2.1. Reagents
The dried rhizomes of Bletilla striata were harvested in Pu′er city, Yunnan province, China. The voucher specimen was identified by Professor Zhi-Kun Wu in Kunming Institute of Botany, Chinese Academy of Sciences. Diethylaminoethanol (DEAE) Sepharose fast flow and high-resolution sephacryl S-200 were from GE Healthcare (Sweden). Monosaccharide standards (glucose [Glc], glucuronic acid [GlcA], mannose [Man], rhamnose [Rha], galactose [Gal], galactose acid [GalA], fucose [Fuc], xylose [Xyl] and araose [Ara]), 1-phenyl-3-methyl-5-pyrazolone (PMP), trifluoroacetic acids (TFA) and dimethyl sulfoxide (DMSO) were from Innochem Co. Ltd (Beijing, China). Lipopolysaccharide (LPS), T-series dextran and 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H tetrazolim (MTS) were from Sigma-Aldrich (USA). The NO-detecting kit was from Beyotime Institute of Biotechnology Co. Ltd (Shanghai, China). Mouse tumor necrosis factor-α (TNF-α), mouse interleukin-6 (IL-6) and mouse interleukin-1β (IL-1β enzyme-linked immunosorbent assay (ELISA) kit was from Boster Biology Engineering Institute (Wuhan, China).
2.2. Extraction and purification of polysaccharide.
3 kg dried rhizomes of planted Bletilla striata were powdered to irregular particles and defatted with 85% ethyl alcohol (EtOH) three times, and were extracted three times by distilled water 1:15 (w/v) at 90 ℃ for 3 h per each time. All of extracts were concentrated with a rotary evaporator at 50 ℃ under reduced pressure and precipitated by adding 95% ethanol to a final concentration of 70% overnight at room temperature. Proteins in the extracts were removed by Sevage method. These protein-free extracts were lyophilized to obtain crude polysaccharide (250 g). A portion of crude polysaccharide (200 mg) which was then dissolved in 10 mg/mL of distilled water. After a centrifugation, the supernatant was harvested and injected into a 2.5×60 cm column of DEAE sepharose fast flow equilibrated with distilled water. The column came through a linear gradient elution with 0, 0.05, 0.3 and 0.5 M NaCl solution at 0.5 mL/min flow rate. Eluent (4 mL/tube) was collected by automatic collector and carbohydrates were monitored by high-performance liquid chromatographic - evaporative light scattering detection (HPLC-ELSD) assay. The eluate was concentrated, dialyzed and lyophilized to obtain sub-fractions of polysaccharide (100 mg). 0.05 M NaCl eluates were further purified by gel-permeation chromatography on a column of Sephacryl S-200 (2.5 × 100 cm) gel respectively. The eluent solvent was water with a flow rate of 0.25 mL/min. PRBS (60 mg) was purified, concentrated, dialyzed, lyophilized and stored at -20 ℃ for further analysis.
2.3. Determination of homogeneity and relative molecular weight.
The homogeneity and average molecular weight of polysaccharide were determined by high performance gel permeation chromatography (HPGPC) in an Agilent 1260 HPLC system (Agilent Co. USA), which was equipped with evaporative light scattering detectors (Alltech ELSD 2000ES, USA) and TSK-GEL G4000 PWxL column (7.8 mm×300 mm). Briefly, 1 mg/mL polysaccharide was eluted with water at 0.8 mL/min flow rate. The column was maintained at 30 ℃. The eluent was detected by an Alltech ELSD 6000 detector. Column calibration was performed with standard T-series dextrans (Mw: 5000, 12,000, 50,000, 150,000, 410,000 and 670,000 Da). The molecular weight was estimated based on the HPLC calibration curve from T-series dextran standards.
2.4. Assay for monosaccharide composition.
Monosaccharide composition of polysaccharide was analyzed by reverse-phase HPLC according to 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization method. 2 mg/mL polysaccharide solution was incubated with 1 mL TFA (4 M) at 120 ℃ for 4 h. These products were dissolved in methanol and evaporated to remove residual TFA, which was repeated four times. 50 μL polysaccharide solution, 100 μL PMP (0.5 M) and 50 μL sodium hydroxide (0.3 M) were mixed and incubated at 70 ℃ for 90 min. After adjusting pH to 7.0 with 0.3 M hydrogen chloride (HCl), 1 mL chloroform was added to the mixture. The bottom chloroform layer was removed using a pipe and the top aqueous layer was harvested for HPLC analysis. PMP-labeled polysaccharide was analyzed using an Agilent technologies 1260 series (Agilent Co. USA) which was equipped with diode array detector (DAD) detectors and Agilent ZORBAX SB-C18 column (250 mm×4.6 mm). The mobile phase was NaH2PO4/Na2HPO4 buffer (pH 6.8) and acetonitrile (v/v, 82:18). The flow rate was 1 mL/min. UV absorbance of effluent was monitored at 245 nm.
2.5. Methylation and chromatography-mass spectrometer (GC–MS) analysis.
Methylation analysis of polysaccharide was carried out. 5 mg polysaccharide was dissolved in 1.5 mL dimethyl sulfoxide (DMSO) with the addition of 100 mg NaOH. The polysaccharide was methylated by adding 1.5 mL methyl iodide (CH3I) followed by a stir for 4 h under a nitrogen protection. The redundant methyl iodide was decomposed by adding deionized water. The methylated polysaccharide was extracted with trichloromethane and evaporated to dryness. Dry methylated polysaccharide was hydrolyzed with 4 M TFA at 100 ℃ for 4 h, reduced with sodium borodeuteride (NaBD4) overnight and acetylated with pyridine and acetic anhydride at 100 ℃ for 1 h. Deionized water and dichloromethane were added into the acetylated derivatives. The organic phase was dried under a nitrogen protection. The dry product was dissolved in dichloromethane and analyzed by GC-MS. Trimethylsilylated derivatives were analyzed by HP 7890/5975C GC-MS system (Agilent Technologies Inc. USA) which was equipped with an iron trap MS detector and HP-5MS quartz capillary column (30 mm×0.25 mm). The temperature program was set as follows: the column at 150 ℃ initial temperature was heated to 200 ℃ at 2 ℃/min, increased to 240 ℃ at 5 ℃/min and held for 5 min. The injection temperature was 230 ℃. The ion source of mass spectrometer was set at 240 ℃.
2.6. Ultraviolet (UV), Infrared Spectroscopy (IR)and NMR spectra analysis.
Ultraviolet spectrum of polysaccharide (2 mg/mL) was analyzed by Shimadzu UV-2700 UV-vis spectrophotometer (Shimadzu, Japan) in a wavelength range of 190-600 nm. FT-IR spectrum was determined using fourier transform infrared spectrophotometer (FT-IR) (Nicolet iS10, Thermo Fisher Scientific Inc. America). Background was collected before every sample and the spectra were recorded from 32 scans at 4.00 cm−1 resolution with wave numbers ranging of 4000–400 cm-1. 1H and 13C NMR spectra were detected by Bruker Avance spectrometer of 600 or 800 MHz (Germany). The sample was pre-dissolved in deuterium (D2O, 99.9%) and lyophilized three times to replace exchangeable protons with D2O. All spectra were recorded with HOD suppression by presaturation. The interpretations of 1H/1H correlated spectroscopy (COSY), total correlation spectroscopy (TOCSY), 1H/13C heteronuclear single-quantum coherence (HSQC) and heteronuclear multiple bond coherence (HMBC) spectra were analyzed using a state-time proportion phase incrementation for quadrature detection in indirect dimension.
2.7. Isothermal titration calorimetry (ITC), transmission electron microscopy (TEM) and dynamic light scattering (DLS) assay.
ITC experiments were performed using a MicroCal PEAQ-ITC. To determine the interactions between polysaccharide and SARS-CoV-2 RBD protein/HIV Env expression plasmid, the entropy and enthalpy were analyzed by ITC[14]. By titrating 0.108 μM (4.9 mg/mL, 60 μL) RBD protein/3.03 μM (3.75 mg/mL, 60 μL) DNA into 0.029 μM (1.25mg/ml, 200 μL)/7.06 μM (5mg/mL, 200 μL) PRBS solution at 25℃ in water, we obtained the critical thermodynamic parameters including binding affinity (K), enthalpy changes (ΔH) and binding stoichiometry (N). For TEM and DLS assay, the nanoscale morphology and structure of polysaccharide-based nanovaccines were performed by TEM (Tecnai G2 F20 U-TWIN TEM system, American FEI). 700 ml nanovaccine aqueous solution (pH 7.4) was added into a DTS1070 disposable capillary cell to measure the zeta potential and hydrate size of nanovaccine using Zetasizer Nano ZS (Malvern, UK) at 25 °C. The data were analyzed using the MicroCal PEAQ-ITC Analysis Software provided by the manufacturer and fitted with a single-site binding model.
2.8. Molecular docking study.
The crystal structure of SARS-CoV-2 RBD protein was from PDB database (http://www.rcsb.org/). The PDB ID of SARS-CoV-2 RBD protein is 7JMO (2.359 Å). The docking simulation was carried out using auto dock 4.2 program. The geometry of RBD and polysaccharide was optimized using Avogadro software[15]. The docking was performed using the Lamarkian genetic algorithm (LGA). The number of GA runs was set to 100 and the highest populated cluster with the lowest energy conformation based on the scoring function was selected as the binding mode. Among all possible spatial conformations and interaction patterns, the conformation with the lowest energy was selected for visual analysis using PyMolv1.6. Auto dock 4.2 program was used for docking calculations between HIV DNA and polysaccharide. The structure of polysaccharide was obtained from Chem3D and the crustal structure of DNA (PDB ID 1BNA) was obtained from Protein Data Bank (http://www.rcsb.org/). The geometry of DNA and polysaccharide was optimized using Avogadro software. The docking was performed using the Lamarkian genetic algorithm (LGA). The number of GA runs was set to 100 and the highest populated cluster with lowest energy conformation based on the scoring function was selected as the binding mode. The conformation with the lowest energy was selected for visual analysis using PyMolv1.6.x.
2.9. Mouse vaccination.
All animal studies were approved by the Animal Ethics Committee of Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, and executed according to guidelines from the Committee of Welfare and Ethics of Laboratory Animals in Yunnan Province. There are six mouse groups in this study, which are (1) blank group consisting of 100 μL saline per each injection, (2) RBD group consisting of 10 μg RBD protein per each injection, (3) PRBS-RBD nanovaccine consisting of 10 μg RBD protein and 50 μg PRBS per each injection, (4) 100 μL blank plasmid group, (5) HIV DNA vaccine group consisting of 10 μg HIV Env expression plasmid and (6) PRBS-HIV DNA nanovaccine group consisting of 10 μg HIV Env expression plasmid and 50 μg PRBS for each injection. Bal B/c mice with 6-8 weeks were intramuscularly injected three vaccines at an interval of two weeks.
2.10. Neutralization assay.
50 μL SARS-CoV-2 pseudovirus (Sino Biological) co-incubated with diluted mouse serum samples at 37 °C for 1 h, and was added into the culture well of ACE2-expressing 293T cells (5×104 cells) for 24 h. Then lysing the cells using a commercial cell lysis buffer (Promega), 10 μL luciferase substrate (Promega) was added and the relative luciferase activity was determined by the luminometer (Bio-Tech). The HOS-CD4-CCR5/CXCR4 cells (8 x 104 cells/well) were inoculated into 6-well plates; 200 μL HIV pseudovirus (SF162.LS) was added to each well, and incubated at 37°C for 1 h; DEAE was added to each well at a final concentration of 17 μg/ml, and after culturing for 5 hours, then added non-resistant complete DMEM medium to each well to a final volume of 1 ml, and incubated for 72 hours. The cells were lysed and the luciferase content was detected. The titer of anti-SARS-CoV-2 and anti-HIV neutralizing antibody was calculated by the 50% inhibitory concentration (IC50).
2.11. Enzyme-Linked Immunosorbent Assay (ELISA).
The 96-well plates (Costar) were coated with purified antigen proteins (RBD and HIV Env protein, 0.01 μg/mL) in phosphate buffer saline (PBS) buffer overnight at 4 °C. ELISA plates were blocked with 5% bovine serum albumin (BSA) in PBS with 0.05% Tween at 37 °C for 2 h. The serum samples were added into each well and incubated for 1 h at 37 °C. The 96-well plates were added into HRP-labeled antibodies with a 1:5000 dilution against mouse IgG, IgG1, IgG2a, IgG2b and IgG3 (Santa Cruz Biotechnology) for an incubation of 1 h at 37 °C. TMB as a chromogenic substrate (Sigma-Aldrich) was added with 100 μL per each well, and incubated for no more than 5 min, following a stop by adding 25 μL 2 M H2SO4. The optical density (OD) was quantified at 450 and 630 nm by an ELISA plate reader (Thermo Life Sciences).
2.12. IFN-γ enzyme-linked immunespot assay (ELISPOT).
The mouse IFN-γ/ELISPOT assay was carried out using the commercial kit (R&D). The mixture of fresh mouse splenocytes (5×105 cells) and RBD/HIV Env epitope peptides (5 μg/mL) were incubated in the 96-well plates pre-coated by IFN-γ capture antibody overnight at 37 °C with 5% CO2. The plates were washed three times with PBS consisting of 0.05% Tween 20 buffer and incubated for 2 h with the biotinylated goat-anti-mouse IFN-γ monoclonal antibody at 2 μg/mL. After 1 h incubation with an avidin horseradish peroxidase complex in PBS/0.05% Tween 20 buffer, the plates were washed three times with PBS and incubated with peroxidase substrate AEC for 30 min. The spots in each well were measured with the ELISPOT Reader System (Bio-Rad). An absolute value (SFU) > 20 in one million cells is considered positive.
2.13. Flow Cytometry Analysis.
Spleens are isolated from mice at 1 week after the last injections. Fresh splenocytes are divided into two parts. One part stimulated with RBD/HIV Env epitope pool (5 μg/mL) for 4−6 h at 37 °C and 5% CO2. The viability of splenocytes was assessed using Zombie NIRIM Fixable Viability Kit, washed by cell staining buffer and blocked with anti-FcR antibodies, and stained with surface antibodies as following: CD3-Billiant Violet 5l0, CD4-FITC, CD8a-Alexa Fluor 700 (Biolegend, the United States). After 30 min incubation at 4 ℃, splenocytes were washed twice by cell staining buffer, fixed and permeabilized using BD Cytofix/Cytoperm Fixation/Permeabilization Kit. Splenocytes were stained 30 min at 4 ℃ with intracellular antibodies of IFN-γ-APC. The viability of the other splenocytes was assessed using Zombie NIRIM Fixable Viability Kit, washed by PBS and blocked with anti-FcR antibodies, and stained with surface antibodies as following: CD45R-B220, H-2kd-FITC, CD11c-BV605, CD80-PE and CD86-PE/cy7 (Biolegend, the United States). After completing the above staining steps, cells were washed twice by PBS with centrifugation. Cells were resuspended in 200 μL cell staining buffer. All samples were acquired on a BD FACS Fortessa and results were analyzed with the FlowJo software v10.7.1.4.
2.14. Cell culture and viability assay.
Macrophage RAW264.7 cell line was from American type culture collection (ATCC), The cells were cultured in 1640 RPMI medium supplemented with 10 % fetal bovine serum and 100 U/mL of penicillin and 100 μg/mL of streptomycin at 37 ºC and 5 % CO2. Polysaccharide with a series of final concentrations (0, 50, 100 and 200 μg/mL) were supplemented into the culture medium of cells and incubated for 24 h. After removing cell-culture medium, the cell viability was detected using the CCK-8 kit (Dojindo Molecular Technologies, Inc.).
2.15. Nitric oxide (NO) assay.
NO was measured by determining the content of nitrite in cell culture supernatant by nitrate/nitrite assay. Marcophages (2×105 cells) was co-incubated with polysaccharide with different concentration (50, 100 and 200 μg/mL) or LPS (1 μg/mL) in 24-well plate for 24 h. Nitrite in the supernatant, as an indicator of NO production, was quantified by Griess reaction.
2.16. Phagocytosis activity assay.
The phagocytosis of macrophages was measured by either neutral red or green fluorescent microspheres assay (Sigma). Macrophages (2×104 cells/well) were co-incubated with polysaccharide with different concentration (50, 100 and 200 μg/mL) or LPS (1 μg/mL) in a 96-well plate at 37 ℃ for 24 h. Either 0.075% neutral red (150 μL/well) or 2 μm green fluorescent microspheres were added and incubated for 1 h at 37 ℃. For neutral red assay, 150 μL cell lying solution (ethanol: glacial acetic acid = 1:1) was added into each well and incubated at room temperature for 1 h. The absorbance value was detected in a microplate reader at 540 nm. The phagocyte phagocytosis rate was calculated according to the equation: phagocytosis rate (%) = (AS/A0) ×100%. A0 is the absorbance value in the control group. AS is the absorbance value in the treatment group. The phagocytosis of DCs was measured by green fluorescent microspheres assay. DCs (1×105 cells/well) were co-cultured with either 50 μg/mL PRBS or 1 μg/mL LPS for 24 h. For green fluorescent microspheres assay, 10 μL of 2 μm green fluorescent microspheres was added into each well and incubated at room temperature for 1 h. The fluorescent intensity was quantified using a BioTek fluorescence analysis system (Agilent).
2.17.TNF-α, IL-6 and IL-1β production.
Macrophages (2×105 cells/well) were seeded in 12-well plates and cultured for 24 h with polysaccharide with different concentration (50, 100 and 200 μg/mL) or LPS (1 μg/mL). TNF-α, IL-6 and IL-1β in supernatant samples were quantified by the commercial ELISA kits (R&D).
2.18. Physiological assay.
Each mouse was intramuscularly injected PRBS for 30 days (400 μg/kg per each day). Fresh mouse blood samples are harvested. Several physiological indicators in mouse blood, such as hemoglobin, platelet, red blood cells, albumin, urea, uric acid, alanine aminotransferase, aspartate transaminase, creatinine and amylase, are quantified by the blood examination instrument (Coulter-JT) and biochemical detector (Roche cobas 6000).
2.19. Immunohistochemical assay.
Each mouse was intramuscularly injected PRBS for 30 days (400 μg/kg per each day). The mouse organs (liver, kidney, spleen, heart and lungs) are harvested and use H&E staining to prepare pathological sections. Pathological sections are diagnosed and imaged using a photo-taking optical microscopy (Leica).
2.20. Statistical analysis.
Data were expressed as mean ± SD (standard deviation) or mean ± SEM (standard error of mean) of triplicate determination. Statistical significance was analyzed by one-way analysis of variation (ANOVA) and Student's t test with GraphPad Prism software (GraphPad, San Diego, CA, USA). P< 0.05 were statistically significant.