it gives you snap shot for vaccine technology

1. Presentation
Topic: 2Modern Vaccines
By Hamze Suleiman H. Nour ( DVM, MSc Candidate).
Tropical Veterinary Medicine
Mekelle Univeristy
CVM-MU, 2019
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2. Steps vaccine production
Selecting for strain for vaccine production
 Virus /bacteria (seed)
Killed or Inactivation of organism
 Solvent / detergent (S/D) in activation
 Pasteurisation
 Acid pH in activation (low pH treatment
 Ultraviolet (UV) inactivation
Formulation of vaccine
 Suspending fluids
 Preservatives and stabilizer
 In activating agents
 Adjuvants /enhancers
Growing the micro-organism
 Various cultures
 Bird embryo
 Live animals inoculation
 Transgenic animals
Isolation and purification of
micro-organism
 Centrifugation
 Chemotherapy
 Filtration
Quality control and
lot release
 Sterility
 Chemistry & Safety
 Residual toxicity
 Efficacy
 Risk environment
 Virulence test
 Batch serial release
for distribution
 Sampling
 Labelling
 Field test (safety e &
efficacy)
 Performance
monitoring
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4. • Vaccines based on synthetic components such as nucleic acids,
nucleotides or carbohydrates synthetic peptides as antigens,
polysaccharides, and Virus-Like Particle and Nanoparticle Vaccines,
are examples of the chemistry synthesis of vaccine.
• Vaccines contain the inactivated part of a disease-forming
microorganism (or antigen) that stimulates the immune system into
recognising the invading organism as 'foreign' and produces antibodies
that attach to the antigen and not only destroy it but also remember it
for future exposure.
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5. • Fragments of the pathogen can also be used as vaccines and, at the
molecular level, those parts of a macromolecule that are recognised by
the immune system are called epitopes. In general, antigens and
epitopes are made up of proteins or polysaccharides.
• Recombinant peptide vaccine consist of protein antigen that have been
produced in a heterologous expression system (e.g. bacteria, yeast).
• The vaccinated person produces antibody to the protein antigen, thus
protecting him/her from the disease.
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6. • In general, to confirm the identity and purity of their vaccine chemists use
mass spectrometry with a 'soft' ionisation technique, such as matrix-assisted
laser desorption ionisation (MALDI).
• The matrix is used to protect the relatively fragile biomolecule from being
destroyed by the laser, which vaporises and ionises the conjugate.
• Gel electrophoresis can be used to analyse synthetic vaccines. In this
method an electric current moves different biomolecules (such as proteins)
through a polymeric gel at different rates depending on their charge, size
and shape.
• A protein carrier will move through the gel at a different rate than the carrier
that is conjugated.
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7. • Step 1: isolate the nucleophile
• Step 2: isolation of electrophile
• Step 3: couple!
• Always depend optimization of individual Epitope-Paratope
pairs through evolution.
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8. • The development of a candidate synthetic peptide vaccine includes the following steps
• Step 1. Selection of immune active peptide fragments of protein antigen(s) of an
infective agent and construction of a peptide antigen (or several antigens).
• Step 2. Chemical synthesis of peptide antigens and their conjugation (if necessary)
with a carrier.
• Step 3. Immunogenicity testing of resultant constructs on laboratory animals,
determination of specificity of antibodies (raised against these constructs) and their
protective properties.
• Step 4. Preclinical trials of selected antigens.
• Step 5. The development of the candidate vaccine and laboratory technology for its
manufacture and production of samples for testing.
• Step 6. Preclinical and clinical trials of the candidate vaccine samples.
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10.  Antibody: protein made by certain white blood cells in response to a foreign substance (antigen).
 Carbohydrate: are sugars or several sugars linked together, contains only C, H, and O, usually in
the ratio 1:2:1.
 Carbohydrate Formula: (C)n+(H2O)n = carbon + water = carbohydrates.
Classic carbohydrate-based vaccine: carbohydrate used in the vaccine is isolated from natural
sources, hence can have heterogeneity and contamination problems.
Alternative: identify carbohydrates and then synthesize them in the laboratory.
۵ Cells of our body have sensors made out of carbohydrates (on outer surface of plasma membrane)
۵ These sensors can detect many kinds of stimuli, and can signal the immune system to respond.
۵ Specific carbohydrates that carry appropriate recognition properties are synthesized and used in
carbohydrate vaccines.
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Condensation reaction.
-OH from the first and H- from the
second sugar are removed.
Glycosidic bond (-O- bond connect. the
two sugars) is formed.
sugar1- OH + HO - sugar2
sugar1- + - O - sugar2 + -OH + H-
sugar1- O - sugar2 + HOH
http://www.bergen.org/ACADEMY/Bio/molbio/LACTOSE_SYNTH/LactoseSynth.html
Synthesis two beta-glucoses
http://www.bergen.org/ACADEMY/Bio/molbio/LACTOSE_SYNTH/LactoseSynth.html

12. • These highly complex synthetic vaccines are made using solid-phase peptide synthesis - each sugar is tethered to an amino acid (bearing a
protecting group on the amino function) that can be linked to a polymeric resin bead.
• The amino group can be de-protected, ready for peptide formation with another sugar-linked amino acid, and the process repeated until the desired
peptide sequence is achieved, which can then be cleaved off the resin and conjugated to the carrier protein.
• Principle:
- Attach one side of the sugar to a secure support, the chain grows at the other side.
- Add monosaccharide one by one.
- After everything is done, cleavage from the solid support
• Main Aspects:
1. Select ‘solid phase’, a polymer inert to all reaction conditions. Most solid-phase is polystyrene, cross-linked with 1%
divinylbenzene.
2. Select linker to attach 1st sugar to the solid support, which inert to all reaction conditions.
3. Select glycosylating agents: such as thioglycosides, anomeric fluorides, trichloro-acetimidates, and sulfoxides.
4. Select protecting group: permanent protection for unoperated hydroxyl, temporary one for involved hydroxyl.
5. Repeat the coupling cycles to get the desired sequence.
6. Remove unreacted reagents at any synthetic step by a wash procedure.3/13/2019 12

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Donor-bound: Dimethyldioxirane (DMDO)
converts the double bond into epoxide. OH
of acceptor 52 reacts w/ 51 to give the
desired -glycoside 53. Repeat this procedure
to get (1 6)-linked tetrasaccharide 55.
Acceptor-bound: Excess of donor is
needed to maximize yield.
Bidirectional: oligosaccharide grows in both
directions. Used to prepare branched structures.
.
http://pubs.acs.org/journals/chreay/100/i12/figures/cr9903104h00012.html
http://pubs.acs.org/journals/chreay/100/i12/figures/cr9903104h00013.html
http://pubs.acs.org/journals/chreay/100/i12/figures/cr9903104h00011.html

14. Definition
• It is a technique used is genetic engineering that involves the
identification, isolation, and insertion of gene of interest into a vector
such as a plasmid or bacteriophage to form recombinant DNA
molecule and production of large quantity of that gene fragment or
product encoded by that gene.
• Derived from the use of recombinant DNA technology.
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15. • Step 1: identification and isolation of gene of interest or DNA
fragment to be cloned.
• Step 2: insertion of this isolated gene in suitable vector.
• Step 3: introduction of this vector into suitable organism / cell called
host (transformation).
• Step 4: selection of the transformed host cell.
• Step 5: multiplication or expression of the introduced gene in the
host.
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16. • From where we get from this
gene of interest.
Genomic library
C-DNA library
Chemical synthesis of gene if
we know sequences
If the number of the copies of
the desired gene is not enough
for gene cloning we can opt for
gene amplification techniques
like PCR.
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17. • The first step is isolation of desired gene from a cell and
the digestion of the bacterial cell wall by enzymatic action.
• This is achieved by treating the bacterial cell / plant or
animal tissue with enzyme like lysosome (bacteria),
cellulose( plant cell), chitinaze (fungus).
• Genes are located on DNA molecules centrifugation with
proteins such as histones.
• RNA can be removed by treatment with ribonuclease,
protein can be removed by protease.
• Other molecules can be removed by appropriate
treatments and further subjected gradient antifugation.
• Ultimately purified DNA is precipitated out after the
addition of chilled ethanol.
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19.  What is a vector?
• Any DNA molecules that has the
ability to replicate inside the host to
which desired gene has integrated for
cloning.
• Vectors include plasmids, and
bacteriophages, cosmids, BAC, yeast
vectors. Shuttle vectors, and etc.
Human DNA + Bacterial plasmid DNA = Recombinant of DNA
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20. • The purified DNA is cut into number of fragments by enzyme called restriction
endonuclease.
• each restriction endonuclease functions by inspecting the length of DNA and
recognizes a specific palindromic nucleotide sequences in that DNA.
• Then it will bind to the DNA and cut each of two stands of the double helix at
specific points.
• These procedure or process is called ‘’molecular scissors.’’ that were first
discovered by the Nathans(1970).
• Over 250 restriction enzymes have been isolated so far. When linear DNA is
treated with restriction enzymes, a large number of DNA fragments are formed.
• The resultant fragment are separated by gel electrophoresis, finally the desired
DNA fragments are selected by southern blotting technique.
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22. • Physical gene transfer methods:
 electroporation
microinjection
Liposome mediated gene transfer
Silicon carbide fibre mediated gene transfer
Ultrasound mediated gene transfer
DNA transfer via pollen
• Chemical gene transfer methods:
 poly ethylene Glycol mediated ( PEG mediated)
Calcium chloride mediated
DEAE dextran mediated gene transfer
• DNA imbibition cells, tissues or organs, transfers.
The process is called transformation3/13/2019 22

23. • Once the desired fragments of DNA (gene) are obtained, they are insert into
suitable vector to produced indefinite number of copies of genes.
• This is known as gene cloning. A cloning vector acts as a vehicle to carry
the desired gene.
• An ideal cloning vector should have the following properties
1. It must have low molecular weight.
2. It must have unique cleavage site for the activity of restriction enzymes at
single point.
3. It must be able to replicate truly inside host cell after its introduction.
4. It must contain genes which provide resistance to antibiotics.
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24. Several types of vectors are used in recombinant DNA technology like plasmids, phages
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25. • The enzyme DNA polymerase helps in replication of DNA is repeated many times. DNA segment can be amplified to
approximately in billion times
• i.e. 1 billion copies are made
• Such repeated replication is achieved by use of thermostable of DNA polymerase which remains active during high
temperature induced denaturation of double stranded DNA.
• The amplified fragment of desired gene can be used to ligate with vector for further cloning.
• To isolate a plamids, the bacteria cell is treated with EDTA ( Ethylene Demine Tetra Acetic Acid). Along with
lysozyme (enzyme) to digest cell wall.
• Then the bacteria cell is subjected to centrifugation in sodium lauryl sulphate solution to separate the plasmids.
• The isolated plasmid DNA is cut with same restriction endonuclease enzyme. The enzymatic cleavage cuts circular
plasmids into a linear molecule having sticky ends.
• The two sticky ends of this linear plasmid are joined to the ends of desired gene.
• The enzyme DNA ligase join the complementary ends of plasmid DNA with that of desired gene by covalent bonding
to regenerate circular Hybrid called recombinant ( r) DNA or Chromic DNA.
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27. Antibiotic resistance in a selective medium.
Visible character
Assay for biological activity
Colony hybridization
Blotting test
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28. • The recombinant plasmid are transferred into a suitable bacterial host cell (generally E.
coli). By a method known as transformation for the expression of desired gene.
• Then the cells into which the recombinant DNA are inserted called transformed cells.
• E.g if r-DNA bearing gene for resistance an antibiotics ( example, ampicillin) is transferred
into E. coli cells, the host cell become transformed into ampicillian resistance cells.
• Only transformants will grow on agar plate containing ampicillin. The transformed
recipient cells will die. The ampicillin resistance gene is called selectable marker.
• The bacteria cell walls are not ordinarily permeable to such recombinant vectors, but
reaping in dilute solution of calcium chloride renders the bacterial cell wall permeable to
the recombinant vectors.
• Inside the host cell, this r-DNA starts replicating. The transformed cell will begin to grow
on medium and divide as a separate units.
• The replicated vectors in each cell are passed on to the daughter cells given rise to
clones.
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31. • Advantages
• Those vectors that are not only safe but also easy to grow and store can be
chosen.
• Antigens which do not elicit protective immunity or which elicit damaging
responses can be eliminated from the vaccine.
• Disadvantages
• Example Cholera toxin A can be safely removed from cholera toxin.
• Since the genes for the desired antigens must be located, cloned, and
expressed efficiently in the new vector, the cost of production is high.
• When engineered vaccinia virus is used to vaccinate, care must be taken to
spare immunodeficient
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32. • Combination vaccines take two or more vaccines that could be given individually and put them into one shot. And
get the same protection as they do from individual vaccines given separately.
 Combined (multivalent) vaccine are immunological products intended for
 Immunisation against different diseases or,
 Immunisation against multi-factorial infectious disease caused by different species, types or variants of
pathogens
• There are two driving forces which lead to the promotion of multivalent (a term which will be used synonymously
with polyvalent) and combined vaccines.
• Combination products simplify vaccine administration and allow for the introduction of new vaccines without
requiring additional health clinic visit and injections.
• Multivalent/polyvalent vector vaccine. Combined antigens from different strains (serotypes/serogroups) of one
pathogen in a single vector to immunize against one disease.
• Multidisease/multipathogen vector vaccine. Key protective antigens from two or more pathogens in a single vector
to immunize against several diseases.
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33. • The potential for vaccine combination to be more reacto-genic then the individual components.
• The effect of increasing toxoid load from conjugates and endotoxin content taken Gram positive
bacteria needs to be considered. For example, in combination for inactivated bacterial vaccines
containing E. Coli
• When an adjuvant is used to augment the immune response to a combine vaccine special
problem may appear, for absorbed vaccines adjuvanticity is usually dependant on each vaccine
component being firmly bound to adjuvant, and the presence nan-occupies sites on the adjuvant.
 quality aspects
 Manafucturing and controlling requirements
 Formulation
 Stability
 Batch testing
 Safety aspects
 Potency aspects
 The development of combined vaccine is not straightforward. Each combination should be
developed and studied individually in term of quality, safety and efficacy.
 correct formulation, the stability and the computability include preservatives, excipients and
stabilizers, inactivating agents and adjuvants.
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35. • Immuno-modulation involves the manipulation of the immune system, altering
how it responds through the action of an immune-modulator.
• An immune-modulator is any molecule or compound capable of such a
manipulation, and includes cytokines, adjuvants and various inflammatory or anti-
inflammatory compounds.
• Depending on the type of immune-modulator, the action employed may involve
the suppression of inflammation, such as in an autoimmune or hypersensitivity
reaction, or may induce presentation of co-stimulatory molecules that promote a
specific type of immune response, enhancing vaccination.
• he inclusion of an adjuvant can alter the responses generated by a VLP vaccine.
For example, the adjuvant α-galactosylceramide can form a composite particle
when combined with RHDV VLP, capable of inducing increased activation of
antigen-specific T cells.
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36. Formulation largely determines
vaccination efficacy
Adjuvant
Any material that increases the immune response
against an antigen (without being immunogenic
by itself).
Delivery system
A device (colloidal particle) that allows
multimeric presentation of antigens (may
contain adjuvants).
NB: An adjuvant may act as a delivery system
vice versa!
A vaccine is more than an one antigen.
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37. • Adjuvant
Colloidal aluminium salts
 Lipid A and derivatives
Muramyl dipeptide (MDP)
Saponins
Cytokines
Cholera toxin, B subunit
CpG
• Characteristics
 Antigen adsorption crucial
 Fragment of bacterial endotoxin
 Fragments of bacterial cell walls
 Plant triterpene glycosides
 Interleukins, Interferon-g
 Mucosal adjuvant
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• Adjuvant mechanisms
Depot function: slow release of the antigen (from the site of injection)
Attraction and stimulation of immunocompetent cells: (eg dendritic cells, lymphocytes) to the
site of injection
Delivery : of the antigen to immunocompetent cells in lymph nodes

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Examples of delivery systems

39.  Recombinant DNA technology
 – First recombinant vaccine in 1985 (Hep B)
 – Live vectors/in clinical trials
 – DNA vaccines/in clinical trials
 Approaches to reduce number of injections for the
needle are Alternatives ways:-
 Combination vaccines
 Single-shot vaccines
 Mucosal (oral, nasal, pulmonary) vaccines
 Epidermal vaccines (immunisation).
 Needle-free injection
Mechanism of uptake and transport of antigen
Lipid carrier system
Oral immunization
Controlled release micro-particle for vaccine
development
Single dose vaccine delivery systems using
biodegradable polymers
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