Chapter 14:  Manipulation of the Immune Response

14-1. Corticosteroids are powerful anti-inflammatory drugs that alter the transcription of many genes

Corticosteroid drugs are powerful anti-inflammatory agents that are used widely to suppress the harmful effects of immune responses of autoimmune or allergic origin, as well as those induced by graft rejection. Corticosteroids are pharmacological derivatives of members of the glucocorticoid family of steroid hormones; one of the most widely used is prednisone, which is a synthetic analogue of cortisol (Fig. 14.1). Cortisol acts through intracellular receptors that are expressed in almost every cell of the body. On binding hormone, these receptors regulate the transcription of specific genes, as illustrated in Fig. 14.2.

The expression of as many as 1% of genes in the genome may be regulated by glucocorticoids, which can either induce or, less commonly, suppress the transcription of responsive genes. The pharmacological effects of corticosteroid drugs result from exposure of the glucocorticoid receptors to supraphysiological concentrations of ligand. The abnormally high level of ligation of glucocorticoid receptors causes exaggerated glucocorticoid-mediated responses, which have both beneficial and toxic effects.

Given the large number of genes regulated by corticosteroids and that different genes are regulated in different tissues, it is hardly surprising that the effects of steroid therapy are very complex. The beneficial effects are antiinflammatory and are summarized in Fig. 14.3; however, there are also many adverse effects, including fluid retention, weight gain, diabetes, bone mineral loss, and thinning of the skin. The use of corticosteroids to control disease requires a careful balance between helping the patient by reducing the inflammatory manifestations of disease and avoiding harm from the toxic side-effects of the drug. For this reason, corticosteroids used in transplant recipients and to treat inflammatory autoimmune and allergic disease are often administered in combination with other drugs in an effort to keep the dose and toxic effects to a minimum. In autoimmunity and allograft rejection, corticosteroids are commonly combined with cytotoxic immunosuppressive drugs.

14-2. Cytotoxic drugs cause immunosuppression by killing dividing cells and have serious side-effects

The two cytotoxic drugs most commonly used as immunosuppressants are azathioprine and cyclophosphamide (Fig. 14.4). Both interfere with DNA synthesis and have their major pharmacological action on dividing tissues. They were developed originally to treat cancer and, after observations that they were cytotoxic to dividing lymphocytes, were found to be immunosuppressive as well. The use of these compounds is limited by a range of toxic effects on tissues that have in common the property of continuous cell division. These effects include decreased immune function, as well as anemia, leukopenia, thrombocytopenia, damage to intestinal epithelium, hair loss, and fetal death or injury. As a result of their toxicity, these drugs are used at high doses only when the aim is to eliminate all dividing lymphocytes, and in these cases treated patients require subsequent bone marrow transplantation to restore their hematopoietic function. They are used at lower doses, and in combination with other drugs such as corticosteroids, to treat unwanted immune responses.

Azathioprine is converted in vivo to a purine antagonist that interferes with the synthesis of nucleic acids and is toxic to dividing cells. It is metabolized to 6-thioinosinic acid, which competes with inosine monophosphate, thereby blocking the synthesis of adenosine monophosphate and guanosine monophosphate and thus inhibiting DNA synthesis. It is less toxic than cyclophosphamide, which is metabolized to phosphoramide mustard, which alkylates DNA. Cyclophosphamide is a member of the nitrogen mustard family of compounds, which were originally developed as chemical weapons. With this pedigree goes a range of highly toxic effects including inflammation of and hemorrhage from the bladder, known as hemorrhagic cystitis, and induction of bladder neoplasia.

14-3. Cyclosporin A, FK506 (tacrolimus), and rapamycin (sirolimus) are powerful immunosuppressive agents that interfere with T-cell signaling

There are now relatively nontoxic alternatives to the cytotoxic class of drugs that can be used for immunosuppression in transplant patients. The systematic study of products from bacteria and fungi has led to the development of a large number of important medicines including the two immunosuppressive drugs cyclosporin A and FK506 or tacrolimus, which are now widely used to treat transplant recipients. Cyclosporin A is a cyclic decapeptide derived from a soil fungus from Norway, Tolypocladium inflatum. FK506, now known as tacrolimus, is a macrolide compound from the filamentous bacterium Streptomyces tsukabaensis found in Japan; macrolides are compounds that contain a many-membered lactone ring to which is attached one or more deoxy sugars. Another Streptomyces macrolide, called rapamycin or sirolimus, is being evaluated in clinical studies and is also likely to become important in the prevention of transplant rejection; rapamycin is derived from Streptomyces hygroscopicus, found on Easter Island (‘Rapa ui’ in Polynesian—hence the name of the drug). All three compounds exert their pharmacological effects by binding to members of a family of intracellular proteins known as the immunophilins, forming complexes that interfere with signaling pathways important for the clonal expansion of lymphocytes (see Chapter 6).

Cyclosporin A and tacrolimus block T-cell proliferation by inhibiting the phosphatase activity of a Ca²⁺-activated enzyme called calcineurin at nanomolar concentrations. Their mechanism of action, which we will discuss further in the next section, revealed a role for calcineurin in transmitting signals from the T-cell receptor to the nucleus. Both drugs reduce the expression of several cytokine genes that are normally induced on T-cell activation (Fig. 14.5). These include interleukin (IL)-2, whose synthesis by T lymphocytes is an important growth signal for T cells. Cyclosporin A and tacrolimus inhibit T-cell proliferation in response to either specific antigens or allogeneic cells and are used extensively in medical practice to prevent the rejection of allogeneic organ grafts. Although the major immunosuppressive effects of both drugs are probably the result of inhibition of T-cell proliferation, they also act on other cells and have a large variety of other immunological effects (see Fig. 14.5), some of which might turn out to be important pharmacologically.

Cyclosporin A and tacrolimus are effective, but they are not problem-free. First, as with the cytotoxic agents, they affect all immune responses indiscriminately. The only way of controlling their immunosuppressive action is by varying the dose; at the time of grafting, high doses are required but, once a graft is established, the dose can be decreased to allow useful protective immune responses while maintaining adequate suppression of the residual response to the grafted tissue. This is a difficult balance that is not always achieved. Furthermore, although T cells are particularly sensitive to the actions of these drugs, their molecular targets are found in other cell types and therefore these drugs have effects on many other tissues. Cyclosporin A and tacrolimus are both toxic to kidneys and other organs. Finally, treatment with these drugs is expensive because they are complex natural products that must be taken for prolonged periods. Thus there is room for improvement in these compounds, and better and less expensive analogues are being sought. Nevertheless, at present, they are the drugs of choice in clinical transplantation, and they are also being tested in a variety of autoimmune diseases, especially those that, like graft rejection, are mediated by T cells.

14-4. Immunosuppressive drugs are valuable probes of intracellular signaling pathways in lymphocytes

The mechanism of action of cyclosporin A and tacrolimus is now fairly well understood. Each binds to a different group of immunophilins: cyclosporin A to the cyclophilins, and tacrolimus to the FK-binding proteins (FKBP). These immunophilins are peptidyl-prolyl cis-trans isomerases but their isomerase activity does not seem to be relevant to the immunosuppressive activity of the drugs that bind them. Rather, the immunophilin:drug complexes bind and inhibit the Ca²⁺-activated serine/threonine phosphatase calcineurin. Calcineurin is activated in T cells when intracellular calcium ion levels rise after T-cell receptor binding; on activation it dephosphorylates the NFATc family of transcription factors in the cytoplasm, allowing them to migrate to the nucleus, where they form complexes with nuclear partners including the transcription factor AP-1, and induce transcription of genes including those for IL-2, CD40 ligand, and Fas ligand (Fig. 14.6, and see Sections 6-11 and 8-10). This pathway is inhibited by cyclosporin A and tacrolimus, which thus inhibit the clonal expansion of activated T cells. Calcineurin is found in other cells besides T cells but at higher levels; T cells are therefore particularly susceptible to the inhibitory effects of these drugs.

Rapamycin has a different mode of action from either cyclosporin A or tacrolimus. Like tacrolimus, it binds to the FKBP family of immunophilins. However, the rapamycin:immunophilin complex has no effect on calcineurin activity but, instead, blocks the signal transduction pathway triggered by ligation of the IL-2 receptor. Rapamycin also inhibits lymphocyte proliferation driven by IL-4 and IL-6, implying a common postreceptor pathway of signaling by these cytokines. The rapamycin:immunophilin complex acts by binding to a protein kinase named mTOR (mammalian target of rapamycin; also known as FRAP, RAFT1, and RAPT1). This kinase phosphorylates two downstream intracellular targets. The first is another kinase, p70 S6 kinase, which in turn regulates the translation of many proteins. The second is PHAS-1, a repressor of protein translation, which is inhibited by phosphorylation mediated by mTOR. Both PHAS-1 and p70 S6 kinase appear to mediate the effects of rapamycin in lymphocytes. Because rapamycin has different pharmacological activities from cyclosporin A and tacrolimus, trials are being undertaken to see if combination therapy involving rapamycin given together with either cyclosporin A or tacrolimus might provide more effective and safer treatment than the use of just one of these drugs. The rationale for such studies is that it may be possible to use lower amounts of each drug when used in combination, compared with the amounts required for treatment with a single agent. This might be a means of reducing unwanted side-effects.

14-5. Antibodies against cell-surface molecules have been used to remove specific lymphocyte subsets or to inhibit cell function

Cytotoxic drugs kill all proliferating cells and therefore indiscriminately affect all types of activated lymphocyte and any other cell that is dividing. Cyclosporin A, tacrolimus, and rapamycin are more selective, but still inhibit most adaptive immune responses. In contrast, antibodies can interfere with immune responses in a nontoxic and much more specific manner. The potential of antibodies for removal of unwanted lymphocytes is demonstrated by anti-lymphocyte globulin, a preparation of immunoglobulin from horses immunized with human lymphocytes, which has been used for many years to treat acute graft rejection episodes. Anti-lymphocyte globulin does not, however, discriminate between useful lymphocytes and those responsible for unwanted responses. Moreover, horse immunoglobulin is highly antigenic in humans and the large doses used in therapy are often followed by the development of serum sickness, caused by the formation of immune complexes of horse immunoglobulin and human anti-horse immunoglobulin antibodies (see Chapter 12). Nevertheless, anti-lymphocyte globulins are still in use to treat acute rejection and have stimulated the quest for monoclonal antibodies to achieve more specifically targeted effects.

Immunosuppressive monoclonal antibodies act by one of two general mechanisms. Some monoclonal antibodies trigger the destruction of lymphocytes in vivo, and are referred to as depleting antibodies, whereas others are nondepleting and act by blocking the function of their target protein without killing the cell that bears it. IgG monoclonal antibodies that cause lymphocyte depletion target these cells to macrophages and NK cells, which bear Fc receptors and which respectively kill the lymphocytes by phagocytosis and antibody-dependent cytotoxicity. Many antibodies are being tested for their ability to inhibit allograft rejection and to modify the expression of autoimmune disease. We will discuss some of these examples after looking at the measures being taken to prepare monoclonal antibodies for therapy in humans.

14-6. Antibodies can be engineered to reduce their immunogenicity in humans

The major impediment to therapy with monoclonal antibodies in humans is that these antibodies are most readily made by using mouse cells, and humans rapidly develop antibody responses to mouse antibodies. This not only blocks the actions of the mouse antibodies but leads to allergic reactions, and if treatment is continued can result in anaphylaxis (see Section 12-10). Once this has happened, future treatment with any mouse monoclonal antibody is ruled out. This problem can, in principle, be avoided by making antibodies that are not recognized as foreign by the human immune system, and three strategies are being explored for their construction. One approach is to clone human V regions into a phage display library and select for binding to human cells, as described in Appendix I (see Section A-13). In this way, monoclonal antibodies that are entirely human in origin can be obtained. Second, mice lacking endogenous immunoglobulin genes can be made transgenic (see Appendix I, Section A-46) for human immunoglobulin heavy- and light-chain loci by using yeast artificial chromosomes. B cells in these mice have receptors encoded by human immunoglobulin genes but are not tolerant to most human proteins. In these mice, it is possible to induce human monoclonal antibodies against epitopes on human cells or proteins.

Finally, one can graft the complementarity-determining regions (CDRs) of a mouse monoclonal antibody, which form the antigen-binding loops, onto the framework of a human immunoglobulin molecule, a process known as humanization. Because antigen-binding specificity is determined by the structure of the CDRs (see Chapter 3), and because the overall frameworks of mouse and human antibodies are so similar, this approach produces a monoclonal antibody that is antigenically identical to human immunoglobulin but binds the same antigen as the mouse monoclonal antibody from which the CDR sequences were derived. These recombinant antibodies are far less immunogenic in humans than the parent mouse monoclonal antibodies, and thus they can be used for the treatment of humans with far less risk of anaphylaxis.

14-7. Monoclonal antibodies can be used to inhibit allograft rejection

Antibodies specific for various physiological targets have been used in attempts to prevent the development of allograft rejection by inhibiting the development of harmful inflammatory and cytotoxic responses. One approach is to perfuse the organ before transplantation with antibodies that react with antigen-presenting cells and thus target them for destruction within the mononuclear phagocytic system. Depletion of antigen-presenting cells in the graft by this method is effective at preventing allograft rejection in animal models, although there is no convincing evidence that it is successful in humans. Antibodies have, however, been used to treat episodes of graft rejection in humans. Anti-CD3 antibodies are moderately effective as an adjunct to immunosuppressive drugs in the treatment of episodes of transplanted kidney rejection.

A further approach to inhibiting allograft rejection is the blockade of the co-stimulatory signals needed to activate T cells that recognize donor antigens. In animal studies of graft rejection, a fusion protein made from CTLA-4 and the Fc portion of human immunoglobulin, which binds to both B7.1 and B7.2 (see Section 8-5), has allowed the long-term survival of certain grafted tissues. Even more effective in a primate model of renal allograft rejection was the use of a humanized monoclonal antibody against the CD40 ligand (CD154), present on T cells (see Section 8-17). CD40 ligand binds to CD40, expressed on dendritic and endothelial cells, stimulating these cells to secrete cytokines such as IL-6, IL-8, and IL-12. The mechanism of the immunosuppressive effect of anti-CD40 ligand antibody is not known, but it is most likely to be a consequence of blocking the activation of dendritic cells by T helper cells recognizing donor antigens.

Monoclonal antibodies against other targets have also had some success in preventing graft rejection in animals. Of particular interest are certain nondepleting anti-CD4 antibodies: when given for a short time during primary exposure to grafted tissue, these lead to a state of tolerance in the recipient (Fig. 14.7). This tolerant state is an example of the dominant immune suppression discussed in Section 13-27. It is long-lived and can be transferred to naive recipients by CD4 T cells producing cytokines typical of T_H2 cells, although T cells producing other patterns of cytokines might also be involved (see Section 14-9). The presence of anti-CD4 antibody at the time of transplantation might favor the development of a nondamaging T_H2 response, rather than an inflammatory T_H1 response, because of a reduced strength of interaction between the graft cell antigens and responding naive T cells, as discussed in Section 10-7.

In human bone marrow transplantation, depleting antibodies directed at mature T lymphocytes have proved particularly useful. Elimination of mature T lymphocytes from donor bone marrow before infusion into a recipient is very effective at reducing the incidence of graft-versus-host disease (see Section 13-21). In this disease, the T lymphocytes in the donor bone marrow recognize the recipient as foreign and mount a damaging alloreaction against the recipient, causing rashes, diarrhea, and pneumonia, which is often fatal.

14-8. Antibodies can be used to alleviate and suppress autoimmune disease

Autoimmune disease is detected only once the autoimmune response has caused tissue damage or has disturbed specific physiological functions. There are three main approaches to treatment. First, anti-inflammatory therapy can reduce tissue injury caused by an inflammatory autoimmune response; second, immunosuppressive therapy can be aimed at reducing the autoimmune response; and third, treatment can be directed specifically at compensating for the result of the damage. For example, diabetes, which is induced by autoimmune attack on pancreatic β cells, is treated by insulin replacement therapy. Anti-inflammatory therapy for autoimmune disease includes the use of anti-cytokine antibodies; anti-TNF-α antibodies induce striking temporary remissions in rheumatoid arthritis (Fig. 14.8). Antibodies can also be used to block cell migration to sites of inflammation; for example, anti-CD18 antibodies prevent leukocytes adhering tightly to vascular endothelium and reduce inflammation in animal models of disease.

The ultimate goal of immunotherapy for autoimmune disease is specific intervention to restore tolerance to the relevant autoantigens. Two experimental approaches are under investigation. The first aims at blocking the specific response to autoantigen. One way to attempt this is to identify the clonally restricted T-cell receptors or immunoglobulin carried by the lymphocytes that cause disease, and to target these with antibodies directed against idiotypic determinants on the relevant antigen receptor. Another way is to identify particular MHC class I or class II molecules responsible for presenting peptides from autoantigens and to inhibit their antigen-presenting function selectively with antibodies or blocking peptides. This approach has been successful in some animal models of autoimmunity, for example experimental autoimmune encephalomyelitis (EAE) (Fig. 14.9), in which it seems that a limited number of clones of T cells, responding to a single peptide, might cause disease. However, autoimmune disease in humans and most animal models is driven by a polyclonal response to autoantigens by T and B lymphocytes. For this reason, immunotherapy based on the identification of specific receptors carried by pathogenic lymphocytes is unlikely to succeed. Immunotherapy based on the identification of the particular MHC molecules that drive an autoimmune response is more likely to be effective, but such therapy would also inhibit some protective immune responses.

14-9. Modulation of the pattern of cytokine expression by T lymphocytes can inhibit autoimmune disease

The second approach to immunotherapy for autoimmune disease is to try to turn a pathological autoimmune response into an innocuous one. This approach is being pursued experimentally because, as we learned in Chapter 13, tolerance to tissue antigens does not always depend on the absence of a T-cell response; instead, it can be actively maintained by T cells secreting cytokines that suppress the development of a harmful, inflammatory T-cell response. As the pattern of cytokines expressed by T lymphocytes is critical in determining the perpetuation and expression of autoimmune disease, the manipulation of cytokine expression offers a way of controlling it. There are various techniques, collectively known as immune modulation, that can affect cytokine expression by T lymphocytes. These involve manipulating the cytokine environment in which T-cell activation takes place, or manipulating the way antigen is presented, as these factors have been observed to influence the differentiation and cytokine-secreting function of the activated T cells (see Sections 8-13, 10-5, and 10-6).

As discussed in earlier chapters, CD4 T lymphocytes can be subdivided into two major subsets, the T_H1 cells, which secrete interferon (IFN)-γ, and the T_H2 cells, which secrete IL-4, IL-5, IL-10, and transforming growth factor (TGF)-β. In many cases, autoimmune disease is associated with the activation of T_H1 cells, which activate macrophages and drive an inflammatory immune response. In animal models of experimentally induced autoimmune disease, such as EAE, the relative activation of the T_H1 and T_H2 subsets of T lymphocytes can be manipulated to give either a T_H1 response and disease, or a T_H2 response that confers protection against disease. The preferential activation of T_H1 or T_H2 cells can be achieved by direct manipulation of the cytokine environment or by administering antigen by particular routes, for example by feeding (see Section 14-10).

Recent evidence shows that patterns of cytokines secreted by T lymphocytes are very complicated and that the T_H1 and T_H2 subdivision of T lymphocytes is a considerable oversimplification. For example, CD4 T cells have been identified that develop in a cytokine environment rich in IL-10, and in turn secrete high levels of IL-10 and little IL-2 and IL-4. This pattern of cytokine secretion has bystander effects on other T cells and suppresses antigen-induced activation of other CD4 T lymphocytes. These cells have been provisionally designated T_r1 cells (T regulatory cells 1).

Another subset of T cells with immunosuppressive bystander effects secretes TGF-β as the dominant cytokine and has been designated T_H3. Such cells might be predominantly of mucosal origin and activated by the mucosal presentation of antigen (see Section 14-10).

A further subset of T cells also seems to be implicated in immunoregulation. These are the NK1.1⁺ CD4 T cells, so named because they bear the receptor NK1.1, which is usually found on NK cells. NK1.1⁺ T cells, which we discussed in Section 10-5, recognize antigens, including lipid antigens, presented by the class I-like molecule CD1 (see Section 5-18) and respond by secreting IL-4. Thus, when stimulated, the NK1.1⁺ T cells can act to promote T_H2 responses. Although there is no direct evidence that NK1.1⁺ T cells are involved in immunomodulation in humans, in mice that suffer spontaneous autoimmune disease this population of cells is either missing or decreased. Furthermore, transfer of NK1.1⁺ T cells into such mice prevents the onset of the autoimmune disease.

Immune modulation aims to alter the balance between different subsets of responding T cells such that helpful responses are promoted and damaging responses are suppressed. As a therapy for autoimmunity it has the advantage that one might not need to know the precise nature of the autoantigen stimulating the autoimmune disease. This is because the administration of cytokines or antigen to modulate the immune response causes changes in the pattern of cytokine expression that have bystander effects on lymphocytes with the presumed autoreactive receptors. However, the drawback of this approach is the unpredictability of the results. In murine models of diseases such as diabetes and EAE, most of the results suggest that a T_H2 response can protect against T_H1-mediated disease, but there is evidence that T_H2 lymphocytes can also contribute to the pathology of these diseases.

An additional problem is the difficulty of modulating established immune responses. Experiments in animals have shown that anti-cytokine antibodies (or recombinant cytokines) present at the time of immunization with an autoantigen can sometimes divert a pathogenic immune response. In contrast, the modification of an ongoing immune response is much harder to achieve with this approach, although there have been some examples of experimental success, as we will see later.

14-10. Controlled administration of antigen can be used to manipulate the nature of an antigen-specific response

When the target antigen of an unwanted response is identified, it is possible to manipulate the response by using antigen directly rather than by using antibodies or relying on the bystander effects discussed in the previous section. This is because the way in which antigen is presented to the immune system affects the nature of the response, and the induction of one type of response to an antigen can inhibit a pathogenic response to the same antigen.

As mentioned in Chapter 12, this principle has been applied with some success to the treatment of allergies caused by an IgE response to very low doses of antigen. Repeated treatment of allergic individuals with higher doses of allergen seems to divert the allergic response to one dominated by T cells that favor the production of IgG and IgA antibodies. These antibodies are thought to desensitize the patient by binding the small amounts of allergen normally encountered and preventing it from binding to IgE.

With T cell-mediated autoimmune disease, there has been considerable interest in using peptide antigens to suppress pathogenic responses. The type of CD4 T-cell response induced by a peptide depends on the way in which it is presented to the immune system. For instance, peptides given orally tend to prime T_H2 T cells that make IL-4 or T cells that make predominantly TGF-β without activating T_H1 cells or inducing a great deal of systemic antibody. These mucosal immune responses have relatively little pathogenic potential. Indeed, experiments in animal models indicate that they can protect against induced autoimmune disease. Experimental autoimmune encephalomyelitis is induced by injection of myelin basic protein in complete Freund's adjuvant and resembles multiple sclerosis, whereas collagen arthritis is similarly induced by injection of collagen type II and has features in common with rheumatoid arthritis. Oral administration of myelin basic protein or type II collagen inhibits the development of disease in animals (see Fig. 13.34), and has some beneficial effects in reducing the activity of preestablished disease. Trials using this approach in humans with multiple sclerosis or rheumatoid arthritis have found marginal therapeutic effects. Intravenous delivery of peptides can also inhibit inflammatory responses stimulated by the same peptide presented in a different context. When a soluble peptide is given intravenously, it binds preferentially to MHC class II molecules on resting B cells and tends to induce anergy in T_H1 cells. Thus, a careful choice of the dose or structure of antigen, or its route of administration, can allow us to control the type of response that results. Whether such approaches can be effective in manipulating the established immune responses driving human autoimmune diseases remains to be seen.

Summary

Existing treatments for unwanted immune responses, such as allergic reactions, autoimmunity, and graft rejection, depend largely on three types of drug. Anti-inflammatory drugs, of which the most potent are the corticosteroids, are used for all three types of response. These have a broad spectrum of actions, however, and a correspondingly wide range of toxic side-effects; their dose must be controlled carefully. They are therefore normally used in combination with either cytotoxic or immunosuppressive drugs. The cytotoxic drugs kill all dividing cells and thereby prevent lymphocyte proliferation, but they suppress all immune responses indiscriminately and also kill other types of dividing cells. The immunosuppressive drugs act by intervening in the intracellular signaling pathways of T cells. They are less generally toxic than the cytotoxic drugs, but they also suppress all immune responses indiscriminately. They are also much more expensive than cytotoxic drugs.

Immunosuppressive drugs are now the drugs of choice in the treatment of transplant patients, where they can be used to suppress the immune response to the graft before it has become established. Autoimmune responses are already well established at the time of diagnosis and, in consequence, are much more difficult to suppress. They are therefore less responsive to the immunosuppressive drugs and, for that reason, they are usually controlled with a combination of corticosteroids and cytotoxic drugs. In animal experiments, attempts have been made to target immunosuppression more specifically, by blocking the response to autoantigen with the use of antibodies or antigenic peptides, or by diverting the immune response into a nonpathogenic pathway by manipulating the cytokine environment, or by administering antigen through the oral route where a nonpathogenic immune response is likely to be invoked. None of these treatments is yet proven in humans, and most require that the relevant antigen be known. For that reason, and because they are relatively ineffective against established immune responses, the promise of these approaches in animal models might be difficult to realize in a clinical context.

14-11. The development of transplantable tumors in mice led to the discovery that mice could mount a protective immune response against tumors

The finding that tumors could be induced in mice after treatment with chemical carcinogens or irradiation, coupled with the development of inbred strains of mice, made it possible to undertake the key experiments that led to the discovery of immune responses to tumors. These tumors could be transplanted between mice, and the experimental study of tumor rejection has generally been based on the use of such tumors. If these bear MHC molecules foreign to the mice into which they are transplanted, the tumor cells are readily recognized and destroyed by the immune system, a fact that was exploited to develop the first MHC-congenic strains of mice. Specific immunity to tumors must therefore be studied within inbred strains, so that host and tumor can be matched for their MHC type.

Transplantable tumors in mice exhibit a variable pattern of growth when injected into syngeneic recipients. Most tumors grow progressively and eventually kill the host. However, if mice are injected with irradiated tumor cells that cannot grow, they are frequently protected against subsequent injection with a normally lethal dose of viable cells of the same tumor. There seems to be a spectrum of immunogenicity among transplantable tumors: injections of irradiated tumor cells seem to induce varying degrees of protective immunity against a challenge injection of viable tumor cells at a distant site. These protective effects are not seen in T-cell deficient mice but can be conferred by adoptive transfer of T cells from immune mice, showing the need for T cells to mediate all these effects.

These observations indicate that the tumors express antigenic peptides that can become targets of a tumor-specific T-cell response. The antigens expressed by experimentally induced murine tumors, often termed tumor-specific transplantation antigens (TSTAs), or tumor rejection antigens (TRAs), are usually specific for an individual tumor. Thus immunization with irradiated tumor cells from tumor X protects a syngeneic mouse from challenge with live cells from tumor X but not from challenge with a different syngeneic tumor Y, and vice versa (Fig. 14.10).

14-12. T lymphocytes can recognize specific antigens on human tumors

Tumor rejection antigens are peptides of tumor-cell proteins that are presented to T cells by MHC molecules. These peptides can become the targets of a tumor-specific T-cell response because they are not displayed on the surface of normal cells, at least not at levels sufficient to be recognized by T cells. Six different categories of tumor rejection antigens can be distinguished and examples of each of these are given in Fig. 14.11. The first category consists of antigens that are strictly tumor specific. These antigens are the result of point mutations or gene rearrangements, which often arise as part of the process of oncogenesis. Point mutations may evoke a T-cell response either by allowing de novo binding of a peptide to MHC class I molecules or by creating a new epitope for T cells by modification of a peptide that already binds class I molecules (Fig. 14.12). A special class of tumor-specific antigen in the case of B- and T-cell tumors, which are derived from single clones of lymphocytes, are the idiotypic sequences unique to the antigen receptor expressed by the clone.

The second category comprises proteins encoded by genes that are normally expressed only in male germ cells, which do not express MHC molecules and therefore cannot present peptides from these molecules to T lymphocytes. Tumor cells show widespread abnormalities of gene expression, including the activation of these genes and thus the presentation of these proteins to T cells; hence, these proteins are effectively tumor specific in their expression as antigens (Fig. 14.13).

The third category of tumor rejection antigen is comprised of differentiation antigens encoded by genes that are only expressed in particular types of tissue. The best examples of these are the differentiation antigens expressed in melanocytes and melanoma cells; a number of these antigens are proteins involved in the pathways of production of the black pigment, melanin. The fourth category is comprised of antigens that are strongly overexpressed in tumor cells compared with their normal counterparts (see Fig. 14.13). An example is HER-2/neu (also known as c-Erb-2), which is a receptor tyrosine kinase homologous to the epidermal growth factor receptor. This receptor is overexpressed in many adenocarcinomas, including breast and ovarian cancers, where it is linked with a poor prognosis. MHC class I-restricted, CD8-positive cytotoxic T lymphocytes have been found infiltrating solid tumors overexpressing HER-2/neu but are not capable of destroying such tumors in vivo. The fifth category of tumor rejection antigens is comprised of molecules that display abnormal posttranslational modifications. An example is underglycosylated mucin, MUC-1, which is expressed by a number of tumors, including breast and pancreatic cancers.

Proteins encoded by viral oncogenes comprise the sixth category of tumor rejection antigen. These oncoviral proteins are viral proteins that may play a critical role in the oncogenic process and, because they are foreign, they can evoke a T-cell response. Examples of such proteins are the human papilloma type 16 virus proteins, E6 and E7, which are expressed in cervical carcinoma.

Although each of these categories of tumor rejection antigen may evoke an anti-tumor response in vitro and in vivo, it is exceptional for such a response to be able to spontaneously eliminate an established tumor. It is the goal of tumor immunotherapy to harness and augment such responses to treat cancer more effectively. In this respect, the spontaneous remission occasionally observed in cases of malignant melanoma and renal cell carcinoma, even when disease is quite advanced, offers hope that this goal is achievable.

In melanoma, tumor-specific antigens were discovered by culturing irradiated tumor cells with autologous lymphocytes, a reaction known as the mixed lymphocyte-tumor cell culture. From such cultures, cytotoxic T lymphocytes could be identified that would kill, in an MHC-restricted fashion, tumor cells bearing the relevant tumor-specific antigen. Melanomas have been studied in detail using this approach. Cytotoxic T cells reactive against melanoma peptides have been cloned and used to characterize melanomas by the array of tumor-specific antigens displayed. These studies have yielded three important findings. The first is that melanomas carry at least five different antigens that can be recognized by cytotoxic T lymphocytes. The second is that cytotoxic T lymphocytes reactive against melanoma antigens are not expanded in vivo, suggesting that these antigens are not immunogenic in vivo. The third is that the expression of these antigens can be selected against in vitro and possibly also in vivo by the presence of specific cytotoxic T cells. These discoveries offer hope for tumor immunotherapy, an indication that these antigens are not naturally strongly immunogenic, and also a caution about the possibility of selecting, in vivo, tumor cells that can escape recognition and killing by cytotoxic T cells.

Consistent with these findings, functional melanoma-specific T cells can be propagated from peripheral blood lymphocytes, from tumor-infiltrating lymphocytes, or by draining the lymph nodes of patients in whom the melanoma is growing. Interestingly, none of the peptides recognized by these T cells derives from the mutant proto-oncogenes or tumor suppressor genes that are likely to be responsible for the initial transformation of the cell into a cancer cell, although a few are the products of mutant genes. The rest derive from normal genes but are displayed at levels detectable by T cells for the first time on tumor cells, as illustrated in Fig. 14.13. Antigens of the MAGE family are not expressed in any normal adult tissues, with the exception of the testis, which is an immunologically privileged site. They probably represent early developmental antigens reexpressed in the process of tumorigenesis. Only a minority of melanoma patients have T cells reactive against the MAGE antigens, indicating that these antigens are either not expressed or are not immunogenic in most cases. The most common melanoma antigens are peptides from the enzyme tyrosinase or from three other proteins—gp100, MART1, and gp75. These are differentiation antigens specific to the melanocyte lineage from which melanomas arise. It is likely that overexpression of these antigens in tumor cells leads to an abnormally high density of specific peptide:MHC complexes and this makes them immunogenic. Although in most cases tumor rejection antigens are presented as peptides complexed with MHC class I molecules, tyrosinase has been shown to stimulate CD4 T-cell responses in some melanoma patients by being ingested and presented by cells expressing MHC class II.

Tumor rejection antigens shared between most examples of a tumor, and against which tolerance can be broken, represent candidate antigens for tumor vaccines. The MAGE antigens are candidates because of their limited tissue distribution and their shared expression by many melanomas. It might seem dangerous to use tumor vaccines based on antigens that are not truly tumor-specific because of the risk of inducing autoimmunity. Often, however, the tissues from which tumors arise are dispensable; the prostate is perhaps the best example of this. With melanoma, however, some melanocyte-specific tumor rejection antigens are also expressed in certain retinal cells, in the inner ear, in the brain, and in the skin. Despite this, melanoma patients receiving immunotherapy with whole tumor cells or tumor-cell extracts, although occasionally developing vitiligo—a destruction of pigmented cells in the skin that correlates well with a good response to the tumor—do not develop abnormalities in the visual, vestibular, and central nervous systems, perhaps because of the low level of expression of MHC class I molecules in these sites.

In addition to the human tumor antigens that have been shown to induce cytotoxic T-cell responses (see Fig. 14.11), many other candidate tumor rejection antigens have been identified by studies of the molecular basis of cancer development. These include the products of mutated cellular oncogenes or tumor suppressors, such as Ras and p53, and also fusion proteins, such as the Bcr-Abl tyrosine kinase that results from the chromosomal translocation (t9;22) found in chronic myeloid leukemia. It is intriguing that, in each of these cases, no specific cytotoxic T-cell response has been identified in cultures of autologous lymphocytes with tumor cells bearing these mutated antigens. However, cytotoxic T lymphocytes specific for these antigens can be developed in vitro by using peptide sequences derived either from the mutated sequence or from the fusion sequence of these common oncogenic proteins; these cytotoxic T cells are able to recognize and kill tumor cells. In chronic myeloid leukemia, it is known that, after treatment and bone marrow transplantation, mature lymphocytes from the bone marrow donor infused into the patient can help to eliminate any residual tumor. At present, it is not clear whether this is a graft-versus-host effect, where the donor lymphocytes are responding to alloantigens expressed on the leukemia cells, or whether there is a specific anti-leukemic response. The ability to prime the donor cells against leukemia-specific peptides offers the prospect of enhancing the anti-leukemic effect while minimizing the risk of graft-versus-host disease. It is a challenge for immunologists to understand why these mutated proteins do not prime cytotoxic T cells in the patients in which the tumors arise. They are excellent targets for therapy, as they are unique to the tumor and have a causal role in oncogenesis.

14-13. Tumors can escape rejection in many ways

Burnet called the ability of the immune system to detect tumor cells and destroy them ‘immune surveillance.’ However, it is difficult to show that tumors are subject to surveillance by the immune system; after all, cancer is a common disease, and most tumors show little evidence of immunological control. The incidence of the common tumors in mice that lack lymphocytes is little different from their incidence in mice with normal immune systems; the same is true for humans deficient in T cells. The major tumor types that occur with increased frequency in immunodeficient mice or humans are virus-associated tumors; immune surveillance thus seems to be critical for control of virus-associated tumors, but the immune system does not normally respond to the novel antigens deriving from the multiple genetic alterations in spontaneous tumors. The goal in the development of anti-cancer vaccines is to break the tolerance of the immune system for antigens expressed mainly or exclusively by the tumor.

It is not surprising that spontaneously arising tumors are rarely rejected by T cells, as in general they probably lack either distinctive antigenic peptides or the adhesion or co-stimulatory molecules needed to elicit a primary T-cell response. Moreover, there are other mechanisms whereby tumors can avoid immune attack or evade it when it occurs (Fig. 14.14). Tumors tend to be genetically unstable and can lose their antigens by mutation; in the event of an immune response, this instability might generate mutants that can escape the immune response. Some tumors, such as colon and cervical cancers, lose the expression of a particular MHC class I molecule (Fig. 14.15), perhaps through immunoselection by T cells specific for a peptide presented by that MHC class I molecule. In experimental studies, when a tumor loses expression of all MHC class I molecules, it can no longer be recognized by cytotoxic T cells, although it might become susceptible to NK cells (Fig. 14.16). However, tumors that lose only one MHC class I molecule might be able to avoid recognition by specific CD8 cytotoxic T cells while remaining resistant to NK cells, conferring a selective advantage in vivo.

Yet another way in which tumors might evade rejection is by making immunosuppressive cytokines. Many tumors make these, although in most cases little is known of their precise nature. TGF-β was first identified in the culture supernatant of a tumor (hence its name, transforming growth factor-β) and, as we have seen, tends to suppress inflammatory T-cell responses and cell-mediated immunity, which are needed to control tumor growth. A number of tumors of different tissue origins, such as melanoma, ovarian carcinoma, and B-cell lymphoma, have been shown to produce the immunosuppressive cytokine IL-10, which can reduce dendritic cell development and activity. Thus, there are many different ways in which tumors avoid recognition and destruction by the immune system.

14-14. Monoclonal antibodies against tumor antigens, alone or linked to toxins, can control tumor growth

The advent of monoclonal antibodies suggested the possibility of targeting and destroying tumors by making antibodies against tumor-specific antigens (Fig. 14.17). This depends on finding a tumor-specific antigen that is a cell-surface molecule. Some of the cell-surface molecules that have been targeted in experimental clinical trials are shown in Fig. 14.18. So far there has been limited success with this approach, although, as an adjunct to other therapies, it holds promise. Some striking initial results have been reported in the treatment of breast cancer with a humanized monoclonal antibody, known as Herceptin, which targets a growth factor receptor, HER-2/neu, that is overexpressed in about a quarter of breast cancer patients. As we discussed in Section 14-12, this overexpression accounts for HER-2/neu evoking an antitumor T-cell response, although HER-2/neu is also associated with a poorer prognosis. It is thought that Herceptin acts by blocking interaction between the receptor and its natural ligand and by downregulating the level of expression of the receptor. The effects of this antibody can be potentiated when it is combined with conventional chemotherapy. A second monoclonal antibody that has promise for the treatment of non-Hodgkin's B-cell lymphoma binds to CD20 and is known as Rituximab. Ligation and clustering of CD20 transduces a signal that causes lymphocyte apoptosis. Monoclonal antibodies coupled to γ-emitting radioisotopes have also been used to image tumors, for the purpose of diagnosis and monitoring tumor spread (Fig. 14.19).

The first reported successful treatment of a tumor with monoclonal antibodies used anti-idiotypic antibodies to target B-cell lymphomas whose surface immunoglobulin expressed the corresponding idiotype. The initial course of treatment usually leads to a remission, but the tumor always reappears in a mutant form that no longer binds to the antibody used for the initial treatment. This case represents a clear example of genetic instability enabling a tumor to evade treatment.

Other problems with tumor-specific or tumor-selective monoclonal antibodies as therapeutic agents include inefficient killing of cells after binding of the monoclonal antibody and inefficient penetration of the antibody into the tumor mass. The first problem can often be circumvented by linking the antibody to a toxin, producing a reagent called an immunotoxin; two favored toxins are ricin A chain and Pseudomonas toxin. Both approaches require the antibody to be internalized to allow the cleavage of the toxin from the antibody in the endocytic compartment, allowing the toxin chain to penetrate and kill the cell.

Two other approaches using monoclonal antibody conjugates involve linking the antibody molecule to chemotherapeutic drugs such as adriamycin or to radioisotopes. In the first case, the specificity of the monoclonal antibody for a cell-surface antigen on the tumor concentrates the drug to the site of the tumor. After internalization, the drug is released in the endosomes and exerts its cytostatic or cytotoxic effect. Monoclonal antibodies linked to radionuclides (see Fig. 14.17) concentrate the radioactive source in the tumor site. Both these approaches have the advantage of also killing neighboring tumor cells, because the released drug or radioactive emissions can affect cells adjacent to those that actually bind the antibody. Ultimately, combinations of toxin-, drug-, or radionuclide-linked monoclonal antibodies, together with vaccination strategies aimed at inducing T cell-mediated immunity, might provide the most effective cancer immunotherapy.

14-15. Enhancing the immunogenicity of tumors holds promise for cancer therapy

Although vaccines based on tumor antigens are, in principle, the ideal approach to T cell-mediated cancer immunotherapy, it may be many decades before the dominant tumor antigens for common cancers are identified. Even then, it is not clear how widely the relevant epitopes will be shared between tumors, and peptides of tumor rejection antigens will be presented only by particular MHC alleles. To be effective, a tumor vaccine may therefore need to include a range of tumor antigens. MAGE-1 antigens, for example, are recognized only by T cells in melanoma patients expressing the HLA-A1 haplotype. However, the range of MAGE-type proteins that has now been characterized encompasses peptide epitopes presented by many HLA class I and II molecules.

Until recently, most cancer vaccines have used the individual patient's tumor removed at surgery as a source of vaccine antigens. These cell-based vaccines are prepared by mixing either irradiated tumor cells or tumor extracts with bacterial adjuvants such as BCG or Corynebacterium parvum, which enhance their immunogenicity (see Appendix I, Section A-4). Such vaccines have generated modest therapeutic results in melanomas but have, in general, been disappointing.

Where candidate tumor rejection antigens have been identified, for example in melanoma, experimental vaccination strategies include the use of whole proteins, peptide vaccines based on sequences recognized by cytotoxic T lymphocytes (either administered alone or presented by the patient's own dendritic cells), and recombinant viruses encoding these peptide epitopes. A novel experimental approach to tumor vaccination is the use of heat-shock proteins isolated from tumor cells. The underlying principle of this therapy is that one of the physiological activities of heat-shock proteins is to act as intracellular chaperones of antigenic peptides. There is evidence for receptors on the surface of professional antigen-presenting cells that take up certain heat-shock proteins together with any bound peptides. Uptake of heat-shock proteins via these receptors delivers the accompanying peptide into the antigen-processing pathways leading to peptide presentation by MHC class I molecules. This experimental technique for tumor vaccination has the advantage that it does not depend on any prior knowledge of the nature of the relevant tumor rejection antigens, but the disadvantage that the heat-shock proteins purified from the cell carry very many peptides, so that any tumor rejection antigen might constitute only a tiny fraction of the peptides bound to the heat-shock protein.

A further experimental approach to tumor vaccination in mice is to increase the immunogenicity of tumor cells by introducing genes that encode co-stimulatory molecules or cytokines. This is intended to make the tumor itself more immunogenic. The basic scheme of such experiments is shown in Fig. 14.20. A tumor cell transfected with the gene encoding the co-stimulatory molecule B7 (see Section 8-5) is implanted in a syngeneic animal. These B7-positive cells can activate tumor-specific naive T cells to become armed effector T cells able to reject the tumor cells. They are also able to stimulate further proliferation of the armed effector cells that reach the site of implantation. These T cells can then target the tumor cells whether they express B7 or not; this can be shown by reimplanting nontransfected tumor cells, which are also rejected.

The second strategy, that of introducing cytokine genes into tumors so that they secrete the relevant cytokine, is aimed at attracting antigen-presenting cells to the tumor and takes advantage of the paracrine nature of cytokines. In mice, the most effective tumor vaccines so far are tumor cells that secrete granulocyte-macrophage colony-stimulating factor (GM-CSF), which induces the differentiation of hematopoietic precursors into dendritic cells and attracts these to the site. GM-CSF is also thought to function as an adjuvant, activating the dendritic cells. It is believed that these cells process the tumor antigens and migrate to the local lymph nodes, where they induce potent anti-tumor responses. The B7-transfected cells seem less potent in inducing anti-tumor responses, perhaps because the bone marrow-derived dendritic cells express more of the molecules required to activate naive T cells than do B7-transfected tumor cells. In addition, the tumor cells do not share the dendritic cells' special ability to migrate into the T-cell areas of the lymph nodes, where they are optimally placed to interact with passing naive T cells (see Section 8-6).

The potency of dendritic cells in activating T-cell responses provides the rationale for yet another strategy for vaccinating against tumors. The use of antigen-pulsed autologous dendritic cells to stimulate therapeutically useful cytotoxic T-cell responses to tumors has been developed in experimental models, and there have been initial trials in humans with cancer.

Clinical trials are in progress to determine the safety and efficacy of many of these approaches in human patients. What is uncertain is whether people with established cancers can generate sufficient T-cell responses to eliminate all their tumor cells under circumstances in which any tumor-specific naive T cells might have been rendered tolerant to the tumor. Moreover, there is always the risk that immunogenic transfectants will elicit an autoimmune response against the normal tissue from which the tumor derived.

Summary

Tumors represent outgrowths of a single abnormal cell, and animal studies have shown that some tumors elicit specific immune responses that suppress their growth. These seem to be directed at MHC-bound peptides derived from antigens that might be mutated, inappropriately expressed, or overexpressed in the tumor cells. T-cell deficient individuals, however, do not develop more tumors than normal individuals. This is probably chiefly because most tumors do not make distinctive antigenic proteins or do not express the co-stimulatory molecules necessary to initiate an adaptive immune response. Tumors also have other ways of avoiding or suppressing immune responses, such as ceasing to express MHC class I molecules, or making immunosuppressive cytokines. Monoclonal antibodies have been developed for tumor immunotherapy by conjugation to toxins or to cytotoxic drugs or radionuclides, which are thereby delivered at high dose specifically to the tumor cells. More recently, attempts have been made to develop vaccines based on tumor cells taken from patients and made immunogenic by the addition of adjuvants, or by pulsing autologous dendritic cells with tumor-cell extracts or tumor antigens. This approach has been extended in animal experiments to transfection of tumor cells with genes encoding co-stimulatory molecules or cytokines that attract and activate dendritic cells.

14-16. There are several requirements for an effective vaccine

The particular requirements for successful vaccination vary according to the nature of the infecting organism. For extracellular organisms, antibody provides the most important adaptive mechanism of host defense, whereas for control of intracellular organisms, an effective CD8 T-lymphocyte response is also essential. The ideal vaccination provides host defense at the point of entry of the infectious agent; stimulation of mucosal immunity is therefore an important goal of vaccination against those many organisms that enter through mucosal surfaces.

Effective protective immunity against some microorganisms requires the presence of preexisting antibody at the time of exposure to the infection. For example, the clinical manifestations of tetanus and diphtheria are entirely due to the effects of extremely powerful exotoxins (see Fig. 9.23). Preexisting antibody against the bacterial exotoxin is necessary to provide a defense against these diseases. Preexisting antibodies are also required to protect against some intracellular pathogens, such as the poliomyelitis virus, which infect critical host cells within a short period after entering the body and are not easily controlled by T lymphocytes once intracellular infection is established.

Immune responses to infectious agents usually involve antibodies directed at multiple epitopes and only some of these antibodies confer protection. The particular T-cell epitopes recognized can also affect the nature of the response. For example, as we saw in Chapter 11, the predominant epitope recognized by T cells after vaccination with respiratory syncytial virus induces a vigorous inflammatory response but fails to elicit neutralizing antibodies and thus causes pathology without protection. Thus, an effective vaccine must lead to the generation of antibodies and T cells directed at the correct epitopes of the infectious agent. For some of the modern vaccine techniques, in which only one or a few epitopes are used, this consideration is particularly important.

A number of very important additional constraints need to be satisified by a successful vaccine (Fig. 14.23). First, it must be safe. Vaccines must be given to huge numbers of people, relatively few of whom are likely to die of, or sometimes even catch, the disease that the vaccine is designed to prevent. This means that even a low level of toxicity is unacceptable. Second, the vaccine must be able to produce protective immunity in a very high proportion of the people to whom it is given. Third, because it is impracticable to give large or dispersed rural populations regular ‘booster’ vaccinations, a successful vaccine must generate long-lived immunological memory. This means that both B and T lymphocytes must be primed by the vaccine. Fourth, vaccines must be very cheap if they are to be administered to large populations. Vaccines are one of the most cost-effective measures in health care, but this benefit is eroded as the cost-per-dose rises.

An effective vaccination program provides herd immunity—by lowering the number of susceptible members of a population, the natural reservoir of infected individuals in that population falls, reducing the probability of transmission of infection. Thus, even nonvaccinated members of a population can be protected from infection if the majority are vaccinated.

14-17. The history of vaccination against Bordetella pertussis illustrates the importance of developing an effective vaccine that is perceived to be safe

The history of vaccination against the bacterium that causes whooping cough, Bordetella pertussis, provides a good example of the challenges of developing and disseminating an effective vaccine. At the turn of the 20th century, whooping cough killed approximately 0.5% of American children under the age of 5 years. In the early 1930s, a trial of a killed, whole bacterial cell vaccine on the Faroe Islands provided evidence of a protective effect. In the United States, systematic use of a whole-cell vaccine in combination with diphtheria and tetanus toxoids (the DPT vaccine) since the 1940s resulted in a decline in the annual infection rate from 200 to less than 2 cases per 100,000 of the population. First vaccination with DPT was typically given at the age of 3 months.

Whole-cell pertussis vaccine causes side-effects, typically redness, pain, and swelling at the site of the injection; less commonly, vaccination is followed by high temperature and persistent crying. Very rarely, fits and a short-lived sleepiness or a floppy unresponsive state ensue. During the 1970s, widespread concern developed after several anecdotal observations that encephalitis leading to irreversible brain damage might very rarely follow pertussis vaccination. In Japan, in 1972, approximately 85% of children were given the pertussis vaccine, and fewer than 300 cases of whooping cough and no deaths were reported. As a result of two deaths after vaccination in Japan in 1975, DPT was temporarily suspended and then reintroduced with the first vaccination at 2 years of age rather than 3 months. In 1979 there were approximately 13,000 cases of whooping cough and 41 deaths. The possibility that pertussis vaccine very rarely causes severe brain damage has been studied extensively and expert consensus is that pertussis vaccine is not a primary cause of brain injury. There is no doubt that there is greater morbidity from whooping cough than from the vaccine.

The public and medical perception that whole-cell pertussis vaccination might be unsafe provided a powerful incentive to develop safer pertussis vaccines. Study of the natural immune response to B. pertussis showed that infection induced antibodies against four components of the bacterium—pertussis toxin, filamentous hemagglutinin, pertactin, and fimbrial antigens. Immunization of mice with these antigens in purified form protected them against challenge with pertussis. This has led to the development of acellular pertussis vaccines, all of which contain purified pertussis toxoid, that is, toxin inactivated by chemical treatment, for example with hydrogen peroxide or formaldehyde, or more recently by genetic engineering of the toxin. Some also contain one or more of the filamentous hemagglutinin, pertactin, and fimbrial antigens. Current evidence shows that these are probably as effective as whole-cell pertussis vaccine and are free of the common minor side-effects of the whole-cell vaccine.

The main messages of the history of pertussis vaccination are, first, that vaccines must be extremely safe and free of side-effects; second, that the public and the medical profession must perceive the vaccine to be safe; and third, that careful study of the nature of the protective immune response can lead to acellular vaccines that are safer than and as effective as whole-cell vaccines.

14-18. Conjugate vaccines have been developed as a result of understanding how T and B cells collaborate in an immune response

Although acellular vaccines are inevitably safer than vaccines based on whole organisms, a fully effective vaccine cannot normally be made from a single isolated constituent of a microorganism, and it is now clear that this is because of the need to activate more than one cell type to initiate an immune response. One consequence of this insight has been the development of conjugate vaccines. We have already described briefly one of the most important of these in Section 9-2.

Many bacteria, including Neisseria meningitidis (meningococcus), Streptococcus pneumoniae (pneumococcus), and Haemophilus species, have an outer capsule composed of polysaccharides that are species- and typespecific for particular strains of the bacterium. The most effective defense against these microorganisms is opsonization of the polysaccharide coat with antibody. The aim of vaccination is therefore to elicit antibodies against the polysaccharide capsules of the bacteria.

Capsular polysaccharides can be harvested from bacterial growth medium and, because they are T-cell independent antigens, they can be used on their own as vaccines. However, young children under the age of 2 years cannot make good T-cell independent antibody responses and cannot be vaccinated effectively with polysaccharide vaccines. An efficient way of overcoming this problem (see Fig. 9.4) is to chemically conjugate bacterial polysaccharides to protein carriers, which provide peptides that can be recognized by antigen-specific T cells, thus converting a T-cell independent response into a T-cell dependent anti-polysaccharide antibody response. By using this approach, various conjugate vaccines have been developed against Haemophilus influenzae, an important cause of serious childhood chest infections and meningitis, and these are now widely applied.

14-19. The use of adjuvants is another important approach to enhancing the immunogenicity of vaccines

Purified antigens are not usually strongly immunogenic on their own and most acellular vaccines require the addition of adjuvants, which are defined as substances that enhance the immunogenicity of antigens (see Appendix I, Section A-4). For example, tetanus toxoid is not immunogenic in the absence of adjuvants, and tetanus toxoid vaccines often contain aluminum salts, which bind polyvalently to the toxoid by ionic interactions and selectively stimulate antibody responses. Pertussis toxin, produced by B. pertussis, has adjuvant properties in its own right and, when given mixed as a toxoid with tetanus and diphtheria toxoids, not only vaccinates against whooping cough but also acts as an adjuvant for the other two toxoids. This mixture makes up the DPT triple vaccine given to infants in the first year of life.

Many important adjuvants are sterile constituents of bacteria, particularly of their cell walls. For example, Freund's complete adjuvant, widely used in experimental animals to augment antibody responses, is an oil and water emulsion containing killed mycobacteria. A complex glycolipid, muramyl dipeptide, which can be extracted from mycobacterial cell walls or synthesized, contains much of the adjuvant activity of whole killed mycobacteria. Other bacterial adjuvants include killed B. pertussis, bacterial polysaccharides, bacterial heat-shock proteins, and bacterial DNA. Many of these adjuvants cause quite marked inflammation and are not suitable for use in vaccines for humans.

It is thought that most, if not all, adjuvants act on antigen-presenting cells, especially on dendritic cells, and reflect the importance of these cells in initiating immune responses. As we learned in Section 8-6, dendritic cells are widely distributed throughout the body, where they act as sentinels to detect potential pathogens at their portals of entry. These tissue dendritic cells take up antigens from their environment by phagocytosis and macropinocytosis, and they are tuned to respond to the presence of infection by migrating into lymphoid tissue and presenting these antigens to T cells. They appear to detect the presence of pathogens in two main ways. The first of these is direct, and follows the ligation and activation of receptors for invading micro-organisms. These include receptors of the complement system, Toll-like receptors (TLRs), and other pattern recognition receptors of the innate immune system. There is much that we still have to learn about the direct mechanisms of detection of infectious agents. For example, bacterial DNA containing unmethylated CpG dinucleotide motifs, bacterial heat-shock proteins, and muramyl dipeptides each have powerful activating effects on antigen-presenting cells, and, while there is indirect evidence that many adjuvants use various TLRs, it is not known how they are detected. When dendritic cells are activated through direct interactions with the products of infectious agents, they respond by secreting cytokines and expressing co-stimulatory molecules, which in turn stimulate the activation and differentiation of antigen-specific T cells.

The second mechanism of stimulation of dendritic cells by invading organisms is indirect and involves their activation by cytokine signals derived from the inflammatory response triggered by infection (see Chapter 2). Cytokines such as GM-CSF are particularly effective in activating dendritic cells to express co-stimulatory signals and, in the context of viral infection, dendritic cells also express interferon (IFN)-α and IL-12.

Adjuvants trick the immune system into responding as though there were an active infection, and just as different classes of infectious agent stimulate different types of immune response (see Chapter 10), different adjuvants may promote different types of response, for example, an inflammatory T_H1 response or an antibody-dominated response. Some adjuvants, for example, pertussis toxin, stimulate mucosal immune responses, which are particularly important in defense against organisms entering through the digestive or respiratory tracts. These adjuvants have been discussed earlier when we described mucosal immunity and will be further discussed in Section 14-26.

Following our increased understanding of the mechanisms of action of adjuvants, rational approaches to improving the activity of vaccines in clinical settings are being implemented. One approach is to coadminister cytokines. For example, IL-12 is a cytokine produced by macrophages, dendritic cells, and B cells that stimulates T lymphocytes and NK cells to release IFN-γ and promotes a T_H1 response. It has been used as an adjuvant to promote protective immunity against the protozoan parasite Leishmania major. Certain strains of mice are susceptible to severe cutaneous and systemic infection by L. major; these mice mount an immune response that is predominantly T_H2 in type and is ineffective in eliminating the organism (see Section 10-6). The coadministration of IL-12 with a vaccine containing leishmania antigens generated a T_H1 response and protected the mice against challenge with L. major. The use of IL-12 to promote a T_H1 response has also proved valuable in reducing the pathogenic consequences of experimental parasitic infection by Schistosoma mansoni and will be considered in Section 14-27. These are important examples of how an understanding of the regulation of immune responses can enable rational intervention to enhance the effectiveness of vaccines.

14-20. Live-attenuated viral vaccines are usually more potent than ‘killed’ vaccines and can be made safer by using recombinant DNA technology

Most antiviral vaccines currently in use consist of inactivated or live attenuated viruses. Inactivated, or ‘killed,’ viral vaccines consist of viruses treated so that they are unable to replicate. Live-attenuated viral vaccines are generally far more potent, perhaps because they elicit a greater number of relevant effector mechanisms, including cytotoxic CD8 T cells: inactivated viruses cannot produce proteins in the cytosol, so peptides from the viral antigens cannot be presented by MHC class I molecules and thus cytotoxic CD8 T cells are not generated by these vaccines. Attenuated viral vaccines are now in use for polio, measles, mumps, rubella, and varicella.

Traditionally, attenuation is achieved by growing the virus in cultured cells. Viruses are usually selected for preferential growth in nonhuman cells and, in the course of selection, become less able to grow in human cells (Fig. 14.24). Because these attenuated strains replicate poorly in human hosts, they induce immunity but not disease when given to people. Although attenuated virus strains contain multiple mutations in genes encoding several of their proteins, it might be possible for a pathogenic virus strain to reemerge by a further series of mutations. For example, the type 3 Sabin polio vaccine strain differs at only 10 of 7429 nucleotides from a wild-type progenitor strain. On extremely rare occasions, reversion of the vaccine to a neurovirulent strain can occur, causing paralytic disease in the unfortunate recipient.

Attenuated viral vaccines can also pose particular risks to immunodeficient recipients in whom they often behave as virulent opportunistic infections. Immunodeficient infants who are vaccinated with live-attenuated polio before their inherited immunoglobulin deficiencies have been diagnosed are at risk because they cannot clear the virus from their gut, and there is therefore an increased chance that mutation of the virus will lead to fatal paralytic disease. For the same reason, patients with immunoglobulin deficiencies show an abnormal susceptibility to chronic infection by opportunistic enteroviruses, and can develop chronic, and ultimately lethal, echovirus encephalitis if mutation of the virus leads to neurovirulence.

An empirical approach to attenuation is still in use but might be superseded by two new approaches that use recombinant DNA technology. One is the isolation and in vitro mutagenesis of specific viral genes. The mutated genes are used to replace the wild-type gene in a reconstituted virus genome, and this deliberately attenuated virus can then be used as a vaccine (Fig. 14.25). The advantage of this approach is that mutations can be engineered so that reversion to wild type is virtually impossible.

Such an approach might be useful in developing live influenza vaccines. As we learned in Chapter 11, the influenza virus can reinfect the same host several times, because it undergoes antigenic shift and thus escapes the original immune response. The current approach to vaccination against influenza is to use a killed virus vaccine that is reformulated annually on the basis of the prevalent strains of virus. The vaccine is moderately effective, reducing mortality in elderly populations and morbidity in healthy adults. The ideal influenza vaccine would be an attenuated live organism that matched the prevalent virus strain. This could be created by first introducing a series of attenuating mutants into the gene encoding a viral polymerase protein, PB2. The mutated gene segment from the attenuated virus could then be substituted for the wild-type gene in a virus carrying the relevant hemagglutinin and neuraminidase antigenic variants of the current epidemic or pandemic strain. This last procedure could be repeated as necessary to keep pace with the antigenic shift of the virus.

14-21. Live-attenuated bacterial vaccines can be developed by selecting nonpathogenic or disabled mutants

Similar approaches are being used for bacterial vaccine development. Salmonella typhi, the causative agent of typhoid, has been manipulated to develop a live vaccine. A strain of wild-type bacteria was mutated using nitrosoguanidine; a new strain was selected to be defective in the enzyme UDP-galactose epimerase, thus blocking the pathway for synthesis of lipopolysaccharide, an important determinant of bacterial pathogenesis. Recent approaches to the rational design of attenuated Salmonella vaccines have involved the specific targeting of genes encoding enzymes in the biosynthetic pathways of amino acids containing aromatic rings, such as tyrosine and phenylalanine. Mutating these genes makes auxotrophic organisms, which are dependent for growth on an external supply of an essential nutrient that wild-type bacteria would be capable of biosynthesizing. These bacteria grow poorly in the gut but should survive long enough as a vaccine to induce an effective immune response.

It is not only vaccination of humans against Salmonella that is important. Modern methods of mass production of chickens for food has led to extensive infection of poultry with Salmonella strains that are pathogenic to humans and an increasingly important cause of food poisoning. Thus, in parts of the world where typhoid is prevalent, vaccinating humans has a high priority. In other parts, where food poisoning caused by Salmonella typhimurium and S. enteritidis infection is common, vaccination of chickens would contribute to public health.

14-22. Attenuated microorganisms can serve as vectors for vaccination against many pathogens

An effective live-attenuated typhoid vaccine would not only be valuable in its own right but could also serve as a vector for presenting antigens from other organisms. Attenuated strains of Salmonella have been used as carriers of heterologous genes encoding tetanus toxoid and antigens from organisms as diverse as Listeria monocytogenes, Bacillus anthracis, Leishmania major, Yersinia pestis, and Schistosoma mansoni. Each of these has been used as an oral vaccine to protect mice against experimental challenge with the respective pathogen.

Viral vectors can similarly be engineered to carry heterologous peptides or proteins from other microorganisms. Although vaccinia is no longer needed to protect against the development of smallpox, it remains a candidate as an avirulent carrier of heterologous antigens. Genes encoding protective antigens from several different organisms could be placed in a single vaccine strain. This approach makes it possible to immunize individuals against several different pathogens at once, but such a vaccine could not be used twice because the vaccinia vector itself generates long-lasting immunity that would neutralize its effectiveness on a second administration; this is an example of the phenomenon called ‘original antigenic sin’ (see Fig. 10.30). The development of successful heterologous vaccines requires the identification of protective antigens; it therefore depends on the analytical power of recombinant DNA methods, as well as their use to manipulate gene structure.

Plant viruses, which are nonpathogenic to humans, have been used as a source of novel vaccine vectors. These viruses can be engineered to incorporate heterologous peptide antigens into chimeric coat proteins. The success of this approach relies on the successful identification of protective peptide antigens as well as the immunogenicity of the vaccine. Using this strategy, mice have been protected against lethal challenge with rabies virus by prior feeding with spinach leaves infected by recombinant alfalfa mosaic virus incorporating a rabies virus peptide. Popeye may need rejuvenation as a role model to encourage children to eat spinach.

14-23. Synthetic peptides of protective antigens can elicit protective immunity

One route to vaccine development is the identification of the T-cell peptide epitopes that stimulate protective immunity. This can be approached in two ways. One possibility is to synthesize systematically overlapping peptides from immunogenic proteins and to test each in turn for its ability to stimulate protective immunity. An alternative, but no less arduous approach—‘reverse’ immunogenetics—has been used in developing a vaccine against malaria (Fig. 14.26).

The immunogenicity of T-cell peptide epitopes depends on their specific associations with particular polymorphic variants of MHC molecules. The starting point for the studies on malaria was an association between the human MHC class I molecule HLA-B53 and resistance to cerebral malaria—a relatively infrequent complication of infection but one that is usually fatal. The hypothesis is that these MHC molecules are protective because they present peptides that are particularly good at evoking cytotoxic T lymphocytes. A direct route to identifying the relevant peptides is to elute them from MHC molecules of cells infected with the pathogen. In HLA-B53, a high proportion of the peptides eluted had proline in the second of nine positions; this information was used to identify candidate protective peptides from four proteins of Plasmodium falciparum expressed in the early phase of hepatocyte infection, an important phase of infection to target in an effective immune response. One of the candidate peptides, from liver stage antigen-1, is recognized by cytotoxic T cells when bound to HLA-B53.

This approach is being extended to other MHC class I and class II molecules associated with protective immune responses against infection. Recently, a protective peptide epitope was eluted from MHC class II molecules in Leishmania-infected macrophages and used as a guide to isolate the gene from Leishmania. The gene was then used to make a protein-based vaccine that primed mice from susceptible strains for responses to Leishmania.

These results show considerable promise, but they also illustrate one of the major drawbacks to the approach. A malaria peptide that is restricted by HLA-B53 might not be immunogenic in an individual lacking HLA-B53: indeed, this presumably accounts for the higher susceptibility of these individuals to natural infections. Because of the very high polymorphism of MHC molecules in humans it will be necessary to identify panels of protective T-cell epitopes and construct vaccines containing arrays of these to develop vaccines that will protect the majority of a susceptible population.

There are other problems with peptide vaccines. Peptides are not strongly immunogenic and it is particularly difficult to generate MHC class I-specific responses by in vivo immunization with peptides. One approach to this problem is to integrate peptides by genetic engineering into carrier proteins within a viral vector, such as hepatitis B core antigen, which are then processed in vivo through natural antigen-processing pathways. A second possible technique is the use of ISCOMs (immune stimulatory complexes). These are lipid carriers that act as adjuvants but have minimal toxicity. They seem to load peptides and proteins into the cell cytoplasm, allowing MHC class I-restricted T-cell responses to peptides to develop (Fig. 14.27). These carriers are being developed for use in human immunization. Another approach to delivering protective peptides, which we discussed in the previous section, is the genetic engineering of infectious microorganisms to create vaccines that stimulate immunity without causing disease.

14-24. The route of vaccination is an important determinant of success

Most vaccines are given by injection. This route has two disadvantages, the first practical, the second immunological. Injections are painful and expensive, requiring needles, syringes, and a trained injector. They are unpopular with the recipient, reducing vaccine uptake, and mass vaccination by this approach is laborious. The immunological drawback is that injection may not be the most effective way of stimulating an appropriate immune response as it does not mimic the usual route of entry of the majority of pathogens against which vaccination is directed.

Many important pathogens infect mucosal surfaces or enter the body through mucosal surfaces. Examples include respiratory microorganisms such as B. pertussis, rhinoviruses and influenza viruses, and enteric microorganisms such as Vibrio cholerae, Salmonella typhi, enteropathogenic Escherichia coli, and Shigella. The enteric microorganisms are particularly important pathogens in underdeveloped countries. It is therefore important to understand how these organisms stimulate mucosal immunity and to develop vaccines that behave similarly. To this end, there are efforts to develop vaccines that can be administered to the mucosa orally or by nasal inhalation.

The power of this approach is illustrated by the effectiveness of live-attenuated polio vaccines. The Sabin polio vaccine consists of three attenuated polio virus strains and is highly immunogenic. Moreover, just as polio itself can be transmitted by fecal contamination of public swimming pools and other failures of hygiene, the vaccine can be transmitted from one individual to another by the orofecal route. Infection with Salmonella likewise stimulates a powerful mucosal and systemic immune response and, as we saw in Section 14-21, has been attenuated for use as a vaccine and carrier of heterologous antigens for presentation to the mucosal immune system.

The rules of mucosal immunity are poorly understood. On the one hand, presentation of soluble protein antigens by the oral route often results in tolerance, which is important given the enormous load of foodborne and airborne antigens presented to the gut and respiratory tract. As discussed in Sections 14-10 and 13-28, the ability to induce tolerance by oral or nasal administration of antigens is being explored as a therapeutic mechanism for reducing unwanted immune responses. On the other hand, the mucosal immune system can respond to and eliminate mucosal infections such as pertussis, cholera, and polio. The proteins from these microorganisms that stimulate immune responses are therefore of special interest. One group of powerfully immunogenic proteins at mucosal surfaces is a group of bacterial toxins that have the property of binding to eukaryotic cells and are protease-resistant. A recent finding of potential practical importance is that certain of these molecules, such as the E. coli heat-labile toxin and pertussis toxin, have adjuvant properties that are retained even when the parent molecule has been engineered to eliminate its toxic properties. These molecules can be used as adjuvants for oral or nasal vaccines. In mice, nasal insufflation of either of these mutant toxins together with tetanus toxoid resulted in the development of protection against lethal challenge with tetanus toxin.

14-25. Protective immunity can be induced by injecting DNA encoding microbial antigens and human cytokines into muscle

The latest development in vaccination has come as a surprise even to the scientists who first developed the method. The story begins with attempts to use nonreplicating bacterial plasmids encoding proteins for gene therapy: proteins expressed in vivo from these plasmids were found to stimulate an immune response. When DNA encoding a viral immunogen is injected intramuscularly, it leads to the development of antibody responses and cytotoxic T cells that allow the mice to reject a later challenge with whole virus (Fig. 14.28). This response does not appear to damage the muscle tissue, is safe and effective, and, because it uses only a single microbial gene, does not carry the risk of active infection. This procedure has been termed ‘DNA vaccination.’ DNA coated onto minute metal projectiles can be administered by ‘biolistic’ (biological ballistic) gun, so that several metal particles penetrate the skin and enter the muscle beneath. This technique has been shown to be effective in animals and might be suitable for mass immunization, although it has yet to be tested in humans. Mixing in plasmids that encode cytokines such as GM-CSF makes immunization with genes encoding protective antigens much more effective, as was seen earlier for tumor immunity.

14-26. The effectiveness of a vaccine can be enhanced by targeting it to sites of antigen presentation

An important way of enhancing the effectiveness of a vaccine is to target it efficiently to antigen-presenting cells. This is an important mechanism of action of vaccine adjuvants. There are three complementary approaches. The first is to prevent proteolysis of the antigen on its way to antigen-presenting cells. Preserving antigen structure is an important reason why so many vaccines are given by injection rather than by the oral route, which exposes the vaccine to digestion in the gut. The second and third approaches are to target the vaccine selectively, once in the body, to antigen-presenting cells and to devise methods of engineering the selective uptake of the vaccine into antigen-processing pathways within the cell.

Techniques to enhance the uptake of antigens by antigen-presenting cells include coating the antigen with mannose to enhance uptake by mannose receptors on antigen-presenting cells, and presenting the antigen as an immune complex to take advantage of antibody and complement binding by Fc and complement receptors. The effects of DNA vaccination have been enhanced experimentally by injecting DNA encoding antigen coupled to CTLA-4, which enables the selective binding of the expressed protein to antigen-presenting cells carrying B7, the receptor for CTLA-4 (see Section 8-5).

A more complicated set of strategies involves targeting vaccine antigens selectively into antigen-presenting pathways within the cell. For example, human papillomavirus E7 antigen has been coupled to the signal peptide that targets a lysosomal-associated membrane protein to lysosomes and endosomes. This directs the E7 antigen directly to the intracellular compartments in which antigens are cleaved to peptides before binding to MHC class II molecules (see Section 5-5). A vaccinia virus incorporating this chimeric antigen induced a greater response in mice to E7 antigen than did vaccinia incorporating wild-type E7 antigen alone. A second approach is the use of ISCOMs, which seem to encourage the entry of peptides into the cytoplasm, thus enhancing the loading of peptides onto MHC class I molecules (see Section 14-23).

An improved understanding of the mechanisms of mucosal immunity (see Chapter 10) has led to the development of techniques to target antigens to M cells overlying Peyer's patches (see Fig. 1.10). These specialized epithelial cells lack the mucin barrier and digestive properties of other mucosal epithelial cells. Instead, they can bind and endocytose macromolecules and micro-organisms, which are transcytosed intact and delivered to the underlying lymphoid tissue. In view of these properties, it is not surprising that some pathogens target M cells to gain entry to the body. The counterattack by immunologists is to gain a detailed molecular understanding of this mechanism of bacterial pathogenesis and subvert it as a delivery system for vaccines. For example, the outer membrane fimbrial proteins of Salmonella typhimurium have a key role in the binding of these bacteria to M cells. It might be possible to use these fimbrial proteins or, ultimately, just their binding motifs, as targeting agents for vaccines. A related strategy to encourage the uptake of mucosal vaccines by M cells is to encapsulate antigens in particulate carriers that are taken up selectively by M cells.

14-27. An important question is whether vaccination can be used therapeutically to control existing chronic infections

There are many chronic diseases in which infection persists because of a failure of the immune system to eliminate disease. These can be divided into two groups, those infections in which there is an obvious immune response that fails to eliminate the organism, and those in which the infection seems to be invisible to the immune system and evokes a barely detectable immune response.

In the first category, the immune response is often partly responsible for the pathogenic effects. Infection by the helminth Schistosoma mansoni is associated with a powerful T_H2-type response, characterized by high IgE levels, circulating and tissue eosinophilia, and a harmful fibrotic response to schistosome ova, leading to hepatic fibrosis. Other common parasites, such as Plasmodium and Leishmania species, cause damage because they are not eliminated effectively by the immune response in many patients. Mycobacteria causing tuberculosis and leprosy cause persistent intracellular infection; a T_H1 response helps to contain these infections but also causes granuloma formation and tissue necrosis (see Fig. 8.43). Among viruses, hepatitis B and hepatitis C infections are commonly followed by persistent viral carriage and hepatic injury, resulting in death from hepatitis or from hepatoma. HIV infection, as we have seen in Chapter 11, persists despite an ongoing immune response.

There is a second category of chronic infection, predominantly viral, in which the immune response fails to clear infection because of the relative invisibility of the infectious agent to the immune system. A good example is herpes simplex type 2, which is transmitted venereally, becomes latent in nerve tissue, and causes genital herpes, which is frequently recurrent. This invisibility seems to be caused by a viral protein, ICP-47, which binds to the TAP complex and inhibits peptide transport into the endoplasmic reticulum in infected cells (see Chapter 4). Thus viral peptides are not presented to the immune system by MHC class I molecules. Another example in this category of chronic infection is genital warts, caused by certain papilloma viruses to which very little immune response is evoked.

There are two main immunological approaches to the treatment of chronic infection. One is to try to boost or change the pattern of the host immune response by using cytokine therapy. The second is to attempt therapeutic vaccination to see whether the host immune response can be supercharged by immunization with antigens from the infectious agent in combination with adjuvant. There has been substantial pharmaceutical investment in therapeutic vaccination but it is too early to know whether the approach will be successful.

Some promise for the cytokine therapy approach comes from the experimental treatment of leprosy: one can clear certain leprosy lesions by the injection of cytokines directly into the lesion, which may cause reversal of the type of leprosy seen. Another example in which cytokine therapy has been shown to be effective in treating an established infection depends on combining a cytokine with an anti-parasitic drug. In a proportion of mice infected with Leishmania and subsequently treated with a combination of drug therapy and IL-12, the immune response deviated from a T_H2 to a T_H1 pattern and the infection was cleared. In most of the animal studies, however, it seems that the anti-cytokine antibody or the cytokine needs to be present at the first encounter with the antigen to modulate the response effectively. For example, in experimental leishmaniasis in mice, susceptible BALB/c mice injected with anti-IL-4 antibody at the time of infection clear their infection (Fig. 14.29). However, if administration of anti-IL-4 antibody is delayed by just one week, there is progressive growth of the parasite and a dominant T_H2 response.

14-28. Modulation of the immune system might be used to inhibit immunopathological responses to infectious agents

We have mentioned several times the possibility of modulating immunity by cytokine manipulation of the immune response. This approach is being explored as a means of inhibiting harmful immune responses to a number of important infections. As we have seen in the preceding section, the liver fibrosis in schistosomiasis results from the powerful T_H2-type response. The coadministration of S. mansoni ova together with IL-12 does not protect mice against subsequent infection with S. mansoni cercariae but has a striking effect in reducing hepatic granuloma formation and fibrosis in response to ova. IgE levels are reduced, with reduced tissue eosinophilia, and the cytokine response indicates the activation of T_H1 rather than T_H2 cells. Although these results indicate that it might be possible to use a combination of antigen and cytokines to vaccinate against the pathology of diseases for which a fully protective vaccine is unavailable, they do not solve the difficulty of applying this approach in patients whose infection is already established.

Summary

The greatest triumphs of modern immunology have come from vaccination, which has eradicated or virtually eliminated several human diseases. It is the single most successful manipulation of the immune system so far, because it takes advantage of the immune system's natural specificity and inducibility. Nevertheless, there are many important infectious diseases for which there is still no effective vaccine. The most effective vaccines are based on attenuated live microorganisms, but these carry some risk and are potentially lethal to immunosuppressed or immunodeficient individuals. Better techniques for developing live-attenuated vaccines, or vaccines that incorporate both immunogenic components and protective antigens of pathogens, are therefore being sought. Most current viral vaccines are based on live attenuated virus, but many bacterial vaccines are based on components of the micro-organism, including components of the toxins that it produces. Protective response to carbohydrate antigens can be enhanced by conjugation to a protein. Vaccines based on peptide epitopes are still at an experimental stage and have the problem that the peptide is likely to be specific for particular variants of the MHC molecules to which they must bind, as well as being only very weakly immunogenic. A vaccine's immunogenicity often depends on adjuvants that can help, directly or indirectly, to activate antigen-presenting cells that are necessary for the initiation of immune responses. The development of oral vaccines is particularly important for stimulating immunity to the many pathogens that enter through the mucosa. Cytokines have been used experimentally as adjuvants to boost the immunogenicity of vaccines or to bias the immune response along a specific path.