Immunologic adjuvant

In immunology, an adjuvant is an agent that may stimulate the immune system and increase the response to a vaccine, without having any specific antigenic effect in itself. [ [http://www.cancer.gov/templates/db_alpha.aspx?CdrID=43987 Definition of Adjuvant,] National Cancer Institute.] The word “adjuvant” comes from the Latin word "adjuvare", meaning to help or aid. [ [http://www.springerlink.com/content/m823465l9p126826/ "DNA Vaccines: Methods and Protocols,"] D.B. Lowrie and R.G. Whalen, Humana Press, 2000. ISBN 978-0-89603-580-5.] "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." [ [http://www.springerlink.com/content/m823465l9p126826/ "The Use of Conventional Immunologic Adjuvants in DNA Vaccine Preparations,"] by Shin Sasaki and Kenji Okuda. In D.B. Lowrie and R.G. Whalen (editors), "DNA Vaccines: Methods and Protocols," Humana Press, 2000. ISBN 978-0-89603-580-5.]

Adjuvants have been called the "dirty little secret" of vaccines [ [http://www.thescientist.com/article/display/39377/ The Scientist] "Deciphering Immunology's Dirty Secret."] in the scientific community, as much about how adjuvants work is a mystery. Known adjuvants include oils, aluminum salts 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 which include liposomes, lipopolysaccharide (LPS), molecular cages for antigen, components of bacterial cell walls, and endocytosed nucleic acids such as double-stranded RNA (dsRNA), single-stranded DNA (ssDNA), and unmethylated CpG dinucleotide-containing DNA. cite journal |author=Gavin A, Hoebe K, Duong B, Ota T, Martin C, Beutler B, Nemazee D |title=Adjuvant-enhanced antibody responses in the absence of toll-like receptor signaling |journal=Science |volume=314 |issue=5807 |pages=1936–8 |year=2006 |pmid=17185603 |url=http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=17185603 |accessdate=2008-04-03 |doi=10.1126/science.1135299] Because immune systems have evolved to recognize these specific antigenic moieties, the presence of adjuvant in conjunction with the vaccine can greatly increase the innate immune response to the antigen by augmenting the activities of dendritic cells (DCs), lymphocytes and macrophages by mimicking a natural infection. [ Majde JA. 1987. Progress in leukocyte biology. Alan R. Liss, Inc. vol. 6.] Furthermore, because adjuvants are attenuated beyond any function of virulence, they pose little or no independent threat to a host organism.

Inorganic adjuvants

Aluminium salts

There are many adjuvants, some of which are inorganic (such as alum), that also carry the potential to augment immunogenicity. [cite journal |author=Clements C, Griffiths E |title=The global impact of vaccines containing aluminium adjuvants |journal=Vaccine |volume=20 Suppl 3 |issue= |pages=S24–33 |year= |pmid=12184361] [Glenny A, Pope C, Waddington H, and Wallace U. 1926. The antigenic value of toxoid precipitated by potassium alum. J Pathol Bacteriol. 29: 38-45.] Two common salts include aluminum phosphate and aluminum hydroxide. These are the most common adjuvants in human vaccines.

Organic adjuvants

While Aluminium salts are popularly used in human vaccines, the organic compound Squalene is also used. However, organic adjuvants are more commonly used in animal vaccines.

Oil-based

Oil-based adjuvants are commonly used in some veterinary vaccines.

Virosomes

Another market-approved adjuvant and carrier system are virosomes. [ [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TD4-3Y44SRT-4K&_user=606406&_coverDate=12%2F31%2F1995&_fmt=full&_orig=search&_cdi=5188&view=c&_acct=C000029798&_version=1&_urlVersion=0&_userid=606406&md5=29035d9b4bf92526c125f3e6e99de680&ref=full "Safety, immunogenicity, and kinetics of the immune response to a single dose of virosome-formulated hepatitis A vaccine in Thais,"] Yong Poovorawana, Apiradee Theamboonlersa, Saowani Chumdermpadetsuka, Reinhard Glückb and Stanley J. Cryz, Jr., "Vaccine," Volume 13, Issue 10, 1995, Pages 891-893.] During the last two decades, a variety of technologies have been investigated to improve the widely-used adjuvants based on aluminum salts. [ [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TD4-3WF7F3Y-8&_user=606406&_coverDate=07%2F31%2F1996&_fmt=full&_orig=search&_cdi=5188&view=c&_acct=C000029798&_version=1&_urlVersion=0&_userid=606406&md5=f0d5695164e9600df29aa0f14f2a0432&ref=full "Immunogenicity and adverse effects of inactivated virosome versus alum-adsorbed hepatitis A vaccine: a randomized controlled trial,"] Benedikt R. Holzer*, Christoph Hatz†, Dagmar Schmidt-Sissolak†, Reinhard Glück‡, Beat Althaus‡ and Matthias Egger, "Vaccine," Volume 14, Issue 10, July 1996, Pages 982-986.] These salts are unfavorable, since they develop their effect by inducing local inflammation, which is also the basis for the extended side-effect pattern of this adjuvant. In contrast, the adjuvant capabilities of virosomes are independent of any inflammatory reaction. Virosomes contain a membrane-bound hemagglutinin and neuraminidase derived from the influenza virus, and serve to amplify fusogenic activity and therefore facilitate the uptake into antigen presenting cells (APC) and induce a natural antigen-processing pathway. The delivery of the antigen by virosomes to the immune system in a way that mimics a natural path may be a reason why virosome-based vaccines stand out due to their excellent safety profile. [ [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TD4-3VW86PK-2G&_user=606406&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000029798&_version=1&_urlVersion=0&_userid=606406&md5=d4e5e5e32cb4b693078ac4464e16459a "Adjuvant activity of immunopotentiating reconstituted influenza virosomes (IRIVs),"] Reinhard Glück, "Vaccine,"Volume 17, Issues 13-14, January 1999, Pages 1782-1787.]

Experimental adjuvants

An increasing number of vaccines with squalene and phosphate adjuvants are being tested on humans. [ [http://www.annalsnyas.org/cgi/content/abstract/754/1/153 "Immunologic adjuvants for modern vaccine formulations,"] F. R. Vogel, "Annals of the New York Academy of Sciences", Vol 754, Issue 1, 1995, pp. 153-160] The compound QS21 is under investigation as a possible immunological adjuvant [cite journal |author=Ghochikyan A, Mkrtichyan M, Petrushina I, Movsesyan N, Karapetyan A, Cribbs D, Agadjanyan M |title=Prototype Alzheimer's disease epitope vaccine induced strong Th2-type anti-Abeta antibody response with Alum to Quil A adjuvant switch |journal=Vaccine |volume=24 |issue=13 |pages=2275–82 |year=2006 |pmid=16368167 |doi=10.1016/j.vaccine.2005.11.039] as is Novartis' (formerly Chiron) MF59. [ [http://www3.niaid.nih.gov/news/newsreleases/2004/h9n2.htm NIH] "Chiron Corporation will produce the H9N2 vaccine at its manufacturing facility in Siena, Italy. The company will prepare different dosages of the vaccine, which is based on an inactivated strain of the H9N2 virus developed by the Centers for Disease Control and Prevention. Some dosages will contain Chiron’s MF59 adjuvant—a substance designed to boost the vaccine’s protective effect." ]

Adjuvants and the adaptive immune response

One misconception concerning adjuvant function is that an adjuvant-enhanced innate immune response should affect only the transitory reaction of the innate immune response and not the more long-lived effects of the adaptive immune response.Fact|date=November 2007 Although it may appear fitting to separate the two systems, it is however important to realize the interconnected nature of the two systems. When the amount of communication that takes place between the innate immune response and the adaptive immune response with the onset of infection is considered it becomes difficult to separate the two systems. 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 DCs engulf pathogens through a process called phagocytosis.
* DCs then migrate to the lymph nodes where T cells (adaptive immune cells) wait for signals to trigger their activation. [cite journal |author=Bousso P, Robey E |title=Dynamics of CD8+ T cell priming by dendritic cells in intact lymph nodes |journal=Nat Immunol |volume=4 |issue=6 |pages=579–85 |year=2003 |pmid=12730692 |doi=10.1038/ni928]
* In the lymph nodes, DCs 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 (MHC).
* T cells can then recognize these clippings and undergo a cellular transformation resulting in its own activation. [cite journal |author=Mempel T, Henrickson S, Von Andrian U |title=T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases |journal=Nature |volume=427 |issue=6970 |pages=154–9 |year=2004 |pmid=14712275 |doi=10.1038/nature02238]
* γδ T cells possess characteristics of both the innate and adaptive immune responses.
* Macrophages can also activate T cells in a similar approach.

This process carried out by both DCs 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. [cite journal |author=Gaboury J, Johnston B, Niu X, Kubes P |title=Mechanisms underlying acute mast cell-induced leukocyte rolling and adhesion in vivo |journal=J Immunol |volume=154 |issue=2 |pages=804–13 |year=1995 |pmid=7814884] [cite journal |author=Kashiwakura J, Yokoi H, Saito H, Okayama Y |title=T cell proliferation by direct cross-talk between OX40 ligand on human mast cells and OX40 on human T cells: comparison of gene expression profiles between human tonsillar and lung-cultured mast cells |journal=J Immunol |volume=173 |issue=8 |pages=5247–57 |year=2004 |pmid=15470070]

Due to the variety of mechanisms and links between the innate and adaptive immune response, an adjuvant-enhanced innate immune response results in an enhanced adaptive immune response. Specifically, a recent study has observed that adjuvants may exert their immune-enhancing effects according to five immune-functional activities. [cite journal |author=Schijns V |title=Immunological concepts of vaccine adjuvant activity |journal=Curr Opin Immunol |volume=12 |issue=4 |pages=456–63 |year=2000 |pmid=10899018 |doi=10.1016/S0952-7915(00)00120-5]
* First, it was found that adjuvants all help in the translocation of antigens to the lymph nodes where they can be recognized by T cells. This will ultimately lead to greater T cell activity resulting in a heightened clearance of pathogen throughout the organism.
* Second, adjuvants provide physical protection to antigens which grants the antigen a prolonged delivery. This means that the organism will be exposed to the antigen for a longer duration, making the immune system more robust as it makes use of the additional time by upregulating the production of B and T cells needed for greater immunological memory in the adaptive immune response.
* Third, adjuvants help to increase the capacity to cause local reactions at the injection site (during vaccination), inducing greater release of danger signals by chemokine releasing cells such as helper T cells and mast cells.
* Fourth, they induce the release of inflammatory cytokines which helps to not only recruit B and T cells at sites of infection but also to increase transcriptional events leading to a net increase of immune cells as a whole.
* Finally, adjuvants are believed to increase the innate immune response to antigen by interacting with pattern recognition receptors (PRRs), specifically Toll-like receptors (TLRs), on accessory cells.

Adjuvants and toll-like receptors

The ability of immune system to recognize molecules that are broadly shared by pathogens is, in part, due to the presence of special Immune receptors called TLRs that are expressed on leukocyte membranes. TLRs were first discovered in drosophila, and are membrane bound pattern recognition receptors (PRRs) responsible for detecting most (although certainly not all) antigen-mediated infections. [cite journal |author=Lemaitre B, Nicolas E, Michaut L, Reichhart J, Hoffmann J |title=The dorsoventral regulatory gene cassette spätzle/Toll/cactus controls the potent antifungal response in Drosophila adults |journal=Cell |volume=86 |issue=6 |pages=973–83 |year=1996 |pmid=8808632 |doi=10.1016/S0092-8674(00)80172-5] [cite journal |author=Beutler B |title=Inferences, questions and possibilities in Toll-like receptor signalling |journal=Nature |volume=430 |issue=6996 |pages=257–63 |year=2004 |pmid=15241424 |doi=10.1038/nature02761] In fact, some studies have shown that in the absence of TLR, leukocytes become unresponsive (no inflammatory responses) to some microbial components such as LPS. [cite journal |author=Poltorak A, He X, Smirnova I, Liu M, Van Huffel C, Du X, Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B, Beutler B |title=Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene |journal=Science |volume=282 |issue=5396 |pages=2085–8 |year=1998 |pmid=9851930 |doi=10.1126/science.282.5396.2085] There are at least thirteen different forms of TLR, each with its own characteristic ligand. Prevailing TLR ligands described to date (all of which elicit adjuvant effects) include many evolutionarily conserved molecules such as LPS, lipoproteins, lipopeptides, flagellin, double-stranded RNA, unmethylated CpG islands and various other forms of DNA and RNA classically released by bacteria and viruses. [cite journal |author=Bültmann B, Finger H, Heymer B, Schachenmayr W, Hof H, Haferkamp O |title=Adjuvancy of streptococcal nucleic acids |journal=Z Immunitatsforsch Exp Klin Immunol |volume=148 |issue=5 |pages=425–30 |year=1975 |pmid=127450] [Capanna SL, Kong YM. 1974. Further studies on the prevention of tolerance induction by poly A:U. Immunology. 27: 647-653.] [cite journal |author=Nauciel C, Fleck J, Martin J, Mock M, Nguyen-Huy H |title=Adjuvant activity of bacterial peptidoglycans on the production of delayed hypersensitivity and on antibody response |journal=Eur J Immunol |volume=4 |issue=5 |pages=352–6 |year=1974 |pmid=4604064 |doi=10.1002/eji.1830040509] [Schmidtke JR, Johnson AG. 1971. Regulation of the immune system by synthetic polynucleotides. I. Characteristics of adjuvant action on antibody synthesis. J. Immunol. 106: 1191-1200.] [Youmans AS, Youmans GP. 1969. Factor affecting immunogenic activity of mycobacterial ribosomal and ribonucleic acid preparations. J. Bacteriol. 99: 42-50.] [cite journal |author=Youmans G, Youmans A |title=Allergenicity of mycobacterial ribosomal and ribonucleic acid preparations in mice and guinea pigs |journal=J Bacteriol |volume=97 |issue=1 |pages=134–9 |year=1969 |pmid=4236903]

The binding of ligand - either in the form of adjuvant used in vaccinations or in the form of invasive moieties during times of natural infection - to the TLR marks the key molecular events that ultimately lead to innate immune responses and the development of antigen-specific acquired immunity. [Kiyoshi Takeda, and Shizuo Akira. 2005. Toll-like receptors in innate immunity. International Immunology. 17(1): 1-14.] cite journal |author=Medzhitov R, Preston-Hurlburt P, Janeway C |title=A human homologue of the Drosophila Toll protein signals activation of adaptive immunity |journal=Nature |volume=388 |issue=6640 |pages=394–7 |year=1997 |pmid=9237759 |doi=10.1038/41131] The very fact that TLR activation leads to adaptive immune responses to foreign entities explains why so many adjuvants used today in vaccinations are developed to mimic TLR ligands.

It is believed that upon activation, TLRs recruit adapter proteins (proteins that mediate other protein-protein interactions) within the cytosol of the immune cell in order to propagate the antigen-induced signal transduction pathway. To date, four adapter proteins have been well-characterized. These proteins are known as MyD88, Trif, Tram and Tirap (also called Mal). [Berczi I, Bertok L, Bereznai T. 1966. Comparative studies on the toxicity of Escherichia coli lipopolysaccharide endotoxin in various animal species. Can. J. Microbiol. 12: 1070-1071.] [Yamamoto,M., Sato,S., Hemmi,H., Hoshino,K., Kaisho,T., Sanjo,H., Takeuchi,O., Sugiyama,M., Okabe,M., Takeda,K. et al. 2003. Role of adapter TRIF in the MyD88-independent Toll-like receptor signaling pathway. Science. 301: 640-643.] [cite journal |author=Yamamoto M, Sato S, Hemmi H, Sanjo H, Uematsu S, Kaisho T, Hoshino K, Takeuchi O, Kobayashi M, Fujita T, Takeda K, Akira S |title=Essential role for TIRAP in activation of the signalling cascade shared by TLR2 and TLR4 |journal=Nature |volume=420 |issue=6913 |pages=324–9 |year=2002 |pmid=12447441 |doi=10.1038/nature01182] [cite journal |author=Yamamoto M, Sato S, Hemmi H, Uematsu S, Hoshino K, Kaisho T, Takeuchi O, Takeda K, Akira S |title=TRAM is specifically involved in the Toll-like receptor 4-mediated MyD88-independent signaling pathway |journal=Nat Immunol |volume=4 |issue=11 |pages=1144–50 |year=2003 |pmid=14556004 |doi=10.1038/ni986] These recruited proteins are then responsible for the subsequent activation of other downstream proteins, including protein kinases (IKKi, IRAK1, IRAK4, and TBK1) that further amplify the signal and ultimately lead to the upregulation or suppression of genes that orchestrate inflammatory responses and other transcriptional events. Some of these events lead to cytokine production, proliferation, and survival, while others lead to greater adaptive immunity. ] The high sensitivity of TLR for microbial ligands is what makes adjuvants that mimic TLR ligands such a prime candidate for enhancing the overall effects of antigen specific vaccinations on immunological memory. Finally, the expression of TLRs is vast as they are found on the cell membranes of innate immune cells (DCs, macrophages, natural killer cells), cells of the adaptive immunity (T and B lymphocytes) and non immune cells (epithelial and endothelial cells, fibroblasts). [cite journal |author=Delneste Y, Beauvillain C, Jeannin P |title=Innate immunity: structure and function of TLRs |journal=Med Sci (Paris) |volume=23 |issue=1 |pages=67–73 |year=2007 |pmid=17212934]

This further substantiates the importance of administering vaccines with adjuvants in the form of TLR ligands as they will be capable of eliciting their positive effects across the entire spectrum of innate and adaptive immunity. Nevertheless, there are certainly adjuvants whose immune-stimulatory function completely bypasses the putative requisite for TLR signaling. In short, all TLR ligands are adjuvants but not all adjuvants are TLR ligands.

Medical complications

Aluminum salts used in many human vaccines are generally regarded as safe; [cite journal |author=Baylor N, Egan W, Richman P |title=Aluminum salts in vaccines--US perspective |journal=Vaccine |volume=20 Suppl 3 |issue= |pages=S18–23 |year=2002 |pmid=12184360 |doi=10.1016/S0264-410X(02)00166-4] however, a recent study revealed that aluminum adjuvants at levels comparable to those administered to Gulf War veterans can cause motor neuron death. [cite journal |author=Petrik MS, Wong MC, Tabata RC, Garry RF, Shaw CA |title=Aluminum adjuvant linked to gulf war illness induces motor neuron death in mice |journal=Neuromolecular Med |volume=9 |issue=1 |pages=83–100 |year=2007 |pmid=17114826 |doi=10.1385/NMM:9:1:83]

In veterinary medicine, particularly in felines, adjuvants have been linked to the induction of sarcomas at the injection site in a small proportion of vaccinations. [ [http://www.comvet.com/print/vax_print.html "Feline Post-Vaccinal Sarcoma - A Literature Review,"] Shane Ryan, "Singapore Veterinary Journal," (1998) 22: 65-73.]

Controversy

Recently, the premise that TLR signaling acts as the key node in antigen-mediated inflammatory responses has been in question as researchers have observed antigen-mediated inflammatory responses in leukocytes in the absence of TLR signaling. ] [cite journal |author=Wickelgren I |title=Immunology. Mouse studies question importance of toll-like receptors to vaccines |journal=Science |volume=314 |issue=5807 |pages=1859–60 |year=2006 |pmid=17185572 |doi=10.1126/science.314.5807.1859a] One researcher found that in the absence of MyD88 and Trif (essential adapter proteins in TLR signaling), they were still able to induce inflammatory responses, increase T cell activation and generate greater B cell abundancy using conventional adjuvants (alum, Freund’s complete adjuvant, Freund’s incomplete adjuvant, and monophosphoryl-lipid A/trehalose dicorynomycolate (Ribi's adjuvant). ]

These observations suggest that although TLR activation can lead to increases in antibody responses, TLR activation is not required to induce enhanced innate and adaptive responses to antigens.

Investigating the mechanisms which underlie TLR signaling has been significant in understanding why adjuvants used during vaccinations are so important in augmenting adaptive immune responses to specific antigens. However, with the knowledge that TLR activation is not required for the immune-enhancing effects caused by common adjuvants, we can conclude that there are, in all likelihood, other receptors besides TLRs that have not yet characterized, opening the door to future research. Perhaps future adjuvants occupying these putative receptors will be employed to bypass the TLR signaling pathway completely in order to circumvent common side effects of adjuvant-activated TLRs such as local inflammation and the general malaise felt because of the costly whole-body immune response to antigen. Surely, such issues will be the subject of much debate for future researchers.

References