Module 02806: Advanced Topics in Immunology
Lecture #9 :
Dr Adrian Mountford
Vaccines and Vaccination.
Aims and objectives;
This lecture will cover different aspects of ‘vaccinology’. It will start with a definition and a perspective on
how vaccines have been effective in combating infectious disease. The lecture will then describe different types
of vaccine from 1st generation (attenuated) vaccines to the most recent 3 rd generation (DNA) experimental
vaccines.
The lecture will require an understanding of the basic mechanisms of the immune system as described in
lectures 1-6. You will be required to use this information to understand how different types of vaccines
operate, and why different vaccination strategies are more applicable than others for certain types of disease.
The lecture will illustrate how the design of an effective vaccine requires a clear appreciation of molecular
biology and immunology.
The lecture will cover the following topics;
 Definition.
 Need for effective vaccines.
 Successful vaccine programmes.
 Designing the optimal vaccine.
 Inactivated vaccines
 Salk v Sabin polio vaccines
 Attenuated vaccines
 Recombinant sub unit vaccines
 Peptide vaccines
 Adjuvants and carriers (IL-12, ISCOMs)
 Live recombinant expression vectors (BCG)
 Naked DNA vaccines
-structure and delivery
-immune response
-adjuvanticity of CpG motifs
-enhancing DNA vaccine efficacy
-alternative vaccination strategies (prime boost)
-safety concerns
References and further reading:
General
Vaccine Design. The subunit and adjuvant approach. Eds Powell and Newman.
1. Bendelac & Medzhitov (2002) Adjuvants of immunity: Harnessing Innate immunity to promote adaptive
immunity. J. Exp. Med 195 F19-23
2. Letvin, N. L. (1998) Science 280: 1875-1880
3. LaCasse, R. A. et al. (1999) Science 283: 357-362 (see also p336 for comment)
4. Schijns V.E.J.C. (2000). Immunological concepts of vaccine adjuvant activity. Curr Opinion in Immunology
12:456-63.
DNA vaccines
1. Gurunathan, S. et al. (2000) DNA vaccines: a key for inducing long-term cellular immunity. Current Opinion
in Immunology. 12: 442-47.
2. Kirman and Seder, (2003) DNA vaccination: the answer to stable, protective memory? Curr Opin Immunol.
15: 471-476. Last half of paper only!!
3. Ramshaw I.A. & Ramsay A.J. (2000). The prime-boost strategy: exciting prospects for improved vaccination.
Immunology Today 21: 163-65
4. Spawasser et al. (1998) Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and
activation of murine dendritic cells. European Journal of Immunology 28: 2045
5. Tighe et al. (1998) Gene vaccination: plasmid DNA is more than just a blueprint. Immunology Today; 19: 89
WWW sources
http://www.york.ac.uk/depts/biol/staff/apm.htm
http://www.who.int/gpv-dvacc
http://www.sabin.georgetown.edu/VACCINE.HTM
http://www. enweb.com/Dnavax/
VACCINES AND VACCINATION:
Definition;
‘A vaccine is a material originating from a microorganism that induces immunologically mediated
protection to disease’
The rationale design of vaccines requires:
 Identification of the protective immune mechanism.
 Identification of the relevant antigen(s).
 Reconstitution to induce protection and not pathology.
The material can also be produced by molecular biological techniques, hence the term recombinant
vaccines.
Vaccination can also be used in the context of:
 Cancer
 Allergy
 Birth control/fertility
BUT, this lecture will deal exclusively with vaccines against infectious disease
Changes in the incidence of disease underlining the incidence of disease
Disease
Maximum Number of
Cases and Year
Diptheria
206,939 in 1921
Measles
894,134 in 1941
Mumps
152,209 in 1968
Pertussis
265,269 in 1934
Polio (paralytic)
21,269 in 1952
Rubella
57,686 in 1969
Tetanus
1,500 in 1923
1993 Cases
% Change
0
-100%
277
1,630
6,132
-99.9%
-98.9%
-97.7%
0
188
9
-100%
-99.7%
-99.4%
History:
Vaccination in various forms has been practiced for many centuries.
“Variolation” used scabs from Leishmania infected individuals to give infection local to a ‘hidden’ area of
skin to new born girls. This gives protection from infection in later life which can affect the face.
In 1796, Edward Jenner vaccinated a local boy James Phipps with cowpox taken from a milkmaid and
showed that the boy was immune to smallpox.
180 years later after an 11 year campaign, smallpox was eliminated worldwide and vaccination was
stopped after 1980.
In the 1970’s the Expanded Program on Immunization (EPI) saw the scope of vaccination reach 80% of
the world’ s children (compared to 5% at the start). Covered vaccines against, diptheria, pertussis, tetanus,
poliomyelitis, measles & tuberculosis.
In 2000, a new initiative “Global Alliance for Vaccines and Immunization” GAVI, was set up to fund new
vaccines and delivery programmes in the worlds poorest countries. Funded largely by Bill Gates
foundation, it will initially target Hepatitis B and Haemophilus influenzae B vaccines.
BASIC PRINCIPLES FOR GOOD VACCINE DESIGN:
1.
2.
3.
4.
5.
6.
7.
8.
9.
Induces an immune response which prevents disease (not necessarily infection).
Long-lasting. A single dose would be optimal.
Induce correct type of immune response. i.e. humoral or cell-mediated?
Induce immune response in the correct tissue location. i.e. gut or lungs.
Include the correct antigens which stimulate immune response to the
disease or infection.
Delivery at the optimum time of life to prevent disease.
Safe with no side effects.
Stable (preferably long lasting on the shelf at ambient temperature)
Cheap for mass production and dosing.
INACTIVATED VACCINES:
Many early vaccines were produced this way.
1. Whole killed cell, treated with formaldehyde or heat-treated e.g. Bordetella pertussis
2. Whole killed virus e.g. Influenza
3. Toxoid (purified inactive toxin) e.g. tetanus toxoid and diptheria toxoid.
4. Polysaccharide cell capsule e.g. Heamophilus influenzae B, meningococcus A & C.
The classic inactive vaccine (IPV) was designed by SALK in 1955 against polio. At the same time an
attenuated vaccine (OPV) was also produced by SABIN.
Comparison of the 2 underlines the advantages and disadvantages of each. See Text books and Handout.
ATTENUATED LIVE VACCINES:
 Irradiation e.g. gamma or UV rays.
Laboratory models of schistosomiasis and malaria vaccines. Parasites die after infection of the host
before inducing pathology.
 Induced mutants. Microorganism cultured in vitro under abnormal conditions e.g. high/low
temperature, pH, poor nutrients. E.g. Influenza or poliomyelitis. Those that survive due to an induced
mutation under these conditions will be non-virulent when used to infect the normal host and so do not
survive.
 Genetic deletion or modification. Delete the gene which confers virulence and ability to replicate in
the normal host. e.g. the nef gene in HIV required for replication and the deletion of B toxin subunit in
cholera renders the vaccine non-pathogenic.
BUT;
 Live attenuated vaccines may revert to the wild type and cause pathology.
 Immunisation with a whole organism involves exposing the host to many hundreds of irrelevant
antigens not required for protection.
 Ultimate aim to immunise only with the antigens required to stimulate protection.
RECOMBINANT OR SUBUNIT VACCINE ANTIGENS:
Prokaryote expression systems. e.g. E. coli
1. Identify antigen of interest using sera or monoclonal antibody
2. Select antigen from cDNA library and determine sequence.
3. Select plasmid and incorporate antibiotic resistance gene.
4. Ligate antigen DNA to plasmid .
5. Transfect E. coli with plasmid containing antigen gene.
6. Grow bacteria in presence of antibiotic.
7. Purify expressed antigen from bacterial lysate.
Advantages
 Well studied system of gene expression and regulation
 Easily manipulated
 Rapid growth/doubling time.
 Large scale production possible.
 Promoters allow strong expression of antigen up to 20% of bacterial protein.
Disadvantages
 Proteins often form insoluble inclusion bodies. Protein remains unfolded and therefore are poorly
immunogenic, particularly important for antibody formation.
 No post-translational modification (e.g. glycosylation). N.B. Carbohydrates are often very important
antibody inducing molecules.
 High levels of endotoxin from bacterial cell wall. Caused production of non-specific inflammatory
response in the host e.g. TNF, IFNg
Can over come the problems of poor solubility by expressing the protein in tandem with a more soluble
bacterial = fusion protein.
The expressed protein can also be tagged with another molecule so that the vaccine antigen can be purified
from the bacterial lysate on an affinity column or similar.
Yeast expression of recombinant antigens:
 Many safe strains available which have low levels of endotoxin (from cell wall) contamination.
 Large scale production possible.
 Good promoters inducing strong expression (up to 20% of yeast protein).
 Good markers to select for expression of recombinants.
 Good post-translational modification e.g. glycosylation.
 Antigen normally produced in secreted form (i.e. soluble).
The only recombinant vaccine so far licensed for use in man is produced in yeast. This is a vaccine made
from the Hepatitis B surface antigen (HBsAg)
and provides excellent protection from infection by the production of neutralising antibodies.
Insect cell lines
 Cells infected with baculovirus vectors.
 Good for post-translational modification i.e. glycosylation.
 High yield.
PEPTIDE VACCINES:
Based entirely on the T or B cell epitope to elicit an immune response.
 Need to establish the DNA sequence of the epitope, the peptide up to about 20 aa can then be
chemically synthesysed.
 Peptide alone can not bind to MHC II molecules, it needs a carrier protein, such as tetanus toxoid, or
something with a strong T cell epitope.
 B cell epitopes need to be in the correct configuration to elicit the appropriate antibody response.
(Peptides alone often lose their 3D configuration).
 T cell epitopes must be recognised by people with a wide range of HLA (MHC II) haplotypes. i.e.
must not select an epitope only recognised by one MHC phenotype.
 Can polymerise the peptide to form a Multiple Antigenic Peptide (MAP) with T and B cell epitopes to
increase immunogenicity.
 Need a strong adjuvant to aid persistence and to boost the immune response.
 Not good if the major antigen is subject to antigenic drift in the normal organism, e.g. flu or HIV.
ADJUVANTS AND CARRIERS:
Adjuvants and carriers are very important in non-replicating vaccines.
Both boost, or potentiate, the immune response to the immunising antigen.
There are important differences:

Adjuvants stimulate the innate immune response via PRRs (i.e. they are PAMPs).

Carriers optimise the configuration of the antigens, and/or aid antigen presentation by targeting
the antigen to the APC. Also act as a ‘depot’ of antigen for slow release.
5 main types of action: (see handout and Ref by Schijns 2000).
1. Transport of antigen to lymphoid tissue (i.e. the sites of signals 1 & 2).(carrier)
2. Act as a depot to cause persistence of antigen (prolongs signal 1). (carrier)
3. Stimulate signal 0 (i.e. activate PRRs on accessory cells of the innate IR). (adjuvant)
4. Induce “danger” signals by causing tissue destruction and necrosis. (carrier and adjuvant)
5. Induce signal 2 (i.e. cytokine and costimulatory activity). (adjuvant)
BUT also avoid stimulating regulatory T cells (i.e. Th3/Tr1) which induce down-regulation or tolerance.
This is almost the complete opposite of the strategies required to break the development of autoimmunity.
Adjuvants:
 Often derivatives of bacterial cell walls which stimulate cells of the innate immune system (i.e. PAMP
-12; Signal 3), which in turn aid the
acquired immune response. Examples include, MDP (muramyl dipeptide), lipid A (purer form of
LPS).
 C3d component of complement (stimulates innate response)
 Unmethylated non-mammalian DNA is a potent adjuvant (see later on DNA vaccines). Stimulates
innate immune response as it is a type of PAMP (Signal 0).
 Can be replaced by adding recombinant cytokines (IL-12), chemokines (SLC: CCL21), or
costimulatory molecules (CD86) which provide extra Signals 2 and 3, or can help by recruiting more
DCs to increase the likelihood of Signal 1. with much the same effect. This has the advantage of
being more controllable.
 Adjuvants are nearly all types of PAMP which bind to receptors of the innate immune response (i.e.
PRRs) Signal 0.
Receptors for adjuvants include:
 TLRs which bind a number of microbial products (see lecture 1).
 Complement receptors (CR2 = CD21) on B cells stimulated by microbial polysaccharides.
 Invariant TCRs on Natural killer T cells (bind a novel adjuvant from sea sponge -GalCer = galactosylceramide). GalCer is a good adjuvant for CD8 T cell responses and has been used to
induce protection against malaria.
 Invariant TCR on  T cells
Adjuvants also formulated into carriers to enhance persistence and presentation of the antigen in an
optimum configuration/structure to achieve maximum antigen processing by APCs.
Carriers:
 Oil (Freund's, or squalane), or alum (ALOH3) used to enhance persistence of the antigen and aid
antigen processing by APCs. Act as a depot to slowly release antigen. May also induce danger signals.
Alum is the only adjuvant licensed for use in humans.
 ISCOMs Immunostimulatory complexes. Comprised of micelles of lipid surrounding the antigen held
in the core region. Facilitate uptake of antigen into APCs. Good for targeting to cells for Class I
presentation.
 Liposomes (operate in a manner similar to above).
 Can include detergents e.g. saponin/QuilA, which aid entry of antigens into cells thus stimulating CD8
responses. May also cause tissue destruction and stimulation of danger signals.
 Microcapsules/biodegradable particles act as slow release carriers of antigen.
LIVE EXPESSION VACCINES:
e.g.
vaccinia (cowpox) virus
canarypox
adenovirus
Mycobacterium bovis (BCG)
Salmonella typhi/typhimurium Aro A strain
Vectors not supposed to cause disease in humans, hence they are non-pathogenic.
This can result from the use of vectors which do not normally infect humans e.g. canarypox, or they can
be attenuated forms of species which can infect man e.g. BCG.
General approach;
 Select plasmid vector and incorporate antibiotic resistance gene.
 Ligate vaccine antigen gene into plasmid.
 Transfect plasmid vector into E.coli.
 Grow in presence of antibiotic and analyse for expression of vaccine antigen
 Isolate plasmid construct and electroporate into BCG.
 Grow in presence of antibiotic and analyse for expression of antigen.
 Immunise host with BCG.
Can incorporate many genes from different organism into the vector in tandem. Also can insert cytokine
genes (e.g. IL-1 or IL-18) to boost the immune response.
Advantages of live vectors e.g. BCG
 Gives long-term expression of antigen to the host's immune system.
 Safe for use in man (already an approved vaccine for tuberculosis).
 Can induce both cell-mediated and antibody immune responses.
 Can incorporate many genes.
 Acts as its own natural adjuvant. (BCG induces IL-12).
Disadvantages of live vectors e.g. BCG
 Slow growing.
 Low level of expression.
 Can reject and expell plasmid containing the vaccine antigen.
 Previous exposure of the host to Mycobacterium can immunise against this recombinant BCG vaccine.
 Post-translational modification may not be optimal.
NAKED DNA VACCINES.
‘Third Generation Vaccines’
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

First described in 1993.
Direct injection of plasmid DNA encoding vaccine antigen gene.
Host cells then express vaccine antigens.
Imitates vaccination with live virus.
Can also use RNA.
Unlike 2nd generation vaccines, do not need to express and purify the recombinant protein, synthesyse
the peptide, or use an adjuvant.
Experimental DNA vaccines exist for;
Viruses Influenza
Hepatitis B
Herpes Simplex Virus
HIV-1
Rabies
Measles
Bacteria
Mycobacterium
Pneumococcus
Salmonella
Clostridium tetani
Protozoa
Plasmodium
Leishmania
Toxoplasma
Essential components of DNA vaccines;
See plasmid diagram on handout
1.
2.
3.
4.
5.
Strong viral promoter (eg pCMV, pRSV) allowing expression in eukaryotic cells.
Polyadenylation/transcriptional termination sequence.
Prokaryote origin of replication (allows growth in bacterial liquid cultures but not in vivo in
eukaryotic cells).
Selectable marker for in vitro growth e.g. antibiotic resistance gene.
The gene of interest!
Delivery of DNA vaccines.
 Intramuscular:
-Antigen taken up and expressed by long-lived myocytes.
-Only a few cells become transfected.
-Use g quantities of DNA in saline.
-Can increase uptake by damaging cells first using a local injection of bupivacaine.
Gene gun / intradermal
- DNA coated onto gold particles and fired at high velocity at the skin.
- Higher transfection rate of keratinocytes
- Use as little as a few ng, but the expression is shorter due to sloughing of
the skin.
- Skin is rich in APCs such as Dendritic cells.
 Oral and intranasal.
- Oral and intranasal route for delivery to the mucosae.
- DNA incorporated into liposomes to prevent degradation.
- Liposomes taken up preferentially by immune cells in the mucosae.
Immune responses to DNA vaccines.
Transfection of host cells by the plasmid DNA is analogous to many viral infections. However, in
comparison the number of ‘infected‘ cells is much lower.
Antibody production;
 Strong neutralising antibodies are elicited, which are long lasting up to 74 weeks in mice.
 Can be boosted by 10-200 fold by a second injection of DNA or recombinant protein.
 Elicited early (2 weeks) with IgM response switching to IgG (4-6 weeks).
 IgE production is inhibited. Indeed all allergic responses are inhibited including reduction of
histamine release, airway hyper-responsiveness. Inhibition is linked to induction of CD8+ cell
response.
CD8 and CD4 immune responses
 Very efficient at inducing cytotoxic T lymphocytes (CD8+) restricted by MHC class I.
 Class II-restricted CD4 response normally Th1-4.
How do the expressed antigens prime cells of the immune response?
 Intracellular processing pathways are poorly understood but cytosolic location of expressed proteins
favors uptake of proteins into ER and association with MHC Class I molecules.
 Expressed antigens also processed using MHC Class II pathways probably following uptake of protein
by phagocytosis or endocytosis.
3 main possibilities for antigen priming;

Do the transfected myocytes/keratinocytes present antigen on their surface to T cells ? (poor
expression of Class II)


Do some professional APCs, such as dendritic cells (DCs) become transfected? (Too scarce?)
Do transfected myocytes prime the APCs with antigen (via Class I or II)?
‘Adjuvanticity’ of DNA
1. Why can such small quantities of DNA elicit potent immune responses?
2. Why do Th1 immune responses dominate?
DNA is a potent stimulator of the innate immune response.
It is a major type of PAMP.
Adjuvant activity is linked to DNA motifs that are
1. rich in CpG dinucleotides
2. the CpG motif should be unmethylated
The motif should be flanked by two purines at 5’ end and two pyrimidines at the 3’ end.
Stimulatory DNA;
TCC ATG ACG TTC
Non-stimulatory DNA;
TCC ATG AGC TTC


CpG motifs occur more frequently 1:16 bases in prokaryotes but 1:50 in eukaryotes.
Less than 5% of CpG bases are methylated in prokaryotes, whereas in eukayotes this is 70-90%.
CpG motifs bind via pattern recognition receptors (PRRs).
In this case, the receptors are the Integrin CD11b/CD18 and the scavenger receptors.
How do CpG motifs induce the innate immune response?
1. cytokine production by APCs (such as DCs or macrophages). These include IL-6, IL-12, IL-18, IFNg
and IFNa which drive the development of naïve T cells to the Th1 phenotype.
2. costimulatory molecules (e.g. CD40, CD80 and CD86).
3. MHC class II expression on dendritic cells and macrophages. This aids the ability of these cells to act
as efficient APCs.
4. B cell proliferation and IgM production (non-specific).
5. NK cell production of IFNg
Improvements to the efficacy of DNA vaccines




Insect DNA is also a good adjuvant. Raises interesting possibilities of vaccine production!
Co-express the antigen in conjunction with cytokine genes to potentiate/enhance the immune response.
Have used plasmids expressing IL-2, IL-4, GM-CSF, ILhave expected immune function. e.g. IL-2 and GM-CSF upregulate both CD4 and CD8 responses
whereas IL-4 down-regulates CD8 responses. IL-10 can be used to downregulate inflammatory
responses such as keratitis of the eye.
Target the expressed antigen to sites of immune priming (i.e. lymph nodes) by engineering antigen in
tandem with adhesion (L-selectin) or costimulatory molecules (CTLA-4).
[L-selectin binds to CD34 on the HEV thus causing antigen to enter the draining lymph node. CTLA4 binds to APCs thus enhancing antigen uptake (Low doses of CTLA-4 are stimulatory but high doses
as used in treatment of autoimmunity require high doses)].
Synthetic CpG motifs can be made (oligodinucleotides;ODNs) which act as potent adjuvants. Can
boost the response to protein antigens.
Alternative immunisation strategies using naked DNA.
 If the appropriate gene is not known, an entire expression library can be used to immunise hosts. Any
dominant immune response to a gene or fragment can be detected using conventional screening
techniques.
 Use attenuated Salmonella as a carrier of the plasmid. This bacterium will target the plasmid to cells
in the mucosa which become transfected when the salmonella die. The plasmid is under the control of
a eukaryotic promoter, so expression will not occur in the salmonella, only in the host cells. Because
the salmonella is attenuated it does not replicate and cause pathology.
 Since dendritic cells are such potent APCs, it has been shown that DCs grown up and transfected in
vitro can be used to immunise hosts. This has been shown to be efficient at protecting against tumor
cell development as a form of vaccine against cancer.
 RNA used instead of DNA. Use gene-gun or alphavirus-based expression vectors. Can express
foreign proteins (e.g. from HIV-1 and papillovirus). Not long-lasting and very unstable.

“Prime boost” strategy heralded as a major advance.
1. Prime first with DNA vaccine encoding the antigen.
2. Boost with modified vaccinia virus encoding the antigen.
 It is thought that the DNA vaccine induce a limited number of specific T cells of high affinity as a
consequence of the encoded antigen being expressed at low but persistent levels.
 The poxvirus which expresses higher levels of the antigen expands the specific high affinity T
cells. Virus on its own would stimulate lots of T cells but only of low affinity. Since the virus
which does not replicate in the host, any immune response is focussed on the specific T cells and
not poxvirus antigens.
 Far more successful than each component on its own; used for influenza, HIV-1 in primates.
Currently in phase II trials in humans for malaria
Safety issues concerning DNA vaccines:
1. Integration of plasmid DNA into the genome of the host cells. Integration may be
mutagenic/carcinogenic. It may also interfere with crucial regulatory genes.
But.
The host cells usually subject to transfection are non-dividing, and the origin of replication is
prokaryotic.
2. Autoimmune responses may be stimulated after destruction of cells expressing the antigen.
But
The number of transfected cells is small.
3. Anti-DNA antibodies may be produced. double stranded DNA does not readily induce antibodies.
DNA needs to be denatured. Anti-DNA antibodies are already present in most individuals without any
adverse reaction.
4. Use RNA vaccines since they only work transiently, do not integrate into chromosomal DNA, and do
not cause insertional mutagenesis.