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 EFFICACY OF SOME CHILDHOOD

VACCINES
Records kept by the Centers for Disease Control
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 ., and Prevention (CDC) since 1912 reveal the num-
Ed i t ors ber 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
V ACCINES AND V ACCINATION poliomyelitis (the last case in the Americas was in
Peru in 1992); over 99 percent in the case of diph-
GORDON ADA, D.SC. theria, measles, mumps, and rubella; and over 97 per-
cent in the case of whooping cough (caused by Borde-
tella pertussis). All these agents undergo little antigenic
variation (or drift) or none at all, showing that un-

M
ORE than 70 bacteria, viruses, parasites,
der virtually ideal conditions, vaccination can be ex-
and fungi are serious human pathogens.1
traordinarily effective.4
Vaccines are available against some of these
Within one year after the introduction in 1999 of
agents and are being developed against almost all
a Neisseria meningitidis serogroup C conjugate vac-
the other bacteria and viruses and about half of the
cine in the United Kingdom, the incidence of men-
parasites. Table 1 lists infections for which there are
ingitis was reduced by 92 percent among young
now licensed vaccines and those for which a candi-
children and by 95 percent among teenagers.5 A Sal-
date vaccine has undergone a phase 3 clinical trial,2
monella typhi Vi conjugate vaccine (Vi-rEPA) reduced
indicating that a vaccine will probably be licensed
the incidence of typhoid fever among two-to-four-
within 5 to 10 years.
year-old children by more than 90 percent.6 Both
Traditionally, attenuated vaccines were made by
findings confirm the remarkable effectiveness of con-
repeated passaging of the infectious agent in tissue
jugate vaccines.
culture or animal hosts until its virulence was greatly
The experience with measles in the United States
decreased but its immunogenicity was retained. Al-
is of interest. From 1912 until 1963, the incidence
ternatively, chemicals such as formalin were used to
never dropped below 100,000 cases per year, and
destroy infectivity. More recently, parts of an infec-
epidemics were common. After the introduction of
tious agent, usually a surface antigen, have been used
the first vaccine in 1963, the number of cases fell to
as a subunit vaccine. The current vaccines against
very low levels and remained so until 1990, when there
hepatitis B virus and Lyme disease rely on recombi-
was an epidemic lasting three years and involving
nant-DNA technology. Bacterial toxins are rendered
nearly 28,000 patients, most of whom were adoles-
nontoxic by chemical treatment, and the resulting tox-
cents or young adults. This resurgence was due to
oid protects against the infectious agent. Protection
inadequate vaccination of these patients at the age of
against some types of encapsulated bacteria has been
one to two years in major urban areas. The recogni-
achieved by immunization with a capsular oligosac-
tion that immunity can wane after vaccination led to
charide or polysaccharide, but these T-cell–independ-
a two-dose vaccination schedule, which prevented the
ent antigens induce only IgM antibodies, which are
transmission of indigenous measles infections within
poorly protective in infants. Conjugating this sac-
the United States, Canada, and Finland.
charide to a protein or proteinaceous complex in-
Sometimes, vaccination can fail, indicating that it
duces IgG antibodies because T cells recognize the
induced a suboptimal immune response. The failure
complex of a peptide with a major-histocompatibil-
to respond or a low level of response to a simple vac-
ity-complex molecule on the antigen-presenting cell
cine, such as the hepatitis B vaccine, can be circum-
(Fig. 1). The Haemophilus influenzae type b conjugate
vented by adding additional helper-T-cell epitopes to
vaccines also induce mucosal immunity, which re-
the vaccine.7 In the case of varicella–zoster virus, like
duces nasal carriage of the bacteria. Such conjugates
other such viruses, which induce latent infections, a
protect infants very effectively.
live attenuated vaccine may not eliminate infection but
does prevent chickenpox.
From the John Curtin School for Medical Research, Australian National SAFETY OF VACCINES
University, Canberra, Australia. Address reprint requests to Dr. Ada at the
John Curtin School for Medical Research, Australian National University, Adverse events associated with childhood vaccines
Box 334, ACT 2601 Canberra, Australia. are sometimes grouped as early and late reactions.

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 ADVA NC ES IN IMMUNOLOGY

TABLE 1. THE STATUS OF VACCINES AGAINST SOME HUMAN PATHOGENS.

TYPES OFTRADITIONAL
INFECTIOUS AGENT VACCINE STATUS* DISEASE VACCINE USE OR TARGET POPULATION

Bacteria
Bacillus anthracis Available Anthrax Inactivated To limit biologic warfare
Bordetella pertussis Available Whooping cough Inactivated, subunit Children and adults
Borrelia burgdorferi Available Lyme disease Subunit Residents of areas of endemic disease
in the United States
Clostridium tetani Available Tetanus Toxoid Children
Corynebacterium diphtheriae Available Diphtheria Toxoid Children
Coxiella burnetii Available Severe fever (Q fever) Inactivated Workers in slaughterhouses and meat-
processing plants
Haemophilus influenzae Available Meningitis, epiglottitis, Conjugated Children
pneumonia type b
Mycobacterium leprae Phase 3 clinical trials Leprosy Inactivated Residents of areas of endemic disease
M. tuberculosis Available Tuberculosis Live attenuated All persons
Neisseria meningitidis
Serogroup B Phase 3 clinical trials Meningitis Subunit, conjugated Children
Serogroup C Available Meningitis Conjugated
Salmonella typhi Available (Ty21a vaccine) Typhoid fever Live attenuated, poly- Residents of and travelers to areas of
saccharide endemic disease, children
Phase 3 clinical trials Typhoid fever Conjugated
(Vi-rEPA vaccine)
Staphylococcus aureus Phase 3 clinical trials Impetigo, toxic shock Conjugated Those at high risk, those with eczema,
syndrome in women those with neutrophil dysfunction
Streptococcus pneumoniae Available Pneumonia, otitis media, Conjugated Elderly persons
meningitis
Vibrio cholerae Available Cholera Live attenuated, inacti- Residents of and travelers to areas of
vated endemic disease
Viruses
Adenovirus Available Respiratory disease Live attenuated Military personnel
Hepatitis A Available Liver disease, cancer Live attenuated, inacti- Residents of and travelers to areas of
vated endemic disease
Hepatitis B Available Liver disease, cancer Subunit All persons
Influenzavirus
A Available Respiratory disease Live attenuated, inacti- Children (live attenuated vaccine
vated, subunit only), elderly persons
B Phase 3 clinical trials
Japanese encephalitis virus Available Brain infection Inactivated Residents of and travelers to areas of
endemic disease
Measles virus Available Respiratory tract infec- Live attenuated Children and adolescents
tion, SSPE†
Mumps virus Available Mumps, meningitis, or- Live attenuated Children and adolescents
chitis
Poliovirus Available Poliomyelitis, paralysis Live attenuated, inacti- Children
vated
Rabies virus Available Rabies Inactivated Exposed persons, residents of areas of
endemic disease
Rubella virus Available German measles, fetal Live attenuated Children
malformations
Vaccinia virus Available Smallpox Live attenuated Laboratory workers
Varicella–zoster virus Available Chickenpox Live attenuated Children
Yellow fever virus Available Jaundice, kidney and liver Live attenuated Residents of areas of endemic disease,
failure particularly children
Parasites
Leishmania Phase 3 clinical trials Kala-azar, tropical sores Live attenuated, inacti- Residents of countries where disease is
vated 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 cry-
ing, syncope, seizures, and rarely, hypotonic, hypo-
responsive 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 dam-
age 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 subse-
quent 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 mil-
lion recipients; the incidence of this complication after
natural measles infection is 1 per 1000.10 The inci-
dence 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 poliomyeli-
tis 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 Amer-
ican Academy of Pediatrics recommended that only
inactivated poliovirus vaccine be used after January 1,
2000.11
Overall, there is no firm scientific or clinical evi-
dence 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 ru-
bella vaccine) and the subsequent occurrence of in-
flammatory 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 small-
pox worldwide was 20 million. The last case of en-
demic 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, sev-
eral 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 polio-
myelitis has already been eliminated from the Amer-
icas, Europe, the western Pacific region, and South-
east Asia, but it will take a few more years to achieve
global eradication.16
Measles is the most contagious infection of hu-
mans and causes 30 percent of all deaths due to vac-
cine-preventable diseases. The successful interruption
of the transmission of measles infection in countries
with very high rates of vaccination coverage is indic-
ative 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 de-
veloping countries from 5 percent in 1974 to an av-
erage 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 acti-
vated, they produce and then secrete IgM antibody molecules. The individual Fab segments of the IgM molecule have only a mod-
erate affinity, but because there are 10 such segments, an IgM molecule has a high avidity. In contrast, in Panel B, some polysac-
charide–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 high-
affinity 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.

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 ADVA NC ES IN IMMUNOLOGY

A
B cell Activated B cell

Polysaccharide
IgM

Activation

B cell
Polysaccharide–protein>
conjugate

Antigen->
presenting>
cell

Interleukin-4, 5, and 6

Th2 cell

Peptide Activation

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 in-
crease rates of childhood vaccination to over 90 per-
cent worldwide, and to include in this coverage vac-
cines 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 mil-
lion to 7 million deaths annually from childhood di-
arrhea, 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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 The Ne w E n g l a nd Jo u r n a l o f Me d ic i ne
human immunodeficiency virus type 1 (HIV-1), tu-
berculosis, and malaria cause 7 million to 8 million
deaths annually, mainly in developing countries. The
incidence of sexually transmitted diseases in devel-
oped countries is steadily increasing.20 Vaccination of-
fers the greatest opportunity to decrease this toll. The
primary purpose of currently available vaccines is to
induce strong antibody responses capable of neutral-
izing infectivity, but attempts to develop such tradi-
tional types of vaccines against many important dis-
eases have been unsuccessful. There is a strong need
for new types of vaccines that not only are more po-
tent overall, but also induce either a stronger humoral
response or a cell-mediated immune response.
IMMUNE RESPONSES AND THE CONTROL
OF INFECTIONS
There are two patterns of infection. All viruses
and some bacteria and parasites are obligate intracel-
lular agents that can live and replicate only inside cells.
Other types of bacteria and parasites exist and repli-
cate 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 infec-
tions 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 con-
trol of infections include the prevention or limitation
of the initial infection and subsequent viremia or bac-
teremia and the killing of infected cells by antibody-
dependent cellular cytotoxicity or complement-medi-
ated lysis.21 In the case of extracellular infections,
specific antibody needs the support of a strong re-
sponse by CD4+ type 1 helper T (Th1) cells.
During intracellular infections, important T-cell re-
sponses 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 cy-
totoxic 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 primar-
ily responsible for controlling and sometimes clear-
ing 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-
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Virus
CD8+ CTs
Antibody-producing>
Titer
cells
4 8 1 2 6 18
Days Months
Figure 2. The Immune Response to Intranasal Infection with In-
fluenzavirus.
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 ti-
ter of infectious virus coincides with the increase in the activity
of cytotoxic T cells, which, in turn, decreases and then disap-
pears within a few days after infectious virus becomes undetect-
able. Large numbers of memory cytotoxic T cells are present at
2 and 6 weeks, and some are still present at 12 months. IgM-pro-
ducing 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 infec-
tion, 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 sus-
ceptible to lysis by specific cytotoxic T cells well be-
fore the release of viral progeny, allowing a window
of time for the effector T cell to seek and destroy in-
fected cells before the progeny arise. In addition to
directly participating in limiting infections by killing
infected cells, cytotoxic T cells secrete potent cyto-
kines with antiviral and macrophage-activating activi-
ties, 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 sero-
negative but possess HIV-1–specific CD8+ cytotox-
ic T cells or interferon-g produced by such cells: ba-
bies born to infected women,25,26 long-term partners
of infected people,27,28 long-term female African pros-
titutes,29 and some health care workers who were ex-
posed once to the virus.30 Furthermore, transfer of
a potent anti-CD8 serum to monkeys before infec-
tion 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-
 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 vac-
cine is administered orally but protects against infec-
tion 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 gen-
ital tract,35 a finding relevant to the development of
vaccines against sexually transmitted diseases. Infec-
tion or vaccination of a mucosal surface not only elic-
its the production of secretory IgA but may also in-
duce cytotoxic T cells to enter the site. For example,
specific cytotoxic T cells are present in specimens ob-
tained 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 in-
accessible epitopes in surface antigens recognized by
neutralizing antibodies and forming a complex be-
tween the microbe and an antibody in order to en-
hance 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 im-
mune-response mechanisms; destruction of CD4+
T cells; down-regulation of the expression of class I
MHC molecules; mutation, which changes viral pep-
tide sequences, rendering existing effector T cells in-
effective; and inhibition of the activity of cytotoxic
T cells. The design of a vaccine that foils the subter-
fuges 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 glyco-
protein that have been produced in transgenic plants
induce IgG antibodies with the correct antigenic spec-
ificity after oral administration to mice. The poten-
tial 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 muco-
sal IgA and serum IgG. Pigs fed transgenic potatoes
expressing the protective protein of transmissible gas-
troenteritis virus had a significant reduction in mor-
bidity and mortality when challenged with infec-
tious virus.
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In addition to vaccine antigens, specific antibodies
(“plantibodies”) have also been made in plants. An
antibody against Streptococcus mutans, which contrib-
utes to tooth decay, was applied to the specially
cleaned mouths of volunteers and prevented recolo-
nization of the mouth by these bacteria for four
months.41 Many such antibodies are now available.42
Transcutaneous Immunization
Transcutaneous immunization involves the appli-
cation of an antigen with an adjuvant, frequently
cholera toxin, to intact skin, prewashed to facilitate
penetration.43 In mice, the reagents enter the epider-
mis, where they come into contact with and are taken
up by Langerhans’ cells, a class of dendritic cells. Dur-
ing their migration through the dermis by means of
afferent lymphatics and thence to the draining lymph
nodes, the dendritic cells mature and become high-
ly 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 an-
tigen, 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 pro-
teolysis. Several administrations, usually with an ad-
juvant, may be required. Conjugating peptide epitopes
to a protein carrier, such as a toxoid, can improve the
production of antibodies. The first simple peptide-
based vaccine, composed of sequences from plasmo-
dium proteins, yielded disappointing results in young
children in countries where malaria is endemic.44
Epitopes of cytotoxic T cells (which are usually non-
amers) can bind to MHC class I molecules on den-
dritic cells. Because dendritic cells also express costim-
ulating 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 immu-
nostimulating complexes, virus-like particles, antigen-
coated beads, or lipid-encapsulated antigen under var-
ious conditions.45-48 Aggregates of linked peptides
are also being tested. For example, preparations be-
ing evaluated in clinical trials include polymers of
 The Ne w E n g l a nd Jo u r n a l o f Me d ic i ne

linked peptides from group A streptococcus as a vac-

cine against rheumatic fever 49 and a plasmodium- Gene gun Soluble>
antigen and>
peptide polymer that also contains a lipopeptide to adjuvant
facilitate interactions between cells and so induce a Gene-coated>
strong immune response against malaria.50 bead
The simplest types of vaccines are often adminis-
tered with adjuvants to enhance their immunogenic-
ity. Alum, the adjuvant most often used, delays the re-
lease of an antigen such as the hepatitis B vaccine and DNA
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 great-
ly increased antibody production in phase 1 clinical Epidermis
trials of a vaccine against malaria.52 Dermis
Live-Agent Vaccines as Vectors of Other Vaccine Antigens
There is wide interest in the use of vaccines com-
posed of attenuated viruses or bacteria as carriers
(vectors) of other antigens.53 Techniques for incorpo- Afferent>
rating DNA encoding an antigen from another in- lymph>
Lymphoid>
fectious agent into vaccinia virus were first described vessel
organ
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 Antigen->
A further dimension was added to this approach presenting cell>
by incorporating DNA encoding interleukin-2 into containing>
antigen and>
a chimeric poxvirus vector.59,60 Cytokines used in this adjuvant
way allow the immune response to be channeled to Antigen->
presenting cell>
induce either a stronger humoral response or a strong- containing>
peptide derived> T cell
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 Ap-
plication 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 (trans- Antibody->
cutaneous immunization). Some antigen reaches the epidermis secreting cell B cell
and also undergoes endocytosis by Langerhans’ cells. During mi-
gration to the draining lymph node through the afferent lymphat-
ics, 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-histocompatibility-
complex antigens. These activated T cells can interact with an
activated B cell to induce a humoral response.

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 ADVA NCES IN IMMUNOLOGY

er cell-mediated immune response. This approach has the same preparation.65 The validity of this “prime–
potential problems, however. Infection with interleu- boost” approach has been confirmed and extended.
kin-4–expressing mousepox (ectromelia virus) caused By priming the immune response with the adminis-
high mortality rates in mice that were genetically re- tration of DNA and boosting the response with the
sistant to infection by ectromelia. Even immunized, re- administration of the highly attenuated Ankara strain
sistant mice infected with the interleukin-4–expressing of vaccinia virus — with each preparation containing
ectromelia had a substantial mortality rate.61 T-cell epitopes from HIV-1 antigens66 or from plas-
Immunization with DNA modium sporozoites67 — very high levels of CD8+
cytotoxic T cells were induced in mice68 and in mon-
Remarkably, DNA that encodes foreign antigens
keys in the case of HIV-1.69 In the malaria experi-
can be inserted together with a suitable promoter in
ment, mice were completely protected against a chal-
a bacterial plasmid. Intramuscular injection of this
lenge with Plasmodium berghei. In contrast, priming
complex induces an immune response to the antigen
the response with the administration of chimeric pox-
encoded by the DNA in mice. Furthermore, the re-
virus and boosting the response with the administra-
sponse is very strong, since bacterial DNA, unlike
tion of DNA were relatively inefficient.
vertebrate DNA, is recognized as foreign by verte-
Monkeys immunized according to an HIV-1–spe-
brates because of its high content of unmethylated
cific or SIV-specific chimeric DNA–fowlpox protocol,
CpG motifs62,63 — namely, GACGTT in the case of
which induced strong cell-mediated but nonprotec-
mice and GTCGTT in the case of humans. Such mo-
tive antibody responses, were protected against per-
tifs, when recognized by a mammalian protein (toll-
sistent infection when later challenged with HIV-156
like receptor 9)64 that is expressed by different cells
or a pathogenic SIV.57 Similarly, monkeys immunized
of the innate immune system, stimulate the produc-
according to an SIV-specific DNA–Ankara (vaccinia)
tion, activation, and maturation of dendritic cells.
protocol were protected against persistent infection
These cells, in turn, preferentially induce a Th1 re-
after a mucosal challenge.70
sponse, which controls many intracellular bacterial
In other successful experiments in monkeys, chi-
infections. The CpG motif preparations are also ef-
meric adenovirus has been used as the booster im-
fective when given mucosally. Clinical trials of these
munization after priming with a DNA construct.71
vaccines are in progress.62
Monkeys immunized with a chimeric DNA construct
Alternatively, a relatively small number of plasmids,
that received interleukin-2–immunoglobulin fusion
adsorbed to tiny beads, are blasted through the skin
protein as a booster were similarly protected against
by a “gene gun.” Some enter dendritic (Langerhans’)
a challenge with a highly pathogenic SIV.72 Clinical
cells directly, apparently bypassing toll-like receptor 9
trials now in progress will establish whether these
and inducing, in mice, a response biased toward the
different DNA–poxvirus protocols induce strong re-
activation of type 2 helper T (Th2) cells and, hence,
sponses from cytotoxic T cells, how long the re-
antibody production (Fig. 3). However, in monkeys,
sponse persists, and whether there is reduced trans-
priming of the immune response by the administra-
mission of virus and long-term protection against
tion of DNA with a gene gun followed by boosting
the development of the acquired immunodeficiency
of the response by the administration of a live chimer-
syndrome in subjects at risk. These results also pro-
ic vector generates a strong Th1 response.56,57
vide a strong case for initiating antiretroviral therapy
This approach has many potential advantages, in-
in persons who have recently been infected with
cluding its low cost, stability, and absence of infec-
HIV and, once viral titers are minimized, vaccinat-
tivity (although the immune response resembles that
ing them to induce a continuing strong cell-mediat-
seen after a natural infection), along with the fact that
ed response.73 This approach might allow drug ther-
the presence of antibody against the antigen to be
apy to be discontinued for long periods.
expressed does not inhibit the response. Potential dis-
A similar approach holds promise for controlling
advantages include the integration of the DNA into
infections with Ebola virus. Monkeys immunized
the genome of the host cell, which might result in
with chimeric DNA followed by chimeric adenovi-
transformation or tumorigenic events, and the for-
rus, each containing DNA coding for Ebola virus
mation of anti-DNA antibodies. Such events have
antigens, have been protected from disease after chal-
not yet been observed.
lenge with a usually lethal dose of Ebola virus.58 En-
Sequential Immunization couraging results have also been obtained with the
Traditionally, immunization is repeated, and the use of the prime–boost approach in mice with tu-
same preparation is given each time. But when mice berculosis.74
were immunized with a preparation of chimeric DNA THE FUTURE OF VACCINATION
and later received as a booster chimeric fowlpox ex-
pressing the same foreign antigen (influenza hemag- Infectious Diseases
glutinin), antihemagglutinin titers were up to 50 Although vaccines are urgently required against
times as high as those found after two injections of many viruses, HIV-1 and a newly emerging reassor-

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Copyright © 2001 Massachusetts Medical Society. All rights reserved.
 The Ne w E n g l a nd Jo u r n a l o f Me d ic i ne
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 vacci-
nation 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, in-
cluding 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 deci-
phered.78 Knowledge of the sequences should pro-
vide a catalogue of the genes that code for every vir-
ulence 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 inter-
est in a “negative” form of vaccination, one that pre-
vents or abrogates a particular immune response. In
animal models of autoimmunity, this approach has
prevented some forms of the disease.80 Studies in hu-
mans include mucosal administration of myelin in mul-
tiple sclerosis, type 2 collagen in rheumatoid arthri-
tis, retinal antigen in uveitis, and insulin in type 1
diabetes. The results of these studies have been equiv-
ocal at best and are mainly disappointing.81 The an-
swer 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 infec-
tion, such as primary hepatocellular carcinoma, which
is due to hepatitis B virus infection; Kaposi’s sarco-
ma, which is associated with herpesvirus infection;
B-cell lymphoma, which is linked to HIV-1 infec-
tion; 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 spon-
taneous tumors such as melanoma that express en-
dogenous tumor antigens.
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Copyright © 2001 Massachusetts Medical Society. All rights reserved.
In the case of the first type, vaccination against the
relevant virus should prevent tumorigenesis. Findings
in relation to hepatitis B virus and primary hepato-
cellular carcinoma are very encouraging. Vaccination
of infants in Taiwan with the hepatitis B vaccine has
reduced the subsequent incidence of primary hepa-
tocellular 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 admin-
istration 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 pap-
illomavirus strains causing cervical cancer are under
way in the United States.
Immunotherapy directed against an established tu-
mor poses a more difficult problem, but there are
now grounds for optimism. Many tumor-associated
antigens have been identified in melanomas. In sev-
eral small clinical trials in which tumor antigen–spe-
cific cytotoxic T cells were generated by different vac-
cination protocols, complete or partial remission of
the tumor was achieved in about 30 percent of pa-
tients.84,85 The aim now is to increase this rate by ex-
ploring different ways of generating very strong and
persistent cytotoxic-T-cell responses against several
tumor-associated antigens. Vaccination with dendrit-
ic cells that have been derived from a patient and load-
ed with killed allogeneic melanoma cells looks prom-
ising.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 specific-
ities produced persistent CD4+ and CD8+ T-cell re-
sponses; the latter cells were capable of lysing the tu-
mor 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 neuro-
toxic plaques in the brains of people with Alzhei-
mer’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 abnor-
malities seen in patients with Alzheimer’s disease.
The early immunization of these mice with amyloid
b prevented the formation of plaques and the sub-
sequent cellular damage. Even more remarkable, most
of the cellular damage disappeared if the vaccine was
administered after plaques had formed.88 Other stud-
ies showed that if the vaccine was administered be-
fore plaques began to form, the otherwise inevitable
loss of memory was averted.89 Long-term nasal ad-
ministration of the protein to the mice also induced
 ADVA NCES IN IMMUNOLOGY
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 spe-
cific 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-regula-
tion of microglia and most plaques had disappeared.
These findings offer hope that a combination of vac-
cination with the amyloid b peptide and immuno-
therapy 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, espe-
cially those administered in childhood, and their im-
pressive 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 in-
duced strong cytotoxic-T-cell responses that prevent-
ed persistent infection in mice and monkeys after a
challenge with HIV-1 and other serious human patho-
gens, 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 vac-
cines. The sequencing of the genome of many bacte-
ria 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 hori-
zons. 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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