evaluation of ovalbumin-specific antibodies in mice after intramuscular administration of cationic nanoemulsion&#45;encapsulated retinoic acid

Evaluering af immunogenicitet af nanoemulsion-indkapslet retinsyre i mus

Adjuvants are often used to enhance the efficacy of a vaccine by stimulating the immune system to respond more strongly to the vaccine, thereby increasing immunity to a particular pathogen1. Nanoemulsion (NE) adjuvant refers to a colloidal dispersion system with thermodynamic stability by emulsifying a certain proportion of oil phase and aqueous phase to produce an emulsion in the form of water-in-oil (W/O) or oil-in-water (O/W)2. O/W nanoemulsion adjuvant can produce cytokines and chemokines at the injection site, induce the rapid aggregation and proliferation of important immune cells such as monocytes, neutrophils, and eosinophils, and enhance the immune response, and improve the immunogenicity of antigens3. Currently, three nanoemulsion adjuvants (MF59, AS03, and AF03) have been licensed for use in vaccines and have shown good safety and efficacy4.
However, mucosal immunity has been poorly addressed by the currently licensed adjuvant formulations in conventional parenteral vaccination5. Retinoic acid (RA) has been reported to induce intestinal homing of immune cells, but its low polarity, poor solubility in water, and poor light and thermal stability limit its use for robust enteric vaccination. Nanoemulsions offer opportunities to increase the bioavailability of highly lipophilic drugs, and the oil core of O/W emulsion adjuvants is suitable for dissolving non-polar substances such as RA6. Therefore, nanoemulsions can be used as carriers for RA in order to achieve the dual response effect of systemic immunity and mucosal immunity.
Compared to neutral or anionic delivery systems, cationic delivery systems have relatively efficient antigen encapsulation and delivery capabilities, which can enhance the immunogenicity of antigens7,8,9. The cationic surface charge of a variety of adjuvant systems is important for their adjuvant effects10,11,12. The cationic charge is an important factor in prolonging vaccine retention at the injection site, increasing antigen presentation and prolonging the stimulation of cellular immunity in the body12.
Based on the above considerations, we developed a cationic nanoemulsion to effectively co-deliver RA and antigens. The particle size and zeta potential of the nanoemulsion were determined using dynamic light scattering (DLS), and the systemic and mucosal immune responses of the nanoemulsion combined with OVA were evaluated by intramuscular injection13.

Forfatter Spotlight: Udvikling og evaluering af et kationisk nanoemulsion-indkapslet retinsyresystem til slimhindevaccination

kationisk nanoemulsionsindkapslet retinsyre som adjuvans for at fremme OVA-specifikke systemiske og slimhinderesponser

We are working on a mucosal vaccine that produces an immune response as the set of infection, which has the potential to prevent infection and interrupt transmission. We have developed a catatonic nanoemulsion-encapsulated retinoic acid delivery system immunization with ovalbumin by intramuscular injection, which triggers both systemic and mucosal immune responses. In future studies, animal models of intestinal-related diseases can be used to further evaluate the effect and the mechanism of a cationic nanoemulsion-encapsulated retinoic acid in enhancing vaccine protection.

Evaluering af ovalbuminspecifikke antistoffer hos mus efter intramuskulær administration af kationisk nanoemulsionsindkapslet retinsyre

For at begynde antigenbelægning fortyndes ovalbumin til fem mikrogram pr. Milliliter med en belægningsbuffer. Overtræk en 96 brønds ELISA-plade med 100 mikroliter ovalbuminopløsning pr. Brønd natten over ved fire grader Celsius. Den næste dag vaskes brøndene fem gange med PBST.Bloker brøndene med 250 mikroliter 1% BSA PBST ved 37 grader Celsius i to timer. For at forberede en 1000 X serumfortynding fortyndes først 20 mikroliter serum til en milliliter ved hjælp af 0,5% BSA PBST. Fjern derefter 25 mikroliter serumalikvote og fortynd det til 500 mikroliter ved hjælp af 0,5% BSA PBST.Tilsæt 200 mikroliter serum fortyndet til 1000 X til den første række af den coatede plade. Derefter tilsættes 100 mikroliter 0,5% BSA PBST til alle brønde undtagen brøndene i første række. Overfør 100 mikroliter fra første række til anden række, og bland 10 gange.For at skabe varierende koncentrationer af serum til IgG-detektion kasseres 100 mikroliter væske fra den sidste række. Dernæst fortyndes mus vaginal skylning, bronchi alveolær skylning, næseskylningsvæsker med 0,5% BSA PBST og tyndtarmsskylningsvæske med 20% føtal kalveserum PBST. Inkuber pladen ved 37 grader Celsius i en time.Vask brøndene fem gange med PBST. Tilsæt 100 mikroliter HRP konjugeret gedeanti-museantistof til de relevante brønde og inkuber ved 37 grader Celsius i 40 minutter. Efter vask af brøndene fem gange med PBST tilsættes 100 mikroliter TMB ELISA-substrat og inkuberes i et lysbeskyttet område i fem minutter.Tilsæt 100 mikroliter 450 nanometer stopopløsning for at afslutte reaktionen. Absorbansen måles ved 450 nanometer på en mikropladelæser. Antistoftiteren beregnes, idet en positiv responsværdi defineres som 2,1 gange serumværdien for kontrolmusegruppen.Nanoemulsionsadjuvansvaccinen øgede signifikant de ovalbuminspecifikke IgG-, IgG1- og IgG2a-antistoftitere i serum. Niveauerne af specifik IgA i vaginal skylningsvæske og tyndtarmsskylningsvæske blev signifikant forbedret, når ovalbumin blev adjuveret med kationisk nanoemulsionsindkapslet retinsyre.

Research

JoVE Journal

Immunology and Infection

Evaluation of Ovalbumin-Specific Antibodies in Mice After Intramuscular Administration of Cationic Nanoemulsion&#45;Encapsulated Retinoic Acid

Cationic Nanoemulsion-Encapsulated Retinoic Acid as an Adjuvant to Promote OVA&#45;Specific Systemic and Mucosal Responses

Cationic nanostructures have emerged as an adjuvant and antigen delivery system that enhances dendritic cell maturation, ROS generation, and antigen ...

Preparation and Characterization of Cationic Nanoemulsion&#45;Encapsulated Retinoic Acid

Fremstilling og karakterisering af kationisk nanoemulsionsindkapslet retinsyre