Part 2 of 3 (Previous - Next)
by Viera Scheibner
Adjuvants, Preservatives And Tissue Fixatives In Vaccines
Vaccines contain a number of substances which can be divided into the following groups:
- Micro-organisms, either bacteria or viruses, thought to be causing certain infectious diseases and which the vaccine is supposed to prevent. These are whole-cell proteins or just the broken-cell protein envelopes, and are called antigens.
- Chemical substances which are supposed to enhance the immune response to the vaccine, called adjuvants.
- Chemical substances which act as preservatives and tissue fixatives, which are supposed to halt any further chemical reactions and putrefaction (decomposition or multiplication) of the live or attenuated (or killed) biological constituents of the vaccine.
All these constituents of vaccines are toxic, and their toxicity may vary, as a rule, from one batch of vaccine to another.
In this article, the first of a two-part series, we shall deal with adjuvants, their expects role and the reactions (side effects).
Adjuvants
The desired immune response to vaccines is the production of antibodies, and this is enhanced by adding certain substances to the vaccines. These are called adjuvants (from the Latin adjuvare, meaning "to help").
The chemical nature of adjuvants, their mode of action and their reactions (side effect) are highly variable. According to Gupta et al. (1993), some of the side effects can be ascribed to an unintentional stimulation of different mechanisms of the immune system whereas others may reflect general adverse pharmacological reactions which are more less expected.
There are several types of adjuvants.
Today the most common adjuvants for human use are
- aluminum hydroxide,
- aluminum phosphate
- and calcium phosphate.
However, there are a number of other adjuvants based on oil emulsions, products from bacteria (their synthetic derivatives as well as liposomes) or gram-negative bacteria, endotoxins, cholesterol, fatty acids, aliphatic amines, paraffinic and vegetable oils.
Recently, monophosphoryl lipid A, ISCOMs with Quil-A, and Syntex adjuvant formulations (SAFs) containing the threonyl derivative or muramyl dipeptide have been under consideration for use in human vaccines.
Chemically, the adjuvants are a highly heterogenous group of compounds with only one thing in common: their ability to enhance the immune response-their adjuvanticity.
They are highly variable in terms of how they affect the immune system and how serious their adverse effects are due to the resultant hyperactivation of the immune system.
The mode of action of adjuvants was described by Chedid (1985) as: the formation of a depot of antigen at the site of inoculation, with slow release; the presentation of antigen immunocompetent cells; and the production of various and different lymphokines (interleukins and tumour necrosis factor).
The choice of any of these adjuvants reflects a compromise between a requirement for adjuvanticity and an acceptable low level of adverse reactions.
The discovery of adjuvants dates back to 1925 and 1926, when Ramon (quoted by Gupta et al., 1993) showed that the antitoxin response to tetanus and diphtheria was increased by injection of these vaccines, together with other compounds such as agar, tapioca, lecithin, starch oil, saponin or even breadcrumbs.
The term adjuvant has been used for any material that can increase the humoral or cellular immune response to an antigen. In the conventional vaccines, adjuvants are used to elicit an early, high and long-lasting immune response. The newly developed purified subunit or synthetic vaccines using biosynthetic, recombinant and other modern technology are poor immunogens and require adjuvants to evoke the immune response.
The use of adjuvants enables the use of less antigen to achieve the desired immune response, and this reduces vaccine production costs.
With a few exceptions, adjuvants are foreign to the body and cause adverse reactions.
Oil Emulsions
In the 1960s, emulsified water-in-oil and water-in-vegetable-oil adjuvant preparations used experimentally showed special promise in providing exalted "immunity" of long duration (Hilleman, 1966). The development of Freund's adjuvants emerged from studies of tuberculosis.
Several researchers noticed that immunological responses in animals to various antigens were enhanced by introduction into the animal of living Mycobacterium tuberculosis. In the presence of Mycobacterium, the reaction obtained was of the delayed type, transferable with leukocytes.
Freund measured the effect of mineral oil in causing delayed-type hypersensitivity to killed mycobacteria. There was a remarkable increase in complement-fixing antibody response as well as in delayed hypersensitivity reaction.
Freund's adjuvant consists of a water-in-oil emulsion of aqueous antigen in paraffin (mineral) oil of low specific gravity and low viscosity. Drakeol 6VR and Arlacel A (mannide monooleate) are commonly used as emulsifiers.
There are two Freund's adjuvants: incomplete and complete.
The incomplete Freund's adjuvant consists of water-in-oil emulsion without added mycobacteria; the complete Freund's adjuvant consists of the same components but with 5 mg of dried, heat-killed Mycobacterium tuberculosis or butyricum added.
The mechanism of action of Freund's adjuvants is associated with the following three phenomena:
1. The establishment of a portion of the antigen in a persistent form at the injection site, enabling a gradual and continuous release of antigen for stimulating the antibody;
2. The provision of a vehicle for transport of emulsified antigen throughout the lymphatic system to distant places, such as lymph nodes and spleen, where new foci of antibody formation can be established; and,
3. Formation and accumulation of cells of the mononuclear series which are appropriate to the production of antibody at the local and distal sites.
The pathologic reaction to the Freund's adjuvants starts at the injection site with mild erythema and swelling followed by tissue necrosis, intense inflammation and the usual progression to the formation of a granulomatous lesion. Scar and abscess formation may occur. The reactions observed following the administration of the complete adjuvant are generally far more extensive than with the incomplete adjuvant.
The earliest cellular response is polymorphonuclear, then it changes into mononuclear and later includes plasmocytes. The adjuvant emulsion may be widely disseminated in varrious organs, depending on the route of inoculation, with the development of focal granulomatous lesions at distal places. Various gram-negative organisms may show a potentiating effect of the adjuvant, similar to that displayed by mycobacteria.
The earliest use of oil emulsion adjuvants was made with the influenza, vaccine by Friedwald (1944) and by Henle and Henle (1945). Following their promising results on animals, Salk (1951) experimented with such adjuvants on soldiers under the auspices of the US Armed Forces Epidemiological Board.
He used a highly refined mineral oil, and developed a purified Arlacel A emulsifier which was free of toxic substances, such as oleic acid which had caused sterile abscesses at the injection site, and he administered the vaccine by intramuscular route.
Subsequently, Miller et al. (1965) reported their, failure to enhance the antibody and protective response to types 3, 4 and 7 adenovirus vaccines in mineral oil adjuvant compared with aqueous vaccine. Unpublished studies have revealed the need for an adequate minimal amount of antigen to trigger an antibody response to the emulsified preparations.
Salk et al. (1953) applied Freund's adjuvant to poliomyelitis vaccine, and later followed with extensive testing of killed crude as well as purified polio virus vaccine in animals and humans, where the reactions in humans were considered inconsequential.
Grayston et al. (1964) reported highly promising results with the trachoma vaccine using an oil adjuvant.
However, the trachoma vaccine lost its relevance because, as demonstrated by Dolin et al. (1997) in their 37 years of research in a sub-Saharan village, the dramatic fall in the disease occurrence was closely connected with improvements in
- sanitation,
- water supply,
- education
- and access to health care.
According to Dolin et al. (1997), the decline in trachoma occurred without any trachoma-specific intervention.
Allergens in Freund's adjuvant deserve special attention because they can be dangerous. These dangers include an overdose, i.e., the immediate release of more than the tolerated amount of properly emulsified vaccine in sensitive persons, or the breaking of the emulsion with the release of all or part of the full content of the allergen within a brief period of time.
Long-term delayed reactions include the development of nodules, cysts or sterile abscesses requiring surgical incision. It is also likely that some allergens used, such as house dust or mould, might have acted like mycobacteria to potentiate the inflammatory response. Such reactions have been reduced with the use of properly tested and standardized reagins.
One must also consider that the first application of Freund's adjuvants was made at a time when modern concepts of safety were nonexistent Indeed, mineral oil adjuvants have not been approved for human use in some countries, including the USA.
Mineral Compounds
Aluminum phosphate or aluminum hydroxide (alum) are the mineral compounds most commonly used as adjuvants in human vaccines. Calcium phosphate is another adjuvant that is used in many vaccines. Mineral salts of metals such as cerium nitrate, zinc sulfate, colloidal iron hydroxide and calcium chloride were observed to increase the antigenicity of' the toxoids, but alum gave the best results.
The use of alum was applied more than 70 years ago by Glenny et al. (1926), who discovered that a suspension of alum-precipitated diphtheria toxoid had a much higher immunogenicity than the fluid toxoid. Even though a number of reports stated that alum-adjuvanted vaccines were no better than plain vaccines (Aprile and Wardlaw, 1966), the use of alum as an adjuvant is now well established.
The most widely used is the antigen solution mixed with pre-formed aluminum hydroxide or aluminum phosohate under controlled conditions. Such vaccines are now called aluminium-adsorbed or aluminium-adjuvanted. However, they are difficult to manufacture in a physico-chemically reproducible way, which results in a batch-to-batch variation of the same vaccine.
Also, the degree of antigen absorption to the gels of aluminum phosphate and aluminum hydroxide varies. To minimize the variation and avoid the non-reproducibility, a specific preparation of aluminum hydroxide (Alhydrogel) was chosen as the standard in 1988 (Gupta et al., 1993).
The aluminum adjuvants allow the slow release of antigen, prolonging the time for interaction between antigen and antigen-presenting cells and lymphocytes. However, in some studies, the potency of adjuvanted pertussis vaccines was more than that of the plain pertussis vaccines, while in others no effect was noted.
The serum agglutinin titres, after vaccination with adjuvanted pertussis vaccines, were higher than those of the plain vaccines, with no difference in regard to protection against the disease (Butler et al., 1962).
Despite these conflicting results, aluminum compounds are universally used as adjuvants for the DPT (diphtheriapertussis-tetanus) vaccine. Hypersensitivity reactions following their administration have been reported which could be attributed to a number of factors, one of which is the production of IgE along with IgG antibodies.
It was suggested that polymerased toxoids, such as the so-called glutaraldehyde-detoxifled purified tetanus and diphtheria toxins, should be used instead of aluminum compounds. They are usually combined with glutaraldehyde-inactivated pertussis vaccine.
Calcium phosphate adjuvant has been used for simultaneous vaccination with diphtheria, pertussis, tetanus, polio, BCG, yellow fever, measles and hepatitis B vaccines and with allergen (Coursaget et al., 1986).
The advantage of this adjuvant has been seen to be that it is a normal constituent of the body and is better tolerated and absorbed than other adjuvants. It entraps antigens very efficiently and allows slow release of the antigen. Additionally, it elicits high amounts of IgG-type antibodies an much less of IgE-type (reaginic) antibodies.
Bacterial Products
Microorganisms in bacterial infections and the administration of vaccines containing whole killed bacteria and some metabolic products and components of various microorganisms have been known to elicit antibody response and act as immunostimulants. The addition of such microorganisms and substances into vaccines augments the immune response to other antigens in such vaccines.
The most commonly used microorganisms, whole or their parts, are
- Bordetella pertussis components,
- Corenybacterium derived P40 component,
- cholera toxin
- and mycobacteria.
B. Pertussis Components
The killed Bordetella pertussis has a strong adjuvant effect on the diptheria and tetanus toxoids in the DPT vaccines. However, there are a number of admitted and well-describe reactions to it, such as
| convulsion | Reye syndrome |
| infantile spasms | Guilain-Barre syndrome |
| epilepsy | sudden infant death syndrome (SIDS) |
| transverse myelitis | cerebral ataxia |
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