current protocols in immunology || oil-based emulsion vaccine adjuvants

UNIT 2.18Oil-Based Emulsion Vaccine AdjuvantsVirgil E.J.C. Schijns,1,2 Marius Strioga,3 and Stephane Ascarateil4

1Cell Biology and Immunology, Wageningen University, Wageningen, The Netherlands2Epitopoietic Research Corporation (ERC), Isnes, Belgium, and ERC, Schaijk, The Netherlands3Department of Immunology and Immunotherapy, Institute of Oncology, Vilnius University,Vilnius, Lithuania4Biologicals and Injectable Division, SEPPIC, Paris, France

ABSTRACT

Vaccine adjuvants are critical components in experimental and licensed vaccines usedin human and veterinary medicine. When aiming to evoke an immune response to apurified antigen, the administration of antigen alone is often insufficient, unless theantigen contains microbial structures or has a natural particulate structure. In mostcases, the rationale to use an adjuvant is obvious to the experimental immunologistor the professional vaccinologist, who is familiar with the nature of the antigen, andthe aim of the vaccine to elicit a specific antibody response and/or a specific typeof T cell response. In this unit, we describe protocols to formulate antigens with oil-based emulsions. Such emulsions represent a major prototype adjuvant category that isfrequently used in experimental preclinical vaccines, as well as veterinary and humanvaccines. Curr. Protoc. Immunol. 106:2.18.1-2.18.7. C© 2014 by John Wiley & Sons, Inc.

Keywords: emulsions � vaccines � adjuvants

INTRODUCTION

In this unit, procedures are described to generate prototype oil-based vaccine adjuvants,including water-in-oil formulation (W/O) formulations (Basic Protocol 1, and AlternateProtocols 1 and 2) and oil-in water (O/W) formulations (Basic Protocol 2 and Alter-nate Protocol 3). These emulsions have different physico-chemical characteristics andare functionally different types of adjuvants, although their exact modes of action re-main debatable (Jansen et al., 2005, 2006; Moreira et al., 2008; Hailemichael et al.,2013).

An emulsion is a mixture of two nonmiscible liquids. One liquid is dispersed, as theinternal phase, in another liquid, the continuous, outer phase. Oil and water normallydo not mix. Hence, an emulsifier is required as a substance that stabilizes the emul-sion by increasing its kinetic stability. An emulsifier often consists of a water-lovinghydrophilic head and an oil-loving hydrophobic tail. The hydrophilic head is directedto the aqueous phase and the hydrophobic tail to the oil phase. One class of emul-sifiers is known as “surface active agents,” or surfactants. The surfactant interfacesbetween the inner and outer phase, thereby stabilizing the emulsion (Ascarateil andDupuis, 2006). Depending on the water-to-oil ratio, the type of emulsifier, and themixing procedure, a water-in-oil (W/O) or an oil-in-water (O/W) emulsion may beproduced.

The procedures used to prepare emulsified vaccines may influence their clinical effects,as demonstrated by Koh et al. (2006) in the case of W/O emulsions; therefore, it isimportant to pay attention to the process and equipment used.

Current Protocols in Immunology 2.18.1-2.18.7, August 2014Published online August 2014 in Wiley Online Library (wileyonlinelibrary.com).DOI: 10.1002/0471142735.im0218s106Copyright C© 2014 John Wiley & Sons, Inc.

Induction ofImmuneResponses

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Supplement 106

BASICPROTOCOL 1

THE SYRINGE-AND-NEEDLE PROCEDURE TO PRODUCE W/OEMULSIONS

The oil phase used in this procedure may be paraffin oil, used in Freund’s adjuvant, or anon-mineral type of oil, in either case associated with a lipophilic surfactant. Surfactantsused as emulsifiers in W/O formulations can include sorbitan esters, such as sorbitanmono- or trioleate, which are esters of sorbitol and oleic acid. They may be used inassociation with ethoxylated grades, such as polysorbates. Mannide oleates, made ofoleic acid and mannitol, are the esters that can be found in the Montanide range ofsurfactants (a registered trademark of Seppic; Ascarateil and Dupuis, 2006).

Materials

Antigen to be emulsified dissolved in aqueous diluent (also referred to as theaqueous phase)

Adjuvant to be used (blend of oil and surfactant also referred to as the oil phase;see introduction to this protocol)

Syringe, latex-, and silicon oil–free (two pieces, luer-lock), e.g.:2-ml Injekt (Braun, ref. 46060701V)5-ml Injekt F (Braun, ref. 4606710VV)2-ml Norm-Ject (Henke Sass Wolf GmbH, ref. 4020.000V0)5-ml Norm-Ject (Henke Sass Wolf GmbH, ref. 4050.000V0)

3-ml crimp vial (e.g., Fisher Scientific)20-G needle

1. Using a sterile syringe, take up 1 ml of aqueous phase and transfer to a 3-ml crimpvial.

2. Perform the same operation as step 1 with the adjuvant, placing it in the same crimpvial.

3. Emulsify by pumping back and forth with a 1-ml syringe, not equipped with rubberplunger (two-piece plastic syringe; see materials list above) and connected to a 20-Gneedle (at a slow rhythm; �10 sec to pump in and 5 sec to flow out, followed by20 cycles at fastest speed, meaning about 7 sec for a complete cycle) to blend theproducts placed in the vial.

A cycle is defined as a complete passage of the emulsion from the vial to the syringe andback.

4. Stop the process once 5 cycles have been completed.

During the process, it is important to avoid injecting air into the formulation by puttingthe open side of the needle toward the vial wall while flushing out through the needle.

ALTERNATEPROTOCOL 1

THE TWO-SYRINGES-AND-CONNECTOR PROCEDURE TO PRODUCEW/O EMULSIONS

This protocol provides steps to produce 2 ml of water-in-oil (W/O) emulsion at a 50:50ratio (1 volume of oil to 1 volume of aqueous phase).

Additional Materials (also see Basic Protocol 1)

Two 1-ml syringes (see Basic Protocol 1 materials list)Syringe connector, e.g.:

Green Peptide connector (Japan; http://www.green-peptide.com/english/news/?action_enews_view=1&nid=22

Didanorm : (France-Europe):http://www.didanorm.fr/achat/index.php?catid=1044

Oil-BasedEmulsion Vaccine

Adjuvants

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Supplement 106 Current Protocols in Immunology

Smiths Medical (U.S.): http://www.smiths-medical.com/catalog/iv-disposables/iv-disposables-components-accessories/caps-connectors-adapters-luer/connectors-components.html

Air-Tite Products (U.S.): http://www.air-tite-shop.com/p-215-double-female-luer-lock-adapter.aspx

Promepla (Worldwide): reference no. ODG0015ST13- or 20-mm vial adapter (West Pharmaceutical Services,

http://www.westpharma.com/ or Promepla, http://www.promepla.com/catalog)or 20-G needle

Sterile needle (usually 26-G) suitable for vaccination procedure

Load the products into the syringes

1. Load 1 ml of the aqueous phase (containing antigen) into one syringe and 1 ml ofoil phase (adjuvant) into a second syringe.

The most easy way to transfer liquid phases from vials into syringes is to use a syringewith a needle or a vial adapter.

CAUTION: Take care to avoid self-injection.

2. Withdraw 1 volume of the aqueous phase (antigen) from the vial into the first syringe,and remove air.

If a vial adapter is used, it is possible to leave the syringe attached to the vial via the vialadaptor until it is used again.

3. Repeat the same operation with the second syringe and the oil phase (adjuvant):withdraw 1 volume of oil.

4. Remove the second syringe (prepared in step 3) and twist it onto the connector.

5. Push the plunger in very slowly in order to drain the maximum possible amount ofair from the system.

6. Remove the first syringe (prepared in step 2) from the vial adapter and twist it ontothe connector.

The system is now ready for emulsification.

Perform phase 1 of the emulsification

The first phase of the emulsification process is carried out at very slow speed.

7. Hold the syringe-connector-syringe apparatus firmly to guarantee a constant con-nection. Use thumbs to push the plungers apart.

Do not push with both of the thumbs simultaneously, to avoid any leakage.

8. Push completely on the plunger of one of the syringes in order to get both phases inone syringe.

9. Start to emulsify by alternatively transferring the formulation from one syringe tothe other very slowly.

One cycle includes the passage of the entire formulation from one container to the otherthrough the connector, and back.

The first 20 cycles are done with a slow rhythm. It requires about 4 sec to transfer thepremix from one side to the other. Hence, a complete cycle will require an average of8 sec. This first part will give a “pre-emulsion,” and the full process should take at least2 min.


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Current Protocols in Immunology Supplement 106

Perform phase 2 of the emulsification

The second phase of the emulsification process is carried out at high speed.

10. Continue to emulsify by alternately transferring the formulation from one syringeto the other very high speed (as fast as possible) for 40 cycles.

11. When the (W/O) emulsion starts to form, a resistance can be felt when applyingpressure to the syringe plunger. The mixture assumes a creamy viscous appearanceat this time.

Total emulsification process should be around 3 min.

Finish the emulsification procedure

12. After the 20 slow-speed and the 40 high-speed cycles of the emulsifying process,transfer the entire emulsion into one syringe. Disconnect the syringe from theconnector.

13. Twist a sterile needle (suitable for the vaccination procedure to be performed; usually26-G) on the syringe, and the homogeneous emulsion is ready for use in a vaccinationprotocol.

If storage is necessary, inject the emulsion into a vial for storage and seal it.

It is recommended to keep the vial at 4°C and away from intense light sources. Theemulsion may be used for up to 2 years.

ALTERNATEPROTOCOL 2

HOMOGENIZER PROCEDURE TO PRODUCE W/O EMULSIONS

Another alternative for generating water-in-oil emulsions is to use a high-shear mixer.For example, an L4RT mixer (Silverson, http://www.silverson.com) equipped with a 1-in.head can be used. This allows for the preparation of at least 20 g of emulsion. To performthis method, combine 10 g of aqueous phase with 10 g of adjuvant (blend of oil andsurfactant) in a 50-ml beaker and mix for 3 min at 5000 rpm. The maximum speed of5000 rpm is reached progressively.

It is useful to swirl the liquid in the beaker around the homogenizer head during the earlyphase of the preparation of emulsion in order to initiate the emulsification process.

BASICPROTOCOL 2

SYRINGE PROCEDURE TO CREATE OIL-IN-WATER (O/W) EMULSIONS

For oil-in-water formulations, often squalene-based adjuvants (e.g., MF59 or AS03) oranother type of oil can be used. Emulsifiers that are used for O/W emulsions can includepolysorbates, sorbitan esters, phospholipids (mainly represented by lecithins), or otherhydrophilic surfactants.

A good, stable oil-in-water (O/W) emulsion has fine oil droplets homogeneously dis-persed throughout the outer water phase. The homogenous dispersion of oil droplets canbe lost if the emulsifier does not match or if an insufficient amount of energy is used tocreate the emulsion. Flocculation and creaming leaves fine oil droplets intact, but theybecome less well distributed throughout the water. This can be reversed by moderatemanual shaking, while coalescence or breaking creates large bodies of oil separatingfrom the water and results in the complete separation of the emulsion.

Preparation of the oil-in-water (O/W) vaccine requires only simple mixing in the recom-mended ratio for utilization of the adjuvant (a blend of oil and surfactant). The oil-to-waterratio may range between 4% and 25%. High shear is less critical; therefore fewer cyclesof emulsification are required.


Adjuvants

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Materials

Antigen to be emulsified dissolved in aqueous diluent (also referred to as theaqueous phase)

Adjuvant to be used (blend of oil and surfactant also referred to as the oil phase;see introduction to this protocol)

1-ml sterile syringe (see Basic Protocol 1 materials list)3-ml crimp vial20-G needle

1. Using a sterile syringe, take up the exact quantity of aqueous antigen phase neededand transfer it to a 3-ml crimp vial.

2. Perform the same operation as in step 1 with the adjuvant (blend of oil and surfactant),placing it into in the same crimp vial.

3. Emulsify by pumping the blend of products that were placed in the vial back andforth, using a syringe that is not equipped with rubber plunger and that is connectedto a 20-G needle.

A cycle is defined as a complete passage of the emulsion form the vial to the syringe andback. The differential rhythms used for pumping the emulsion back and forth, describedfor the W/O emulsions in Basic Protocol 1 and Alternate Protocol 1, are not critical inthis protocol.

4. Stop the process once 10 cycles have been completed.

ALTERNATEPROTOCOL 3

HOMOGENIZER PROCEDURE TO PRODUCE O/W EMULSIONS

Homogenization of the antigen and adjuvant to generate O/W emulsions can be performedusing a simple low-shear homogenizer (IKA). The homogenizer should be equipped witha three- or four-blade marine-style propeller. Strong shear stress for a prolonged periodshould be avoided, as it is not necessary and can produce foam. Very low viscosity of thefinal vaccine allows the use of classical stirring devices used for water-based emulsions. Acritical point in performing the homogenization is that mixing time should be sufficientto ensure perfect homogeneity of the complete batch, which depends on the volumeof the product and the efficacy of the mixing. As an example, for lab-scale vaccines,preparation with a four-blade propeller should be performed at the maximum speedwithout introduction of air (to avoid air bubbles). The aqueous phase is first placed intoa beaker under 500 rpm stirring. The adjuvant phase is introduced slowly, and agitationof the blend is maintained for 10 min at 1000 rpm. An additional rest time of 30 minshould be observed (without agitation) to allow stabilization of interfacial phenomenaand disappearance of air bubbles.

COMMENTARY

Background InformationModern vaccine antigens are often highly

purified antigens (for example, recombinantproteins or peptides), which require co-formulation of a vaccine adjuvant in order tobecome immunogenic and trigger a decent im-mune response. A vaccine adjuvant is definedas a component that potentiates the immuneresponse to an antigen and/or modulates it to-ward the desired immune responses, as firstobserved by Ramon (1925). Hence, vaccineadjuvants accelerate, increase, and prolong im-

mune responses, compensating for the poorimmune responsiveness of the antigen. Ad-juvants may also reduce the dose of antigenrequired for protection, which is very usefulwhere antigen is scarce. In addition, adjuvantsmay strongly influence the quality of the ensu-ing immune reaction, such as a type 1 or type 2immune pathway (Schijns and Lavelle, 2011).Many different molecules and formulations fitthis broad definition, but they differ in theirability to elicit the desired type and strength ofimmune response, which makes it difficult to


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choose the right type of adjuvant (Dang et al.,2012; Flemming, 2013; Hailemichael et al.,2013). Hence, the choice of the right adjuvantand its correct formulation largely determinethe success of the vaccine.

Well-known adjuvants include, e.g., Fre-und’s complete or incomplete adjuvant, alu-minum salt–based formulations, saponins, nat-ural or synthetic agonists of innate immunecell receptors, and various types of particu-late structures. Until recently, only aluminumsalt–based formulations have been used in li-censed human vaccines, since other choicesare considered too toxic for use in a healthyhuman population, although recently the oil-in-water adjuvant MF59 has been licensed forhuman vaccines (Ott et al., 1995). By contrast,in the veterinary field many different typesof adjuvants have been licensed (Schijns andO’Hagan, 2006).

Strategies of Adjuvant ChoiceIn general, the first consideration is which

type of immune pathway is expected to pro-vide the desired vaccine efficacy. For exam-ple, should the immune response preferablyevoke antigen-specific antibodies or effector Tcells, which recognize processed antigen frag-ments presented in MHC molecules of targetcells? The type of desired immune reactiondepends on the type of disease indicationand the known or expected immunologi-cal correlates of protection. In general, mi-croorganisms that are vulnerable to antibod-ies during an extracellular stage of theirlife cycle may be targeted by vaccine-elicited antibodies specific to an accessibleextracellular antigen. By contrast, intracel-lular microorganisms, with a predominantlyintracellular life-cycle, as well as tumors,require clonally expanded antigen-specificT cells for elimination.

Choice of the right adjuvantThe choice of the right type of adjuvant is

critical in order to evoke the desired type andstrength of immune response. Many vaccinefailures result from the wrong choice, and,unfortunately, are seldom published. Also,comparative adjuvant studies with the sameantigen are rare. The impact of the type ofadjuvant is obvious in view of its effect onimmune signals 2, 3, and 4 (Schijns et al.,2014). In attempts to evoke a T cell response,Dang et al. (2012) showed that addition of theTLR agonist imiquimod (a trigger of immunesignal 2 and 3), administered transdermallyat the vaccine site immediately after the

antigen administration, in combination withthe injected adjuvant granulocyte macrophagestimulating factor (GM-CSF), increases sys-temic levels of MDSC and regulatory T cells,which results in a reduced immune responseand more interleukin-10 production. Anotherclear example of how adjuvants influenceimmune cell homing (signal 4) has beenpublished by Hailemichael and co-workers,who elegantly demonstrated that Freund’sincomplete adjuvant (IFA), a W/O formula-tion prepared from mineral oil, readily evokespeptide-specific T cells; however, it resultedin sequestration of specific CD8+ T cells atthe injection site, and less available systemiceffector function relative to a non-depot ad-juvant (Hailemichael et al., 2013). This studydescribes an example that may be specific tocancer and may also refer to a specific peptidelength.

Critical ParametersAntigenic concentration and quality can

have an influence on physico-chemical param-eters of the final emulsions generated using theprotocols described in this unit. It is recom-mended that some pilot preparations be gen-erated initially using a placebo aqueous phasein lieu of the antigen prior to use, in order tobecome familiar with these protocols.

Rapid quality controls on water-in-oilemulsions

No accelerated test exists for stability pre-diction, since various physicochemical param-eters are involved in creating the emulsion.However, different controls can be done toassess efficacy of the emulsification process,including visual inspection, viscosity mea-surement, and microscopic observation. In ad-dition, dispersed phase droplet size measure-ment by the laser light scattering method canhelp to characterize the emulsion and predictits stability.

The composite of all observations togethergives a good impression of the quality of theemulsion and of the process that was used toperform it.

As a very easy and quick control, it is possi-ble to check if a W/O emulsion has really beenachieved using the so-called drop test. A dropof emulsion is placed into a beaker of water bymeans of a pipet. In the case of a W/O emul-sion, this drop will never mix with the water,even after stirring.

from a more long-term point of view, andfor stability assessment, the following controlscan be performed at determined timelines.


Adjuvants

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Visual inspectionThe emulsion should be homogeneous and

present a white color. However, some defectscan appear over the time. Some are critical,such as breakage, which is a separation of thetwo phases. Others are noncritical and accept-able, such as an oil residue on the top of theformulation, or creaming presenting two dif-ferent tones of white.

Microscopic inspectionA droplet of emulsion can placed under the

microscope and observed at 600× magnifica-tion. This gives information on how fine orcoarse the emulsion is.

Laser-light scatteringA drop of the prepared emulsion is placed

into the chamber of a Malvern Mastersizer(http://www.malvern.com), running with oil-phase solvent. The equipment is linked to com-puter software, and events are statistically re-ported. It allows definition of a value calledDv50, where that the volume occupied by 50%of dispersed phase is made of droplets smallerthan the indicated value. For a stable emulsion,this value should not evolve over the time.

Quality controls on oil-in-wateremulsions

The so-called drop test can also be used forO/W emulsions. A drop of emulsion is placedinto a beaker of water by means of a pipet. Incase of a O/W emulsion, this drop is immedi-ately dispersed into the water.

Visual inspection of the emulsion, micro-scopic observation, and laser-light scatteringare also quality controls that can be appliedfor O/W formulations. For laser-light scatter-ing, however, the equipment should be runwith water solvent before including the dropof the emulsion and performing drop-sizemeasurements.

Literature CitedAscarateil, S. and Dupuis, S. 2006. Surfactants in

vaccine adjuvants: Descriptions and perspec-tives. Vaccine 24:S283-S285

Dang, Y., Wagner, W.M., Gad, E., Rastetter, L.,Berger, C.M., Holt, G.E., and Disis, M.L. 2012.

Dendritic cell-activating vaccine adjuvants dif-fer in the ability to elicit antitumor immunity dueto an adjuvant-specific induction of immunosup-pressive cells. Clin. Cancer Res. 18:3122-3131.

Flemming, A. 2013. Tumour vaccination: Devil inthe details of adjuvant choice. Nat. Rev. DrugDiscov. 12:343.

Hailemichael, Y., Dai, Z., Jaffarzad, N., Ye, Y.,Medina, M.A., Huang, X.F., Dorta-Estremera,S.M., Greeley, N.R., Nitti, G., Peng, W., Liu,C., Lou, Y., Wang, Z., Ma, W., Rabinovich, B.,Schluns, K.S., Davis, R.E., Hwu, P., and Over-wijk, W.W. 2013. Persistent antigen at vaccina-tion sites induces tumor-specific CD8+ T cell se-questration, dysfunction and deletion. Nat. Med.19:465-472.

Jansen, T., Hofmans, M.P.M., Theelen, M.J.G., andSchijns, V.E.J.C. 2005. Structure–activity rela-tions of water-in-oil vaccine formulations andinduced antigen-specific antibody responses.Vaccine 23:1053-1060.

Jansen, T., Hofmans, M.P.M., Theelen, M.J.G.,Manders, F., and Schijns, V.E.J.C. 2006.Structure-and oil type-based efficacy of emul-sion adjuvants. Vaccine 24:5400-5405.

Koh, Y., Higgins, S.A., Weber, J.S., and Kast,W.M. 2006. Immunological consequences ofusing three different clinical/laboratory tech-niques of emulsifying peptide-based vaccines inincomplete Freund’s adjuvant. J. Transl. Med.4:42.

Moreira, L.O., Smith, A.M., DeFreitas, A.A.,Qualls, J.E., El Kasmi, K.C., and Murray, P.J.2008. Modulation of adaptive immunity by dif-ferent adjuvant-antigen combinations in micelacking Nod2. Vaccine 26:5808-5813.

Ott, G., Barchfeld, G.L., Chernoff, D., Radhakr-ishnan, R., van Hoogevest, P., and Van Nest,G. 1995. MF59. Design and evaluation of a safeand potent adjuvant for human vaccines. Pharm.Biotechnol. 6:277-296.

Ramon, G. 1925. Sur l’aumentation anormale del’antitoxine chez les chevaux producteurs deserum antidiphterique. Bull. Soc. Centr. Med.Vet. 101:227-234.

Schijns, V.E.J.C. and Lavelle, E.C. 2011. Trends invaccine adjuvants. Expert Rev. Vaccines 10:539-550.

Schijns, V.E.J.C. and O’Hagan, D. (Editors). 2006.Immunopotentiators in Modern Vaccines. Else-vier/Academic Press, London.

Schijns, V., Tartour, E., Michalek, J., Stathopoulos,A., Dobrovolskiene, N.T., and Strioga, M. 2014.Immune adjuvants as critical guides directingimmunity triggered by therapeutic cancer vac-cines. Cytotherapy 16:427-439.


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current protocols in immunology || oil-based emulsion vaccine adjuvants

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