Attenuated live vaccines are not always practical or available for many infectious diseases. This is especially the case when natural infections do not confer adequate protective immunity. If these infections are to be prevented, then killed, inactivated or subunit vaccines must be used, and in many case these vaccines are poorly immunogenic. They therefore require additional components called adjuvants to enhance their immunogenicity, prolong their effect, and provide adequate protection. Adjuvants are also essential for the effectiveness of many recombinant vaccines (Table 7.1) TABLE 7.1 ■ Emulsigen-D MVP Laboratories MF59 (Novartis) ISCOMs ISCOMATRIX MetaStim Fort Dodge Havlogen Merck Animal Health AS04 Glaxo Smith Kline AS03 Glaxo Smith Kline TS6, Boehringer Ingelheim TLR, Toll-like receptor. In 1924, Gaston Ramon, a French veterinarian working at the Pasteur Institute, observed that the antibody levels in horses immunized with tetanus or diphtheria toxoids were higher in animals that developed injection site abscesses. Ramon then induced sterile abscesses by injecting starch, breadcrumbs, or tapioca together with the toxoids and was able to enhance antibody production still further. Thus substances that induced inflammation at the injection site promoted antibody formation. In 1926, Alexander Glenny did essentially the same thing by injecting a foreign antigen together with alum (aluminum potassium sulfate). As a result, the addition of aluminum salts to enhance vaccine efficacy became a standard procedure. To maximize the effectiveness of vaccines, especially those containing killed organisms or highly purified antigens, it is now common practice to add substances, called adjuvants, to a vaccine (adjuvare is the Latin verb for “to help”). These adjuvants trigger innate responses that in turn promote the adaptive responses and so provide long-term protection. Adjuvants can increase the speed or the magnitude of the adaptive response to vaccines. They may permit a reduction in the dose of antigen injected or in the numbers of doses needed to induce satisfactory immunity. Adjuvants have also been used to induce appropriate bias in the adaptive response (toward a type 1 or type 2 response), and they are essential if long-term memory is to be established to soluble antigens. On the other hand, it is sometimes difficult to find the correct balance between adjuvant toxicity and immune stimulation to optimize safety and efficacy. Until recently little attention has been paid to how adjuvants work and their mechanisms of action were speculative. Vaccine adjuvants were defined by what they do and the “science” of adjuvants has been empirical. In other words, stuff was added to vaccines to see if it improved either the strength or duration of the immune response. As a result, adjuvants appear, at first sight, to be an eclectic mixture of natural extracts and inorganic salts, and also particles such as emulsions, nanoparticles, and liposomes. Innate immune responses are needed to initiate protective adaptive immunity. The early innate immune response plays a key role in determining the magnitude, quality, and duration of the adaptive immune responses. Very highly purified antigens make poor vaccines because they lack the signals that trigger innate immune responses and as a result cannot generate the downstream signaling required to enhance adaptive responses. Conversely, modified live vaccines, when mimicking natural infections, cause cell damage, trigger the release of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), and promote strong innate and adaptive responses. In effect therefore, adjuvants trigger the mandatory innate response needed to optimize the adaptive responses and promote the uptake of vaccine antigens by antigen-presenting cells—essentially dendritic cells (Fig. 7.1). They do this in two ways. First, they trigger innate immune responses that provide a stimulus for dendritic cell function and antigen presentation. Alternatively (or additionally) they deliver the antigen in a form optimized for dendritic cell processing and antigen presentation. Innate immune responses are triggered when pattern-recognition receptors detect microbial invasion and tissue damage. Molecules released by tissue damage (DAMPs) or molecules from foreign microbes (PAMPs) trigger innate responses through pattern-recognition receptors (PRRs) (Fig. 7.2). The activation of PRRs and other cellular receptors on dendritic cells triggers cytokine release. These cytokines promote helper T cell responses, while these in turn activate B and T cells and so promote adaptive immunity. Some adjuvants cause cell and tissue damage and so provide the body with DAMPs. These DAMP-type adjuvants act by chemical irritation or have direct toxic effects. Thus adjuvants such as the saponins and some emulsions cause cell lysis at the injection site. Saponins are amphipathic soap-like glycosides that can form complexes with cell membrane cholesterol resulting in membrane destruction. The toxicity of emulsions is caused by the presence of short-chain detergent-like molecules that lyse cell membranes. Longer chains are less toxic but poorer adjuvants. The emulsifiers used in water/oil emulsions may have similar toxic effects. Aluminum salts are also cytotoxic and cause the release of DNA, uric acid, and adenosine from dying cells. All these adjuvants release DAMPs that bind to receptors on antigen presenting cells and activate their inflammasome pathway. This generates cytokines leading to helper T cell activation. (Inflammasomes are multiprotein complexes whose activation stimulates the production of cytokines such as interleukin (IL)-1 and IL-18 and so promote both innate and adaptive immunity.) A second type of adjuvant contains microbial products, PAMPs, the essential “danger” signals that also trigger innate immune processes. These PAMP-type adjuvants also provide signals through pattern recognition receptors such as the toll-like receptors (TLRs) and so activate dendritic cells. PAMP-type adjuvants contain killed bacteria, or microbial molecules such as flagellin (a TLR5 ligand), lipopolysaccharide (LPS) (a TLR 4/2 ligand), DNA containing CpG oligodeoxynucleotides (a TLR 9 ligand), their analogs such as monophosphoryl lipid A (MPLA, a TLR4/2 ligand), or synthetic TLR ligands. They may contain bacterial toxins like cholera toxin (CT) or Escherichia coli labile toxin (LT). These PAMP-type adjuvants directly activate PRRs on dendritic cells and so cause the release of proinflammatory cytokines such as IL-1, IL-6, and tumor necrosis factor-α (TNF-α). They also stimulate the production of neutrophil-attracting chemokines such as CCL-3, -4, -8, and -20. Many adjuvants contain components that engage both pathways by using a mixture of PAMPs and DAMPs. For example, TLR agonists synergize with cell-damaging squalene oil-in-water emulsions to induce strong innate responses. This increases the release of the stimulatory cytokines and chemokines. These in turn recruit antigen-presenting cells to the injection site. They enhance antigen uptake as well as the activation and maturation of the antigen-presenting cells (Fig. 7.3). Note, however, that because they stimulate innate immunity, adjuvants also promote inflammation. This occurs immediately after vaccination and accounts for the commonly observed transient local inflammatory reactions at the injection site. Depression, fever, and malaise, occasionally encountered after animals are vaccinated, result from cytokines at the injection site overflowing into the circulation and acting on the brain. Thus one of the challenges in developing new adjuvanted vaccines is to generate the most potent ones and minimizing their adverse effects, especially inflammation. Antigen-processing cells are the key to effective adjuvant action. Once activated, these cells take up antigen based on its size, its charge, and its hydrophobicity. They then mature, express high levels of major histocompatibility complex (MHC) molecules, and effectively present the antigen to helper T cells. These cells are critical in determining the nature of the immune response and can be directly influenced by adjuvants. Therefore a third type of adjuvant consists of particles optimized for ingestion and processing by antigen-presenting cells. The use of particles coated with antigen, cytokines, and costimulatory molecules as adjuvants has led to encouraging improvements in vaccine efficacy and provides a framework for future advances. It was long believed that adjuvants, such as the aluminum salts, acted as antigen depots, slowly releasing the antigen into the body to trigger a prolonged immune response. This effect has probably been exaggerated. Surgical removal of the antigen-alum depot at two hours after injection has no influence on its adjuvanticity. Similar studies have shown a similar lack of depot effect for the oil-based adjuvant MF59 and for ISCOMS (immune stimulating complexes). To mount an effective immune response, B cells need to generate at least 20,000 plasma cells, and many more T cells are needed to mount a cell-mediated response. Most modern adjuvants can generate sufficient B cells but not enough CD8 T cells. The few adjuvants that can stimulate adequate T cell responses rely on signaling through antigen processing cells. Aluminum adjuvants have been used since the 1920s and they are by far the most widely employed DAMP-type adjuvants. They are administered to millions of people annually and are both safe and cost-effective. Until recently alum (aluminum potassium sulfate AlK(SO4)2, was the only adjuvant globally licensed for human use. This has now generally been replaced by aluminum oxyhydroxide (AlO[OH], aluminum hydroxyphosphate (HAlO5P), or aluminum phosphate (Al[PO4]3). At a neutral pH the hydroxide binds to negatively charged proteins, whereas the phosphate binds positively charged proteins. These aluminum adjuvants either adsorb antigen to the salt nanoparticles or they can be coprecipitated with the antigen. Either method ensures that the antigen is tightly bound to the mineral matrix. It thus makes soluble antigens particulate so that they can be endocytosed and effectively processed. Calcium phosphate is also used as an adjuvant. It is less irritating than the aluminum salts and has been used in experimental vaccines. Aluminum-adjuvanted vaccines induce tissue damage and cell death at the injection site. This releases DAMPs including DNA, uric acid, ATP, heat shock protein 70, and interleukins 1 and 33. These DAMPs then attract neutrophils with some eosinophils and lymphocytes. Alum causes dying neutrophils to release DNA to form extracellular traps and enhance dendritic cell T cell interactions. Recruitment of mature dendritic cells to the sites of injection is also enhanced. Subsequently macrophages are also attracted to these sites and these macrophages may then develop into dendritic cells. Alum appears to affect lipids in the plasma membrane and promotes dendritic cell homing to lymph nodes. Alum also stimulates the production of chemokines that attract neutrophils and eosinophils. Thus alum has multiple effects largely based on its tissue damaging properties. DNA is known to accumulate at sites of alum deposition and is apparently important because local DNase treatment blocks this adjuvant activity. It has been suggested that alum kills cells at the injection site, releasing DAMPs, which in turn activate host DCs. Recently it has also been demonstrated that alum signals through inflammasomes. DCs or macrophages stimulated with alum plus lipopolysaccharide induced IL-1β and IL-18 release. Alum however, is not good at generating Th1 CD8+ cells and does not induce strong cytotoxic T cell responses. Alum promotes the production of IL-4 enhancing Th2 responses to protein antigens and generates large numbers of B cells. As a result, while promoting antibody responses, these adjuvants have little effect on cell-mediated responses. Aluminum adjuvants greatly influence the primary immune response but have much less effect on secondary immune responses. Saponins are natural triterpene glycosides derived from plants. Their glycosides are hydrophilic whereas the triterpenes are lipophilic, and so saponins act like soap or detergents. The most important of the adjuvant saponins is Quil-A, a mixture of 23 different saponins derived from the inner bark of the South American soapbark tree (Quillaja saponaria). It is a potent DAMP-type adjuvant, but crude Quil-A is too toxic for use in humans. As a result, Quil-A has been fractionated and its active fractions identified. The most abundant of these fractions is QS-21. QS-21 combines the most potent adjuvant activity with minimal toxicity. Saponin-based adjuvants selectively stimulate Th1 and cytotoxic T cell responses because they direct antigens into endogenous processing pathways and enhance IFN-γ release by dendritic cells. The saponins cause tissue damage and so activate inflammasomes. Saponins are employed as adjuvants for foot-and-mouth disease vaccines and recombinant feline leukemia vaccines in addition to experimental porcine respiratory and reproductive system virus (PRRSV) vaccines for pigs. Toxic saponin mixtures are used in anthrax vaccines, where they lyse tissue at the site of injection so that the anthrax vaccine spores may germinate. Immune stimulating complexes (ISCOMs) are stable matrixes containing cholesterol, phospholipids, and a mixture of Quil-A saponins, and antigen. They assemble into very stable, spherical 40 nm cage-like structures with multiple copies of the antigen exposed on their surface. ISCOMs act as both DAMP-type and particulate adjuvants combined within the same particle. In general, the antibody response to an antigen incorporated in an ISCOM is about tenfold that of the same antigen in saline. They are highly effective in targeting antigens to dendritic cells. The sugar groups on the saponin bind to cell surface lectins on DCs and activate them. Saponins deliver proteins, not only to endosomes, but also to the cytosol of DCs so that they can be presented on MHC class I molecules. In addition, the saponins promote cytokine production and the expression of costimulatory molecules. Depending on the antigen employed, ISCOMs can stimulate either Th1 or Th2 responses. ISCOMATRIX is a particulate adjuvant consisting of cholesterol, phospholipid, and saponin without incorporated antigen. MATRIX-M also consists of nanoparticles made from purified saponins, cholesterol, and phospholipid. Emulsions are generated when two immiscible liquids are mixed together. They occur in several different forms (Fig. 7.4). For example, a water-in-oil (W/O) emulsion consists of aqueous droplets suspended in a continuous oil phase. The best example of this is Freund’s adjuvant. Because antigens are water soluble, the droplets are slowly released as the oil breaks down and this slows the degradation of the antigen. An alternative is to use an oil-in-water (O/W) emulsion, where oil droplets are suspended in a continuous aqueous phase. The best example of this is MF59, a commercial adjuvant used in human vaccines.
Adjuvants and adjuvanticity
Adjuvant Name
Type
Contents
Oil in water emulsion
Mineral oil plus dimethyldioctadecyl ammonium bromide
Oil in water emulsion
Squalene, Tween 80, Span 85, citrate buffer
Nanoparticles
Cholesterol, phospholipids, saponins
Combination
Saponin, Cholesterol, dipalmitoyl phosphatidylcholine
Oil emulsion
Oil with emulsifier
Polymer
Carbopol
Alum adsorbed TLR agonist
Alum, Monophosphoryl Lipid A (MPLA)
Oil in water emulsion
Squalene, Polysorbate 80, α-tocopherol
Oil in water emulsion
Light mineral oil plus multiple lipophilic and hydrophilic surfactants
How adjuvants work
The depot effect
Types of adjuvants
Damage-associated molecular patterns-type adjuvants
Aluminum adjuvants
Saponin-based adjuvants
Emulsion adjuvants
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Adjuvants and adjuvanticity
Some Commonly Used Adjuvants in Veterinary Vaccines
