1. Recombinant DNA technology allows for the creation of vaccines by expressing immunogenic proteins or peptides from pathogens in benign systems like bacteria or yeast. 2. Different types of vaccines produced through recombinant DNA include subunit vaccines using purified proteins, peptide vaccines using short antigen sequences, and vector vaccines that use viruses to deliver foreign DNA encoding antigens. 3. These modern vaccines produced through genetic engineering overcome many of the safety and production challenges of earlier vaccine technologies.

1. Vaccine
Presented by
Arunkumar Rengaraj

2.  British physician Edward Jenner, who in 1796 used the cowpox virus (Latin variola vaccinia) to confer protection against smallpox.
 In 1885 the French microbiologist Louis Pasteur and Emile Roux developed the first vaccine against rabies.
 A vaccine is a biological preparation that improves immunity to a particular disease.
 It contains certain agents that not only resembles a disease-causing microorganism but it also stimulates body’s immune system recognize the foreign
agents.
History:
Definition:
(Ref: www.wikipedia.org, www.britannica.com, www.pathmicro.med.sc.edu)
INTRODUCTION

3. Edward Jenner used the cowpox virus to vaccinate individuals against smallpox virus in 1796
See http://www.youtube.com/watch?v=jJwGNPRmyTI
Smallpox
INTRODUCTION

4. Old Technology:
 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
Drawbacks
 Can’t grow all organisms in culture
 Safety to lab personnel
 Expense
 Insufficient attentuation
 Reversion to infectious state
 Need refrigeration
 Do not work for all infectious agents
 Infants/children receive them – immature immunity
INTRODUCTION

5. http://davidpratt.info/vaccin.htm
INTRODUCTION

6. INTRODUCTION

7. Recombinant DNA technology
 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)
Types of recombinant vaccine
 Subunit vaccine
 Peptide vaccine
 DNA vaccine
 Attenuated vaccine
 Vector vaccine
INTRODUCTION

8. Subunit vaccine
 Vaccines that use components of a pathogenic organism
Advantages
 Using a purified protein(s) as an immunogen ensures that the preparation is stable and safe
 Precisely defined chemically, and is free of extraneous proteins and nucleic acids
Disadvantage
 Purification of a specific protein can be costly
 Isolated protein may not have the same conformation as it does in situ
SUBUNIT VACCINE

9. Note: capsid and envelope proteins can elicit neutralizing
antibodies
HSV type 1 (HSV-1)
envelope glycoprotein D (gD)
Herpes Simplex Virus
 Herpes simplex virus (HSV) has been implicated as a cancer-causing
(oncogenic) agent
 3 to 200 kb ( Double or Single – DNA or RNA)
 Sexually transmitted disease
 Severe eye infections
 Encephalitis,
SUBUNIT VACCINE

10. A subunit vaccine against Herpes Simplex Virus (HSV)
CHO cell = Chinese Hamster Ovary cell
gD = glycoprotein D
SUBUNIT VACCINE

11. SARS (severe acute respiratory syndrome)
 Southern people’s republic of china, in November 2002.
 Spread to 29 countries on five continents.
 Until mid-july 2003, 8,096 sars cases and 774 associated deaths
Property of this virus
 single-stranded plus-sense RNA genome of approximately 30 kb
 Spike protein (i.E., Amino acids 318 to 510)
SUBUNIT VACCINE

12.  Complete nucleotide sequence of the SARS virus in 2003
 Codon-optimized version of this 192-amino-acid peptide in CHO cells
 Also included a mammalian secretion signal, an n-terminal (staphylococcus aureus) protein A purification tag, and a tobacco etch virus protease
cleavage site
 CHO cells was secreted into the growth medium
 Purified by affinity chromatography on a column containing immobilized immunoglobulin G,
 Tobacco etch virus protease to remove the protein A purification tag
 Using this construct, the spike protein fragment was readily synthesized and purified
rDNA technology
SUBUNIT VACCINE

13. Staphylococcus aureus
 Major cause of hospital-acquired infection.
 Infections of the bloodstream, lower respiratory tract, and skin
 The gram-positive bacterium S. Aureus produces a pore-forming toxin;
 Whole-cell attenuated or killed vaccine was not effective ( outer surface protein )
 23 bacterial outer surface proteins
 Polymerase chain reaction (PCR) amplified and cloned into plasmid vectors
 Enabled the proteins to be expressed in E. Coli
SUBUNIT VACCINE

14. Peptide Vaccines
Small discrete portion (domain) of a protein can act as an effective subunit vaccine
Limitations to using short peptides as vaccines:
• To be effective, an epitope must consist of a short stretch of contiguous amino acids, which does not always occur naturally.
• The peptide must be able to assume the same conformation as the epitope in the intact viral particle.
• A single epitope may not be sufficiently immunogenic
PEPTIDE VACCINES

15. Structure of a peptide vaccine, representing yet another rDNA approach
Foot-and-Mouth Disease
 Antigenic protein FMDV VP1 is responsible for FMDV.
 This protein consists of different peptides
 141 to 160, 151 to 160, and 200 to 213, which are located near the c-terminal end of vp1
 9 to 24, 17 to 32 and 25 to 41, which are located near the n-terminal end of VP1
 141 to 160 elicited sufficient antibody to protect animals against subsequent challenges with FMDV
 DNA encoding FMDV VP1 peptide 142 to 160 was linked to the gene encoding a highly immunogenic
carrier molecule, hepatitis B virus core antigen
 After expression the protein molecules self-assembled into stable “27-nm particles,”
with the FMDV VP1 peptide located on the outer surface of the particle.
These particles are highly immunogenic
PEPTIDE VACCINES

16. DNA Vaccines
 Create a recombinant plasmid containing a gene encoding a specific antigen.
 Engineer in sequences
 Enabling it to be expressed in humans
 Passaged through bacteria
 Introduce it into humans
 Let the human cells produce the antigen
 This method induces both humoral and cellular immunity
 Proteins –correctly posttranslational modified
DNA VACCINES

17. 17
DNA Vaccines:
Fig: Use of DNA vaccines raises both humoral and cellular immunity
DNA VACCINES

18. (with gene encoding the antigenic protein under the control of an animal virus promoter)
Slowly released over a period of 2 to 3 weeks after inoculation of an animal
A biolistic system or direct injection is used to introduce this DNA microparticle into animals
DNA VACCINES

19.  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
 More effective than killed or subunit vaccines.
 Example: the enterotoxin gene for the a1 peptide of v. 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)
Attenuated vaccines
ATTENUATED VACCINES

20. Cholera
Cholera, caused by the bacterium V. cholerae, is a fastacting intestinal disease characterized by fever,
dehydration, abdominal pain, and diarrhea
It is transmitted by drinking water contaminated with fecal matter
1. A plasmid containing the cloned DNA segment for the A1 peptide was digested with the restriction
enzymes ClaI and XbaI, each of which cut only within the A1 peptide-coding sequence of the insert.
2. To recircularize the plasmid, an XbaI linker was added to the ClaI site and then cut with XbaI.
3. T4 DNA ligase was used to join the plasmid at the XbaI sites, thereby deleting a 550-base-pair segment
from the middle of the A1 peptide-coding region.
4. Then, by conjugation, the plasmid containing the deleted A1 peptide-coding sequence was transferred into
the V. cholerae strain
6. After growth for a number of generations, the extrachromosomal plasmid, which is unstable in V.
cholerae, was spontaneously lost.
7. Cells with an integrated defective A1 peptide were selected on the basis of their tetracycline sensitivity.
The desired cells no longer had the tetracycline resistance gene but carried the A1 peptide sequence with the
deletion

21. ATTENUATED VACCINES

22. Vector vaccines
 Here the idea is to use a benign virus as a vector to carry your favorite antigen gene from some pathogenic agent
 The vaccinia virus is one such benign virus and has been used to express such antigens (smallpox)
 Properties of the vaccinia virus:
 187kb dsDNA genome, encodes ~200 different proteins, replicates in the cytoplasm with its own replication machinery, broad host range, stable for years after drying
 However, the virus genome is very large and lacks unique RE sites,
 so gene encoding specific antigens must be introduced into the viral genome by homologous recombination
VECTOR VACCINES

23. DNA sequence coding for a specific antigen, such as HBcAg, 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 .
2. This plasmid is used to transfect thymidine kinase-negative animal cells in culture, usually chicken embryo
fibroblasts, that have previously been infected with wild-type vaccinia virus, which produces a functional
thymidine kinase.
3. Recombination between DNA sequences that flank the promoter and the neutralizing antigen gene on the
plasmid and the homologous sequences on the viral genome results in the incorporation of the cloned gene
into the viral DNA activity in the host cells and the disruption of the thymidine kinase gene in the recombined
virus render the host cells resistant to the
otherwise toxic effects of bromodeoxyuridine. This selection scheme enriches for cell lines that carry a
recombinant vacciniavirus.
4. The definitive selection of cells with a recombinant vaccinia virus is made by DNA hybridization with a
probe for the antigen gene

24. VECTOR VACCINES
Bacteria as Antigen Delivery systems
 Cholera toxin B subunit was inserted into a portion of the salmonella flagellin gene
 which consisted of amino acid residues 50 to 64 of the cholera toxin B subunit
 The chimeric flagellin functioned normally. Furthermore, the epitope was present at the flagellum surface.
 Attenuated Salmonella strains can be administered orally

25. Summary
 Recombinant DNA technology has been used in various ways to create reliable vaccines.
 Immunologically active, noninfectious agents are produced by deleting the genes that cause virulence
 The genes or segments of genes that encode the major antigenic determinants of pathogenic organisms can be cloned into expression vectors, and large amounts of
the product can be harvested, purified, and used as a vaccine.
 With the last strategy, complete genes produce subunit vaccines, and cloned domains of the major antigenic determinants produce peptide vaccines.
 Peptide vaccines may also be produced by chemical peptide synthesis.
 As an alternative to using cloned antigenic proteins or peptides for inoculation, DNA constructs encoding the antigenic protein or peptide may be utilized.
 These DNA constructs may be delivered directly to animals or humans
SUMMARY