Slide 1

Vaccines

Watch: http://www.youtube.com/watch?v=jJwGNPRmyTI

 A vaccine is a biological preparation that improves
immunity to a particular disease. 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, purified, and either inactivated or
attenuated.
 Edward Jenner used the cowpox virus to vaccinate
individuals against smallpox virus in 1796.
PHRM-521-5th-E-01

Smallpox

1


Slide 2

Mechanism of vaccine

2


Slide 3

Traditional vaccines
 Traditional vaccines consist of either inactivated (killed) or
live but attenuated (avirulent) bacterial cells or viral
particles.
 Killed (inactivated): Some vaccines contain killed
microorganisms (but previously virulent) that have been
destroyed with chemicals, heat, or antibiotics.
 Attenuated: Some vaccines contain live, attenuated
microorganisms. Many of these are live viruses that have
been cultivated under conditions that disable their
virulent properties. Many vaccines contain closely related
but less dangerous organisms to produce a broad
immune response.
3


Slide 4

Limitations to traditional vaccines
 Not all infectious agents can be grown in culture.
Therefore, many vaccines have not been
developed for a number of diseases.
 Production of animal and human virus requires
animal cell culture, which is expensive.
 Both the yield and rate of production of animal
and human viruses in culture are often quite low,
making vaccine production costly.
 Extensive safety precautions are necessary to
ensure that laboratory and production personnel
are not exposed to the pathogenic agents.
4


Slide 5

Limitations to traditional vaccines (contd.)
 Batches of vaccines may not be killed or may
be insufficiently attenuated during production
process, thereby introducing virulent
organisms into the vaccine and inadvertently
spreading the disease.
 Attenuated strains may revert, a possibility
that requires continual testing to ensure that
the reacquisition of virulence has not
occurred.
 Not all diseases (e.g., AIDS) are preventable
through the use of traditional vaccine.

5


Slide 6

Production of traditional vaccine in USA

6


Slide 7

For understanding influenza, visit:
http://www.lung.org/lung-disease/influenza/understanding-influenza.html
For information about swine flu, visit: http://www.cdc.gov/flu/swineflu/
7


Slide 8

Recombinant DNA technology can
create better, safer, reliable vaccines
 Immunologically active, non-infectious agents can be
produced by deleting virulence genes.
 A gene(s) encoding a major antigenic determinant(s)
can be cloned into a benign carrier organisms (virus
or bacteria).
 Genes or portions of genes encoding major antigenic

determinants can be cloned in expression vectors
and large amounts of the product are purified, which
are used as a subunit or peptide vaccine.
8


Slide 9

Some human disease agents for which rDNA vaccines are being developed

9


Slide 10

Some human disease agents for which rDNA vaccines are being developed(contd.)

10


Slide 11

Subunit Vaccines

 For disease causing viruses, it has been shown that purified
outer surface viral proteins, either capsid or envelope proteins are often sufficient for eliciting neutralizing antibodies in
the host organism.
 Vaccines that use components of a pathogenic organism
rather than whole organism are called “subunit” vaccines.11


Slide 12

Schematic representation of the development of a
subunit vaccine against Herpes Simplex Virus (HSV)

 The isolated HSV envelope glycoprotein D (gD) gene is
used to transfect CHO cells. Then, the transfected cells are
grown in culture that produced gD protein.
 Mice inoculated with the purified gD protein are protected
12
against infection HSV.


Slide 13

A similar approach was used to create a subunit
vaccine against foot-and-mouth disease virus
(FMDV) and Human Papillovmavirus (HPV)
 FMDV has a devastating effect on cattle and swine.
 The successful subunit vaccine is based on the expression
of the capsid viral protein 1 (VP1) as a fusion protein with
the bacteriophage MS2 replicase protein in E. coli.

 The FMDV genome consists of a 8kb ssRNA; a cDNA was
made to this genome and the VP1 region was identified
immunologically.
 A subunit vaccine (Gardasil) was developed against human
papillomavirus; this virus causes genital warts and is associated with the development of cervical cancers; used the
capsid proteins from four HPV types (type 6, 11,16 and 18).
13


Slide 14

Peptide Vaccines

Envelope bound protein with
external epitopes (1 to 5)

 Use discrete portion (domain)
of a surface protein as vaccine.
 These domains are ‘epitopes’
(antigenic determinants) and
capable to elicit an immune
response.

 In some cases, small peptides are
often degraded and therefore show

poor or no immune response.
 When these peptides are fused with
a inert carrier proteins, vaccine
exhibits stronger immune response.
14


Slide 15

Advantage and disadvantage of subunit vaccines
Advantages
 Using a purified protein(s) as an immunogen ensures that

the preparation is stable and safe, is precisely defined and
is free of extraneous proteins and nucleic acids that can
initiate undesirable side effects in the host organisms.

Disadvantages
 Purification of a specific protein can be costly and in certain
instances, an isolated protein may not have the same

confirmation as it does in situ (within the viral capsid or
envelope) with the result that its antigenicity is decreased.
15


Slide 16

Genetic Immunization: DNA Vaccines
Genetic approaches to
vaccine development.
 One or more genes encoding
critical pathogen-specific
antigens are isolated and
recombined with a harmless
or disabled vector for
delivery by injection, or
incorporated into food plants
for ingestion, or modified for
injection as naked DNA.
 Subunit antigens can also be
produced by genetic engineering for direct injection.
16


Slide 17

Advantages of genetic immunization over
conventional vaccines

17


Slide 18

Problems with the use of DNA vaccines
 In large animals and humans, the transfection efficiency of
introduced plasmid DNA is often insufficient to generate a
protective immune response.
 To alleviate this problem, biodegradable microscopic (0.3to 1.5 µm) polymeric particles with a cationic surface that
binds the plasmid DNA have been used to deliver foreign
DNA to animal cells.
 A biolistic system or direct
injection is used to introduce this
DNA microparticle into animals
where plasmid DNA carrying an
antigenic gene under the control
of an animal virus promoter.

18


Slide 19

Attenuated vaccines
 Attenuated vaccines traditionally use nonpathogenic
bacteria or viruses related to their pathogenic
counterparts.
 Genetic manipulation may also be used to create
attenuated vaccines by deleting a key disease causing
gene from the pathogenic agent.
 Example: the enterotoxin gene for the A1 peptide of
Vibrio cholerae, the causative agent of cholera, was
deleted; the resulting bacteria was non-pathogenic
and yet elicits a good immunoprotection (some side
effects noted however).
19


Slide 20

Strategy for deleting part of the cholera toxin A1 peptide DNA sequence


A plasmid containing the cloned DNA
segment for the A1 peptide was digested
with ClaI and XbaI.



To recircularize the plasmid, an XbaI linker
was added for the ClaI site and then cut with
XbaI.



T4 DNA ligase was used to join the plasmid
at the XbaI site, thereby deleting a 550 bp
segment from the middle to the A1 peptide
coding region.



Then, by conjugation, the plasmid containing
the deleted A1 peptide coding sequence was
transferred into the V. cholerae strain
carrying the TetR gene within its A1 peptide
DNA sequence.



After recombination, the homologous
segment on the plasmid carrying the deletion
replaced the Tet-disrupted A1 peptide gene
on the chromosome.



Cells with an integraed defective A1 peptide
were selected based on Tet sensitivity, The
desired cells die in the presence of tet.

20


Slide 21

Vector vaccines
 Here, the idea is to use benign viruses or harmless bacterial
strains as a vector to deliver and express cloned genes
encoding antigens that elicit neutralizing antibodies against
pathogenic agents.
 The vaccinia virus is one such benign virus and has been used
to the eradication of smallpox globally.
 Properties of the vaccinia virus: 187kb dsDNA genome,
encodes ~200 different proteins, replicates in the cytoplasm
(not in the nucleus) with its own replication machinery, broad
host range, stable for years after lyophilization (freeze drying).
 However, the virus genome is very large and lacks unique RE
sites, so genes encoding specific antigens must be introduced
into the viral genome by homologous recombination.
21


Slide 22

Strategy for the integration of antigen gene into vaccinia virus DNA
1.

2.

3.

4.

The DNA sequence coding for a specific
gene is inserted into a plasmid vector
immediately downstream of a cloned
vaccinia virus promoter and in the middle
of a nonessential vaccinia virus gene such
as the gene for the enzyme thymidine
kinase (TK) as illustrated in Fig.A.
This plasmid is used to transfect chicken
embryo fibroblast that have previously
infected with wild-type vaccinia virus,
which produces functional TK.
Recombination between the plasmid and
vaccinia virus chromosomal DNA results in
transfer of antigen gene from the
recombinant plasmid to the vaccinia virus
DNA (Fig. B). Primary selection is based
on bromodeoxyuridine sensitivity test.
The definitive selection of cells with
recombinant vaccinia virus is made by
DNA hybridization with a probe for the
antigen gene before using this modified
virus as a vaccine.

22


Slide 23

Bacteria as antigen delivery systems
Antigen gene

Attenuated bacterium

Antigenic proteins
expressed in bacteria

Vaccinate patient

 Use live nonpathogenic bacterium
such as attenuated Salmonella,
which contains plasmid DNA
encoding flagellin-cholera toxin B
subunit fusion protein.
 Plasmid DNA containing antigenic
gene fused with flagellin gene is
transformed into flagellin negative
Salmonella strain.
 Epitope (antigenic determinant) is
expressed on the flagellum surface.
 Here, flagellin engineered bacteria
is the vaccine against cholera.
 Advantage – this vaccine can be
administered orally.

23


Slide 24

Application of 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.
 Such attenuated Salmonella strains have been used as
an antigen delivery system to provide host immunity
against cholera and Helicobacter pylori.
24
24


Slide 25

Summary of recombinant vaccines

25