用于重组流感疫苗开发的佐剂纳米脂质体制备方法

Zachary R. Sia; Wei-Chiao Huang; Matthew Willadsen; Hilliard L. Kutscher; Jonathan F. Lovell; Bruce A. Davidson

doi:10.21769/BioProtoc.5662

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Peer-reviewed

Preparing Adjuvanted Nanoliposomes for Applications Toward Recombinant Influenza Vaccine Development

用于重组流感疫苗开发的佐剂纳米脂质体制备方法

ZS Zachary R. Sia

WH Wei-Chiao Huang

MW Matthew Willadsen

HK Hilliard L. Kutscher

JL Jonathan F. Lovell

Bruce A. Davidson email

发布: 2026年04月05日第16卷第7期 DOI: 10.21769/BioProtoc.5662 浏览次数: 389

评审: Navnita DuttaChhuttan L MeenaAnonymous reviewer(s)

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Cover of cell reports medicine, featuring study using the protocol.

Mar 2024

Special Issue on Protocols for Organoid Research

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

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Abstract

Nanoparticle vaccines can provide advantages over traditional vaccine methodologies, including adjuvant delivery to enhance the effectiveness of recombinant antigens. Many approaches exist to formulate different vaccine nanoparticles, which are designed for different biomolecular cargos, adjuvant compositions, and disease targets. Here, a protocol is described to produce nanoliposomes whose surface is decorated with recombinant protein influenza antigens with monophosphoryl lipid A and QS-21 adjuvants incorporated into the lipid bilayer for protection against influenza infection. This protocol includes methods for producing adjuvanted liposomes and coupling with His-tagged antigens for surface decoration of the particle. This allows for a rapid methodology of producing immunogenic antigen-presenting liposomes that can be tailored to display a combination of influenza surface antigens.

Key features

• This protocol uses recombinant proteins, which can be adapted to the desired strain sequence.

• Production of this influenza vaccine formulation uses in vitro techniques, eliminating the need for virus growth in eggs.

• Additional lipid adjuvants can be included in the liposome production step to be incorporated into the surface membrane.

• Cobalt-porphyrin phospholipid provides a flexible platform for binding His-tag-bearing proteins.

Keywords: Influenza (流感)

Nanoparticle vaccine (纳米颗粒疫苗)

Nanoliposomes (纳米脂质体)

Recombinant protein vaccine (重组蛋白疫苗)

Vaccine adjuvant (疫苗佐剂)

Graphical overview

Application of liposomes as a platform for multistrain influenza vaccines. Cobalt-porphyrin liposomes present multiple surface antigens through spontaneous associations between cobalt ions and histidine residues on recombinant proteins. Figure reproduced under Creative Commons license from [1].

Background

The rapid rate of mutation among the viruses in the Orthomyxoviridae family, commonly known as influenza, requires vaccines to be updated on an annual basis to effectively provide protection against seasonal epidemics. Furthermore, genetic transfers between zoonotic strains, such as avian influenza, with human competent strains pose a significant threat of spontaneously forming novel influenza strains, which could expand into a global pandemic with a high mortality rate [2]. The adaptability of influenza viruses is primarily due to two major surface proteins on the virion, hemagglutinin and neuraminidase, which serve to facilitate viral uptake into and release from host cells, respectively [3–5]. These antigens undergo continuous mutation and reassortment between antigenically distinct strains, resulting in annual epidemics [6]. To combat the ever-evolving landscape of influenza viruses, an epidemic response demands a robust vaccine, which can rapidly shift production to utilize newly emerging antigens, providing the most up-to-date protection possible. Traditional influenza vaccines produce whole viruses within chicken eggs, which are split with a detergent or attenuated for use in immunizations. These methods incur numerous challenges, including production bottlenecks based on egg supply, a time-intensive process of selecting and cultivating egg-adapted viruses, the possibility of egg-specific mutations replacing epitopes found in human-competent viruses, and anaphylaxis in egg-allergic individuals. Recombinant protein production would allow for faster production and greater quality control, yet the technique remains relatively underutilized due to the limited antigenicity of isolated surface proteins, requiring high doses of antigen to be effective [7]. Adjuvants provide a critical supportive component to recombinant vaccines by increasing the immune response to these antigens through the activation of immunologic signaling pathways and increased uptake into antigen-presenting cells [8,9]. The nanoliposomes described in this protocol provide a robust adjuvant and vehicle for recombinant vaccines. The combination of surface presentation of influenza antigens and the effect of lipid adjuvants has been shown to significantly increase protection against lethal influenza infections in preclinical animal studies on mice and ferrets; additionally, these liposomes have been shown to increase immunological responses against a wide range of hemagglutinin and neuraminidase variants, providing a straightforward admixture method for producing multiplexed vaccine particles [1,10]. The surface binding properties of these liposomes are facilitated by the cobalt-porphyrin phospholipids (CoPoP) embedded in the bilayer membrane, which bind to chains of histidine residues called His-tags, which are routinely added to recombinant proteins for purification (Graphical overview). This platform is compatible with a wide array of antigenic proteins, including other viral infections such as SARS-CoV-19 [11,12], surface proteins of the malaria parasite [13,14], and cancer markers [15,16]. This protocol describes the process for generating CoPoP, formulating ~100 nm diameter adjuvanted liposomes with CoPoP-bearing membranes, and incubating these liposomes with recombinant influenza antigens to produce immunogenic vaccine liposomes for preclinical testing.

Materials and reagents

Biological materials

1. Cobalt porphyrin-phospholipid (CoPoP) (POP Biotechnologies, Inc.)

2. Recombinant influenza surface antigens bearing 6×-His-tag (purchased or produced)

3. Cholesterol (Evonik, catalog number: 00078928)

4. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (CordenPharma, catalog number: LP-R4-070)

5. Monophosphoryl hexa-acyl lipid A, 3-deacyl (3D6A-PHAD) (Avanti, catalog number: 699855)

6. QS-21 (Quillaja Saponaria derivative) (Desert King)

7. Anti-mouse IgG with HRP conjugate (Thermo Fisher, catalog number: 62-6520)

8. Chicken whole blood with Alsevers (Pel-Freez, catalog number: 33133-1)

9. Receptor destroying enzyme (RDE), lyophilized powder (Sigma-Aldrich, catalog number: C8772-1VL)

Reagents

1. Hydrochloric acid (HCl) (Millipore Sigma, catalog number: 320331)

2. Phosphate-buffered saline, pH 7.4 (1× PBS and 10× PBS) (Sigma-Aldrich, catalog number: P3813)

3. 200-proof ethanol, pure research grade (EtOH) (Sigma-Aldrich, catalog number: 459844)

4. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 795429)

5. Deionized water, lab grade (Thermo Fisher, catalog number: A57775)

6. 5× ELISA coating buffer (Bio-Rad, catalog number: BUF030C)

7. SuperBlock^TM blocking buffer (Thermo Fisher, catalog number: 37515)

8. Bovine serum albumin (BSA), heat shock fraction, pH 7, >98% (Sigma-Aldrich, catalog number: A9647)

9. Tween 20 washing buffer, 1% in 10× PBS (Thermo Fisher, catalog number: J63314.K3)

10. 1-Step^TM Ultra TMB-blotting substrate solution (Thermo Fisher, catalog number: 37574)

Solutions

1. CPQ liposomes (see Recipes)

Recipes

1. CPQ liposomes

Reagent	Amount	Molar weight (g/mol)	Final concentration (mg/mL, mM)
CoPoP	12.5 mg	1068.5	2.5 mg/mL, 2.34 mM
Cholesterol	62.5 mg	386.65	12.5 mg/mL, 32.3 mM
DOPC	250.0 mg	734.04	50.0 mg/mL, 68 mM
3D6A-PHAD	5.0 mg	1763.47	1.0 mg/mL, 0.57 mM
QS-21*	5.0 mg	1990.1	1.0 mg/mL, 0.5 mM
PBS	4.0 mL		10 mM phosphate, pH 7.4
Ethanol	1.0 mL
Total	5 mL

*QS-21 is added to the liposomes following ethanol removal.

CP and CA can be made by omitting QS-21 and QS-21/3D6A-PHAD, respectively.

Laboratory supplies

1. Snap-top microcentrifuge tubes, 0.6 mL sterile (Thermo Fisher, catalog number: 3449)

2. Snap-top microcentrifuge tubes, 1.5 mL sterile (Thermo Fisher, catalog number: 3451)

3. 0.2 μm syringe filter (Foxx Life Sciences, catalog number: 379-2215-OEM)

4. 15 mL centrifuge tube (Thermo Scientific, catalog number: 339651)

5. 20 mL glass vials (DWK Life Sciences, catalog number: 986532)

6. 5 mL syringe (Becton, Dickinson & Co., catalog number: 309646)

7. 50 mL centrifuge tube (Thermo Scientific, catalog number: 339653)

8. Serological pipettes, various volumes (VWR, catalog number: 75816)

9. Micropipette tips, various sizes (VWR, catalog number: 76322)

10. 96-well flat-bottom plate (clear) (Greiner Bio-one, catalog number: 655801)

11. 96-well V-bottom plate (clear) (Greiner Bio-One, catalog number: 651261)

12. Clear flat-bottom Immuno 96-well plates, MaxiSorp coating (Thermo Fisher, catalog number: 439454)

13. Aluminum foil (Reynold’s wrap)

14. Dialysis clips (Spectrum Chemical, catalog number: 888-11584-PK)

15. Dialysis tubing 12,000–14,000 MWCO (Fisherbrand, catalog number: 21-152-16)

16. Cuvettes (BrandTech, catalog number: 759075D)

17. Parafilm (Amcor, catalog number: PM-996)

18. Becton-Dickinson ultra-fine insulin syringe, 0.3 mL, 31G × 8 mm (BD, catalog number: 320440)

19. Microtainer^® capillary blood collector, gold, serum separator tube (Fisher Scientific, catalog number: 02-675-185)

Equipment

1. Pipette controller (e.g., VWR, catalog number: 612-7179)

2. Single-channel 0.5–10 μL micropipette (VWR, catalog number: 89079-962)

3. Single-channel 2–20 μL micropipette (VWR, catalog number: 89079-964)

4. Single-channel 20–200 μL micropipette (VWR, catalog number: 89079-970)

5. Single-channel 100–1,000 μL micropipette (VWR, catalog number: 89079-974)

6. 1 L graduated cylinder (Eisco, catalog number: CH0344O)

7. 0.08 μm extruder filter (Cytiva, catalog number: 10419306)

8. 0.1 μm extruder filter (Cytiva, catalog number: 10419506)

9. 0.2 μm extruder filter (Cytiva, catalog number: 10417006)

10. 10 mL Lipex extruder (Evonik, catalog number: 002108)

11. 1 L glass beaker (Eisco, catalog number: CH0126KPK6)

12. 2 L laboratory glass bottle (VWR, catalog number: 10754-822)

13. Analytical balance (Mettler Toledo, catalog number: XSR104)

14. Antistatic spatulas (VWR, catalog number: 80081-194)

15. Bath sonicator (Branson, catalog number: CPX2800H)

16. Heated circulating bath (VWR, catalog number: 1130A)

17. Hot water bath (IKA, catalog number: HB10S098)

18. Magnetic stir bar, 20 and 30 mm length (VWR, catalog numbers: 442-4523 and 442-4525)

19. Microcentrifuge (Thermo Scientific, catalog number: 75002447)

20. Compressed nitrogen gas (Praxair, catalog number: AR LC160-230)

21. Plate reader (abs/fluorescence) (Tecan, catalog number: F129013)

22. Serological pipetter (Drummond Scientific)

23. Stir plate (VWR, catalog number: 97042-714)

24. Vortexer (VWR, catalog number: 58816-121)

25. Dynamic Light Scattering (DLS) machine (BeNano)

26. UV-VIS spectrophotometer (Perkin Elmer LAMBDA 365+)

27. Incubating microplate shaker (e.g., Fisher Scientific, catalog number: 02-217-760)

28. Class II biosafety cabinet (e.g., Labconco Type A2 model)

Software and datasets

1. PerkinElmer UV WinLab software

2. BeNano particle solutions software

Procedure

English

中文翻译

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稿件历史记录

提交日期: Jan 13, 2026

接收日期: Mar 3, 2026

在线发布日期: Mar 26, 2026

出版日期: Apr 5, 2026

版权信息

如何引用

Sia, Z. R., Huang, W., Willadsen, M., Kutscher, H. L., Lovell, J. F. and Davidson, B. A. (2026). Preparing Adjuvanted Nanoliposomes for Applications Toward Recombinant Influenza Vaccine Development. Bio-protocol 16(7): e5662. DOI: 10.21769/BioProtoc.5662.