In immunology, an adjuvant is a substance that increases or modulates the immune response to a vaccine. The word "adjuvant" comes from the Latin word , meaning to help or aid. "An immunologic adjuvant is defined as any substance that acts to accelerate, prolong, or enhance antigen-specific immune responses when used in combination with specific vaccine antigens."
In the early days of vaccine manufacture, significant variations in the efficacy of different batches of the same vaccine were correctly assumed to be caused by contamination of the reaction vessels. However, it was soon found that more scrupulous cleaning actually seemed to reduce the effectiveness of the vaccines, and some contaminants actually enhanced the immune response.
There are many known adjuvants in widespread use, including potassium alum, various plant and animal derived oils and virosomes.
Overview Adjuvants in immunology are often used to modify or augment the effects of a vaccine by stimulating the immune system to respond to the vaccine more vigorously, and thus providing increased immunity to a particular disease. Adjuvants accomplish this task by mimicking specific sets of evolutionarily conserved molecules, so called pathogen-associated molecular patterns, which include liposomes, lipopolysaccharide, molecular cages for antigens, components of bacterial cell walls, and endocytosed nucleic acids such as RNA, double-stranded RNA, single-stranded DNA, and unmethylated CpG dinucleotide-containing DNA. Because immune systems have evolved to recognize these specific antigenic moieties, the presence of an adjuvant in conjunction with the vaccine can greatly increase the innate immune response to the antigen by augmenting the activities of dendritic cells, lymphocytes, and macrophages by mimicking a natural infection.
Types Inorganic compounds: potassium alum, aluminium hydroxide, aluminium phosphate, calcium phosphate hydroxide Oils: paraffin oil, propolis (only in preclinical studies). Adjuvant 65 (based on peanut oil) was tested in influenza vaccines in the 1970s, but was never released commercially. Freund's incomplete adjuvant is a water-in-oil emulsion, a classical adjuvant. Water-in-oils are generally too reactogenic to be used on humans, however. Squalene is a natural oil made by human bodies. MF59 is a oil-in-water emulsion based on squalene. Bacterial products: killed bacteria of the species Bordetella pertussis, Mycobacterium bovis, toxoids. MPL (Monophosphorylated lipid A) is a modified form of a bacterial lipid A protein that is used in several vaccines. Plant saponins from Quillaia (soap bark tree), soybean and Polygala senega Cytokines: IL-1, IL-2, IL-12 CpG oligonucleotides Combinations: Freund's complete adjuvant (incomplete + dead Mycobacterium), AS01 (combining MPL and Quillaia saponins), Matrix-M (combining Quillaia saponins and two types of fat) Small molecules: TLR7/8 agonists (imidazoquinolines, imidazopyrimidines) Physical methods: radiofrequency heating (only tested in mice), microneedles (only tested in mice)
Inorganic adjuvants Aluminium salts There are many adjuvants, some of which are inorganic, that carry the potential to augment immunogenicity. Alum (hydrated potassium double sulfate) was the first aluminium (Al) salt used for this purpose, but has been almost completely replaced by aluminium hydroxide and aluminium phosphate for commercial vaccines. These newer types are also customarily called "alum" in some contexts (distinguished in this article by capitalization). Al salts are the most commonly used adjuvants in human vaccines. Their adjuvant activity was described in 1926.
The precise mechanism of Al salts ("Alum") remains unclear but some insights have been gained. It was formerly thought that they function as delivery systems by generating depots that trap antigens at the injection site, providing a slow release that continues to stimulate the immune system. However, studies have shown that surgical removal of these depots had no impact on the magnitude of IgG1 response. Depot formation is downstream to the adsorption of antigens onto the molecular structure of the adjuvant driven by electrostatic interactions and phosphate exchange. Conventional wisdom holds that one should maximize the adsorbed % of antigens when using an Alum. There is mixed evidence regarding the role of adsorption in the action of Alum, with some studies showing no effect and others showing a positive effect with low antigen doses in antigens of 10–100 kDa. Also, more strongly adsorbing Alums may paradoxically generate a weaker response, possibly because not enough antigen is being shown to the immune system.
Al can trigger dendritic cells and other immune cells to secrete Interleukin 1 beta (IL1β), an immune signal that promotes antibody production. Al adheres to the cell's plasma membrane and rearranges certain lipids there. Spurred into action, the dendritic cells pick up the antigen and speed to lymph nodes, where they stick tightly to a helper T cell and presumably induce an immune response. A second mechanism depends on alum killing immune cells at the injection site although researchers aren't sure exactly how alum kills these cells. It has been speculated that the dying cells release DNA which serves as an immune alarm. Some studies found that DNA from dying cells causes them to adhere more tightly to helper T cells which ultimately leads to an increased release of antibodies by B cells. No matter what the mechanism is, Al is not a perfect adjuvant because it does not work with all antigens (e.g. malaria and tuberculosis). However, recent research indicates that alum formulated in a nanoparticle form rather than microparticles can broaden the utility of alum adjuvants and promote stronger adjuvant effects.
Aluminum salt adjuvants can be damaged by freezing, making the vaccine not as effective. Accidental freezing is a concern in vaccine storage, especially in places with poor cold chains. One possible explanation is that freezing causes the loss of hydroxyl groups on the Alum, destabilizing its association with the antigen. Another explanation is the agglomeration of adjuvant-antigen complexes, forming larger particles of lower surface area. In either case, there are a number of cryoprotectants such as glycerine that can be added to the typical vial of aluminum liquid suspension vaccine to reduce the loss of potency. Some scientists are also working on making Alum-containg vaccines suitable for freeze-drying. Freeze-dried vaccines exist as a stable dry powder containing nothing to freeze.
Organic adjuvants
Emulsions The so-called "oil" adjuvants are really oils intended to be made into an emulsion with water, either oil-in-water (O/W) or water-in-oil (W/O). They would not work when moved out of the droplet form.
Freund's complete adjuvant is a solution of inactivated Mycobacterium tuberculosis in mineral oil and/or lanolin developed in 1930. It is used as water-in-oil, It is not safe enough for human use. A safer version without the bacteria is known as Freund's incomplete adjuvant, but its effect is weaker. In either case, the W/O structure probably helps vaccines release antigens for a longer time. Despite the side effects, its potential benefit has led to a few clinical trials, including a few attempts at adding other microbial-derived parts to balance safety and strength. (A common side effect for both versions is long-term retention of the oil in tissue.)
All "oil" adjuvants approved for humans are oil-in-water. They adsorb antigens to their surface and induce immune cell recruitment. All of these human-approved O/W adjuvants use squalene, an oil produced by human liver and skin. MF59 is an oil-in-water emulsion of squalene adjuvant used in some human vaccines. As of 2021, over 22 million doses of one vaccine with squalene, FLUAD, have been administered with no severe adverse effects reported. In addition, squalene-based O/W emulsions have also been shown to stably incorporate small molecule TLR7/8 adjuvants (e.g. PVP-037) and lead to enhanced adjuvanticity via synergism.
An interesting choice of oil in O/W emusions is vitamin E. The α-tocopheryl acetate form can replace the mineral oil in Fruend's adjuvants and is generally very efficacious in animals. The related α-tocopherol is used in AS03 in addition to squalene.Pandemrix – Summary of product characteristics , European Medicines Agency website European Medicines Agency website Just as its makers had hoped, the AS03-based flu vaccine strong immunity despite using very little antigen; but the same vaccine also caused narcolepsy in a very small amount of people, all sharing one specific gene variant.
O/W emulsions need to be stabilized so they do not revert into one large blob of oil. The more common way is by using a surfactant such as Tween-80. Another way is to use an aluminum microgel.
Liposomes Much like the droplets in an emulsion, liposomes are created when watery and oily things mix. However, they are made from phospholipids, which unlike other oil have a water-loving "head" part. This allows it to form lipid bilayers similar to cell membranes. When it encloses a aqueous volume, the result is a liposome.
The plant extract QS-21 is a liposome loaded with saponins extracted from the tree Quillaja saponaria.
The combination of QS-21, cholesterol and MPL forms the liposome adjuvant AS01 which is used in the Shingrix vaccine approved in 2017, as well as in the approved malaria vaccine Mosquirix.
Others
Monophosphoryl lipid A (MPL), a detoxified version of the lipopolysaccharide toxin from the bacterium Salmonella Minnesota, interacts with the receptor TLR4 to enhance immune response.
Several unmethylated cytosine phosphoguanosine (CpG) oligonucleotides activate the TLR9 receptor that is present in a number of cell types of the immune system. The adjuvant CpG 1018 is used in an approved Hepatitis B vaccine.
The adjuvant Matrix-M is an immune stimulating complex (ISCOM; a specific patented kind of liposome) consisting of nanospheres made of QS-21, cholesterol and phospholipids. It is used in the approved Novavax Covid-19 vaccine and in the malaria vaccine R21/Matrix-M.
Adaptive immune response In order to understand the links between the innate immune response and the adaptive immune response to help substantiate an adjuvant function in enhancing adaptive immune responses to the specific antigen of a vaccine, the following points should be considered: Innate immune response cells such as dendritic cells engulf pathogens through a process called phagocytosis. Dendritic cells then migrate to the lymph nodes where T cells (adaptive immune cells) wait for signals to trigger their activation. In the lymph nodes, dendritic cells mince the engulfed pathogen and then express the pathogen clippings as antigen on their cell surface by coupling them to a special receptor known as a major histocompatibility complex. T cells can then recognize these clippings and undergo a cellular transformation resulting in their own activation. γδ T cells possess characteristics of both the innate and adaptive immune responses. Macrophages can also activate T cells in a similar approach (but do not do so naturally).
This process carried out by both dendritic cells and macrophages is termed antigen presentation and represents a physical link between the innate and adaptive immune responses.
Upon activation, mast cells release heparin and histamine to effectively increase trafficking to and seal off the site of infection to allow immune cells of both systems to clear the area of pathogens. In addition, mast cells also release chemokines which result in the positive chemotaxis of other immune cells of both the innate and adaptive immune responses to the infected area.