Laboratory-Scale Emulsification Process of a Recombinant Adenovirus Vaccine with a Water-In-Oil-In-Water Adjuvant

Summary

This manuscript describes a simple method for the formulation and control of vaccines with a water-in-oil-in-water adjuvant at the laboratory scale, compatible with the safety requirements of live recombinant vaccines.

Abstract

Adjuvants play an important role to enhance the efficacy of vaccines and are often required to direct immune responses toward specific long-term protection. Several vaccination trials have described promising results with the combination of recombinant adenoviruses and water-in-oil-in-water (W/O/W) adjuvants. Specifically, the antibody response elicited by vaccines based on canine adenovirus type 2 (CAV2) vectors steadily increases after being formulated in a W/O/W emulsion. Thus, the production process directly impacts its physical properties, which are crucial to obtain stable, safe, and efficient vaccine emulsions. This article describes a lab-scale process for the formulation of O-206, a W/O/W adjuvant, in a total volume of 1 mL and 10 mL, that is compatible with safety requirements of live vaccines based on recombinant adenovirus. Moreover, this article provides reliable and simple quality control analyses of the W/O/W vaccine emulsion formulated with recombinant adenoviruses.

Introduction

In the context of growing populations, secure access to uncontaminated food while improving animal and human health will become increasingly important. The expression "One World, One Health" describes a multidisciplinary international cooperation associating animal and human health to notably better prevent and control zoonotic agents¹. Indeed, 60% of emerging infectious diseases in humans are transmitted by animals².

Nowadays, vaccination is still the most effective way to prevent and control infectious diseases in humans and animals. In comparison, only drinking water allows such a r....

Protocol

1. Emulsification process of 1 mL formulation for rodents

1. Gently shake the vial of O-206 before opening and transfer 560 µL of O-206 into a 2 mL microtube using a positive displacement pipette.
2. Add 440 µL of aqueous phase containing purified canine recombinant adenovirus (CAV2) at the desired concentration in a 2 mL microtube.
   CAUTION: CAV2 recombinant vectors are genetically modified organisms (GMOs) derived from canine adenovirus type 2, classified as a Risk Group II pathogenic mi.......

Discussion

The protocol in this study details two lab-scale processes adapted to the safety requirements of live vaccines based on recombinant adenoviruses formulated with W/O/W adjuvant.

These optimized protocols allow a robust and reproducible formulation. However, it is crucial to scrupulously respect certain critical steps. Firstly, it is important to obtain a final adjuvant/aqueous phase ratio of 50/50 weight/weight. Secondly, warming of the oil and aqueous phases before their assembly must ensure a.......

Materials

Name	Company	Catalog Number	Comments
15 mL conical tube	FALCON	352096
20 mL dispersing tube	IKA	3700600	Tube with rotor-stator element. Sterile model, with pierceable membrane
50 mL dispersing tube	IKA	3701600	Tube with rotor-stator element. Sterile model, with pierceable membrane
DMEM (1x) + GlutaMAX-I	GIBCO	61965-026
Fetal Calf Serum	EUROBIO	CVFSVF00-01
Homogenizer	IKA	3646000
Laser diffraction particle size analyzer	Malvern Panalytical	Mastersizer 3000
MDCK (NBL-2)	ATCC	CCL-34
Microtube 2 mL	EPPENDORF	30120094
O-201	SEPPIC	MONTANIDE ISA 201 VG
O-206	SEPPIC	MONTANIDE ISA 206 VG
Penicillin (10,000 U/mL) Streptomycin (10,000 µg/mL)	GIBCO	15140-212
Pipette Tips C POSD 1000 µL S 180/3	RAININ	17008609	100 μL – 1000 μL
Positive-Displacement Pipette MR-1000	RAININ	17008580	100 μL – 1000 μL
Sodium Chloride 0.9% injectable	BBRAUN
Sodium pyruvate 100 mM (100x)	GIBCO	11360-039
Sterile glass vial	WEST PHARMACEUTICAL SERVICES	8072035	Optional
Syringe 1 mL	BBRAUN	9166017V	Syringe without rubber
Syringe 10 mL	BBRAUN	4606728V	Syringe without rubber
Syringe 2 mL	BBRAUN	4606701V	Syringe without rubber
Syringe 5 mL	BBRAUN	4606710V	Syringe without rubber
Vial adapter	WEST PHARMACEUTICAL SERVICES	8072035	Optional
Vortex mixer	SCIENTIFIC INDUSTRIES	SI-0256	Vortex-Genie 2