Experimental design
The objective of this study is to develop a new generation of influenza vaccines that are safe, efficient and virus-independent and that induce cross-protection. We developed ferritin-based nanoparticle vaccines which were fused with the ectodomain of the influenza HA protein, three sequential repeats of highly conserved M2e epitopes, and the M-cell-targeting ligand Co4B. To validate the immunoenhancing effects of the nanoparticle vaccine, we studied differences in the induction of humoral immune responses and T-cell-mediated cellular immune responses by comparing the nanoparticle vaccine with soluble HA (rHA). We further evaluated the prophylactic protection against both homologous and heterologous influenza strains via intranasal vaccination with the ferritin-based nanoparticle in mice. Statistical analyses were conducted when applicable and are explained in the figure legend. All animal procedures were performed according to the guidelines of Northwest A&F University and approved by the Institutional Animal Care and Use Committee (IACUC).
Mice, cells and viruses
Female 6-8-week-old specific pathogen-free BALB/c and C57BL/6 mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Liaoning, China). The mice were housed in individually ventilated cage (IVC) systems (Fengshi Group, Suzhou, China) and supplied ad libitum with feed and water.
MDCK cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Cytiva, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA). DC2.4 cells were cultured in 10% FBS (Gibco, USA) RPMI 1640 medium (Gibco, USA) containing 1% penicillin/streptomycin (Cytiva, USA). Sf9 and Hi5 cells were cultured in IB905 serum-free medium (Yishengke, China).
A/Puerto Rico/8/34 (PR8, H1N1), H3N2 (CVCC AV1520), A/duck/Jiangsu/k1203/2010 (H5N8) and A/Chicken/Jiangsu/7/2002 (H9N2) were cultured in pathogen-free 10-day-old embryonic chicken eggs (Boehringer Ingelheim Witte Biotechnology Co., Ltd., Beijing, China). The 50% lethal dose (LD50) of the viruses was calculated via the Reed & Muench method. The swine virus strain H3N2 (CVCC AV1520) was obtained from the Centers for Veterinary Culture Collection of China (China Institute of Veterinary Drug Control, Beijing, China). The other virus strains were stored in our laboratory.
Vector construction
The genes encoding the H1N1 (A/victoria/2570/2019 pdm09-like virus) HA protein were downloaded from the GISAID Epiflu database with accession number EPI_ISL_401903. The genes encoding the influenza HA protein were codon optimized for enhanced expression in Spodoptera frugiperda (Sf9) insect cells and they were synthesized biochemically by GenScript (Nanjing, China). On this basis, various gene combinations, such as HM and CHM, were constructed via overlapping polymerase chain reaction (PCR). HM fusion genes were generated by fusing three sequential repeats of M2e to the C-terminus of the ectodomain of HA (residues HA1 1–HA2 174) with a Glu-Ala-Ala-Ala-Lys linker. The genes encoding the Co4B peptide were attached to the N-terminus of HM fusion genes via a Gly-Gly-Gly-Gly-Ser linker to generate CHM. All the genes were separately fused to the N-terminus of Helicobacter pylori ferritin (residues 5-167, GenBank: NP_223316) via a (G4S)3 linker, and denoted as HA-f, HM-f and CHM-f. And a 6x His tag was added to the C-terminus of ferritin. The soluble HA protein (rHA) in the trimeric form (trimeric HA) contains the full-length HA gene. It is used as a control against nanoparticle vaccines. All the genes mentioned above were cloned and inserted into pBacPAK9 baculovirus transfer vectors between the BamHI- NdeI sites downstream of the polyhedrin promoter.
Protein expression and purification
To produce rHA, HA-f, HM-f, CHM-f and ferritin-only nanoparticles, the plasmids were co-transfected into Sf9 cells with qBac Bacmid (Shaanxi Bacmid Biotechnology Co., Ltd., Yangling, China) and Sofast Transfection reagent (Sunma, Xiamen, China). Six days after transfection, the recombinant baculovirus (rBV) was passaged to amplify the viral load to the 3rd generation, and the 3rd generation of rBV was used to infect Hi5 cells to express the recombinant protein. Forty-eight hours after infection, the medium supernatant was harvested via centrifugation at 10,000 rpm for 20 min. The target proteins were then concentrated, and the buffer was exchanged with phosphate buffer (25 mM PB, 150 mM NaCl, 0.05% Tween 20, pH 7.0) via cross-flow ultrafiltration.
A two-step purification process was employed to purify the target proteins. Initially, the proteins were purified through size exclusion and binding chromatography using a Capto™ Core 400 column (Cytia, USA), with the effluent collected. The collected effluent underwent further purification via ion-exchange chromatography on an Nuvia™ HP-Q column (Bio-Rad, USA), with the effluent corresponding to each signal peak being collected. After overnight dialysis, the purified proteins were filtered through a 0.22-µm membrane, and the protein concentration was determined with a BCA protein assay kit (Thermo, USA).
Characterization of the nanoparticles
The purified proteins were verified by reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The morphological features of the nanoparticles were analyzed via transmission electron microscopy. Initially, the purified protein was diluted to 0.2 mg/mL with phosphate buffer. Subsequently, 10 µL of the diluted solution was added dropwise to carbon-coated copper grids. The grids were allowed to dry at room temperature for 2 min, followed by staining with 2% phosphotungstic acid for another 2 min, and the excess liquid was then removed via filter paper. Upon drying, images were recorded on an HT7800 microscope (Hitachi, Japan) at 80 kV.
The particle sizes of the nanoparticles were measured at 25 °C via dynamic light scattering (DLS) using a Zetasizer Nano ZS ZEN3600 (Malvern Instruments Ltd., UK). The purified protein was first diluted to an appropriate concentration in phosphate-buffered saline prior to analysis. The diluted sample was subsequently placed in a plastic cuvette and measured at a fixed scattering angle of 90° for particle size detection.
Stimulation of DCs by nanoparticles in vitro
Murine bone-marrow-derived DCs (BMDCs) were prepared from bone marrow of 6-8-week-old C57BL/6 and cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA), 1% penicillin/streptomycin (Cytiva, USA), 20 ng/mL of GM-CSF (R&D Systems, USA) and 10 ng/mL of IL-4 (R&D Systems, USA) at 37 °C and 5% CO2. Following 6 days of culture, BMDCs were harvested and transferred to 24-well plates (5 × 105 cells/well), and cultured overnight. And BMDCs were treated with LPS (1 µg/mL, Sigma-Aldrich, USA), rHA (5 µg/mL), CHM-f (5 µg/mL) or PBS (10 µL) respectively for another 24 h for further assays.
During the experiment to assess DCs maturation, the antigen-treated BMDCs were collected and washed three times with PBS. Then, the mature BMDCs were stained with fluorescent antibodies, containing CD11c-PerCP-Cy5.5 (BD, USA), CD80-PE (BD USA), and MHC II-FITC (BD, USA), for 30 min at 4 ℃. Following samples being washed three times with PBS, DCs maturation markers were measured with a BD FACSAria™ III flow cytometer.
Cellular uptake of nanoparticles in vitro
The antigen uptake capacity by cells was evaluated by doing a dextran uptake experiment. 5 × 105/mL antigen-treated mature BMDCs were collected, then centrifuged at 1000 g for 5 minutes, and washed three times with PBS. The cells were incubated with 1 mg/mL FITC-dextran (MW 40 000; Sigma-Aldrich, USA) at 37 °C or4 °C for 2 h. Mean fluorescence intensity (MFI) was measured by flow cytometry. ∆MFI (increase in mean fluorescent intensity) was calculated as follows: ∆MFI = MFI (37 °C treatment) – MFI (4 °C treatment).
To assess the uptake of nanoparticles by cells, CHM-f was labeled using an FITC-conjugation kit (MCE, USA), and NHS-FITC-tagged CHM-f was evaluated by confocal fluorescence imaging. DC2.4 cells were cultured at low density in 35 mm confocal dishes (Biosharp, China) overnight, and FITC-labeled CHM-f was then added (30 µg/well) and incubated for 24 h. Following the incubation, supernatants were gently discarded and the plates washed three times with PBS. Cells were fixed with 4% paraformaldehyde (Biosharp, China) for 15 min, washed 3 times with PBS, and permeabilized with 0.1% Triton X-100/PBS (Biosharp, China) for 5 min. Phalloidin-CoraLite 594 (Proteintech, China) and DAPI (Beyotime, China) were used to stain of F-actin and nuclei, respectively. And confocal images were acquired using an Nikon A1 + A1R + confocal microscope. Fluorescence intensity was measured using Image J software.
Animal study
Female BALB/c mice (6–8 weeks old) were randomly divided into 10 groups. The mice were intranasally (i.n.) immunized with 50 µL of rHA, HA-f, HM-f or CHM-f in saline (15 µg of H1 protein per mouse). We previously conducted studies on the immune-enhancing effects of different doses of CpG complex adjuvants on vaccines, and the results showed that 20 µg CpG induced the optimal immunity [35]. For the adjuvant groups, all the proteins were mixed with 20 µg of CpG IAMA-002 (JSIAMA, China) before immunization. Additionally, serving as positive controls, mice were intramuscularly (i.m.) immunized with a quadrivalent inactivated influenza vaccine (QIV) containing the same 2019 pdm-like HA at equimolar concentrations of HA. Mice as negative controls were immunized with 50 µL of PBS. The mice were vaccinated twice at an interval of 4 weeks.
Serum samples were collected 3 weeks post-priming and post-boosting immunizations. Nasal and lung washes were performed 3 weeks after immunization and was done by flushing the nostrils or lungs with 1 mL of pre-cooled sterile PBS, followed by centrifugation at 12,000 rpm for 3 min. The spleens of the immunized mice were collected 3 weeks after boosting immunization, and the lymphocytes were harvested using the Mouse 1× Lymphocyte Separation Medium (DAKEWE, China).
Four weeks after immunization, the immunized mice were challenged intranasally (i.n.) with a 15× median lethal dose (LD50) of A/PR/8 (H1N1), a 10× LD50 of H3N2 (CVCC AV1520), a 10× LD50 of A/duck/Jiangsu/k1203/2010 (H5N8) or a 10× LD50 of A/Chicken/Jiangsu/7/2002 (H9N2). Mice were monitored daily for body weight loss and survival rate for 14 days following the challenge. Weight loss greater than 25% of the initial weight was used as the humane endpoint. Five days postinfection, four mice were euthanized, and their lung tissues were harvested for histopathological examination and lung viral titre determination. All viruses and infectious samples were handled in a biosafety level 3 (BSL-3) containment facility except for H1N1 and H3N2, which were handled under BSL-2 laboratory conditions.
Enzyme-linked immunosorbent assay
Anti-HA/M2e IgG, IgG subclasses and IgA antibody titres were determined via antibody ELISA in sera, BALF and nasal wash samples. Briefly, HA or M2e peptide was coated on 96-well plates at a concentration of 1 µg/mL, 100 µL per well. The plates were then incubated at 4 °C overnight. After washing three times with PBST, the plates were blocked with 5% nonfat milk at 37 °C for 2 h and washed with PBST three times. The immune sera were then serially diluted 2-fold, 100 µL was added to each well of the plates, and the plates were incubated at 37 °C for 1 h. Subsequently, HRP-conjugated goat anti-mouse IgG (Biodragon, China), HRP-conjugated goat anti-mouse IgG1 (Biodragon, China), HRP-conjugated goat anti-mouse IgG2a (Biodragon, China) or HRP-conjugated goat anti-mouse IgA (Proteintech, China) at a 5000-fold dilution was added, and the mixture was incubated at 37 °C for 1 h. Then, 100 µL of 3,3′,5,5′-tetramethylbenzidine solution (TMB, Beyotime, China) was added, the mixture was incubated at 37 °C for 15 min, and the stop solution was then added. The absorbance at 450 nm was measured via a Prolong DNM-9602 Microplate Reader (Pro-long New Technology Co., Ltd., China), and the highest dilution with an OD450 value of more than twice that of the PBS group was regarded as the endpoint of the antibody titre.
HAI and viral neutralization assay
HAI titres of immunized mouse serum samples were determined using 1% chicken erythrocytes and 4 HA units of A/victoria/2570/2019 (IVR-215) antigen. Before performing the HAI assay, the serum samples were treated with receptor destroying enzyme (RDE; Denka Seiken Co., Ltd., Japan) overnight at 37 °C and then heated at 56 °C for 30 min to remove nonspecific inhibitors. Finally, the concentrated chicken erythrocytes were added to RDE-treated sera at a 1:20 volume ratio to remove nonspecific agglutinins. The treated sera were diluted 2-fold serially with saline in 96-well plates, and the highest dilution of serum that inhibited virus haemagglutination was deemed the HAI titre.
A neutralization assay was used to determine the neutralizing antibody titres in the serum. First, the mouse serum samples were inactivated at 56 °C for 30 min, and two-fold serial dilutions were then performed and mixed with 100 × TCID50 of influenza virus and incubated at 37 °C for 2 h. Afterward, the mixture was added to MDCK cells (1.5 × 105/mL, 100 µL/well, with 2 µg/mL TPCK-trypsin) in 96-well plates and incubated for 3 days at 37 °C. The neutralization titres were determined by the combination of the cytopathic effect on the cells and the standard haemagglutination assay.
Flow cytometry analysis
The CD3+CD4+ and CD3+CD8+ T-cell subpopulations and intracellular cytokines in the spleen were detected by flow cytometry. The spleens of immunized mice were collected 3 weeks after boost immunization, and lymphocytes were harvested using the Mouse 1× Lymphocyte Separation Medium. The isolated lymphocytes were resuspended in RPMI 1640 medium, transferred to 24-well plates and incubated at 37 °C for 30 min. Then, individual peptide pools (15-mer overlapping by 11 residues, 2.5 µg/mL for each peptide), which cover the HA protein of H1N1, along with 1 µL protein transport inhibitor (BFA, BD, USA), were added to stimulate the cells at 37 °C for 5–6 h. Stimulated lymphocytes were harvested and stained with 50 µL of live/dead stain containing 50 µL of FCS (PBS with 1% FBS) and 1 µL of fixable viability stain 620 (BD, USA) for 15 min at RT and then centrifuged at 600 × g for 5 min, after which the supernatant was discarded. Subsequently, 50 µL of Fc blocker (BD, USA) was added to each sample and incubated at 4 °C for 10 min to eliminate nonspecific binding. After incubation, cell surface staining was performed for 30 min at 4 °C in the dark with APC-Cy7-antimouse-CD3 (BD, USA), PerCP-Cy5.5-antimouse-CD4 (BD, USA), and AmCyan-anti-CD8α antibodies (BD, USA). After cell surface staining, cell fixation and membrane breaking were performed by adding fixation/permeabilization solution (BD, USA), followed by intracellular cytokine staining (ICS) with FITC-conjugated rat anti-mouse IFN-γ (BD), PE-Cy™7-conjugated rat anti-mouse IL-4 (BD), PE-conjugated rat anti-mouse IL-2 (BD), CD154 (CD40 ligand) monoclonal antibodies and eFluor™ 450 (Thermo, USA) and incubation at RT for 40 min. After washing once with 100 µL of 1× Perm Wash solution (BD), the cells were resuspended in 150–200 µL of FCS. The stained cells were tested with a BD FACSAria™ III flow cytometer and analyzed via FlowJo software.
Histopathological analysis of the lungs
Five days after the challenge, four mice per group were euthanized, and lung tissues were collected from the infected mice. The right lung lobes were fixed in 4% paraformaldehyde overnight at 4 °C and then embedded in paraffin, followed by sectioning and staining with haematoxylin and eosin (H&E). The images of the lung sections were recorded by Chengdu Lilai Biotechnology Co., Ltd.
Determination of lung virus titres
Lung tissues were homogenized, and the supernatants were obtained via centrifugation at 12,000 rpm for 5 min. Total viral RNA was extracted from the supernatants using a viral genome DNA/RNA extraction kit (TIANGEN, China), after which the RNA was reverse transcribed to cDNA using a reverse transcription kit (TransGen, China). Then, the copy numbers of the viral genome were detected via real-time fluorescence quantitative PCR (qPCR).
Statistical analysis
All the data were analyzed using GraphPad Prism software (version 8.0.2) and presented as the means ± SEMs. The unpaired t-test was used to estimate the statistical significance of differences between two groups, and one-way ANOVA with Tukey’s multiple comparison test was used to compare the statistical significance between multiple groups. P