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Chap 7 Vaccines
I. Introduction
Vaccination:

Vaccine induces the host to generate immunity against the disease-causing organism
(bacteria or virus), so upon future exposures the infectious agent is neutralized or killed.

The first vaccine was developed 200 years ago, against smallpox (Edward Jenner) by
using exudates (滲出物) from a cowpox pustule (膿苞).

In 1885, Louis Pasteur developed a vaccine against rabies. He also developed a vaccine
against anthrax using a heat-attenuated strain of Anthrax bacillius.

Emil von Behring (in 1890) demonstrated that serum from animals previously
immunized to diptheria could transfer the immunity to unimmunized animals.
Traditional Vaccines:

The organism is grown in culture, purified then inactivated or attenuated without losing
the ability to evoke an immune response.
1.
Killed vaccine (inactivated by heat or chemicals such as formalin, formaldehyde, ether..).

Fluarix (GlaxoSmithKline) is a trivalent (two type A strains and one type B strain)
inactivated influenza vaccine prepared from influenza viruses propagated in
embryonated chicken eggs. After harvesting the virus containing fluid, each
influenza virus is concentrated and purified by ultracentrifugation using a linear
sucrose density gradient solution containing detergent to disrupt the viruses. Each
influenza virus is inactivated by sodium deoxycholate and formaldehyde leading to
the production of a split virus. The vaccine contains 15 g of viral hemagglutinin
(HA) per 0.5 ml from each of the strain entering into the composition.
2.
Live attenuated vaccine (attenuated by repeated passage of an infectious agent).

Vaccinia virus (VV) Ankara strain (MVA) is generated by repeated passage in chick
embryo fibroblasts and is extensively used in smallpox eradication program.
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
FluMistTM is a trivalent flu vaccine (two type A strains and one type B strain)
delivered intranasally as a spray. The influenza viruses are cold-adapted to grow
well at 25C by passaging the virus at successively lower temperatures. These
viruses are reliably attenuated because the cold-adaptation to 25C allows only
minimal replication in the nasopharyngal respiratory tract, but no replication in the
lungs of infected animals. FluMistTM is also produced in eggs.
Selected Vaccines introduced in routine immunization programs before the 1980s

Disease
Year
Diphtheria (白喉)
1939
Tetanus (破傷風)
1949
Pertussis (百日咳)
1953
Influenza (流行性感冒)
1958
Measles (痲疹)
1963
Mumps (流行性腮腺炎)
1966
Limitations:

Not all infectious agents can be grown in culture (∴ no vaccines for such diseases, e.g. HCV)

Safety: most flu vaccines are produced using embryonated chicken eggs, high-risk
individuals such as persons suffering from allergies and patients with asthma are
prone to developing side effects such as allergic reactions to chicken proteins. In
many cases, even a small quantity of chicken protein is enough to provoke lifethreatening hypersensitive, allergic reactions.


Not all diseases (e.g. AIDS) are preventable by traditional vaccines
rDNA technology has provided a means of creating a new generation of vaccines.
II. Immune System [1, 2]
1. Innate (nonspecific) immunity:

A set of defense mechanisms that is not specific to a particular pathogen, mediated by
phagocytic cells (e.g. macrophage, neutrophil) and others (e.g. lysozyme cleaves bacterial
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cell wall, low pH of the skin (3-5) retards microbial growth)), provides the first line of
defense.

Macrophages (see Appendix) encountering microbes in tissues are triggered to release
cytokines that increase the permeability of blood vessels, allowing fluid and proteins to
pass into the tissues. They also produce chemokines that direct the migration of
neutrophils to the site of infection.

Like macrophages, neutrophils have surface receptors for common bacterial constituents
and for complement and are the principal cells that engulf and destroy the invading
microorganisms. The accumulation of fluid and cells at the site of infection causes the
redness, swelling, heat and pain known collectively as inflammation.
Local
inflammation and phagocytosis may also be triggered by the activation of complement.
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
Complement is a system of plasma proteins that activate a cascade of proteolytic
reactions on microbial surfaces  cell death and lysis.

However, if the innate immunity does not completely eliminate infectious
microorganisms, the inflammatory response increases the flow of lymph, which contains
the antigen-bearing cells
(macrophage and dendritic cells),
into lymphoid tissueAdaptive
immunity.
2. Adaptive (Specific) immunity:

If an invading microorganism
eludes the innate immunity, the
specific immunity is triggered
within 5-6 days after initial
exposure. Involves two major
groups of cells: lymphocytes and
antigen-presenting cells.
Lymphocyte: includes B and T lymphocytes (and NK cells in the innate immunity arm, see Appendix)

White blood cells that arise in the bone marrow and differentiate in the central lymphoid
organs (yellow, B cells mature in bone marrow and T cells mature in thymus).

Migrate from these tissues and are carried in the bloodstream to the peripheral organs
(blue): the lymphoid nodes, spleen, and lymphoid tissues associated with mucosa (e.g.
the gut-associated tonsils, Peyer’s patches). The peripheral lymphoid organs are the sites
of lymphocyte activation by antigen (Ag1, and lymphocytes circulate between the blood
and these organs until they encounter their specific Ag.

The spleen is a fist-sized organ behind the stomach that collects Ag from the blood. The
gut-associated lymphoid tissues (GALT), which include the tonsils, adenoids (腺樣增值
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An antigen (Ag) is a molecule that binds to Ag-specific receptors, but cannot necessarily induce an immune response in the
body by itself. Antigens are usually peptides, polysaccharides or lipids.
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體), appendix (闌尾) and Peyer’s patches in the small intestine, collect Ag from
epithelial surfaces of the gastrointestinal tract (in Peyer’s patches, the Ag is collected by
M cells). Albeit different in appearance, the lymph nodes, spleen and GALT operate on
the same principle, trapping Ag ingested by dendritic cells (DC) or macrophages from
sites of infection and presenting it to migratory lymphocytes, thus inducing adaptive
immune responses.
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
B lymphocyte:
naïve B cells
(with Ab on the
membrane)
Ag binding to the
receptor (Ab)
rapid cell division
memory B cell
plasma B cells
* Memory B cells have a longer life span and continue to express the same membranebound Ab. Instead, plasma cells express secreted Ab and survive only a few days.

T lymphocyte: TH (CD4+), Tc (CD8+) and Treg (CD4+CD25+FoxP3+).

B cell can recognize the Ag alone, but T-cell receptor can recognize only Ag that is
presented by the major histocompatibility complex (MHC).

TH cells recognize Ag presented
by MHC class II (expressed mainly on APC)
whereas Tc cells recognize Ag2
presented by MHC class I (expressed
on nearly all nucleated cells).

Treg: can suppress immune
responses by repressing TH and Tc
cells functions.
Antigen Presenting Cells (APC):

Include macrophage, B lymphocytes
and dendritic cells (DC).
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The term “antigen” originally describes a structural molecule that binds specifically to an antibody. It expands to refer to
any molecule or a linear molecular fragment that can be recognized by highly variable antigen receptors (B-cell receptor or
T-cell receptor) of the adaptive immune system
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
Express MHC class II molecules and co-stimulatory signals for TH cell activation.
A virus (thymus-dependent Ag) is recognized by B cells via the epitope and then
internalized. The epitope is then displayed on the surface by MHC II. The epitopeMHC complex can be recognized by pre-stimulated Th cells, which secrete
cytokines (e.g. IL-4, IL-5) to activate B cells.
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Functions of Humoral and Cell-Mediated Responses
Humoral: Ab is the main effector

The immunity can be transferred to a nonimmune
individual by administration of serum Ab from an
immune individual.

Ab binding to Agelimination of the Ag in several
ways (see Fig. 1-24):

Primary response (prime): has a lag of 5-7 days due to
the time required for the activation of naïve B cells. Ab
levels peak at about day 14 and then drops as the
plasma cells begin to die.

Secondary response (boost): the lag is much shorter and
Ab levels are much higher (100-1000-fold higher)
because of the presence of memory cells.
Cell-mediated:

Immunity that can be transferred only by administration
of T cells from an immune individual.

CTL participate in the killing of altered self-cells (e.g.
virus-infected or tumor cells)


CD4+ Th cells include two subsets:

Th1 cells: mainly lead to cell-mediated immunity and the production of IgG

Th2 cells: mainly lead to humoral immunity, especially IgM, IgA and IgE.
Th1 cells can:

activate macrophage, enabling them to destroy intracellular microorganism.

activate B cells to produce IgG (IgG1 and IgG3 in humans, IgG2a and IgG2b in mouse).

secrete IFN- (which is the main macrophage-activating cytokine that can enhance the virus degradation) and
TNF- (which activates macrophages and inhibits B cells).

secrete IL-2 which induces T cell proliferation.
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[3]
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III.Subunit Vaccines:

Vaccines may not need the whole organism to induce Ab.

Purified recombinant proteins are used as the subunit vaccine.
Pros:

Chemically defined

Less contaminating proteins, carbohydrates and nucleic acids that can cause side
effects.
Cons:

Purified protein may not have the same conformation and immunogenicity is altered
(because some epitopes are conformation-dependent)

Soluble proteins usually elicit predominantly humoral immunity, but not strong
cellular immunity.
To enhance the immunogenicity, the antigen may be administered with adjuvants.
Currently the adjuvants approved for human use include alum (aluminum hydroxide
(Al(OH)3), aluminum phosphate (AlPO4)) and MF59 (triggers the innate immunity).
The recombinant antigen may be co-administered with immunostimulating cytokines such
as GM-CSF (which acts as a growth and maturation factor for macrophages and dendritic
cells) or IL-2 (which acts as a growth and maturation factor for T-lymphocytes), or a
combination of the two (or IL-12, IL-18…). The antigen and cytokines may be
encapsulated within liposomes/nanoparticles.
Examples:

Subunit vaccines against HBV (rHBsAg), B. pertussis (rPT) and porcine circovirus 2
(PCV2).
IV.

Conjugate Vaccines
Polysaccharides on the encapsulated bacteria have been purified and used as vaccines
against Neisseria meningitidis (腦膜炎球菌=meningococcus), Hib, Salmonella typhi and
S. pneumonia (see Appendix).

Poor immunogenicity and efficacy in infants. No immunological memory and repeated
immunization are unable to increase Ab titers.
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
Immunogenicity is improved by conjugation to carrier proteins. Conjugation converts Tindependent Ag into T-dependent Ag by providing a source of appropriate T-cell
epitopes (present in the carrier protein).
Ex:
Conjugated vaccine against S. Pneumoniae (licensed in the US in 2000) contains
polysaccharides from seven serotypes (4, 6B, 9V, 14, 18C, 19F and 23F). The polysaccharides
are chemically conjugated to diphtheria toxin 197 (CRM, a mutant toxin whose toxicity is
deleted).
V. Peptide vaccines

Subunit vaccines are normally whole proteins, on which only some domains (epitopes)
induce the immune response

May synthesize short peptides that mimic the epitopes
Ex: FMDV VP1

FMDV VP1 is the most immunogenic protein in the virus.

Peptides corresponding to different epitopes in VP1 are
chemically synthesized

Very small peptides are degraded shortly unless they are
bound to a large carrier

The carrier can be conjugated with single or multiple types of
peptides (broaden the spectrum, e.g. some epitopes may target
B cell, some may target Th and some may target Tc).
Carrier (e.g. HBcAg as the carrier protein)

DNA encoding FMDV VP1 peptide 142 to 160 (more immunogenic than other
regions) is linked to the gene of Hepatitis B core antigen (HBcAg, which can form
particles) and expressed.

Fusion protein self-assembles into stable 27-nm particles, with VP1 on the outer
surface  very immunogenic.
Drawbacks of synthetic peptides:

May need to resume the same conformation of the epitope
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
A single peptide may not be sufficiently immunogenic.

The amount needed is >> the inactivated FMDV.
VI.

DNA vaccine
DNA encoded in a plasmid is directly injected (e.g. most commonly into the muscle).
 The plasmids are taken up by the cells
 the proteins (immunogen, usually under the control of a strong virus promoter) are expressed inside the
cells.
 elicit the immune response
Pros:

No need to produce and purify recombinant proteins.

Proteins produced by DNA vaccine are more likely to be correctly processed (posttranslationally modified)

Endogenously synthesized proteins readily enter the processing pathways for the loading
of peptide epitopes onto MHC-I (induce CTL responses) and MHC-II (induce Th cells),
thus inducing both cell-mediated and humoral responses.
e.g. influenza A virus nucleoprotein (a core protein)
 is encoded in a plasmid and injected into mice in the muscle.
 injected mice were also protected against different strains of flu virus
 cross-protection (∵ core proteins are less variant) traditional vaccines (surface antigen) usually are
directed against surface antigens and hence only one strain of flu virus. NP-specific Ab has no virus neutralizing activity, but
NP is an important target for CTL which are cross-reactive with all type A viruses.

DNA vaccine can trigger the adaptive response only against the encoded protein but not
on the plasmid itself (but plasmid DNA can evoke innate immune responses by TLR9).
 ∴ same vector can be used to deliver different genes
Cons:

Transfection efficiency is low (large amounts of DNA are needed, with the aid of microparticles such as polymer or
liposomes, the amount of DNA can be reduced >100-fold)
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VII. Recombinant viruses as vaccines

Some benign viruses expressing effective immunogens (may co-express cytokines such
as IL-2, IFN- or GM-CSF, etc.) are used as vaccines.

Examples:

NYVAC: a vaccinia virus (VV) that has 18 ORF deleted and highly
attenuatedNYVAC vectors encoding a wide range of pathogen-derived genes
have been constructed.

rMVA expressing influenza haemagglutin and nucleoproteins are shown to protect
animals from influenza virus challenge.

Fowlpox virus: Fowlpox virus expressing hemagglutinin from the H5 subtype
protects chickens against avian influenza virus (H5N2) infection and is currently
licensed and used as an avian influenza vaccine in Mexico.

Adenovirus (Ad): noneveloped, icosahedral virus, 80-90 nm, spiked, 3 subtypes (A,
B, C), subtypes A and B may be oncogenic in mice, while subtype C causes mild
respiratory diseases in humans, accounts for 5-15% of “common cold”. Ad can be
administered orally and live vaccines are already in use.

Baculovirus: can enter mammalian and avian cells and express the antigens as long
as the genes are driven by the promoter active in the cells. Baculovirus expressing
the influenza HA has been tested as a vaccine.
VIII. Attenuated Vaccines

Bacteria or viruses are genetically engineered to modify or delete the virulence genes.

The important antigenic determinants are retained with correct conformation.

Immunogenicity retained.

Live vaccine, generally much more effective than killed or subunit vaccines.
Ex: attenuated virus vaccine for IBDV (via Reverse Genetics)

IBDV (infectious bursal disease virus) causes severe immunodepression in young
chickens death, economic loss.

current vaccine: killed vaccine
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
genome consists of two dsRNA segments:

segment B: encodes VP1 (polymerase and capping enzyme)

segment A: encodes a polyprotein which after translation is processed into VP2, VP3
by VP4 (VP2, VP3 are major structural proteins of the virus particle)
NS
VP2
VP4
VP3

NS gene precedes and partially overlaps with VP2 and represents another ORF.

NS gene encodes VP5 (a nonstructural protein, not in the virion), but involves in viral
pathogenesis (induces lesion).
Yao et al. constructed the cDNA clones of segments A and B, in which the
ATG start codon of NS is mutated to TAG (stop codon)
The cDNA clones of segment A and B are inserted into the plasmids
under the control of T7 promoter in vitro transcription using T7
RNA polymerase
segment A and B transcripts
transfection of both transcripts into Vero
viable IBDV progeny
 resultant mutant IBDV:

Lacks NS gene expression

Cytotoxic and apoptotic effects decreased in cell culture

No signs of lesion or disease in the infected animal

Induce Ab level similar to the original virus

No reversion of mutation was detected

Replicate slightly more slowly than original virus
 Can be used as live-attenuated vaccines for IBDV
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IX.

VLP (Virus-like Particle)
Virus: composed of proteins (some form the coat)
and the nucleic acid.

VLP: empty particles consisting of only the viral
proteins but do not contain viral DNA or
RNAnon-infectious.

Some VLPs are naturally occurring, e.g. HBV empty particles or poliovirus-like particles.
VLP structural proteins can elicit immune responses, hence VLPs are excellent vaccine
candidates.

VLP generates higher titers of antibodies than subunit vaccines in some animal tests,
presumably due to the maintenance of conformation and display of multiple functional
epitopes. VLP also induces potent cellular immunity.
Ex: Enterovirus 71 (EV71)

Enterovirus 71 is a single-stranded, positive-sense RNA virus whose genome (≈7500
nucleotides) consists of one open reading frame (ORF, ≈6600 nt) flanked by 5’ and 3’
untranslated regions (5’ and 3’ UTR).

The ORF is expressed as large polyprotein that can be divided into 3 regions: P1, P2 and
P3. The four structural proteins, VP1, VP2, VP3 and VP4, are encoded within P1 region
while the non-structural proteins, such as proteases 2A, 3Cpro, 3CD, and other proteins
responsible for virus replication and virulence, are encoded within P2 and P3 regions.

Upon translation, 2A undergoes self-cleavage at its N-terminus to liberate P1 from the
entire polyprotein and then the protease 3CD cleaves P1 into VP1, VP3 and VP0 in trans.

VP1, VP3 and VP0 spontaneously assemble into an icosahedral procapsid (VLP).
Enterovirus RNA is then encapsidated in the central cavity of the procapsid to form
provirions.
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
P1 and 3CD can be cloned into a single baculovirus for co-expression in insect cells.
The expressed 3CD cleaves P1 into VP1, VP3 and VP0, leading to the VLP selfassembly within the cells.

Injection of the EV71 VLP into animals induce potent and long-lasting humoral and
cellular immune responses.
Commercial products:

Hepatitis B vaccine: Recombivax-HB (Merck); Engerix-B (GSK)

Human papillomavirus (HPV) vaccine: VLP consisting of HPV L1 protein (e.g.
Gardasil (Merck), Cervarix (GSK).
[4]
References:
[1]
[2]
[3]
[4]
Janeway CA, Travers P, Walport M, Shlomchik MJ. Immunobiology. New York:
Garland Science, 2005.
Goldsby RA, Kindt TJ, Osborne BA. Kuby Immunology. New York: W.H. Freeman
and Co., 2000.
Mills KH. Regulatory T cells: friend or foe in immunity to infection? Nat Rev
Immunol 2004; 4:841-55.
Mena JA, Kamen AA. Insect cell technology is a versatile and robust vaccine
manufacturing platform. Expert Rev Vaccines 2011; 10:1063-81.
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X. Appendix
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Selected Vaccines Licensed after 1980
Disease/pathogen
Year
Hepatitis B (B 型肝炎)
1986
Haemophilus influenzae type b (嗜血性流感, 1990
Hib, Conjugate)
Salmonella typhi (傷寒, live attenuated,
1991
capsular polysaccharide)
Hepatitis A (A 型肝炎)
1994
Varicella (水痘)
1995
Pertussis (recombinant subunit vaccine)
1996
Influenza (adjuvanted)
1997
Lyme disease
1998
Meningococcus (腦膜炎球菌, Conjugate)
2000
Pneumococcus (肺炎雙球菌, Conjugate)
2000
Rotavirus (輪狀病毒)
2006
HPV (人類乳頭瘤病毒)
2006
HPV Vaccine FDA has approved Merck's human papillomavirus vaccine Gardasil for
sale and marketing to girls and women ages nine to 26. Merck in October 2005 announced
that Gardasil in clinical trials was 100% effective in preventing infection with HPV strains 16
and 18, which together cause about 70% of cervical cancer cases. The trials showed the
vaccine -- given in three injections over six months -- also was about 99% effective in
preventing HPV strains 6 and 11, which together with strains 16 and 18 cause about 90% of
genital wart cases. Gardasil also protects against vaginal and vulvar cancers, two other
gynecological cancers that also are linked to HPV. An FDA advisory panel in May (2006)
voted that the vaccine is safe and effective for girls and women ages nine to 26. Gardasil in
studies has been shown to be effective for three-and-a-half years. Merck said the full treatment
course will cost $360.
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