISA was performed to quantify total IgA immunoglobulin in the BALF supernatant using the Invitrogen kit (Thermo Fisher, USA) in accordance with the manufacturer’s recommendations. Another part of the BALF samples was used for cell quantification. Trypan blue exclusion method using a Neubauer chamber was applied for cell quantification. For differential cytological analysis, a dried cell smear was prepared with an aliquot of the suspension, and stained with May-Grünwald-Giemsa staining. The slides were analyzed by light optical microscopy. At least 500 leukocytes were counted per high-power field, and the absolute differential cell counts were calculated by multiplying the percentage of each given cell type by the total cell count.
Serology
Mice were anesthetized and blood was collected, through the retro-orbital plexus, at days 0, 36 and 255. Then, sera were evaluated for the presence of IAV-specific antibodies by hemagglutination inhibition (HI) assay [35]. The same IAV strains used in the virosomal vaccine composition were used as antigens in the HI assay. Results were reported as geometric mean antibody titers.
Isolation of white blood cells from the spleen
The complete spleen from each mouse was aseptically collected in RPMI 1640 medium (Gibco) at necropsy (days 36 and 255). Each spleen was mechanically dissociated, and filtered through a nylon filter (70 μm). Then, red blood cells were lysed with Pharm Lyse™ buffer (BD Biosciences). The lysis reaction was stopped by adding of RPMI 1640 medium with 2% FBS, and the cells were washed twice. The cells were resuspended in complete RPMI 1640 medium, supplemented with 10% FBS (Gibco, Brazil), 1 mM GlutaMAX (Gibco, Brazil), 25 mM HEPES (Sigma-Aldrich, USA), 1 mM sodium pyruvate (Sigma-Aldrich, USA), 50 M 2-mercaptoethanol (Gibco, USA) and 100 U/mL penicillin-streptomycin (Sigma-Aldrich, USA). Finally, the cell number was counted with 0.4% trypan blue to determine the viable cell concentration. In general, the mice spleens yielded around 8–10 × 107 viable splenocytes. The cells were resuspended in 95% FBS + 5% DMSO (Sigma-Aldrich) and cryopreserved at a final concentration of 2 × 106 cells/mL.
In vitro cell proliferation assay
Viable spleen cells were thawed and suspended in DPBS at a concentration of 5 × 106 cells/mL and labeled with 2.5 µM carboxyfluorescein succinimidyl ester (CFSE) by applying the CellTrace™ CFSE Cell Proliferation kit (Invitrogen), according to previous reports [36]. After CFSE labeling, splenocytes were resuspended in complete RPMI 1640 medium, plated in 24-well plates (5 × 106 cells/well). Subsequently, the cells were stimulated in vitro by adding 8000 TCID50/mL of the three vaccine viruses (H1N1, H1N2 and H3N2) for 96 h at 37 °C, under 5% CO2, in the dark. For the negative control, only culture medium was added to cells (non-virus-stimulated cells), and for the positive control, the cells were stimulated separately with 5 µg/mL of Concanavalin A from Canavalia ensiformis (Sigma-Aldrich). After in vitro stimulation of cells with H1N1, H1N2 and H3N2 viruses, lymphocyte proliferation from spleens was measured as an indicator of T and B-cell responses at 21 days after the boost immunization.
Cell staining and flow cytometry
CFSE in combination with monoclonal antibodies (mAbs) enabled concomitant access to cell proliferation and activation status of cell subpopulations. Proliferation was detected by loss of CFSE fluorescence [36]. Flow cytometry analysis was performed to identify and quantify lymphocyte subpopulations (CD3e, CD4, CD8α, CD19, CD45R/B220 and sIgM mAbs), to measure the levels of cellular activation and mature resting marker expression (CD23, CD25 and CD69 mAbs), and cellular memory marker expression (CD62L and CD44 mAbs) (Becton Dickinson; Table 1). Cell densities were calculated and transferred to flow cytometry tubes (approximately 1 × 106/well). Cells were treated with a blocking solution (10% v/v normal mouse serum) to block unoccupied binding sites on the second antibody, and thus cells were labeled for 30 min at room temperature in a dark room with a cocktail of specific mAbs (Table 1), 7-aminoactinomycin D (7-AAD) and isotype controls (BD Biosciences). Antibody concentrations used were in accordance with the manufacturer’s instructions.
Before sample analysis, the flow cytometer settings were checked using Cytometer Setup and Tracking beads (CS&T beads, BD), as described in the manufacturer’s instructions. Compensation beads were used with single stains of each antibody to establish the compensation settings. The compensation matrix was identically applied to all samples. The side scatter (SSC) threshold level was set at 8,000 units to eliminate debris. Gates considered indicating positive and negative staining cells were set based on fluorescence minus one (FMO) tests of samples and these gates were applied consistently to each sample, allowing minor adjustments for SSC variability. 7-Aminoactinomycin D (7-AAD) staining was used to distinguish dead from viable cells by flow cytometry. In the preliminary procedure to set up instrument technical parameters, isotype controls were used to evaluate fluorochrome unspecific staining. Buffer for flow cytometry was prepared in PBS containing 0.01% w/v sodium azide (Sigma-Aldrich), 2% v/v FBS (Gibco) and 2% w/v bovine serum albumin (BSA, Sigma-Aldrich).
A total of 100,000 events per tube were acquired in the flow cytometer (Accuri C6 Plus and FACSCanto, Becton-Dickinson, USA) and analyzed using the FlowJo software (Becton-Dickinson, USA). The lymphocyte gate was set on light-scatter properties (Forward Scatter vs. Side Scatter). Proliferation by CFSE (reflected by successive reduction of fluorescence intensities by dye distribution to daughter cells) was measured by flow cytometry. Results were expressed as percentages of stained cells.
Statistical analysis
Differences between vaccinated groups (intranasal – IN and intramuscular – IM routes) and non-vaccinated groups (NV) in biochemical data, immunoglobulins, apoptosis rate, and BALF cell count were analyzed through ANOVA, using the MIXED procedure of Statistical Analysis System (SAS – Cary, North Carolina, USA). In addition, differences between these groups in the in vitro cell proliferation assay were evaluated using the two-sided Student’s t test. Analysis of variance (F test) was carried out to assess the effect of the administration route and age in the in vitro cell proliferation assay, applying the Tukey test whenever a significant effect (P ≤ 0.05) of virosome was detected. For the analysis of HI, the descriptive level of probability of Fisher’s exact test was used; percentages followed by distinct letters on the lines differ significantly according to Fisher’s exact test. P values ≤ 0.05 were considered statistically significant [37].