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Adjuvants for vaccines, a quest
International Immunopharmacology 3 (2003) 1187 – 1193 www.elsevier.com/locate/intimp
Adjuvants for vaccines, a quest Francßoise Audibert 22 rue EMERIAU, 75015 Paris, France Received 5 November 2002; received in revised form 5 November 2002; accepted 5 November 2002
Abstract Efficient vaccines comprise a specific moiety: the structures presenting the protective antigenic determinants, and a nonspecific moiety: the adjuvant components. Dramatic advances have been reported concerning the specific moiety and new and highly purified immunogens have been defined and prepared. The use of vaccines is no longer restricted to the prevention of infections, they are now considered as therapeutic tools especially in cancer immunotherapy. In contrast, alum is still the only adjuvant suitable for clinical application. The success of the new avenues opened in vaccinology depends on the availability of appropriate immunomodulating preparations. For each given type of vaccine, the optimal profile of activity of the adjuvant moiety has to be defined, according to the response required to provide protection or cure. Thus, it is urgent to design and develop adjuvants active not only on the humoral responses but also on the cellular immune responses. This adjuvant function must have the capacity of turning on the innate responses, which play a decisive and instructive role in emanating the adaptive immune responses. These considerations encourage one to finalize immunomodulating procedures rather than to look only for new adjuvant compounds. Manipulations of dendritic cells (DCs), use of heat-shock proteins (HSPs) as carriers endowed of adjuvant activity or introduction of varying immunostimulating motives in genetic vaccines represent examples illustrating this new rationale. D 2003 Published by Elsevier Science B.V. Keywords: Adjuvants; Vaccines; Moiety
1. Introduction Immunoadjuvants belong to the history of basic and applied immunology. Ramon administered tapioca to horses to produce strong antitoxic sera, the effectiveness of Freund’s adjuvants allowed many scientific advances and for decades alum has played an obvious role in the clinical success of vaccination. Currently, great deals of changes are happening in the field of vaccines. A number of new protective antigens have been characterised and lots of well-defined and safe
E-mail address: [email protected] (F. Audibert).
preparations have become available. Moreover, vaccines are now considered not only for prevention but also as therapeutic agents. Some of these recent advances concern infectious diseases, but the most active field is cancerology and specific immunotherapy starts to represent a potential weapon against tumours. Thus, a dramatic evolution of the specific moiety of vaccines has been observed. In contrast, as far as the nonspecific moiety is concerned, alum is still the only adjuvant available in clinics. This situation is totally irrational since alum is far from fulfilling the latest requirements for an efficient immunopotentiation. Furthermore, the administration of aluminium-containing vaccines might be associated
1567-5769/03/$ - see front matter D 2003 Published by Elsevier Science B.V. doi:10.1016/S1567-5769(03)00011-0
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with the emergence of macrophagic myofasciitis (MMF), an inflammatory myopathy described recently [1,2]. Even if the relationship between alum and this disorder is ruled out by the epidemiological studies in progress, this potential hazard is another incentive to develop new immunostimulants. Indeed, the refinements brought to the chemical structure of many immunogens and the availability of new antigens able to elicit protective and, even more, therapeutic responses would require novel classes of adjuvants. For decades, in the concluding remarks of reports describing potential vaccinating preparations there is a leitmotiv likely to be ‘‘This very promising approach should have a future in clinics but would require the use of an appropriate adjuvant’’. In the following review, some prerequisites for performing relevant studies in the domain of immunomodulation will be recorded as well as examples of approaches under investigation at the present time.
2. An appropriate adjuvant Classically, a quantitative role is expected from an adjuvant to establish high and long-lasting immune responses. Actually, an appropriate adjuvant must also be able to promote the induction of the best biologically active responses by modulation of the immune system. The characteristics of the responses correlating with effectiveness are different according to the aims of each particular vaccine: prevention or therapy, against circulating or intracellular pathogens or against tumours. Its adjuvant moiety should provide an antigenic stimulus to all the components it lacks to induce suitable responses. Therefore, it is not possible to describe a universal and appropriate adjuvant. Moreover, the term adjuvant must apply not only to compounds endowed of immunomodulating properties but also to combinations of procedures which can be carried out by the required adjuvant function.
3. Preventive and curative immune responses The immune response comprises two branches leading either to humoral immunity or to cell-mediated immunity (CMI). Existing successful clinical vaccines, capable of mediating long-term protection, have been
obtained when the protection is mediated by humoral antibodies (diphtheria, tetanus, several viruses, etc.). Today, novel vaccines are needed [3] to fight a number of pathogens: parasites such as plasmodium, viruses such as the Human Immunodeficiency Virus (HIV), or the Hepatitis C Virus (HCV), bacteria such as Mycobacterium tuberculosis of which the resurgence may necessitate the replacement of BCG [4,5]. Moreover, therapeutic vaccines in the field of cancer are actively studied and suitable immunogens have been obtained [6 –8]. Although humoral immunity can also play a positive role in some cases, long-lived CMI is central to achieve efficacy against all these diseases. It has to be acknowledged that vaccines inducing CMI do not exist or are not uniformly effective [3]. Moreover, most potential adjuvants which have been described recently are mainly good stimulants of the humoral responses when given under conditions suitable for clinical use. The acquired cellular immune response comprises CD4+ and CD8+ T cells. CD4+ T cells are activated by antigens (generally proteins or peptides) after their processing by antigen-presenting cells (APC). These cells may be dendritic cells (DC), macrophages or B cells. CD4+ T cells recognize antigens processed through the exogenous pathway by APCs expressing major histocompatibility complex (MHC) class II molecules. This recognition leads to the differentiation of CD4+ T cells into the functional subsets T helper 1 (TH1) and TH2. The signature cytokines is interferon (IFN)-g for TH1 and interleukin (IL)-4 for TH2 cells. The TH2 cell subset mediates the production of specific antibodies by sensitized B-cells. TH1 cells mediate the killing of organisms responsible for a variety of intracellular infections through their production of IFN-g. The induction of a functional TH1 response is crucially dependent upon another cytokine, IL-12, which is produced by APC especially DCs. Thus, IL-12 can be considered as the cytokine inducer of TH1 cells while IFN-g is the effector cytokine mediating their efficacy. CD8+ T cells recognize antigens which are processed through the endogenous pathway and presented by APC cells expressing MHC class I molecules. CD8+ T cells mediate their effector function by producing IFN-g and tumour necrosis factor (TNF)-a. They can also kill their target cells through a direct cytolytic mechanism. These processes concern protective immunity against intracellular infections but
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the same mechanisms apply to tumor cells following sensitization of the immune cells to tumor-associated antigens [9]. Many other factors are involved in the modulation of these cascades of events, such as the nature of epitopes, the induction of co-stimulatory factors, etc. But, to delineate the potential points of impact for adjuvant activity, it is sufficient to point out that the obtaining of a full CMI requires the activation of the TH1 cell population and the sensitization of CD8+ T cells. B and T lymphocytes respond to antigens with high degree of specificity but they do not have the capacity to select the nature of the immune response. The choice is founded on the cytokine environment which is linked to the characteristics of the antigenic stimulus and to DCs activation [10]. When higher organisms are exposed to pathogenic microorganisms innate responses occur immediately both in terms of cell activation and inflammation. Pathogens are phagocytosed or endocytosed and subsequently destroyed or degraded, then the innate immune cells, macrophages or DCs are acutely activated resulting in a series of events [11]. The synthesis of co-stimulatory cell surface molecules and of MHC class I and II molecules are up-regulated, pro-inflammatory cytokines (TNF, IL1) and effector cytokines (IL-12, IFN g) are produced. The presentation to antigen-reactive T-cells of the antigenic peptides produced by pathogen degradation via the MHC class I or II presentation pathway is enhanced. Thus, innate immune cells represent not only a first line of defence towards infections but they also control the characteristic of the following adaptive response. It follows that to induce efficient immunity, vaccines must strongly promote appropriate innate responses. Highly purified antigenic preparations as well as tumour antigens do not carry a sufficient capacity of turning on the innate response and this deficiency represents an important need for adjuvant help.
4. Adjuvant interventions It is instructive to classify adjuvants according to the immunological events they evoke [12]. Certain vehicles of administration such as ISCOMs, Quil A, Al(OH)3, liposomes, etc. can facilitate APCs activities. Others allow a prolonged delivery of antigens because
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they help form a depot, this action is achieved by oil emulsions, Al(OH)3, polymers, microspheres, gels, etc. Some immunomodulators play an important role in activating the innate immune cells, interestingly they often represent compounds isolated from pathogenic microorganisms or mimicking certain of their structures. Unmethylated cytosine – phosphate – guanosine (CpG)-oligonucleotides motifs, LPS, and muramyl peptides belong to this category. Stress or stressrelated structures such as heat-shock proteins (HSPs) can also activate the innate responses. Finally, mediators of the immune system such as selected cytokines or co-stimulatory molecules have been also used with vaccines and act directly on T and B cells and on the maturation of APCs. This classification emphasizes the key role played by the first events of the immune responses to modulate their strength and their nature. It can help in defining the appropriate use of these tools in the strategies of adjuvantation currently submitted to experimental and clinical testing.
5. Manipulation of dendritic cells DCs have been described as ‘‘nature’s adjuvants’’ because of their unique capacity to turn on naive T cells [13]. They are involved in the initiation of both innate and adaptive immunity. DCs can achieve cross priming, a phenomenon which consists of the capacity to present exogenous antigens onto MHC class I molecules [14]. Thus, DCs are the most potent APCs and are clearly central to the regulation, maturation and maintenance of a cellular immune response to antigens needing CMI and particularly in the field of cancer. Several strategies are under experimentation to deliver tumor antigens into DCs [15,16]. Their mobilization in vivo has been tried with the help of GM-CSF or of costimulatory signals [17,18]. Most often, DCs are manipulated ex vivo to increase their tumoral antigen load. Monocytes are isolated from circulating blood, they are incubated with IL-4 to promote their differentiation into DCs. The cultured cells are then pulsed with tumor lysates, purified peptides [19], apoptotic tumors, RNA or DNA encoding known tumor antigens. DCs can be also genetically modified to express tumor antigens. Co-expression of known melanoma antigens with GM-CSF, IL-12 or IFN-a has been
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found promising in mouse models [18,20]. Viral vectors are very often used, and they have the advantage of stimulating DCs activation [21]. The manipulated cells are then reintroduced not only into the host with the hope that the antigens will be presented at the cell surface as peptide – MHC class II complexes eliciting CD4+ T cell responses but also onto MHC class I molecules thanks to cross priming. Thus, DCs can stimulate both T cells and B cells by a specialized processing of exogenous antigens. The use of DCs-based immunotherapy in clinical trials has not been highly successful. Nevertheless, some encouraging results support the need for future studies in the field. It will be important to increase the therapeutic potential in favoring T cell survival and specificity since clinical responsiveness will depend on the enhancement of T cell immunity [9,16].
6. HSPs as carriers and adjuvants Heat-shock proteins (HSPs) are among the most highly conserved molecules of the biosphere. They are found in eukaryotes, in prokaryotes and even in plants. Their functions are essential for life since they behave as molecular chaperones [22]. Under normal conditions, they help to achieve suitable protein folding and subunit assembly. In stress situations, HSPs synthesis is increased to protect cells, they prevent the aggregation of partially denatured proteins and initiate their refolding or their proteolytic degradation if the unfolding is irreversible. HSPs are very important in the control of protective immunity [23]. Thanks to their chaperone function they participate in the assembly of antibody molecules, they are involved in the stabilization of MHC class I and class II molecules and they can stimulate the synthesis of cytokines [24]. It has been even proposed to name one of them ‘‘chaperokine’’ [25]. HSP – peptides complexes may form inside the cells allowing the transport of peptides resulting from the proteolytic cleavage of endogenous proteins. The chaperoning allows the protection, the transport and the delivery of the peptides and the end of the process is their loading onto MHC class I molecules at the surface of the cells. Cell death leads to the release of biologically potent HSPs loaded with peptides. The more recent results indicate that necrotic cells are the
most able to induce the maturation of immunostimulatory DCs [26] in contrast with apoptotic cells [27]. Specialized receptors present on DCs allow the cross presentation of the HSP –peptide complexes [28,29]. Very interestingly, after processing HSP – peptide complexes are presented on MHC class I molecules. Cross presentation allows the activation of professional APCs to secrete cytokines and induce expression of antigen-presenting cells and co-stimulatory molecules on the dendritic cells. Extremely small quantities of peptides (nano or picograms) are sufficient for eliciting potent CD8+ T cells responses but only if the peptides are chaperoned by HSPs. Immunization with HSP – peptide complexes increases the number of antigen reactive T-cells and can elicit not only CTL but also antibodies [29,30]. Thus, HSPs represent the first adjuvants of mammalian origin. HSPs isolated from tumors have been shown to carry minute amounts of tumor peptides and their use is seriously considered in the domain of specific immunotherapy of tumors [31,32]. The choice of HSP –peptide complexes generated in vivo as sources of tumor antigens represents several advantages. Their choice circumvents the need to identify tumor-specific antigens [33]. Such peptides associated with tumorderived HSPs represent an aggregation of epitopes corresponding to numerous human lymphocyte antigen specificities. On the contrary, purified or synthetic peptides are restricted to specific molecules of the MHC [34]. These HSP –peptide complexes generated in vivo may reduce immunological escape variants by providing the entire antigenic repertoire of the tumor resulting in the generation of T effector cells directed against different tumor epitopes. On the contrary, immunization with a specific tumor-associated antigen may lead to outgrowth of tumor cells not expressing this particular antigen [22]. Early clinical trials using HSPs isolated from tumors (particularly gp 96) have been performed including patients with cancers of the stomach, pancreas, kidney, colon, melanoma and lymphoma [35,36]. The encouraging results obtained allowed the initiation of phase III clinical trials currently in progress [37,38]. HSPs produced in stress situations are endowed with carrier and adjuvant functions [39] and they are ‘‘sticky’’ structures binding small peptides strongly. These peptides not only can originate from the tissues from which HSPs are isolated but they can also be
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exogenous peptides and the complexes be reconstituted artificially in vitro. Viral and bacterial antigenic peptides have been associated with HSPs of various origins and very promising results have been obtained in animal models [40,41]. HSPs are also important in the field of infectious vaccines not only as potential carrier-adjuvants but also as pathogen-derived immunogens. Indeed, HSPs synthesis occurs in pathogens during the invading phase of infection and their induction is vital for many pathogens’ survival. HSPs may also be produced when intracellular pathogens are trapped into phagocytes, for example M. tuberculosis into the macrophages of the tubercle lesions [42]. Thus, HSPs represent major protective antigens in a wide spectrum of infectious agents and have been shown to induce strong biologically active humoral and cellular responses [42].
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the inclusion of genes coding for co-stimulatory molecules such as B7.1 or 2, CD40, etc. B7 signals are required to activate both CD4+ and CD8+ T cells and several models have demonstrated that their use leads to an increase in the protection from subsequent tumor challenge or the induction of CTL against viral antigens [52]. The use of HSPs may also have a future in genetic vaccines since it has been shown in mice that plasmids coding simultaneously for HSPs and different viral or bacterial antigens evoke biologically active immunity [53]. The relevance of these approaches for the next generations of genetic vaccines will have to be extensively assessed. In addition, it can be expected that the delivery of these adjuvanted DNA vaccines will require additional safety evaluation [54].
8. Concluding remarks 7. DNA vaccines and CMI DNA vaccines result in antigen synthesis in vivo following administration of plasmids coding for antigen structures. In various species including humans, this method of vaccination can induce CTL responses through direct expression of the antigenic structures within APCs [43], or by cross presentation of these antigens following their release by somatic cells [44]. DNA vaccines have shown their potential value in animal models of infectious diseases and of cancers. So far, this potential has not been confirmed in clinical trials since the protective epitopes did not induce uniformly an efficient level of immunity. Thus, several avenues are currently being explored to optimize DNA vaccines. One of the most promising is the introduction into the plasmids of stimulatory motifs, unmethylated CpG oligonucleotides [45,46]. These sequences activate the innate immunity through a specific receptor present on DCs, the Toll-like receptor 9 (TLR9) [47 – 49]. Other sequences, known as neutralizing motives, which can counteract stimulating CpG motifs, may be also found in DNA vaccines. Thus, the efficacy of a DNA vaccine might be improved through the removal of neutralizing motifs and addition of species-specific stimulatory CpG motifs [50]. Another way of modulating the immune response to DNA immunization may be the co-administration of biological adjuvants such as GM-CSF and IL-12 [51] or
For decades, basic research in Immunology has been focused mostly on the specific facets of the immune response. The mechanisms of nonspecific immunity were not considered and empiricism prevailed in the search for vaccine adjuvants. Progress in the understanding of the mechanisms of immunity has indicated clearly that the key points to modulate the characteristics of the immune response are its first steps. A strong innate response capable of instructing the adaptive responses through APCs especially DCs is essential. The presentation of antigens through the MHC class I or II pathways is controlled at this stage, leading, respectively, to the activation of CD8+ T cells or CD4+ T cells. In addition, the cytokine environment induced at the very beginning of the adaptive response dictates the ratio TH1 versus TH2. Recently, potential new preventive vaccines have been described and specific immunotherapy is foreseen as a therapeutic weapon against cancer. These new trends necessitate the design of adjuvants able to stimulate strongly the innate responses and to induce preferentially CMI. To fulfil these requirements, it is perhaps more fruitful to build appropriate strategies of adjuvantation rather than to design novel compounds. Several examples of current investigations illustrate this approach, e.g. the manipulation of DCs ex vivo, the utilisation of the unique properties of HSPs, and the engineering of genetic vaccines.
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The search for appropriate adjuvant strategies is based more and more on sound scientific grounds. It is most interesting to see that totally different avenues could lead to an efficient control of the nonspecific facets of the human immune system. Nevertheless, many experimental and clinical studies will have to be performed to derive full benefit of this knowledge.
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Adjuvants for vaccines, a quest Francßoise Audibert 22 rue EMERIAU, 75015 Paris, France Received 5 November 2002; received in revised form 5 November 2002; accepted 5 November 2002
Abstract Efficient vaccines comprise a specific moiety: the structures presenting the protective antigenic determinants, and a nonspecific moiety: the adjuvant components. Dramatic advances have been reported concerning the specific moiety and new and highly purified immunogens have been defined and prepared. The use of vaccines is no longer restricted to the prevention of infections, they are now considered as therapeutic tools especially in cancer immunotherapy. In contrast, alum is still the only adjuvant suitable for clinical application. The success of the new avenues opened in vaccinology depends on the availability of appropriate immunomodulating preparations. For each given type of vaccine, the optimal profile of activity of the adjuvant moiety has to be defined, according to the response required to provide protection or cure. Thus, it is urgent to design and develop adjuvants active not only on the humoral responses but also on the cellular immune responses. This adjuvant function must have the capacity of turning on the innate responses, which play a decisive and instructive role in emanating the adaptive immune responses. These considerations encourage one to finalize immunomodulating procedures rather than to look only for new adjuvant compounds. Manipulations of dendritic cells (DCs), use of heat-shock proteins (HSPs) as carriers endowed of adjuvant activity or introduction of varying immunostimulating motives in genetic vaccines represent examples illustrating this new rationale. D 2003 Published by Elsevier Science B.V. Keywords: Adjuvants; Vaccines; Moiety
1. Introduction Immunoadjuvants belong to the history of basic and applied immunology. Ramon administered tapioca to horses to produce strong antitoxic sera, the effectiveness of Freund’s adjuvants allowed many scientific advances and for decades alum has played an obvious role in the clinical success of vaccination. Currently, great deals of changes are happening in the field of vaccines. A number of new protective antigens have been characterised and lots of well-defined and safe
E-mail address: [email protected] (F. Audibert).
preparations have become available. Moreover, vaccines are now considered not only for prevention but also as therapeutic agents. Some of these recent advances concern infectious diseases, but the most active field is cancerology and specific immunotherapy starts to represent a potential weapon against tumours. Thus, a dramatic evolution of the specific moiety of vaccines has been observed. In contrast, as far as the nonspecific moiety is concerned, alum is still the only adjuvant available in clinics. This situation is totally irrational since alum is far from fulfilling the latest requirements for an efficient immunopotentiation. Furthermore, the administration of aluminium-containing vaccines might be associated
1567-5769/03/$ - see front matter D 2003 Published by Elsevier Science B.V. doi:10.1016/S1567-5769(03)00011-0
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with the emergence of macrophagic myofasciitis (MMF), an inflammatory myopathy described recently [1,2]. Even if the relationship between alum and this disorder is ruled out by the epidemiological studies in progress, this potential hazard is another incentive to develop new immunostimulants. Indeed, the refinements brought to the chemical structure of many immunogens and the availability of new antigens able to elicit protective and, even more, therapeutic responses would require novel classes of adjuvants. For decades, in the concluding remarks of reports describing potential vaccinating preparations there is a leitmotiv likely to be ‘‘This very promising approach should have a future in clinics but would require the use of an appropriate adjuvant’’. In the following review, some prerequisites for performing relevant studies in the domain of immunomodulation will be recorded as well as examples of approaches under investigation at the present time.
2. An appropriate adjuvant Classically, a quantitative role is expected from an adjuvant to establish high and long-lasting immune responses. Actually, an appropriate adjuvant must also be able to promote the induction of the best biologically active responses by modulation of the immune system. The characteristics of the responses correlating with effectiveness are different according to the aims of each particular vaccine: prevention or therapy, against circulating or intracellular pathogens or against tumours. Its adjuvant moiety should provide an antigenic stimulus to all the components it lacks to induce suitable responses. Therefore, it is not possible to describe a universal and appropriate adjuvant. Moreover, the term adjuvant must apply not only to compounds endowed of immunomodulating properties but also to combinations of procedures which can be carried out by the required adjuvant function.
3. Preventive and curative immune responses The immune response comprises two branches leading either to humoral immunity or to cell-mediated immunity (CMI). Existing successful clinical vaccines, capable of mediating long-term protection, have been
obtained when the protection is mediated by humoral antibodies (diphtheria, tetanus, several viruses, etc.). Today, novel vaccines are needed [3] to fight a number of pathogens: parasites such as plasmodium, viruses such as the Human Immunodeficiency Virus (HIV), or the Hepatitis C Virus (HCV), bacteria such as Mycobacterium tuberculosis of which the resurgence may necessitate the replacement of BCG [4,5]. Moreover, therapeutic vaccines in the field of cancer are actively studied and suitable immunogens have been obtained [6 –8]. Although humoral immunity can also play a positive role in some cases, long-lived CMI is central to achieve efficacy against all these diseases. It has to be acknowledged that vaccines inducing CMI do not exist or are not uniformly effective [3]. Moreover, most potential adjuvants which have been described recently are mainly good stimulants of the humoral responses when given under conditions suitable for clinical use. The acquired cellular immune response comprises CD4+ and CD8+ T cells. CD4+ T cells are activated by antigens (generally proteins or peptides) after their processing by antigen-presenting cells (APC). These cells may be dendritic cells (DC), macrophages or B cells. CD4+ T cells recognize antigens processed through the exogenous pathway by APCs expressing major histocompatibility complex (MHC) class II molecules. This recognition leads to the differentiation of CD4+ T cells into the functional subsets T helper 1 (TH1) and TH2. The signature cytokines is interferon (IFN)-g for TH1 and interleukin (IL)-4 for TH2 cells. The TH2 cell subset mediates the production of specific antibodies by sensitized B-cells. TH1 cells mediate the killing of organisms responsible for a variety of intracellular infections through their production of IFN-g. The induction of a functional TH1 response is crucially dependent upon another cytokine, IL-12, which is produced by APC especially DCs. Thus, IL-12 can be considered as the cytokine inducer of TH1 cells while IFN-g is the effector cytokine mediating their efficacy. CD8+ T cells recognize antigens which are processed through the endogenous pathway and presented by APC cells expressing MHC class I molecules. CD8+ T cells mediate their effector function by producing IFN-g and tumour necrosis factor (TNF)-a. They can also kill their target cells through a direct cytolytic mechanism. These processes concern protective immunity against intracellular infections but
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the same mechanisms apply to tumor cells following sensitization of the immune cells to tumor-associated antigens [9]. Many other factors are involved in the modulation of these cascades of events, such as the nature of epitopes, the induction of co-stimulatory factors, etc. But, to delineate the potential points of impact for adjuvant activity, it is sufficient to point out that the obtaining of a full CMI requires the activation of the TH1 cell population and the sensitization of CD8+ T cells. B and T lymphocytes respond to antigens with high degree of specificity but they do not have the capacity to select the nature of the immune response. The choice is founded on the cytokine environment which is linked to the characteristics of the antigenic stimulus and to DCs activation [10]. When higher organisms are exposed to pathogenic microorganisms innate responses occur immediately both in terms of cell activation and inflammation. Pathogens are phagocytosed or endocytosed and subsequently destroyed or degraded, then the innate immune cells, macrophages or DCs are acutely activated resulting in a series of events [11]. The synthesis of co-stimulatory cell surface molecules and of MHC class I and II molecules are up-regulated, pro-inflammatory cytokines (TNF, IL1) and effector cytokines (IL-12, IFN g) are produced. The presentation to antigen-reactive T-cells of the antigenic peptides produced by pathogen degradation via the MHC class I or II presentation pathway is enhanced. Thus, innate immune cells represent not only a first line of defence towards infections but they also control the characteristic of the following adaptive response. It follows that to induce efficient immunity, vaccines must strongly promote appropriate innate responses. Highly purified antigenic preparations as well as tumour antigens do not carry a sufficient capacity of turning on the innate response and this deficiency represents an important need for adjuvant help.
4. Adjuvant interventions It is instructive to classify adjuvants according to the immunological events they evoke [12]. Certain vehicles of administration such as ISCOMs, Quil A, Al(OH)3, liposomes, etc. can facilitate APCs activities. Others allow a prolonged delivery of antigens because
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they help form a depot, this action is achieved by oil emulsions, Al(OH)3, polymers, microspheres, gels, etc. Some immunomodulators play an important role in activating the innate immune cells, interestingly they often represent compounds isolated from pathogenic microorganisms or mimicking certain of their structures. Unmethylated cytosine – phosphate – guanosine (CpG)-oligonucleotides motifs, LPS, and muramyl peptides belong to this category. Stress or stressrelated structures such as heat-shock proteins (HSPs) can also activate the innate responses. Finally, mediators of the immune system such as selected cytokines or co-stimulatory molecules have been also used with vaccines and act directly on T and B cells and on the maturation of APCs. This classification emphasizes the key role played by the first events of the immune responses to modulate their strength and their nature. It can help in defining the appropriate use of these tools in the strategies of adjuvantation currently submitted to experimental and clinical testing.
5. Manipulation of dendritic cells DCs have been described as ‘‘nature’s adjuvants’’ because of their unique capacity to turn on naive T cells [13]. They are involved in the initiation of both innate and adaptive immunity. DCs can achieve cross priming, a phenomenon which consists of the capacity to present exogenous antigens onto MHC class I molecules [14]. Thus, DCs are the most potent APCs and are clearly central to the regulation, maturation and maintenance of a cellular immune response to antigens needing CMI and particularly in the field of cancer. Several strategies are under experimentation to deliver tumor antigens into DCs [15,16]. Their mobilization in vivo has been tried with the help of GM-CSF or of costimulatory signals [17,18]. Most often, DCs are manipulated ex vivo to increase their tumoral antigen load. Monocytes are isolated from circulating blood, they are incubated with IL-4 to promote their differentiation into DCs. The cultured cells are then pulsed with tumor lysates, purified peptides [19], apoptotic tumors, RNA or DNA encoding known tumor antigens. DCs can be also genetically modified to express tumor antigens. Co-expression of known melanoma antigens with GM-CSF, IL-12 or IFN-a has been
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found promising in mouse models [18,20]. Viral vectors are very often used, and they have the advantage of stimulating DCs activation [21]. The manipulated cells are then reintroduced not only into the host with the hope that the antigens will be presented at the cell surface as peptide – MHC class II complexes eliciting CD4+ T cell responses but also onto MHC class I molecules thanks to cross priming. Thus, DCs can stimulate both T cells and B cells by a specialized processing of exogenous antigens. The use of DCs-based immunotherapy in clinical trials has not been highly successful. Nevertheless, some encouraging results support the need for future studies in the field. It will be important to increase the therapeutic potential in favoring T cell survival and specificity since clinical responsiveness will depend on the enhancement of T cell immunity [9,16].
6. HSPs as carriers and adjuvants Heat-shock proteins (HSPs) are among the most highly conserved molecules of the biosphere. They are found in eukaryotes, in prokaryotes and even in plants. Their functions are essential for life since they behave as molecular chaperones [22]. Under normal conditions, they help to achieve suitable protein folding and subunit assembly. In stress situations, HSPs synthesis is increased to protect cells, they prevent the aggregation of partially denatured proteins and initiate their refolding or their proteolytic degradation if the unfolding is irreversible. HSPs are very important in the control of protective immunity [23]. Thanks to their chaperone function they participate in the assembly of antibody molecules, they are involved in the stabilization of MHC class I and class II molecules and they can stimulate the synthesis of cytokines [24]. It has been even proposed to name one of them ‘‘chaperokine’’ [25]. HSP – peptides complexes may form inside the cells allowing the transport of peptides resulting from the proteolytic cleavage of endogenous proteins. The chaperoning allows the protection, the transport and the delivery of the peptides and the end of the process is their loading onto MHC class I molecules at the surface of the cells. Cell death leads to the release of biologically potent HSPs loaded with peptides. The more recent results indicate that necrotic cells are the
most able to induce the maturation of immunostimulatory DCs [26] in contrast with apoptotic cells [27]. Specialized receptors present on DCs allow the cross presentation of the HSP –peptide complexes [28,29]. Very interestingly, after processing HSP – peptide complexes are presented on MHC class I molecules. Cross presentation allows the activation of professional APCs to secrete cytokines and induce expression of antigen-presenting cells and co-stimulatory molecules on the dendritic cells. Extremely small quantities of peptides (nano or picograms) are sufficient for eliciting potent CD8+ T cells responses but only if the peptides are chaperoned by HSPs. Immunization with HSP – peptide complexes increases the number of antigen reactive T-cells and can elicit not only CTL but also antibodies [29,30]. Thus, HSPs represent the first adjuvants of mammalian origin. HSPs isolated from tumors have been shown to carry minute amounts of tumor peptides and their use is seriously considered in the domain of specific immunotherapy of tumors [31,32]. The choice of HSP –peptide complexes generated in vivo as sources of tumor antigens represents several advantages. Their choice circumvents the need to identify tumor-specific antigens [33]. Such peptides associated with tumorderived HSPs represent an aggregation of epitopes corresponding to numerous human lymphocyte antigen specificities. On the contrary, purified or synthetic peptides are restricted to specific molecules of the MHC [34]. These HSP –peptide complexes generated in vivo may reduce immunological escape variants by providing the entire antigenic repertoire of the tumor resulting in the generation of T effector cells directed against different tumor epitopes. On the contrary, immunization with a specific tumor-associated antigen may lead to outgrowth of tumor cells not expressing this particular antigen [22]. Early clinical trials using HSPs isolated from tumors (particularly gp 96) have been performed including patients with cancers of the stomach, pancreas, kidney, colon, melanoma and lymphoma [35,36]. The encouraging results obtained allowed the initiation of phase III clinical trials currently in progress [37,38]. HSPs produced in stress situations are endowed with carrier and adjuvant functions [39] and they are ‘‘sticky’’ structures binding small peptides strongly. These peptides not only can originate from the tissues from which HSPs are isolated but they can also be
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exogenous peptides and the complexes be reconstituted artificially in vitro. Viral and bacterial antigenic peptides have been associated with HSPs of various origins and very promising results have been obtained in animal models [40,41]. HSPs are also important in the field of infectious vaccines not only as potential carrier-adjuvants but also as pathogen-derived immunogens. Indeed, HSPs synthesis occurs in pathogens during the invading phase of infection and their induction is vital for many pathogens’ survival. HSPs may also be produced when intracellular pathogens are trapped into phagocytes, for example M. tuberculosis into the macrophages of the tubercle lesions [42]. Thus, HSPs represent major protective antigens in a wide spectrum of infectious agents and have been shown to induce strong biologically active humoral and cellular responses [42].
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the inclusion of genes coding for co-stimulatory molecules such as B7.1 or 2, CD40, etc. B7 signals are required to activate both CD4+ and CD8+ T cells and several models have demonstrated that their use leads to an increase in the protection from subsequent tumor challenge or the induction of CTL against viral antigens [52]. The use of HSPs may also have a future in genetic vaccines since it has been shown in mice that plasmids coding simultaneously for HSPs and different viral or bacterial antigens evoke biologically active immunity [53]. The relevance of these approaches for the next generations of genetic vaccines will have to be extensively assessed. In addition, it can be expected that the delivery of these adjuvanted DNA vaccines will require additional safety evaluation [54].
8. Concluding remarks 7. DNA vaccines and CMI DNA vaccines result in antigen synthesis in vivo following administration of plasmids coding for antigen structures. In various species including humans, this method of vaccination can induce CTL responses through direct expression of the antigenic structures within APCs [43], or by cross presentation of these antigens following their release by somatic cells [44]. DNA vaccines have shown their potential value in animal models of infectious diseases and of cancers. So far, this potential has not been confirmed in clinical trials since the protective epitopes did not induce uniformly an efficient level of immunity. Thus, several avenues are currently being explored to optimize DNA vaccines. One of the most promising is the introduction into the plasmids of stimulatory motifs, unmethylated CpG oligonucleotides [45,46]. These sequences activate the innate immunity through a specific receptor present on DCs, the Toll-like receptor 9 (TLR9) [47 – 49]. Other sequences, known as neutralizing motives, which can counteract stimulating CpG motifs, may be also found in DNA vaccines. Thus, the efficacy of a DNA vaccine might be improved through the removal of neutralizing motifs and addition of species-specific stimulatory CpG motifs [50]. Another way of modulating the immune response to DNA immunization may be the co-administration of biological adjuvants such as GM-CSF and IL-12 [51] or
For decades, basic research in Immunology has been focused mostly on the specific facets of the immune response. The mechanisms of nonspecific immunity were not considered and empiricism prevailed in the search for vaccine adjuvants. Progress in the understanding of the mechanisms of immunity has indicated clearly that the key points to modulate the characteristics of the immune response are its first steps. A strong innate response capable of instructing the adaptive responses through APCs especially DCs is essential. The presentation of antigens through the MHC class I or II pathways is controlled at this stage, leading, respectively, to the activation of CD8+ T cells or CD4+ T cells. In addition, the cytokine environment induced at the very beginning of the adaptive response dictates the ratio TH1 versus TH2. Recently, potential new preventive vaccines have been described and specific immunotherapy is foreseen as a therapeutic weapon against cancer. These new trends necessitate the design of adjuvants able to stimulate strongly the innate responses and to induce preferentially CMI. To fulfil these requirements, it is perhaps more fruitful to build appropriate strategies of adjuvantation rather than to design novel compounds. Several examples of current investigations illustrate this approach, e.g. the manipulation of DCs ex vivo, the utilisation of the unique properties of HSPs, and the engineering of genetic vaccines.
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The search for appropriate adjuvant strategies is based more and more on sound scientific grounds. It is most interesting to see that totally different avenues could lead to an efficient control of the nonspecific facets of the human immune system. Nevertheless, many experimental and clinical studies will have to be performed to derive full benefit of this knowledge.
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