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Janeway CA Jr, Travers P, Walport M, et al. Immunobiology: The Immune System in Health and Disease. 5th edition. New York: Garland Science; 2001.

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Immunobiology: The Immune System in Health and Disease. 5th edition.

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Manipulating the immune response to fight infection

Infection is the leading cause of death in the human population. The two most important contributions to public health in the past 100 years have been sanitation and vaccination, which together have dramatically reduced deaths from infectious disease. Modern immunology grew from the success of Jenner's and Pasteur's vaccines against smallpox and chicken cholera, respectively, and its greatest triumph has been the global eradication of smallpox, announced by the World Health Organization in 1980. A global campaign to eradicate polio is now under way.

Adaptive immunity to a specific infectious agent can be achieved in several ways. One early strategy was to deliberately cause a mild infection with the unmodified pathogen. This was the principle of variolation, in which the inoculation of a small amount of dried material from a smallpox pustule would cause a mild infection followed by long-lasting protection against reinfection. However, infection following variolation was not always mild: fatal smallpox ensued in about 3% of cases, which would not meet modern criteria for safety. Jenner's achievement was the realization that infection with a bovine analogue of smallpox, vaccinia (from vacca—a cow), which caused cowpox, would provide protective immunity against smallpox in humans without the risk of significant disease. He named the process vaccination, and Pasteur, in his honour, extended the term to the stimulation of protection to other infectious agents. Humans are not a natural host of vaccinia, which establishes only a brief and limited subcutaneous infection but contains antigens that stimulate an immune response that is cross-reactive with smallpox antigens and thereby confers protection from the human disease.

This established the general principles of safe and effective vaccination, and vaccine development in the early part of the 20th century followed two empirical pathways. The first was the search for attenuated organisms with reduced pathogenicity that would stimulate protective immunity; the second was the development of vaccines based on killed organisms and, subsequently, purified components of organisms that would be as effective as live whole organisms. Killed vaccines were desirable because any live vaccine, including vaccinia, can cause lethal systemic infection in the immunosuppressed.

Immunization is now considered so safe and so important that most states in the United States require all children to be immunized against measles, mumps, and polio viruses with live attenuated vaccines, as well as against tetanus (caused by Clostridium tetani), diphtheria (caused by Coryne- bacterium diphtheriae), and whooping cough (caused by Bordetella pertussis), with inactivated toxins or toxoids prepared from these bacteria (see Fig. 1.33). More recently, a vaccine has become available against Haemophilus B, one of the causative agents of meningitis. Current vaccination schedules for children in the United States are shown in Fig. 14.21. Impressive as these accomplishments are, there are still many diseases for which we lack effective vaccines, as shown in Fig. 14.22. Even where a vaccine such as measles or polio can be used effectively in developed countries, technical and economic problems can prevent its widespread use in developing countries, where mortality from these diseases is still high. The development of vaccines therefore remains an important goal of immunology and the latter half of the 20th century saw a shift to a more rational approach, based on a detailed molecular understanding of microbial pathogenicity, analysis of the protective host response to pathogenic organisms, and the understanding of the regulation of the immune system to generate effective T- and B-lymphocyte responses.

Figure 14.21. Recommended childhood vaccination schedules (in red) in the United States.

Figure 14.21

Recommended childhood vaccination schedules (in red) in the United States. Each red bar denotes a time range during which a vaccine dose should be given. Bars spanning multiple months indicate a range of times during which the vaccine may be given. * (more...)

Figure 14.22. Diseases for which effective vaccines are still needed.

Figure 14.22

Diseases for which effective vaccines are still needed. *The number of people infected is estimated at ~200 million, of which 20 million have severe disease. †Current measles vaccines are effective but heat-sensitive, which makes their use difficult (more...)

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.

Figure 14.23. There are several criteria for an effective vaccine.

Figure 14.23

There are several criteria for an effective vaccine.

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 TH1 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 TH1 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 TH2 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 TH1 response and protected the mice against challenge with L. major. The use of IL-12 to promote a TH1 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.

Figure 14.24. Viruses are traditionally attenuated by selecting for growth in nonhuman cells.

Figure 14.24

Viruses are traditionally attenuated by selecting for growth in nonhuman cells. To produce an attenuated virus, the virus must first be isolated by growing it in cultured human cells. The adaptation to growth in cultured human cells can cause some attenuation (more...)

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.

Figure 14.25. Attenuation can be achieved more rapidly and reliably with recombinant DNA techniques.

Figure 14.25

Attenuation can be achieved more rapidly and reliably with recombinant DNA techniques. If a gene in the virus that is required for virulence but not for growth or immunogenicity can be identified, this gene can be either multiply mutated (left lower panel) (more...)

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).

Figure 14.26. ‘Reverse’ immunogenetics can be used to identify protective T-cell epitopes against infectious diseases.

Figure 14.26

‘Reverse’ immunogenetics can be used to identify protective T-cell epitopes against infectious diseases. Population studies show that the MHC class I variant HLA-B53 is associated with resistance to cerebral malaria. Self nonapeptides were (more...)

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.

Figure 14.27. ISCOMs can be used to deliver peptides to the MHC class I processing pathway.

Figure 14.27

ISCOMs can be used to deliver peptides to the MHC class I processing pathway. ISCOMs (immune stimulatory complexes) are lipid micelles that will fuse with cell membranes. Peptides trapped in ISCOMs can be delivered to the cytosol of an antigen-presenting (more...)

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.

Figure 14.28. DNA vaccination by injection of DNA encoding a protective antigen and cytokines directly into muscle.

Figure 14.28

DNA vaccination by injection of DNA encoding a protective antigen and cytokines directly into muscle. Influenza hemagglutinin contains both B- and T-cell epitopes. When a DNA plasmid containing the gene for hemagglutinin is injected directly into muscle, (more...)

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 TH2-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 TH1 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 TH2 to a TH1 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 TH2 response.

Figure 14.29. Treatment with anti-IL-4 antibody at the time of infection with Leishmania major allows normally susceptible mice to clear the infection.

Figure 14.29

Treatment with anti-IL-4 antibody at the time of infection with Leishmania major allows normally susceptible mice to clear the infection. The top panel shows a hematoxylin-eosin-stained section through the footpad of a mouse of the BALB/c strain infected (more...)

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 TH2-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 TH1 rather than TH2 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.

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Copyright © 2001, Garland Science.
Bookshelf ID: NBK27131

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