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New developments in adjuvants
Mechanisms of Ageing and Development 93 (1997) 171 – 177
New developments in adjuvants John J. Donnelly* Department of Virus and Cell Biology, WP16 -327, Merck Research Laboratories, West Point, PA 19486, USA Received 16 October 1996
Abstract The development of improved methods for the production of vaccines have fostered the use of purified subunits in place of live attenuated and whole inactivated organisms. Greater understanding of immune mechanisms has stimulated efforts to design vaccines that elicit specific mechanisms of resistance to particular pathogens. Together these forces have driven a search for novel adjuvants with specific properties optimally suited to vaccines for particular diseases. At the First International Conference on Immunology and Aging, a mini-symposium was devoted to the topic of new adjuvants. This review and the following two papers summarize the proceedings of the mini-symposium and illustrate the potential for the development of new adjuvants of greater efficacy and specificity, and the complexities involved in bringing new discoveries in adjuvants from the laboratory to the clinic. © 1997 Elsevier Science Ireland Ltd. Keywords: Adjuvants; Vaccines; Immune mechanisms
1. Introduction Adjuvants (from the Latin ‘adjuvare’, to aid or help) have been considered to be an essential component of most inactivated vaccines for the last five decades. Where * Corresponding author. Tel.: + 1 215 6523683; fax: + 1 215 6528127; e-mail: john – [email protected] 0047-6374/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 0 4 7 - 6 3 7 4 ( 9 6 ) 0 1 8 1 0 - 6
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live attenuated vaccines may trigger endogenous mechanisms that enhance immune responses, non-living materials do not generally exhibit this capability and may require the addition of exogenous agents to achieve appropriate levels of immunogenicity. In addition to stimulating immune responses, adjuvants can serve other purposes, such as improving the stability of vaccines containing more than one component or reducing the toxicity of vaccine constituents. Inorganic gels, consisting of aluminum or calcium phosphate, sulfate, oxide or hydroxide in varying proportions, were among the first adjuvants and still are the only ones licensed for human use [1]. The development of recombinant methods for the production of proteins, and improvements in large-scale purification of proteins and polysaccharides, have allowed vaccines to move away from the use of whole killed bacteria and viruses and toward the use of purified subunits. Greater understanding of immune mechanisms has stimulated efforts to target vaccines to elicit specific mechanisms of resistance to particular pathogens. Together these forces have driven a search for novel adjuvants with specific physical and immunological properties optimally suited to vaccines for particular diseases. This review and the following two papers illustrate the potential for the development of new adjuvants of greater efficacy and specificity, and the complexities involved in bringing new discoveries in adjuvants from the laboratory to the clinic. 2. What are the desired effects of adjuvants? The principal reason for which most adjuvants are included in most vaccines is to increase the magnitude of the immune response to vaccination. For most traditional vaccines this is manifested as an increase in average serum antibody titers across individuals, and as an increase in the proportion of individuals making a sufficiently high immune response to be considered as protected [2]. A secondary goal is to increase the duration of the protective response. Antibody titers elicited by vaccination generally reach a peak within several months and then decay with time. In most instances after vaccination with non-living material, serum antibodies against the immunogen are no longer detectable by 5–10 years after immunization unless environmental contact with the pathogen continues to stimulate antibody production. Although immunological memory in the form of primed B-cells and T-cells can persist for much longer, and can provide protection by means of a recall response after exposure to the pathogen, this is not readily measured by the clinician and thus without revaccination it can be difficult to ascertain which vaccinees have developed long-term protection and which have not. Increasing the peak antibody titer (assuming a constant decay rate) can lengthen the period after vaccination during which antibodies can be detected in the serum. For pathogens capable of causing serious disease before the recall response can occur, maintenance of serum antibody titers may be essential for long-term protection. Thus the use of adjuvants to increase immune responses can both increase the number of vaccines making a protective response and lengthen the duration of the period over which protection can be assumed.
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Recent developments in immunology have defined two general patterns of immune responses to immunization or infection, distinguished by the types of cytokines produced by helper T-lymphocytes during the early stages of the immune response [3]. Helper T-cell responses characterized as Type 1 (Th1) are distinguished by predominant production of the cytokines IFN-g and interleukin-2 (IL-2) by antigen-specific helper T-cells upon antigen stimulation. Type 2 helper T-cell (Th2) responses are characterized by the production of interleukins 4 and 5 (IL-4 and IL-5) by antigen-specific helper T-cells. Th1 type responses are associated with cellular immunity, including delayed-type hypersensitivity (DTH) and cytotoxic T-cell (CTL) responses, while Th2 responses are associated with production of high levels of antibody, with mucosal immune responses, and with a lower cell-mediated immune component. In mice, Th1 responses are associated with serum antibody responses in which the IgG2a isotype predominates, while in Th2 responses the predominant isotype is IgG1. In humans, the relationship between the particular Th response type and the distribution of antibody isotypes in serum, is less clear, although the differences in cytokine production by Th-cells are demonstrable. Particular adjuvants selectively can increase one type of immune response over the other. For example, aluminum adjuvants are associated with Th2 type responses [3] while saponin adjuvants such as QS-21 [4] and lipopolysaccharides such monophosphoryl lipid A (MPL) are associated with Th1 responses [5]. Thus if a particular type of immune response is desired an adjuvant can be selected to maximize this response. Increasing the magnitude of immune responses through the use of adjuvants also offers practical advantages for the use of vaccine as public health measures. If the use of a particular adjuvant can reduce the number of immunizations needed to produce the required level of immunity, compliance will be improved as the number of individuals successfully completing the course of immunization will increase. The cost of administering the vaccine also will be lower if the number of physician visits required for vaccination can be reduced. Increasing the potency of vaccines by the use of adjuvants also may reduce the dose of vaccine required to elicit a protective immune response. For example, approximately 1/10 as much hepatitis-B surface antigen is required to induce an antibody response when the antigen is given on aluminum adjuvant as would be required for the antigen alone (M.J. Caulfield, D.B. Volkin, J.J. Donnelly, unpublished observations). Thus the overall cost of the vaccine can be reduced if the relatively inexpensive adjuvant component can be substituted for the more expensive recombinant protein. Where vaccines are complex mixtures, reducing the dose of vaccine required also can reduce side effects as the amount of potentially toxic material admnistered is decreased. Vaccines targeted for use in elderly populations, including influenza, S. pneumoniae, respiratory syncytial virus and parainfluenza virus, may particularly benefit from the use of adjuvants to increase the magnitude and duration of immune responses.
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3. Challenges of adjuvant research A number of factors, such as slow release of antigen (depot effect), more efficient delivery of antigen to draining lymph nodes, nonspecific activation of antigen-presenting cells or of B and/or T-lymphocytes, increased uptake of antigen by antigen-presenting cells, or increased recruitment of immune cells to the site where the antigen is present, can contribute to increased immune responses to immunization. Many of these factors involve the interaction of various immune system components and specific anatomical features, making them difficult to replicate in model systems in vitro. Furthermore, the overall effect of an adjuvant may result from the interaction of multiple factors. For example, an oil–water emulsion [6,7] might provide more effective delivery of antigens to draining lymph nodes, increased uptake by antigen-presenting cells, and activation of antigen-presenting cells or T-cells, all of which may contribute to its effect. Thus the ability to develop mechanism-based in vitro screens for novel adjuvant compounds is severely limited at present. Most adjuvants have been identified on the basis of testing in vivo. Species differences in responsiveness also may complicate the search for novel adjuvants. Not all adjuvants that are active in mice, for example, are equally active in nonhuman primates or in humans.
4. Examples of adjuvants that currently are under investigation
4.1. Aluminum Aluminum adjuvants are the only adjuvants currently licensed for clincal use [1]. In general, aluminum adjuvants are effective only if the antigen is incorporated in or on the particles of gel by coprecipitation with the aluminum, or adhered to the particles by ionic interactions. Since the interaction of proteins with the aluminum particles depends on the protein and the aluminum gel being of opposite charges, the pI of the protein species and of the aluminum are important determinants of the stability of the antigen/aluminum complex and therefore of the effectiveness of the vaccine [1]. Various types of aluminum gels, incorporating anions including phosphates, hydroxides, and oxides, continue to be investigated to refine and improve existing vaccines and to facilitate combination vaccines.
4.2. Emulsions Mixtures of a metabolizable oil such as squalane or squalene and an aqueous phase, including a detergent such as Tween and emulsifiers such as sorbitan monooleate (Span 85) to stabilize the oil droplets, are micro-fluidized to produce an oil-in-water emulsion with droplets in the 0.1–10 mm size range [7]. Some versions also incorporate a block copolymer, consisting of a hydrophobic core region flanked by hydrophilic regions [6]. Antigens can be incorporated in the emulsion during the emulsification process or added to the emulsion after it has been formed.
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4.3. Lipopolysaccharides (LPS) LPS are found in the outer membranes of gram-negative bacteria and have long been known to have adjuvant activities. These materials are thought to bind to LPS-binding proteins and activate B and T-cells and antigen-presenting cells. However, they also exhibit significant toxicity which appears to be mediated by IL-1 and TNF released from macrophages. Various chemical modifications of the lipid A portion of LPS, such as removing one phosphate to produce monophosphoryl lipid A, have been found to resuce the toxic effects of LPS while retaining adjuvant activity [5].
4.4. Polymers Various synthetic polymers have been developed for use as adjuvants. Some, such as poly-lactide-co-glycolide (PLGA) are dissolved in organic solvents, mixed with antigen, and the solvent then is removed to yield microspheres with the antigen encapsulated within them [8,9]. The antigen can be incorporated as an aqueous solution, in which case the antigen solution is emulsified in the organic solvent containing the polymer, producing a water-in-oil emulsion, or in particulate form as lyophilized powder in which case no emulsification is required [8]. The microspheres degrade in vivo, due to breakdown of the polymer, releasing the entrapped antigen. Polymers such as PLGA have little inherent immune stimulating activity and serve primarily as a reservoir of antigen [9]. Other polymers, such as block copolymers [10] and polyphosphazenes [11], encapsulate antigen in solution by ionic or hydrophobic interactions. The resulting complexes may provide a slow release of antigen but also appear to possess immune stimulating activity, perhaps by facilitating the uptake of antigen by antigen-presenting cells.
4.5. Saponins Extracts of the bark of the tree Quillaja saponaria were found to have adjuvant activity [4]. The extracts contain a number of components that have been purified and found to exhibit adjuvant activity individually. Saponins share a terpene core structure and vary in their glycosylation and in the addition of an acyl side chain linked to the carbohydrate moiety. They exhibit hemolytic activity and are thought to displace cholesterol from membranes. Their adjuvant activities include the delivery of antigens to the endogenous pathway of antigen processing for presentation by major histocompatibility complex (MHC) class I molecules, and they are thought to act by delivering antigen across the membrane directly to the cytosol of antigen-presenting cells [4]. In the presence of cholesterol, phosphatidyl ethanolamine, and phosphatidyl serine, saponins can assemble into polyhedral cage-like structures termed immune-stimulating complexes (ISCOMs) [12]. ISCOMs also are capable in some instances of delivering antigen to the cytosolic pathway of antigen processing [12].
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4.6. Liposomes Various techniques have been used to entrap antigens in vesicles formed from lipids. The constituents of these vesicles generally are charged diacyl lipids similar to those found in cell membranes, such as dioleyl phosphatidylcholine and phosphatidylethanolamine [13]. Non-polar lipids such as cholesterol also can be included. Liposomes can be made as single bimolecular leaflet structures in which a droplet of aqueous solution is entrapped within a membrane composed of two layers of lipids, or as multilamellar structures with many concentric layers of lipids. Integral membrane proteins insert into the liposome membrane, giving these antigens a conformation similar to their native state [13]. Hydrophilic proteins may be entrapped in the aqueous phase inside the vesicles. Like ISCOMs, liposomes can be used to stimulate MHC Class I-restricted CTL responses, presulably by delivering antigens to the cytosolic pathway of antigen processing [14]. In addition to antigens, other additives that are amphiphilic or lipid-soluble, such as MPL, also can be incorporated into liposomes to increase their adjuvant effect [13].
4.7. Cytokines Proteins produced by activated T-cells, B-cells, or APCs also can be used as adjuvants. Administration of cytokines with vaccines is intended to increase or redirect immune responses by providing intercellular signals that normally would be produced in an immune response, but at an earlier time, at higher concentrations, or in an atypical location. Examples of cytokines that have been used in animal models for this purpose are interleukin-12 (IL-12) [15] and granulocyte-macrophage colony-simulating factor (GM-CSF) [16]. IL-12 normally is produced by macrophages and induces the production of IFN-g by natural killer (NK) cells, driving the Th response toward a Th1 phenotype [15]. GM-CSF is normally produced by macrophages and NK cells and enhances the differentiation and antigen presentation functions of macrophages and dendritic cells. GM-CSF has been found to increase T-cell responses in a number of laboratory animal models [16]. Cytokines may be rapidly inactivated by inhibitors present in normal serum and tissue fluid and may act over narrow concentration gradients. Therefore multiple injections, delivery using a viral vector, or delivery in a slow-release system such as PLGA, are required to produce effects using cytokines as adjuvants in experimental systems. The delivery of cytokines for clinical use in vaccines will require further refinement.
5. Conclusions This review has summarized a few of many adjuvants that are under investigation for potential use in human vaccines [17]. Improved adjuvants may increase the effectiveness of current vaccines and also may be essential for novel vaccines against pathogens such as HIV, herpes simplex virus, and human papillomavirus. In the
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elderly, the use of novel adjuvants may increase response rates and extend the duration of protection offered by current vaccines for diseases such as influenza and pneumococcal infection.
References [1] S. Shirodkar, R.L. Hutchinson, D.L. Perry, J.L. White and S.L. Hem, Aluminum compounds used as adjuvants in vaccines. Pharm Res, 7 (1990) 1282 – 1288. [2] P.J. Kniskern and W.L. Miller, Hepatitis B vaccines: Blueprints for vaccines of the future. In R.W. Ellis (ed.), Vaccines: New approaches to immunological problems, Butterworth – Heinemann, Boston, 1992, pp. 177–204. [3] T.R. Mosmann and R.L. Coffman, Th1 and Th2 cells: Different patterns of lymphokine secretion lead to different functional properties. Ann. Re6. Immunol., 7 (1989) 145 – 173. [4] N.E. Jacobsen, W.J. Fairbrother, C.R. Kensil, A. Lim, D.A. Wheeler and M.F. Powell, Structure of the saponin adjuvant QS-21 and its base-catalyzed isomerization product by 1H and natural abundance 13C NMR spectroscopy. Carbohydr. Res., 280 (1996) 1 – 14. [5] J.T. Ulrich and K.R. Myers, Monophosphoryl lipid A as an adjuvant, past experiences and new directions. Pharm. Biotechnol., 6 (1995) 495 – 524. [6] A.C. Allison and N.E. Byars, Syntex adjuvant formulation. Res. Immunol., 143 (1992) 519 – 525. [7] G. Ott, G.L. Barchfeld and G. Van Nest, Enhancement of humoral response against human influenza vaccine with the simple submicron oil/water emulsion adjuvant MF59. Vaccine, 13 (1995) 1557–1562. [8] D.T. O’Hagan, J.P. McGee, J. Holmgren, A.M. Mowat, A.M. Donachie, K.H. Mills, W. Gaisford, D. Rahman and S.J. Challacombe, Biodegradable microparticles as controlled release antigen delivery systems. Vaccine, 11 (1993) 149 – 154. [9] J.L. Cleland, L. Barron, P.W. Berman, A. Daugherty, T. Gregory, A. Lim, J. Vennari, T. Wrin, and M.F. Powell, Development of a single-shot subunit vacine for HIV-1: Part 2. Defining optimal autoboost characteristics to maximize the humoral immune repsonse. J. Pharm. Sci., 60 (1996) 2289–2295. [10] R.L. Hunter and B. Bennett, The adjuvant activity of nonionic block polymer surfactants. II. Antibody formation and inflammation related to the structure of triblocks and octablock copolymers. J. Immunol., 133 (1984) 3167 – 3175. [11] A.K. Andrianov and M.P. LeGolvan, Characterization of poly[di(carboxylatophenoxy)phosphazene] by an aqueous gel permeation chromatography. J. Appl. Polymer Sci., 60 (1996) 2289–2295. [12] H. Takahashi, T. Takeshita, B. Morein, S. Putney, R.N. Germain and J.A. Berzofsky, Induction of CD8 + cytotoxic T-cells by immunization with purified HIV-1 envelope protein in ISCOMs. Nature, 344 (1990) 873–875. [13] W.I. White, D.R. Cassatt, J. Madsen, S.J. Burke, R.M. Woods, N.M. Wassef, C.R. Alving and S. Koenig, Antibody and cytotoxic-T-lymphocyte responses to a single liposome-associated peptide antigen. Vaccine, 13 (1995) 1111 – 1122. [14] D.C. Powers, M.C. Manning, P.J. Hanscome and P.J.F. Pietrobon, Cytotoxic-T-lymphocyte responses to a liposome-adjuvanted influenza A virus vaccine in elderly adults. J. Infect. Dis., 172 (1995) 1103–1107. [15] L.C. Afonso, T.M. Scharton, L.Q. Vieira, M. Wysocka, G. Trinchieri and P. Scott, Administration of recombinant IL-12 in a vaccine against Leishmania major. Science, 263 (1994) 235 – 237. [16] M.H. Tao and R. Levy, Idiotype/granulocyte-macrophage colony-stimulating factor fusion protein as a vaccine for B-cell lymphoma. Nature, 362 (1993) 755 – 758. [17] F.R. Vogel and M.F. Powell, A compendium of vaccine adjuvants and excipients. In M.F. Powell and M.J. Newman (eds.), Vaccine Design: The Subunit and Adju6ant Aproach, Plenum Press, N.Y., 1995, pp. 141–228.
New developments in adjuvants John J. Donnelly* Department of Virus and Cell Biology, WP16 -327, Merck Research Laboratories, West Point, PA 19486, USA Received 16 October 1996
Abstract The development of improved methods for the production of vaccines have fostered the use of purified subunits in place of live attenuated and whole inactivated organisms. Greater understanding of immune mechanisms has stimulated efforts to design vaccines that elicit specific mechanisms of resistance to particular pathogens. Together these forces have driven a search for novel adjuvants with specific properties optimally suited to vaccines for particular diseases. At the First International Conference on Immunology and Aging, a mini-symposium was devoted to the topic of new adjuvants. This review and the following two papers summarize the proceedings of the mini-symposium and illustrate the potential for the development of new adjuvants of greater efficacy and specificity, and the complexities involved in bringing new discoveries in adjuvants from the laboratory to the clinic. © 1997 Elsevier Science Ireland Ltd. Keywords: Adjuvants; Vaccines; Immune mechanisms
1. Introduction Adjuvants (from the Latin ‘adjuvare’, to aid or help) have been considered to be an essential component of most inactivated vaccines for the last five decades. Where * Corresponding author. Tel.: + 1 215 6523683; fax: + 1 215 6528127; e-mail: john – [email protected] 0047-6374/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 0 4 7 - 6 3 7 4 ( 9 6 ) 0 1 8 1 0 - 6
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live attenuated vaccines may trigger endogenous mechanisms that enhance immune responses, non-living materials do not generally exhibit this capability and may require the addition of exogenous agents to achieve appropriate levels of immunogenicity. In addition to stimulating immune responses, adjuvants can serve other purposes, such as improving the stability of vaccines containing more than one component or reducing the toxicity of vaccine constituents. Inorganic gels, consisting of aluminum or calcium phosphate, sulfate, oxide or hydroxide in varying proportions, were among the first adjuvants and still are the only ones licensed for human use [1]. The development of recombinant methods for the production of proteins, and improvements in large-scale purification of proteins and polysaccharides, have allowed vaccines to move away from the use of whole killed bacteria and viruses and toward the use of purified subunits. Greater understanding of immune mechanisms has stimulated efforts to target vaccines to elicit specific mechanisms of resistance to particular pathogens. Together these forces have driven a search for novel adjuvants with specific physical and immunological properties optimally suited to vaccines for particular diseases. This review and the following two papers illustrate the potential for the development of new adjuvants of greater efficacy and specificity, and the complexities involved in bringing new discoveries in adjuvants from the laboratory to the clinic. 2. What are the desired effects of adjuvants? The principal reason for which most adjuvants are included in most vaccines is to increase the magnitude of the immune response to vaccination. For most traditional vaccines this is manifested as an increase in average serum antibody titers across individuals, and as an increase in the proportion of individuals making a sufficiently high immune response to be considered as protected [2]. A secondary goal is to increase the duration of the protective response. Antibody titers elicited by vaccination generally reach a peak within several months and then decay with time. In most instances after vaccination with non-living material, serum antibodies against the immunogen are no longer detectable by 5–10 years after immunization unless environmental contact with the pathogen continues to stimulate antibody production. Although immunological memory in the form of primed B-cells and T-cells can persist for much longer, and can provide protection by means of a recall response after exposure to the pathogen, this is not readily measured by the clinician and thus without revaccination it can be difficult to ascertain which vaccinees have developed long-term protection and which have not. Increasing the peak antibody titer (assuming a constant decay rate) can lengthen the period after vaccination during which antibodies can be detected in the serum. For pathogens capable of causing serious disease before the recall response can occur, maintenance of serum antibody titers may be essential for long-term protection. Thus the use of adjuvants to increase immune responses can both increase the number of vaccines making a protective response and lengthen the duration of the period over which protection can be assumed.
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Recent developments in immunology have defined two general patterns of immune responses to immunization or infection, distinguished by the types of cytokines produced by helper T-lymphocytes during the early stages of the immune response [3]. Helper T-cell responses characterized as Type 1 (Th1) are distinguished by predominant production of the cytokines IFN-g and interleukin-2 (IL-2) by antigen-specific helper T-cells upon antigen stimulation. Type 2 helper T-cell (Th2) responses are characterized by the production of interleukins 4 and 5 (IL-4 and IL-5) by antigen-specific helper T-cells. Th1 type responses are associated with cellular immunity, including delayed-type hypersensitivity (DTH) and cytotoxic T-cell (CTL) responses, while Th2 responses are associated with production of high levels of antibody, with mucosal immune responses, and with a lower cell-mediated immune component. In mice, Th1 responses are associated with serum antibody responses in which the IgG2a isotype predominates, while in Th2 responses the predominant isotype is IgG1. In humans, the relationship between the particular Th response type and the distribution of antibody isotypes in serum, is less clear, although the differences in cytokine production by Th-cells are demonstrable. Particular adjuvants selectively can increase one type of immune response over the other. For example, aluminum adjuvants are associated with Th2 type responses [3] while saponin adjuvants such as QS-21 [4] and lipopolysaccharides such monophosphoryl lipid A (MPL) are associated with Th1 responses [5]. Thus if a particular type of immune response is desired an adjuvant can be selected to maximize this response. Increasing the magnitude of immune responses through the use of adjuvants also offers practical advantages for the use of vaccine as public health measures. If the use of a particular adjuvant can reduce the number of immunizations needed to produce the required level of immunity, compliance will be improved as the number of individuals successfully completing the course of immunization will increase. The cost of administering the vaccine also will be lower if the number of physician visits required for vaccination can be reduced. Increasing the potency of vaccines by the use of adjuvants also may reduce the dose of vaccine required to elicit a protective immune response. For example, approximately 1/10 as much hepatitis-B surface antigen is required to induce an antibody response when the antigen is given on aluminum adjuvant as would be required for the antigen alone (M.J. Caulfield, D.B. Volkin, J.J. Donnelly, unpublished observations). Thus the overall cost of the vaccine can be reduced if the relatively inexpensive adjuvant component can be substituted for the more expensive recombinant protein. Where vaccines are complex mixtures, reducing the dose of vaccine required also can reduce side effects as the amount of potentially toxic material admnistered is decreased. Vaccines targeted for use in elderly populations, including influenza, S. pneumoniae, respiratory syncytial virus and parainfluenza virus, may particularly benefit from the use of adjuvants to increase the magnitude and duration of immune responses.
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3. Challenges of adjuvant research A number of factors, such as slow release of antigen (depot effect), more efficient delivery of antigen to draining lymph nodes, nonspecific activation of antigen-presenting cells or of B and/or T-lymphocytes, increased uptake of antigen by antigen-presenting cells, or increased recruitment of immune cells to the site where the antigen is present, can contribute to increased immune responses to immunization. Many of these factors involve the interaction of various immune system components and specific anatomical features, making them difficult to replicate in model systems in vitro. Furthermore, the overall effect of an adjuvant may result from the interaction of multiple factors. For example, an oil–water emulsion [6,7] might provide more effective delivery of antigens to draining lymph nodes, increased uptake by antigen-presenting cells, and activation of antigen-presenting cells or T-cells, all of which may contribute to its effect. Thus the ability to develop mechanism-based in vitro screens for novel adjuvant compounds is severely limited at present. Most adjuvants have been identified on the basis of testing in vivo. Species differences in responsiveness also may complicate the search for novel adjuvants. Not all adjuvants that are active in mice, for example, are equally active in nonhuman primates or in humans.
4. Examples of adjuvants that currently are under investigation
4.1. Aluminum Aluminum adjuvants are the only adjuvants currently licensed for clincal use [1]. In general, aluminum adjuvants are effective only if the antigen is incorporated in or on the particles of gel by coprecipitation with the aluminum, or adhered to the particles by ionic interactions. Since the interaction of proteins with the aluminum particles depends on the protein and the aluminum gel being of opposite charges, the pI of the protein species and of the aluminum are important determinants of the stability of the antigen/aluminum complex and therefore of the effectiveness of the vaccine [1]. Various types of aluminum gels, incorporating anions including phosphates, hydroxides, and oxides, continue to be investigated to refine and improve existing vaccines and to facilitate combination vaccines.
4.2. Emulsions Mixtures of a metabolizable oil such as squalane or squalene and an aqueous phase, including a detergent such as Tween and emulsifiers such as sorbitan monooleate (Span 85) to stabilize the oil droplets, are micro-fluidized to produce an oil-in-water emulsion with droplets in the 0.1–10 mm size range [7]. Some versions also incorporate a block copolymer, consisting of a hydrophobic core region flanked by hydrophilic regions [6]. Antigens can be incorporated in the emulsion during the emulsification process or added to the emulsion after it has been formed.
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4.3. Lipopolysaccharides (LPS) LPS are found in the outer membranes of gram-negative bacteria and have long been known to have adjuvant activities. These materials are thought to bind to LPS-binding proteins and activate B and T-cells and antigen-presenting cells. However, they also exhibit significant toxicity which appears to be mediated by IL-1 and TNF released from macrophages. Various chemical modifications of the lipid A portion of LPS, such as removing one phosphate to produce monophosphoryl lipid A, have been found to resuce the toxic effects of LPS while retaining adjuvant activity [5].
4.4. Polymers Various synthetic polymers have been developed for use as adjuvants. Some, such as poly-lactide-co-glycolide (PLGA) are dissolved in organic solvents, mixed with antigen, and the solvent then is removed to yield microspheres with the antigen encapsulated within them [8,9]. The antigen can be incorporated as an aqueous solution, in which case the antigen solution is emulsified in the organic solvent containing the polymer, producing a water-in-oil emulsion, or in particulate form as lyophilized powder in which case no emulsification is required [8]. The microspheres degrade in vivo, due to breakdown of the polymer, releasing the entrapped antigen. Polymers such as PLGA have little inherent immune stimulating activity and serve primarily as a reservoir of antigen [9]. Other polymers, such as block copolymers [10] and polyphosphazenes [11], encapsulate antigen in solution by ionic or hydrophobic interactions. The resulting complexes may provide a slow release of antigen but also appear to possess immune stimulating activity, perhaps by facilitating the uptake of antigen by antigen-presenting cells.
4.5. Saponins Extracts of the bark of the tree Quillaja saponaria were found to have adjuvant activity [4]. The extracts contain a number of components that have been purified and found to exhibit adjuvant activity individually. Saponins share a terpene core structure and vary in their glycosylation and in the addition of an acyl side chain linked to the carbohydrate moiety. They exhibit hemolytic activity and are thought to displace cholesterol from membranes. Their adjuvant activities include the delivery of antigens to the endogenous pathway of antigen processing for presentation by major histocompatibility complex (MHC) class I molecules, and they are thought to act by delivering antigen across the membrane directly to the cytosol of antigen-presenting cells [4]. In the presence of cholesterol, phosphatidyl ethanolamine, and phosphatidyl serine, saponins can assemble into polyhedral cage-like structures termed immune-stimulating complexes (ISCOMs) [12]. ISCOMs also are capable in some instances of delivering antigen to the cytosolic pathway of antigen processing [12].
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4.6. Liposomes Various techniques have been used to entrap antigens in vesicles formed from lipids. The constituents of these vesicles generally are charged diacyl lipids similar to those found in cell membranes, such as dioleyl phosphatidylcholine and phosphatidylethanolamine [13]. Non-polar lipids such as cholesterol also can be included. Liposomes can be made as single bimolecular leaflet structures in which a droplet of aqueous solution is entrapped within a membrane composed of two layers of lipids, or as multilamellar structures with many concentric layers of lipids. Integral membrane proteins insert into the liposome membrane, giving these antigens a conformation similar to their native state [13]. Hydrophilic proteins may be entrapped in the aqueous phase inside the vesicles. Like ISCOMs, liposomes can be used to stimulate MHC Class I-restricted CTL responses, presulably by delivering antigens to the cytosolic pathway of antigen processing [14]. In addition to antigens, other additives that are amphiphilic or lipid-soluble, such as MPL, also can be incorporated into liposomes to increase their adjuvant effect [13].
4.7. Cytokines Proteins produced by activated T-cells, B-cells, or APCs also can be used as adjuvants. Administration of cytokines with vaccines is intended to increase or redirect immune responses by providing intercellular signals that normally would be produced in an immune response, but at an earlier time, at higher concentrations, or in an atypical location. Examples of cytokines that have been used in animal models for this purpose are interleukin-12 (IL-12) [15] and granulocyte-macrophage colony-simulating factor (GM-CSF) [16]. IL-12 normally is produced by macrophages and induces the production of IFN-g by natural killer (NK) cells, driving the Th response toward a Th1 phenotype [15]. GM-CSF is normally produced by macrophages and NK cells and enhances the differentiation and antigen presentation functions of macrophages and dendritic cells. GM-CSF has been found to increase T-cell responses in a number of laboratory animal models [16]. Cytokines may be rapidly inactivated by inhibitors present in normal serum and tissue fluid and may act over narrow concentration gradients. Therefore multiple injections, delivery using a viral vector, or delivery in a slow-release system such as PLGA, are required to produce effects using cytokines as adjuvants in experimental systems. The delivery of cytokines for clinical use in vaccines will require further refinement.
5. Conclusions This review has summarized a few of many adjuvants that are under investigation for potential use in human vaccines [17]. Improved adjuvants may increase the effectiveness of current vaccines and also may be essential for novel vaccines against pathogens such as HIV, herpes simplex virus, and human papillomavirus. In the
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elderly, the use of novel adjuvants may increase response rates and extend the duration of protection offered by current vaccines for diseases such as influenza and pneumococcal infection.
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