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The Lancet 1997; 350:1316-1319
DOI:10.1016/S0140-6736(97)03257-1
Host immunobiology and vaccine development
Prof Gustav JV NossalMD a
Summary
Interacting cells in immune responses
Afferent limb of immune response
Cell pathway to antibody formation and immunological memory
Influence of host age
Challenges for the vaccine developer
References
Summary
As the rules of immunoregulation become clearer, the design of vaccines and adjuvants is becoming more scientific. To understand these rules, the interactions between three kinds of cells need to be grasped. Antigen-presenting cells (APCs) initiate the immunoglobulin cascade. The most important of these are dendritic cells, which must first capture antigen, a process aided by particulate matter, the presence of natural or acquired antibodies, or the capacity to activate complement. Then T cells become activated through conjoint action of processed antigenic peptides and APC surface and secreted molecules. T cells mediate inflammation, develop cytotoxic capacity, and help in antibody formation. Whether cells of type 1 or type 2 predominate influences the direction of both cellular and humoral responses. B cells are then activated, leading to antibody formation and often to better antigen presentation. Both T and B cell memory, embedded in long-lived lymphocyte populations, aid heightened immune reactivity when the antigen is re-encountered. The best vaccines stimulate strong memory.
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In the golden age of immunology (1870?1910), the time of Pasteur, von Behring, Ehrlich, Koch, and Metchnikoff, the study of immune mechanisms was intimately linked to clinical microbiology and to practical problems in the prevention, diagnosis, and treatment of infectious diseases. As the science matured, it became apparent that immunology was about far more than just infection. Antibodies could be made to foreign cells and to chemical substances synthesised in the test tube, with a specificity that was amazing. This discovery raised the question of how such a vast diversity of antibodies could be formed by a single individual. Antibodies were found to be involved in allergy and some forms of autoimmune disease. A different kind of immune response, mediated by lymphocyte cells, was recognised to be at the root of the rejection of skin and organ grafts. Animals were found to have strong immune responses against some cancers. The diversity of lymphocytes, their origins in thymus or bone marrow, and the nature of their receptors for antigen were progressively revealed. The unique mechanisms for the creation of T and B lymphocyte repertoires through the somatic assembly, cell by cell, of variable region minigenes were uncovered. Great strides were made towards understanding self-recognition, and its limitations. The unexpected capacity of T cells to see short peptide fragments from the inside of viruses, not in isolation but always within a special groove on a MHC molecule, was gradually described in both cellular and molecular terms. The ready availability of lymphocyte cells and the capacity to grow them in vitro with preservation of most functions made them favourite tools for the study of cellular receptors, ligand interactions, signalling cascades, malignant transformation, cell-cycle control, and programmed cell death. Little wonder that in the second golden age of immunology (1955 to present), the academic aspects of the subject wandered far from medical microbiology. It was left to virologists and bacteriologists to pioneer new vaccines.
In the past 10?15 years, this indifference began to change. The emergence of new communicable diseases, the increase in antibiotic resistance, the lure of the possibility of total disease eradication, and a general cry for more relevance in research all played their part. The urgent calls for help from the World Health Organization to bring the fruits of immunology to developing countries touched many academics, as did availability of funding. Above all, success in understanding the cells and molecules of the immune system left as an open challenge the question of how antigens guide the response down one or more of the many diverse pathways of immune response. This new concern with immunoregulation fits naturally with the search for more effective vaccines. Whatever the reasons it is now possible to recognise the science of vaccinology, in which basic immunology is combined with several sister sciences, as a powerful new force. In the early years of the new millennium, this will usher in the third golden age of immunology, with a panoply of new vaccines and combinations to protect humanity not only against a much larger range of communicable diseases but also against the ravages of some cancers, autoimmune diseases, allergies, and other disorders. The former black box separating vaccine injection from resultant antibody or cell-mediated immunity will be demystified. Host immunobiology will provide the answers (panel).
Concepts of host immunobiology
?When a foreign antigen is introduced into the body, immunity is not the only possible response. Indeed sometimes the opposite result is obtained, namely immunological tolerance or an impairment of late immune response capacity to that specific antigen.1 Under some circumstances, the antigen may be completely ignored, having no discernible effect. Occasionally, one or more antigens included in a vaccine can engender an immunopathological result, leading to an increased susceptibility to the pathogen in question.
?The form of an immune response can vary. Antigens can provoke one or more antibodies to be formed from different classes of antibody; IgM, IgG, IgA, and IgE antibodies have strikingly different effects. Antigens can also provoke T-cell based or cell-mediated immunity, and different subsets of T cells do quite different tasks.
?The effective duration of an immune response can vary strikingly as can the memory of an encounter with antigen.
?While the chemical nature of the antigen has an influence, the most important variables bearing on the host response include the physical form of the antigen, its route of entry into the body, and the nature of any immune-strengthening or adjuvant substance included in the vaccine.
?Both B and T cells develop such that one cell carries only one specificity?ie, one immunoglobulin with its unique combining site or one kind of T-cell receptor. An antigen must thus find the cell bearing a receptor with which it can react.2 Such encounters happen most readily within the confines of lymphoid tissues, where millions of lymphocytes traffic constantly.
Interacting cells in immune responses
The three key interacting elements in immune responses are antigen-presenting cells (APCs), thymus-derived lymphocytes (T cells), and bone-marrow derived lymphocytes (B cells; figure 1).
Click to enlarge image
Figure 1. The three types of cells that collaborate in immune responses
APCs come in various forms. The most important are dendritic cells.3 These cells are specialised for the capture of antigens, for the processing of antigens into small fragments (usually peptides), and for the presentation of these fragments at the cell surface, in association with MHC molecules so that an appropriate T cell can recognise the peptide-MHC complex and be activated. Dendritic cells are present literally all over the body. In the skin, they are in the form of Langerhans cells. In most parenchymal tissues, immunohistology can readily identify them lying dispersed but relatively closely-spaced between the specialised cells of the tissue. In the brain, largely shielded from antigens by the blood-brain barrier, astrocytes can carry out antigen-presenting functions. In lymphatic tissues, dendritic cells are richly scattered in the zones where T cells circulate. Dendritic cells are mobile, being bone-marrow derived, circulating in reasonable numbers in the blood (from which they can be purified and cultured) and they also move in afferent lymph from the tissues to the lymph nodes, bringing their antigenic load to where there is the optimal chance for T-cell stimulation. Dendritic cells activate T cells not only by engaging and cross-linking the T-cell receptors, but also by using several costimulatory cell
surface-bound ligands. Of several cognate interactions, that between B7?1/B7?2 and CD28 is among the more important.4 Activated B cells and macrophages are other powerful APCs. Critical cells known as follicular dendritic cells lie in lymphoid follicles.5,6 These cells have the peculiar property of being able to retain antigen on their surface in an unprocessed form for many months (figure 2). The antigen is either of an immune complex and thus held by an Fc receptor, or has activated the alternative complement pathway and is then held via a C3 receptor on the follicular dendritic cell. The follicular-dendritic-cell-bound antigen is essential for the formation of germinal centres and memory B cells.
Click to enlarge image
Figure 2. Electronmicroscopic autoradiograph of antigen capture (black dots) on the surface of a process of a follicular dendritic cell
The next cell in the activation cascade is the T cell. These emerge from the thymus as CD4+ or CD8+ cells. The former are involved in a helper function to B cells, in cell-mediated immune responses in lymphokine secretion. The latter are specialised for cytotoxic killing of other cells, particularly virus-infected cells or, at least in experimental circumstances, tumour cells. There is some overlap in function, as CD8+ cells certainly secrete lymphokines and CD4+ cells can act as killers. The most important distinction is that CD4+ cells see antigenic peptides in association with MHC class II molecules whereas CD8+ cells react with peptide plus MHC class I. Usually, class II molecules bear peptides derived from material that the APC has pinocytosed and fragmented within its endosomes, whereas class I molecules attach to their groove peptides derived from within that particular cell?eg, self peptides or peptides derived from viral proteins being assembled by that cell.7 Thus the CD8+ cell can exercise a surveillance function against virus-infected cells, cytotoxically killing the cell before the virus has completed its life cycle, thereby impeding virus spread; and against cells with mutated (and therefore possibly defective) proteins?eg, tumour cells. The above description applies to standard T cells possessing an αβ heterodimeric T-cell receptor. There are other T cells, such as γδ T cells, CD1-restricted T cells and NK1-1+ T cells, the functions of which are just being discovered. CD4+ T cells, when activated, develop into T cells secreting a large variety of cytokines.8 However, as the immune response matures, there are many instances where either a T helper (Th)-1 response or a tH-2 response becomes dominant.9 The former involves secretion of interleukin (IL)-2, γ-interferon, and lymphotoxin, the latter IL-4, IL-5, IL-6, and IL-10. Whereas the distinction between Th-1 and Th-2 cells is not absolute at the single-cell level,10 the short-hand description is nevertheless useful as the Th-1 response leads to inflammatory phenomena and the Th-2 response to antibody formation, including IgG1 and IgE formation.11
B cells are responsible for antibody formation.12 The early IgM response of B cells may be triggered directly by antigen in a T-cell-independent manner, but most long-lasting immune responses, involving IgG, IgA, or IgE antibodies, need the help of activated T cells.13 The T cell stimulates the B cell not only by secreting cytokines which activate the B cell and aid its multiplication and differentiation, but also by means of cognate co-stimulatory interactions?eg, between the CD40 ligand molecule on the T cell and the CD40 molecule on the B cell.14 There is no convincing evidence of inherently different B-cell subsets emerging from the marrow but B cells can be stimulated to develop along very different lines.
Classically, lymphocyte physiology has been defined in terms of: primary lymphoid organs (the thymus and the bone marrow in mammals), where T and B cells are formed; and secondary lymphoid organs (lymph nodes, spleen, and lymphoid collections), where lymphocytes encounter the world of antigens. It is now important to subdivide the latter into lymph nodes and spleen on the one hand and mucosal lymphoid tissue (Peyer's patches, tonsils, adenoids, appendix, and all gut and bronchus-associated lymphoid tissue) on the other, as the two compartments follow distinctly different rules.15
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Afferent limb of immune response
The afferent limb of the immune response comprises all those processes whereby antigen reaches the sites where it can stimulate immune responses. The physical form of the antigen is of prime importance. Particulate antigens such as micro-organisms or virus-like assemblies (eg, the spontaneously formed hepatitis B surface antigen) readily go to APCs. Their uptake is facilitated by natural antibodies and other opsonins. Natural antibodies are antibodies present before the introduction of the antigen in question, which arise through cross reactivities, particularly between carbohydrate antigens. Furthermore, particles with closely spaced antigen epitopes may activate the alternate complement pathway. As dendritic cells and macrophages
display C3 receptors, this aids removal. Finally, APCs possess various scavenger receptors that recognise common microbial surface components. A recent example of just how important facilitation of APC uptake can be is the demonstration that covalent linkage of C3d to a protein antigen can enhance its capacity to induce IgG1 antibody formation 10 000-fold, raising the possibility of using such procedures as a form of natural adjuvant.16 The route of entry is important. Intradermal or subcutaneous administration of antigen facilitates uptake by dendritic cells, and thus immune activation. Intravenous injection of soluble, highly deaggregated antigen can lead to tolerance in both T and B cells.17
Entry via a mucosal surface (eg, oral feeding or intranasal administration) can lead to uptake by specialised intramucosal cells known as M cells and a subsequent immune response of IgA and IgE antibody formation. An exciting finding is that the mucosal route can lead to a particular type of T-cell activation which involves secretion of tumour growth factor-β and other immunosuppressive cytokines, as a result of which standard Th-1-type responses are damped down.18 This so-called oral tolerance is actually a form of immune deviation, in which an active immune response suppresses other, and in the case of autoimmunity, more dangerous immune responses.
The nature of the adjuvant given, influences the direction of an immune response. Unfortunately, our understanding of this is only at an empirical level. The commonly used technique of adsorbing antigens to aluminium salts (alum precipitation) leads to a predominantly Th-2-type T-cell response and thus to antibody formation. Adjuvants, such as polyanionic block co-polymers of lipid A, can promote a strong Th-1-type response. It is probable that a dominant determinant is the nature of the cytokine first produced by a particular antigen-adjuvant combination. There is a tendency for T-cell responses to get locked in to a certain direction. There is a powerful positive feedback loop whereby the presence of IL-4 leads to the formation of more IL-4 secreting cells. At the same time, there is a cross-inhibition. IL-4 impedes the formation of interferon-γ and other Th-1-type cytokines.19 In contrast, IL-12 promotes Th-1 type T cell development and inhibits Th-2-type cytokine production.20 Whether it will be possible to use interleukins directly as adjuvants, thus guiding the immune response in particular directions, remains to be seen because these products are expensive and by no means lacking in toxicity. Some substances act as strong mucosal adjuvants. For example, the non-toxic B subunit of cholera toxin can be co-administered with oral antigens to give a major lift in mucosal immunity.21 This forms the principle behind one highly effective new cholera vaccine. Moreover, when the B subunit is covalently bound to a protein designed to act as an oral toleragen, the requisite effect is obtained with vastly less antigen.
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Cell pathway to antibody formation and immunological memory
The cellular events after the injection of an antigen adsorbed to alum are now well understood.22 Antigen is taken up by dendritic cells, many of which move in afferent lymph to the draining lymph nodes. In the T-cell zones of lymph nodes (or spleen, if the antigen reaches the spleen) the dendritic cells will stimulate CD4+ T cells. These in turn will activate appropriate B cells. For such T-cell help to B cells, it is important that the T-cell stimulatory part of the antigen, the epitope, is molecularly associated with the relevant B-cell epitope. It is this principle which makes conjugate vaccines (eg, against Haemophilus influenzae type B [Hib]) so effective. The stimulated B cell then takes one of two paths. It can embark directly on antibody formation, which involves sequential divisions and the emergence of immunohistologically identifiable foci of plasmablasts and plasma cells. These express unmutated germ-line antibody genes and are usually of relatively low affinity.23 Alternatively, the activated B cell can move toward a lymphoid follicle and into the vicinity of a follicular-dendritic-cell-bound antigen. There, extensive localised B-cell division takes place with the creation of a characteristic histological structure known as a germinal centre (figure 3). Within this specialised micro-environment, extensive mutation of the variable portion of the immunoglobulin light-chain and heavy-chain genes occurs.24 An efficient process of positive selection, dependent on follicular-dendritic-cell-bound antigen, ensures the selective survival of those mutants with a raised affinity for antigen. Other mutant B cells die locally by apoptosis and are quickly engulfed by macrophages. The process is iterative, so a B cell can easily end up with 50 mutations in its V, or variable region, genes. High affinity B cells leave the germinal centre and generate both high-affinity antibody on restimulation.
Click to enlarge image
Figure 3. A typical germinal centreNote that follicular-dendritic-cell-bound antigen has been pushed into a crescent cap at one pole of germinal centre.
Immunological memory, which is so important to a successful vaccine, depends on many factors. Long-term persistence of antigen on follicular dendritic cells in lymphoid follicles is clearly one such factor.25 A subset of antibody-forming plasma cells is very long-lived, and tiny amounts of antibody can be protective if of sufficiently high affinity. The primary immune response generates both T and B cells of the requisite specificity in considerable numbers, and these memory cells are long-lived. A second entry of the antigen thus encounters many more lymphocytes of the right specificity. Furthermore, these memory T and B cells may have a lowered threshold for activation. Cross-reactions are common in immunology, and it is possible that memory cells are stimulated by cross-reacting environmental antigens. A clinically inapparent re-encounter with the relevant pathogen may also reinforce memory. This gives rise to the very practical concern that, as herd immunity lowers the circulation of particular pathogens in the community, such reinforcement will not happen, stressing the need for booster doses.
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Influence of host age
The immunobiology of host responses is influenced by age, with problems at both ends of the spectrum. The newborn infant is relatively immature immunologically, yet possesses sufficient T and B cells to make immunisation against some diseases worthwhile. Both BCG and hepatitis B (first dose) are frequently given at birth. Infant immunisation is clearly a compromise between suboptimal responses and the need to protect the very young. 2 months of age is judged the right time for a first dose of diphtheria-tetanus-pertussis, Hib, and poliomyelitis vaccines. Passive antibodies from the mother can hinder the uptake of some live attenuated vaccines; measles vaccine is not given before age 9 months even in developing countries, and measles-mumps-rubella not before age 1 year. The older form of polysaccharide Hib vaccine or of presently available polyvalent pneumococcal vaccine are ineffective before 2 years.
In the aging individual, it is clear that no new export of T cells from the thymus can take place, as the thymic cortex has been largely replaced by fatty tissue. Moreover, the export of new B cells from the bone marrow is also sharply reduced. Nevertheless, old people usually have sufficient memory-type T and B cells to make immunisation worthwhile despite responses which are quantitatively inferior to those of younger adults. There is a need to develop a consensus as to what booster vaccination is desirable in old age. For the moment, a reasonable case can be made for immunisation against influenza, pneumococci, and herpes zoster.
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Challenges for the vaccine developer
The vaccine developer faces some difficult challenges. On one hand, conventional vaccines are coming through the clinical research pipeline very quickly. These could transform the situation with respect to many diarrhoeal, acute respiratory, sexually-transmitted, and parasitic diseases. If vaccination schedules are not to become unduly burdensome, new combination vaccines will have to be devised. On the other hand, basic immunology is providing some radical new solutions. These include powerful new adjuvants not yet fully tested in humans; recombinant vaccines in which harmless micro-organisms act as Trojan horses for genes coding for important antigens;26 and novel micro-encapsulation and other one-shot techniques. Perhaps most excitingly, plasmids have been engineered to contain genes for antigens behind strong promoters. Such nucleic acid vaccines can be injected intradermally or intramuscularly. The genes are expressed, and proteins thus made provoke both T and B cell responses.27 It is imperative that preclinical and clinical studies on these unconventional approaches proceed in parallel with standard vaccine development so that the use of possibly superior agents is not delayed. Once the research and development process has run its course, there remains the ineffable challenge of how to pay for these powerful new tools in developing countries in order to protect all the world's children.
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<!--start tail=--> References
1. Billingham RE, Brent L, Medawar PB. Actively acquired tolerance of foreign cells. Nature 1953; 172: 603-606. CrossRef
2. Burnet FM. A modification of Jerne's theory of antibody production using the concept of clonal selection. Austral J Sci 1957; 20: 67-69.
3. Sprent J, Webb SR. Function and specificity of T cell subsets in the mouse. Adv Immunol 1987; 41: 39-133. MEDLINE
4. Hathcock KS, Laszlo G, Dickler HB, Bradsh?w J, Linsley P, Hodes RJ. Identification of an alternative CTLA-4 ligand costimulatory for T cell activation. Science 1993; 262: 905-907. MEDLINE
5. Nossal GJV, Abbot A, Mitchell J, Lummus Z. Antigens in immunity XV: ultrastructural features of antigen capture in primary and secondary lymphoid follicles. J Exp Med 1968; 127: 277-290. MEDLINE | CrossRef
6. Szakal AK, Kosco MH, Tew JG. Microanatomy of lymphoid tissue during humoral immune responses: structure: function relationships. Annu Rev Immunol 1989; 7: 91-109. MEDLINE
7. York IA, Rock KL. Antigen processing and presentation by the Class I major histocompatibility complex. Annu Rev Immunol 1996; 14: 369-396. MEDLINE | CrossRef
8. Kelso A, Troutt AB, Maraskovsky E, et al. Heterogeneity in lymphokine profiles of CD4+ and CD8+ T cells and clones activated in vivo and in vitro. Immunol Rev 1991; 123: 85-113. MEDLINE
9. Mosmann TR, Coffman RL. Th1 and Th2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol 1989; 7: 145-173. MEDLINE
10. Kelso A. Th1 and Th2 subsets: paradigms lost?. Immunol Today 1995; 16: 374-379. MEDLINE | CrossRef
11. Finkelman FD, Holmes J, Katona IM, et al. Lymphokine control of in vivo immunoglobulin isotype selection. Annu Rev Immunol 1990; 8: 303-333. MEDLINE
12. Nossal GJV, Cunningham A, Mitchell GF, Miller JFAP. Cell to cell interaction in the immune response. III. Chromosomal marker analysis of single antibody-forming cells in reconstituted irradiated or thymectomized mice. J Exp Med 1968; 128: 839-853. MEDLINE | CrossRef
13. Miller JFAP. Lymphocyte interactions in immune responses. Int Rev Cytol 1972; 33: 77-130. MEDLINE
14. Clark EA, Ledbetter JA. How B and T cells talk to each other. Nature 1994; 367: 425-428. MEDLINE | CrossRef
15. Neutra MR, Pringault E, Krachenbuhl J-P. Antigen sampling across epithelial barriers and induction of mucosal immune responses. Annu Rev Immunol 1996; 14: 275-300. MEDLINE | CrossRef
16. Dempsey PW, Allison MED, Akkarajn S, Goodnow CC, Fearon DT. C3d of complement as a molecular adjuvant bridging innate and acquired immunity. Science 1996; 271: 348-350. MEDLINE
17. Chiller JM, Habicht GS, Weigle WO. Cellular sites of immunologic unresponsiveness. Proc Natl Acad Sci USA 1970; 65: 551-556. MEDLINE | CrossRef
18. Weiner HL, Friedman A, Miller A, et al. Oral tolerance: immunologic mechanisms of animal and human organ-specific autoimmune diseases by oral administration of autoantigens. Annu Rev Immunol 1994; 12: 809-837. MEDLINE
19. Gross A, Ben-Sasson SS, Paul WE. Anti-IL4 diminishes in vivo priming for antigen-specific IL4 production by T cells. J Immunol 1993; 150: 2112-2120. MEDLINE
20. Heinzel FP, Schoenhaut DS, Rerko RM, Rosser LE, Cately MK. Recombinant interleukin 12 cures mice infected with Leishmania major. J Exp Med 1993; 177: 1505-1509. MEDLINE | CrossRef
21. McKenzie SJ, Halsey JF. Cholera toxin B subunit as a carrier protein to stimulate a mucosal immune response. J Immunol 1984; 133: 1818. MEDLINE
22. Jacob J, Kassir R, Kelso G. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl) acetyl. J Exp Med 1991; 173: 1165-1175. MEDLINE | CrossRef
23. Kelsoe G. The germinal center reaction. Immunol Today 1995; 16: 324-326. MEDLINE | CrossRef
24. Berck C, Berger A, Apel M. Maturation of the immune response in germinal centers. Cell 1991; 67: 1121-1129. MEDLINE | CrossRef
25. Nossal GJV, Ada GL. In: Antigens, lymphoid cells and the immune response. New York: Academic Press, 1971: 324.
26. Moss B, Flexner C. Vaccinia virus expression vectors. Annu Rev Immunol 1987; 5: 305-324. MEDLINE
27. Donnelly JJ, Ulmer JB, Liu MA. Immunization with DNA. J Immunol Meth 1994; 176: 145-152. MEDLINE | CrossRef
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DOI:10.1016/S0140-6736(97)03257-1
Host immunobiology and vaccine development
Prof Gustav JV NossalMD a
Summary
Interacting cells in immune responses
Afferent limb of immune response
Cell pathway to antibody formation and immunological memory
Influence of host age
Challenges for the vaccine developer
References
Summary
As the rules of immunoregulation become clearer, the design of vaccines and adjuvants is becoming more scientific. To understand these rules, the interactions between three kinds of cells need to be grasped. Antigen-presenting cells (APCs) initiate the immunoglobulin cascade. The most important of these are dendritic cells, which must first capture antigen, a process aided by particulate matter, the presence of natural or acquired antibodies, or the capacity to activate complement. Then T cells become activated through conjoint action of processed antigenic peptides and APC surface and secreted molecules. T cells mediate inflammation, develop cytotoxic capacity, and help in antibody formation. Whether cells of type 1 or type 2 predominate influences the direction of both cellular and humoral responses. B cells are then activated, leading to antibody formation and often to better antigen presentation. Both T and B cell memory, embedded in long-lived lymphocyte populations, aid heightened immune reactivity when the antigen is re-encountered. The best vaccines stimulate strong memory.
Back to top
In the golden age of immunology (1870?1910), the time of Pasteur, von Behring, Ehrlich, Koch, and Metchnikoff, the study of immune mechanisms was intimately linked to clinical microbiology and to practical problems in the prevention, diagnosis, and treatment of infectious diseases. As the science matured, it became apparent that immunology was about far more than just infection. Antibodies could be made to foreign cells and to chemical substances synthesised in the test tube, with a specificity that was amazing. This discovery raised the question of how such a vast diversity of antibodies could be formed by a single individual. Antibodies were found to be involved in allergy and some forms of autoimmune disease. A different kind of immune response, mediated by lymphocyte cells, was recognised to be at the root of the rejection of skin and organ grafts. Animals were found to have strong immune responses against some cancers. The diversity of lymphocytes, their origins in thymus or bone marrow, and the nature of their receptors for antigen were progressively revealed. The unique mechanisms for the creation of T and B lymphocyte repertoires through the somatic assembly, cell by cell, of variable region minigenes were uncovered. Great strides were made towards understanding self-recognition, and its limitations. The unexpected capacity of T cells to see short peptide fragments from the inside of viruses, not in isolation but always within a special groove on a MHC molecule, was gradually described in both cellular and molecular terms. The ready availability of lymphocyte cells and the capacity to grow them in vitro with preservation of most functions made them favourite tools for the study of cellular receptors, ligand interactions, signalling cascades, malignant transformation, cell-cycle control, and programmed cell death. Little wonder that in the second golden age of immunology (1955 to present), the academic aspects of the subject wandered far from medical microbiology. It was left to virologists and bacteriologists to pioneer new vaccines.
In the past 10?15 years, this indifference began to change. The emergence of new communicable diseases, the increase in antibiotic resistance, the lure of the possibility of total disease eradication, and a general cry for more relevance in research all played their part. The urgent calls for help from the World Health Organization to bring the fruits of immunology to developing countries touched many academics, as did availability of funding. Above all, success in understanding the cells and molecules of the immune system left as an open challenge the question of how antigens guide the response down one or more of the many diverse pathways of immune response. This new concern with immunoregulation fits naturally with the search for more effective vaccines. Whatever the reasons it is now possible to recognise the science of vaccinology, in which basic immunology is combined with several sister sciences, as a powerful new force. In the early years of the new millennium, this will usher in the third golden age of immunology, with a panoply of new vaccines and combinations to protect humanity not only against a much larger range of communicable diseases but also against the ravages of some cancers, autoimmune diseases, allergies, and other disorders. The former black box separating vaccine injection from resultant antibody or cell-mediated immunity will be demystified. Host immunobiology will provide the answers (panel).
Concepts of host immunobiology
?When a foreign antigen is introduced into the body, immunity is not the only possible response. Indeed sometimes the opposite result is obtained, namely immunological tolerance or an impairment of late immune response capacity to that specific antigen.1 Under some circumstances, the antigen may be completely ignored, having no discernible effect. Occasionally, one or more antigens included in a vaccine can engender an immunopathological result, leading to an increased susceptibility to the pathogen in question.
?The form of an immune response can vary. Antigens can provoke one or more antibodies to be formed from different classes of antibody; IgM, IgG, IgA, and IgE antibodies have strikingly different effects. Antigens can also provoke T-cell based or cell-mediated immunity, and different subsets of T cells do quite different tasks.
?The effective duration of an immune response can vary strikingly as can the memory of an encounter with antigen.
?While the chemical nature of the antigen has an influence, the most important variables bearing on the host response include the physical form of the antigen, its route of entry into the body, and the nature of any immune-strengthening or adjuvant substance included in the vaccine.
?Both B and T cells develop such that one cell carries only one specificity?ie, one immunoglobulin with its unique combining site or one kind of T-cell receptor. An antigen must thus find the cell bearing a receptor with which it can react.2 Such encounters happen most readily within the confines of lymphoid tissues, where millions of lymphocytes traffic constantly.
Interacting cells in immune responses
The three key interacting elements in immune responses are antigen-presenting cells (APCs), thymus-derived lymphocytes (T cells), and bone-marrow derived lymphocytes (B cells; figure 1).
Click to enlarge image
Figure 1. The three types of cells that collaborate in immune responses
APCs come in various forms. The most important are dendritic cells.3 These cells are specialised for the capture of antigens, for the processing of antigens into small fragments (usually peptides), and for the presentation of these fragments at the cell surface, in association with MHC molecules so that an appropriate T cell can recognise the peptide-MHC complex and be activated. Dendritic cells are present literally all over the body. In the skin, they are in the form of Langerhans cells. In most parenchymal tissues, immunohistology can readily identify them lying dispersed but relatively closely-spaced between the specialised cells of the tissue. In the brain, largely shielded from antigens by the blood-brain barrier, astrocytes can carry out antigen-presenting functions. In lymphatic tissues, dendritic cells are richly scattered in the zones where T cells circulate. Dendritic cells are mobile, being bone-marrow derived, circulating in reasonable numbers in the blood (from which they can be purified and cultured) and they also move in afferent lymph from the tissues to the lymph nodes, bringing their antigenic load to where there is the optimal chance for T-cell stimulation. Dendritic cells activate T cells not only by engaging and cross-linking the T-cell receptors, but also by using several costimulatory cell
surface-bound ligands. Of several cognate interactions, that between B7?1/B7?2 and CD28 is among the more important.4 Activated B cells and macrophages are other powerful APCs. Critical cells known as follicular dendritic cells lie in lymphoid follicles.5,6 These cells have the peculiar property of being able to retain antigen on their surface in an unprocessed form for many months (figure 2). The antigen is either of an immune complex and thus held by an Fc receptor, or has activated the alternative complement pathway and is then held via a C3 receptor on the follicular dendritic cell. The follicular-dendritic-cell-bound antigen is essential for the formation of germinal centres and memory B cells.
Click to enlarge image
Figure 2. Electronmicroscopic autoradiograph of antigen capture (black dots) on the surface of a process of a follicular dendritic cell
The next cell in the activation cascade is the T cell. These emerge from the thymus as CD4+ or CD8+ cells. The former are involved in a helper function to B cells, in cell-mediated immune responses in lymphokine secretion. The latter are specialised for cytotoxic killing of other cells, particularly virus-infected cells or, at least in experimental circumstances, tumour cells. There is some overlap in function, as CD8+ cells certainly secrete lymphokines and CD4+ cells can act as killers. The most important distinction is that CD4+ cells see antigenic peptides in association with MHC class II molecules whereas CD8+ cells react with peptide plus MHC class I. Usually, class II molecules bear peptides derived from material that the APC has pinocytosed and fragmented within its endosomes, whereas class I molecules attach to their groove peptides derived from within that particular cell?eg, self peptides or peptides derived from viral proteins being assembled by that cell.7 Thus the CD8+ cell can exercise a surveillance function against virus-infected cells, cytotoxically killing the cell before the virus has completed its life cycle, thereby impeding virus spread; and against cells with mutated (and therefore possibly defective) proteins?eg, tumour cells. The above description applies to standard T cells possessing an αβ heterodimeric T-cell receptor. There are other T cells, such as γδ T cells, CD1-restricted T cells and NK1-1+ T cells, the functions of which are just being discovered. CD4+ T cells, when activated, develop into T cells secreting a large variety of cytokines.8 However, as the immune response matures, there are many instances where either a T helper (Th)-1 response or a tH-2 response becomes dominant.9 The former involves secretion of interleukin (IL)-2, γ-interferon, and lymphotoxin, the latter IL-4, IL-5, IL-6, and IL-10. Whereas the distinction between Th-1 and Th-2 cells is not absolute at the single-cell level,10 the short-hand description is nevertheless useful as the Th-1 response leads to inflammatory phenomena and the Th-2 response to antibody formation, including IgG1 and IgE formation.11
B cells are responsible for antibody formation.12 The early IgM response of B cells may be triggered directly by antigen in a T-cell-independent manner, but most long-lasting immune responses, involving IgG, IgA, or IgE antibodies, need the help of activated T cells.13 The T cell stimulates the B cell not only by secreting cytokines which activate the B cell and aid its multiplication and differentiation, but also by means of cognate co-stimulatory interactions?eg, between the CD40 ligand molecule on the T cell and the CD40 molecule on the B cell.14 There is no convincing evidence of inherently different B-cell subsets emerging from the marrow but B cells can be stimulated to develop along very different lines.
Classically, lymphocyte physiology has been defined in terms of: primary lymphoid organs (the thymus and the bone marrow in mammals), where T and B cells are formed; and secondary lymphoid organs (lymph nodes, spleen, and lymphoid collections), where lymphocytes encounter the world of antigens. It is now important to subdivide the latter into lymph nodes and spleen on the one hand and mucosal lymphoid tissue (Peyer's patches, tonsils, adenoids, appendix, and all gut and bronchus-associated lymphoid tissue) on the other, as the two compartments follow distinctly different rules.15
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Afferent limb of immune response
The afferent limb of the immune response comprises all those processes whereby antigen reaches the sites where it can stimulate immune responses. The physical form of the antigen is of prime importance. Particulate antigens such as micro-organisms or virus-like assemblies (eg, the spontaneously formed hepatitis B surface antigen) readily go to APCs. Their uptake is facilitated by natural antibodies and other opsonins. Natural antibodies are antibodies present before the introduction of the antigen in question, which arise through cross reactivities, particularly between carbohydrate antigens. Furthermore, particles with closely spaced antigen epitopes may activate the alternate complement pathway. As dendritic cells and macrophages
display C3 receptors, this aids removal. Finally, APCs possess various scavenger receptors that recognise common microbial surface components. A recent example of just how important facilitation of APC uptake can be is the demonstration that covalent linkage of C3d to a protein antigen can enhance its capacity to induce IgG1 antibody formation 10 000-fold, raising the possibility of using such procedures as a form of natural adjuvant.16 The route of entry is important. Intradermal or subcutaneous administration of antigen facilitates uptake by dendritic cells, and thus immune activation. Intravenous injection of soluble, highly deaggregated antigen can lead to tolerance in both T and B cells.17
Entry via a mucosal surface (eg, oral feeding or intranasal administration) can lead to uptake by specialised intramucosal cells known as M cells and a subsequent immune response of IgA and IgE antibody formation. An exciting finding is that the mucosal route can lead to a particular type of T-cell activation which involves secretion of tumour growth factor-β and other immunosuppressive cytokines, as a result of which standard Th-1-type responses are damped down.18 This so-called oral tolerance is actually a form of immune deviation, in which an active immune response suppresses other, and in the case of autoimmunity, more dangerous immune responses.
The nature of the adjuvant given, influences the direction of an immune response. Unfortunately, our understanding of this is only at an empirical level. The commonly used technique of adsorbing antigens to aluminium salts (alum precipitation) leads to a predominantly Th-2-type T-cell response and thus to antibody formation. Adjuvants, such as polyanionic block co-polymers of lipid A, can promote a strong Th-1-type response. It is probable that a dominant determinant is the nature of the cytokine first produced by a particular antigen-adjuvant combination. There is a tendency for T-cell responses to get locked in to a certain direction. There is a powerful positive feedback loop whereby the presence of IL-4 leads to the formation of more IL-4 secreting cells. At the same time, there is a cross-inhibition. IL-4 impedes the formation of interferon-γ and other Th-1-type cytokines.19 In contrast, IL-12 promotes Th-1 type T cell development and inhibits Th-2-type cytokine production.20 Whether it will be possible to use interleukins directly as adjuvants, thus guiding the immune response in particular directions, remains to be seen because these products are expensive and by no means lacking in toxicity. Some substances act as strong mucosal adjuvants. For example, the non-toxic B subunit of cholera toxin can be co-administered with oral antigens to give a major lift in mucosal immunity.21 This forms the principle behind one highly effective new cholera vaccine. Moreover, when the B subunit is covalently bound to a protein designed to act as an oral toleragen, the requisite effect is obtained with vastly less antigen.
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Cell pathway to antibody formation and immunological memory
The cellular events after the injection of an antigen adsorbed to alum are now well understood.22 Antigen is taken up by dendritic cells, many of which move in afferent lymph to the draining lymph nodes. In the T-cell zones of lymph nodes (or spleen, if the antigen reaches the spleen) the dendritic cells will stimulate CD4+ T cells. These in turn will activate appropriate B cells. For such T-cell help to B cells, it is important that the T-cell stimulatory part of the antigen, the epitope, is molecularly associated with the relevant B-cell epitope. It is this principle which makes conjugate vaccines (eg, against Haemophilus influenzae type B [Hib]) so effective. The stimulated B cell then takes one of two paths. It can embark directly on antibody formation, which involves sequential divisions and the emergence of immunohistologically identifiable foci of plasmablasts and plasma cells. These express unmutated germ-line antibody genes and are usually of relatively low affinity.23 Alternatively, the activated B cell can move toward a lymphoid follicle and into the vicinity of a follicular-dendritic-cell-bound antigen. There, extensive localised B-cell division takes place with the creation of a characteristic histological structure known as a germinal centre (figure 3). Within this specialised micro-environment, extensive mutation of the variable portion of the immunoglobulin light-chain and heavy-chain genes occurs.24 An efficient process of positive selection, dependent on follicular-dendritic-cell-bound antigen, ensures the selective survival of those mutants with a raised affinity for antigen. Other mutant B cells die locally by apoptosis and are quickly engulfed by macrophages. The process is iterative, so a B cell can easily end up with 50 mutations in its V, or variable region, genes. High affinity B cells leave the germinal centre and generate both high-affinity antibody on restimulation.
Click to enlarge image
Figure 3. A typical germinal centreNote that follicular-dendritic-cell-bound antigen has been pushed into a crescent cap at one pole of germinal centre.
Immunological memory, which is so important to a successful vaccine, depends on many factors. Long-term persistence of antigen on follicular dendritic cells in lymphoid follicles is clearly one such factor.25 A subset of antibody-forming plasma cells is very long-lived, and tiny amounts of antibody can be protective if of sufficiently high affinity. The primary immune response generates both T and B cells of the requisite specificity in considerable numbers, and these memory cells are long-lived. A second entry of the antigen thus encounters many more lymphocytes of the right specificity. Furthermore, these memory T and B cells may have a lowered threshold for activation. Cross-reactions are common in immunology, and it is possible that memory cells are stimulated by cross-reacting environmental antigens. A clinically inapparent re-encounter with the relevant pathogen may also reinforce memory. This gives rise to the very practical concern that, as herd immunity lowers the circulation of particular pathogens in the community, such reinforcement will not happen, stressing the need for booster doses.
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Influence of host age
The immunobiology of host responses is influenced by age, with problems at both ends of the spectrum. The newborn infant is relatively immature immunologically, yet possesses sufficient T and B cells to make immunisation against some diseases worthwhile. Both BCG and hepatitis B (first dose) are frequently given at birth. Infant immunisation is clearly a compromise between suboptimal responses and the need to protect the very young. 2 months of age is judged the right time for a first dose of diphtheria-tetanus-pertussis, Hib, and poliomyelitis vaccines. Passive antibodies from the mother can hinder the uptake of some live attenuated vaccines; measles vaccine is not given before age 9 months even in developing countries, and measles-mumps-rubella not before age 1 year. The older form of polysaccharide Hib vaccine or of presently available polyvalent pneumococcal vaccine are ineffective before 2 years.
In the aging individual, it is clear that no new export of T cells from the thymus can take place, as the thymic cortex has been largely replaced by fatty tissue. Moreover, the export of new B cells from the bone marrow is also sharply reduced. Nevertheless, old people usually have sufficient memory-type T and B cells to make immunisation worthwhile despite responses which are quantitatively inferior to those of younger adults. There is a need to develop a consensus as to what booster vaccination is desirable in old age. For the moment, a reasonable case can be made for immunisation against influenza, pneumococci, and herpes zoster.
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Challenges for the vaccine developer
The vaccine developer faces some difficult challenges. On one hand, conventional vaccines are coming through the clinical research pipeline very quickly. These could transform the situation with respect to many diarrhoeal, acute respiratory, sexually-transmitted, and parasitic diseases. If vaccination schedules are not to become unduly burdensome, new combination vaccines will have to be devised. On the other hand, basic immunology is providing some radical new solutions. These include powerful new adjuvants not yet fully tested in humans; recombinant vaccines in which harmless micro-organisms act as Trojan horses for genes coding for important antigens;26 and novel micro-encapsulation and other one-shot techniques. Perhaps most excitingly, plasmids have been engineered to contain genes for antigens behind strong promoters. Such nucleic acid vaccines can be injected intradermally or intramuscularly. The genes are expressed, and proteins thus made provoke both T and B cell responses.27 It is imperative that preclinical and clinical studies on these unconventional approaches proceed in parallel with standard vaccine development so that the use of possibly superior agents is not delayed. Once the research and development process has run its course, there remains the ineffable challenge of how to pay for these powerful new tools in developing countries in order to protect all the world's children.
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