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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Immunology and Infection

Antigenic Liposomes for Generation of Disease-specific Antibodies

Published: October 25th, 2018

DOI:

10.3791/58285

1Janssen R&D, 2Department of Microbiology and Immunology, University of North Carolina, 3UNC Food Allergy Initiative, University of North Carolina, 4Department of Pediatrics, University of North Carolina, 5Department of Chemistry, University of Alberta, 6Department of Molecular Medicine, Scripps Research Institute, 7Department of Medical Microbiology and Immunology, University of Alberta
* These authors contributed equally

Described is the preparation of antigenic liposomal nanoparticles and their use in stimulating B-cell activation in vitro and in vivo. Consistent and robust antibody responses led to the development of a new peanut allergy model. The protocol for generating antigenic liposomes can be extended to different antigens and immunization models.

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for vaccination as well as understand how pathogenic antibody responses develop in allergies and autoimmune disease. Generating robust antigen-specific antibody responses is not always trivial. In mouse models, it often requires multiple rounds of immunizations with adjuvant that leads to a great deal of variability in the levels of induced antibodies. One example is in mouse models of peanut allergies where more robust and reproducible models that minimize mouse numbers and the use of adjuvant would be beneficial. Presented here is a highly reproducible mouse model of peanut allergy anaphylaxis. This new model relies on two key factors: (1) antigen-specific splenocytes are adoptively transferred from a peanut-sensitized mouse into a naïve recipient mouse, normalizing the number of antigen-specific memory B- and T-cells across a large number of mice; and (2) recipient mice are subsequently boosted with a strong multivalent immunogen in the form of liposomal nanoparticles displaying the major peanut allergen (Ara h 2). The major advantage of this model is its reproducibility, which ultimately lowers the number of animals used in each study, while minimizing the number of animals receiving multiple injections of adjuvant. The modular assembly of these immunogenic liposomes provides relatively facile adaptability to other allergic or autoimmune models that involve pathogenic antibodies.

Food allergy affects 8% of children in the United States, and has increased in prevalence over the past decade1. Allergy to peanut affects 1% of children and is not typically outgrown2. Although several promising clinical trials are underway for the treatment of food allergy, including oral immunotherapy (OIT), sublingual immunotherapy (SLIT), and epicutaneous immunotherapy (EPIT), there are currently no FDA-approved treatment strategies for desensitizing peanut-allergic individuals3,4,5,6....

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The general method of coupling protein to lipid and incorporating into liposomes is based largely on earlier work15. All animal procedures described below have been approved by the University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee (IACUC). All mice used in the peanut allergy model are BALB/cJ females purchased from at 3 weeks of age. The University of Alberta Animal Care and Use Committee (ACUC) has approved experiments involving use of mouse spleens for e.......

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Conjugation of the protein of interest with DSPE-PEG(2000) can be demonstrated by running a reducing showing an increase in molecular weight compared to the unconjugated protein. Figure 1A shows a representative gel of anti-mouse IgM F(ab) fragment conjugation to PEG-DSPE, which shows a 2–3 kDa bandshift for the denatured protein. Note that approximately 50% of the protein appears to be modified, which is expected given that 1:1 stoichiometry was achiev.......

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The methods outlined here are a general protocol for the conjugation of a protein to a lipid which enables the display of the protein on liposomal nanoparticles. For very large multi-subunit proteins, this protocol may have limited utility. The ideal method would be the introduction of a site-specific tag that enables a biorthogonal chemical linking strategy to be used. If expressing the protein recombinantly, this can be possible using available site-specific strategies17, and a wide array of fun.......

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This research was supported by grants from the Department of Defense (W81XWH-16-1-0302 and W81XWH-16-1-0303).

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Name Company Catalog Number Comments
Model 2110 Fraction Collector BioRad 7318122
Cholestrol Sigma C8667 Sigma grade 99%
SPDP Thermo Fisher Scientific 21857
DSPC Avanti 850365
DSPE-PEG 18:0 Avanti 880120
DSPE-PEG Maleimide Avanti 880126
Extruder Avanti 610000 1mL syringe with holder/heating block
Filters 0.1 µm Avanti 610005
Filters 0.8 µm Avanti 610009
10mm Filter Supports Avanti 6100014
Glass Round Bottom Flask Sigma Z100633
Turnover stoppers Thermo Fisher Scientific P-301398
Tubing Thermo Fisher Scientific P-198194
Leur Lock Thermo Fisher Scientific k4201634503
Sephadex G50 Beads GE Life Sciences 17004201
Sephadex G100 Beads GE Life Sciences 17006001
Heat Inactivated Fetal Calf Serum Thermo Fisher Scientific 10082147
HEPES (1M) Thermo Fisher Scientific 15630080
EGTA Sigma E3889
Penicillin-Streptomycin (10,000 U/mL) Thermo Fisher Scientific 15140122
1x RBC lysis Buffer Thermo Fisher Scientific 00-4333-57
Indo-1 Invitrogen I1203
CD5-PE BioLegend 100608
B220-PE-Cy7 BioLegend 103222
HBSS Thermo Fisher Scientific 14170112 without calcium and magnesium
MgCl2 Sigma M8266
CaCl2 Sigma C4901
Fab anti-mouse IgM Jackson ImmunoResearch 115-007-020
F(ab')2 anti-mouse IgM Jackson ImmunoResearch 115-006-020
Peanut flour Golden Peanut Co. 521271 12% fat light roast, 50% protein
Animal feeding needles Cadence Science 7920 22g x 1.5", 1.25 mm - straight
Microprobe thermometer Physitemp BAT-12
Rectal probe for mice Physitemp Ret-3
Cholera toxin, from vibrio cholera List Biological Laboratories, Inc. 100B Azide free
BCA Protein Assay Kit Pierce 23225
Carbonate-bicarbonate buffer Sigma C3041
TMB Stop Solution KPL 50-85-06
SureBlue TMB Microwell Peroxidase Substrate KPL 5120-0077
96 well Immulon 4HBX plate Thermo Scientific 3855
Purified soluble Ara h 2 N/A N/A purified as in: Sen, et al., 2002, Journal of Immunology
HSA-DNP Sigma A-6661
Mouse IgE anti-DNP Accurate Chemical BYA60251
Sheep anti-Mouse IgE The Binding Site PC284
Biotinylated Donkey anti-Sheep IgG Accurate Chemical JNS065003
NeutrAvidin Protein, HRP ThermoFisher Scientific 31001
Mouse IgG1 anti-DNP Accurate Chemical MADNP105
HRP Goat anti-mouse IgG1 Southern Biotech 1070-05
1 mL Insulin Syringes BD 329412 U-100 Insulin, 0.40 mm(27G) x 16.0 mm (5/8")
Superfrost Microscope Slides Fisher Scientific 12-550-14 25 x 75 x 1.0 mm
ACK Lysing Buffer gibco by Life Technologies A10492-01 100 mL
RPMI 1640 Medium Thermo Fisher Scientific 11875093 500 mL
Cell Strainer Corning 352350 70 μm Nylon, White, Sterile, Individually packaged
NuPAGE 4-12% Bis-Tris Protein Gels Invitrogen NP0322BOX 10 gels
NuPAGE LDS buffer, 4X Invitrogen NP0008 250 mL
SeeBlue Plus2 Pre-stained standard Invitrogen LC5925 500 µL
NuPAGE MES/SDS running buffer, 20X Invitrogen NP0002 500 mL
GelCode Blue Stain Thermo Scientific 24590 500 mL

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