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Jan 2020

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A Potent Vaccine Delivery System
一个有效的疫苗输送系统   

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Abstract

Most vaccines require co-delivery of an adjuvant in order to generate the desired immune responses. However, many currently available adjuvants are non-biodegradable, have limited efficacy, and/or poor safety profile. Thus, new adjuvants, or self-adjuvanting vaccine delivery systems, are required. Here, we proposed a self-adjuvanting delivery system that is fully defined, biodegradable, and non-toxic. The system is produced by conjugation of polyleucine to peptide antigen, followed by self-assembly of the conjugate into nanoparticles. The protocol includes solid-phase peptide synthesis of the vaccine conjugate, purification, self-assembly and physicochemical characterization of the product. Overall, this protocol describes, in detail, the production of a well-defined and effective self-adjuvanting delivery system for peptide antigens, along with tips for troubleshooting.

Keywords: Poly (hydrophobic amino acid) (聚(疏水氨基酸)), Delivery system (分配系统), Subunit vaccine (亚单位疫苗), Peptide synthesis (肽合成), Particle self-assembly (粒子自我组装), Self-adjuvanting nanoparticles (自佐剂纳米颗粒), Polyleucine (聚亮氨酸), Secondary structure (二级结构)

Background

Peptide subunit vaccines, which use the small antigen fragments (epitopes) to trigger protective immune responses against infectious diseases, are one of the most promising vaccine technologies to have emerged in recent decades (Skwarczynski and Toth, 2016; Malonis et al., 2020). However, as peptides, themselves, are always poorly immunogenic, they need to be co-administered with an adjuvant (immune stimulator) and/or delivery system (Azmi et al., 2014; Nevagi et al., 2018). Currently, only a few options exist when it comes to adjuvants that are safe enough to be administered to humans. While more numerous in options, experimental adjuvants are often poorly defined, toxic, or have limited efficacy (Shi et al., 2019). One of the most recent strategies developed to deliver vaccines utilizes nanostructures with self-adjuvanting properties (Skwarczynski and Toth, 2014). Self-assembling polymers, in particular, have been widely investigated (Zhao et al., 2017; Nevagi et al., 2019). However, the structures of these polymers are not fully defined (number of units, stereochemistry) and, therefore, batch variability may affect vaccine activity and safety profile.


We have conceptualized, designed, and developed a new vaccine adjuvant/delivery system to overcome the disadvantages outlined above. This system is based on fully-defined and biodegradable polymers built from our own natural hydrophobic amino acids. The lead vaccine candidate produced based on this system was able to stimulate the production of highly opsonic antibodies against six clinical isolate strains of group A streptococcus in mice (Skwarczynski et al., 2020). The compound was more efficient than the powerful, but toxic, “gold standard” Complete Freund’s Adjuvant and did not induce undesired inflammatory responses. The strategy to deliver antigenic epitopes attached to self-adjuvanting amino acid-based polymer described here offers an attractive, safe alternative to conventional vaccine adjuvants. Importantly, this approach can be fully customized to match the properties of the antigen of choice. The procedure on how to produce this vaccine candidate (Figure 1) is presented here, with reference to the published vaccine study (Skwarczynski et al., 2020). Notes provide additional helpful information.



Figure 1. Flowchart of vaccine candidate synthesis, purification and characterization steps

Materials and Reagents

Note: All chemicals should be analytical grade, unless stated otherwise.

  1. Vaccine candidate synthesis

    1. Chemical resistance gloves (Ansell, catalog number: 02-100)

    2. Rink amide p-methyl-benzhydrylamine hydrochloride (pMBHA·HCl) resin (substitution: 0.59 mmol/g; 100-200 mesh; Peptides International, catalog number: RMB-1045-PI)

    3. N,N-dimethylformamide (DMF; Merck, catalog number: 227056) (see Note 1)

    4. N,N-diisopropylethylamine (DIPEA; 6.2 equivalent; Merck, catalog number: 387649)

    5. Trifluoroacetic acid (TFA; Merck, catalog number: 302031)

    6. Butyloxycarbonyl (Boc)–protected l-amino acids (0.84 mmol/g; 4.2 equivalent; Novabiochem Merck Chemicals and Mimotopes)

    7. 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU; 0.5 M; 4 equivalent; Mimotopes, catalog number: 148893-10-1) solution: 9.5 g HATU in 50 ml DMF (store solution at 0 °C for no longer than 1 week after preparation) (see Note 2)

    8. Capping solution: 5% acetic anhydride (Sigma-Aldrich, catalog number: 320102), 5% DIPEA, and 90% DMF (v/v/v)

    9. Dichloromethane (DCM; Merck, catalog number: 270997)

    10. Piperidine deprotection solution: 20% piperidine (Sigma-Aldrich, catalog number: 8.22299) and 80% DMF (v/v)

    11. Methanol (Merck, catalog number: 34860)

    12. p-Cresol (Sigma-Aldrich, catalog number: C85751)

    13. Hydrofluoric acid (HF; Ghc Gerling, Holz & Co. Handels gmbh, catalog number: 3100, Hydrogen Fluoride [99.95%])

    14. Diethyl ether (Sigma-Aldrich, catalog number: 91238)

    15. n-hexane (Merck, catalog number: 1.04367)

    16. Acetonitrile (Merck, catalog number: 271004)

    17. Endotoxin-free Milli-Q water (sensitivity of 18.2 MΩ.cm at 25 °C and total organic content below 5 parts per billion)

    18. Solvent A: 100% Milli-Q water and 0.1% TFA (v/v; solution can be stored at room temperature for up to 3 months)

    19. Solvent B: 90% acetonitrile, 10% Milli-Q water, and 0.1% TFA (v/v/v; solution can be stored at room temperature for up to 3 months)


  2. Vaccine candidate purification

    1. Phenex syringe filter (0.45 µm; Phenomenex, catalog number: AF3-3107-52)

    2. Reagents listed previously (Solvent A and B)


  3. Vaccine candidate characterization

    1. Disposable capillary cuvettes (Malvern Analytical, model: DTS1070)

    2. Whatman filter paper (Merck, catalog number: WHA1005090)

    3. Phosphate-buffered saline (PBS; ThermoFisher Scientific, catalog number: 10010031)

    4. Phosphotungstic acid stain (2%): 2 mg phosphotungstic acid hydrate (Sigma-Aldrich, catalog number: P4006-25G) in 100 ml Milli-Q water (stir for 1 h, then filter; solution can be stored at 2-8 °C for up to 3 months)

Equipment

  1. Vaccine candidate synthesis

    1. Laboratory glassware

    2. CEM Discover Solid Phase Synthesis (SPS) reactor (CEM Corporation, model: Discover SPS; see Software 1) (see Note 3)

    3. Peptide synthesis vessel (CEM Corporation, catalog number: 170897) (see Note 4)

    4. Glass peptide synthesis vessel (Sigma-Aldrich, catalog number: Z41,850-1)

    5. CEM Discovery SPS vacuum manifold filtration apparatus (CME Corporation, catalog number: 167993) (see Note 4)

    6. Scintillation vials (Merck, catalog number: DWK986568)

    7. Vortex mixer (Phoenix Instruments, model: RS-VA 1) or sonicator (Baranson Ultrasonicator Corporation, catalog number: 2510E-MTH)

    8. Rotary mixer (Ratek Instruments, catalog number: RSM7DC)

    9. Desiccator

    10. Hydrofluoric acid (HF)-reaction apparatus (including HF reaction vessel) for peptide cleavage from the resin (refer to Jadhav et al., 2020)

    11. Alpha 2-4 LD freeze dryer (John Morris Scientific, catalog number: 101521)


  1. Vaccine candidate purification

    1. Shimadzu preparative reverse-phase HPLC (RP-HPLC) instrument (Shimadzu, models: LC-20AP × 2, CBM-20A, SPD-20A, FRC-10A) with a 20.0 ml/min flow rate (see Software 2)

    2. Vydac C4 (Hichrom, catalog number: 214TP54, 5 µm, 4.6 × 250 mm; and 214TP1022, 10 µm, 22 × 250 mm) or C8 columns (Hichrom, catalog number: 208TP54; 5 μm, 4.6 × 250 mm)

    3. Perkin-Elmer-Sciex API3000 electrospray ionization mass spectrometry (ESI-MS) instrument (Applied Biosystems/MDS Sciex, model: Sciex API3000; see Software 3)

    4. Shimadzu analytical RP-HPLC instrument (Shimadzu, models: DGU-20A5, LC-20AB, SIL-20ACHT, SPD-M10AVP) with a 1.0 ml/min flow rate (see Software 2)


  1. Vaccine candidate characterization

    1. Malvern Zetasizer dynamic light scattering (DLS; Malvern Instruments, model: Nano ZS; see Software 4)

    2. JEM-1010 transmission electron microscope (TEM; JEOL, see Software 5)

    3. Carbon-coated copper grids (Pure Carbon Film 200 mesh, Ted Pella, catalog number: 01840-F)

    4. Jasco J710 circular dichroism (CD) spectrometer (JASCO Corporation, model: J710; see Software 6)

    5. CD 1.0 mm cell (Starna, catalog number: 21/Q/1/CD)

Software

  1. SynergyTM (CME Corporation, North Carolina, USA, www.cemsynthesis.com)

  2. LabSolutions (Shimadzu, Kyoto, Japan, www.shimadzu.com)

  3. Analyst® 1.6 (Applied Biosystems/MDS Sciex, Toronto, Canada, www.sciex.com)

  4. Malvern Zetasizer Analyzer 6.2 (Malvern Instruments, Worcestershire, UK, www.malvernpanalytical.com)

  5. Olympus Soft Imaging Solutions (Olympus Corporation, Tokyo, Japan, www.olympus-global.com)

  6. Spectra ManagerTM (JASCO Corporation, Tokyo, Japan, www.jascoinc.com)

Procedure

  1. Vaccine candidate synthesis (see Note 5)

    1. Resin swelling: Weight out 339 mg pMBHA•HCl resin (0.2 mmol equivalent) into a peptide synthesis vessel and add 10 ml of DMF and 0.216 ml of DIPEA (see Note 6). Let the resin swell for at least 2.5 h (see Note 7).

    2. Resin washing: Drain the solvent using vacuum filtration and wash the resin three times using DMF (~5 ml per wash; see Notes 8-9).

    3. Boc deprotection (see Note 9): Drain all of the solvent before adding ~5 ml of neat TFA. Stir the resin gently for 2 min with a stirring rod (see Note 10).

    4. Drain the TFA, and repeat Step A3.

    5. Resin washing: Drain the solvent using vacuum filtration and wash the resin five times with DMF (~5 ml per wash; see Notes 8-9).

    6. Amino acid activation: Weight out each amino acid in scintillation vials. Add 1.6 ml of HATU and 0.181 ml of DIPEA (see Note 11).

    7. Amino acid coupling: Drain all of the solvent off before adding the preactivated amino acid to the washed resin and stir gently (see Note 12). Insert the vessel, with temperature probe, into the microwave and heat for 5 min at 70 °C (see Note 13).

    8. Resin washing: Drain the solvent using vacuum filtration and wash the resin three times with DMF (~5 ml per wash; see Note 8).

    9. Repeat Steps A6-A7 for the second coupling (10 min at 70 °C).

    10. Resin washing: Drain the solvent using vacuum filtration and wash the resin five times with DMF (~5 ml per wash; see Note 8).

    11. Acetylation: Drain the solvent completely, then add 5 ml of capping solution to the resin. Stir gently, then insert the vessel, with temperature probe, into the microwave and heat the mixture for 10 min at 70 °C (see Note 14).

    12. Resin washing: Drain the solvent using vacuum filtration and wash the resin three times with DMF (~5 ml per wash; see Note 8).

    13. Repeat Step A11 for the second acetylation.

    14. Resin washing: Drain the solvent using vacuum filtration and wash the resin five times with DMF (~5 ml per wash; see Note 8).

    15. Repeat Steps A2-A10 for the following amino acid sequence, until the Peptide 1 (Figure 2) sequence is finished (Table 1, entry 1-42).



      Figure 2. Synthesis of the vaccine candidate Peptide 2, which is comprised of (i) GAS J8 B-cell epitope, and (ii) PADRE T-helper cell epitope, branched with (iii) a leucine poly(hydrophobic amino acid) (pHAA) unit via lysine linker


      Table 1. List of amino acids used for the synthesis of vaccine candidate Peptide 2

      PADRE 1 Boc-Ala-OH A J8
      (continue)
      30 Boc-Lys(2Cl-Z)-OH K
      2 Boc-Ala-OH A 31 Boc-Ala-OH A
      3 Boc-Ala-OH A 32 Boc-Glu(OcHx)-OH E
      4 Boc-Lys(2Cl-Z)-OH K 33 Boc-Arg(Tos)-OH R
      5 Boc-Leu-OH H2O L 34 Boc-Ser(Bzl)-OH S
      6 Boc-Thr(Bzl)-OH T 35 Boc-Gln-OH Q
      7 Boc-Trp(For)-OH W 36 Boc-Lys(2Cl-Z)-OH K
      8 Boc-Ala-OH A 37 Boc-Val-OH V
      9 Boc-Ala-OH A 38 Boc-Lys(2Cl-Z)-OH K
      10 Boc-Val-OH V 39 Boc-Asp(OcHx)-OH D
      11 Boc-Phe-OH F 40 Boc-Glu(OcHx)-OH E
      12 Boc-Lys(2Cl-Z)-OH K 41 Boc-Ala-OH A
      13 Boc-Ala-OH A 42 Boc-Gln-OH Q
      Branching moiety 14 Boc-Lys(Fmoc)-OH K Leucine
      pHAA
      unit
      43 Boc-Leu-OH H2O L
      J8 15 Boc-Gln-OH Q 44 Boc-Leu-OH H2O L
      16 Boc-Val-OH V 45 Boc-Leu-OH H2O L
      17 Boc-Lys(2Cl-Z)-OH K 46 Boc-Leu-OH H2O L
      18 Boc-Asp(OcHx)-OH D 47 Boc-Leu-OH H2O L
      19 Boc-Glu(OcHx)-OH E 48 Boc-Leu-OH H2O L
      20 Boc-Leu-OH H2O L 49 Boc-Leu-OH H2O L
      21 Boc-Gln-OH Q 50 Boc-Leu-OH H2O L
      22 Boc-Lys(2Cl-Z)-OH K 51 Boc-Leu-OH H2O L
      23 Boc-Leu-OH H2O L 52 Boc-Leu-OH H2O L
      24 Boc-Ala-OH A 53 Boc-Leu-OH H2O L
      25 Boc-Lys(2Cl-Z)-OH K 54 Boc-Leu-OH H2O L
      26 Boc-Glu(OcHx)-OH E 55 Boc-Leu-OH H2O L
      27 Boc-Val-OH V 56 Boc-Leu-OH H2O L
      28 Boc-Gln-OH Q 57 Boc-Leu-OH H2O L
      29 Boc-Lys(2Cl-Z)-OH K

      After coupling the final amino acid of Peptide 1, repeat Steps A2-A5 (washing and Boc deprotection), then proceed with Steps A11-A14 (Peptide 1 acetylation at the N-terminus; see Note 15).

    16. Fmoc group deprotection: Drain all of the solvent, then add 5 ml of piperidine deprotection solution to the resin and stir gently. Insert the vessel and temperature probe into the microwave. Heat to 70 °C for 2 min.

    17. Resin washing: Drain the solvent using vacuum filtration, then wash the resin three times with DMF (~5 ml per wash; see Note 8).

    18. Repeat Step A16 for the second Fmoc group deprotection (5 min at 70 °C; see Note 16).

    19. Resin washing: Drain the solvent using vacuum filtration, then wash the resin five times with DMF (~5 ml per wash; see Note 8).

    20. Repeat Steps A6-A10 for the first leucine in the pHAAs.

    21. Repeat Steps A2-A10 for the remaining leucine’s, until the Peptide 2 (Figure 2) sequence is complete (Table 1, entry 43-57).

    22. After the final amino acid coupling of Peptide 2, repeat Steps A2-A5 (washing and Boc deprotection), then proceed with Steps A11-A14 (Peptide 2 acetylation at the N-terminus; see Note 15).

    23. Resin drying: Drain the solvent using vacuum filtration, then wash the resin using ~5 ml of DMF (three times), DCM (three times), then methanol (once). Remove all solvent and leave the resin under vacuum filtration for 5 min.

    24. Leave the vessel with the resin in a desiccator under reduced pressure overnight to fully remove the solvents.

    25. Measure the finished resin-Peptide 2, then weigh out 500 mg of the resin into a 15 ml Falcon tube (see Note 17).

    26. Peptide cleavage: Transfer the 500 mg of resin into a HF reaction vessel, then add 0.25 ml of p-cresol scavenger (0.5 ml/g of resin) and 5 ml of HF (10 ml/g of resin). Cleave the peptide from the resin following the HF-cleavage protocol (see Note 18 and Jadhav et al., 2020).

    27. Reaction work up: Wash the resin with 30 ml of cold diethyl ether:n-hexane (4:1; v/v) twice, then remove the solution through filtration. Dissolve the white precipitate using 50% Solvent A and 50% Solvent B (v/v), then filter the solution (from the resin) into a round-bottom flask. Wash the resin with Solvent B and collect the filtrate into a round-bottom flask. Freeze-dry the filtrate to obtain a yellowish-white powder (crude Peptide 2).


  2. Vaccine candidate purification

    1. Purification (see Note 19): Dissolve 30 mg crude Peptide 2 in 2 ml of 50% Solvent A and 50% Solvent B (v/v). Load the solution into a 5 ml syringe and filter the solution through syringe-filter into a scintillation vial. Wash the filter with 2 ml of 50% Solvent A and 50% Solvent B (v/v). Run the filtered crude Peptide 2 using preparative RP-HPLC on a C4 column with solvent B gradients (65-85%) for 25 min, with compound detection at 214 nm.

    2. Analyze the fractions of the purified Peptide 2 using ESI-MS and analytical RP-HPLC on a C4 column with a 0-100% gradient of solvent B for 40 min and compound detection at 214 nm (see Note 20).

    3. Combine and freeze-dry the fraction based on its purity (as analyzed by ESI-MS and analytical RP-HPLC; Figure 3; Note 21). The final product should be a white solid. Molecular weight: 6521.03. ESI-MS [M + 4H]4+ m/z 1631.8 (calc. 1631.3), [M + 5H]5+ m/z 1305.6 (calc. 1305.2), [M + 6H]6+ m/z 1088.0 (calc. 1087.8), [M + 7H]7+ m/z 932.9 (calc. 932.6), [M + 8H]8+ m/z 816.3 (calc. 816.1), [M + 9H]9+ m/z 725.6 (calc. 725.6). tR = 30.9 min (0 to 100% solvent B; C4 column); purity ≥ 99%.



      Figure 3. Analysis of the purified vaccine candidate Peptide 2 by ESI-MS (left) and analytical RP-HPLC (right)


  3. Vaccine candidate characterization

    1. Measure 1.5 mg of pure Peptide 2 into a 2 ml microcentrifuge tube. Add 1 ml of PBS to prepare a 1.5 mg/ml solution (concentration of the vaccine candidate for the in vivo study). Vortex the mixture for 2-30 min (with 1-min intervals) until none of the solid peptide is visible (see Note 22).

    2. Size and PDI:

      1. Transfer 0.5 ml of self-assembled vaccine candidate (from Step C1) into a new 2 ml microcentrifuge tube, then add 1 ml of PBS to prepare a 0.5 mg/ml solution (1:3 dilution) for DLS analysis.

      2. Turn on the Malvern Zetasizer DLS, open the Analyzer software, and connect with the instrument before starting measurements.

      3. Fill the disposable cell with diluted solution (~800 µl) and insert the cell into the instrument.

      4. Perform measurements (size, PDI) at 25 °C with non-invasive backscatter at a backscattering angle of 173°. Correlation times are based on 10 s per run; at least 10 consecutive runs should be made per measurement. Take at least five measurements per sample, and measure PBS as a blank (negative control).

      5. Calculate the mean ± standard deviation for each sample using the five measurements.

      6. In general, compounds should self-assemble into a mixture of small nanoparticles (10-30 nm) and larger aggregates with high polydispersity indexes (PDI > 0.3, according to DLS).

    1. Morphology:

      1. Pipette 5 µl of the 1:3 diluted solution (the same as what was used for DLS) onto a glow-discharged carbon-coated copper grid and leave for 2 min to dry (see Note 23).

      2. Gently drain the excess liquid with a piece of filter paper and allow the grid to dry.

      3. Add one drop (~5 µl) of 2% phosphotungstic acid to the dried grid for 20 s to negatively-stain the sample, then drain the excess stain solution off with a piece of filter paper.

      4. Air-dry the grid for 5 min before observing it under TEM. Take images at an accelerating voltage of 100 kV (Figure 4).

      5. Distinct nanoparticles and chain-like aggregates of nanoparticles (CLAN) should be visible in Peptide 2 when viewed under TEM (Figure 4).



        Figure 4. Particle image of vaccine candidate Peptide 2 captured by TEM [bar 500 nm; the dark areas are a result of the negative stain (2% phosphotungstic acid)]


    1. Secondary structure:

      1. Transfer 0.1 ml of the self-assembled vaccine candidate (non-diluted sample from Step C1) into a new 2 ml microcentrifuge tube and add 1.4 ml PBS to prepare 0.1 mg/ml solution (1:15 dilution) for CD analysis (see Note 24).

      2. Set up the Spectra Manager software with the following parameters: bandwidth, 5 nm; scan rate, 50 nm/min; response time, 2 s; interval, 1 nm over the wavelength range of 195 to 260 nm.

      3. Insert the cell into the instrument and run a quick scan on an empty CD cell to make sure the cell is clean (the flat, horizontal line should be visible without substantial deviation from zero mdeg; see Note 25). Wash the cell if needed (see Note 26).

      4. Fill the CD cell with diluted solution (~200 µl) and insert the cell into the instrument. Take at least six accumulations per measurement. Run PBS or water as a blank (negative control). After all measurements, clean the cell with water or methanol, only.

      5. Using Spectra Manager, subtract the baseline spectra (PBS blank reading) from the vaccine candidate spectra before converting millidegrees (mdeg) to molar ellipticity ([θ]; degcm2dmol−1).

      6. The conversion is done via the following formula:


        [θ] = mdeg/(l × c × n) × 1000

        where:

        l = path length (0.1 cm),

        c = peptide concentration (mM), and

        n = number of amino acids in the peptide.


      7. The vaccine candidate Peptide 2 should adopt a helical conformation with a minimum at 222 nm and a lower-intensity minimum at 208 nm (Figure 5).



        Figure 5. Circular dichroism (CD) spectra of vaccine candidate Peptide 2

Notes

  1. Normal nitrile or latex gloves provide poor protection against DMF. Chemical resistance gloves should instead be worn when dealing with DMF.

  2. HATU/HBTU/HCTU can induce an allergic response if it comes in contact with skin; caution should be taken when handling these chemicals.

  3. The reactor is equipped with a vacuum manifold for liquid transfers and a fiber optic temperature probe. Each microwave system usually has its own complementary peptide synthesis vessels and vacuum filtration apparatus. Consult with your microwave provider for further information.

  4. This peptide synthesis vessel is a 20 ml, microwave-safe, open-vessel apparatus equipped with porous frit (to allow filtration of the peptide from the resin) and a cap at the bottom. Alternatively, synthesis can be done without the assistance of a microwave (heat); a glass peptide synthesis vessel may be used instead. Draining and washing of this glass vessel need to be done through filtration under vacuum. A rotary mixer is also required to provide continuous mixing.

  5. Synthesis goes from the C- to N-terminus of the peptide amino acid sequence. Make sure the sequence is correct before starting. The information provided here is calculated for a 0.2 mmol synthesis scale.

  6. During resin swelling and peptide synthesis, the resin should remain submerged in DMF solvent, with solvent level at least 2 cm higher than the resin. A rotary mixer can be used to improve swelling by continuous mixing.

  7. The resin can be left to swell for up to 24 h. Alternatively, insert the resin-containing vessel into the microwave, together with the temperature probe, and heat to 70 °C for 10 min for fast swelling.

  8. Perform each wash carefully to ensure complete removal of all reagents. Inadequate washing could result in the formation of side products during synthesis. If the microwave was used, remove the vessel from the microwave, then wash the temperature probe along with the resin and stirring rod (used in Boc deprotection).

  9. Wash the resin five times with DCM (~5 ml for each wash; instead of DMF) before and after TFA deprotection of Boc from Boc-Gln(Xan)-OH. This prevents the cyclization of glutamine.

  10. Alternatively, if the synthesis is done in a glass peptide synthesis vessel (for synthesis without microwave (heat) assistance), stirring can be done by placing the TFA/resin mixture on a rotary mixer for 2 min. Make sure the vessel is tightly closed on both ends after adding the TFA.

  11. Each amino acid should be preactivated 2-5 min before reaction. Dissolve the amino acid fully before coupling to the resin. Use a sonicator or vortex to speed up the process.

  12. Add additional DMF to the vessel to make sure the resin stays submerged.

  13. Boc-amino acids are commonly heated at 70 °C for 10 min during peptide synthesis. The double-couplings are done for 5 min, then 10 min. However, cystine, histidine, and arginine must be coupled for 15 min, twice, at 50 °C. Alternatively, for coupling without microwave (heat) assistance, place the glass vessel onto a rotary mixer for 10 min (first coupling), then 20 min (second coupling), or 20 min each for cystine, histidine, and arginine. Make sure the vessel is tightly closed on both ends.

  14. Double-acetylations are done for 10 min, each. The acetylation is performed after the first amino acid coupling to cap the unreacted site to the resin to avoid the formation of side products, which affect the purity of the final product. Do not remove Boc prior to this acetylation (non-N terminus acetylation), as it can impede any additional amino acid coupling. Alternatively, for acetylation without microwave (heat) assistance, place the glass vessel on a rotary mixer for 20 min. Make sure the vessel is tightly closed on both ends.

  15. After all amino acids have been coupled, remove the Boc protective group of the final amino acid before proceeding with acetylation at the N-terminus of the peptide.

  16. Double-deprotections are done for 2 min, then 5 min. Make sure that these deprotection steps are done after the acetylation of the N-terminus. The Fmoc protective group on the lysine (branching moiety) is removed using 20% piperidine. Piperidine also removes the formyl group of tryptophan (Boc-Trp(For)-OH; PADRE sequence). According to our experience, this does not trigger the production of side products. Alternatively, if the synthesis is done in a glass peptide synthesis vessel (for synthesis without microwave (heat) assistance), stirring can be done by placing the piperidine/resin mixture on a rotary mixer for 5 min (first deprotection), then 10 min (second deprotection). Make sure the vessel is tightly closed on both ends.

  17. 500 mg is the maximum amount of resin for efficient HF cleavage. A 15 ml Falcon tube allows for easy transfer of the resin into a HF reaction vessel.

  18. HF is highly toxic and corrosive. Follow the protocol attached to the HF apparatus precisely. If there is cysteine or methionine in the peptide sequence, add additional p-thiocresol scavenger (0.25 ml; 0.5 ml/g of resin) to the p-cresol/resin mixture. Make sure the HF is completely evaporated before proceeding to workup.

  19. Make sure the peptide is fully soluble before filtration. Add a few drops of Solvent B and use a sonicator or vortex to aid solubility. Always filter the solution before running it through ESI-MS and RP-HPLC machines. Peptide 2 is hydrophobic and needs to be run through a C4 column. More hydrophilic peptides may be purified using a C8 column for better separation. The crude compound can degrade easily and is not stable; therefore, it must be purified for long-term storage.

  20. Analytical RP-HPLC graphs show pure compounds in a single peak. The mass from this peak must match the mass of the desired peptide in ESI-MS. Peptide 2 is hydrophobic and needs to be run through a C4 column. Run more hydrophilic peptides through C8 columns (as described above).

  21. Analyze the combined fraction through analytical RP-HPLC and ESI-MS to confirm purity. Keep the pure compound in powder form at -20 °C to ease storage and prolong shelf-life.

  22. The incorporation of a polyleucine tail increases the hydrophobicity of the compound and makes it difficult to dissolve. Using both sonication and vortex can help with the process. The end product should be a white, semi-cloudy or clear solution, which contains the self-assembled vaccine candidate. Vortex the solution before use.

  23. The appropriate dilution is necessary for visualization; sample that contains too high concentration of nanoparticles will result in overlapping particles, which affects the visibility of particle morphology. Dilution ratio variability depends on the particular sample, but a concentration of 0.5 mg/ml is a good place to start.

  24. Dilution is required to avoid measurement with high tension (HT) > 600 V.

  25. A significant deviation from zero (> or < 1 mdeg) indicates that the CD cell is contaminated.

  26. Clean the cell by filling the cell with 5M nitric acid (nitric acid doesn’t damage the quartz) and leave it for a few hours or overnight. A sonicator can be used to speed up the process. Rinse the cell with water before a final wash with methanol. Remove the contents and dry the cell completely before use or storage. Clean the outside of the cell with Kimwipes, only.

Acknowledgments

This work was supported by the National Health and Medical Research Council (Program Grant APP1132975) and an Australian Government Research Training Program (RTP) Scholarship. We acknowledge the original research paper “Poly (amino acids) as a potent self-adjuvanting delivery system for peptide-based nanovaccines”, from which this protocol was derived. We also acknowledge the facilities and the scientific and technical assistance of the Australian Microscopy and Microanalysis Research Facility at the Centre for Microscopy and Microanalysis, The University of Queensland.

Competing interests

M.S., G.Z., and I.T. are co-inventors in a patent application entitled “Self assembling, self adjuvanting system for delivery of vaccines” filed by The University of Queensland (application number AU 2019900328). The remaining authors declare that they have no competing interests.

References

  1. Azmi, F., Ahmad Fuaad, A. A., Skwarczynski, M. and Toth, I. (2014). Recent progress in adjuvant discovery for peptide-based subunit vaccines. Hum Vaccin Immunother 10: 778-796.
  2. Jadhav, K. B., Woolcock, K. J. and Muttenthaler, M. (2020). Anhydrous Hydrogen Fluoride Cleavage in Boc Solid Phase Peptide Synthesis. Methods Mol Biol 2103: 41-57
  3. Malonis, R., Lai, J. R. and Vergnolle, O. (2020). Peptide-Based Vaccines: Current Progress and Future Challenges. Chem Rev 120: 3210-3229.
  4. Nevagi, R. J., Skwarczynski, M. and Toth, I. (2019). Polymers for subunit vaccine delivery. Eur Polym J 114: 397-410.
  5. Nevagi, R. J., Toth, I. and Skwarczynski, M. (2018). Peptide Applications in Biomedicine, Biotechnology and Bioengineering. Woodhead Publishing 327-358.
  6. Shi, S., Zhu, H., Xia, X., Liang, Z., Ma, X. and Sun, B. (2019). Vaccine adjuvants: Understanding the structure and mechanism of adjuvanticity.Vaccine 37: 3167-3178.
  7. Skwarczynski, M. and Toth, I. (2014). Recent advances in peptide-based subunit nanovaccines. Nanomedicine (Lond) 9: 2657-2669.
  8. Skwarczynski, M. and Toth, I. (2016). Peptide-based synthetic vaccines. Chem Sci 7: 842-854.
  9. Skwarczynski, M., Zhao, G., Boer, J., Ozberk, V., Azuar, A., Cruz, J. G., Giddam, A. K., Khalil, Z., Pandey, M., Shibu, M., et al. (2020). Poly(amino acids) as a potent self-adjuvanting delivery system for peptide-based nanovaccines. Sci Adv 6: eaax2285.
  10. Zhao, G., Chandrudu, S., Skwarczynski, M. and Toth, I. (2017). The application of self-assembled nanostructures in peptide-based subunit vaccine development. Eur Polym J 93: 670-681.

简介

[摘要]大多数疫苗需要共同递送佐剂,以产生所需的免疫应答。但是,许多当前可用的佐剂是不可生物降解的,功效有限,和/或安全性较差。因此,需要新的佐剂或自佐剂疫苗递送系统。在这里,我们提出了一种完全定义,可生物降解且无毒的自佐剂递送系统。该系统是通过将多亮氨酸与肽抗原缀合,然后将缀合物自组装成纳米颗粒而产生的。该方案包括疫苗结合物的固相肽合成,产物的纯化,自组装和理化表征。总体而言,该协议详细描述了针对肽抗原的定义明确且有效的自佐剂递送系统的生产,以及疑难解答的技巧。


[背景]肽亚基疫苗使用小抗原片段(表位)来引发针对传染病的保护性免疫反应,是近几十年来出现的最有希望的疫苗技术之一(Skwarczynski和Toth ,2016; Malonis等, 2020)。但是,由于肽本身总是很差 由于它们具有免疫原性,因此需要与佐剂(免疫刺激剂)和/或递送系统共同给药(Azmi等人,2014; Nevagi等人,2018)。当前,当涉及足以安全地施用于人类的佐剂时,仅存在几种选择。尽管有更多选择,但实验性佐剂的定义往往不明确,有毒或功效有限(Shi等人,2019)。开发来提供疫苗的最新策略之一是利用具有自佐剂特性的纳米结构(Skwarczynski和Toth,2014年)。特别是自组装聚合物已被广泛研究(Zhao等人,2017; Nevagi等人,2019 )。但是,这些聚合物的结构尚未完全定义(单位数,立体化学),因此,批次差异可能会影响疫苗的活性和安全性。

我们已经概念化,设计并开发了一种新的疫苗佐剂/递送系统,以克服上述缺点。该系统基于完全定义的,可生物降解的聚合物,这些聚合物是由我们自己的天然疏水性氨基酸构建而成的。基于该系统产生的候选铅候选疫苗能够刺激小鼠抗六种临床分离的A组链球菌菌株的高声调抗体的产生(Skwarczynski等,2020 )。该化合物比功能强大但有毒的“金标准”完全弗氏佐剂更有效,并且不会引起不良的炎症反应。本文所述的递送附着至基于自佐剂的氨基酸聚合物的抗原表位的策略为常规疫苗佐剂提供了一种有吸引力的,安全的替代方法。重要的是,可以完全定制该方法以匹配所选抗原的特性。参考已发表的疫苗研究报告(Skwarczynski等,2020 ),此处介绍了如何生产该候选疫苗的步骤(图1 )。注释提供了其他有用的信息。

图1.候选疫苗的合成,纯化和表征步骤流程图

关键字:聚(疏水氨基酸), 分配系统, 亚单位疫苗, 肽合成, 粒子自我组装, 自佐剂纳米颗粒, 聚亮氨酸, 二级结构



材料和试剂


注意:除非另有说明,否则所有化学药品均应为分析纯。


A.疫苗候选者综合     
耐化学手套(Ansell,目录号:02-100)
Rink酰胺对甲基苯甲酰盐胺盐酸盐(pMBHA · HCl)树脂(取代:0.59 mmol / g; 100-200目;Peptides International,目录号:RMB-1045-PI)
N,N-二甲基甲酰胺(DMF;默克(Merck),目录号 227056)(请参阅注1)
N,N-二异丙基乙胺(DIPEA;6.2当量; 默克(Merck),货号:387649 )
三氟乙酸(TFA; 默克(Merck),货号: 302031)
丁氧羰基(Boc)保护的1-氨基酸(0.84 mmol / g; 4.2当量; Novabiochem默克化工和Mimotopes)
2-(7-氮杂-1H-苯并三唑-1-基)-1,1,3,3-四甲基脲六氟磷酸盐(HATU; 50万;4当量; 拟人动物,目录号: 148893-10-1)溶液:溶于50 ml DMF的9.5 g HATU(溶液在0°C下储存,制备后不得超过1周)(请参见注释2)
封盖溶液:5%的乙酸酐(Sigma-Aldrich,目录号:320102),5%的DIPEA和90%的DMF(v / v / v)
二氯甲烷(DCM;默克(Merck),货号: 270997)
哌啶脱保护溶液:20%哌啶(Sigma-Aldrich,目录号:8.22299)和80%DMF(v / v)
甲醇(默克(Merck),货号: 34860)
对甲酚(西格玛奥德里奇(Sigma-Aldrich),目录号: C85751)
氢氟酸(HF; Ghc Gerling,Holz&Co. Handels GmbH,目录号:3100,氟化氢[ 99.95%] )
乙醚(Sigma-Aldrich,目录号:91238 )
正己烷(默克(Merck),目录号:1.04367)
乙腈(Merck,目录号:271004)
不含内毒素的Milli-Q水(25°C时灵敏度为18.2MΩ.cm °C和总有机物含量低于十亿分之5)
溶剂A:100%Milli-Q水和0.1%TFA(v / v;溶液在室温下最多可保存3个月)
溶剂B:90%乙腈,10%Milli-Q水和0.1%TFA(v / v / v;溶液在室温下最多可保存3个月)

B.        候选疫苗的纯化


Phenex针筒式过滤器(0.45 µm; Phenomenex,目录号:AF3-3107-52 )
先前列出的试剂(溶剂A和B)

C.候选疫苗的表征     
一次性毛细管比色皿(Malvern Analytical,型号:DTS1070)
Whatman滤纸(默克公司,目录号:WHA1005090)
磷酸盐缓冲盐水(PBS; ThermoFisher Scientific,目录号:10010031)
磷钨酸染色剂(2%):2毫克磷钨酸水合物(Sigma-Aldrich,目录号:P4006-25G)在100毫升Milli-Q水中(搅拌1小时,然后过滤;溶液可在2-8 °C下保存)C长达3个月)

设备


A.候选疫苗的合成     
实验室玻璃器皿
CEM Discover固相合成(SPS)反应器(CEM Corporation,型号:Discover SPS;请参阅软件1)(请参阅注释3)
肽合成容器(CEM Corporation,目录号:170897 )(请参阅注释4)
玻璃肽合成容器(Sigma-Aldrich,目录号:Z41,850-1)
CEM Discovery SPS真空歧管过滤设备(CME Corporation,目录号:167993 )(请参阅注释4)
闪烁瓶(默克,目录号:DWK986568)
涡旋混合器(Phoenix Instruments,型号:RS-VA 1 )或超声仪(Baranson Ultrasonicator Corporation,目录号:2510E-MTH)
旋转混合器(Ratek仪器,目录号:RSM7DC)
干燥器
氢氟酸(HF)反应 用于从树脂上裂解肽的设备(包括HF反应容器)(请参阅Jadhav等人,2020年)
阿尔法2 - 4 LD冷冻干燥机(约翰莫里斯科学,目录号:101521)

B.候选疫苗的纯化     
Shimadzu制备型反相HPLC(RP-HPLC)仪器(Shimadzu,型号:LC-20AP × 2,CBM-20A,SPD-20A,FRC-10A ),流速为20.0 ml / min(请参见软件2)
Vydac C4(Hichrom,目录号:214TP54,5 µm,4.6 × 250 mm;和214TP1022,10μm,22 × 250 mm)或C8色谱柱(Hichrom,目录号:208TP54; 5μm,4.6 × 250 mm)
Perkin-Elmer-Sciex API3000电喷雾电离质谱(ESI-MS)仪器(Applied Biosystems / MDS Sciex,型号:Sciex API3000;请参阅软件3)
Shimadzu分析型RP-HPLC仪器(Shimadzu,型号:DGU-20A5,LC-20AB,SIL-20ACHT,SPD-M10AVP),流速为1.0 ml / min(请参见软件2)

C.候选疫苗的表征     
Malvern Zetasizer动态光散射(DLS; Malvern Instruments,模型:Nano ZS;请参阅软件4 )
JEM-1010透射电子显微镜(TEM; JEOL,请参阅软件5 )
碳涂层铜栅格(纯碳膜200目,Ted Pella,目录号:01840-F)
Jasco J710圆二色性(CD)光谱仪(JASCO Corporation,型号:J710;请参阅软件6 )
CD 1.0毫米电池(斯塔纳,目录号:21 / Q / 1 / CD)

软件


Synergy TM (美国北卡罗莱纳州CME公司,www.cemsynthesis.com)
LabSolutions(Shimadzu,日本京都,www.shimadzu.com)
分析师® 1.6(Applied Biosystems公司/ MDS Sciex公司,加拿大多伦多,www.sciex.com) 
Malvern Zetasizer Analyzer 6.2(Malvern Instruments,Worcestershire,UK,www.malvernpanalytical.com)
奥林巴斯软成像解决方案(奥林巴斯公司,日本东京,www.olympus-global.com)
光谱经理TM (JASCO公司,东京,日本,WWW。jascoinc.com)

程序


候选疫苗的合成(见注5)
树脂溶胀:称量339 mg pMBHA•HCl树脂(0.2 mmol当量)肽合成容器中,加入10毫升DMF和0.216毫升DIPEA(参见注释6)。让树脂溶胀至少2.5小时(请参见注释7)。
树脂洗涤:使用真空过滤沥干溶剂和洗涤树脂使用DMF三次(约5毫升,每洗涤;见注解8 - 9)。
Boc脱保护(请参阅注9):在加入约5 ml纯净TFA之前,先排干所有溶剂。用搅拌棒将树脂轻轻搅拌2分钟(请参见注释10)。
排空TFA,然后重复步骤A 3 。
树脂清洗:使用真空过滤器排干溶剂,并用DMF清洗树脂五次(每次清洗约5毫升;请参见注释8-9)。
氨基酸激活:称量闪烁瓶中的每个氨基酸。加入1.6 ml的HATU和0.181 ml的DIPEA(请参见注释11)。
氨基酸偶联:排空所有溶剂,然后将预活化的氨基酸添加到洗涤过的树脂中并轻轻搅拌(请参见注释12)。将带有温度探头的容器插入微波炉中,并在70°C下加热5分钟(请参见注释13)。
树脂清洗:使用真空过滤器排干溶剂,并用DMF清洗树脂3次(每次清洗约5毫升;请参见注释8)。
重复小号TEPS甲6-甲7 F或所述第二耦合器(在70℃下10分钟)。
树脂清洗:使用真空过滤器排干溶剂,并用DMF清洗树脂五次(每次清洗约5毫升;请参见注释8)。
乙酰化:完全排干溶剂,然后向树脂中加入5 ml的加盖溶液。轻轻搅拌,然后将带有温度探头的容器插入微波炉中,并在70°C下加热混合物10分钟(请参见注释14)。
树脂清洗:使用真空过滤器排干溶剂,并用DMF清洗树脂3次(每次清洗约5毫升;请参见注释8)。
REPE在小号TEP甲11用于第二乙酰化。
树脂清洗:使用真空过滤器排干溶剂,并用DMF清洗树脂五次(每次清洗约5毫升;请参见注释8)。
重复S teps A 2- A 10 f或以下氨基酸序列,直到完成肽1 (图2 )序列(表1 ,条目1-42)。





图2.疫苗候选肽2的合成,该肽由(i)GAS J8 B细胞表位和(ii)PADRE T辅助细胞表位组成,并与(iii)亮氨酸聚(疏水氨基酸)( pHAA)单元通过赖氨酸接头


表1.用于合成候选疫苗肽2的氨基酸列表


帕德雷


1个


Boc-Ala-OH


一种


J8


(继续)


30


Boc-Lys(2Cl-Z)-OH


ķ


2个


Boc-Ala-OH


一种


31


Boc-Ala-OH


一种


3


Boc-Ala-OH


一种


32


Boc-Glu(OcHx)-OH


Ë


4


Boc-Lys(2Cl-Z)-OH


ķ


33


Boc-Arg(Tos)-OH


[R


5


Boc-Leu-OH H 2 O


大号


34


Boc-Ser(Bzl)-OH


小号


6


Boc-Thr(Bzl)-OH


Ť


35


Boc-Gln-OH





7


Boc-Trp(对于)-OH


w ^


36


Boc-Lys(2Cl-Z)-OH


ķ


8


Boc-Ala-OH


一种


37


Boc-Val-OH


伏特


9


Boc-Ala-OH


一种


38


Boc-Lys(2Cl-Z)-OH


ķ


10


Boc-Val-OH


伏特


39


Boc-Asp(OcHx)-OH


d


11


Boc-Phe-OH


F


40


Boc-Glu(OcHx)-OH


Ë


12


Boc-Lys(2Cl-Z)-OH


ķ


41


Boc-Ala-OH


一种


13


Boc-Ala-OH


一种


42


Boc-Gln-OH





支链部分


14


Boc-Lys(Fmoc)-OH


ķ


亮氨酸


酸度


单元


43


Boc-Leu-OH H 2 O


大号


J8


15


Boc-Gln-OH





44


Boc-Leu-OH H 2 O


大号


16


Boc-Val-OH


伏特


45


Boc-Leu-OH H 2 O


大号


17


Boc-Lys(2Cl-Z)-OH


ķ


46


Boc-Leu-OH H 2 O


大号


18岁


Boc-Asp(OcHx)-OH


d


47


Boc-Leu-OH H 2 O


大号


19


Boc-Glu(OcHx)-OH


Ë


48


Boc-Leu-OH H 2 O


大号


20


Boc-Leu-OH H 2 O


大号


49


Boc-Leu-OH H 2 O


大号


21岁


Boc-Gln-OH





50


Boc-Leu-OH H 2 O


大号


22


Boc-Lys(2Cl-Z)-OH


ķ


51


Boc-Leu-OH H 2 O


大号


23


Boc-Leu-OH H 2 O


大号


52


Boc-Leu-OH H 2 O


大号


24


Boc-Ala-OH


一种


53


Boc-Leu-OH H 2 O


大号


25


Boc-Lys(2Cl-Z)-OH


ķ


54


Boc-Leu-OH H 2 O


大号


26


Boc-Glu(OcHx)-OH


Ë


55


Boc-Leu-OH H 2 O


大号


27


Boc-Val-OH


伏特


56


Boc-Leu-OH H 2 O


大号


28岁


Boc-Gln-OH





57


Boc-Leu-OH H 2 O


大号


29


Boc-Lys(2Cl-Z)-OH


ķ


偶合后的最终氨基酸Pe的ptide 1 ,重复š TEPS甲2-甲5(洗涤和Boc脱保护),则继续进行与小号TEPS甲-11-甲14(打气潮1乙酰化在N末端;见注15 )。


Fmoc基团的脱保护:排空所有的溶剂,接着将5ml添加的哌啶去保护溶液到树脂中,轻轻搅拌。将容器和温度探头插入微波炉。加热至70°C持续2分钟。
树脂清洗:使用真空过滤器排干溶剂,然后用DMF清洗树脂3次(每次清洗约5毫升;请参见注释8)。
重复š TE p阿1 6用于第二Fmoc基团脱保护(在70 5分钟℃;见注16)。
树脂清洗:使用真空过滤器排干溶剂,然后用DMF清洗树脂五次(每次清洗约5毫升;请参见注释8)。
重复小号TEPS一个6-一10在pHAAs第一亮氨酸。
重复小号TEPS甲2-甲10对剩余的亮氨酸的,直至肽2 (图2 )序列完成(表1,条目43-57)。
在肽2的最终氨基酸偶联之后,重复S teps A 2 -A 5(洗涤和Boc脱保护),然后继续进行S teps A 11- A 14(N末端的Pe ptide 2乙酰化;请参见注释15)。 。
树脂干燥:使用真空过滤器排干溶剂,然后依次使用〜5 ml DMF(三次),DCM(三次),甲醇(一次)洗涤树脂。除去所有溶剂,并将树脂真空过滤5分钟。
将装有树脂的容器放在干燥器中减压放置过夜,以完全除去溶剂。
测量完成的树脂-肽2 ,然后在15 ml Falcon管中称出500 mg树脂(参见注释17)。
肽裂解:将500 mg树脂转移到HF反应容器中,然后添加0.25 ml对甲酚清除剂(0.5 ml / g树脂)和5 ml HF(10 ml / g树脂)。切割从树脂上肽之后的HF裂解协议(见注18和贾达夫等人,2020) 。
反应后处理:洗涤用30毫升树脂的正己烷:冷乙醚(4:1;体积/体积)两次,然后通过过滤除去该溶液。使用50%溶剂A和50%溶剂B(v / v)溶解白色沉淀物,然后将溶液(来自树脂)过滤到圆底烧瓶中。用溶剂B洗涤树脂,并将滤液收集到圆底烧瓶中。冷冻干燥滤液,获得黄白色粉末(粗肽2 )。

候选疫苗的纯化
纯化(参见注释19):将30 mg粗肽2溶解在2 ml的50%溶剂A和50%溶剂B(v / v)中。将溶液装入5 ml注射器中,然后通过注射器过滤器将溶液过滤到闪烁瓶中。用2 ml的50%溶剂A和50%溶剂B(v / v)清洗过滤器。使用制备型RP-HPLC在C4色谱柱上使用溶剂B梯度(65-85%)进行25分钟的过滤后的粗肽2电泳,运行25分钟,并在214 nm处检测化合物。
使用ESI-MS和分析型RP-HPLC在C4色谱柱上以0-100%的溶剂B梯度洗脱40分钟并在214 nm处进行化合物检测,以分析纯化的肽2的馏分(参见注释20)。
根据其纯度合并级分并冻干(通过ESI-MS和分析型RP-HPLC分析;图3 ;注21)。最终产品应为白色固体。分子量:652.13。ESI-MS [M + 4H] 4+ m / z 1631.8(计算1631.3),[M + 5H] 5+ m / z 1305.6(计算1305.2),[M + 6H] 6+ m / z 1088.0(计算。1087.8),[M + 7H] 7+ m / z 932.9(计算932.6),[M + 8H] 8+ m / z 816.3(计算816.1),[M + 9H] 9+ m / z 725.6(计算725.6)。t R = 30.9分钟(0至100%溶剂B; C4柱);t R = 30.9min。纯度≥99%。





图3.通过ESI-MS(左)和分析型RP-HPLC(右)对纯化的候选疫苗肽2的分析


候选疫苗 表征
将1.5 mg的纯肽2放入2 ml微量离心管中。加入1ml的PBS中以制备1.5毫克/毫升溶液(浓度的候选疫苗的体内研究)。涡旋混合物2 - ,直到没有固体肽是可见的30分钟(以1分钟的间隔)(见注22)。             
大小和PDI:
转移0.5毫升自组装候选疫苗(从小号TEP Ç 1)到新的2ml微量离心管中,再加入1毫升的PBS来制备0.5mg / ml的溶液(1:3稀释)DLS分析。
打开Malvern Zetasizer DLS,打开分析仪软件,并在开始测量之前与仪器连接。
用稀释溶液(〜800 µl)填充一次性试管,然后将试管插入仪器中。
在25°C下以173°的反向散射角进行非侵入性反向散射测量(尺寸,PDI)。相关时间基于每次运行10 s;每次测量至少应连续运行10次。每个样品至少进行五次测量,并将PBS作为空白(阴性对照)。
使用五次测量计算每个样品的平均值±标准偏差。
通常,化合物应自组装成小的纳米颗粒(10-30 nm)和具有高多分散性指数(根据DLS的PDI > 0.3 )的较大聚集体的混合物。
形态学:
用移液管吸取5 µl 1:3稀释溶液(与用于DLS的溶液相同)到辉光放电的碳涂层铜网上,并静置2分钟使其干燥(请参见注释23)。
用一张滤纸轻轻地沥干多余的液体,然后让网格干燥。
一滴(添加〜2%磷钨酸5微升)到干燥网格20s至负染色样品,然后沥干多余的染色液断用一张滤纸。
风干格栅5分钟,然后在TEM下观察。在100 kV的加速电压下拍摄图像(图4)。
在TEM下观察时,不同的纳米颗粒和纳米颗粒的链状聚集体(CLAN)在肽2中应该可见(图4 )。





图4. TEM捕获的疫苗候选肽2的颗粒图像[ bar 500 nm; 深色区域是负染色(2%磷钨酸)的结果]


二级结构:
将0.1 ml自组装的候选疫苗(来自S tep C 1的未稀释样品)转移到新的2 ml微量离心管中,并加入1.4 ml PBS以制备0.1 mg / ml溶液(稀释度为1:15)用于CD分析(见注24)。              
使用以下参数设置Spectra Manager软件:带宽,5 nm;扫描速度50 nm / min; 响应时间2 s;在195至260 nm的波长范围内间隔为1 nm。
将电池插入仪器中,并在空的CD电池上进行快速扫描,以确保电池清洁(可以看到平坦的水平线,且与0 mdeg的偏差不大;请参见注释25)。如果需要,请清洗电池(请参阅注释26)。
用稀释溶液(〜200 µl)填充CD池,然后将池插入仪器中。每次测量至少要进行六次累加。运行PBS或水作为空白(阴性对照)。完成所有测量后,仅用水或甲醇清洁电池。
使用Spectra Manager,从疫苗候选光谱中减去基线光谱(PBS空白读数),然后将毫度(mdeg)转换为摩尔椭圆率([θ]; deg∙cm 2 ∙dmol -1 )。
通过以下公式完成转换:

[θ] = mdeg /(l×c×n)× 1000


在哪里:


l =路径长度(0.1厘米),


c =肽浓度(mM),和


n =肽中氨基酸的数量。


疫苗候选肽2应采用螺旋构象,其最小波长为222 nm,强度最低的波长为208 nm(图5 )。

             
图5.候选疫苗肽2的圆二色性(CD)光谱


笔记


普通的丁腈或乳胶手套对DMF的防护效果很差。在处理DMF时,应戴上耐化学药品的手套。
如果与皮肤接触,HATU / HBTU / HCTU会引起过敏反应。处理这些化学药品时应格外小心。
该反应器配备有用于液体传输的真空歧管和一个光纤温度探头。每个微波系统通常都有自己的互补肽合成容器和真空过滤设备。请咨询您的微波提供商以获取更多信息。
该肽合成容器是20毫升,微波安全,开放容器的设备,配有多孔玻璃料(允许从树脂中过滤出肽)和底部的盖子。或者,可以在没有微波(加热)帮助的情况下进行合成。可以代替使用玻璃肽合成容器。需要通过在真空下过滤来排干和洗涤该玻璃容器。还需要旋转混合器以提供连续的混合。
合成从肽氨基酸序列的C-端到N-端。开始之前,请确保顺序正确。此处提供的信息是针对0.2 mmol的合成规模计算的。
在树脂溶胀和肽合成过程中,树脂应保持浸没在DMF溶剂中,溶剂水平至少应比树脂高2 cm。旋转混合器可用于通过连续混合来改善溶胀。
树脂可以膨胀最多24小时。或者,将含树脂的容器与温度探头一起插入微波中,并加热到70°C持续10分钟以快速溶胀。
仔细进行每次清洗,以确保完全去除所有试剂。洗涤不充分可能导致合成过程中形成副产物。如果使用微波,请从微波中移出容器,然后将温度探针与树脂和搅拌棒(用于Boc脱保护)一起洗涤。
在TFA使Boc从Boc-Gln(Xan)-OH脱保护之前和之后,用DCM(每次洗涤约5 ml;而不是DMF)洗涤树脂五次。这防止了谷氨酰胺的环化。
或者,如果在玻璃肽合成容器中进行合成(用于无微波(热)辅助的合成),则可以通过将TFA /树脂混合物在旋转混合器上放置2分钟来进行搅拌。添加TFA后,确保容器的两端均密闭。
每个氨基酸应预活化2 -反应前5分钟。在与树脂偶联之前,应将氨基酸充分溶解。使用超声波仪或涡旋仪可以加快该过程。
向容器中添加其他DMF,以确保树脂保持浸没状态。
在肽合成过程中,通常将Boc氨基酸在70°C下加热10分钟。双重偶联进行5分钟,然后进行10分钟。但是,胱氨酸,组氨酸和精氨酸必须在50°C下偶联15分钟,两次。或者,对于没有微波(热)辅助的偶联,将玻璃容器放在旋转混合器上10分钟(第一次偶联),然后20分钟(第二次偶联),或者对于胱氨酸,组氨酸和精氨酸各20分钟。确保容器的两端均密闭。
每次进行两次乙酰化10分钟。在第一次氨基酸偶联后进行乙酰化反应,以将未反应的位点封端到树脂上,以避免形成副产物,副产物影响最终产物的纯度。在此乙酰化(非N末端乙酰化)之前,请勿除去Boc ,因为它会阻碍任何其他氨基酸偶联。或者,要在没有微波(热)辅助的情况下进行乙酰化,请将玻璃容器放在旋转混合器上20分钟。确保容器的两端均密闭。
偶联所有氨基酸后,除去最终氨基酸的Boc保护基,然后在肽的N端进行乙酰化。
双脱保护完成2分钟,然后5分钟。确保在N末端乙酰化后完成这些脱保护步骤。使用20%的哌啶除去赖氨酸(支链部分)上的Fmoc保护基。哌啶还去除了色氨酸的甲酰基(Boc-Trp(For)-OH; PADRE序列)。根据我们的经验,这不会触发副产品的生产。或者,如果合成是在玻璃肽合成容器中进行的(无需微波(热)辅助进行合成),则可通过将哌啶/树脂混合物置于旋转混合器上5分钟(首先脱保护),然后进行10分钟来进行搅拌(第二次解除保护)。确保容器的两端均密闭。
500 mg是有效裂解HF的最大树脂量。15毫升的Falcon管可轻松将树脂转移到HF反应容器中。
HF具有剧毒和腐蚀性。严格遵循HF设备附带的协议。如果肽序列中存在半胱氨酸或蛋氨酸,则向对甲酚/树脂混合物中添加额外的对硫代甲酚清除剂(0.25 ml; 0.5 ml / g树脂)。在进行后处理之前,请确保HF已完全蒸发。
在过滤之前,请确保肽完全溶解。加入几滴溶剂B,并使用超声仪或涡旋仪帮助溶解。在通过ESI-MS和RP-HPLC机器运行溶液之前,请始终对其进行过滤。肽2是疏水的,需要通过C4色谱柱。可以使用C8柱纯化更多的亲水性肽,以实现更好的分离。粗化合物容易降解且不稳定。因此,必须对其进行纯化才能长期保存。
RP-HPLC分析图显示单个峰中存在纯化合物。该峰的质量必须与ESI-MS中所需肽的质量相匹配。肽2是疏水的,需要通过C4色谱柱。通过C8色谱柱运行更多亲水性肽(如上所述)。
通过分析型RP-HPLC和ESI-MS分析合并的馏分,以确认纯度。将纯化合物以粉末形式保存在-20°C下,以利于保存并延长保质期。
聚亮氨酸尾部的引入增加了化合物的疏水性并使其难于溶解。同时使用超声处理和涡旋处理可以帮助完成该过程。最终产品应为白色,半混浊或澄清的溶液,其中应包含自组装的候选疫苗。使用前涡旋溶液。
进行可视化需要适当的稀释;纳米颗粒浓度过高的样品会导致颗粒重叠,从而影响颗粒形态的可见性。稀释比的可变性取决于特定的样品,但是0.5 mg / ml的浓度是一个很好的起点。
为了避免在高电压(HT)> 600 V时进行测量,需要进行稀释。
与零(>或<1 mdeg)的显着偏差表明CD细胞受到污染。
通过用5M硝酸填充电池(硝酸不会损坏石英)来清洁电池,并将其放置几个小时或一整夜。超声仪可用于加快该过程。用水冲洗细胞,最后用甲醇洗涤。在使用或存放之前,请取出内容物并彻底干燥电池。仅使用Kimwipes清洁电池的外部。

致谢


这项工作得到了美国国家卫生和医学研究委员会(计划拨款APP1132975)和澳大利亚政府研究训练计划(RTP)奖学金的支持。我们承认最初的研究论文“聚(氨基酸)是一种有效的基于肽的纳米疫苗的自佐剂递送系统”,该协议是从中得出的。我们还感谢昆士兰大学显微镜和微分析中心的澳大利亚显微镜和微分析研究设施所提供的设施和科学技术援助。


利益争夺


MS,GZ和IT是昆士兰大学提交的名为“用于疫苗交付的自组装,自佐剂系统”的专利申请(申请号AU 2019900328)的共同发明人。其余作者声明他们没有竞争利益。


参考


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Jadhav,KB ,Woolcock,KJ和Muttenthaler,M。(2020)。Boc固相肽合成中的无水氟化氢裂解。方法中号醇乙IOL 2103 :41-57
Malonis,R.,Lai,JR和Vergnolle,O. (2020)。基于肽的疫苗:当前的进展和未来的挑战。Chem Rev 120 :3210-3229。
              Nevagi,RJ ,Skwarczynski,M.和Toth,I. (2019年)。用于亚单位疫苗递送的聚合物。Eur Polym J 114 :397 -410。
Nevagi,RJ,Toth,I.和Skwarczynski,M. (2018)。肽在生物医学,生物技术和生物工程中的应用。伍德海德出版社327-358。
Shi,S.,Zhu,H.,Xia,X.,Liang,Z.,Ma,X. and Sun,B.(2019年)。疫苗佐剂:了解佐剂的结构和机理。疫苗37:3167-3178。
Skwarczynski,M.和Toth,I.(2014年)。基于肽的亚单位纳米疫苗的最新进展。纳米医学(Lond)9:2657-2669。
Skwarczynski,M.和Toth,I.(2016年)。基于肽的合成疫苗。Chem Sci 7:842-854。
Skwarczynski,M.,Zhao,G.,Boer,J.,Ozberk,V.,Azuar,A.,Cruz,JG,Giddam,AK,Khalil,Z.,Pandey,M.,Shibu,M.等。(2020)。聚氨基酸作为一种有效的基于肽的纳米疫苗的自佐剂递送系统。科学建议6:eaax2285。             
Zhao,G.,Chandrudu,S.,Skwarczynski,M.和Toth,I.(2017年)。自组装纳米结构在基于肽的亚基疫苗开发中的应用。Eur Polym J 93:670-681。
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Zhao, G., Azuar, A., Toth, I. and Skwarczynski, M. (2021). A Potent Vaccine Delivery System. Bio-protocol 11(7): e3973. DOI: 10.21769/BioProtoc.3973.
  2. Skwarczynski, M., Zhao, G., Boer, J., Ozberk, V., Azuar, A., Cruz, J. G., Giddam, A. K., Khalil, Z., Pandey, M., Shibu, M., et al. (2020). Poly(amino acids) as a potent self-adjuvanting delivery system for peptide-based nanovaccines. Sci Adv 6: eaax2285.
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