Name: laboratory-scale emulsification process of a recombinant adenovirus vaccine with a water-in-oil-in-water adjuvant
Uploaded: 2021-04-16T14:31:34
Description: Adjuvants play an important role to enhance the efficacy of vaccines and are often required to direct immune responses toward specific long-term ...

Manon Broutin was funded by a CIFRE PhD fellowship (2017/1080) from the French Association Nationale de la Recherche et de la Technologie (ANRT) in the framework of a public-private partnership between Ecole Nationale V&#233;t&#233;rinaire d&#39;Alfort and SEPPIC, part of Air Liquid Healthcare.

1. Emulsification process of 1 mL formulation for rodents
<ol>
	<li>Gently shake the vial of O-206 before opening and transfer 560 &#181;L of O-206 into a 2 mL microtube using a positive displacement pipette.</li>
	<li>Add 440 &#181;L 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...

<ol>
	<li>Gibbs, E. P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+One+Health:+a+decade+of+progress+and+challenges+for+the+future.">The evolution of One Health: a decade of progress and challenges for the future.</a> Veterinary Record. 174 (4), 85-91 (2014).</li><li>Jones, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+trends+in+emerging+infectious+diseases.">Global trends in emerging infectious diseases.</a> Nature. 451 (7181), 990-993 (2008).</li><li>Plotkin, S. L., Plotkin, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Short+History+of+Vaccination.">A Short History of Vaccination.</a> Plotkin's Vaccines (Seventh Edition). , 1-15 (2018).</li><li>Krammer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SARS-CoV-2+vaccines+in+development.">SARS-CoV-2 vaccines in development.</a> Nature. 586 (7830), 516-527 (2020).</li><li>Reed, S. G., Orr, M. T., Fox, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Key+roles+of+adjuvants+in+modern+vaccines.">Key roles of adjuvants in modern vaccines.</a> Nature Medicine. 19 (12), 1597-1608 (2013).</li><li>Ganne, V., Eloit, M., Laval, A., Adam, M., Trouve, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+the+efficacy+of+a+replication-defective+adenovirus-vectored+vaccine+by+the+addition+of+oil+adjuvants.">Enhancement of the efficacy of a replication-defective adenovirus-vectored vaccine by the addition of oil adjuvants.</a> Vaccine. 12 (13), 1190-1196 (1994).</li><li>Jouneau, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibody+response+induced+FMDV+vaccines+in+sheep+correlates+with+early+transcriptomic+responses+in+blood.">The antibody response induced FMDV vaccines in sheep correlates with early transcriptomic responses in blood.</a> NPJ Vaccines. 5 (1), 1-11 (2020).</li><li>Cubillos-Zapata, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+viral+vectors+on+migratory+afferent+lymph+dendritic+cells+in+vitro+predict+enhanced+immunogenicity+in+vivo.">Differential effects of viral vectors on migratory afferent lymph dendritic cells in vitro predict enhanced immunogenicity in vivo.</a> Journal of Virology. 85 (18), 9385-9394 (2011).</li><li>Aucouturier, J., Dupuis, L., Ganne, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adjuvants+designed+for+veterinary+and+human+vaccines.">Adjuvants designed for veterinary and human vaccines.</a> Vaccine. 19 (17), 2666-2672 (2001).</li><li>Szelechowski, M., Bergeron, C., Gonzalez-Dunia, D., Klonjkowski, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+purification+of+non+replicative+canine+adenovirus+type+2+derived+vectors.">Production and purification of non replicative canine adenovirus type 2 derived vectors.</a> Journal of Visualized Experiments: JoVE. (82), e50833 (2013).</li><li>Li, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comparison+of+the+efficacy+of+swine+FMD+vaccine+emulsified+with+oil+adjuvant+of+ISA+201+VG+or+ISA+206+VG.">The comparison of the efficacy of swine FMD vaccine emulsified with oil adjuvant of ISA 201 VG or ISA 206 VG.</a> Journal of Biosciences and Medicines. 01, 22-25 (2013).</li><li>Park, M. -. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+immune+responses+of+foot-and-mouth+disease+vaccine+using+new+oil/gel+adjuvant+mixtures+in+pigs+and+goats.">Enhanced immune responses of foot-and-mouth disease vaccine using new oil/gel adjuvant mixtures in pigs and goats.</a> Vaccine. 32 (40), 5221-5227 (2014).</li><li>Dar, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Montanide+ISA+201+adjuvanted+FMD+vaccine+induces+improved+immune+responses+and+protection+in+cattle.">Montanide ISA 201 adjuvanted FMD vaccine induces improved immune responses and protection in cattle.</a> Vaccine. 31 (33), 3327-3332 (2013).</li><li>Ibrahim, E. E. -. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+on+the+immunopotentiator+effect+of+ISA+201,+ISA+61,+ISA+50,+ISA+206+used+in+trivalent+foot+and+mouth+disease+vaccine.">Comparative study on the immunopotentiator effect of ISA 201, ISA 61, ISA 50, ISA 206 used in trivalent foot and mouth disease vaccine.</a> Veterinary World. 8 (10), 1189-1198 (2015).</li></ol>

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...

In the context of growing populations, secure access to uncontaminated food while improving animal and human health will become increasingly important. The expression &#34;One World, One Health&#34; 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&#39;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)&#160;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&#160;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL conical tube</td>
			<td>FALCON</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL dispersing tube</td>
			<td>IKA</td>
			<td>3700600</td>
			<td>Tube with rotor-stator element. Sterile model, with pierceable membrane</td>
		</tr>
		<tr>
			<td>50 mL dispersing tube</td>
			<td>IKA</td>
			<td>3701600</td>
			<td>Tube with rotor-stator element. Sterile model, with pierceable membrane</td>
		</tr>
		<tr>
			<td>DMEM (1x) + GlutaMAX-I</td>
			<td>GIBCO</td>
			<td>61965-026</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum</td>
			<td>EUROBIO</td>
			<td>CVFSVF00-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td>IKA</td>
			<td>3646000</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser diffraction particle size analyzer</td>
			<td>Malvern Panalytical</td>
			<td>Mastersizer 3000</td>
			<td></td>
		</tr>
		<tr>
			<td>MDCK (NBL-2)</td>
			<td>ATCC</td>
			<td>CCL-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtube 2 mL</td>
			<td>EPPENDORF</td>
			<td>30120094</td>
			<td></td>
		</tr>
		<tr>
			<td>O-201</td>
			<td>SEPPIC</td>
			<td>MONTANIDE ISA 201 VG</td>
			<td></td>
		</tr>
		<tr>
			<td>O-206</td>
			<td>SEPPIC</td>
			<td>MONTANIDE ISA 206 VG</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin (10,000 U/mL) Streptomycin (10,000 &#181;g/mL)</td>
			<td>GIBCO</td>
			<td>15140-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips C POSD 1000 &#181;L S 180/3</td>
			<td>RAININ</td>
			<td>17008609</td>
			<td>100 &#956;L &#8211; 1000 &#956;L</td>
		</tr>
		<tr>
			<td>Positive-Displacement Pipette MR-1000</td>
			<td>RAININ</td>
			<td>17008580</td>
			<td>100 &#956;L &#8211; 1000 &#956;L</td>
		</tr>
		<tr>
			<td>Sodium Chloride 0.9% injectable</td>
			<td>BBRAUN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate 100 mM (100x)</td>
			<td>GIBCO</td>
			<td>11360-039</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile glass vial</td>
			<td>WEST PHARMACEUTICAL SERVICES</td>
			<td>8072035</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Syringe 1 mL</td>
			<td>BBRAUN</td>
			<td>9166017V</td>
			<td>Syringe without rubber</td>
		</tr>
		<tr>
			<td>Syringe 10 mL</td>
			<td>BBRAUN</td>
			<td>4606728V</td>
			<td>Syringe without rubber</td>
		</tr>
		<tr>
			<td>Syringe 2 mL</td>
			<td>BBRAUN</td>
			<td>4606701V</td>
			<td>Syringe without rubber</td>
		</tr>
		<tr>
			<td>Syringe 5 mL</td>
			<td>BBRAUN</td>
			<td>4606710V</td>
			<td>Syringe without rubber</td>
		</tr>
		<tr>
			<td>Vial adapter</td>
			<td>WEST PHARMACEUTICAL SERVICES</td>
			<td>8072035</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>SCIENTIFIC INDUSTRIES</td>
			<td>SI-0256</td>
			<td>Vortex-Genie 2</td>
		</tr>
	</tbody>
</table>

laboratory-scale emulsification process of a recombinant adenovirus vaccine with a water-in-oil-in-water adjuvant

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.

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...

This manuscript describes a simple method for the formulation and control of vaccines with&#160;a water-in-oil-in-water adjuvant at the laboratory scale, compatible with the safety requirements of live recombinant vaccines.

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