More about adjuvants
Historically the goal of vaccination has been to induce both a fast onset and long-lasting protective immunity to prevent infectious diseases...
Nowadays, vaccines are also included in therapeutic treatments fighting diseases such as cancer, malaria, AIDS or autoimmune disorders. Initial generations of vaccines were based on inactivated bacterial whole-cells or viruses. Such formulations were efficacious but could present some side-effects. To reduce reactogenicity, innovative vaccines containing recombinant protein have been developed but those highly purified proteins are often linked to lower immunogenicity. Vaccines with a more defined antigenic composition require increasingly powerful adjuvants (1). Vaccine adjuvants (Latin verb adjuvare meaning “to help”) are substances which increase the immune response intensity to a co-administered antigen. Therefore, adjuvants are required to assist vaccines to induce potent and persistent immune responses, with the additional benefits that fewer antigens and fewer injections are needed.
The immune system is composed of cells and soluble proteins which respond quickly to antigens. A secondary system composed of a different cell population ensures a long term and highly specific memory response. Those two systems are called innate and adaptive immune system, respectively. Mainly based on monocytes, macrophages (MØ) and dendritic cells (DCs), all of which are forms of antigen presenting cells (APC), the innate immune system activates the adaptive immune system cell population composed of B and T lymphocytes. Immunocompetent cells from both systems can be circulating in blood or lymph flow but are also based in the effector organs of the immune response: the spleen and lymph nodes.
Innate immunity has regained a primary role, not only chronologically but also functionally, through its importance in shaping the adaptive response. Applied research has benefited directly from these basic findings, thanks in particular to the discovery of the Tolllike receptors (TLRs) and other pattern-recognition receptors (PRRs). Following host invasion, microorganisms expressing pathogen-associated molecular patterns (PAMPs) are recognized by innate immune cells through specific and non-polymorphic PRRs. Among the PRRs, TLRs have been shown to play a crucial role in the early stages of the immune response to infection. The innate immunity process of antigens prior to their presentation to lymphocytes has a great influence on adaptive response. Some innate cells such as DCs and MØ have long been known to be mandatory for initiating immune responses by presenting antigens to T cells. Among these APCs, DCs are the key effector because it is now clear that many agents with adjuvant activity on the immune response boost immunity through induction of DC maturation (2-5). DCs have an immature phenotype in peripheral tissues specialized for antigen uptake but upon recognition of foreign material they migrate to the lymph node, where they arrive as mature cells (6). The DCs bridge innate and adaptive immunity (7). DCs have the potential to recognize a foreign antigen, process it into small peptides for presentation onto major histocompatibility (MHC) molecules to the T-cell receptor (TCR), and to provide the essential costimulatory molecules for activation of naive CD4+ and CD8+ T cells (8). T CD4+ cells lead to three main T helper (Th) cell responses in association with class II MHC:
- The Th1-type response leads to secretion of interleukin-2 (IL-2) and interferon-γ (IFN-γ), favouring cell-mediated immune responses, activation of CD8+ cytotoxic T cells (CTLs)
- The Th2-type response is characterized by secretion of IL-4, IL-5, IL-10 and IL-13, providing B-cell help and so antibody production by plasma cells
- Treg-type response expresses TGF-β and IL-10. This response is essential to maintain immune tolerance because in this case lymphocytes inhibit immune activity
Others profiles exist, such as Th17 and Th9 responses involving auto-immune disease or proinflammatory response.
Used for more than 70 years, adjuvants include different classes of compounds, such as microbial products, mineral salts, emulsions, microparticles and liposomes. The term adjuvant is often used as a synonym for immunostimulant. However, whereas immunostimulants are generally single compounds with intrinsic immunostimulant and/or immunomodulatory properties, adjuvants can be composed of different constituents with different functions and activities.
The current challenge facing adjuvant research is to find the “perfect mix” for efficacious, stable and safe formulation.
We detail below the main immune system activation pathways through vaccine:
A- Inflammatory response to injection
B- Depot effect, protection of the antigen from enzymatic fast degradation and longlasting antigen release
C- Prolonged inflammatory response due to vaccine remnants
D- Recruitment of competent cells, innate then adaptive immune system
Oil adjuvants and aluminium hydroxide and are among the most frequently used adjuvants in vaccines for humans and animals. Nevertheless, many other adjuvants are used in research.
- Oil adjuvant for vaccine: emulsions
Oil adjuvants are mixtures of injectable surfactants and oils. Such adjuvants are used for emulsification when mixed to an aqueous antigenic phase. According to the type of surfactant system, emulsions can be water-in-oil (W/O), oil-in-water (O/W) or water-in-oil-in-water (W/O/W). The oil nature (mineral, vegetable or synthetic) can also be a key parameter to optimize the safety and efficacy balance of the final vaccine (9, 10). Compared to traditional Incomplete Freund’s Adjuvants (IFA), modern oil adjuvants such as the MONTANIDE ISA ranges have been developed to improve both the safety and efficacy of the formulated vaccines and also provide vaccine emulsion properties (stability, viscosity, seringeability, etc.) (11). MONTANIDE adjuvants have been applied in veterinary and human therapeutic vaccines (12). Veterinary applications have been used in foot-and-mouth disease (FMD) eradication programmes worldwide for more than 40 years. Human applications have existed since 1992 for therapeutic vaccines and recently led to a cancer treatment breakthrough with the first cancer vaccine license based on MONTANIDE ISA 51 VG (10).
The mechanism of action of oil adjuvant-based vaccines includes the formation of a depot at the injection site, enabling the slow release of the antigen and the stimulation of antibody-producing plasma cells (13). These formulations promote antigen uptake by APCs. Formulated antigens are concentrated and protected against degradation, while phagocytosis is stimulated by different ligand/receptor interactions.
MONTANIDE ISA ranges have been shown to induce high antibody titers and CTL responses (14-19). In many cases, the response was greater than that achieved using other types of adjuvants
- Mineral salts
The adjuvant activity of aluminium salts was first demonstrated in 1926. So far, aluminium-based compounds (principally aluminium hydroxide or phosphate) have been the most widely used adjuvants in humans (20). Alum salts stimulate antibody production by a Th2 response, however they show relatively poor impact in many situations, particularly in inducing cellular immune responses (21-24). This adjuvant is also known to induce short-term immunity, therefore implying multiple booster injections. The mechanism whereby aluminium salts work remains unknown although one suggestion is that they work by the formation of an aluminium/antigen aggregate, inducing a depot at the inoculation site. Granulomas and necrosis in injected muscle are common when alum is administered by the intramuscular route. Mineral salts can directly stimulate non-specific cells such as macrophages, and are able to activate the complement (25).
- Example of other type of adjuvant formulation
Saponins are derived from the bark of the South American tree Quillaja saponaria Molina and have been tested and used in both veterinary and human medicine for decades. Saponins also constitute the active components of particulate formulations such as ISCOMS, which are cage-like structures containing antigen, cholesterol, phospholipids and saponin. We can also quote liposomes and microparticles. These particulate compounds can either encapsulate an antigen or carry it on their surface through adsorption or covalent linkage.
Meat quality and animal welfare concerns demand well tolerated and efficient vaccines. The “universal” adjuvant does not exist, because each adjuvant and its targeted antigens will have unique requirements. The vaccine formulation and its protocol are critical to finding the best compromise between the efficacy, safety and cost effectiveness of the vaccination. Selecting the appropriate adjuvant technology during a vaccine development is one of the key points ensuring the success of vaccination in the field. The choice of the adjuvant formula has to consider critical criteria such as antigen type, target animals, safety profile expected, specificity and the kinetics of the immune response targeted.
1. Petrovsky, N., and J. C. Aguilar. 2004. Vaccine adjuvants: current state and future trends. Immunol Cell Biol 82:488-496.
2. De Smedt, T., B. Pajak, E. Muraille, L. Lespagnard, E. Heinen, P. De Baetselier, J. Urbain, O. Leo, and M. Moser. 1996. Regulation of dendritic cell numbers and maturation by lipopolysaccharide in vivo. J Exp Med 184:1413-1424.
3. De Becker, G., V. Moulin, B. Pajak, C. Bruck, M. Francotte, C. Thiriart, J. Urbain, and M. Moser. 2000. The adjuvant monophosphoryl lipid A increases the function of antigenpresenting cells. Int Immunol 12:807-815.
4. Shah, J. A., P. A. Darrah, D. R. Ambrozak, T. N. Turon, S. Mendez, J. Kirman, C. Y. Wu, N. Glaichenhaus, and R. A. Seder. 2003. Dendritic cells are responsible for the capacity of CpG oligodeoxynucleotides to act as an adjuvant for protective vaccine immunity against Leishmania major in mice. J Exp Med 198:281-291.
5. Fujii, S., K. Shimizu, C. Smith, L. Bonifaz, and R. M. Steinman. 2003. Activation of natural killer T cells by alpha-galactosylceramide rapidly induces the full maturation of dendritic cells in vivo and thereby acts as an adjuvant for combined CD4 and CD8 T cell immunity to a coadministered protein. J Exp Med 198:267-279.
6. Shi, Y., J. E. Evans, and K. L. Rock. 2003. Molecular identification of a danger signal that alerts the immune system to dying cells. Nature 425:516-521.
7. Bendelac, A., and R. Medzhitov. 2002. Adjuvants of immunity: harnessing innate immunity to promote adaptive immunity. J Exp Med 195:F19-23.
8. Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392:245-252.
9. Aucouturier, J., L. Dupuis, and V. Ganne. 2001. Adjuvants designed for veterinary and human vaccines. Vaccine 19:2666-2672.
10. Aucouturier, J., S. Deville, C. Perret, I. Vallee, and P. Boireau. 2001. Assessment of efficacy and safety of various adjuvant formulations with a total soluble extract of Trichinella spiralis. Parasite 8:S126-132. 11. Aucouturier, J., S. Ascarateil, and L. Dupuis. 2006. The use of oil adjuvants in therapeutic vaccines. Vaccine 24 Suppl 2:S2-44-45.
12. Dupuis, L., S. Ascarateil, J. Aucouturier, and V. Ganne. 2006. SEPPIC vaccine adjuvants for poultry. Ann N Y Acad Sci 1081:202-205.
13. Freund, J. 1956. The mode of action of immunologic adjuvants. Bibl Tuberc:130-148.
14. Oliveira, G. A., K. Wetzel, J. M. Calvo-Calle, R. Nussenzweig, A. Schmidt, A. Birkett, F. Dubovsky, E. Tierney, C. H. Gleiter, G. Boehmer, A. J. Luty, M. Ramharter, G. B. Thornton, P. G. Kremsner, and E. H. Nardin. 2005. Safety and enhanced immunogenicity of a hepatitis B core particle Plasmodium falciparum malaria vaccine formulated in adjuvant MONTANIDE ISA 720 in a phase I trial. Infect Immun 73:3587-3597.
15. Miles, A. P., H. A. McClellan, K. M. Rausch, D. Zhu, M. D. Whitmore, S. Singh, L. B. Martin, Y. Wu, B. K. Giersing, A. W. Stowers, C. A. Long, and A. Saul. 2005. MONTANIDE ISA 720 vaccines: quality control of emulsions, stability of formulated antigens, and comparative immunogenicity of vaccine formulations. Vaccine 23:2530-2539.
16. Hersey, P., S. W. Menzies, B. Coventry, T. Nguyen, M. Farrelly, S. Collins, D. Hirst, and H. Johnson. 2005. Phase I/II study of immunotherapy with T-cell peptide epitopes in patients with stage IV melanoma. Cancer Immunol Immunother 54:208-218.
17. Hirunpetcharat, C., J. Wipasa, S. Sakkhachornphop, T. Nitkumhan, Y. Z. Zheng, S. Pichyangkul, A. M. Krieg, D. S. Walsh, D. G. Heppner, and M. F. Good. 2003. CpG oligodeoxynucleotide enhances immunity against blood-stage malaria infection in mice parenterally immunized with a yeast-expressed 19 kDa carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein-1 (MSP1(19)) formulated in oil-based Montanides. Vaccine 21:2923-2932. 18. Perlaza, B. L., M. Arevalo-Herrera, K. Brahimi, G. Quintero, J. C. Palomino, H. Gras-Masse, A. Tartar, P. Druilhe, and S. Herrera. 1998. Immunogenicity of four Plasmodium falciparum preerythrocytic antigens in Aotus lemurinus monkeys. Infect Immun 66:3423-3428.
19. Roestenberg, M., E. Remarque, E. de Jonge, R. Hermsen, H. Blythman, O. Leroy, E. Imoukhuede, S. Jepsen, O. Ofori-Anyinam, B. Faber, C. H. Kocken, M. Arnold, V. Walraven, K Teelen, W. Roeffen, Q. de Mast, W. R. Ballou, J. Cohen, M. C. Dubois, S. Ascarateil, A. van der Ven, A. Thomas, and R. Sauerwein. 2008. Safety and immunogenicity of a recombinant Plasmodium falciparum AMA1 malaria vaccine adjuvanted with Alhydrogel, MONTANIDE ISA 720 or AS02. PLoS One 3:e3960.
20. Allison, A. C., and N. E. Byars. 1991. Immunological adjuvants: desirable properties and sideeffects. Mol Immunol 28:279-284.
21. Schirmbeck, R., K. Melber, T. Mertens, and J. Reimann. 1994. Antibody and cytotoxic T-cell responses to soluble hepatitis B virus (HBV) S antigen in mice: implication for the pathogenesis of HBV-induced hepatitis. J Virol 68:1418-1425.
22. Traquina, P., M. Morandi, M. Contorni, and G. Van Nest. 1996. MF59 adjuvant enhances the antibody response to recombinant hepatitis B surface antigen vaccine in primates. J Infect Dis 174:1168-1175.
23. Brewer, J. M., M. Conacher, A. Satoskar, H. Bluethmann, and J. Alexander. 1996. In interleukin-4-deficient mice, alum not only generates T helper 1 responses equivalent to freund's complete adjuvant, but continues to induce T helper 2 cytokine production. Eur J Immunol 26:2062-2066.
24. Nicklas, W. 1992. Aluminum salts. Res Immunol 143:489-494; discussion 574.
25. HogenEsch, H. 2002. Mechanisms of stimulation of the immune response by aluminum adjuvants. Vaccine 20 Suppl 3:S34-39.