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 reduction in mortality³. Today, the global effort to contain the COVID-19 pandemic underlies the overriding need for a vaccine. The expected benefits of a vaccine on our public health and society stimulate the development of new vaccines at unprecedented breadth and speed. Among the numerous vaccines in development, traditional approaches (inactivated or live-attenuated virus vaccines) have to cohabit with new vaccine technologies (recombinant protein, DNA or RNA fragment, viral vector, etc.), which are widely used and show promising results⁴. Thus, vaccines carrying the genetic information encoding a foreign antigen, including viral vectors as well as nucleic acids (DNA plasmid or mRNA), are strategies that are increasingly being developed.

Adjuvants are also expected to play an important role in the efficacy of advanced vaccines by triggering stronger immune responses. The panel of available adjuvants constitutes a wide range of precious tools to enhance and/or shape immune responses toward specific long-term protection. However, there is no universal adjuvant, and their mode of action is still partially understood, as it often relies on several mechanisms. The selection and formulation of adjuvants must consider a wide range of criteria such as the target population (species, age, etc.), the type of antigen, the route of inoculation, and the expected immune mediators of protection. Expected benefits of an adjuvantation include vaccine dose sparing, faster immune response, broadening of immune response profiles, greater magnitude and functionality of antibody responses, or specific targeting of effective T cell responses⁵. Thus, tomorrow's vaccines are likely to be more sophisticated, combining new vaccine and adjuvant technologies to achieve the best balance between efficacy and safety. The development of new technologies will improve both human and animal health.

In this article, a protocol to prepare a vaccine formulation containing an adenovirus-vectored vaccine with the oily O-206 adjuvant is proposed. The resulting W/O/W emulsion consists of a continuous aqueous phase within which oil droplets contain a secondary aqueous phase. Stable, fluid, and safe, W/O/W emulsion showed promising results in several vaccination trials, associating human adenovirus type 5 (Ad5) and O-206. Different oily adjuvants and formulations (water-in-oil; oil-in-water; water-in-oil-in-water) were evaluated in mice with a non-replicative recombinant adenovirus vaccine expressing pseudorabies virus gp50 (Ad5-pg50). O-206 based formulation induced higher IgG titers (IgG2a) and stimulated IL6 production, even at low viral vector concentrations⁶. Formulation of O-206 with an Ad5-expressing foot-and-mouth disease virus antigens (Ad5-FMDV) enhanced the antibody response in sheep to a protective level⁷. Formulation of O-206 with an Ad5 vector encoding the green fluorescent protein (GFP) improved GFP expression during the first hours after transduction in bovine migrating DCs. Thus, the adjuvant potentially stimulated the recruitment of DCs at the site of injection, reinforcing antigen uptake and migration to draining lymph nodes. Beyond that, the frequency of CD4+ T cells increased following calves immunization with Ad5-FMDV and O-206⁸.

Initially, W/O/W adjuvant was developed for cattle, swine, and small ruminants in association with non-immunoreactive antigens such as inactivated vaccines, purified proteins, or synthetic peptides. Fluid and easy to use, W/O/W emulsions enhance short- and long-term immune responses against various antigens. Because of the double emulsion structure, the antigens in the outer aqueous phase are immediately available to the immune system, while the antigens in the inner aqueous phase are protected against enzymatic degradation and have a sustained release. Multiphasic emulsions are also known to act through a variety of mechanisms, including a depot effect at the injection site, local inflammation stimulating the recruitment of antigen-presenting cells, and a contribution to the transport of antigens throughout the lymphatic system accompanied by an accumulation of lymphocytes in the draining lymph nodes⁹.

The vaccine formulation process with W/O/W adjuvant directly impacts on its physical properties, which are crucial to obtain a stable, safe, and efficient vaccine emulsion. Emulsion stability is highly sensitive to changes and the formulation protocol needs to be optimized for any proposed modification in the production scale. The protocol here details two laboratory processes for the O-206 adjuvant that are suitable for testing small-scale immunization and compatible with the safety requirements of live vaccines based on recombinant adenoviruses. These optimized protocols allow a robust and reproducible formulation. They can be used for other compatible antigens, not interacting with O-206 surfactant. Moreover, they are adapted to the requirements of research laboratories: the first is particularly suitable for the vaccination of rodents, with a formulation volume of 1 mL; the second is well-adapted to the vaccination of large animals such as swine, small ruminants, and cattle, with a formulation volume of 10 mL.

A non-replicative canine adenovirus vector expressing the GFP is used throughout this study. It is not properly designated as a vaccine vector, but the expression of the reporter gene provides a useful approach for assessing the biological activity of a CAV2-derived gene transfer vector.

Reliable and simple quality control analyses of the W/O/W vaccine emulsion formulated with recombinant adenoviruses are also provided. These quality control tests should be considered an integral part of the process.

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 microorganism. The handling of CAV2 vectors and the treatment of its waste must meet local biohazard management requirements.
   NOTE: Non replicative canine adenovirus type 2 derived vectors were produced and purified as previously described by Szelechowski, M. et al.¹⁰.
3. Warm both the microtubes containing adjuvant and aqueous phase in a water bath or incubator at 37 °C for at least 20 min.
4. Transfer 440 µL of the pre-warmed aqueous phase in the tube containing 560 µL of O-206. Immediately mix by vortexing at 2,500 rpm ± 50 rpm for 1.5 min at room temperature (18-25 °C).
   NOTE: It is crucial to get a final ratio adjuvant/aqueous phase of 50/50 weight/weight. Because the temperature of the oil and aqueous phases during mixing (32 °C ± 1°C) is a critical step of the process, perform the assembly as fast as possible. Optimal and accurate agitation speed and mixing time are also critical parameters. Use the recommended containers and the process exactly as described here.
5. Cool the emulsion for at least 1 h at 20 °C (or 4 °C if 20 °C storage is not available) with minimal turbulence.
   ​NOTE: This step is an integral and critical part of the process. Insufficient cooling will affect vaccine stability. The same protocol is suitable for larger formulations of 5 mL, using 15 mL conical tubes.

2. Emulsification process of 10 mL formulation for large animals

1. Transfer 4.6 g of O-206 into a 20 mL gamma sterilized dispersing tube (see Table of Materials).
2. Prepare 4.6 mL of aqueous phase containing purified canine recombinant adenovirus at the desired concentration in a 15 mL tube.
   CAUTION: CAV2 recombinant vectors are genetically modified organisms (GMOs) derived from canine adenovirus type 2, classified as a Risk Group II pathogenic microorganism. The handling of CAV2 vectors and the treatment of its waste must meet local biohazard management requirements.
   NOTE: Non-replicative canine adenovirus type 2 derived vectors were produced and purified as previously described by Szelechowski, M. et al.¹⁰.
3. Warm both tubes containing O-206 and aqueous phase in an incubator (or a water bath) at 37 °C for at least 20 min.
4. Transfer 4.6 mL of the pre-warmed aqueous phase in the 20 mL dispersing tube containing 4.6 g of O-206. Immediately place the 20 mL dispersing tube on the homogenizer and mix at 1,100 rpm (speed 3) for 3 min at room temperature (18-25 °C).
   NOTE: It is crucial to get a final ratio adjuvant/aqueous phase of 50/50 weight/weight. Because the temperature of the oil and aqueous phases during mixing (32 °C ± 1°C) is a critical step of the process, perform the assembly as fast as possible. Optimal and accurate agitation speed and mixing time are also critical parameters. Use the recommended containers and the process exactly as described here.
5. Cool the 20 mL dispersing tube containing the emulsion for at least 1 h at 20 °C (or 4 °C if 20 °C storage is not available) with minimal turbulence.
   ​NOTE: This step is an integral and critical part of the process. Insufficient cooling will affect the stability of the vaccine. The same protocol is suitable for larger volumes. For 10-15 mL, use a 20 mL dispersing tube; for 20-40 mL, use a 50 mL dispersing tube.

3. Storage (optional)

1. If storage is required, transfer the emulsion into a sterile glass vial after the cooling step. Use a pierceable sterile cap to seal it.
   ​NOTE: The emulsion can be stored for up to 1 week at 4 °C without significant loss of the CAV2 vector-mediated gene delivery. To preserve the vaccine, ensure that the packaging materials do not interact physically or chemically with the finished product (e.g., do not use rubber). Elevated temperatures may alter the partition characteristics of the emulsifiers and result in instability.

4. Quality control tests

NOTE: Perform the quality control tests after one-night storage at 4 °C.

1. Appearance
   1. On the day of the formulation, keep the emulsion in a transparent tube at 4 °C. Handle the tube with minimal turbulence. Do not shake the emulsion container for this test.
   2. The next day, arrange a light directed throughout the emulsion in a transparent tube.
   3. Check for the absence of critical defaults in the appearance of the resulting emulsion (Figure 1).
2. Drop test
   1. Gently shake the vaccine container with the emulsion to ensure that it is well mixed.
   2. Add a drop of the emulsion in a 50 mL screw cap bottle containing 30-40 mL of water.
   3. Gently shake and immediately observe the repartition of the droplet in water (Figure 2).
3. Microscopic observation
   1. Gently shake the tube containing the vaccine to ensure it is well mixed.
   2. Place a small drop of the vaccine in the middle of a Petri dish.
   3. Carefully place a coverslip on the drop, without introducing air bubbles or crushing it.
   4. Close the Petri dish and immediately observe the vaccine drop under a microscope using brightfield (Figure 3).
4. Biological activity
   1. A day before inoculation, seed MDCK cells on a 6-well plate. Grow cells at 37 °C in 5% CO₂, in Dulbecco's Modified Eagle Medium (DMEM) with high glucose, supplemented with 7% heat-inactivated fetal calf serum, 1 mM of sodium pyruvate and 100 U/mL of penicillin/100 µg/mL of streptomycin. Allow the cells to become 70-80% confluent the following day for inoculation.
   2. On the day of the biological activity assay, gently shake the tube containing the formulated vaccine. Then, add up to 10 µL of the W/O/W emulsion per well in a 6-well plate.
      ​NOTE: This test is scalable in 24-well plates by adding up to 2 µL of vaccine formulation per well.
   3. Gently shake the plates and incubate at 37 °C in 5% CO₂ for 24 h.
   4. The expression of the adenovirus-encoded antigen is sought in transduced cells by immunocytochemistry or other appropriate methods.

5. Vaccination

NOTE: The vaccine can be administered if the formulation passes at least the appearance, dilution, and microscopic observation tests. Evaluation of biological activity is not mandatory for every formulation. However, it is strongly recommended to ensure, at least once, the biological activity of the formulated viral-vectored vaccine.

1. Gently shake the vaccine container with the emulsion.
2. If the vaccine is packaged in a container with a pierceable cap, install a vial adapter and mount a syringe with a rubber-free piston. Pull-up the required volume of vaccine and then place the needle.
3. Otherwise, place a needle on a syringe with a rubber-free piston, pierce the vial or the cap of the 20 mL dispersing tube and then pull-up the required volume of the vaccine.
4. Administer the vaccine formulation in vivo directly by the intramuscular or subcutaneous route.

Results

Typical results obtained from the appearance test are shown in Figure 1. An emulsion is stable if there is no default or non-critical defaults. A default is considered non-critical when the difference of both color and phase is low. Non-critical defaults are hand reversible because the properties of the emulsion are conserved (e.g., whitish phase at the surface). A default is considered critical when the physical properties of the emulsion are permanently altered: when there is a change in d...

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