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Evolution of Adjuvants Across the Centuries
Adjuvants are substances that are added to vaccine antigens to enhance and modulate the immunogenicity of its antigen. The first adjuvants developed focused on increasing antibody responses, and this has often been sufficient for the vaccines considered. During the last three decades, however, it has been realized that simply increasing antibody responses is not always sufficient for candidate vaccines to be effective. It has been observed that adjuvants can be used very effectively to:
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provide a strong priming response in naïve populations, effectively reducing the number of doses required to induce protection.
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increase the duration of the immune response.
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enhance specific arms of the immune response such as cell-mediated immunity (CMI), a critical target for many of the remaining infectious diseases for which we do not have vaccines yet.
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increase the breadth of the immune response to variable antigens, enabling broader cross-protection.
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enhance the immune response in poorly responsive populations, such as elderly and immunosuppressed populations.
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allow for dose sparing of antigens where antigen supply is limited.
Generally speaking, adjuvants are useful for antigens such as inactivated, subunit, and recombinant proteins, which can lose, during the purification process, some of the immunological properties present in the pathogen that are needed to trigger an immune response. They have not yet been required for live attenuated vaccines, which carry the necessary immune-stimulating signals themselves. As discussed later, however, some preliminary research suggests that adjuvants can have an effect on live attenuated vaccines as well.
CHANCE AND NECESSITY: THE DISCOVERY OF ADJUVANTS
The use of adjuvants has been known for more than a century, and it is during the last decade that their mechanism of action was elucidated, in part owing to the progress in microbiology and immunology. The first recorded observation of immune potentiation by “adjuvants” is probably that of Coley, who in 1893 observed that administration of killed bacteria (Coley toxins) could in some cases, cure certain forms of cancer. It was only in the 1990s that it was determined that this effect was the result of immune stimulation mediated by bacterial DNA. From there on, the specific oligonucleotide sequences that could enhance the immune response to a coadministered antigen were discovered.
It took another two decades to recognize the usefulness of adjuvants to enhance humoral immunity. In 1925, Ramon observed that administering diphtheria toxoid to horses with a variety of substances, including starch, plant extracts, or fish oils, substantially enhanced the antibody response to the toxoid. A year later, Glenny observed a similar effect with aluminum potassium sulfate, or alum. Alum was used thereafter as an adjuvant for numerous human vaccines, and to this day, other aluminum salts, in the form of aluminum oxyhydroxide or hydroxyphosphate, are the most widely used adjuvants in human vaccines. The starch and fish oils shown by Ramon to act as adjuvants have, in recent decades, been tested in vaccines in the form of inulin and squalene, respectively.
During the 80 years following the first use of aluminum salts as adjuvants, a wide variety of substances were tested, but many of them failed to be acceptable for human use. In the 1940s, Jules Freund developed a water-in-oil emulsion, the Freund adjuvant, in which the vaccine antigen is emulsified as water droplets in a continuous mineral oil phase, containing killed mycobacterium (Freund complete adjuvant) or not (Freund incomplete adjuvant). The latter was briefly used for a commercial influenza vaccine in the United Kingdom in the 1960s but was soon withdrawn owing to unacceptable reactogenicity. This, however, led to the development of oil-in-water emulsions, in which oil droplets are present in a continuous aqueous phase.
The first oil-in-water emulsions as distinct from mineral oils as in the original Freund adjuvant, were based on a nonmetabolizable oil (squalane) and replaced later with metabolizable oils (squalene). A water-in-oil emulsion similar in structure to the Freund adjuvant has been introduced in a cancer vaccine, using mineral oil with a higher degree of purity that allows for use in human vaccine candidates.
In the 1970s, liposomes and virosomes that adsorb or encapsulate antigen were developed. Liposomes consist of lipid layers that form nanospheres or microspheres and can encapsulate or integrate antigens into their membranes. Several licensed vaccines contain virosomes, which are reconstituted empty envelopes of influenza viruses similar in structure to liposomes.
A TURNING POINT: BETTER UNDERSTANDING OF IMMUNOLOGY AND ITS IMPACT ON THE DEVELOPMENT OF ADJUVANTS
For most of the 20th century, adjuvant discovery and development was based on observations and experimentation with no clear immunological knowledge of the mechanism behind the adjuvant effect. This, however, dramatically changed in 1996 with the discovery of the Toll-like receptors (TLR) family in Drosophila and their relation to fungal resistance by Lemaitre and colleagues. One year later, in 1997, Janeway identified the link between human TLR4 and its key role in initiating an adaptive immune response, the first necessary step to a long-lasting immune response. The discovery by Poltorak and colleagues that TLR4 functioned as a lipopolysaccharide (LPS)-sensing receptor and, hence, the use of LPSs or their derivatives as adjuvants, brought the final piece to the understanding of the mechanism of actions of TLR agonist molecules.
In the early 1980s, Edgar Ribi established that it was possible to produce a molecule that retained the immune potentiation activity of LPS without the associated toxicity. Approximately 30 years later, in 2009, monophosphoryl lipid (MPL) A was the first new adjuvant in a vaccine (Cervarix vaccine against human papillomavirus [HPV]) approved for use by the U.S. Food and Drug Administration.
It is well understood now that the immune system uses pathogen-associated molecular patterns (PAMPs) to activate pathogen-recognition receptors such as TLR. A host of other more recently discovered receptors acts according the same principle: retinoic-acid–inducible gene-based-I–like receptors (RLRs), and cytosolic nucleotide oligomerization domain (NOD)-like receptors (NLRs). , These receptors bind various pathogen ligands (ranging from, e.g., bacterial cell wall and cell membrane components to bacterial or viral nucleotides, to fungal lipids) to trigger different types of immune responses and, if combined with an antigen, can initiate and enhance specific arms of the immune responses to that antigen. For the purpose of brevity, readers are directed to other sources for a detailed description of how pathogen components stimulate various cytokine pathways and how they direct different arms of the immune response ; Fig. 7.1 shows this information schematically. As shown in Fig. 7.1 , different TLRs, located on the plasma membrane or intracellularly, respond to different pathogen-derived signals to induce proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, or type 1 interferon, leading to a predominantly T-helper cell 1 (Th1) response. Certain TLR2 agonists, however, have been reported to activate Th2 responses.
Table 7.1 lists the various agonists that activate TLRs, as well as the adaptor molecules, also known as TLR agonists, and examples of adjuvants, which function through these receptors.
TABLE 7.1
Agonists That Activate the TLRs, Adaptor Molecules, and Examples of Adjuvants
| TLR | Ligand | Ligand Location | Adaptor | Adjuvant |
|---|---|---|---|---|
| 1 | Tripalmitoyl-cysteine lipopeptides | Bacterial membranes | MyD88 MAL | |
| 2 | Lipopeptides, β-glucan, glycolipids | Bacterial membranes | MyD88 MAL | |
| 3 | Double-stranded RNA | Viral RNA | TRIF | Poly(I:C); poly(A:U) |
| 4/MD2 | Lipopolysaccharide | Bacterial membranes | MyD88 MAL/TRIF | MPL, GLA, E6020, RC529 |
| 5 | Flagellin | Bacterial outer surface | MyD88 | VaxInnate |
| 6 | Dipalmitoyl-cysteine lipopeptides | Bacterial membranes | MyD88 MAL | |
| 7 | Single-stranded RNA | MyD88 | Imidazoquinolines: imiquimod, loxoribine | |
| 8 | Single-stranded RNA | MyD88 | R848 | |
| 9 | Bacterial DNA, unmethylated CpG DNA sequences, poly(dI:dC) | Bacteria | MyD88 | CpG; IC-31 CpG 1018 |
CpG, cytosine phosphate guanine; GLA, glucopyranosyl lipid adjuvant; MAL, MyD88 adaptor-like; MPL, monophosphoryl lipid A; MyD88, myeloid differentiation 88; TLR, Toll-like receptor; TRIF, TIR domain-containing adaptor-inducing IFN-β.
Based on this understanding, it is possible to recognize today how most of the adjuvants function. This knowledge should allow rapid screening of compound libraries for molecules that bind these receptors and that may have adjuvant activity leading to the rational design of new adjuvants aimed at stimulating specific arms of the immune response. The level of knowledge associated with the mechanism of action of those specific molecules and, as a consequence, the pattern of cytokines induced should in turn allow an assessment of the impact of an adjuvant on the safety of a vaccine. This has not yet led to the discovery of new molecules with defined activities but in certain cases has enabled a better understanding of the mode of action of specific adjuvants and helped support their safety profile within a given vaccine. A clear understanding of the pathogenesis of immune-mediated disorders and their triggers is required, however, to ascertain the potential negative impact of the adjuvant. Progress has been made in understanding the mechanism of action of non-PAMPs adjuvants. QS21, an essential part of the adjuvants known as AS01 (Zoster vaccine), has been established, albeit in a specific liposomal formulation that abrogate its inherent local reactogenicity when use alone. For several adjuvants the exact mechanism of action remains elusive or may present multiple modes of action (such as aluminum salts).
This is only the first step in defining the value of a molecule as an adjuvant. Further evaluation of the compound in vivo and its safety profile will define its real potential as an adjuvant for vaccines. ,
DEFINING ADJUVANTS: CLASSIFICATION AND EVALUATION
This section is limited to a review of the types of adjuvants that are incorporated in licensed vaccine formulations or for which there is extensive clinical experience.
A Categorization of Adjuvants Based on Mechanism of Action
The majority of adjuvant reviews during recent decades have tried to classify adjuvants according to their mechanism of action and typically classified adjuvants as vehicles or immunostimulants . Immunostimulants are substances that act directly on the immune system, such as TLR ligands. Vehicles are thought to act primarily by presenting antigens to the immune system. In this group, there are various aluminum salts, emulsions, immunostimulatory immune complexes (ISCOMs), and biodegradable microparticles. It is now known that most of the vehicles act directly on the immune system, and antigen presentation may be only a minor component of the adjuvant activity (e.g., see the later discussions of modes of action of aluminum salts [“Aluminum Salt Adjuvants”] and oil-in-water emulsions [“Oil-in-Water Emulsions”]). This classification seems outdated, and it may be preferable to classify adjuvants according to their receptor or, when the receptor is unknown, by their physical or chemical nature.
Currently 12 adjuvants are approved for use in vaccines (three aluminum salts with different counterions, four oil-in-water emulsions, aluminum/MPL combination, virosomes, polyoxidonium, MPL/QS21 in liposome combination and CpG). Table 7.2 lists the approved vaccines containing these adjuvants. Numerous other adjuvants are in vaccines that are under development. AS01 is part of the zoster vaccine Shingrix, and was approved in 2017 in the United States. CpG was approved the same year in the hepatitis B virus vaccine, Heplisav B. These and their classifications by receptor or physicochemical nature are presented in Table 7.3 and discussed in the following sections. Many more adjuvants are in preclinical development; however, they are too numerous to discuss in this chapter.
TABLE 7.2
Types of Licensed Vaccines Containing Adjuvants
| Vaccine | Trade Name a | Adjuvant |
|---|---|---|
| Diphtheria and tetanus vaccine (DT) | Diphtheria and Tetanus Toxoid Adsorbed USP (1) | Aluminum potassium phosphate |
| DT acellular pertussis (DTaP) | Tripedia (1) | Aluminum potassium phosphate |
| Hemophilus influenzae type b (Hib) | Liquid PedvaxHIB (2) | Aluminum hydroxyphosphate sulfate |
| DTaP + Hib | TriHIBit (1) | Aluminum potassium phosphate |
| Hepatitis B | Recombivax HB (2) | Aluminum hydroxyphosphate sulfate |
| Hepatitis B | Engerix-B (3) | Aluminum hydroxide |
| Hepatitis B + Hib | Comvax (2) | Aluminum hydroxyphosphate sulfate |
| Hepatitis A | Havrix (3) | Aluminum hydroxide |
| Hepatitis A | Epaxal (6) | Virosomes |
| Hepatitis A + hepatitis B | Twinrix (3) | Aluminum hydroxide/phosphate |
| Pneumococcal conjugate vaccine | Prevnar (4) Synflorix (3) |
Aluminum phosphate |
| Influenza vaccine | FLUAD (4) b | MF59 |
| Influenza vaccine | Inflexal V (6) b | Virosomes |
| Pandemic influenza vaccine | Pandemrix | AS03 |
| Pandemic influenza vaccine | Focetria | MF59 |
| Pandemic influenza vaccine | Humenza | AF03 |
| Human papillomavirus (HPV) | Gardasil (2) | Aluminum hydroxyphosphate sulfate |
| HPV | Cervarix (3) | Aluminum hydroxide + MPL |
| Hepatitis B | Fendrix (3) b | AS04 (MPL + aluminum phosphate) |
| Hepatitis B | SUPERVAX (7) b , c | RC529 |
| Hepatitis B | Heplisav | CpG |
MPL, monophosphoryl lipid A.
a Manufacturers are as follows: 1, Sanofi Pasteur; 2, Merck; 3, GlaxoSmithKline; 4, Wyeth now Pfizer; 5, Novartis; 6, Crucell; 7, Dynavax Europe.
TABLE 7.3
Nonexhaustive List of Adjuvants in Vaccines Under Development or Approved
| Class (By MOA) | Component | Adjuvant Name and Other Compounds | Manufacturer | Phase I | Phase II | Phase III | Licensed |
|---|---|---|---|---|---|---|---|
| Hiltonol (polylysine) | Oncovir | Cancer | |||||
| TLR4 | MPL | AS04 (alum) | GSK | HPV, HBV | |||
| MPL | AS02 (emulsion, QS21) | GSK | Cancer | ||||
| MPL | AS01 (liposome, QS21) | GSK | Malaria, zoster | ||||
| MPL | AS15 (liposomes QS21, CpG) | GSK | Cancer | ||||
| MPL | (tyrosine) | ALT | Allergy | ||||
| RC529 | RC529 (alum) | GSK | HBV | ||||
| GLA | SE-GLA (emulsion) | IDRI | Influenza | ||||
| Stimuvax | Biomira/Oncothyreon | Cancer | |||||
| E6020 | Esai | ||||||
| TLR5 | Flagellin | Fusion to influenza hemagglutinin | VaxInnate | Influenza | |||
| TLR7 | Imiquimod | Topical | Cancer | ||||
| Imiquimod | Combined with ISA51 | Cancer | |||||
| TLR9 | CpG | 1018 ISS | Dynavax | Allergy | |||
| CpG 7909 | Coley/Pfizer | HBV | Cancer | ||||
| CpG 7909 + alum | NIAID | Malaria | |||||
| AS15 (liposomes, MPL, QS21) | GSK | Cancer | |||||
| dI:dC | IC31 (cationic peptide) | Intercell | Influenza, TB | ||||
| Saponins | QS21 | AS01 (liposome, MPL) | GSK | Malaria | |||
| QS21 | AS15 (MPL, CpG) | GSK | Cancer | ||||
| QS21 | QS21 | Universities | HIV, influenza | Cancer | |||
| Quil fractions | ISCOM (cholesterol) | CSL | |||||
| Iscomatrix (cholesterol) | CSL | HPV, influenza | Cancer | ||||
| GPI-0100 | Cancer | ||||||
| Oil-in-water emulsion | Squalene | MF59 | Novartis | HIV | HBV, CMV | Seasonal influenza, pandemic influenza | |
| Squalene | AF03 | Sanofi Pasteur | Influenza | ||||
| SE | IDRI | Influenza | |||||
| Tocopherol | AS03 (squalene) | GSK | Pandemic influenza | ||||
| Squalane | CoVaccine (acyl sucrose sulfate) | Protherics | Angiotensin | ||||
| Nobilon | Influenza | ||||||
| Water-in-oil emulsion | Squalene | ISA 720 | Seppic | Malaria, cancer | |||
| ISA 51 | Seppic | Malaria | Cancer | ||||
| Polysaccharides | Inulin | Advax (alum) | Vaxine | HBV, influenza | |||
| Cationic liposomes | DDA | CAF (TDM) | SSI | TB | |||
| JVRS-100 (DNA) | Juvaris | Influenza | |||||
| Virosomes | Crucell | HAV, influenza | |||||
| Pevione | Malaria | ||||||
| Polyelectrolytes | Polyoxidonium | Microgen | Influenza |
Classified according to adjuvant receptor or adjuvant physicochemical nature (alum-derived adjuvants excluded for clarity).
ALT, alanine aminotransferase; CMV, cytomegalovirus; CpG, cytosine phosphate guanine; DDA, dimethyldioctadecylammonium; GLA, glucopyranosyl lipid adjuvant; GSK, GlaxoSmithKline; HAV, hepatitis A virus; HBV, hepatitis B virus; HPV, human papillomavirus; IDRI, Infectious Disease Research Institute; ISCOM, immunostimulatory immune complex; MOA, mechanism of action; MPL, monophosphoryl lipid A; NIAID, National Institute of Allergy and Infectious Diseases; SE, stable emulsion; TB, tuberculosis; TLR, Toll-like receptor.
Aluminum Salt Adjuvants
Aluminum-containing adjuvants have historically served as immunostimulants in vaccines and continue to be the most widely used adjuvants. Several aluminum compounds are used and known, such as aluminum hydroxide, aluminum phosphate, and alum. All three of these commonly used names are scientific misnomers. Although this family of adjuvants is used the longest, it is only recently that we have begun to understand their mechanism of action and the complexity of formulating them with antigens.
The following sections summarize the structure and properties of different aluminum salts, the mechanisms by which they stimulate the immune response, and the effect of freezing on aluminum-adjuvanted vaccines.
Structure and Properties
Aluminum hydroxide adjuvant is not Al(OH) 3 , but rather crystalline aluminum oxyhydroxide (AlOOH). This difference is important, as crystalline aluminum hydroxide has a low surface area (approximately 20–50 m 2 /g) and, as such, is a poor adsorbent. Crystalline aluminum oxyhydroxide has a surface area of approximately 500 m 2 /g, which makes it an excellent adsorbent. This high surface area is a result of its morphology. The primary particles are fibers having dimensions of approximately 5 × 2 × 200 nm.
Aluminum oxyhydroxide is a stoichiometric compound. The surface is composed of Al–OH and Al–O–Al groups. The Al–OH surface groups can accept a proton, resulting in a positive surface charge, or donate a proton, resulting in a negative surface charge. As shown in Fig. 7.2 , the isoelectric point (IEP) of Al–OH is 11.4. Thus, aluminum oxyhydroxide exhibits a positive surface charge at pH 7.4, which is the pH of interstitial fluid.
Aluminum phosphate adjuvant is a chemically amorphous aluminum hydroxyphosphate in which some of the hydroxyl groups of aluminum hydroxide are replaced by phosphate groups. The disordered, amorphous state is responsible for the high surface area and high adsorptive capacity.
The surface of aluminum phosphate adjuvant is composed of Al–OH and Al–OPO 3 groups. The IEP varies from 9.4 to 4.5 depending on the degree of phosphate substitution. Commercial aluminum phosphate adjuvants have IEP values in the 4.5–5.5 range. In contrast with aluminum oxyhydroxide, commercial aluminum phosphate adjuvants are negatively charged at pH 7.4 (see Fig. 7.2 ).
Alum, which is water-soluble, is chemically aluminum potassium sulfate, AlK(SO 4 ) 2 . The earliest vaccines containing aluminum adjuvants were prepared by in situ precipitation. A solution of alum is mixed with a solution of the antigen dissolved in a phosphate buffer. It is common practice to refer to the adjuvant produced by in situ precipitation as alum. The precipitate is known as amorphous aluminum hydroxyphosphate and has a composition and properties similar to aluminum phosphate adjuvant. ,
Techniques to be used to characterize aluminum-containing adjuvants have been reviewed by White and Hem.
Effect of Freezing
Vaccines containing aluminum hydroxide adjuvant or aluminum phosphate adjuvant should not be allowed to freeze and should not be used if suspected of having been exposed to freezing temperatures. Freezing may affect the aluminum-containing adjuvant and the adsorbed antigen. Coagulates, which cannot be redispersed by shaking, form when aluminum-containing adjuvants are frozen. Thermostability of vaccines is of increasing importance and, as demonstrated for aluminum salts, concerns not only excursion at high temperature but also at low temperature, such as freezing.
Adsorption Mechanisms
The major mechanisms responsible for the adsorption of antigens are electrostatic attraction, hydrophobic forces, and ligand exchange. Electrostatic attraction is probably the most frequently used adsorption mechanism.
Electrostatic attraction can be optimized by determining the IEP of the antigen and then selecting an adjuvant that will have the opposite surface charge at the desired pH. For example, at pH 7.4, aluminum hydroxide adjuvant (IEP = 11.4) adsorbs albumin (IEP = 4.8) but does not adsorb lysozyme (IEP = 11.0). In contrast, aluminum phosphate adjuvant (IEP = 4.0) adsorbs lysozyme but not albumin at pH 7.4.
Care must be taken in selecting a buffer for an aluminum hydroxide adjuvant-containing vaccine. Electrostatic attraction for an acidic antigen may be reduced or reversed if a phosphate buffer is used. Acetate and tromethamine (TRIS) are examples of buffers that do not alter the IEP of aluminum hydroxide adjuvant.
Aluminum hydroxide adjuvant can also be pretreated to lower the IEP to optimize electrostatic adsorption of basic antigens.
Hydrophobic forces can also contribute to the adsorption of antigens by aluminum-containing adjuvants. The contribution of hydrophobic attractive forces can be determined by observing the effect of ethylene glycol on adsorption. Ethylene glycol stabilizes the hydration layer of proteins, which renders hydrophobic interactions thermodynamically unfavorable.
Mechanisms of Action of Aluminum Salt Adjuvants
There is still no consensus regarding the mechanisms by which aluminum-containing adjuvants potentiate the immune response. Several mechanisms are frequently cited to explain how aluminum-containing adjuvants increase antibody production. The depot mechanism was initially thought of as the dominant one; later the promotion of uptake of antigens by antigen-presenting cells (APCs) and, more recently, a direct immune-stimulating mechanism were proposed.
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The depot mechanism postulates that the aluminum-containing adjuvant and the adsorbed antigen remain at the site of injection. The antigen is released slowly to stimulate the production of antibodies. This hypothesis is supported by the observation with some antigens that stronger binding to the aluminum salt crystals can result in higher immune responses. This hypothesis is, however, inconsistent with the observation that alum injection sites can be excised shortly after injection with no impact on immunogenicity.
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It has also been proposed that adsorption of antigen to aluminum-containing adjuvants converts the soluble antigen to a particulate form. APCs take up particulate matter more efficiently by phagocytosis. Thus, antigen, which remains adsorbed, is taken into macrophages and dendritic cells. A dendritic cell culture study revealed that antigens that elute from the aluminum-containing adjuvants are internalized by macropinocytosis, while those that remain adsorbed are internalized by phagocytosis. Antigen internalization by dendritic cells was enhanced when the antigen remained adsorbed to the aluminum-containing adjuvant following administration and when the aggregate size of the adjuvant was smaller than dendritic cells.
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Several groups have identified a role for a direct stimulation of the immune system through innate immune receptors and identified activation of the Natch domain, leucine-rich repeat, and PYD-containing protein (NALP)-3 inflammasome pathway by alum as the mechanism of action. The precise mechanism by which alum stimulated NALP3 remained unknown. This has been refined and alum crystals have been shown to interact directly with membrane lipids on the surface of dendritic cells. The resulting lipid sorting triggers signaling cascades, independent of the inflammasome, that promote CD4 + T-cell activation.
It is likely that all three proposed mechanisms contribute to the immunostimulation produced by aluminum-containing adjuvants.
Safety of Aluminum-Containing Vaccines
Aluminum salts as adjuvants have the longest and largest safety track record of all adjuvanted vaccines, with more than 3 billion vaccine doses used during the past 90 years with a positive benefit-to-risk ratio. Focal histologic lesions have been observed in patients with diffuse muscular symptoms that included persistent myalgias, arthralgias, and fatigue. In the approximately 130 cases studied, these lesions were identified as macrophagic myofasciitis (MMF). Intracytoplasmic inclusions in the infiltrating macrophages have been identified as containing aluminum by electron microscopy, microanalysis, and atomic adsorption spectroscopy. The presence of aluminum in the deltoid muscle biopsies suggested to Gherardi and colleagues that the source of the aluminum was aluminum hydroxide adjuvant. However, no relationship between the presence of aluminum and MMF and the clinical symptoms has been established. The Vaccine Safety Advisory Committee of the World Health Organization (WHO) reviewed MMF at a meeting in 1999. The committee found that there was no basis for recommending a change in vaccination practices involving vaccine selection, schedule, delivery practices, or information involving aluminum-containing vaccines. The committee recommended that “research studies be undertaken to evaluate the clinical, epidemiological, and basic science aspects of MMF.” The U.S. Food and Drug Administration, while recognizing the desirability of new adjuvants, confirmed its support of aluminum salts in vaccines. Research studies undertaken to assess the neurotoxicity of aluminum when it is administered intramuscularly or in a vaccine showed a difference between the control group and the aluminum-based vaccine tested. A repeat of the experiment, however, did not confirm any differences between the control group and the two vaccines containing aluminum. To date, even though it is established that aluminum salt can be recovered at the injection site months or years after intramuscular injections, no link between the presence of aluminum salt and the MMF syndrome has been clearly established.
Water-in-Oil Emulsions
Water-in-oil emulsions, of which the Freund adjuvant is the best-known example, were included in a commercial influenza vaccine in the United Kingdom in the 1960s. The vaccine was later withdrawn owing to occasional abscesses observed at the site of injection. Initial large-scale clinical studies on 18,000 military recruits conducted in 1953 , resulted in some nodules at the injection sites, which were attributed to impurities (short-chain fatty acids) in the Arlacel-A surfactant. However, when this surfactant was purified, the incidence of cysts was reduced. A 10-year follow-up on these volunteers showed that cyst-like reactions had required hospitalization in 0.1–0.6% of the volunteers, but otherwise there were no adverse effects of the vaccine observed. A subsequent 35-year follow-up demonstrated that not only were there no adverse correlations with different diagnoses, including autoimmune diseases, but also, for some of the disease categories, there was decreased mortality. In contrast with these data from a large clinical trial, studies in rodents in 1972 demonstrated that when male Swiss mice were injected with mineral oil–based emulsions, the mice developed tumors. As a consequence, mineral oil–based emulsions were determined to be unacceptable for human use.
As a result, water-in-oil emulsions based on metabolizable oil were developed using squalene instead of mineral oil. The best-known examples of these are the Montanide adjuvants such as ISA 720 produced by the company Seppic. ISA 720 water-in-oil emulsion has been widely tested in more than 70 clinical trials in which it was often shown to induce immune responses rarely surpassed by other adjuvants. However, as with mineral oil–based emulsions, cysts or sterile abscesses at the site of injection were not infrequent and tend to increase in frequency on boosting. In addition, instability of the antigen in contact with the emulsion was observed. Finally, the difficulty in performing reproducible formulation at the time of administration led to a preformulation that may be incompatible with antigen stability. These challenges suggest that for prophylactic vaccines, researchers should, when possible, avoid using water-in-oil emulsions. For therapeutic vaccines, however, the risk of cysts and the challenges of formulation may be less significant. Such a vaccine (CIMAvax), which contains a mineral oil–based water-in oil-emulsion, Montanide ISA 51, has been licensed in Cuba for non–small-cell lung cancer.
Oil-in-Water Emulsions
Oil-in-water emulsion adjuvants were initially developed as an alternative to water-in-oil emulsions; the lower viscosity makes them easier to inject. The first emulsion of this class to be developed for human use was the SAF adjuvant made by Syntex Corporation. This emulsion was based on nonbiodegradable squalane, the catalytically hydrogenated form of squalene, and was designed as a vehicle to carry a synthetic immunostimulant, threonyl muramyl dipeptide (MDP). The SAF adjuvant, while displaying strong adjuvant activity, was too reactogenic for use, partially because of MDP. However, because it was less effective without the immunostimulant, the emulsion was abandoned. Later, Chiron Corporation developed a range of oil-in-water emulsions by replacing squalane with squalene, a biodegradable lipid, and removing muramyl derivatives. One of these emulsions (MF59) demonstrated some adjuvant properties and further evaluated. MF59 and the majority of the later-developed oil-in-water emulsions used squalene, a natural, metabolizable product found in all plant and animal cells where it is a precursor of cholesterol. The commercial source is generally from shark liver, where it is abundant; alternative sources such as phytosqualene or hemisynthetic squalene are being explored. However, to date, only squalene from shark origin allows for a product with a purity level acceptable for human use.
Despite extensive clinical studies with a wide range of antigens, MF59 is approved only in one vaccine, Fluad, an influenza vaccine for older adults, and licensed in several European countries from 1997 onward. While there was benefit of the adjuvanted vaccine in terms of antibody response to the influenza hemagglutinin in the target population, the really significant benefit of MF59 and other oil-in-water emulsions became clear during investigations on pandemic influenza vaccines. The emergence of avian H5N1 influenza with occasional human-to-human transmission and the fear that this could become a pandemic led to intensive research in academic and pharmaceutical environments for ways to immunize a largely immunologically naïve population in the context of limited antigen supplies. This is especially critical as for an H5N1 pandemic strain, sixfold more antigen was required to induce an immune response to a level equivalent to the seasonal influenza vaccine (90 µg compared with 15 µg). MF59 enabled an immune response with significantly reduced doses of antigen, down to 7.5 µg, nearly a 12-fold dose reduction.
In parallel to the development of MF59, several other oil-in-water emulsions were developed. AS03, an oil-in-water emulsion containing α-tocopherol as the immunostimulating compound, was formulated by GlaxoSmithKline. This emulsion was tested earlier as part of the initial development of a malaria vaccine, alone (AS03) or in combination with the immunostimulants MPL and QS21 (described later). AS03 demonstrated potent dose-sparing potential for pandemic influenza antigens, allowing for dose sparing down to 3.75 µg.
In response to the need for dose sparing and the demonstrated potential of oil-in-water emulsions, Sanofi Pasteur developed AF03. This adjuvant is also squalene-based; however, unlike the other emulsions, which are made by microfluidization of the components, the emulsification of AF03 is achieved without mechanical energy and uses a temperature-induced self-emulsification process (PCT application WO2007080308).
With the outbreak of the H1N1 influenza pandemic, European regulatory authorities approved three oil-in-water emulsions containing pandemic influenza vaccines, with MF59, AS03, and AF03 as adjuvants.
Other emulsions are under development, such as SE (stable emulsion), a squalene-based emulsion, originally developed by researchers at Corixa as a vehicle for MPL and synthetic TLR4 agonists. This emulsion differs from the others in that the emulsifier is a natural phospholipid rather than a surfactant such as Tween-80. SE has been tested in clinical trials in combination with MPL in a Leishmania vaccine. It has also been tested in combination with the TLR-4 agonist GLA (glucopyranosyl lipid adjuvant) in a schistosomiasis vaccine, and an influenza vaccine. CoVaccine is an experimental adjuvant comprising sucrose fatty acid sulfate ester, combined with squalane, in the form of an oil-in-water emulsion. This adjuvant has been reported to allow for dose sparing in the context of influenza vaccines. A single immunization with CoVaccine HT-adjuvanted H5N1 influenza virus vaccine induces protective cellular and humoral immune responses in ferrets and is undergoing clinical evaluation. Emulsions that replicate or diverge from MF59 are been developed during the past 5 years such as ADDAVAX, or SWE both developed by VFI (Vaccine Formulation Institute).
Table 7.4 gives an overview of oil-in-water emulsions used as adjuvants in licensed and investigational vaccines.
TABLE 7.4
Composition of Various Oil-in-Water Emulsions
| Name | Components | Regulatory Status |
|---|---|---|
| SAF | Squalane; block copolymer; MDP | Abandoned |
| MF59 | Squalene; Tween-80; Span 85 | Approved for seasonal influenza for elderly people; approved for pandemic influenza (by the EMA); clinical benefit demonstrated for seasonal influenza in infants |
| AS03 | Tocopherol; squalene; Tween-80 | Approved for pandemic influenza (by the EMA) |
| AF03 | Squalene; Tween-80; trometamol | Approved in pandemic influenza (by the EMA) |
| SE | Squalene; lecithin; block copolymer; glycerol; vitamin E | Clinical evaluation for Leishmania , influenza |
| CoVaccine | Squalane; sucrose fatty acid sulfate; Tween-80 | Clinical evaluation for hepatitis |
EMA, European Medicines Agency; MDP, muramyl dipeptide; SE, stable emulsion.
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