Vaccines
Wim Jiskoot and Gideon F.A. Kersten
Introduction
VACCINES are the most commonly administered
immunotherapeutics. Supported by great improve-
ments in sanitation facilities such as safe drinking
water, vaccination has been the most effective meas-
ure to control a diversity of life-threatening infectious
diseases in the 20th century. The most impressive
success of vaccination has been the global eradica-
tion of smallpox in the 1970s. Moreover, the inci-
dence of many other infectious diseases, such as
diphtheria, tetanus, pertussis, poliomyelitis, measles,
mumps, and rubella, has been drastically reduced
thanks to extensive vaccination programmes.
Upon a natural infection with a pathogen, an
unprotected person usually falls ill before the
immunological defence system is able to respond
adequately. Vaccination aims to stimulate the specif-
ic immune response against a pathogen by the
administration of attenuated or killed organisms, or
fractions thereof. If vaccination is successful and the
host comes into contact with the pathogen after-
wards, the specific immune response will be imme-
diate and sufficiently strong to kill the invading
organism before it has the opportunity to multiply
and cause disease.Thus, in a strict sense VACCINES are
immunoprophylactics rather than therapeutics.
In most cases, repeated doses of the vaccine are
given to boost the immune response. Apart from the
number of doses, several other factors determine
vaccine EFFICACY, as summarised in Table 1. If a high
enough proportion of a population is immunised,
vaccination not only protects the immunised individ-
uals, but also may help to protect the community as
it decreases the chance that non-immunised persons
encounter the pathogen. This is referred to as herd
immunity [1].
C1
A brief history of vaccination will be given below.
Next, current vaccine categories for human use will
be addressed. Finally, new developments in vaccinol-
ogy will be outlined.
Historical background
Vaccination has a long history [1,2].The most promi-
nent milestones of vaccinology are listed in Table 2.
The first attempts to become immune probably date
back to as early as the 7th century, when Indian Bud-
dhists drank snake venom and may thus have
become immune against this toxin. Written reports
bear witness to the practice of variolation, i.e., the
administration of scabs or pustule preparations
obtained from patients recovered from smallpox,
ever since about 1000 A.D. in various parts of the
world, amongst others in China, India, North Africa,
and England.
Variolation was widely applied until Edward
Jenner introduced cowpox vaccination at the end of
the 18th century. His practice was based on the
recognition that milkmaids were frequently subject-
ed to mild pox infection acquired from the cows
they milked, but were spared from disease during
smallpox epidemics. The first demonstration that the
principle of immunisation works was Jenner’s anec-
dotal experiment with an 8-year old boy who
remained healthy when challenged with smallpox
virus after he had been immunised with cowpox
virus. It was Jenner who introduced the terms “vac-
cine” for cowpox preparations (derived from the
Latin vacca = cow) and “vaccination” for the admin-
istration thereof. Later, in honour of Jenner, Louis
Pasteur generalised the meaning of vaccination to
immunisation with agents other than cowpox.
During the 19th century vaccination with live cow-
232 Vaccines

TABLE 1. FACTORS DETERMINING VACCINE EFFICACY

Pathogen-dependent Host-dependent Vaccine-dependent Vaccination schedule-dependent

Port of entry Species Nature of antigenic Route of administration

component(s)
Localisation in host Age Antigen content Number of doses
Antigenic variation Genetic factors Delivery systems Immunisation intervals
Mutation frequency Physical state Adjuvants Simultaneous administration of other
vaccines (administered separately)
Immune status Combination with other
vaccine components
(in one vial or syringe)

pox virus became common practice.In the 20th cen-
tury, vaccinia virus, which is closely related to cow-
pox virus [1], became widely used as a live vaccine
until smallpox was eradicated (Tab. 2).
Pasteur gave a new impetus to vaccinology in the
last quarter of the 19th century. He showed that the
virulence (i.e., infectivity) of pathogens could be
reduced by successive passage in culture. Vaccin-
ation with attenuated strains thus obtained could
confer protection without causing disease. The
efforts of Louis Pasteur and others led to the develop-
ment of live ATTENUATED VACCINES against cholera,
anthrax, and rabies. Along with the introduction of
ATTENUATED VACCINES, it became apparent that infec-
tion with live material was not essential to induce
immunity. The procedure of killing bacteria by heat
and subsequent stabilisation with phenol was devel-
oped, resulting in the introduction of heat-inactivat-
ed whole-cell VACCINES against cholera, typhoid and
plague at the end of the 19th century.
At the beginning of the 20th century the develop-
ment and introduction of new live (tuberculosis, yel-
low fever) and killed VACCINES (pertussis, influenza,
rickettsia) followed. Moreover, it was being recog-
nised that some components of a micro-organism
were more relevant for protection than others, and
the concept of SUBUNIT VACCINES was born. This and
the discovery of chemical inactivation of bacterial
toxins with formaldehyde led to the introduction of
SUBUNIT VACCINES against diphtheria (1923) and
tetanus (1927).
In the early 1950s tissue-culture techniques for
virus propagation were developed. This resulted in
the licensing of Salk’s inactivated polio vaccine
(IPV) in 1955. In the same period Sabin developed
an oral polio vaccine (OPV) consisting of live atten-
uated viruses, which became available in the U.S.A.
in 1961. Several other viral VACCINES derived from tis-
sue cultures followed. Furthermore, several bacterial
SUBUNIT VACCINES based on purified proteins or poly-
saccharides were introduced since the 1970s. The
first vaccine based on rDNA technology was market-
ed in 1986.
Current vaccine categories
Classification
The currently available VACCINES for human use are of
either bacterial or viral origin and can be divided
into several categories (see Table 3).These categories
will be discussed below. For a more detailed descrip-
tion of individual VACCINES currently in use, the read-
er is referred to the textbook of Plotkin & Orenstein
[1] (Tab. 3).
 Current vaccine categories 233

TABLE 2. MILESTONES IN VACCINE HISTORY1

Year Event2
ca. 1000 Intranasal administration of preparations of scabs from smallpox patients in China
16th–17th century Parenteral variolation in India by Hindus
17th century Oral administration of white cow flea pills for smallpox prevention in China
1796 Immunisation of 8-year old boy with cowpox virus and challenging with smallpox virus
(Edward Jenner)
1798 Initiation of general cowpox immunisation with Jenner’s variola vaccine
1870s Discovery of attenuation of fowl cholera bacteria (Louis Pasteur)
1884 Attenuated Vibrio cholerae: the first bacterial vaccine used in humans (Robert Koch)
1885 First administration to humans of attenuated rabies vaccine (Louis Pasteur)
1896-1897 Introduction of the first heat-inactivated vaccines against typhoid, cholera and plague
1923 Introduction of the first subunit vaccine: formaldehyde-treated diphtheria toxin
1927 Introduction of BCG, attenuated tuberculosis vaccine
1955 Introduction of inactivated poliovirus vaccine (Salk): the first vaccine developed with
tissue-culture technique
1961 Attenuated poliovaccine (Sabin) as the first licensed oral vaccine
1980 Declaration of the eradication of smallpox by the WHO
1986 Licensing of the first rDNA vaccine: recombinant HBsAg
1987 Licensing of the first conjugate vaccine against Hib: PRP-T

1Sources: [1, 2]
2Abbreviations: BCG, bacille Calmette-Guérin; HBsAg, hepatitis B surface antigen; Hib, Haemophilus influenzae type
b; PRP-T, polyribosylribitol phosphate-tetanus toxoid conjugate vaccine; WHO, World Health Organisation

Live attenuated vaccines
Attenuation through serial passage and selection of
less virulent and less toxic variants has been applied
to obtain safe vaccine strains. Once a suitable strain
has been obtained, master and working seed lots are
prepared. The seed lot system provides the basis for
the reproducible production of live (and other) VAC-
CINES. The dose of live VACCINES is determined on the
basis of the number of viable organisms.
Live VACCINES have a number of advantages over
non-living VACCINES. Although attenuation generally
means reduced infectivity, attenuated strains will
replicate to some extent in the recipient.This furnish-
es a sustained Ag dose, inducing strong immune
responses even after a single dose. In general, live
VACCINES generate higher cell-mediated immune
responses than inactivated VACCINES. A single immu-
nisation with a live vaccine can provide lifelong
immunity.
The major drawback of live VACCINES is the risk of
reversion to pathogenicity. For instance, the occur-
rence of vaccine-associated paralytic poliomyelitis
after the introduction of OPV has been reported [1,
3]. Furthermore, live VACCINES sometimes cause mild
symptoms resembling the disease caused by the
pathogen. Live VACCINES should never be given to
immunosuppressed persons, because they lack the
ability to respond even to infections by attenuated
organisms.
Attenuated viral vaccines
Examples of live viral VACCINES are polio, measles,
mumps and rubella VACCINES. Attenuated polio vac-
cine is administered orally. Poliovirus is a non-
234 Vaccines

TABLE 3. CLASSIFICATION AND EXAMPLES OF CURRENT VACCINES

Category Example Vaccine characteristics

Live attenuated organisms

viral oral polio vaccine (Sabin) attenuated viruses, serotypes 1-3; oral vaccine
measles vaccine attenuated virus
mumps vaccine attenuated virus
rubella vaccine attenuated virus
yellow fever vaccine attenuated virus
bacterial tuberculosis vaccine attenuated Mycobacterium bovis
typhoid fever vaccine attenuated bacteria, oral vaccine

Killed whole organisms

viral Inactivated polio vaccine (Salk)
rabies vaccine
hepatitis A vaccine
Japanese encephalitis vaccine
bacterial whooping cough vaccine
cholera vaccine
typhoid fever vaccine
formaldehyde-inactivated viruses, serotypes 1–3
β-propiolactone-inactivated virus
formaldehyde-inactivated virus
formaldehyde-inactivated virus
heat-inactivated bacteria
phenol-inactivated bacteria
heat-inactivated bacteria

Subunit vaccines
viral influenza vaccine influenza surface antigens
hepatitis B vaccine recombinant hepatitis B surface antigen
bacterial diphtheria vaccine formaldehyde-treated toxin
tetanus vaccine formaldehyde-treated toxin
whooping cough vaccine mixture of purified proteins
meningococcal vaccine purified capsular polysaccharides
pneumococcal vaccine purified capsular polysaccharides
Hib1 vaccine polysaccharide-protein conjugates

1Haemophilus influenzae type b

enveloped single-strand RNA virus. Its four structural
proteins (VP1-4) form a regular three-dimensional
structure with a diameter of 28 nm.OPV contains the
three existing serotypes (1, 2 and 3), which differ
from each other in a number of distinct EPITOPES rele-
vant for protection. The virus is relatively stable and
additives such as magnesium chloride or sorbitol fur-
ther enhance the thermal stability of the vaccine.
OPV is included in many childhood immunisation
programmes. It is usually administered to infants in a
4-dose scheme and probably provides lifelong pro-
tection.
Attenuated mumps, measles and rubella viruses
are often combined in a COMBINATION VACCINE (MMR
vaccine). These attenuated RNA viruses vary in size
and number of structural proteins. Measles, mumps
and rubella VACCINES,whether separate or combined,
are lyophilised preparations stabilised with sucrose,
sorbitol, hydrolysed gelatine and/or amino acids.
They contain the antibiotic neomycin and have to

be kept refrigerated. The three vaccine components
have in common that one single s.c.administration is
probably sufficient for lifelong protection.
Nevertheless, in some countries the first dose given
at 12–15 months of age is followed by a second vac-
cination at the age of 4–6 or 11–12 years. Both
humoral and cell-mediated immunity are important
for protection. VACCINE EFFICACY is estimated to be at
least 90% and combining the components does not
seem to influence their effectiveness. Side-effects are
generally mild and usually occur 7–12 days after vac-
cination. MMR VACCINES are not indicated for infants
below the age of one year,because circulating mater-
nal Ab impair vaccine EFFICACY in this age group.
Attenuated bacterial vaccines
The most well-known attenuated bacterial vaccine is
tuberculosis vaccine, which has been incorporated
in many immunisation programmes as of the 1930s.
The vaccine is based on Mycobacterium bovis bacte-
ria, which primarily infect cattle but can also infect
humans. The vaccine consists of lyophilised attenuat-
ed M. bovis, known as bacille Calmette-Guérin
(BCG), and is administered i.d. to infants and older
children. Current vaccine strains vary in the extent of
attenuation and the dosage varies among vaccine
suppliers. The immunisation schedule varies signifi-
cantly among nations. The nature of the immune
response is not known in detail, but cell-mediated
immune mechanisms are probably involved in pro-
tection,whereas Ab do not seem to play a substantial
role. Besides strain variations,the lack of reliable tests
for immunity and the poorly understood MECHANISM
OF ACTION contribute to the fact that estimates of vac-
cine EFFICACY vary from 0 to 80% [1].
Oral attenuated Salmonella typhi VACCINES are
indicated for high-risk groups, such as children in
endemic areas and travellers, to prevent typhoid
fever.The only licensed strain is Ty21a, whose attenu-
ation has been stimulated by using nitrosoguani-
dine, a chemical mutagenic agent. Strain Ty21a lacks
the ability to synthesise capsular polysaccharides,
which are essential for virulence. In order to protect
the bacteria against peptic digestion, the vaccine is
formulated as lyophilised bacteria in enteric-coated
capsules. Protection is achieved through 3–4 doses
Current vaccine categories 235
administered every other day. The vaccine provides
significant protection by inducing relatively strong
intestinal IgA and cell-mediated responses, and a
weak systemic Ab response. Protective Ab are direct-
ed against flagelli and LPS. The duration of the pro-
tection is relatively short (3–5 years).
Killed whole organisms
Inactivated bacterial and viral VACCINES are obtained
from virulent strains by heat treatment or by chemi-
cal inactivation, usually with formaldehyde. Since
killed pathogens are not able to propagate after
administration, these VACCINES usually are less
immunogenic than live VACCINES. An advantage over
the latter is the inability to revert to virulence. On the
other hand, deficient inactivation has caused vac-
cine-related accidents. For instance, immunisation
with insufficiently inactivated polio vaccine in 1955
resulted in cases of paralytic disease [1,2].Examples
of this category include inactivated polio vaccine
(IPV) and whole-cell pertussis vaccine, which are
discussed below.
Inactivated polio vaccine
IPV is currently used in several countries. The vac-
cine consists of formaldehyde-inactivated poliovirus
and includes the three serotypes. The dose is deter-
mined on the basis of Ag contents. Advantages of IPV
over OPV are a better temperature stability, the lower
risk of vaccine-related disease and the possibility of
combination with diphtheria, tetanus and pertussis
components in one formulation (DTP-IPV vaccine).
In contrast to OPV, IPV does not elicit substantial
amounts of secretory IgA Ab, but its effect relies on
the induction of virus-neutralising serum IgG. A vac-
cination regimen with both IPV and OPV combines
the advantages of both VACCINES and is applied in
Denmark [1].
Whole-cell pertussis vaccine
Pertussis (or whooping cough) vaccine consists of
heat-inactivated B. pertussis cells. The dose is deter-
mined on the basis of the opacity of the inactivated
236 Vaccines
cell suspension. The vaccine potency is tested by
protection assays in mice. The protective EFFICACY of
whole-cell pertussis VACCINES is probably based on
Ab against several pertussis Ag, such as pertussis
toxin, filamentous haemagglutinin and LPS. Whole-
cell pertussis VACCINES are notorious for their fre-
quent side-reactions, mostly fever and irritability.
Other side reactions include excessive sleepless-
ness, persistent inconsolable crying and shock-like
phenomena. The adverse effects of whole-cell per-
tussis VACCINES are largely due to the LPS present in
B. pertussis’s outer membrane. The adverse effects
are stronger in older children and adults, so that
whole-cell pertussis VACCINES are not indicated for
these age groups. Although protection is probably
restricted to a period of about 10 years, this is not a
significant problem, because whooping cough
infections are most dangerous in toddlers and
infants. Adolescents and adults in general (but not
always) have relatively mild symptoms.The vaccine
contains colloidal aluminium salt as ADJUVANT and is
usually combined with diphtheria and tetanus vac-
cine components (DTP vaccine). The vaccine is
given in 4-5 i.m. doses.
Subunit vaccines
SUBUNIT VACCINES contain one or more selected Ag
(subunits) significant for protection against the
pathogen they are derived from. SUBUNIT VACCINES
have better defined physicochemical characteristics
and show fewer side-effects than VACCINES consisting
of attenuated or inactivated organisms. ANTIGENS
used for current SUBUNIT VACCINES include viral and
bacterial proteins as well as bacterial capsular poly-
saccharides.
Proteins
Protection against diphtheria or tetanus is mainly
based on the presence of Ab directed against the
respective toxins. These toxins are water-soluble pro-
teins and form the basis of diphtheria and tetanus
VACCINES. In order to eliminate the toxicity of diphthe-
ria and tetanus toxin, they are incubated with
formaldehyde. This process is called toxoidation and
the resulting products are referred to as toxoids.
Formaldehyde forms covalent bonds with the toxin,
which is initiated by a reversible reaction of
formaldehyde with primary amino groups, followed
by an irreversible reaction with other amino acid
residues [2]:
R-NH2 + CH2O ↔ R-NH-CH2OH ↔ R-N = CH2
+ H2O (1)
R-N = CH2 + R’-H → R-NH-CH2-R’ (2)
where R is the toxin and R’ can be a reactive amino
acid residue (e.g., lysine, arginine, tryptophan, tyro-
sine, histidine) in the toxin molecule (or possibly a
neighbouring toxin molecule) or a free amino acid
present in the medium. Thus, stable crosslinks are
formed, yielding a heterogeneous product with
respect to number and sites of formaldehyde
adducts and molecular weight. The degree of toxoi-
dation is highly dependent on the reaction condi-
tions, including formaldehyde concentration, pH,
temperature and the presence of other components.
The toxoidation process must be a compromise
between sufficient detoxification and preservation of
relevant EPITOPES. To enhance the relatively poor
immunogenicity of toxoids,they are adsorbed to alu-
minium salt suspensions.
A solution to the risk of residual toxicity and loss
of immunogenic sites has been found by the intro-
duction of genetic toxoidation, which has been
applied to pertussis toxin, a crucial component in
recently developed SUBUNIT VACCINES against pertus-
sis. Genetic toxoidation of pertussis toxin was
achieved by site-directed mutagenesis of the site
responsible for toxicity. The resultant toxoid is devoid
of toxicity and well defined, does not bear the risk of
reversal to toxicity and is more immunogenic than
chemically detoxified pertussis toxin [4]. Curiously,
genetically obtained pertussis toxoid is stabilised
with low concentrations of formaldehyde.
Acellular pertussis VACCINES have been intro-
duced in Japan in the early 1980s as alternatives to
the whole-cell VACCINES for the immunisation of older
children. Although pertussis toxoid alone may be
sufficient for protection,most acellular pertussis VAC-
CINES contain at least two proteins important for the
virulence of B. pertussis, including (inactivated) per-

tussis toxin, filamentous haemagglutinin, fimbriae
and pertactin. Recent field trials indicate that the
EFFICACY of these VACCINES is comparable to that of
whole-cell VACCINES, whereas the acellular VACCINES
induce virtually no adverse effects [5]. This makes
them suitable for immunisation of older children
and adults. Acellular pertussis VACCINES have now
been introduced in many national immunisation
programmes, mostly combined with diphtheria and
tetanus components (DTaP vaccine).
Hepatitis B vaccine was the first marketed recom-
binant vaccine, which has replaced hepatitis B VAC-
CINES obtained from plasma of infected humans.
Recombinant hepatitis B VACCINES are composed of
hepatitis B surface Ag (HBsAg) derived from yeast or
mammalian cells. Introduction of the genes for
HBsAg in eukaryotic cells has been accomplished
by inserting the protein gene into a plasmid, which
was then used to transform the host cells. Purified
HBsAg self-assembles to 22-nm particles identical to
those excreted by cells infected with the native virus.
Advantages of the rDNA vaccine as compared to the
plasma-derived product are safety, high yields and
consistent quality. The ease of production has made
the vaccine available worldwide. Hepatitis B vaccine
has been included in several national immunisation
programmes [1]. For countries with endemic hepati-
tis (many developing countries),WHO recommends
immunisation of all infants.
Capsular polysaccharides
Many bacteria have a capsule consisting of high-
molecular-weight polysaccharides, which act as viru-
lence factors. Capsule-forming species include both
Gram-positive (e.g., pneumococci) and Gram-nega-
tive bacteria (e.g., meningococci). The polysaccha-
rides of the different species are composed of linear
repeat oligosaccharide units that vary in sugar com-
position and chain length. The host defence against
encapsulated bacteria relies on anti-polysaccharide
Ab interacting with complement to opsonise the
organisms and prepare them for PHAGOCYTOSIS and
CLEARANCE. Licensed capsular polysaccharide VAC-
CINES include meningococcal (serogroups A, C, W-
135, Y), pneumococcal (up to 23 serotypes) and
Haemophilus influenzae type b (Hib) VACCINES.
Pharmacological effects of vaccination 237
The main disadvantage of capsular polysaccha-
ride VACCINES is their T CELL independency, which
implies that they do not elicit immunological memo-
ry. Moreover, infants up to 2 years of age show very
weak, non-protective immune responses, whereas
they belong to the highest risk groups for infections
with the encapsulated bacteria mentioned above.
Polysaccharide-protein conjugate vaccines
The poor immunogenicity of plain polysaccharides
can be overcome by covalent coupling to carrier
proteins containing T CELL EPITOPES. These helper EPI-
TOPES make them T CELL-dependent and enables the
induction of strong immune responses and immuno-
logical memory in all age groups, including infants.
Conjugate VACCINES licensed so far are Hib, meningo-
coccal group C and pneumococcal VACCINES. The
Hib polysaccharide consists of repeat units of ribo-
syl(1-1)ribitol phosphate. An effective Haemophilus
influenzae vaccine is relatively easy to produce,
because – in contrast with the diversity of pathogen-
ic meningococcal and pneumococcal strains – Hib
is responsible for about 95% of infections with
Haemophilus species, so only one polysaccharide
type has to be included in the vaccine.Table 4 shows
that the four licensed Hib conjugate VACCINES vary in
composition, owing to differences in polysaccharide
length, carrier protein, coupling procedure and poly-
saccharide-to-protein ratio.As a result, these VACCINES
differ with respect to immunogenicity and EFFICACY
[6]. The VACCINES are incorporated in many child-
hood immunisation programmes and are normally
administered i.m. in a multi-dose schedule with DTP,
either separate or as a combined DTP-Hib vaccine.
Another example of a licensed conjugate vaccine is
a mixture of polysaccharides from seven types of
pneumococci conjugated to diphtheria toxin. Finally,
several manufacturers produce meningococcus
group C conjugate vaccine.
Pharmacological effects of vaccination
The EFFICACY of a vaccine is difficult to estimate,
because the relationship between immune response
and degree of protection is not straightforward.
238 Vaccines

TABLE 4. CHARACTERISTICS OF LICENSED HAEMOPHILUS INFLUENZAE TYPE B CONJUGATE VACCINES1

Vaccine2
Property PRP-D HbOC PRP-OMP PRP-T

Polysaccharide size medium small medium large

Polysaccharide content (µg) 25 10 15 10
Carrier protein Diphtheria Diphtheria Meningococcal group B Tetanus toxoid
toxoid toxin mutant outer membrane proteins
Protein content (µg) 18 20 250 20
Linkage via spacer direct via spacer via spacer
Formulation aqueous aqueous lyophilised, reconstituted lyophilised, reconstituted
solution solution with alum salt suspension with aqueous buffer

1Source: [1]
2Abbreviations: PRP, polyribosylribitol phosphate; PRP-D, PRP-diphtheria toxoid conjugate vaccine; HbOC,
Haemophilus type b oligosaccharide conjugate vaccine; PRP-OMP, PRP-outer membrane protein conjugate vaccine;
PRP-T, PRP-tetanus toxoid conjugate vaccine

Seroconversion, i.e., the increase in the level of spe-
cific circulating Ab, is commonly determined as a
measure for the immunogenicity. Moreover, the pro-
tective quality of these Ab can be measured with
assays for bactericidal activity, i.e., their ability to kill
bacteria in the presence of complement (e.g.,
meningococcal VACCINES), virus-neutralising activity
(e.g., polio VACCINES) or toxin-neutralising activity
(e.g., diphtheria and tetanus VACCINES). However, it is
hard to correlate the level and persistence of circu-
lating Ab to protective EFFICACY. Moreover, the extent
of cell-mediated immunity may in some instances
be a better measure for protection, e.g., against
tuberculosis, but is more difficult to measure. The
effectiveness of vaccination is most clearly demon-
strated by the reduction of disease after introduc-
tion of a vaccine in national immunisation pro-
grammes.Recent examples are the drastic reduction
in incidence of Hib infections observed in those
areas where routine vaccination in infants was intro-
duced and a similar effect after the introduction of
meningococcus group C vaccination in the UK.
There is much indirect evidence of vaccine EFFICACY.
For instance, in The Netherlands, where the use of
IPV has effectively protected most of the population,
two significant outbreaks of poliomyelitis in 1978
and 1992 were restricted to communities which
refuse vaccination on religious grounds [3]. Con-
cerns about the safety of whole-cell pertussis VAC-
CINES in the 1970s were the cause of a sharp decline
in vaccination levels in Great Britain, which in turn
resulted in epidemics in 1978 and 1982; after
renewed public acceptance of the vaccine the inci-
dence of disease dropped again [1].
Since the target groups of VACCINES in many cases
include healthy infants and young children, vaccine
safety is of particular importance.The occurrence of
side-effects may be due to the antigenic components
(e.g., LPS in whole-cell pertussis vaccine), impurities
derived from the production process,(e.g.,chick pro-
tein from the cell substrate used for measles vaccine
production), or to additives used in a vaccine formu-
lation (e.g., neomycin or gelatine in MMR VACCINES,
aluminium salts in SUBUNIT VACCINES). Before a new
vaccine candidate is licensed, its safety is investigat-
ed in phase I, II and III field trials. Phase I trials
include a small number of healthy adults and serve
to collect preliminary safety data and to assess vac-
cine dosage. In phase II studies safety and immuno-
genicity are determined in a larger number of volun-

teers.Phase III trials are meant to evaluate safety and
EFFICACY in large target populations.
New developments
Introduction
Notwithstanding the success of vaccination, several
infectious diseases remain against which an effective
vaccine is not yet available. New VACCINES against
bacterial (e.g., group B meningococci), viral (e.g.,
human immunodeficiency virus) and parasitic (e.g.,
malaria) infections are under development. Ideally,
these VACCINES should provide lifelong protection in
any individual of any age, be absolutely safe, easy to
produce in unlimited quantities, stable under vary-
ing conditions, easy to administer and cheap. As yet
the design of a vaccine with all these ideal character-
istics combined remains an important challenge for
developers of new and better VACCINES.
Apart from new prophylactic VACCINES, current
research is also focused on the development of ther-
apeutic VACCINES, especially for the treatment of
chronic diseases such as AIDS and cancer. The
rationale of administering VACCINES to patients
already suffering from disease is to specifically boost
the IMMUNE SYSTEM weakened by the disease.
The number of VACCINES routinely applied is
expected to increase, which demands efforts to
reduce the number of injections. An obvious way to
achieve this is combining separate vaccine compo-
nents into one vial or syringe.Examples of such COM-
BINATION VACCINES have been given before. Simply
mixing vaccine components, however, may not only
pose pharmaceutical problems (e.g., incompatibility
of vaccine components and/or excipients), but also
bears the risk of immunological interference. For
instance, Hib conjugate was reported to be less
immunogenic when mixed with DTaP vaccine [7].
Modern technologies
Whereas traditional vaccine development has large-
ly been dependent on empirical methods, a better
insight into immune mechanisms and immunogenic
New developments 239
structures of infectious organisms has led to a better
understanding of what would be the optimal vac-
cine composition as related to the desired immuno-
logical effect. Recent advances in genomics, pro-
teomics and bioinformatics are now being applied
to identify putative Ag [8]. Moreover, the advent of
(bio)technological advances has enabled scientists
to translate the improved immunological knowledge
into the rational design of new VACCINES against a
variety of life-threatening and chronic diseases. Sev-
eral classical and modern approaches to the devel-
opment of a variety of new VACCINES are currently
being explored, some of which are schematically
shown in Figure 1.Most of these approaches offer the
following common advantages over classical VAC-
CINES: (1) relevant EPITOPES of pathogenic organisms
or cancer cells are obtained by safer means and (2)
in greater quantities, (3) the products are better
defined, and (4) EPITOPES of a single or multiple path-
ogenic agents can be combined easily in one vac-
cine. Approaches not yet addressed before are
briefly discussed below. For more detailed informa-
tion about modern vaccine technology the reader is
referred to specialised textbooks [1, 2, 9, 10].
Recombinant live vaccines
Non-pathogenic or attenuated organisms can be
used as carriers for heterologous protein Ag. Such
live carriers are called vectors (Fig. 1c). They are
obtained by cloning the desired gene and introduc-
ing it into an appropriate carrier organism. Both viral
(e.g., vaccinia virus, attenuated poliovirus) and bac-
terial vectors (e.g., Salmonella species, BCG) are
being explored as carriers to express a variety of Ag.
The properties of recombinant live VACCINES are com-
parable to those of classical ATTENUATED VACCINES.
Fusion proteins
Fusion proteins are non-toxic proteins containing
inserted EPITOPES, larger protein fragments or even
entire proteins derived from pathogenic species (Fig.
1e). They are obtained by the insertion of DNA
sequences encoding EPITOPES in the gene of the carri-
er protein, such as HBsAg [11] or a fusion partner
with the capability to target the fused Ag to APC (see
240 Vaccines

A B

bacterium
or
virus isolation of
antigens

Live or inactivated organism Subunit vaccine

C D

bacterium
or isolation of
virus antigens

Live vector Recombinant

subunit vaccine

E F

Fusion protein Anti-idiotype

antibody

G H

Synthetic peptide Plasmid DNA

FIGURE 1. SCHEMATIC REPRESENTATION OF (A-B) CLASSICAL VACCINE COMPONENTS AND (C-G) NEW GENERATION
VACCINE COMPONENTS
(A) Whole bacterium or virus, live attenuated or killed, with protein antigens (grey objects) containing protective epi-
tope (black semi-circle); (B) subunit vaccine: antigen isolated from pathogenic organism; (C) live vector: antigenic pro-
teins derived from pathogenic organism expressed by live, non-pathogenic bacterium or virus; (D) recombinant sub-
unit vaccine: antigenic protein isolated from heterologous expression system; (E) fusion protein: non-toxic protein con-
taining epitope of protein from pathogen isolated from non-pathogenic organism; (F) anti-idiotype antibody: antibody
containing antigen-binding sites mimicking epitope of antigen from pathogen; (G) synthetic peptide with amino acid
sequence mimicking epitope of antigen from pathogen; (H) nucleic acid vaccine: plasmid DNA containing the gene
encoding antigenic protein or epitope from pathogen.
 New developments 241

BOX 1. FUSION PROTEINS TARGETED TO APC

Fusion of Ag to Ig-binding proteins can strongly increase the immunogenicity of the fused Ag. Snake toxins, normally
weak Ag, have been genetically fused to a carrier protein called ZZ, which is the Ig-binding fragment of staphylococcal
protein A (see cartoon).

Fusion constructs with ZZ have the capability to target the fused Ag to APC via two pathways: (1) after immunisation
the ZZ-fused Ag are targeted to surface Ig of APC by their ZZ moiety; (2) the construct is, before immunisation, com-
plexed with Ig (mAb) directed against APC surface structures such as CD11c; after immunisation,the ZZ-Ag/Ig complex
is targeted to APC via the APC-specific Ig. Using this approach, non-adjuvanted ZZ-Ag/Ig complexes were shown to elic-
it Ab responses in mice ~1000-fold higher than free snake toxin and also enhanced the Ag-specific T CELL response [12].

Box 1) [12].The recombinant gene is expressed in a
suitable organism and the fusion protein is then puri-
fied.A drawback of this genetic fusion technology is
potential misfolding of the EPITOPE when incorporat-
ed in the carrier protein, which would lead to irrele-
vant immune responses.
Anti-idiotype antibodies
The concept of anti-IDIOTYPE Ab VACCINES involves the
generation of a mAb (Ab-1) recognising a relevant
EPITOPE of the pathogenic agent, followed by the gen-
eration of a second mAb (Ab-2) directed against the
IDIOTYPE, i.e., the Ag-binding site of Ab-1. Ab-2 thus
mimics the original EPITOPE (Fig. 1f) and can be used
for vaccination, thereby eliciting protective Ab (Abs-
3). Virtually any desired EPITOPE can be structurally
mimicked by anti-IDIOTYPE Ab, whether it be continu-
ous or discontinuous EPITOPES of either protein or
polysaccharide nature [1].
Synthetic peptide vaccines
Chemically synthesised peptides belong to the best-
defined vaccine components currently under inves-
tigation.The synthetic peptide technology allows for
the design of VACCINES consisting of selected EPITOPES
free from irrelevant or unwanted structures. Large
amounts of linear peptides resembling T CELL or B
CELL EPITOPES (Fig. 1g) can be prepared by automated
methods. The immunogenicity of synthetic peptide
Ag is weak, but can be enhanced by conjugation to
carrier proteins (analogous to polysaccharide-pro-
tein conjugates) or to lipids,or by the construction of
multiple different B and T CELL EPITOPES [10,11].These
options offer the possibility to render synthetic B CELL
EPITOPES T CELL-dependent. Furthermore, the confor-
mational freedom of small linear peptides can be
restricted by cyclisation, which aims to force them
into a conformation reflecting the native structure,
which is especially important for peptide analogues
of B CELL EPITOPES [13].
Edible vaccines
Since the 1990s attempts have been made to use
plants as heterologous expression systems for ANTI-
GENS by integrating genes encoding the Ag of interest
into the plant genome [14, 15].The goal is to express
the Ag preferably in edible parts of plants, such as
242 Vaccines

BOX 2. PRESENTATION FORMS

Adjuvants comprise a large number of substances of variable chemistry and origin. Examples are colloidal salts, lipid
matrices, surface-active compounds and emulsions of mineral, bacterial, vegetable or synthetic nature.Adjuvants have
in common that they are not immunogenic per se, but enhance the immunogenicity of co-administered Ag.
Vaccine delivery systems are colloidal carriers with a size which can vary from ~50 nm to the micrometer range, allow-
ing multimeric Ag presentation at their surface.Thereby they mimic the natural presentation of Ag on viral or bacterial
surfaces. In general, multimeric presentation of Ag strongly improves their immunogenicity. Colloidal carriers can func-
tion as a depot at the administration site, resulting in sustained delivery and a reduction of the number of doses
required. Moreover, they can enhance humoral and/or cellular immune reactions, because colloidal particles are taken
up more efficiently by APC (in particular dendritic cells) as compared to free Ag. Uptake of colloidal particles by den-
dritic cells can be further promoted by coupling targeting moieties, specifically recognising dendritic cell receptors, to
their surface.As M cells present in mucosal membranes are specialised in the uptake of particulate material and subse-
quent presentation to immune cells, colloidal particles are also suitable Ag carriers when mucosal immunisation is pur-
sued. In addition, they may protect the Ag from proteolytic attack (e.g., in oral vaccine formulations). Besides Ag, adju-
vants are sometimes incorporated into these carrier systems, and many carrier systems have intrinsic adjuvant activity.

leaves or fruits. Among the numerous examples of
plants investigated for this purpose (e.g., potato,
tobacco, tomato), bananas seem to be most suitable
for human applications.This approach offers the pos-
sibility of cheap and local production in developing
countries, using the standard growing methods of a
given region, thereby alleviating logistical and eco-
nomic problems.Moreover,being edible,the VACCINES
would require no syringes and are safe to administer
in a patient-friendly way. Examples of ANTIGENS
expressed in plants include HBsAg, heat-labile
enterotoxin from E. coli, and rabies virus glycopro-
tein. Studies carried out in animals over the past
decade, and small tests in people, give hope that edi-
ble VACCINES can work. Major obstacles for the use of
edible VACCINES are related to quality control.
Nucleic acid vaccines
A development of potential clinical use is genetic
immunisation, i.e., direct administration of DNA
encoding one or more Ag of interest (Fig. 1h). Upon
i.m. immunisation with non-replicating plasmid
DNA, the protein encoded is produced and ex-
pressed by the host cell. RNA may also be used, but
is less suitable because it is rapidly degraded in vivo
and more expensive to produce. Nucleic acid VAC-
CINES are capable of eliciting both humoral and cel-
lular immunity. A sustained production of Ag after a
single immunisation is expected to provide long-
term protection [16].
Route of administration
The EFFICACY of vaccination programmes would be
enhanced greatly through the availability of needle-
free immunisation methods. Several alternative
routes of administration have therefore been
explored, including mucosal (oral, intranasal, pul-
monary) or dermal immunisation.
Mucosal immunisation routes are attractive alter-
natives to parenteral routes, not only because of the
ease of administration, but also because both sys-
temic and mucosal (secretory IgA) responses are
induced.The latter is advantageous, because mucos-
al surfaces are the common port of entrance of
many organisms and a strong local immune
response may hamper entry into the host by prevent-
ing adherence to and colonisation in mucosal sur-
faces. However, although mucosal immunisation
belongs to the oldest means of vaccination (Tab. 2),
the number of VACCINES suitable for mucosal immuni-
sation is limited to a few oral VACCINES. Poliovirus can
 Summary 243

BOX 3. VIROSOMES

Virosomes are unilamellar vesicles (typical diameter 150 nm) prepared from viral envelopes consisting of phospho-
lipids and viral membrane proteins.Virosomal delivery of membrane proteins generally induces enhanced immune
responses against these antigens. In addition, virosomes are useful not only for parenteral delivery but also as Ag carri-
ers for nasal administration. In particular, virosomes derived from influenza virus (also called immunopotentiating
reconstituted influenza virosomes,IRIV) have been widely investigated.The presence of viral proteins (haemagglutinin,
neuraminidase) has been shown to provide intrinsic APC targeting properties, to promote cell entry and to improve
(cytosolic) Ag delivery. This makes IRIV suitable as a delivery system for Ag other than influenza proteins. IRIV-based
hepatitis A and influenza vaccines are licensed and are examples of this approach.

be given orally because the virus is relatively resist-
ant to low pH, whereas oral typhoid vaccine is pro-
tected from gastric breakdown by formulation in
enteric-coated capsules. Other mucosal administra-
tion routes,such as nasal or pulmonary delivery,have
the advantage that the harsh conditions in the gas-
trointestinal tract are circumvented. Approaches to
augment the immunogenicity of future mucosal VAC-
CINES include the use of vectors (e.g., Salmonella) or
VACCINE DELIVERY SYSTEMS and co-administration of
ADJUVANTS [17, 18].
TOPICAL administration of Ag to the skin is an
appealing delivery route because the skin is a very
immune active organ containing a large number of
specialised APC, the Langerhans cells [19]. Until
recently, the skin was considered almost imperme-
able for large ANTIGENS. However, chemical penetra-
tion enhancers (e.g., surfactants and liposomes) as
well as physical approaches (e.g., iontophoresis) to
enhance the permeability of the skin for macromol-
ecules have shown some success. Glenn and co-
workers have shown that TOPICAL application of Ag-
containing patches generate potent immune
responses in man [20]. Moreover, targeting to epider-
mal Langerhans cells is possible by epidermal pow-
der immunization [21].
into enhancing the immune response to SUBUNIT VAC-
CINE components by suitable presentation forms,
including sophisticated VACCINE DELIVERY SYSTEMS and
ADJUVANTS (see Box 2) [9, 10, 22]. Carrier proteins and
live vectors are delivery systems already discussed.
Other delivery systems include particulate carriers,
such as biodegradable microcarriers, nanoparticles,
liposomes, immune-stimulating complexes (ISCOMs)
and virosomes (see Box 3) [22]. The traditional alu-
minium phosphate and aluminium hydroxide colloid
salts, which are the only ADJUVANTS currently used in
licensed products for human use, only stimulate
humoral immune responses. Also, they direct the
immune response mainly to type 2 (T-helper 2)
responses. Often a more balanced Th1/Th2 response
is needed for protection. Many novel candidate ADJU-
VANTS, such as saponines, lipid-A derivatives and bac-
terial DNA sequences containing CpG-oligodinu-
cleotides, also augment cellular immune responses
and mediate their effect through non-specific induc-
tion of several CYTOKINES, resulting in balanced
Th1/Th2 responses. The B subunits of cholera toxin
and of E. coli heat-labile enterotoxin are examples of
powerful mucosal ADJUVANTS. CYTOKINES such as IL-2,
IL-12 and IFN-γ have become of interest as more spe-
cific ADJUVANTS, especially in the search for potent
VACCINES against AIDS and cancer.

Vaccine delivery systems and adjuvants

Summary
In general it can be stated that the smaller the size of
a vaccine component, the weaker its immunogenici- VACCINES have been very successful in the prevention
ty. Therefore, a lot of effort has been and is being put of infectious diseases. Traditional VACCINES consist of
244 Vaccines
whole (live or killed) bacteria or viruses or compo-
nents thereof. Several modern approaches, most of
which are based on rDNA technologies, are emerg-
ing with the aim to generate more effective and safer
VACCINES. As a result of the tendency to design small-
er, better-defined antigenic components, proper Ag
presentation forms are becoming increasingly
important. Moreover, to limit the increasing number
of injections, needle-free immunisation routes and
ways to combine multiple vaccine components in
one vial are being explored. It is expected that VAC-
CINES to be marketed in the near future will be based
on some of the modern vaccine technologies dis-
cussed in this chapter and – like the conventional
VACCINES – will make a significant contribution to the
improvement of public health.
Selected readings
Plotkin SA, Orenstein WA (eds) (2004) Vaccines, fourth edi-
tion.WB Saunders Company, Philadelphia
Levine MM,Woodrow GC,Kaper JB,Cobon GS (eds) (1997)
New Generation Vaccines, second edition. Marcel
Dekker Inc., New York
Kaufman S (ed) (2004) Novel Vaccination Strategies.Wiley-
VCH, Berlin
Recommended websites
NIAID Net News, Infectious Diseases: http://www.niaid.
nih.gov/final/infds/infds.htm (Accessed March 2005)
The Vaccine Page,Vaccine News and Database: http://vac-
cines.org/ (Accessed March 2005)
World Health Organization; Immunization, Vaccines and
Biologicals: http://www.who.int/vaccines/ (Acces-
sed March 2005)
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