VACCINE TECHNOLOGY

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Definitions
◼Originally derived from the Latin word ‘‘vacca’’
meaning cow
◼Pharmaceutical suspension or solution of an
immunogenic substance or compound(s) that is intended
to induce active immunity
◼Preparations containing antigenic substances that have
been shown to be capable of inducing a specific and
active immunity in man
 VACCINES

• Immunization: a procedure designed to increase

concentrations of antibodies and/or effector T-cells
which are reactive against infection (or cancer).

• Immunization procedure called vaccination and the

immunizing agent called vaccine (or “serum” in
historical references)

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 Principles of Vaccination
Immunity
• Self vs. non-self
• Protection from infectious disease
• Usually indicated by the presence of antibody
• Very specific to a single antigen
 Principles of Vaccination
Active Immunity
• Protection produced by the person's own
immune system
• Usually permanent

Passive Immunity
• Protection transferred from another person
or animal as antibody
 Principles of Vaccination
inject or administer

Antigen
• A live or inactivated substance (e.g., protein,
polysaccharide) capable of producing an
immune response
Antibody
• Protein molecules (immunoglobulin)
produced by B lymphocytes to help eliminate
an antigen
 Passive Immunity
• Transfer of antibody from an exogenous
source

• Transplacental most important source in

infancy

• Temporary protection
 Sources of Passive Immunity
• Almost all blood or blood products

• Homologous pooled human antibody

(immune globulin)

• Homologous human hyperimmune globulin

• Heterologous hyperimmune serum (antitoxin)

Classification of Vaccines
• Live attenuated
– viral
– bacterial

• Inactivated
kill the virus -> test can we make the vaccine or not
 Inactivated Vaccines
Whole
we can prepare from:

• virus
• bacteria
Fractional
a component that include in inactivated vaccines

• protein-based
– subunit what is it? go home study it for exam
– toxoid some of them help you prevent disease
• polysaccharide-based some can cause immune response
(hypersensitivity) -> need to purify

– pure
carefully (phải high purity)

bc polysaccharide is very weak, easy to hydrolyze ->

– conjugate have to conjugate -> need to protect polysaccharide in
living system -> stable in the body
 Live Attenuated Vaccines
this vaccine required the microbes to replicate -> survive -> need to remove
virulant

• Attenuated (weakened) form of the "wild"

virus or bacteria remove the virulence -> replicate
replicate because for vaccine to
maintain long life in body
if not might can't live in body
• Must replicate to be effective
can they give the co-stimulation: combine with your antibody but no activity or decrease the action

• Immune response similar to natural infection

• Usually effective with one dose because long half-life and can
replicate
 Live Attenuated Vaccines
• Severe reactions possible because replicate, they may become
dangerous again (become virulant
again)

• Interference from circulating antibody

antibody will recognize and then they kill the microbe, therefore the activity of vaccine
will slow down or even nothing.

• Unstable
 Live Attenuated Vaccines
• Viral measles, mumps, rubella,
vaccinia, varicella, yellow
fever (oral polio) (rotavirus)
(influenza)

• BacterialBCG, oral typhoid

Vaccines in (parenthesis) are not available in the United States.

 Inactivated Vaccines
• Cannot replicate because inactivated
• Minimal interference from circulating
antibody no antigen at all
• Generally not as effective as live vaccines

• Generally require 3-5 doses short halflife

• Immune response mostly humoral

• Antibody titer falls over time
 Inactivated Vaccines
Whole cell vaccines
• Viral polio, hepatitis A,
rabies (influenza)

• Bacterial (pertussis) (typhoid)

(cholera) (plague)

Vaccines in (parenthesis) are not available in the United States.

 Inactivated Vaccines
Fractional vaccines
• Subunit hepatitis B, influenza,
acellular pertussis,
typhoid Vi (Lyme)

• Toxoid diphtheria, tetanus

 Polysaccharide Vaccines
have a lot of hydroxyl group, can conjugate
if no conjugate, then consider the quality of
Pure polysaccharide polysaccharide

• pneumococcal
• meningococcal
• Haemophilus influenzae type b

Conjugate polysaccharide
• Haemophilus influenzae type b
• pneumococcal
 Pure Polysaccharide Vaccines
• Not consistently immunogenic in
children <2 years of age

• No booster response

• Antibody with less functional activity

• Immunogenicity improved by
conjugation
 VACCINES IN Immunization

Passive versus active immunization:

-> TYPE ACQUIRED THROUGH Passive Immunization –
-> Natural maternal serum/milk
-> Artificial immune serum

-> Type ACQURIED THROUGH Active Immunization –

-> Natural infection
-> Artificial infection*:
Attenuated organisms (live)
inactivated organisms (dead)
Cloned genes of microbiological antigens
Purified microbial macromolecules
Synthetic peptides
DNA
*Artificial refers to steps involving human intervention

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 Mechanisms of VACCINES

Establish resistance to virus/pathological organism by evoking an immune

response

1. Give host a foreign organism/protein in non-infectious form -> active

immunization

2. Antibodies are generated Ab binds to surface proteins of organism

-> passive immunization

Other vaccination components:

let the product release slowly, prolong the effect
• Adjuvant: chemicals in the vaccine solution that enhance the immune response
– Alum – Ag in the vaccine clumps with the alum such that the Ag is released
– slowly, like a time-release capsule
– gives more time for memory cells to form

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 Vaccine preparation

• A vaccine renders the recipient resistant to infection.

• During vaccination a vaccine is injected or given orally.

• The host produces antibodies for a particular pathogen.

• Upon further exposure the pathogen is inactivated by the antibodies

and disease state prevented.

• Generally to produce a vaccine the pathogen is grown in culture and

inactivated or nonvirulent forms are used for vaccination.

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 Traditional preparation

– Grow in animals (vaccinia in

calves for smallpox; rabbit
brains for rabies)

– Simple bacterial culture

(Cholera vibrio) then
inactivation

– Grow in eggs (influenza,

vaccinia) then inactivate

>100 million eggs used for

influenza in the USA every year
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 Improved Preparation

• Recombinant DNA technology is being used to produce a new

generation of vaccines.

 Virulence genes are deleted and organism is still able to stimulate an

immune response.
 Live nonpathogenic strains can carry antigenic determinants from
pathogenic strains.
 If the agent cannot be maintained in culture, genes of proteins for
antigenic determinants can be cloned and expressed in an alternative
host e.g. E. coli.

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 Recombinant Vaccines

1. Subunit Vaccines
peptide vaccines
Genetic immunization

2. Attenuated Vaccines

3. Vector Vaccines

4.Antigen Delivery Systems

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 VACCINES
Principle of Vaccination

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 VACCINES
Subunit/Peptide Vaccines

•Do NOT use entire virus or bacteria (pathogenic agent)

•Use components of pathogenic organism instead of whole organism

•Advantage: no extraneous pathogenic particles i.e. DNA

•Disadvantage: Is protein the same as in situ? -> Cost?

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 VACCINES
Subunit/Peptide Vaccines

Development of Subunit vaccines based on the following observation:

• It has been showed that the capsid or envelope proteins are enough to
cause an immune response:
 Herpes simplex virus envelop glycoprotein O.
 Foot and mouth disease virus capsid protein (VP1)
 Extracellular proteins produced by Mycobacterium tuberculosis.

• Subunit Vaccines
• Antibodies usually bind to surface proteins of the pathogen or proteins
generated after the disruption of the pathogen.
• Binding of antibodies to these proteins will stimulate an immune response.
• Therefore proteins can be use to stimulate an immune response.

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 VACCINES
Subunit/Peptide Vaccines

Example for a subunit vaccine -> Tuberculosis

• Tuberculosis is caused by Mycobacterium tuberculosis.
• The bacterium forms lessions in the tissues and organs -> causing cell death. Often
the lung is affected.
• About 2 billion people are infected and there are 3 million deaths/year.
• Currently tuberculosis is controlled by a vaccine called BCG (Bacillus Calmette-
Guerin) which is a strain of M. bovis.
• M. bovis often responds to diagnostic test for M. tuberculosis.

• Six extracellular proteins of M. tuberculosis were purified.

• Separately and in combinations these proteins were used to immunized guinea
pigs.
• These animals were then challenged with M. tuberculosis.
• After 9-10 weeks examination showed that some combinations of the purified
proteins provided the same level of protection as the BCG vaccine.

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 VACCINES
Subunit/Peptide Vaccines

Selection & delivery of vaccine peptides:

Use discrete portion (domain) of a surface protein

as Vaccine

These domains are ‘epitopes’ (antigenic

determinants) -> are recognized by antibodies

CARRIER PROTEINS

Problem -> Small Peptides are often Digested

-> no strong immune response

-> Carrier Proteins Make more Stable + stronger

immune response

Make fusion protein of carrier + vaccine peptide

-> inert carrier or highly immunogenic carrier (hepatitis B
core protein)
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 VACCINES need to remove virulence

Attenuated Vaccines

• Attenuated vaccines often consists of a pathogenic strains in which the

virulent genes are deleted or modified.

• Live vaccines are more effective than a killed or subunit (protein)

vaccines.

Example -> Vaccine against Cholera

• cholera is caused by infection withVibrio cholerae and is transmitted through
contaminated water.
need to remove
• V. cholerae produces a enterotoxin with an A1 subunit and 5 B subunits -> causes
disease

• Presently the cholera vaccine consist of a phenol-killed V. cholerae and it only last 3-6
months.

• A live vaccine would be more effective. 30

 VACCINES
Attenuated Vaccines
Example -> Vaccine against Cholera

550 bases deleted

of A1 peptide The final result is V. cholerae with a 550 bp of the A peptide
deleted.

-> Currently being tested. 31

 VACCINES
Vector Vaccines

Virus as Antigen Gene Delivery System !!!

Vaccinia good candidate for a live recombinant viral vaccine

•benign virus
•replicate in cytoplasm (viral replication genes)
•easy to store
-> The vaccinia virus is generally nonpathogenic.

The procedure involves:

• The DNA sequence for the specific antigen is inserted into a plasmid beside
the vaccinia virus promoter in the middle of a non-essential gene e.g.
thymidine kinase.

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 VACCINES
Vector Vaccines

The procedure involves:

• The plasmid is used to transform thymdine kinase

negative cells which were previously infected with
the vaccinia virus.

• Recombination between the plasmid and vaccinia

virus chromosomal DNA results in transfer of
antigen gene from the recombinant plasmid to the
vaccinia virus.

• Thus virus can now be used as a vaccine for the

specific antigen.

-> Recombinant Virus

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 VACCINES
Vector Vaccines

• A number of antigen genes have been inserted into the vaccinia virus
genome e.g.

❖ Rabies virus G protein

❖ Hepatitis B surface antigen
❖ Influenza virus NP and HA proteins.

• A recombinant vaccinia virus vaccine for rabies is able to elicit

neutralizing antibodies in foxes which is a major carrier of the
disease.

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 VACCINES
Vector Vaccines

Control of Viral Vaccines Post Innoculation

•Vaccinia virus is resistant to

interferon -> due to presence of
K3L protein

•Use an interferon-sensitive strain of

vaccinia virus -> delete K3L gene to
create mutant

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 VACCINES
Bacterial Antigen Delivery Systems

-> Use live nonpathogenic bacterium

Antigen Gene which contains antigen (Salmonella or
epitope from cholera)

•Insert antigen gene into flagellin gene

Bacterium
•Epitope is expressed on the flagellum
surface

Antigen Proteins made on Bacterial cell -> Flagellin-engineered bacteria is

VACCINE

Advantage - Oral Administration

Vaccinate Patient

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 VACCINES
Vaccine Approval

• Done by CBER (Center for Biologics Evaluation and Research),

an arm of the FDA

• Generally same clinical trial evaluation as other biologics and

drugs

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Vaccine Technology
Vaccine Technology
Vaccine Technology
 Future Development scope of
vaccines
(a) Vaccine against Alzheimeir’s disease
(b) Vaccine for Meningitis C
(c) Super vaccine
(d) Immunomodulators
(e) Vaccination with Gas-Lighter
(f) Vaccine against cervical-cancer
(g) Vaccination without needles
VACCINE DESIGN
 Polytope optimization
•Successful immunization can be obtained only if the
epitopes encoded by the polytope are correctly
processed and presented.
•The design of a polytope can be done in an effective
way by modifying the sequential order of the different
epitopes, and by inserting specific amino acids that will
favor optimal cleavage as linkers between the epitopes.
Polytope starting configuration
 Polytope optimization Algorithm
• Optimization of four measures:
1. The number of poor C-terminal cleavage sites of
epitopes (predicted cleavage < 0.9)
2. The number of internal cleavage sites (within
epitope cleavages with a prediction larger than the
predicted C-terminal cleavage)
3. The number of new epitopes (number of
processed and presented epitopes in the fusing
regions spanning the epitopes)
4. The length of the linker region inserted between
epitopes.
Polytope final configuation
 Prediction of antigens
• Protective antigens
• Functional definition (phenotype)
• Which antigens will be protective (genotype)?
• They must be recognized by the immune system
• Predict epitopes (include processing)
• CTL (MHC class I)
• Helper (MHC class II)
• Antibody
 Function and conservation
• Some of the epitopes must exist in the wild type
• Conservation (http://www.ncbi.nlm.nih.gov/BLAST)
• Function
• When is it expressed?
• Where is it trafficked to?
• SecretomeP
Non-classical and leaderless secretion of eukaryotic proteins.
SignalP
Signal peptide and cleavage sites in gram+, gram-
and eukaryotic amino acid sequences.
TargetP
Subcellular location of proteins: mitochondrial,
chloroplastic, secretory pathway, or other.
• Expression level?
 Selection of antigens
• Epitopes
• Polytope
• Proteins
• Helper epitopes
• Does it contain any
• Can they be added
• Hide epitopes
• Immunodominant and variable ones
Examples of antigen selections
Clustering of HLA alleles
Inside out:
1. Position in RNA
2. Translated regions (blue)
3. Observed variable spots
4. Predicted proteasomal cleavage
5. Predicted A1 epitopes
6. Predicted A*0204 epitopes
7. Predicted A*1101 epitopes
8. Predicted A24 epitopes
9. Predicted B7 epitopes
10. Predicted B27 epitopes
11. Predicted B44 epitopes
12. Predicted B58 epitopes
13. Predicted B62 epitopes
 Strategy for the
quantitative ELISA assay
C. Sylvester-Hvid, et al., Tissue antigens, 2002: 59:251

• Step I: Folding of MHC class I molecules in solution

b2m
Heavy chain Peptide-MHC
peptide Incubation complex

• Step II: Detection of de novo folded MHC class I molecules by ELISA

Development

C Sylvester-Hvid et al., Tissue Antigens. 2002 59:251-8

 Flow Chart of ELISPOT Assay

PBMCs + Culture in vitro for 10 days + peptide

Peptide

+ peptide - peptide
Incubating in anti IFN- pre-coated plate for 18-20 h
•Coating Ab:
–Human IFN- MAb
(ENDOGEN, Pierce
Washing off the cells Biotechnology, Inc)
•Detection Ab:
–Human IFN- MAb, Biotin
Adding Biotin-anti IFN- labeled
–(ENDOGEN, Pierce
Biotechnology, Inc)

Adding Streptavidin-HRP after washing the plate

Adding a substrate

Automatical counting
 Selected pathogens

Pathogen HLA binding ELISPOT

Influenza X X
Variola major (smallpox) vaccine strain X X/VRC, NIH
Yersinia pestis X
Francisella tularensis (tularemia) X (X) A
Sjostedt
LCM X
Lassa Fever X
Hantaan virus (Korean hemorrhagic fever X
virus)
Rift Valley Fever X
Dengue X (X) T
August
Ebola X
Marburg X
Multi-drug resistant TB (BCG vaccine) X X
Yellow fever X (X) T
August
Prediction of Class II epitopes
 Prediction of MHC Class II binding
• Virtual matrices
– TEPITOPE: Hammer, J., Current Opinion in Immunology 7, 263-269, 1995,
– PROPRED: Singh H, Raghava GP Bioinformatics 2001 Dec;17(12):1236-7

• Web interface
http://www.imtech.res.in/raghava/propred
• Prediction Results
 Prediction of Antibody epitopes
• Linear
– Hydrophilicity scales (average in ~7 window)
• Hoop and Woods (1981)
• Kyte and Doolittle (1982)
• Parker et al. (1986)
– Other scales & combinations
• Pellequer and van Regenmortel
• Alix
– New improved method (Pontoppidan et al. in preparation)
• http://www.cbs.dtu.dk/services/BepiPred/
• Discontinuous
– Protrusion (Novotny, Thornton, 1986)
 Prediction of proteins structure
• Homology modeling
• Secondary structure prediction