2.  A vaccine is any preparation of dead or
attenuated pathogens, or their products, that
when introduced into the body, stimulates the
production of protective antibodies or T-cells
without causing the disease

3.  The terms vaccination and vaccine derive from the
work of Edward Jenner who, over 200 years ago,
showed that inoculating people with material from
skin lesions caused by cowpox (L. vaccinus, of cows;
vacca, cow) protected them from the highly contagious
and frequently fatal disease smallpox
 He tested his theory in 1796 by inoculating 8-year-old
James Phipps with liquid from cowpox pustule
 Subsequent inoculation of the boy with smallpox
produced no disease
 Since Jenner's time, the term has been retained for any
preparation intended to attain the same

4. Edward Jenner (1749-1823)

5.  However, this approach dates back, well before
Jenner’s time in India and China where they performed
what is known as variolation
 Variola virus=smallpox virus
 Variolation, a procedure developed in China and India
1000 AD used a live smallpox vaccine to generate
immunity
 Employing several different techniques ‘well
individuals’ were exposed to variolous material from a
human
 with a milder form of smallpox—presumably in the
expectation that this would cause less severe disease in
the recipient—an early form of ‘attenuation

6.  Whole organism
 Live/attenuated vaccines
 Killed/inactivated vaccines
 Toxoids
 Peptide vaccines
 Recombinant vaccines
 DNA vaccines

7.  Live/attenuated vaccines make up the bulk of
successful viral vaccines
 Are prepared from attenuated strains that are
almost or completely devoid of pathogenicity
but are still immunogenic
 They multiply in the human host and provide
continuous antigenic stimulation over a period
of time

8.  Use of a related virus from another animal –
e.g. the use of cowpox to prevent smallpox
 Administration of pathogenic or partially
attenuated virus by an unnatural route
 the virulence of the virus is often reduced when
administered by an unnatural route
 immunization of military recruits against adult
respiratory distress syndrome using enterically
coated live adenovirus type 4, 7 and (21).

9.  Passage of the virus in an "unnatural host" or
host cell –
 the major vaccines used in man and animals have all
been derived this way
 After repeated passages, the virus is administered to
the natural host
 The initial passages are made in healthy animals or
in primary cell cultures
 Examples: the 17D strain of yellow fever (in mice
and then in chick embryos), Polioviruses (in monkey
kidney cells) and measles (in chick embryo
fibroblasts)
 Development of temperature sensitive
mutants
 this method may be used in conjunction with the
above method

10.  The vaccine is injected sc/im, virions enter
various cell types (APCs) using receptor-
mediated endocytosis
 Proteolytic degradation of viral proteins
occurs, the peptides produced are then loaded
onto MHC I molecules
 The complex is displayed on the cell surface
 Circulating cytotoxic T cells recognize the
complex, become activated and and release
cytokines

11.  The cytokines trigger apoptosis (programmed
suicide) of the infected cells
 Some Tc become memory cells but the basis of
this is incompletely understood
 Additionally, immature DCs will phagocytose
virus vaccine initiating a series of events that
leads to the production of plasma cells,
neutralizing IgG antibodies and memory B
cells

12.  One important advantage of live/ attenuated
vaccines is that they are sufficiently
immunogenic therefore primary vaccine failure
are uncommon and are usually the result of
inadequate storage or administration

13.  Several potential safety problems exist with
live/attenuated vaccines:
 Underattenuation
 Mutation leading to reversion to virulence
 Preparation instability
 Contaminating viruses in cultured cells
 Heat lability
 administration to immunocompromized or
pregnant patients may be dangerous

14.  The term killed generally refers to bacterial
vaccines whereas inactivated relates to viral
vaccines
 An inactivated whole organism vaccine uses
pathogens which are killed and are no longer
capable of replicating within the host
 The pathogens are inactivated by heat or
chemical means while assuring that the surface
antigens are intact
 Inactivated vaccines are generally safe, but are
not entirely risk free

15. Organism Method of inactivation
Rabies β-propiolactone
Influenza β-propiolactone
Polio Formaldehyde
Hepatits A formaldehyde

16. Organism Method of inactivation
Salmonella typhi Heat plus phenol or
acetone
Vibrio cholera Heat
Bordetella pertusis Heat or formaldehyde
E.Coli (experimental) Colicin
Yersinia pestis Formaldehyde

17.  Following injection, the whole organism is
phagocytosed by immature dendritic cells
 Processed peptides will be presented on the cell
surface as separate MHC II:antigenic fragment
complexes
 Th2, each with a TCR for a separate antigenic
fragment will be activated
 B cells, each with a BCR for a separate antigenic
fragment will bind antigens that drain along
lymph channels

18.  The separate antigens will be internalized and
presented as an MHC II:antigenic fragment
 This will lead to linked recognition with the
appropriate Th2
 Activated Th2 will release IL2, IL4 IL5 and IL6,
inducing B-cell activation, differentiation and
proliferation with subsequent isotype switch
(IgM to IgG) and memory B cell formation.

19.  First, they are safe because they cannot cause
the disease they prevent and there is no
possibility of reversion to virulence
 Second, because the vaccine antigens are not
actively multiplying, they cannot spread to
unimmunized individuals
 Third, they are usually stable and long lasting
as they are less susceptible to changes in
temperature, humidity and light which can
result when vaccines are used out in the
community
 Fourth, all the antigens associated with
infection are present and will result in
antibodies being produced against each of
them

20.  Contamination by toxins or chemicals
 Allergic reactions
 Surface endotoxins on inactivated pertussis
vaccine occasionally induce DTH responses,
and influenza virus has been linked to
similar reactions, though this may be due
more to the immunogenicity of the egg
whites in which the virions are raised
 Autoimmunity

21.  Also, inactivated vaccines do not always
induce protective immunity.
 Multiple boosters and an adjuvant are
usually necessary for continual antigen
exposure
 the dead organism is incapable of sustaining
itself in the host, and is quickly cleared by the
immune sysytem
 Furthermore, inactivated vaccines are generally
capable of inducing humoral immunity rather
than cellular immunity.

22.  A toxoid is a chemically or physically modified
toxin that is no longer harmful but retains
immunogenicity
 Certain pathogens cause disease by secreting
an exotoxin:
 these include tetanus, diphtheria, botulism and
cholera
 In addition, some infections, for example
pertussis, appear to be partly toxin mediated
 Specific physical or chemical modification of
the toxins produces a toxoid, which is a vaccine

23.  The principal toxin is tetanospasmin
 Binds to specific membrane receptors located
only on presynaptic motor nerve cells
 Internalization and migration of this toxin to the
CNS blocks the metabolism of glycine which is
essential for the normal functioning of gama
amino butyric acid (GABA) neurons
 GABA neurons are inhibitory for motor neurons
 Their non-functioning results in excess activity
in motor neurons
 This gives rise to muscle spasms, a characteristic
feature of tetanus

24.  Manufactured by growing a highly toxigenic
strain of Clostridium tetani in a semi-synthetic
medium
 Bacterial growth and subsequent lysis release
the toxin into the supernatant
 Formaldehyde treatment converts the toxin to a
toxoid by altering particular amino acids and
inducing minor molecular conformational
changes

25.  Ultrafiltration then removes unnecessary
proteins residues
 The toxoid is physicochemically similar to the
native toxin thus inducing cross-reacting
antibodies
 But, the changes induced by formaldehyde
treatment render it non-toxigenic

26.  Upon administration (sc/im) the toxoid
molecules are taken up at the vaccination site
by immature dendritic cells
 Within this cell, they are processed through the
endosomal pathway where they are bound to
MHC II molecules
 The MHC II:toxoid complex then migrates to
the cell surface
 mature DCs migrate along lymph channels to
the draining lymph node
 There , they encounter naïve Th 2 cells
 Identifying and then binding of the MHC
II:toxoid to the specific Th2 receptor then
activates the naive T cell, causing it to
proliferate

27.  Some toxoid molecules not taken up by DCs
pass along lymph channels to the same
draining lymph nodes
 There, they come into contact with B cells
 Binding to the B cell through the specific
immunoglobulin receptor that recognizes the
toxoid
 The toxoid is internalized, processing through
the endosomal pathway and presented on the
cell surface as an MHC II:toxoid complex as
happens in the DCs

28.  These two processes occur in the same part of the
lymph node
 The B cell with the MHC II:toxoid complex on its
surface now comes into contact with the activated Th2
whose receptors are specific for this complex
 This process is called linked recognition
 The Th2 activates the B cell to become a plasma cell
with the production initially of IgM, and then there is
an isotype switch to IgG
 In addition, a subset of B cells becomes memory cells

29.  The rationale of toxoid vaccination is
production of antibodies with enhanced
capacity to bind the toxins
 They thus form complexes with the toxins
preventing then to interact with toxin receptors
on the nerve cells (tetanus)
 This is referred to as neutralization of the
toxins by antibodies

30.  There are three principal advantages:
 First, they are safe because they cannot cause
the disease they prevent and there is no
possibility of reversion to virulence.
 Second, because the vaccine antigens are not
actively multiplying, they cannot spread to
unimmunized individuals.
 Third, they are usually stable and long
lasting as they are less susceptible to changes
in temperature, humidity and light which
can result when vaccines are used out in the
community

31.  First, they usually need an adjuvant and
require several doses (otherwise less
immunogenic)
 Second, local reactions at the vaccine site are
more common—this may be due to the
adjuvant or a type III (Arthus) reaction
 The reaction generally starts as redness and
induration at the injection site several hours after the
vaccination and resolve usually within 48–72 h
 The reaction results from excess antibody at the site
complexing with toxoid molecules
 activating complement by the classical pathway causing an
acute local inflammatory reaction

32.  A recombinant vaccine contains either a
protein or a gene encoding a protein of a
pathogen origin that is immunogenic and
critical to the pathogen function
 The vaccine is produced using recombinant
DNA technology
 The vaccines based on recombinant proteins
are also called subunit vaccines
 e.g. RTS,S malaria vaccine, passed phase II now
entering phase III

33.  The logic of such vaccines, in simple terms, is
as follows:
 Proteins are generally immunogenic, and many of
them are critical for the pathogenic organism
 The genes encoding such proteins can be identified
and isolated from a pathogen and expressed in E.
coli or some other suitable host for a mass
production of the proteins
 The proteins of interest are then purified and mixed
with suitable stabilizers and adjuvants, if required,
and used for immunization

34.  The first step is to identify a protein that is both
immunogenic and critical for the pathogen
 The gene encoding this protein is then
identified and isolated
 The gene is integrated into a suitable
expression vector and introduced into a
suitable host where it expresses the protein in
large quantities
 The protein is then isolated and purified from
the culture system
 It is used for the preparation of vaccine

35. 1. Genetically engineered microorganisms, e.g., yeast for the
expression of hepatitis B surface antigen (HBsAg) used as
vaccine against hepatitis B virus
2. Cultured animal cells, e.g., HBsAg expressed in CHO
(Chinese hamster ovary) cell line and C-127 cell line
4. Transgenic plants, e.g., HBsAg, HIV-l (human
immunodeficiency virus-I) epitope (in experimental
stages)
5. Insect larvae; the gene is integrated into a bacculovirus
genome, which is used to infect insect larvae. Often a
very high quantity of the recombinant protein is
produced .

36.  An alternative application or recombinant
technology is the production of hybrid virus
vaccines e.g. HBsAg vaccine
 Here, vaccinia virus is used as a carrier for
genes that encode antigenic proteins of interest
 The genes may be derived from organisms
which are difficult to grow or inherently
dangerous, and the constructs themselves are
replication deficient, nonintegrating, stable and
relatively easy to prepare

37.  DNA sequence coding for the foreign gene is
inserted into the plasmid vector along with a
vaccinia virus promoter and vaccinia
thymidine kinase sequences
 The resultant recombination vector is then
introduced into cells infected with vaccinia
virus to generate a virus that expresses the
foreign gene
 The recombinant virus vaccine can then
multiply in infected cells and produce the
antigens of a wide range of viruses
 The genes of several viruses can be inserted, so
the potential exists for producing polyvalent
live vaccines

38.  Proteins encoded by these genes are appropriately
expressed in vivo with respect to glycosylation
and secretion
 They are processed for major histocompatibility
complex (MHC) presentation by the infected cells,
thus effectively endowing the host with both
humoral immunity and CMI

40.  Small peptide sequences corresponding to
important epitopes on a microbial antigen can
be synthesized readily economically
 Some long ones are also being invented, but are
more expensive to manufacture
 eg. Pf MSP-3 long synthetic peptide (now in phase II)

41.  Synthetic peptides can be highly immunogenic
in their free form provided they contain, in
addition to the B cell epitope, T- cell epitopes
recognized by T-helper cells
 The T-cell epitope must be linked to the B-cell
epitope
 Such T-cell epitopes can be provided by carrier
protein molecules, foreign antigens or within
the synthetic peptide molecule itself

42.  The antigens are precisely defined and free from
unnecessary components which may be associated
with side effects
 They are stable and relatively cheap to manufacture
 Furthermore, less quality assurance is required
 Feasible even if the pathogen cannot be cultivated
 Changes due to natural variation of the virus can be
readily accommodated

43.  DNA vaccines are usually circular plasmids
(supercoiled) that include a gene encoding the
target antigen (or antigens) under the
transcriptional control of a promoter region
active in human cells
 With DNA vaccines, the subject is not injected
with the antigen but with DNA encoding the
antigen

44.  DNA vaccines are composed of a bacterial
plasmids
 Expression plasmids used in DNA-based
vaccination normally contain two units:
 Antigen expression unit composed of
promoter/enhancer sequences followed by antigen-
encoding and polyadenylation sequences
 The production unit composed of of bacterial
sequences necessary for plasmid amplification and
selection
 The construction of bacterial plasmids with
vaccine inserts is accomplished using
recombinant DNA technology

45.  Sometimes DNA sequences encoding
 costimulatory molecules
 sequences that target the expressed protein to
specific intracellular locations (e.g., endoplasmic
reticulum)
 Once constructed, the vaccine plasmid is
transformed into bacteria, where bacterial
growth produces multiple plasmid copies
 The plasmid DNA is then purified from the
bacteria, by separating the circular plasmid
from the much larger bacterial DNA and other
bacterial impurities

46.  This purified DNA acts as the vaccine
 The DNA vaccine can be injected into a muscle
just as conventional vaccines are
 Using ordinary syringe or gene gun
 DNA vaccines elicit cell-mediated as well as
antibody-mediated immune responses

47.  A plasmid vector that expresses the protein of
interest (e.g. viral protein) under the control of an
appropriate promoter is injected into the skin or
muscle of the the host
 After uptake of the plasmid, the protein is
produced endogenously
 The protein is processed intracellularly into small
antigenic peptides by the host proteases and
presented to the cell surface with MHC I
 Subsequent CD8+ cytotoxic T cells (CTL) are
stimulated and they evoke cell-mediated
immunity

48.  CTLs inhibit viruses through both cytolysis of infected
cells and noncytolysis mechanisms such as cytokine
production
 The foreign protein can also be presented by the MHC
class II pathway by APCs which elicit helper T cells
(CD4+) responses
 Depending on the the type of CD4+ cell that binds to
the complex, B cells are stimulated to produce
antibodies production
 This is the same manner in which traditional vaccines
work

49.  The plasmid is taken up by an antigen-
presenting cell (APC) like a dendritic cell
 The gene(s) encoding the various components
are transcribed and translated
 The protein products are degraded into
peptides
 These are exposed at the cell surface nestled in
class I histocompatibility molecules
 MHC I-peptide complex serves as a powerful
stimulant for the development of cell-mediated
immunity

50.  If the plasmid is taken up by other cells (e.g.
muscle cells)
 The proteins synthesized are released and can
be engulfed by antigen-presenting cells
(including B cells)
 In this case, the proteins are degraded in the
class II pathway and presented to helper T cells
 These secrete lymphokines that aid B cells to
produce antibodies

54.  So far, most of the work on DNA vaccines has
been done in mice where they have proved
able to protect them against tuberculosis,
SARS, smallpox, and other intracellular
pathogens
 Different DNA vaccines against HIV-1 — the
cause of AIDS — are in clinical trials

55.  Delivery of the DNA to cells is still not optimal,
particularly in larger animals
 The possibility that exists with all gene
therapy, that the vaccine's DNA will be
integrated into host chromosomes and will
 either turn on oncogenes
 or turn off tumor suppressor genes
 Antibiotic resistance?
 Extended immuno-stimulation by the foreign
antigen could in theory provoke chronic
inflammation or autoantibody production

56.  Thousands of individual steps (summary)
 Recognize the disease and Identify the etiologic
agent
 Attempt to grow the agent in laboratory
 Establish an animal model for disease
 Identify an immunologic correlate for immunity to
the disease- Usually a serum antibody
 choose antigen (in laboratory)
 Prepare candidate vaccine following Good
Manufacturing Practice

57.  Evaluate candidate vaccine for ability to protect
animal model
 Prepare protocol for human studies
 Phase I human trials- Safety and
immunogenicity using small group (phase Ia in
company area)
 Phase II trials- Safety and immunogenicity using
relatively larger group
 Phase III trial- Efficacy and safety
 Long and complicated process
 Usually takes 10–15 years
 Many vaccine candidates fail for every success

58. The vaccine:
• Attenuated organisms revert to wild type (e.g. polio types 2,3)
• ‘Killed’ organisms not properly killed (has happened with polio)
• Inclusion of toxic material (e.g. typhoid, pertussis)
• Contamination by animal viruses
• Contamination by egg proteins (hypersensitivity)
• Cross-reaction with ‘self’ (autoimmunity)
The patient:
• Immunodeficiency (attenuated organisms may cause serious/fatal disease)
• Local inflammatory reactions, often to the adjuvant
• Worsening of disease by increasing immunopathology (risk of therapy)
• Hypersensitivity to vaccine (e.g. tetanus)
• Interference between vaccines given together (not always)
• Induction of inappropriate response (e.g. dengue)

59. •Public aversion to the risks of adverse effects pushes towards sub-unit vaccines
rather than live attenuated
•Pathogens change their antigens (or have distinct life cycle stages)
a) makes initial composition of the vaccine components complicated
b) make a successful vaccine redundant eg. antigenic drift/shift in influenza, (HIV)
•Success so far has been where naturally occurring immunity is strong
eg childhood viruses
BUT this is not the case for HIV, TB, malaria etc –so vaccine has to be even better
•Successes to date mostly with antibody inducing vaccines- not cell mediated!
albendazole
•Confounding effects in tropical populations-
eg AIDS, malnutrition, pre-existing exposure
to environmental organisms (eg re BCG)
- parasitic diseases (Th1 vs Th2 balances)
Does deworming prior to vaccination enhance BCG vaccine?
Elias et al., Clin Exp Immunol. 2001 123: 219-25

60. Summary
•Early vaccines were mostly whole dead or live
attenuated
•These provide their own integrated adjuvant/
antigens
•Now the shift is towards defined subunit-based
vaccines
•This requires you to select which adjuvant/
antigen to include
•Immunology is now having a greater impact on
vaccine design:-optimized activation (e.g. TLR
synergy)-temporary removal of immune-
regulation? (e.g. anti-IL10)
•Safety, efficacy and memory are the key!

61.  Peter Delves, Seamus Martin, Dennis Burton and Ivan Roitt: Roitt’s
Essential immunology, 11th edition 2006 page 287-311
 David Baxter. Active and passive immunity, vaccine types,
excipients and licensing: Occupational Medicine 2007;57:552–556
 Medical immunology 2006: edited by Gabriel Virella. ‑‑ 6th ed
 Goering RV, Dockrell HM, Zuckerman M, Wakelin D, Roitt IM,
Chiodini PL and Mims C; Mims’ Medical Microbiology 4th Edition,
Philadelphia Elsevier (2008) page 519-542
 http://www.brown.edu/Courses/Bio_160/Projects1999/vaccineovervie
 http://www.microvet.arizona.edu/Courses/MIC419/Tutorials/vac
cines.html
 www.google.com/(specific topic)