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common usage. Experience has shown that not every vaccine candidate that was successful in laboratory and animal stud- ies prevents disease in humans. Some potential vaccines cause unacceptable side effects, and some may even worsen the disease they were meant to prevent. Live virus vaccines pose a special threat to those with primary or acquired im- munodeficiency (see Chapter 19). Stringent testing is an ab- solute necessity, because vaccines will be given to large numbers of well persons. Adverse side effects, even those that occur at very low frequency, must be balanced against the po- tential benefit of protection by the vaccine. Vaccine development begins with basic research. Recent advances in immunology and molecular biology have led to effective new vaccines and to promising strategies for finding new vaccine candidates. Knowledge of the differences in epi- topes recognized by T cells and B cells has enabled immunol- ogists to begin to design vaccine candidates to maximize activation of both arms of the immune system. As differences in antigen-processing pathways became evident, scientists began to design vaccines and to use adjuvants that maximize antigen presentation with class I or class II MHC molecules.
chapter 18
■ Active and Passive Immunization ■ Designing Vaccines for Active Immunization ■ Whole-Organism Vaccines ■ Purified Macromolecules as Vaccines ■ Recombinant-Vector Vaccines ■ DNA Vaccines ■ Multivalent Subunit Vaccines
Vaccines
T
the early vaccination trials of Edward Jenner and Louis Pasteur. Since those pioneering efforts, vac- cines have been developed for many diseases that were once major afflictions of mankind. The incidence of diseases such as diphtheria, measles, mumps, pertussis (whooping cough), rubella (German measles), poliomyelitis, and tetanus has de- clined dramatically as vaccination has become more com- mon. Clearly, vaccination is a cost-effective weapon for disease prevention. Perhaps in no other case have the bene- fits of vaccination been as dramatically evident as in the eradication of smallpox, one of mankind’s long-standing and most terrible scourges. Since October 1977, not a single naturally acquired smallpox case has been reported any- where in the world. Equally encouraging is the predicted eradication of polio. The last recorded case of naturally ac- quired polio in the Western Hemisphere occurred in Peru in 1991, and the World Health Organization (WHO) predicts that paralytic polio will be eradicated throughout the world within the next few years. A new addition to the weapons against childhood disease is a vaccine against bacterial pneu- monia, a major cause of infant death. A crying need remains for vaccines against other diseases. Every year, millions throughout the world die from malaria, tuberculosis, and AIDS, diseases for which there are no effec- tive vaccines. It is estimated by the World Health Organiza- tion that 16,000 individuals a day, or 5.8 million a year, become infected with HIV-1, the virus that causes AIDS. An effective vaccine could have an immense impact on the con- trol of this tragic spread of death and disaster. In addition to the challenges presented by diseases for which no vaccines ex- ist, there remains the need to improve the safety and efficacy of present vaccines and to find ways to lower their cost and deliver them efficiently to all who need them, especially in de- veloping countries of the world. The WHO estimates that millions of infant deaths in the world are due to diseases that could be prevented by existing vaccines (see Clinical Focus). The road to successful development of a vaccine that can be approved for human use, manufactured at reasonable cost, and efficiently delivered to at-risk populations is costly, long, and tedious. Procedures for manufacture of materials that can be tested in humans and the ways they are tested in clinical trials are regulated closely. Even those candidate vac- cines that survive initial scrutiny and are approved for use in human trials are not guaranteed to find their way into
Vaccination with DNA
414 PART I V The Immune System in Health and Disease
recently of a causal relationship between vaccination and autism, a condition of unknown etiology. Most such reports are based solely on the coincidental tim- ing of vaccination and onset of disease, or on limited sampling and poor statisti- cal analyses. So far, no alleged asso- ciations have withstood scrutiny that included large population samples and acceptable statistical methods. While children in this country are pro- tected against a variety of once-deadly diseases, this protection depends on continuation of our immunization pro- grams. Dependency on herd immunity is dangerous for both the individual and society. Adverse reactions to vaccines must be examined thoroughly, of course, and if a vaccine causes unacceptable side reactions, the vaccination program must be reconsidered. At the same time, anecdotal reports of disease brought on
by vaccines, and unsupported beliefs, such as the contention that vaccines weaken the immune system, must be countered by correct information from trusted sources. To retreat from our progress in immunization by noncom- pliance will return us to the age when measles, mumps, whooping cough, and polio were part of the risk of growing up. Children in the developing world suf- fer from a problem different from those in the United States. Examination of in- fant deaths worldwide shows that exist- ing vaccines could save the lives of millions of children. As seen in the table, there are safe, effective vaccines for five of the top ten killers of children. Al- though the list of diseases in the table in- cludes HIV, TB, and malaria, for which no vaccines are available, administration of the vaccines that are recommended for infants in the United States could cut child mortality in the world by approxi- mately half. What barriers exist to the achievement of worldwide vaccination and complete eradication of many childhood diseases? The inability to achieve higher levels of
Many previously common childhood diseases are seldom seen in the United States, a testament to the ef- fectiveness of vaccination. A major bar- rier to similar success in the rest of the world is the difficulty of delivering vac- cines to all children. However, even at home the U.S. is becoming a victim of its own success. Some parents who have never encountered diseases now nearly vanquished in the U.S. do not consider it important to have their in- fants vaccinated or they may be lax in adhering to recommended schedules of immunization. Others hold the unin- formed belief that the risks associated with vaccination outweigh the risk of in- fection. This flawed reasoning is fueled by periodic allegations of linkage be- tween vaccination and various disor- ders, such as the report circulating
CL I N I CA L F O CU S
Vaccination: Challenges in the
U.S. and Developing Countries
Genetic engineering techniques can be used to develop vac- cines to maximize the immune response to selected epitopes and to simplify delivery of the vaccines. This chapter de- scribes the vaccines now in use and describes vaccine strate- gies, including experimental designs that may lead to the vaccines of the future.
Active and Passive Immunization
Immunity to infectious microorganisms can be achieved by active or passive immunization. In each case, immunity can be acquired either by natural processes (usually by transfer from mother to fetus or by previous infection by the organ- ism) or by artificial means such as injection of antibodies or vaccines (Table 18-1, on page 416). The agents used for in- ducing passive immunity include antibodies from humans or animals, whereas active immunization is achieved by inocu- lation with microbial pathogens that induce immunity but
do not cause disease or with antigenic components from the pathogens. This section describes current usage of passive and active immunization techniques.
Passive Immunization Involves Transfer of Preformed Antibodies Jenner and Pasteur are recognized as the pioneers of vaccina- tion, or induction of active immunity, but similar recogni- tion is due to Emil von Behring and Hidesaburo Kitasato for their contributions to passive immunity. These investigators were the first to show that immunity elicited in one animal can be transferred to another by injecting it with serum from the first (see Clinical Focus, Chapter 4). Passive immunization, in which preformed antibodies are transferred to a recipient, occurs naturally by transfer of ma- ternal antibodies across the placenta to the developing fetus. Maternal antibodies to diphtheria, tetanus, streptococci, rubeola, rubella, mumps, and poliovirus all afford pas- sively acquired protection to the developing fetus. Maternal
immunization is the best preventative currently available. A monoclonal antibody or a combination of two monoclonal antibodies may be administered to children at risk for RSV disease. These monoclonal antibodies are prepared in mice but have been “humanized” by splicing the constant regions of human IgG to the mouse variable regions (see Chapter 5). This modification prevents many of the complications that may follow a second injection of the complete mouse anti- body, which is a highly immunogenic foreign protein. Although passive immunization may be an effective treatment, it should be used with caution because certain risks are associated with the injection of preformed antibody. If the antibody was produced in another species, such as a horse, the recipient can mount a strong response to the isotypic determinants of the foreign antibody. This anti-isotype response can cause serious complications. Some individuals, for example, produce IgE antibody
specific for determinants on the injected antibody. Immune complexes of this IgE bound to the passively administered antibody can mediate systemic mast cell degranulation, leading to systemic anaphylaxis. Other individuals produce IgG or IgM antibodies specific for the foreign antibody, which form complement-activating immune complexes. The deposition of these complexes in the tissues can lead to type III hypersensitive reactions. Even when human gamma globulin is administered passively, the recipient can gener- ate an anti-allotype response to the human immunoglobu- lin, although its intensity is usually much less than that of an anti-isotype response.
Active Immunization Elicits Long-Term Protection Whereas the aim of passive immunization is transient pro- tection or alleviation of an existing condition, the goal of ac- tive immunization is to elicit protective immunity and immunologic memory. When active immunization is suc- cessful, a subsequent exposure to the pathogenic agent elicits a heightened immune response that successfully eliminates the pathogen or prevents disease mediated by its products. Active immunization can be achieved by natural infection with a microorganism, or it can be acquired artificially by ad- ministration of a vaccine (see Table 18-1). In active immu- nization, as the name implies, the immune system plays an active role—proliferation of antigen-reactive T and B cells results in the formation of memory cells. Active immuniza- tion with various types of vaccines has played an important
416 PART I V The Immune System in Health and Disease
TABLE 18-
Acquisition of passive and active
immunity
Type Acquired through
Passive immunity Natural maternal antibody Immune globulin* Humanized monoclonal antibody Antitoxin† Active immunity Natural infection Vaccines‡ Attenuated organisms Inactivated organisms Purified microbial macromolecules Cloned microbial antigens Expressed as recombinant protein As cloned DNA alone or in virus vectors Multivalent complexes Toxoid§ *An antibody-containing solution derived from human blood, obtained by cold ethanol fractionation of large pools of plasma; available in intramuscu- lar and intravenous preparations. †An antibody derived from the serum of animals that have been stimulated with specific antigens. ‡A suspension of attenuated live or killed microorganisms, or antigenic por- tions of them, presented to a potential host to induce immunity and prevent disease. §A bacterial toxin that has been modified to be nontoxic but retains the capacity to stimulate the formation of antitoxin.
TABLE 18-2 Common agents used for passive
immunization
Disease Agent
Black widow spider bite Horse antivenin Botulism Horse antitoxin Diphtheria Horse antitoxin Hepatitis A and B Pooled human immune gamma globulin Measles Pooled human immune gamma globulin Rabies Pooled human immune gamma globulin Respiratory disease Monoclonal anti-RSV*
Snake bite Horse antivenin Tetanus Pooled human immune gamma globulin or horse antitoxin *Respiratory syncytial virus
role in the reduction of deaths from infectious diseases, espe- cially among children. Vaccination of children is begun at about 2 months of age. The recommended program of childhood immunizations in this country, updated in 2002 by the American Academy of Pediatrics, is outlined in Table 18-3. The program includes the following vaccines:
■ (^) Hepatitis B vaccine
■ (^) Diphtheria-pertussis (acellular)-tetanus (DPaT) combined vaccine
■ (^) Inactivated (Salk) polio vaccine (IPV); the oral (Sabin) vaccine is no longer recommended for use in the United States
■ (^) Measles-mumps-rubella (MMR) combined vaccine
■ (^) Haemophilus influenzae (Hib) vaccine
■ (^) Varicella zoster (Var) vaccine for chickenpox
■ (^) Pneumococcal conjugate vaccine (PCV); a new addition to the list.
In addition, hepatitis A vaccine at 18 months and influenza vaccines after 6 months are recommended for infants in high-risk populations. The introduction and spreading use of various vaccines for childhood immunization has led to a dramatic decrease in the incidence of common childhood diseases in the United States (Figure 18-1). The comparisons of disease incidence in 1999 to that reported in the peak years show dramatic drops and, in one case, complete elimination of the disease in the United States. As long as widespread, effective immunization programs are maintained, the incidence of these childhood diseases should remain low. However, the occurrence of side reactions to a vaccine may cause a drop in its use, which can lead to re-emergence of that disease. For example, the side ef- fects from the pertussis attenuated bacterial vaccine included seizures, encephalitis, brain damage, and even death. De- creased usage of the vaccine led to an increase in the inci-
Vaccines CH APTER 18 417
TABLE 18-3 Recommended childhood immunization schedule in the United States, 2002
AGE
Vaccine * Birth 1 mo 2 mos 4 mos 6 mos 12 mos 15 mos 18 mos 4–6 yrs
Hepatitis B†^ Diphtheria, tetanus, pertussis‡ H. influenzae, type b Inactivated polio§^ Pneumococcal conjugate Measles, mumps, rubella Varicella#^ *This schedule indicates the recommended ages for routine administration of currently licensed childhood vaccines. Bars indicate ranges of recommended ages. Any dose not given at the recommended age should be given as a “ catch-up” immunization at any subsequent visit when indicated and feasible. †Different schedules exist depending upon the HBsAg status of the mother. A first vaccination after the first month is recommended only if the mother is HBsAg negative. ‡DTaP (diphtheria and tetanus toxoids and acellular pertussis vaccine) is the preferred vaccine for all doses in the immunization series. Td (tetanus and diphtheria toxoids) is recommended at 11–12 years of age if at least 5 years have elapsed since the last dose. §Only inactivated poliovirus (IPV) vaccine is now recommended for use in the United States. However, OPV remains the vaccine of choice for mass immunization campaigns to control outbreaks due to wild poliovirus. #Varicella (Var) vaccine is recommended at any visit on or after the first birthday for susceptible children, i.e., those who lack a reliable history of chickenpox (as judged by a health-care provider) and who have not been immunized. Susceptible persons 13 years of age or older should receive 2 doses, given at least 4 weeks apart. SOURCE: Adapted from the ECBT Web site (see references); approved by the American Academy of Pediatrics.
Designing Vaccines for
Active Immunization
Several factors must be kept in mind in developing a success- ful vaccine. First and foremost, the development of an im- mune response does not necessarily mean that a state of protective immunity has been achieved. What is often critical is which branch of the immune system is activated, and therefore vaccine designers must recognize the important differences between activation of the humoral and the cell- mediated branches. A second factor is the development of immunologic memory. For example, a vaccine that induces a protective primary response may fail to induce the formation of memory cells, leaving the host unprotected after the pri- mary response to the vaccine subsides. The role of memory cells in immunity depends, in part, on the incubation period of the pathogen. In the case of influenza virus, which has a very short incubation period ( or 2 days), disease symptoms are already under way by the time memory cells are activated. Effective protection against influenza therefore depends on maintaining high levels of neutralizing antibody by repeated immunizations; those at highest risk are immunized each year. For pathogens with a longer incubation period, maintaining detectable neutraliz- ing antibody at the time of infection is not necessary. The poliovirus, for example, requires more than 3 days to begin to infect the central nervous system. An incubation period of this length gives the memory B cells time to respond by producing high levels of serum antibody. Thus, the vaccine for polio is designed to induce high levels of immunologic memory. After immunization with the Salk vaccine, serum antibody levels peak within 2 weeks and then decline, but the
memory response continues to climb, reaching maximal lev- els at 6 months and persisting for years (Figure 18-3). If an immunized individual is later exposed to the poliovirus, these memory cells will respond by differentiating into plasma cells that produce high levels of serum antibody, which defend the individual from the infection. In the remainder of this chapter, various approaches to the design of vaccines—both currently used vaccines and ex- perimental ones—are described, with an examination of their ability to induce humoral and cell-mediated immunity and the production of memory cells.
Vaccines CH APTER 18 419
FIGURE 18-2 Introduction of the measles vaccine in 1962 led to a dramatic decrease in the annual incidence of this disease in the United States. Occasional outbreaks of measles in the 1980s ( inset )
occurred mainly among unvaccinated young children and among college students; most of the latter had been vaccinated, but only once, when they were young. [Data from Centers for Disease Control.]
80 81 82 83 84 85 86 87 88
15 10 5 0 Number of cases,in thousands
1950 52 54 56 58 60 62 64 66 68 70 72 74 76 78 80 82 84 86 88
Number of cases, in thousands
1, 900 800 700 600 500 400 300 200 100 0 Year
Vaccine licensed
Year
2048 1024 512 256 128 64 32 16 8 4
Mean antibody titer
Immunologic memory
Serum antibody 1 6 12 Vaccine Time, months
FIGURE 18-3 Immunization with a single dose of the Salk polio vaccine induces a rapid increase in serum antibody levels, which peak by 2 weeks and then decline. Induction of immunologic mem- ory follows a slower time course, reaching maximal levels 6 months after vaccination. The persistence of the memory response for years after primary vaccination is responsible for immunity to polio- myelitis. [From M. Zanetti et al., 1987, Immunol. Today 8: 18.]
Whole-Organism Vaccines
As Table 18-4 indicates, many of the common vaccines cur- rently in use consist of inactivated (killed) or live but attenu- ated (avirulent) bacterial cells or viral particles. The primary characteristics of these two types of vaccines are compared in Table 18-5 to one another and to DNA vaccines that are cur- rently being tested for use in humans.
Attenuated Viruses and Bacteria Cause
Immunity Without Disease
In some cases, microorganisms can be attenuated so that they lose their ability to cause significant disease (pathogenicity) but retain their capacity for transient growth within an inoc- ulated host. Attenuation often can be achieved by growing a pathogenic bacterium or virus for prolonged periods under abnormal culture conditions. This procedure selects mutants that are better suited to growth in the abnormal culture con- ditions and are therefore less capable of growth in the natural host. For example, an attenuated strain of Mycobacterium bo- vis called Bacillus Calmette-Guerin (BCG) was developed by growing M. bovis on a medium containing increasing con- centrations of bile. After 13 years, this strain had adapted to growth in strong bile and had become sufficiently attenuated that it was suitable as a vaccine for tuberculosis. The Sabin polio vaccine and the measles vaccine both consist of attenu- ated viral strains. The poliovirus used in the Sabin vaccine was attenuated by growth in monkey kidney epithelial cells. The measles vaccine contains a strain of rubella virus that was grown in duck embryo cells and later in human cell lines. Attenuated vaccines have advantages and disadvantages. Because of their capacity for transient growth, such vaccines provide prolonged immune-system exposure to the individ- ual epitopes on the attenuated organisms, resulting in in- creased immunogenicity and production of memory cells. As a consequence, these vaccines often require only a single im- munization, eliminating the need for repeated boosters. This property is a major advantage in Third World countries, where epidemiologic studies have shown that roughly 20% of individuals fail to return for each subsequent booster. The ability of many attenuated vaccines to replicate within host cells makes them particularly suitable for inducing a cell- mediated response. The Sabin polio vaccine, consisting of three attenuated strains of poliovirus, is administered orally to children on a sugar cube or in sugar liquid. The attenuated viruses colonize the intestine and induce protective immunity to all three strains of virulent poliovirus. Sabin vaccine in the intestines induces production of secretory IgA, which serves as an im- portant defense against naturally acquired poliovirus. The vaccine also induces IgM and IgG classes of antibody. Unlike most other attenuated vaccines, which require a single im- munizing dose, the Sabin polio vaccine requires boosters,
420 PART I V The Immune System in Health and Disease
TABLE 18-4 Classification of common vaccines
for humans
Disease or pathogen Type of vaccine
WHOLE ORGANISMS
Bacterial cells Anthrax Inactivated Cholera Inactivated Pertussis* Inactivated Plague Inactivated Tuberculosis Live attenuated BCG† Typhoid Live attenuated
Viral particles Hepatitis A Inactivated Influenza Inactivated Measles Live attenuated Mumps Live attenuated Polio (Sabin) Live attenuated Polio (Salk) Inactivated Rabies Inactivated Rotavirus Live attenuated Rubella Inactivated Varicella zoster (chickenpox) Live attenuated Yellow fever Live attenuated
PURIFIED MACROMOLECULES
Toxoids Diphtheria Inactivated exotoxin Tetanus Inactivated exotoxin
Capsular polysaccharides Haemophilus influenzae Polysaccharide type b protein carrier Neissera meningitidis Polysaccharide Streptococcus pneumoniae 23 distinct capsular polysaccharides Surface antigen Hepatitis B Recombinant surface antigen (HBsAg) *There is an now also an acellular pertussis vaccine consisting of toxoids and inactivated bacteria components. †Bacillus Calmette-Guerin (BCG) is an avirulent strain of Mycobacterium bovis.
cell-mediated immunity and in eliciting a secretory IgA response. Even though they contain killed pathogens, inactivated whole-organism vaccines are still associated with certain
risks. A serious complication with the first Salk vaccines arose when formaldehyde failed to kill all the virus in two vaccine lots, which caused paralytic polio in a high percent- age of recipients.
422 PART I V The Immune System in Health and Disease
FIGURE 18-4 Progress toward the worldwide eradication of polio. Comparison of infection numbers for 1988 with those for 1998 show considerable progress in most parts of the world. Some experts question whether the use of live attenuated oral polio vaccine will
cause reversion to pathogenic forms at a rate sufficiently high to pre- vent total eradication of this once prevalent crippling disease. [Data from WHO.]
0 cases
Reported polio cases 1988
1–10 cases
More than 10 cases
No report
0 cases
1998
1–10 cases
More than 10 cases
No report
Purified Macromolecules as Vaccines
Some of the risks associated with attenuated or killed whole- organism vaccines can be avoided with vaccines that consist of specific, purified macromolecules derived from patho- gens. Three general forms of such vaccines are in current use: inactivated exotoxins, capsular polysaccharides, and recom- binant microbial antigens (see Table 18-4).
Bacterial Polysaccharide Capsules
Are Used as Vaccines
The virulence of some pathogenic bacteria depends primarily on the antiphagocytic properties of their hydrophilic polysac- charide capsule. Coating of the capsule with antibodies and/ or complement greatly increases the ability of macrophages and neutrophils to phagocytose such pathogens. These find- ings provide the rationale for vaccines consisting of purified capsular polysaccharides. The current vaccine for Streptococcus pneumoniae , which causes pneumococcal pneumonia, consists of 23 antigeni- cally different capsular polysaccharides. The vaccine induces formation of opsonizing antibodies and is now on the list of vaccines recommended for all infants. The vaccine for Neisse- ria meningitidis , a common cause of bacterial meningitis, also consists of purified capsular polysaccharides. One limitation of polysaccharide vaccines is their inabil- ity to activate TH cells. They activate B cells in a thymus- independent type 2 (TI-2) manner, resulting in IgM produc- tion but little class switching, no affinity maturation, and little, if any, development of memory cells. Several investigators have
reported the induction of IgA-secreting plasma cells in hu- mans receiving subcutaneous immunization with the pneu- mococcal polysaccharide vaccine. In this case, since TH cells are not involved in the response, the vaccine may activate IgA- specific memory B cells previously generated by naturally- occurring bacterial antigens at mucosal surfaces. Because these bacteria have both polysaccharide and protein epitopes, they would activate TH cells, which in turn could mediate class switching and memory-cell formation. One way to involve TH cells directly in the response to a polysaccharide antigen is to conjugate the antigen to some sort of protein carrier. For example, the vaccine for Haemo- philus influenzae type b (Hib), the major cause of bacterial meningitis in children less than 5 years of age, consists of type b capsular polysaccharide covalently linked to a protein carrier, tetanus toxoid. The polysaccharide-protein conjugate is considerably more immunogenic than the polysaccharide alone, and because it activates TH cells, it enables class switch- ing from IgM to IgG. Although this type of vaccine can in- duce memory B cells, it cannot induce memory T cells specific for the pathogen. In the case of the Hib vaccine, it ap- pears that the memory B cells can be activated to some degree in the absence of a population of memory TH cells, thus ac- counting for the efficacy of this vaccine.
Toxoids Are Manufactured from Bacterial Toxins Some bacterial pathogens, including those that cause diph- theria and tetanus, produce exotoxins. These exotoxins produce many of the disease symptoms that result from
Vaccines CH APTER 18 423
TABLE 18-
Risk of complications from natural measles infection compared with known risks
of vaccination with a live attenuated virus in immunocompetent individuals
Complication Risk after natural disease * Risk after vaccination†
Otitis media 7–9% 0 Pneumonia 1–6% 0 Diarrhea 66% 0 Post-infectious encephalomyelitis 0.5–1 per 1000 1 per 1,000, SSPE 1 per 100,000 0 Thrombocytopenia —‡^ 1 per 30,000§ Death 0.1–1 per 1000 (up to 5–15% in 0 developing countries) *Risk after natural measles are calculated in terms of events per number of cases. †Risks after vaccination are calculated in terms of events per number of doses. ‡Although there have been several reports of thrombocytopenia occurring after measles including bleeding, the risk has not been properly quantified. §This risk has been reported after MMR vaccination and cannot only be attributed to the measles component. MMR measles, mumps, and rubella. SSPE subacute sclerosing panencephalitis.
Vaccines CH APTER 18 425
Tissue-culture cells
Restriction-enzyme Vaccinia cleavage site promoter TK gene TK gene
Vaccinia promoter TK gene TK gene
Recombinant plasmid
DNA encoding Ag from pathogen
Cleavage and ligation
Gene from pathogen
Transfection Infection
Vaccinia virus
Homologous recombination
BUdr selection
Recombinant vaccinia vector vaccine
Plasmid
FIGURE 18-5 Production of vaccinia vector vaccine. The gene that encodes the desired antigen (orange) is inserted into a plasmid vec- tor adjacent to a vaccinia promoter (pink) and flanked on either side by the vaccinia thymidine kinase ( TK ) gene (green). When tissue- culture cells are incubated simultaneously with vaccinia virus and the recombinant plasmid, the antigen gene and promoter are inserted into the vaccinia virus genome by homologous recombination at the site of the nonessential TK gene, resulting in a TK ^ recombinant virus. Cells containing the recombinant vaccinia virus are selected by addi- tion of bromodeoxyuridine (BUdr), which kills TK^ cells. [Adapted from B. Moss, 1985, Immunol. Today 6: 243.]
genes. Unlike vaccinia, the canarypox virus does not appear to be virulent even in individuals with severe immune suppres- sion. Another possible vector is an attenuated strain of Salmo- nella typhimurium , which has been engineered with genes
from the bacterium that causes cholera. The advantage of this vector vaccine is that Salmonella infects cells of the mucosal lining of the gut and therefore will induce secretory IgA pro- duction. Effective immunity against a number of diseases, including cholera and gonorrhea, depends on increased pro- duction of secretory IgA at mucous membrane surfaces. Simi- lar strategies using bacteria that are a normal part of oral flora are in development. The strategy would involve introduction of genes encoding antigens from pathogenic organisms into bacterial strains that inhabit the oral cavity or respiratory tract. Eliciting immunity at the mucosal surface could provide excel- lent protection at the portal used by the pathogen.
DNA Vaccines In a recently developed vaccination strategy, plasmid DNA encoding antigenic proteins is injected directly into the mus- cle of the recipient. Muscle cells take up the DNA and the encoded protein antigen is expressed, leading to both a humoral antibody response and a cell-mediated response. What is most surprising about this finding is that the injected DNA is taken up and expressed by the muscle cells with much greater efficiency than in tissue culture. The DNA ap- pears either to integrate into the chromosomal DNA or to be maintained for long periods in an episomal form. The viral antigen is expressed not only by the muscle cells but also by dendritic cells in the area that take up the plasmid DNA and express the viral antigen. The fact that muscle cells express low levels of class I MHC molecules and do not express co- stimulatory molecules suggests that local dendritic cells may be crucial to the development of antigenic responses to DNA vaccines (Figure 18-6). DNA vaccines offer advantages over many of the existing vaccines. For example, the encoded protein is expressed in the host in its natural form—there is no denaturation or modifi- cation. The immune response is therefore directed to the anti- gen exactly as it is expressed by the pathogen. DNA vaccines also induce both humoral and cell-mediated immunity; to stimulate both arms of the immune response with non-DNA vaccines normally requires immunization with a live attenu- ated preparation, which introduces additional elements of risk. Finally, DNA vaccines cause prolonged expression of the antigen, which generates significant immunological memory. The practical aspects of DNA vaccines are also very promising (Table 18-5). Refrigeration is not required for the handling and storage of the plasmid DNA, a feature that greatly lowers the cost and complexity of delivery. The same plasmid vector can be custom tailored to make a variety of proteins, so that the same manufacturing techniques can be used for different DNA vaccines, each encoding an antigen from a different pathogen. An improved method for admin- istering these vaccines entails coating microscopic gold beads with the plasmid DNA and then delivering the coated parti- cles through the skin into the underlying muscle with an air gun (called a gene gun ). This will allow rapid delivery of a
426 PART I V The Immune System in Health and Disease
FIGURE 18-6 Use of DNA vaccines raises both humoral and cel- lular immunity. The injected gene is expressed in the injected muscle cell and in nearby APCs. The peptides from the protein encoded by the DNA are expressed on the surface of both cell types after pro- cessing as an endogenous antigen by the MHC class I pathway. Cells that present the antigen in the context of class I MHC molecules
stimulate development of cytotoxic T cells. The protein encoded by the injected DNA is also expressed as a soluble, secreted protein, which is taken up, processed, and presented in the context of class II MHC molecules. This pathway stimulates B-cell immunity and gen- erates antibodies and B-cell memory against the protein. [Adapted from D. B. Weiner and R. C. Kennedy, 1999, Sci. Am. 281: 50.]
TH 1 cytokines
Cytokines prime cytotoxic T cells for action
Cytokine receptor
Gene for antigenic protein
Plasmid vaccine
Helper T cell
Activated B cell
Activated cytotoxic T cells
Dying inoculated cell
Helper T cell
Cytokines help to activate B cells
MHC I MHC II
Co-stimulatory molecule
Antigen binding triggers display of cytokine receptors
Antigen binding triggers release of cytokines
Local antigen-presenting cell Local antigen-presenting cell
Cytotoxic T cell
Antigen binding causes cytotoxic T cells to mutiply and attack inoculated cells
Antigen binding triggers release of antibodies
PRIMING OF CELLULAR IMMUNE RESPONSE PRIMING OF HUMORAL IMMUNE RESPONSE
Antibodies
Memory B cell
Memory T cell
Some T cells become memory cells
Some B cells become memory cells
Antigenic peptides
Plasmids yield antigenic protein
Antigen TH 2 cytokines
One approach is to prepare solid matrix–antibody- antigen (SMAA) complexes by attaching monoclonal anti- bodies to particulate solid matrices and then saturating the antibody with the desired antigen. The resulting complexes are then used as vaccines. By attaching different monoclonal antibodies to the solid matrix, it is possible to bind a mixture of peptides or proteins, composing immunodominant epi- topes for both T cells and B cells, to the solid matrix (see Fig- ure 18-7a). These multivalent complexes have been shown to induce vigorous humoral and cell-mediated responses. Their particulate nature contributes to their increased immuno- genicity by facilitating phagocytosis by phagocytic cells. Another means of producing a multivalent vaccine is to use detergent to incorporate protein antigens into protein mi- celles, lipid vesicles (called liposomes), or immunostimulat- ing complexes (see Figure 18-7b). Mixing proteins in deter- gent and then removing the detergent forms micelles. The in- dividual proteins orient themselves with their hydrophilic residues toward the aqueous environment and the hydropho- bic residues at the center so as to exclude their interaction with the aqueous environment. Liposomes containing pro- tein antigens are prepared by mixing the proteins with a sus- pension of phospholipids under conditions that form vesicles bounded by a bilayer. The proteins are incorporated into the bilayer with the hydrophilic residues exposed. Immunostimu- lating complexes (ISCOMs) are lipid carriers prepared by mixing protein with detergent and a glycoside called Quil A. Membrane proteins from various pathogens, including in- fluenza virus, measles virus, hepatitis B virus, and HIV have been incorporated into micelles, liposomes, and ISCOMs and are currently being assessed as potential vaccines. In addition to their increased immunogenicity, liposomes and ISCOMs appear to fuse with the plasma membrane to deliver the anti- gen intracellularly, where it can be processed by the cytosolic pathway and thus induce a cell-mediated response (see Figure 18-7c).
SUMMARY
■ (^) A state of immunity can be induced by passive or active immunization a) Short-term passive immunization is induced by transfer of preformed antibodies. b) Infection or inoculation achieves long-term active immu- nization.
■ (^) Three types of vaccines are currently used in humans: at- tenuated (avirulent) microorganisms, inactivated (killed) microorganisms, or purified macromolecules.
■ (^) Protein components of pathogens expressed in cell culture may be effective vaccines.
■ (^) Recombinant vectors, including viruses or bacteria, engi- neered to carry genes from infectious microorganisms, maximize cell-mediated immunity to the encoded antigens.
■ (^) Plasmid DNA encoding a protein antigen from a pathogen can serve as an effective vaccine inducing both humoral and cell-mediated immunity. ■ (^) Realizing the optimum benefit of vaccines will require cheaper manufacture and improved delivery methods for existing vaccines.
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USEFUL WEB SITES
http:/ / www.VaccineAlliance.org/ Homepage of global alliance for vaccines and immunization (GAVI), a source of information about vaccines in developing countries and worldwide efforts at disease eradication. http:/ / www.ecbt.org/ Every Child by Two offers information on childhood vaccina- tion.
428 PART I V The Immune System in Health and Disease
Study Questions
CLINICAL FOCUS QUESTION A connection between the new pneu- mococcus vaccine and a relatively rare form of arthritis has been reported. What data would you need to validate this report? How would you proceed to evaluate this possible connection?
1. Indicate whether each of the following statements is true or false. If you think a statement is false, explain why. a. Transplacental transfer of maternal IgG antibodies against measles confers short-term immunity on the fetus. b. Attenuated vaccines are more likely to induce cell-mediated immunity than killed vaccines are. c. Multivalent subunit vaccines generally induce a greater re- sponse than synthetic peptide vaccines. d. One disadvantage of DNA vaccines is that they don’t gen- erate significant immunologic memory. e. Macromolecules generally contain a large number of potential epitopes. f. A DNA vaccine only induces a response to a single epitope. 2. What are the advantages and disadvantages of using attenu- ated organisms as vaccines? 3. A young girl who had never been immunized to tetanus stepped on a rusty nail and got a deep puncture wound. The doctor cleaned out the wound and gave the child an injection of tetanus antitoxin.
a. Why was antitoxin given instead of a booster shot of tetanus toxoid? b. If the girl receives no further treatment and steps on a rusty nail again 3 years later, will she be immune to tetanus?
4. What are the advantages of the Sabin polio vaccine compared with the Salk vaccine? Why is the Sabin vaccine no longer rec- ommended for use in the United States? 5. In an attempt to develop a synthetic peptide vaccine, you have analyzed the amino acid sequence of a protein antigen for (a) hydrophobic peptides and (b) strongly hydrophilic peptides. How might peptides of each type be used as a vaccine to in- duce different immune responses? 6. Explain the phenomenon of herd immunity. How does this phenomenon relate to the appearance of certain epidemics? 7. You have identified a bacterial protein antigen that confers protective immunity to a pathogenic bacterium and have cloned the gene that encodes it. The choices are either to ex- press the protein in yeast and use this recombinant protein as a vaccine, or to use the gene for the protein to prepare a DNA vaccine. Which approach would you take and why? 8. Explain the relationship between the incubation period of a pathogen and the approach needed to achieve effective active immunization. 9. List the three types of purified macromolecules that are cur- rently used as vaccines.
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