Table Of Content1
Overview of Vaccines
Gordon Ada
1, Patterns of Infectious Processes
Most vaccines are designed as a prophylactic measure, that is, to stimulate
the immune response so that on subsequent exposure to the particular infec-
tious agent, the extent of infection in the vaccinated individual is so low that
disease does not occur. There is also increasing interest in designing vaccines
that may be effective as a therapeutic measure. There are two contrasting types
of infectious processes.
1.1. Intracellular vs Extracellular Patterns
Some organisms, including all viruses and some bacteria, are obligate intrac-
ellular parasites in that they only replicate inside a susceptible cell. Some para-
sites, e.g., malarta, have an intracellular phase as one part of their life cycle. In
contrast, many bacteria and parasites replicate extracellularly. Because of these
differences, the immune responses required to control the infection may differ.
1.2. Acute vs Persistent Infections
In the case of an acute infection, exposure of a naive individual to a suble-
thal dose of the infectious agent may cause disease, but the immune response
so generated will clear the infection within days or weeks. Death may occur if
the infecting dose is so high that the immune response is qualitatively or quan-
titatively insufficient to prevent continuing replication of the agent so that the
host is overwhelmed. In contrast, many infections persist for months or years if
the process of infection by the agent results in the evasion or the subversion of
what would normally be an effective immune control reaction(s).
Most of the vaccines registered for use in developed countries, and discussed
briefly in the next section, are designed to prevent/control acute human infections.
From. Methods m Molecular Medme: Vaccrne Protocols
Edited by A Robmson, G Farrar, and C Wlblrn Humana Press Inc , Totowa, NJ
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Table 1
Currently Registered Viral and Bacterial Vaccines
Viral Bacterial
Love attenuated
Vaccmia BCG
PO110 (OPV) Salmonella (Ty2 1 a)
Measles
Adeno
Yellow fever
Mumps
Rubella
Inactivated, whole organism
Influenza Vibno cholerae
Rabies Bordetella pertussis
Japanese encephalitis Yersinia pestis
Polio (IPV)
Subunit
Hepatitis B Streptococcus pneumonzae
Influenza Salmonella typhl VI carbohydrate
Haemophilus vzfluenzae, type b
Acellular B pertusszs
Nelsseria meningldltis (A,C)
Conjugates (polysaccharides/protem carrier) H injluenzae, type b (Hib)
Toxoids Clostridlum tetanl
Corynebactenum diphthenae
Combmations
Measles, mumps, rubella (MMR) Diphtheria, pertussis, tetanus
VT)
DPT, H injluenzae, type b (Hib)
General reference (43)
2. Types of Vaccines
Almost all of the vaccines m use today are against viral or bacterial mfec-
tions (Table 1). They are of three types-live, attenuated microorganisms;
inactivated whole microorganisms; and subunit preparations.
2.1. Live, Attenuated Microorganisms
Some live viral vaccines are regarded by many as the most successful of all
human vaccines, with one or two administrations conferring long-lasting
immumty. Four general approaches to develop such vaccines have been used:
1. One approach, pioneered by Edward Jenner, is to use a vuus that is a natural
pathogen in another host as a vaccine in humans. Examples of this approach are
Overview of Vaccine 3
the use of cowpox and parainfluenza viruses in humans and the turkey herpes
virus in chickens.M ore recently, the use of avipox viruses, such as fowlpox and
canarypox,w hich undergo an abortive infection in humans,h as given encourag-
ing results in human trials (I).
2. The polio, measles,a nd yellow fever vaccines typify the second approach. The
wild-type viruses are extensively passagedin tissue-culture/ammalh ostsu ntil a
balance is reached between loss of virulence and retention of immunogenic-
ity m humans.
3. Type 2 poho vnus is a naturally occurring attenuateds train that has been highly
successfulM ore recently, rotavirus strainso f low virulence have been recovered
from children’s nurseriesd uring epidemics (2).
4. A fourth approach has been to select mutants that will grow at low temperatures
and very poorly above 37’C (Chapter 2). The cold-adapted strains of influenza
vuus grow at 25°C and have mutations in four of the internal viral genes (3).
Although such strainsw ere first describedi n the late 1960s and have since under-
gone extensive clnncal trials in adults and children, they are not yet registeredf or
human use.
In contrast to the above successes, BCG for the control of tuberculosis
remained until comparatively recently the only example of a live attenuated
bacterial vaccine. Although still widely used in the WHO Expanded Programme
of Immunization (EPI) for children, it has given very variable results in adult
human trials. However, prolonged studies to make other attenuated bacterial
vaccines, especially against Salmonella infections, have led more recently to a
general approach involving the selective deletion or inactivation of groups of
genes (4 and see Chapter 4). The first successw as with the strain Ty2la, which
has a faulty galactose metabolism, the successo f which led to the development
of strains with other gene deletions. This approach also shows promise for
complex viruses. Thus, 18 open reading frames have been selectively deleted
from the Copenhagen strain of vaccinia virus, including six genes involved in
nucleotide metabolism, to form a preparation that 1s of very low virulence, but
retains immunogenicity (5). The selective deletion of specific nucleic acid
sequences is also being tried with simian immunodeficiency virus with some
Initial success( 6). This approach offers the prospect of a selective and repro-
ducible means of producing adequately attenuated viral and bacterial prepara-
tions. Live attenuated vaccines have the potential of stimulating the widest
range of different immune responses, which may be effective in preventing,
controlling, and clearing a later infection.
2.2. Inactivated Whole Microorganisms
Inactivation of viruses, such as polio, influenza, rabies, and Japanese
encephalitis vu-uses, and some bacteria, including Bordetella pertussis and
Vzbrio cholerae, is the basis of vaccines with varying efficacy. Compared to
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the attenuated preparations, these vaccines need to be administered in substan-
tially larger doses and sometimes more frequently. The viral vaccines are gen-
erally effective in preventing disease, the low efficacy (70%) of the influenza
viral vaccine being in part owing to the continuing antigenic drift to which this
virus is subject. In contrast, the only bacterial vaccine of this nature still in
wide use is the pertussis vaccine, which is highly effective, but has already
been replaced by a subunit preparation in some countries because of adverse
side effects attributed to the whole-cell vaccine (7).
Inactivated whole vaccines generally induce many of the desirable immune
responses, particularly infectivity-neutralizing antibody, but generally do not
generate a class I MIX-restricted cytotoxic T-cell (CTL) response, which has
been shown to be the major response required to clear intracellular infections
by many viruses and some bacteria and parasites.
2.3. Subunit Vaccines
The generation of antibody that prevents infection by both intra- and extra-
cellular microorganisms has been regarded as the prime requirement of a vac-
cine. The epitopes recognized by such antibodies are most usually confined to
one or a few proteins or carbohydrate moieties present at the external surface
of the microorganism. Isolation (or synthesis) of such components formed the
basis of the first viral and bacterial subunit vaccines. Viral vaccines were com-
posed of the influenza surface antigens, the hemagglutinin and neuraminidase,
and the hepatitis B surface antigen (HBsAg). Bacterial vaccines contained the
different oligosaccharide-based preparations from encapsulated bacteria
(Chapter 8). In the latter case, immunogenicity was greatly increased, espe-
cially for infants, by coupling the haptenic moiety (carbohydrate) to a protein
carrier, thereby ensuring the involvement of T helper cells (Th-ceils) in the
production of different classes of immunoglobulin (Ig), particularly IgG. The
two bacterial toxoids, tetanus and diphtheria, represent a special situation
where the primary requirement was neutralization of the activity of the toxin
secreted by the invading bacteria (Chapter 7).
HBsAg is present as such in the blood of hepatitis B virus-infected people,
which was the source of antigen for the first vaccines. A major advance
occurred when the same product was made from yeast cells transfected with
DNA coding for this antigen, initiating the era of genetically engineered
vaccines (8). Up to 17% of adults receiving this vaccine turn out to be poor or
nonresponders, because of the age of the recipients and their genetic makeup (9).
3. Vaccine Safety
All available data concerning the efficacy and safety of a candidate vaccine
are reviewed by regulatory authorities before registration (Chapter 20). At that
Overview of Vaccine 5
stage, potential safety hazards, which occur at a frequency of perhaps l/10,000,
should have been detected. There are examples of undesirable side effects
occurring at much lower frequencies, which are seen only during immuno-
surveillance following registration, but these may be so low that their occur-
rence as a consequence of vaccination is difficult to prove. For example,
following the mass vaccmation program of people in the United States with
swine influenza vaccine m 1976-1977, a small proportion developed the
Guillain-Barre syndrome (10). This has turned out to be an isolated event.
In the prolonged absence of frequent outbreaks of diseaseb y specific vaccine-
preventable infections following successful vaccination campaigns, the occur-
rence of low levels of undesirable side effects following vaccination gains
notoriety. The evidence bearing on causality and specific adverse health out-
comes following vaccination against a number of childhood viral and bacterial
infections, mainly in the United States,h as recently been evaluated by an expert
committee for the Institute of Medicine in the United States( II). The possibility
of adverse neurological effects was of particular concern, and evidence for these,
as well as several immunological reactions, such as anaphylaxis and delayed-
type hypersensitivity (DTH), was examined in detail. In the great majority of
cases,t here was insufficient evidence to support a causal relationship, and where
the data were more persuasive, the risk was considered to be extraordinarily low.
Measles has provided an interesting example of vaccine safety. The WHO/EPI
has provided data illustrating the remarkable safety of the standard vaccine
(12). Furthermore, although natural measles infection induces an immunosup-
pressive state from which most children recover, the above study recorded only
two cases of immunosuppression in tmmunocompromised children following
vaccination (II). In many developing countries, measles vaccmation is given
at 9 mo of age: This delay is necessary to allow a sufficient decay of maternally
acquired antibody. This decay to low levels occurs earlier in some infants, allow-
mg an opportunity for infection by circulating wild-type virus before 9 mo. This
factor contributes significantly to the l-2 million deaths/yr from measles infec-
tion worldwide. To lessen this risk, “high-titer” measles vaccines were devel-
oped that could be effective in 54mo-old children. Trials in several countries
showed their apparent safety and ability to induce satisfactory immune respon-
ses in this age group, so their general use was authorized by the WHO in 1989.
Unfortunately, reports later appeared recording unexpected cases of mortality
following vaccination, especially in young girls in disadvantaged populations
(13), leading to the withdrawal of these vaccines from use. One possibility is
that the high-titer vaccine caused a degree of immunosuppression sufficient to
allow infections by other agents to occur.
Inactivation of a whole microorganism, even a relatively sample virus, does
not guarantee safety, Immunization of infants with mactivated measles or res-
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piratory syncytial vnus (RSV) preparations sensitized some recipients to severe
reactions when they were later exposed to the wild-type virus (e.g., 14). Never-
theless, the great safety record of the subunit viral vaccines is one factor con-
tributing to the attractiveness of the subunit approach to vaccine development.
4. Efficacy
There could be no more persuasive evidence of the worth of an immuniza-
tion program as a very effective public health procedure than the eradication/
elimination of an mfectious agent. Global eradication was first achieved in
1977 when the last case of endemic smallpox was detected, slightly more than
10 years after the intensified WHO campaign was initiated. Followmg mten-
sive immunization campaigns, the last case of endemic polio m the Americas
was detected more than three years ago (1.5). Clearly, the smallpox and poho
vn-us vaccines used in these campaigns are/were highly efficacious, although
both elicited some undesirable side effects (16,17). The eradication of polio in
the Americas is of itself remarkable and has led to intensified efforts in other
regions, although it is recognized that global eradication of polio is a substan-
tially greater challenge compared to smallpox. Nevertheless, the successi n the
Americas with poliomyelitis has led to the next challenge in that region-can
measles, another viral infection specific for humans, also be eliminated in the
Americas (18)?
These achievements, together with the emergence of such diseases as AIDS,
have greatly increased interest in all aspects of “vaccinology.” The following
sections discuss the need for improved and new vaccmes against a variety of
infectious agents, some of the new approaches now available for vaccine
development, the properties and functions of different immune responses, and
some of the obstacles that still face the vaccine developer.
5. Opportunities for Improved and New Vaccines
There are clearly two possible requirements for vaccine development. One
1st o develop improved vaccines to replace some existing vaccines. The other,
even more pressing need, is for vaccines against the many infectious agents
that still cause considerable morbidity and in some cases mortality. Table 2
lists examples of diseases where improved vaccines are desirable, and some
viral, bacterial, and other infections for which vaccines are not yet available.
The rationale for the need for improved compared to current vaccines var-
ies. For example, despite the efficacy and safety of the standard measles vac-
cine, there is a need for an (additional?) vaccine that would be effective in the
presence of maternal antibody. A genetically more stable type 3 live polio virus
and a means to make the oral polio vaccine and other live vaccines more heat-
stable would be desirable. The standard Japanese encephalitis viral vaccine is
Overview of Vaccine 7
Table 2
Opportunities for Improved and New Vaccines
Improved New
Viral
Influenza A and B Corona
Japanese encephalms Cytomegalo
Polio Dengue
Rabies Hepatitis A and C
Measles HIVland2
Hantan
Herpes
Norwalk agent
Papilloma
Parainfluenza
Respriatory syncytial
Rota
Varicella
Bacterial
Cholera Chlamydia
Meningococcus E. coli
M. tuberculosis Group A and B streptococcus
B. pertussis Haemophilus ducreyi
Mycobacteria leprae
Menmgococcus B
Neisseria gonorrhoeae
Shigella
Others Malaria
Schistosomiasis
Giardia
Filariasis
Treponema
B. burgdorferi
produced from infected baby mouse brains, surely now an out-of-date
approach. However, above all, fulfillment of the aim of the Children’s Vac-
cine Initiative, i.e., to produce a formulation of children’s vaccines that can
be administered at a smgle visit at or near birth and provide effective
immunity against numerous diseases (19), is likely in the long term to result in
major changes.
Vaccines against many of the other agents in Table 2 are unlikely to be made
using traditional techniques. For example, Mycobacterium leprue cannot be
produced in sufficient quantity to make a whole-organism vaccine to admmis-
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ter to >lOO million people. It may also be impractical to produce large quanti-
ties of some viruses to form the basis of a vaccine, but above all, some of the
new approaches to develop vaccines hold out so much promise that they are
bound to influence future manufacturing practices greatly.
6. New Approaches to Vaccine Development
There are basically three new approaches that are being investigated.
1. The use of anti-idiotype antibody preparationst o mimic B-cell epitopes.
2. The synthesiso f ohgo/polypeptides,w hich reflect naturally occurrmg ammo acid
sequencesm proteins of the pathogen( Chapter 6).
3. The use of recombinant DNA (rDNA) technology (Chapter 5) to obtain DNA/
cDNA coding for antigen(s) of different pathogens or other factors, such as
cytokines,a nd to use thesem mainly three different ways:
a. To transfectc ells so that the inserted DNA/cDNA is translateda nd expressed.
b. To insert the DNA/cDNA into the genome of other viruses or bacteria,w hich
are usually chosen as vectors becauseo f then record as effective and safe
vaccines.S uchc lnmencc onstructsa re potential new vaccines (Chapters 3-5)
c. A plasmid contannng the DNA/cDNA can be directly injected into cells in
viva, where it is translated and expresseda nd immune responsesn ntiated
(Chapter 21).
6.7. Anti-ldiofypes
The attractions of this approach included the fact that the anti-idiotype
should mimic (1) both carbohydrate and peptide-based epitopes; and (2) the
conformation of the epitope in question. Despite such advantages, this approach
has never really prospered.
6.2. O/igo/Po/ypepfides
The sequences may contain either B- or both B-cell epitopes and T-cell
determinants. Sequences containing B-cell epitopes may be conjugated to car-
rier proteins that frequently act as a source of T-cell determinants or assembled
in different configurations to achieve particular configurations or produce mul-
tiple determinants. Some of the obvious advantages of this approach include
the fact that the final product contains the critical components of the antigen,
which offers the possibility of removal of segmentsm imicking host sequences.
Multimerm constructs, such as Multiple Antigemc Peptide Systems (MAPS)
can be highly immunogenic (20). In addition, recent work has shown that
immunogemcity of important “cryptic” sequences may sometimes be enhanced
by deletion of other segments of a molecule (21), and new methods of synthe-
sis offer the possibility of more closely mimicking conformational patterns in
the original protein (e.g., 22). This is now a very active field, and peptide-based
vaccines seem to be assured of a significant share of the future vaccine market.
Overview of Vaccine
Table 3
Some Live Viral and Bacterial Vectors
Viruses
Vaccinia, fowlpox, canarypox,
adenovlrus, polio, herpes, influenza
Bacteria
BCG, Salmonella, E. colt
6.3. Transfection of Cells with DNA/cDNA
Three cell types have been used-prokaryotes; lower eukaryotes, mainly
yeast; and mammalian cells, either primary cells (e.g., monkey kidney), cell
strains (with a finite replicating ability), or cell lines (immortalized cells, such
as Chinese hamster ovary [CHO] cells). Each has its own advantages, and bac-
terial, yeast, and different mammalian cells are now widely used. As a general
rule, other bacterial proteins should preferably be made in transfected bacterial
cells and human viral antigens, especially glycoproteins, m mammalian cells,
because of the substantial differences in properties, such as posttranslational
modification in different cell types (23).
6.4. Live Viral and Bacterial Vectors
Table 3 lists the viruses and bacteria mostly used for this purpose. Most
experience has been with vaccinia virus, since it is very convenient to use, has
a wide host range, possesses about 100 different promoters, and, as already
stated, substantial amounts of DNA can be removed from it, leaving room for
inserted DNA coding for at least 10 average-sized proteins. Several of the vec-
tors, such as adenovirus, polio virus, and SuZmonelZu, should be ideal for deliv-
ery via a mucosal route, although both vaccinia and BCG have also been given
orally and intranasally.
Making chimeric vectors has also been an effective way of assessing the
potential role in immune processes of different cytokines. Inserting cDNA cod-
ing for a particular cytokine as well as that for the foreign antigen results in
synthesis and secretion of the cytokine at the site of infection. One of the more
interesting recent findings is that inclusion of the cDNA coding for IL-6 greatly
enhances production of sIgA specific for the viral antigens (24).
6.5. “Naked” DNA
The most fascinating of recent approaches has been the injection of plas-
mids containing the DNA of interest, either directly into muscle cells or as
DNA-coated microgold particles via a “gene-gun” into skin cells. In the latter
case, some beads are taken up by dendritic-like cells and transported to the
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Table 4
Properties and Functions
of Different Components of the Immune Response
Stageso f infectious process
Type of Type of Cytokine
response infection profile Prevent Limit Reduce Clear
Nonadaptive I - ++ + -
E - ? ? -
Adaptive
Antibody I +++ ++ ++ H-
E +++ +++ +++ ++-I-
CD4+ Th2 I IL-3,4,5,6,10,13
E TNFa
CD4+ Thl I IL-2,3, IFNy, TNFa - ++ ++ ++?
E J-w - +++ +-k+ +++
CD8+CTLs I IL-2, IFNy, TNFP _ +++ +++ ++-I-
E - - - -
I, mtracellular mfectlon, E, extracellular mfectlon, IL, mnterleukm, IFN, Interferon, TNF,
tumor necrosis factor
draining lymph nodes. This procedure has resulted in quite prolonged humoral
and cell-mediated immune responses. One of the potential benefits is that the
induction of such responses should also occur in the presence of specific anti-
body. The fact that a recent issue of a relevant scientific journal consists entirely
of articles describing the use of this approach reflects the widespread interest
in this approach (25).
7. Properties and Functions
of Different Components of the Immune Response
7.1. Classes of Lymphocytes
Our knowledge of the properties of lymphocytes, the cell type of major
importance in vaccine development, has increased enormously in recent years.
The major role of B-lymphocytes 1s the production of antibodies of different
isotypes and, of course, specrficity. The other class of lymphocytes, the T-cells,
consist of two main types. One, with the cell-surface marker CD4, exists in two
subclasses, the Thl- and ThZcells (h standing for helper activity). A major
role of Th2-cells is to “help” B-cells differentiate, replicate, and secrete anti-
body. They do this in part by the secretion of different cytokines (interleukins,
ILs), which are listed in Table 4. Thl-cells also have a small, but important
role in helping B-cells produce antibody of certain isotypes, but the overall
pattern of cytokine secretion is markedly different. Such factors as IEN-7, TNF-a,
