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Approaches to vaccine development.. Pptx
- 1. 1 Vaccine Development Traditional andModern Approaches
- 2. • Immunization: aprocedure 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 • Immunization procedure called vaccination and the immunizing agent called vaccine (or “serum” in historical references) VACCINES 2
- 3. 3 Purpose of Vaccination •Protect the individual from disease. • Reduce the severity of disease. • Protect the community. • Eradication of the disease.
- 4. 4 Basic concept ofvaccines • Deliver to the body some part or all of the disease organism that IMITATES the pathogen but is not pathogenic. –Induce protective immune response. Polysaccharide Surface proteins Intracellular proteins Toxins Entire organism • live (attenuated) • killed LPS capsular
- 5. 5 Vaccine manufacture Antigen Production Eggs –Influenza – Bacterial / Yeast fermentation – Whole organism (e.g. Cholera) – Subunit vaccines (e.g. Capsular polysaccharide, Tetanus and Diphtheria toxoid) – Genetically engineered proteins (e.g. Hepatitis B and HPV vaccines) – Cell culture – Viral vaccines either whole virus or subunit – Genetically engineered proteins
- 6. 6 Antigen concentration. Removal ofunwanted foreign components. – Host proteins – Host DNA – Adventitious agents Removal of unnecessary Bacterial or Viral components. – LPS – Unwanted proteins (not involved in immunity) – Unwanted nucleic acid Change of diluting solution. Addition of other components. – Adjuvants, stabilizers, preservatives etc Vaccine Processing
- 7. 7 Types of Vaccines •Inactivated toxins • Inactivated whole bacteria or viruses • Live attenuated bacteria or viruses • Subunit vaccines • Genetically engineered proteins • Polysaccharide vaccines • Conjugated vaccines • Recombinant DNA modified organisms • DNA vaccines
- 8. 8 Whole Killed Whole pathogengrown and killed –Heat, chemical modification (formaldehyde) –Pertussis, cholera. –IPV (Inactivated Polio), Influenza, Hepatitis A. Advantage –Relatively easy –Generally safe to administer - no risk of reversion, infection.
- 9. 9 Whole Killed • Disadvantages –Numerousinjections normally required –No or limited cellular immunity –Immunity often shorter-lasting than live vaccines –Reactogenicity from LPS, lipids.. –Adjuvant often required (alum, emulsions) –Risk of growing pathogenic organism –Risk of incomplete inactivation –Inappropriate immune response ? eg RSV –Can not focus immunity on protective antigen.
- 10. 10 Inactivated Whole CellCholera Vaccine Manufacturing Process Fermentation Concentration And Washing Inactivation Bulk Monovalent Formulation SBL Vaccine Cholera toxin B subunit
- 11. 11 Subunit • Purify aprotein or proteins from pathogen –Eliminate reactogenic contaminants (eg LPS) –Selective presentation of 'protective' antigens • Pertussis –Pertussis toxin + filamentous haemagglutinin + pertactin (no LPS) • Influenza (subunit) –Mainly haemaglutinin + neuraminidase • Disadvantages –Requires growing the pathogen and purifying protective (antigens) subunits.
- 12. 12 Polysaccharide • Many bacteriaproduce a strain-specific capsular polysaccharide on their surface. • Antibody to these antigens are protective. –Streptococcus pneumoniae, Haemophilus Type B, Meningitis A,C,W,Y (not B!) Typhoid (Vi). • Can be easily purified. • Immunogenic in older children / adults. • But poorly immunogenic in infants • T-cell independent responses • Short lived • Low antibody responses
- 13. 13 Different forms ofPolysaccharide LPS Capsular polysaccharide
- 14. 14 Live Attenuated • Mostcommon type of vaccine. • OPV (oral polio), measles, mumps, rubella, yellow-fever, rotavirus, influenza, smallpox, • BCG, typhoid, anthrax, Live pathogen selected or genetically modified causes no disease or only mild disease. • Derived from non-human source (eg cows, monkeys) • Selected mild strain from humans • Passaged (tissue adapted) strain from
- 15. 15 Live Attenuated • Advantages: –Mimicsnatural infection –Humoral and cellular response. –Immunological memory. –Generally cheap. • Disadvantages: –Not suitable for all organisms –May revert to a virulent form –Circulating antibody may interfere Immunity to non-protective antigens –Safety in immuno-suppressed individuals?
- 16. 16 Live Attenuated Vaccines Typhoidbacteria Measles virus
- 17. 17 Measles Vaccine • KilledInactivated vaccine was abandoned in 1967 as it did not provide complete protection. • An attenuated vaccine was also being developed and first licensed in 1963. This was found to be too reactogenic for routine use. • Further attenuation produced an acceptable vaccine.
- 18. 18 Attenuation of Measles EDMONSTONB STRAIN • 24 passages in primary kidney cells • 28 passages in human amnion cells • Passages in chick embryo cells • Vaccine immunogenic but too reactive. SCHWARZ • 85 additional passages in chick embryo cells • Vaccine 90-95% effective, low reactogenicity.
- 19. 19 Recombinant Protein produced bygenetic engineering • Express protective antigen in safe easy-to- grow organism –Hepatitis B (HBsAg expressed in yeast) –HPV (papilloma L1 expressed in yeast) VIRUS LIKE PARTICLES
- 20. 20 Recombinant • Advantage –Safe. Growthin non pathogenic yeast cells –Easier – in case of difficult to grow viruses like hepB, HPV. • Disadvantages: –Need to identify protective antigen/s –Obtaining antigen in 'correct' conformation –Usually poorly immunogenic alone –Poor CMI – requires adjuvant.
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- 23. 23 Recombinant Vaccine • Thegene coding for HBsAg was discovered in 1970. • The gene has been inserted into a yeast cell. • As the yeast cell grows it produces large amounts of HBsAg. • The HBsAg is extracted and purified then incorporated into the vaccine.
- 24. 24 Recombinant Vaccine • Advantagesof the recombinant vaccines. –Produced more quickly. –In larger quantities. –Free from infectious virus particles.
- 25. 25 Recombinant DNA modified organisms LiveVectors • Cloning of genetic material from one organism into another. • The non virulent parent organism expresses the antigens of the cloned genetic material. • A vaccine would elicit a response against the introduced antigen as well as the original organism.
- 26. 26 Recombinant DNA modifiedorganisms Vaccinia virus expressing papilloma virus antigens on its surface.
- 27. 27 DNA Vaccines • Involvesthe injection of naked DNA coding for one or more genes. • The gene is grafted onto another piece of DNA which acts as a vector. • Injected into muscle tissue, once in the cell the gene prompts the cell to produce antigen. • The immune system then mounts an immune response.
- 28. 28 Injected DNA coding fora specific antigen Antibody-producing cell Cytotoxic T-lymphocyte Class1 MHC mRNA Viral protein Nucleus DNA Vaccines
- 29. 29 • Clinical trialsto date with naked DNA vaccines have not proved to be that successful • DNA vaccines may be useful as a priming dose in prime-boost regimes due to their ability to induce cell mediated immune responses. DNA Vaccines
- 30. Attenuated Vaccines 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. 30
- 31. Vector Vaccines VACCINES Virus asAntigen 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 31
- 32. Vector Vaccines VACCINES The procedureinvolves: • 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 32
- 33. Vector Vaccines VACCINES • Anumber 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. 33
- 34. INTRODUCTION DNA vaccineis DNA sequence used as a vaccine. This DNA Sequence code for antigenic protein of pathogen. As this DNA inserted into cells it is translated to form antigenic protein. As this protein is foreign to cells , so immune response raised against this protein. In this way ,DNA vaccine provide immunity against that pathogen.
- 35. DNA vaccines VsTraditional vaccines Uses only the DNA from infectious organisms. Avoid the risk of using actual infectious organism. Provide both Humoral & Cell mediated immunity Refrigeration is not required Uses weakened or killed form of infectious organism. Create possible risk of the vaccine being fatal. Provide primarily Humoral immunity Usually requires Refrigeration. DNA vaccines Traditional vaccines
- 36. HOW DNA VACCINEIS MADE Viral gene Expression plasmid Plasmid with foreign gene Recombinant DNA Technology
- 37. Bacterial cell Transform into bacterialcell Plasmid DNA
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- 40. METHODS OF DELIVERY Syringedelivery:- Either intramuscularly or Intradermally
- 41. Contd.. Gene gun delivery:- Adsorbedplasmid DNA into gold particles Ballastically accelerated into body with gene gun.
- 42. HOW DNA VACCINEWORKS BY TWO PATHWAYS ENDOGENOUS :- Antigenic Protein is presented by cell in which it is produced EXOGENOUS :- Antigenic Protein is formed in one cell but presented by different cell
- 43. HOW DNA VACCINESWORK Muscle Cells Plasmid DNA +
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- 45. Multiply Memory T cells T-Helper Cell
- 46. EXOGENOUS PATHWAY Antigenic Proteincome outside
- 47. Phagocytosed Antigen Presenting Cell AntigenicPeptides T- Helper Cell Cytokines Activated B-Cell Memory B-Cell Plasma B-Cell Memory Antibodies MHC-II
- 48. WHEN VIRUS ENTERIN THE BODY Viral Protein Memory T-Cell Antibodies
- 49. ADVANTAGES Elicit both Humoral& cell mediated immunity Focused on Antigen of interest Long term immunity Refrigeration is not required Stable for storage
- 50. DISADVANTAGES Limited to proteinimmunogen only Extended immunostimulation leads to chronic inflammation Some antigen require processing which sometime does not occur
- 51. CURRENT CLINICAL TRIALS June2006,DNA vaccine examined on horse Horse acquired immunity against west nile viruses August 2007,DNA vaccination against multiple Sclerosis was reported as being effective
- 52. Genetic Toxicity Integration ofDNA vaccine into host Genome Insertional mutagenesis Chromosome instability Turn ON Oncogenes Turn OFF Tumor suppressor genes
- 53. Over Expression ofDNA vaccine Acute or chronic inflammatory responses Destruction of normal tisues
- 54. Generation of Autoimmunediseases Anti DNA Antibodies Autoimmune diseases Autoimmune Myositis
- 55. Antibiotic Resistance Plasmid usedis resistance to antibiotics for selection Raise the resistance to same antibiotic in the host
- 56. FUTURE PROSPECTS Plasmid withmultiple genes provide immunity against many diseases in one booster DNA vaccines against infectious diseases such as AIDS, Rabies, Malaria can be available
- 57. CONCLUSION DNA vaccines arein their early phase. There are no DNA vaccines in market at present. But this just the beginning . DNA vaccines are going to be the vaccines of next generation.