types of vaccines, preparation of vaccine, immuno-modulation, environmental role of immuno-modulation

1. VACCINES &
IMMUNOMODULATION
Dr Alok Tripathi
Department of Biotechnology
aquaimmuno@yahoo.co.in

2. Objectives
 The role of immunological memory in protection against infectious diseases
 The main differences between memory and naïve T and B cells
 How vaccines can be used to manipulate the immune response to induce
immunological memory
 Examples of attenuated vaccines are and how they are produced.
 Examples of killed vaccines are and how they are produced.
 Examples of recombinant vaccines are and how they are produced
 Current experimental approaches to design vaccines and their associated
problems :
 Recombinant plant vaccines
 Live recombinant vaccines
 Genetically attenuated vaccines
 Peptide vaccines
 DNA vaccines

3.  What vaccine adjuvants are and their
functions in vaccine formulations
 Which adjuvants are used in human
vaccines
 The types of responses induced by
adjuvants
 The current status of research into HIV,
Malaria and TB

4. Definition
 Vaccines are a manipulation of the
adaptive immune response whereby the
host is induced to generate a protective
immune response to a pathogenic
organism without actually experiencing
the full infection.
 Vaccination involves deliberate
exposure to antigen under conditions
where disease should not result.

5. Role of Memory in Vaccine induced
protection to infection
 During this primary challenge, the immune response responds by
producing not only effector cells but also memory cells Compared
with naïve T cells, memory T cells: Are Long Lived
 Have Increased frequency
 Proliferate more rapidly
 Have reduced co-stimulatory requirements (signal 2)
 Compared with naïve B cells, memory B cells : Are Long Lived
 Have Increased frequency
 Proliferate more rapidly
 Produce more Ab
 Produce higher affinity Ab
 Produce antibodies with better effector functions (IgG & IgA)

6. secondary antibody response to antigen.
 Memory T and B cells induced during vaccination facilitate
more rapid control of subsequent infections with pathogens.
This can be seen most clearly in the secondary antibody
response to antigen. Secondary antibody responses are T cell
dependent. During the primary response, expansion of T
cells specific for protein antigens is sufficient for the host to
mount a secondary antibody response on subsequent
exposure to that antigen, plus any other B cell antigen which
is physically associated with it (e.g. a hapten). This is known
as the carrier effect - it means that the secondary antibody
response to a vaccine can be induced by conjugating the B
cell antigen of choice to a T cell antigen to which people have
already been exposed (see the Hib vaccine below)


7. History of Vaccination
 Jenner performed the definitive experiments to demonstrate
effective protection against smallpox (causative agent,
Variola) by prior inoculation or ‘vaccination’ with cowpox
(containing the closely related Vaccinia virus) in 1796. The
‘golden age of microbiology’ brought about the rapid
isolation of the pathogenic agents of many diseases and
subsequent attempts to produce vaccines against them. In
the 1880’s Pasteur generated attenuated versions of polio
virus and anthrax by in vitro culture. Subsequent methods of
producing vaccines included heat or chemical inactivation of
pathogens. After improvements in living conditions, vaccines
are the most effective public health measure introduced. In
Glasgow in the 1900’s, 1 in 5 deaths were due to smallpox
infection. In May 1980, the WHO officially announced the
global eradication of the smallpox virus.

8. Currentstatus
ThefollowingvaccinesarerecommendedforroutinevaccinationintheUK
• Diphtheria/
Tetanus/Pertussis
(DTP)
1st @ 2 months
2nd @ 3 months
3rd @ 4 months
• DT boost and
Polio boost
13-18 years
• Haemophilus
influenzae B (Hib)
schedule as
above
• Hepatitis A high risk groups
• Polio schedule as
above
• Hepatitis B high risk groups
• Measles/
Mumps/Rubella
12 - 15 months • Influenza high risk groups
• DT boost, MMR
boost, and Polio
3-5 years • Pneumococcal
vaccines
high risk groups
• BCG 10-14 years

9. Producing Vaccines

The whole point of vaccination is to induce a
protective immune response as would be elicited
by infection, with minimal disease associated
with the infection. This can be achieved by
producing attenuated strains of bacteria or
viruses, for example: host range mutants
where bacteria or viruses (e.g. BCG or Sabin
polio virus) are selected for in vitro growth in
non-human cells
 cold attenuated mutants where viruses are
grown at temperatures of 32-34ûC, well below
body temperature e.g. Measles.

10. Procedures to produce undefined mutations
the bacteria or virus
allow it to grow
well under all
possible condition
in vitro specific
conditions.
After a great deal of selection for safety and stability, the mutated,
attenuated virus or bacteria will not grow as well in humans as the
wild type organism and can be used as a vaccine.
Over many years the
BCG vaccine has
been selected for
reduced local
reactions at the site
of injection.
This has raised concerns that due to
selection, the vaccine is now of limited
effectiveness in conferring protection against
TB

11. Other techniques for vaccine production are:
• Usually using chemicals such as formaldehyde
Killed
vaccines
• Toxins are the pathogenic fragments of bacteria
• Antibodies to toxins can protect against infection and disease
• Chemically inactivated toxins are called toxoids
Subunit
Vaccines
• Polysaccharides are T cell independent (no memory)
• Hib vaccine is conjugated to tetanus or diphtheria toxoid
Conjugate
vaccines
• Hepatitis B is the only recombinant vaccine
• HBsAg expressed in yeast
Recombinant
vaccines
These methods have been used to produce a
number of vaccines,

12. Further Developments in Vaccine Production
Improvements in using attenuated
pathogens as vaccines have been made
available through developments in rDNA
Tech.
Using these methods, defined genetic lesions can be
introduced into pathogens to specifically attenuate
them (e.g. in Salmonella and Cholera).
Future directions also include recombinant vaccines
where genes encoding protective antigens from
pathogens are engineered by recombinant DNA
technology into existing attenuated virus or bacteria
which act as carriers.

13. Viruses
 • Vaccinia : the first recombinant virus was
produced and tested in 1982.
 Since then it has been extensively used as a
recombinant carrier for over 100 different antigens.
 The extremely large genome of the poxviruses has
facilitated the insertion of larger parasite and
bacterial genes into Vaccinia.
 NYVAC, a highly attenuated form of Vaccinia, has
been used as a recombinant carrier for HIV vaccine
and clinical trials are underway.
 Constructs have also been made incorporating
cytokine genes (ALVAC). (see Letvin, Science (1998)
280 (Jun 19) 1875-80)

14. Adenovirus
 have also been used as a carriers for HIV
vaccines, but with limited success.
 This is probably due to the highly cytolytic
nature of adenovirus. Letvin, Science
(1998) 280 (Jun 19) 1875-80)

15.  Bacteria
• BCG : Used in vaccines against: Malaria (Merozoite surface protein -
1), HIV-1 (Letvin, Science (1998) 280 (Jun 19) 1875-80), Borrelia
burgdorferi and Streptococcus pneumoniae
• Salmonella : The EvansTy21a attenuated strain used in vaccines
was produced by chemical mutagenesis. Recently, aro mutants, which
lack enzymes involved in the production of aromatic amino acids have
extensively studied as genetically attenuated alternative and as carrier
for other vaccines (reviewed Chatfield et al.Vaccine (1989) 7, 495-498).
• Cholera : The Cholera toxin A (CTA) subunit has ADP ribosylating
activity and is responsible for Cholera induced diarrhoea. The CTB
subunit is highly immunogenic, especially when given orally. Attempts
to produce a vaccine have therefore concentrated on attenuating the A
subunit (clinical trials with CVD103Hg-R are underway)
• Bordetella pertussis: intranasal administration of a recombinant
vaccine encoding the 28-kDa glutathione S-transferase antigen from
Schistosoma mansonii has been demonstrated to mediate protection
to infection.

16.  Problems associated with attenuated vaccines:
• undefined genetic lesions (not the case with
genetically attenuated vaccines)
• possibly of reversion to virulence/toxicity (e.g.
in HIV/SIV vaccines: Johnson, Nature Medicine
(1999), 5(2), 154-155)
• cannot be used in immunocompromised hosts
• possibility of zoonosis
• loss of replication due to interference by other
infections
• public acceptance of genetically modified
vaccines

17.  Subunit vaccines
A number of subunit vaccines are already
licensed for use (See Section 2) and interest
in producing new subunit vaccines continues,
mainly due to safety concerns. For example,
the immunogenicity and toxicity of
Bordetella pertussis vaccine, an inactivated
whole cell vaccine is greatly affected by
culture conditions. Therefore development of
acellular whooping cough vaccines,
containing pertussis toxin and fimbral HA,
are of interest.

18. Recombinant subunit vaccines
 To avoid the problems involved in bulk
culture of pathogens and to increase the yield
of protective antigens, recombinant vaccines
have been introduced.
 Hepatitis B was the first recombinant vaccine
licensed for human use.
 The surface antigen (HBsAg) was expressed
in yeast, the antigen thus produced
spontaneously forms multimeric particles
similar in appearance to the non-infectious
Dane particles produced during hepatitis
infection.

19. Recombinant plant vaccines
 Hepatitis B surface Ag expressed in
potato/tobacco is immunogenic when fed to
mice.
 This has also been the case when Cholera toxin
B subunit and E.coli enterotoxin B subunit (LT-
B) were expressed in plants.
 Furthermore, a clinical trial of LT-B expressed
in potato (Tacket et al.Nature Medicine 1998, 4
(May), 607-9), demonstrated that
administration of 3 doses of 50g of potato (4 -
15 ug of LT-B) produced :

20. antibody secreting cells in
peripheral blood circulating IgG
induced to LT-B (91% had 4-
fold increase - 1 non responder)
73%
developed
neutralising
Ab
55% had 4
fold
increase in
serum IgA 50% in
secretory
IgA

21. Peptide Vaccines
 Whole proteins only contain a handful of
protective epitopes, these can be
synthesised and produced in large scale by
peptide synthesis techniques.
 Information from the primary sequence of
proteins can be used to predict which
sequences may be T cell epitopes.
 Relative merits of peptide vaccines

22. Advantages Disadvantages
• Chemical purity/safety • Need to know amino acid sequence
• Unlimited source of material • Need to identify of T and B cell
epitopes
• Costs • Genetic restriction of T cell
recognition
• Stability, storage and delivery • Lack of Immunogenicity
• Defined immunogen
• Exclusion of adverse epitopes

23. Adjuvants
 One approach to increase the immunogenicity of a protein is to
formulate it with a vaccine adjuvant. Adjuvants are described in the
Dictionary of Immunology as "Agents which act non-specifically to
increase the specific immune response or responses to an antigen"
or alternatively "the immunologists dirty little secret" by Charles
Janeway.
 Essentially, adjuvants appear be able to provide signal 2 to T cells,
a feature absent in purified proteins. The effects of adjuvants are
mainly mediated indirectly via antigen presenting cells.
 The only adjuvant currently licenced for use in humans in the UK
are the Aluminium compounds. Their adjuvant activity was first
described in 1926 and they form a component of vaccines against
Hepatitis A, Hepatitis B, Anthrax and DTP (Diphtheria, Tetanus,
Pertussis).
 However one of the major limitations of Aluminium compounds is
that they only stimulate the induction of Th2 responses. In contrast
some experimental adjuvants (such as Freund's Complete Adjuvant
(FCA)) can stimulate Th1 responses, but are too toxic to be used
clinically.

24.  The ability to modulate Th1 or Th2 responses, has been assigned to a
third signal in T cells, therefore adjuvants can clearly influence the
provision of signal 3. This is a major problem as induction of Th1
responses are thought to be required for vaccine induced protection
against the big 3 pathogens (HIV, Tuberculosis and Malaria). Therefore
a number of new adjuvants are under development by various
companies and institutes to try and induce strong antigen specific Th1
responses to vaccines.
 Particles
 Liposomes (Swiss Serum and Vaccine Institute)
These are composed of lipid bilayers (like plasma membranes) which can have
antigens entrapped inside
 ISCOMs
These contain a glycoside extract (Quil A) prepared from tree bark. When mixed
with virus spike proteins (surface proteins) they form micelles
 Bacterially derived adjuvants
 Monophosphoryl lipid A (SKB)
Is a relatively de-toxified derivative of lethally toxic endotoxin from gram
negative bacteria (LPS)
 Natural/Synthetic surfactants
 QS21 (SKB)
A more purified version of Quil A, but not micellar like ISCOMs
 Oil/Water emulsions
 MF-59 (Chiron)
 These adjuvants are highly diverse

25. DNA vaccines (Genetic Immunisation)
 Previous studies have used molecular biology to express genes
encoding protective antigens in expression vectors. These antigens,
such as the Hepatitis B vaccine expressed in yeast cells, are then
isolated and used as vaccines. More recent approaches have used
naked plasmid DNA containing genes encoding the protective
antigen to actually transfect the host. A typical plasmid vector such
as pcDNA, contains strong promoters to induce transcription of the
protective antigen DNA. Host cells then express the protein antigen
in situ, and host immune responses are generated to the foreign
protein. Methods used to introduce DNA include :
 Intramuscular injection
 Intradermal injection
 Gene Gun
 DNA bound to gold particles and shot under gas pressure at high speed into
epidermis
 Jet injection
 Using even higher pressure and speed, it is possible to shoot DNA (or
proteins) into epidermal cells without the requirement for gold particles

26. DNA vaccines have been applied to numerous
infectious agents
 DNA vaccines have been applied to
numerous infectious agents and has been
frequently successful in the mouse. This is
partly due to plasmid DNA having its own
built-in adjuvant. Prokaryotic DNA
contains CpG motifs which are largely
absent in mammalian DNA, however,
CpGs are recognised as foreign by the
mammalian immune system and directly
activate it.

27. Advantages Disadvantages
Simple : Doesn't require an expression system
Limited effectiveness
in human trials
Has its own inbuilt adjuvant (CpG)
Lack of control of
antigen expression:
Low doses of antigen
could lead to tolerance
or Th2 induction
Long term stimulus from a single injection (not
intramuscular injection)
Questions over
integration of plasmid
DNA
Effective (in mice, less so in humans, although this
may be due to characteristics of CpG flanking
sequences)
Public acceptance
Can express multiple antigens in a single plasmid
Can include adjuvants in plasmid, e.g.
• genes encoding costimulatory molecules
• genes encoding adjuvant active cytokines (eg. IL-12)
• genes targetting antigen localisation in APCs (e.g. to
Class II compartments)

28. Experimental Approaches to
Vaccine Development
 With our expanded understanding of how the
immune system works, experimental approaches
to vaccine development are aimed at developing
vaccines that target protective immune
responses. One area of interest is the
development of better vaccines to stimulate
mucosal immunity, since most pathogens enter
the body through mucosal membranes. The oral
polio vaccine is an example of a vaccine that
enters by the pathogen's normal route and
stimulates protective neutralizing antibody.
Difficulties with oral vaccine administration
include antigen destruction in the stomach or
intestines and risk of inducing tolerance.

29.  Another area of vigorous research is targeting antigens
to APC. Antigens have been covered with mannose to
bind macrophage mannose receptor and made into
immune complexes to stimulate uptake by FcR+ cells.
Pathogen DNA has been complexed with CTLA-4 to
promote its uptake and expression by B7+ APC.
ISCOMs target antigen to Class I MHC, while antigen
coupled to a particular signal peptide can be used to
move antigen into endosomes for processing and
presentation on Class II MHC. The outer membrane
protein of Salmonella typhimurium binds M cells and
may be useful for targeting antigen to the mucosal
immune system.

30.  Finally, the ability of vaccination or cytokine
administration to control ongoing infection is
being studied. Chronic infections occur with
Herpes simplex viruses, hepatitis B and C
viruses, Mycobacterium tuberculosis and M.
leprae, and the parasites Leishmania,
Plasmodium, and Schistosoma. Persisting
infections lead to prolonged infectivity, tissue
damage from immune hypersensitivity, and
tumor development. Established immune
responses are very difficult to modify or
eliminate; but there is hope that with a properly-
targeted vaccine boost the immune system may
be able to completely eliminate pathogen.