The Ne w E n g l a nd Jo u r n a l o f Me d ic i ne

Review Article

Advances in Immunology I A N R . M A C K A Y , M. D . , A N D F R E D S . R O S E N , M .D ., Ed i t ors

V ACCINES

AND

V ACCINATION

GORDON ADA, D.SC.

M

ORE than 70 bacteria, viruses, parasites, and fungi are serious human pathogens.1 Vaccines are available against some of these agents and are being developed against almost all the other bacteria and viruses and about half of the parasites. Table 1 lists infections for which there are now licensed vaccines and those for which a candidate vaccine has undergone a phase 3 clinical trial,2 indicating that a vaccine will probably be licensed within 5 to 10 years. Traditionally, attenuated vaccines were made by repeated passaging of the infectious agent in tissue culture or animal hosts until its virulence was greatly decreased but its immunogenicity was retained. Alternatively, chemicals such as formalin were used to destroy infectivity. More recently, parts of an infectious agent, usually a surface antigen, have been used as a subunit vaccine. The current vaccines against hepatitis B virus and Lyme disease rely on recombinant-DNA technology. Bacterial toxins are rendered nontoxic by chemical treatment, and the resulting toxoid protects against the infectious agent. Protection against some types of encapsulated bacteria has been achieved by immunization with a capsular oligosaccharide or polysaccharide, but these T-cell–independent antigens induce only IgM antibodies, which are poorly protective in infants. Conjugating this saccharide to a protein or proteinaceous complex induces IgG antibodies because T cells recognize the complex of a peptide with a major-histocompatibility-complex molecule on the antigen-presenting cell (Fig. 1). The Haemophilus influenzae type b conjugate vaccines also induce mucosal immunity, which reduces nasal carriage of the bacteria. Such conjugates protect infants very effectively.

From the John Curtin School for Medical Research, Australian National University, Canberra, Australia. Address reprint requests to Dr. Ada at the John Curtin School for Medical Research, Australian National University, Box 334, ACT 2601 Canberra, Australia.

EFFICACY OF SOME CHILDHOOD VACCINES

Records kept by the Centers for Disease Control and Prevention (CDC) since 1912 reveal the number of reported cases of an infectious disease before and after a vaccine became available.4 The decrease is remarkable: 100 percent in the case of indigenous poliomyelitis (the last case in the Americas was in Peru in 1992); over 99 percent in the case of diphtheria, measles, mumps, and rubella; and over 97 percent in the case of whooping cough (caused by Bordetella pertussis). All these agents undergo little antigenic variation (or drift) or none at all, showing that under virtually ideal conditions, vaccination can be extraordinarily effective.4 Within one year after the introduction in 1999 of a Neisseria meningitidis serogroup C conjugate vaccine in the United Kingdom, the incidence of meningitis was reduced by 92 percent among young children and by 95 percent among teenagers.5 A Salmonella typhi Vi conjugate vaccine (Vi-rEPA) reduced the incidence of typhoid fever among two-to-fouryear-old children by more than 90 percent.6 Both findings confirm the remarkable effectiveness of conjugate vaccines. The experience with measles in the United States is of interest. From 1912 until 1963, the incidence never dropped below 100,000 cases per year, and epidemics were common. After the introduction of the first vaccine in 1963, the number of cases fell to very low levels and remained so until 1990, when there was an epidemic lasting three years and involving nearly 28,000 patients, most of whom were adolescents or young adults. This resurgence was due to inadequate vaccination of these patients at the age of one to two years in major urban areas. The recognition that immunity can wane after vaccination led to a two-dose vaccination schedule, which prevented the transmission of indigenous measles infections within the United States, Canada, and Finland. Sometimes, vaccination can fail, indicating that it induced a suboptimal immune response. The failure to respond or a low level of response to a simple vaccine, such as the hepatitis B vaccine, can be circumvented by adding additional helper-T-cell epitopes to the vaccine.7 In the case of varicella–zoster virus, like other such viruses, which induce latent infections, a live attenuated vaccine may not eliminate infection but does prevent chickenpox. SAFETY OF VACCINES

Adverse events associated with childhood vaccines are sometimes grouped as early and late reactions.

1042 · N Engl J Med, Vol. 345, No. 14 · October 4, 2001 · www.nejm.org Downloaded from www.nejm.org at UNIVERSITA GD ANNUNZIO on March 12, 2010 . Copyright © 2001 Massachusetts Medical Society. All rights reserved.

ADVA NC ES IN IMMUNOLOGY

TABLE 1. THE STATUS

OF

VACCINES

AGAINST

SOME HUMAN PATHOGENS. TYPES

INFECTIOUS AGENT

VACCINE STATUS*

DISEASE

TRADITIONAL VACCINE

OF

USE

OR

TARGET POPULATION

Bacteria Bacillus anthracis Bordetella pertussis Borrelia burgdorferi

Available Available Available

Anthrax Whooping cough Lyme disease

Inactivated Inactivated, subunit Subunit

Clostridium tetani Corynebacterium diphtheriae Coxiella burnetii

Available Available Available

Tetanus Diphtheria Severe fever (Q fever)

Toxoid Toxoid Inactivated

Haemophilus influenzae

Available

Meningitis, epiglottitis, pneumonia type b Leprosy Tuberculosis

Conjugated

To limit biologic warfare Children and adults Residents of areas of endemic disease in the United States Children Children Workers in slaughterhouses and meatprocessing plants Children

Inactivated Live attenuated

Residents of areas of endemic disease All persons

Subunit, conjugated Conjugated Live attenuated, polysaccharide Conjugated

Children

Mycobacterium leprae M. tuberculosis Neisseria meningitidis Serogroup B Serogroup C Salmonella typhi

Phase 3 clinical trials Available Available (Ty21a vaccine)

Meningitis Meningitis Typhoid fever Typhoid fever

Staphylococcus aureus

Phase 3 clinical trials (Vi-rEPA vaccine) Phase 3 clinical trials

Phase 3 clinical trials Available

Streptococcus pneumoniae

Available

Vibrio cholerae

Available

Impetigo, toxic shock syndrome in women Pneumonia, otitis media, meningitis Cholera

Viruses Adenovirus Hepatitis A

Available Available

Respiratory disease Liver disease, cancer

Available

Liver disease, cancer

Available

Hepatitis B Influenzavirus A B Japanese encephalitis virus

Phase 3 clinical trials Available

Conjugated

Those at high risk, those with eczema, those with neutrophil dysfunction Elderly persons

Live attenuated, inactivated

Residents of and travelers to areas of endemic disease

Live attenuated Live attenuated, inactivated Subunit

Military personnel Residents of and travelers to areas of endemic disease All persons

Respiratory disease

Live attenuated, inactivated, subunit

Children (live attenuated vaccine only), elderly persons

Brain infection

Inactivated Live attenuated

Residents of and travelers to areas of endemic disease Children and adolescents

Live attenuated

Children and adolescents

Live attenuated, inactivated Inactivated

Children

Measles virus

Available

Mumps virus

Available

Poliovirus

Available

Respiratory tract infection, SSPE† Mumps, meningitis, orchitis Poliomyelitis, paralysis

Rabies virus

Available

Rabies

Rubella virus

Available

Vaccinia virus Varicella–zoster virus Yellow fever virus

Available Available Available

German measles, fetal malformations Smallpox Chickenpox Jaundice, kidney and liver failure

Conjugated

Residents of and travelers to areas of endemic disease, children

Live attenuated

Exposed persons, residents of areas of endemic disease Children

Live attenuated Live attenuated Live attenuated

Laboratory workers Children Residents of areas of endemic disease, particularly children

Parasites Leishmania

Phase 3 clinical trials

Kala-azar, tropical sores

Live attenuated, inactivated

Residents of countries where disease is endemic

Fungus Coccidioides immitis

Phase 3 clinical trials

Lung infection

Inactivated

Residents of countries where disease is endemic

*Vaccines that have been evaluated in phase 3 clinical trials should be available in 5 to 10 years. †SSPE denotes subacute sclerosing panencephalitis.

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The Ne w E n g l a nd Jo u r n a l o f Me d ic i ne

Reactions within the first 24 hours include erythema and swelling at the injection site, fever, prolonged crying, syncope, seizures, and rarely, hypotonic, hyporesponsive episodes or anaphylaxis. Reactions that occur within a few weeks after vaccination include encephalitis and encephalopathy and sometimes lead to clinically significant brain damage. In the United Kingdom in the 1970s, fears that the whole-cell pertussis vaccine induced brain damage caused vaccination levels to drop to approximately 30 percent. Two subsequent outbreaks of whooping cough caused more than 30 deaths, and many of the infected children suffered brain damage.8 A subsequent review of the evidence failed to substantiate the association between the vaccine and brain damage. Evidence of an adverse reaction may appear only after a vaccine has been licensed. This was the case with a rotavirus vaccine in the United States, which was associated with an unacceptably high incidence of intussusception.9 A demyelinating encephalopathy occurs after measles vaccination in about 1 in a million recipients; the incidence of this complication after natural measles infection is 1 per 1000.10 The incidence of subacute sclerosing panencephalitis, in which the measles virus is directly involved, decreased by at least 90 percent after measles vaccination became widespread. The Guillain–Barré syndrome develops in about 1 in a million recipients of the influenza vaccine. The oral poliovirus vaccine eradicated poliomyelitis from the Americas, but in the United States, the vaccine caused about 1 case of paralysis in a vaccinee or close contact per million doses of the vaccine, as a result of the reversion of the type 3 virus strain used in the vaccine to virulence. The CDC Advisory Committee on Immunization Practices and the American Academy of Pediatrics recommended that only inactivated poliovirus vaccine be used after January 1, 2000.11 Overall, there is no firm scientific or clinical evidence that the administration of any vaccine causes a specific allergy, asthma, autism, multiple sclerosis, or the sudden infant death syndrome. A widely cited report claimed an association between vaccination

against measles (usually with measles, mumps, and rubella vaccine) and the subsequent occurrence of inflammatory bowel disease or autism.12 At least 10 studies13,14 found no evidence to substantiate such an association. GLOBAL VACCINE DELIVERY

Four conditions are essential for the success of a vaccine-based eradication program: the infection must be limited to humans, with no animal reservoir; in the case of viral infections, there must be only one or a few strains of the virus and these must have constant antigenic properties; the virus must not persist in the infected host; and there must be an effective vaccine. In 1966 the estimated number of cases of smallpox worldwide was 20 million. The last case of endemic smallpox occurred in 1977, and eradication of the disease was announced in 1980.15 Poliomyelitis became the next target for eradication, through the administration of the oral poliovirus vaccine. This task is more difficult because the vaccine is heat labile, several doses are required, the vaccine itself can induce paralysis (although very rarely), and unlike the case with smallpox, there is no simple test to indicate that vaccination has been successful. Indigenous poliomyelitis has already been eliminated from the Americas, Europe, the western Pacific region, and Southeast Asia, but it will take a few more years to achieve global eradication.16 Measles is the most contagious infection of humans and causes 30 percent of all deaths due to vaccine-preventable diseases. The successful interruption of the transmission of measles infection in countries with very high rates of vaccination coverage is indicative of the progress being made toward the goal of eliminating measles from the Americas. The World Health Organization (WHO) Expanded Programme on Immunization increased the level of vaccination to control tetanus, diphtheria, whooping cough, tuberculosis, measles, and poliomyelitis in developing countries from 5 percent in 1974 to an average of 80 percent by the 1980s, and it has since remained at about that level. The goals of an impor-

Figure 1 (facing page). Antibody Responses to Polysaccharide Antigens and Polysaccharide–Protein Conjugates. In Panel A, a polysaccharide antigen binds to an IgM receptor on the surface of a B cell in lymphoid tissues. Once B cells are activated, they produce and then secrete IgM antibody molecules. The individual Fab segments of the IgM molecule have only a moderate affinity, but because there are 10 such segments, an IgM molecule has a high avidity. In contrast, in Panel B, some polysaccharide–protein conjugates will be taken up by dendritic cells, which present peptides from the protein portion of the conjugate to type 2 helper T (Th2) cells. Other conjugate molecules bind to B cells that have IgM receptors specific for the carbohydrate moiety and will undergo endocytosis and be processed by the B cell; the resulting peptides will be expressed with class II MHC molecules on the surface of the B cell. This complex is recognized by the activated Th2 cell, which then secretes interleukin-4, interleukin-5, and interleukin-6. These cause the B cell to differentiate and express IgG molecules with polysaccharide specificity. These cells mature in the lymphoid follicles; only cells that express very-high-affinity IgG molecules become plasma cells and secrete highaffinity IgG that binds strongly to the encapsulated bacteria and mediates opsonic activity and complement-mediated bactericidal activities. A recent study3 suggests that the formation of memory B cells is a critical component of protective immunity against infection with Haemophilus influenzae type b.

1044 · N Engl J Med, Vol. 345, No. 14 · October 4, 2001 · www.nejm.org Downloaded from www.nejm.org at UNIVERSITA GD ANNUNZIO on March 12, 2010 . Copyright © 2001 Massachusetts Medical Society. All rights reserved.

ADVA NC ES IN IMMUNOLOGY

A B cell

Activated B cell

Polysaccharide IgM Activation B B cell

Polysaccharide–protein> conjugate Antigen-> presenting> cell

Interleukin-4, 5, and 6 Th2 cell Activation

Peptide

Differentiation> >

MHC–peptide > complex

Plasma cell Memory B cell IgG

tant new group, the Global Alliance for Vaccines and Immunization, are to eradicate poliomyelitis, to increase rates of childhood vaccination to over 90 percent worldwide, and to include in this coverage vaccines against hepatitis B and H. influenzae type b infections.17

A WHO world health report in 199818 stated that there were about 5 billion cases of disease and 6 million to 7 million deaths annually from childhood diarrhea, including those due to rotavirus infections,19 and acute respiratory tract infections, especially those caused by respiratory syncytial virus.2 Infections with

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IMMUNE RESPONSES AND THE CONTROL OF INFECTIONS

There are two patterns of infection. All viruses and some bacteria and parasites are obligate intracellular agents that can live and replicate only inside cells. Other types of bacteria and parasites exist and replicate extracellularly. Also, infections are either acute or persistent. Infections are considered acute when a sublethal dose of the agent is controlled and rapidly cleared by the immune system; most current vaccines are directed against such infections. Persistent infections occur when the agent succeeds in evading or subverting elements of the immune response. The functions of different classes of antibody — IgM, IgE, IgA, and subclasses of IgG — in the control of infections include the prevention or limitation of the initial infection and subsequent viremia or bacteremia and the killing of infected cells by antibodydependent cellular cytotoxicity or complement-mediated lysis.21 In the case of extracellular infections, specific antibody needs the support of a strong response by CD4+ type 1 helper T (Th1) cells. During intracellular infections, important T-cell responses precede the formation of substantial levels of antimicrobial antibody. The sequence of two main responses, that of cytotoxic T cells and the response of cells that secrete specific antibody in the lungs of mice infected intranasally with influenzavirus, is shown in Figure 2. The decrease in the level of infectious virus coincides with the increase in the activity of cytotoxic T cells.22 In humans infected with HIV-1, the decrease in the levels of infectious virus in the blood also coincides with the appearance of virus-specific cytotoxic T cells and occurs long before neutralizing antibody appears.23,24 That effector T cells are primarily responsible for controlling and sometimes clearing many different intracellular infections has been fully documented for many infections. Class I–Restricted CD8+ Cytotoxic T Cells

Since all types of cells, except gametes, neurons, red cells, and cells of the trophoblast, express MHC class I antigens, most infected cells should be recog-

Virus CD8+ CTs Antibody-producing> cells

Titer

human immunodeficiency virus type 1 (HIV-1), tuberculosis, and malaria cause 7 million to 8 million deaths annually, mainly in developing countries. The incidence of sexually transmitted diseases in developed countries is steadily increasing.20 Vaccination offers the greatest opportunity to decrease this toll. The primary purpose of currently available vaccines is to induce strong antibody responses capable of neutralizing infectivity, but attempts to develop such traditional types of vaccines against many important diseases have been unsuccessful. There is a strong need for new types of vaccines that not only are more potent overall, but also induce either a stronger humoral response or a cell-mediated immune response.

4

8

1

2

Days

6

18

Months

Figure 2. The Immune Response to Intranasal Infection with Influenzavirus. During the first few days after the inoculation of influenzavirus, the titer of infectious virus and the levels of CD8+ cytotoxic T cells (CTs) rise. Approximately four days after infection, the levels of antibody-producing cells begin to rise. Cytotoxic T cells appear before antibody-producing cells. The decrease in the titer of infectious virus coincides with the increase in the activity of cytotoxic T cells, which, in turn, decreases and then disappears within a few days after infectious virus becomes undetectable. Large numbers of memory cytotoxic T cells are present at 2 and 6 weeks, and some are still present at 12 months. IgM-producing cells precede the appearance of IgA- and IgG-producing cells by a few days, and these are present in large numbers for about 12 months, after which only IgG-producing cells are found; these are found in steadily decreasing numbers. The numbers of memory B cells are highest approximately 2 months after infection, but these cells persist in the spleen for at least 18 months. Data are from Ada and Jones.22

nized by cytotoxic T cells. Infected cells become susceptible to lysis by specific cytotoxic T cells well before the release of viral progeny, allowing a window of time for the effector T cell to seek and destroy infected cells before the progeny arise. In addition to directly participating in limiting infections by killing infected cells, cytotoxic T cells secrete potent cytokines with antiviral and macrophage-activating activities, such as interferon-g and tumor necrosis factor a. There are four situations in which persons who have been exposed to HIV-1 but not treated with antiviral agents are negative for the virus and seronegative but possess HIV-1–specific CD8+ cytotoxic T cells or interferon-g produced by such cells: babies born to infected women,25,26 long-term partners of infected people,27,28 long-term female African prostitutes,29 and some health care workers who were exposed once to the virus.30 Furthermore, transfer of a potent anti-CD8 serum to monkeys before infection with simian immunodeficiency virus (SIV) and shortly thereafter caused a large, transient increase in viral levels31-33 and a decrease in the levels of CD4+ T cells.31 Regional Immunity

Mucosal surfaces cover an area far greater than that of the skin and are well endowed with associated lymphoid tissues. The exception is the vagina, which is normally colonized by about six different bacteria that maintain an environment hostile to coloniza-

1046 · N Engl J Med, Vol. 345, No. 14 · October 4, 2001 · www.nejm.org Downloaded from www.nejm.org at UNIVERSITA GD ANNUNZIO on March 12, 2010 . Copyright © 2001 Massachusetts Medical Society. All rights reserved.

ADVA NC ES IN IMMUNOLOGY

tion by other bacteria. There is a common mucosal system so that immunization at one site may afford protection at another site. Thus, the adenovirus vaccine is administered orally but protects against infection of the respiratory tract.34 In mice and monkeys, immunization by means of the respiratory tract is an effective way to induce a strong response in the genital tract,35 a finding relevant to the development of vaccines against sexually transmitted diseases. Infection or vaccination of a mucosal surface not only elicits the production of secretory IgA but may also induce cytotoxic T cells to enter the site. For example, specific cytotoxic T cells are present in specimens obtained with a cytobrush from the cervix in some HIV-1–infected women.36 Evasion, Suppression, and Subversion of Immune Responses in Intracellular Infections

Microbes subvert humoral immunity mainly by varying the sequence of surface antigens.37 Other tactics include having weak immunogenicity and inaccessible epitopes in surface antigens recognized by neutralizing antibodies and forming a complex between the microbe and an antibody in order to enhance the likelihood of infected cells that express Fc or complement receptors, such as macrophages. HIV-1 uses all three strategies.38 A persistent infection usually occurs when the cell-mediated response is suppressed or subverted. HIV-1 employs all of the following strategies38: latent infection; infection of sites that are inaccessible to immune-response mechanisms; destruction of CD4+ T cells; down-regulation of the expression of class I MHC molecules; mutation, which changes viral peptide sequences, rendering existing effector T cells ineffective; and inhibition of the activity of cytotoxic T cells. The design of a vaccine that foils the subterfuges of HIV-1 will not be easy, but new forms of vaccine technology may overcome the difficulties. NEW APPROACHES TO VACCINES Production of Antigens and Antibodies in Transgenic Plants

Viral and bacterial antigens have been produced in transgenic plants.39,40 Hepatitis B surface antigen, Escherichia coli enterotoxin, and rabies virus glycoprotein that have been produced in transgenic plants induce IgG antibodies with the correct antigenic specificity after oral administration to mice. The potential advantages of this approach include its low cost and the ability to effect vaccination simply by having a person eat a selected part of the transgenic plant. For example, mice fed potato tubers containing one or more foreign antigens had antigen-specific mucosal IgA and serum IgG. Pigs fed transgenic potatoes expressing the protective protein of transmissible gastroenteritis virus had a significant reduction in morbidity and mortality when challenged with infectious virus.

In addition to vaccine antigens, specific antibodies (“plantibodies”) have also been made in plants. An antibody against Streptococcus mutans, which contributes to tooth decay, was applied to the specially cleaned mouths of volunteers and prevented recolonization of the mouth by these bacteria for four months.41 Many such antibodies are now available.42 Transcutaneous Immunization

Transcutaneous immunization involves the application of an antigen with an adjuvant, frequently cholera toxin, to intact skin, prewashed to facilitate penetration.43 In mice, the reagents enter the epidermis, where they come into contact with and are taken up by Langerhans’ cells, a class of dendritic cells. During their migration through the dermis by means of afferent lymphatics and thence to the draining lymph nodes, the dendritic cells mature and become highly effective antigen-presenting cells (Fig. 3). In the lymph node, they contact and activate T cells, thus initiating a strong antibody response to antigens such as diphtheria toxoid. VACCINE DEVELOPMENT Peptides, Subunits, and Adjuvants

The use of peptides, which are only parts of an antigen, as vaccines has many advantages and some drawbacks.1 The advantages include the fact that the product is chemically defined, stable, and safe and contains only important B-cell and T-cell epitopes. The drawbacks include difficulty in mimicking the conformation of antigen polymers found with many viruses, the fact that B-cell epitopes recognized by neutralizing antibodies are sometimes discontinuous sequences, and the susceptibility of peptides to proteolysis. Several administrations, usually with an adjuvant, may be required. Conjugating peptide epitopes to a protein carrier, such as a toxoid, can improve the production of antibodies. The first simple peptidebased vaccine, composed of sequences from plasmodium proteins, yielded disappointing results in young children in countries where malaria is endemic.44 Epitopes of cytotoxic T cells (which are usually nonamers) can bind to MHC class I molecules on dendritic cells. Because dendritic cells also express costimulating molecules, they can directly react with and activate naive CD8+ T cells. This approach is being used in cancer immunotherapy, as described below. Subunit vaccines are frequently made with the use of recombinant-DNA technology. Immunogenicity can be enhanced and the immune response directed to induce both cell-mediated and humoral responses through the formation of aggregates such as immunostimulating complexes, virus-like particles, antigencoated beads, or lipid-encapsulated antigen under various conditions.45-48 Aggregates of linked peptides are also being tested. For example, preparations being evaluated in clinical trials include polymers of

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linked peptides from group A streptococcus as a vaccine against rheumatic fever 49 and a plasmodiumpeptide polymer that also contains a lipopeptide to facilitate interactions between cells and so induce a strong immune response against malaria.50 The simplest types of vaccines are often administered with adjuvants to enhance their immunogenicity. Alum, the adjuvant most often used, delays the release of an antigen such as the hepatitis B vaccine and enhances the production of antibodies. The range of other products is very wide. Some are being tested in humans.51 Two adjuvants, alum and QS21, have greatly increased antibody production in phase 1 clinical trials of a vaccine against malaria.52

Gene gun Gene-coated> bead

Soluble> antigen and> adjuvant

DNA

Epidermis Dermis

Live-Agent Vaccines as Vectors of Other Vaccine Antigens

There is wide interest in the use of vaccines composed of attenuated viruses or bacteria as carriers (vectors) of other antigens.53 Techniques for incorporating DNA encoding an antigen from another infectious agent into vaccinia virus were first described in 1982.54,55 On infecting cells or an animal with the chimeric poxvirus, antigen encoded by the foreign DNA was expressed and the animal was protected from infection by the donor of the foreign DNA. More than 20 different RNA and DNA viruses as well as bacteria are used experimentally as vectors. Leading candidates include poxviruses, especially the highly attenuated strain Ankara, as well as fowlpox and canarypox, which infect but do not replicate in human cells.56,57 Since approximately 10 percent of the large poxvirus genome can be replaced by foreign DNA, these vectors have the potential to become multivalent vaccines. Adenovirus and Sal. typhi can be used as vectors if a mucosal response is desired.58 A further dimension was added to this approach by incorporating DNA encoding interleukin-2 into a chimeric poxvirus vector.59,60 Cytokines used in this way allow the immune response to be channeled to induce either a stronger humoral response or a strong-

Afferent> lymph> vessel

Lymphoid> organ

Antigen-> presenting cell> containing> antigen and> adjuvant Antigen-> presenting cell> containing> peptide derived> from the gene

Figure 3. Activation of Helper T Cells after the Application of Antigen-Coated Beads with the Aid of a “Gene Gun” or the Application of Soluble Antigen to the Skin as an Alternative to Vaccination with a Needle. A gene gun is used ballistically to accelerate the transdermal passage of microscopic gold beads coated with DNA plasmids (about 600 copies per bead) through the stratum corneum into the epidermis, where some are taken up by resident dendritic (Langerhans’) cells. Alternatively, a soluble antigen together with an adjuvant, usually cholera toxin, is applied to the skin (transcutaneous immunization). Some antigen reaches the epidermis and also undergoes endocytosis by Langerhans’ cells. During migration to the draining lymph node through the afferent lymphatics, these cells mature and express receptors for chemokines. The foreign DNA is expressed, and the antigens are degraded to polypeptides, some of which bind to major-histocompatibilitycomplex antigens. These activated T cells can interact with an activated B cell to induce a humoral response.

T cell

Antibody-> secreting cell

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B cell

ADVA NCES IN IMMUNOLOGY

er cell-mediated immune response. This approach has potential problems, however. Infection with interleukin-4–expressing mousepox (ectromelia virus) caused high mortality rates in mice that were genetically resistant to infection by ectromelia. Even immunized, resistant mice infected with the interleukin-4–expressing ectromelia had a substantial mortality rate.61 Immunization with DNA

Remarkably, DNA that encodes foreign antigens can be inserted together with a suitable promoter in a bacterial plasmid. Intramuscular injection of this complex induces an immune response to the antigen encoded by the DNA in mice. Furthermore, the response is very strong, since bacterial DNA, unlike vertebrate DNA, is recognized as foreign by vertebrates because of its high content of unmethylated CpG motifs62,63 — namely, GACGTT in the case of mice and GTCGTT in the case of humans. Such motifs, when recognized by a mammalian protein (tolllike receptor 9)64 that is expressed by different cells of the innate immune system, stimulate the production, activation, and maturation of dendritic cells. These cells, in turn, preferentially induce a Th1 response, which controls many intracellular bacterial infections. The CpG motif preparations are also effective when given mucosally. Clinical trials of these vaccines are in progress.62 Alternatively, a relatively small number of plasmids, adsorbed to tiny beads, are blasted through the skin by a “gene gun.” Some enter dendritic (Langerhans’) cells directly, apparently bypassing toll-like receptor 9 and inducing, in mice, a response biased toward the activation of type 2 helper T (Th2) cells and, hence, antibody production (Fig. 3). However, in monkeys, priming of the immune response by the administration of DNA with a gene gun followed by boosting of the response by the administration of a live chimeric vector generates a strong Th1 response.56,57 This approach has many potential advantages, including its low cost, stability, and absence of infectivity (although the immune response resembles that seen after a natural infection), along with the fact that the presence of antibody against the antigen to be expressed does not inhibit the response. Potential disadvantages include the integration of the DNA into the genome of the host cell, which might result in transformation or tumorigenic events, and the formation of anti-DNA antibodies. Such events have not yet been observed. Sequential Immunization

Traditionally, immunization is repeated, and the same preparation is given each time. But when mice were immunized with a preparation of chimeric DNA and later received as a booster chimeric fowlpox expressing the same foreign antigen (influenza hemagglutinin), antihemagglutinin titers were up to 50 times as high as those found after two injections of

the same preparation.65 The validity of this “prime– boost” approach has been confirmed and extended. By priming the immune response with the administration of DNA and boosting the response with the administration of the highly attenuated Ankara strain of vaccinia virus — with each preparation containing T-cell epitopes from HIV-1 antigens66 or from plasmodium sporozoites67 — very high levels of CD8+ cytotoxic T cells were induced in mice68 and in monkeys in the case of HIV-1.69 In the malaria experiment, mice were completely protected against a challenge with Plasmodium berghei. In contrast, priming the response with the administration of chimeric poxvirus and boosting the response with the administration of DNA were relatively inefficient. Monkeys immunized according to an HIV-1–specific or SIV-specific chimeric DNA–fowlpox protocol, which induced strong cell-mediated but nonprotective antibody responses, were protected against persistent infection when later challenged with HIV-156 or a pathogenic SIV.57 Similarly, monkeys immunized according to an SIV-specific DNA–Ankara (vaccinia) protocol were protected against persistent infection after a mucosal challenge.70 In other successful experiments in monkeys, chimeric adenovirus has been used as the booster immunization after priming with a DNA construct.71 Monkeys immunized with a chimeric DNA construct that received interleukin-2–immunoglobulin fusion protein as a booster were similarly protected against a challenge with a highly pathogenic SIV.72 Clinical trials now in progress will establish whether these different DNA–poxvirus protocols induce strong responses from cytotoxic T cells, how long the response persists, and whether there is reduced transmission of virus and long-term protection against the development of the acquired immunodeficiency syndrome in subjects at risk. These results also provide a strong case for initiating antiretroviral therapy in persons who have recently been infected with HIV and, once viral titers are minimized, vaccinating them to induce a continuing strong cell-mediated response.73 This approach might allow drug therapy to be discontinued for long periods. A similar approach holds promise for controlling infections with Ebola virus. Monkeys immunized with chimeric DNA followed by chimeric adenovirus, each containing DNA coding for Ebola virus antigens, have been protected from disease after challenge with a usually lethal dose of Ebola virus.58 Encouraging results have also been obtained with the use of the prime–boost approach in mice with tuberculosis.74 THE FUTURE OF VACCINATION Infectious Diseases

Although vaccines are urgently required against many viruses, HIV-1 and a newly emerging reassor-

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tant influenza A virus pose the greatest threat.75 In the case of HIV-176 and influenzavirus,77 it may still be possible to induce a widely neutralizing antibody response. But if the prime–boost protocol for vaccination against HIV-1 infection, malaria, and Ebola virus infection induces strong, persistent cytotoxic T-cell responses against HIV-1 in humans, the same approach could be applied to many other viruses, including pandemic influenza. The complete DNA sequences of many bacteria, including Mycobacterium tuberculosis and Chlamydia trachomatis, are known, and those of Leishmania major and Plasmodium falciparum are being deciphered.78 Knowledge of the sequences should provide a catalogue of the genes that code for every virulence factor and of potential immunogens, either as a target for neutralizing antibodies, on the basis of their structural characteristics, or as a source of T-cell epitopes for common regional class I and II HLA alleles. As an example, some bacteria have been found to contain a gene encoding DNA adenine methylase. Salmonella lacking this gene are much less virulent but still induce a strong immune response.79 Noncommunicable Diseases Autoimmune Diseases

The high prevalence of and serious health deficits resulting from autoimmunity have prompted interest in a “negative” form of vaccination, one that prevents or abrogates a particular immune response. In animal models of autoimmunity, this approach has prevented some forms of the disease.80 Studies in humans include mucosal administration of myelin in multiple sclerosis, type 2 collagen in rheumatoid arthritis, retinal antigen in uveitis, and insulin in type 1 diabetes. The results of these studies have been equivocal at best and are mainly disappointing.81 The answer may lie in being able to identify persons who are at high genetic risk or who are in the earliest stage of an autoimmune disease, a time when tolerance could be more readily achieved. Cancer

There are two types of cancer from the vaccine standpoint. One type is associated with a viral infection, such as primary hepatocellular carcinoma, which is due to hepatitis B virus infection; Kaposi’s sarcoma, which is associated with herpesvirus infection; B-cell lymphoma, which is linked to HIV-1 infection; genital and squamous-cell carcinomas, which are associated with papillomavirus infection; Burkitt’s lymphoma and nasopharyngeal carcinoma, which are linked to Epstein–Barr virus infection; and adult T-cell leukemia, which is linked to infection with human T-cell lymphotropic virus types I and II. The other type of cancers amenable to vaccines includes spontaneous tumors such as melanoma that express endogenous tumor antigens.

In the case of the first type, vaccination against the relevant virus should prevent tumorigenesis. Findings in relation to hepatitis B virus and primary hepatocellular carcinoma are very encouraging. Vaccination of infants in Taiwan with the hepatitis B vaccine has reduced the subsequent incidence of primary hepatocellular carcinoma among children 6 to 14 years of age by 50 percent and the incidence of death from cancer by 70 percent.82 In another study, the administration of one preparation consisting of virus-like particles containing the L1 antigen of papillomavirus strains 6 and 11 induced strong antibody responses, and there was complete regression of genital warts in 22 of 33 subjects.83 Clinical trials of vaccines against the transforming protein E7 of various papillomavirus strains causing cervical cancer are under way in the United States. Immunotherapy directed against an established tumor poses a more difficult problem, but there are now grounds for optimism. Many tumor-associated antigens have been identified in melanomas. In several small clinical trials in which tumor antigen–specific cytotoxic T cells were generated by different vaccination protocols, complete or partial remission of the tumor was achieved in about 30 percent of patients.84,85 The aim now is to increase this rate by exploring different ways of generating very strong and persistent cytotoxic-T-cell responses against several tumor-associated antigens. Vaccination with dendritic cells that have been derived from a patient and loaded with killed allogeneic melanoma cells looks promising.86 Immunization of HLA-A2–positive women with breast cancers that overexpress HER-2/neu with an antigen-specific polypeptide containing CD4+ and CD8+ T-cell epitopes of appropriate HLA specificities produced persistent CD4+ and CD8+ T-cell responses; the latter cells were capable of lysing the tumor cells.87 Alzheimer’s Disease

Alzheimer’s disease is the result of the formation of a small mutant protein, amyloid b peptide (Ab42). The deposition of this protein in the form of neurotoxic plaques in the brains of people with Alzheimer’s disease causes the loss of mental function. Transgenic mice that express DNA that encodes this protein provide an excellent model, since these mice have brain plaques and many of the cellular abnormalities seen in patients with Alzheimer’s disease. The early immunization of these mice with amyloid b prevented the formation of plaques and the subsequent cellular damage. Even more remarkable, most of the cellular damage disappeared if the vaccine was administered after plaques had formed.88 Other studies showed that if the vaccine was administered before plaques began to form, the otherwise inevitable loss of memory was averted.89 Long-term nasal administration of the protein to the mice also induced

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the synthesis of antiinflammatory cytokines in the brain and the formation of antibodies.90 These results strengthen the case for the accumulation of amyloid b as the cause of dementia in Alzheimer’s disease.91,92 Using a highly sensitive multiphoton microscope, researchers saw tightly focused images of plaques in the brains of living transgenic mice; it was the first time such plaques have been observed in a living host.93 They first labeled a monoclonal antibody specific for amyloid b with fluorescein and then applied it in vivo to the cortex, revealing numerous deposits of amyloid b. Three days later, there was up-regulation of microglia and most plaques had disappeared. These findings offer hope that a combination of vaccination with the amyloid b peptide and immunotherapy with a humanized monoclonal antibody could mitigate and perhaps even reverse the abnormalities caused by Alzheimer’s disease. CONCLUSIONS

The remarkable success of many vaccines, especially those administered in childhood, and their impressive safety record, together with the eradication of smallpox, are regarded among the greatest public health achievements of the 20th century. But there are still many serious diseases caused by pathogens that evade or subvert control by a specific humoral or cell-mediated immune response. An immunization protocol involving a prime–boost approach (usually DNA followed by a chimeric live virus vector) has induced strong cytotoxic-T-cell responses that prevented persistent infection in mice and monkeys after a challenge with HIV-1 and other serious human pathogens, including Ebola virus and plasmodium. The success of the current clinical trials of vaccination against HIV-1 with the use of this protocol would signal a paradigm shift in the development of vaccines. The sequencing of the genome of many bacteria is also an important step forward. Efforts to use vaccination and immunotherapeutic techniques to battle noncommunicable diseases, especially cancer and Alzheimer’s disease, are also expanding our horizons. The achievements of the 21st century may be as spectacular as those of the 20th century.

I am indebted to several colleagues for their useful comments.

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