Fundamentals of Biotechnology

1. Fundamentals of Biotechnology Vaccines

2. Immunization • 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. • In 1796 by Dr. Edward Jenner protect the James Phipps against Small Pox.

3. Immunization (cont’d) • When performed before exposure to an infectious agent (or soon after exposure in certain cases), it is called immunoprophylaxis, • intended to prevent the infection. • When performed during an active infection (or existing cancer), it is called immunotherapy, intending to cure the infection (or cancer)

4. Types of Immunity • Two mechanisms by which immunization can be achieved • Passive immunization: • Protective Abs --> non immune recipient • No immunological memory w/o Th cells. • Active immunization: • Induction of adaptive immune response, with protection and memory.

5. Passive and 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

6. Passive Immunity Naturally- transplacental transfer of maternal IgG Abs to developing fetus; transfer of IgG + IgA Abs in milk during breast-feeding of newborn Medically - injection of immune globulin Performed prophylactically, either after diagnosis of exposure to toxin/virus or as a short term preventive procedure, e.g. if one is traveling to an endemic area Blocking- prevent hemolytic anemia of the newborn: Rhogam injected into pregnant Rh- mother prior to delivery of each baby conceived with Rh+ father.

7. Active Immunization Naturally- following exposure to an infection Medically- by vaccination: Performed either by i.m. injection of killed or attenuated antigens (often with adjuvant) or by ingestion of attenuated live organisms. Blocking - Reversal of auto-immune response Anti-cancer- Reactivation of tumor-stimulated T lymphocytes.

8. Mechanism of Vaccination Establish resistance to virus/pathological organism by evoking an immune response 1. Give host a foreign organism/protein in non-infectious form 2. Antibodies are generated Ab binds to surface proteins of organism

9. Structure of a Virus particle

10. Traditional I. Types A. Inactivated (Killed) B. Live C. Attentuated (Live, Non-infectious) LIVE MORE EFFECTIVE THAN KILLED II. Pathogens A. Bacteria B. Virus C. Parasites

11. Types of Vaccines • Attenuated– live microbe (usually virus) which has a vital function inactivated by heat, chemicals or genetic manipulation • e.g. Rabies virus vaccine, MMR (Measles, Mumps and Rubella) • BCG (Bacillus Calmette Guerin vaccine for Mycobacterium tuberculosis • Risk it could revert back to infectious agent • will stimulate both cell mediated and antibody mediated immune responses

12. Types of Vaccines (cont’d) • Inactivated– uses toxoid – inactivated toxins which are purified proteins • stimulates the antibody mediated response only • e.g. DPT (diphtheria, pertussis, tetanus toxoids) • stimulates the antibody mediated response only

13. Types of Vaccines (cont’d) • Component (subunit)– contains purified components from bacteria and viruses • How recombinant viruses are made • Hepatitis B vaccine – purified viral coat protein • Streptococcus pneumoniae(PneumoShot) – capsular polysaccharide • Hemophilusinfluenzae(HiB) – capsular polysaccharide, part of DPTPolio- • Hib vaccine given to infants • Nesseriameningiditis– capsular polysaccharide • stimulates the antibody mediated response only

14. Other vaccinations/components • Booster Shots: same vaccine given at a later date (e.g. DT given every 10 years • to refresh the memory cell population • 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

15. Antibody Titer • A test to measures the presence and amount of antibodies in blood against a particular type of tissue, cell, or substance • Titer determines if you have adequate protection against a disease • May need to give booster if titer too low • e.g., happens with HepB vaccine

16. Possible Limitations of Traditional Vaccine Production • Not all infectious agents can be grown in culture • Animal/human cell culture expensive if needed • Yield of viruses from cultures can be low • Safety precautions for culture of live agents • Insufficient killing/attenuation of agents • Reversion of attenuated agents • Traditional vaccines don’t work for all agents • Traditional vaccines may have shorter shelf-lives (storage problems)

17. New Strategies • Delete virulence genes • Reversion nearly impossible • Use live nonpathogenic carriers for immunization (unrelated pathogenic agent) • Clone antigenic determinants into alternative host systems for production (non cultureable) • Address autoimmune system response/problems • Kill infected cells that wouldn’t naturally die • Target killing system with fusion proteins

18. Human Diseases for Which Recombinant Vaccines Are Currently Being Developed

19. Recombinant Vaccines Recombinant Vaccines • Subunit Vaccines • peptide vaccines • Genetic immunization • 3. Attenuated Vaccines • 4. Vector Vaccines • 5. Bacterial Antigen Delivery Systems

20. Subunit vaccines • Do NOT use entire virus or bacteria (pathogenic agent) • Use components of pathogenic organism instead of whole organism • Advantage: no extraneous pathogenic particles ie DNA • Disadvantage: Is protein same as in situ? • Cost

21. Examples of Subunit Vaccines • A. HSV • Problem with Traditional vaccine- HSV is oncogenic • envelope glycoprotein D (gD) elicits Ab response • Clone gene for gD into vector • Express in mammalian cells

22. Transmembrane protein • modify gene to remove TM portion

23. Subunit Vaccine Development • Clone HSV glycoprotein gD gene • Transfect into chinese hamster ovary (CHO) cells • Isolate secreted glycoprotein • Purify and use as vaccine • Protects mice from HSV

24. Other Subunit Vaccines B. Tuberculosis Mycobacterium tuberculosis antibiotic resistant strains use purified extracellular (secreted) proteins as Vaccine C. Foot -and-Mouth Disease virus (FMDV) cattle/pigs VP1 capsid viral protein elicits response - used this protein as Vaccine

25. Peptide Vaccines Use discrete portion (domain) of a surface protein as Vaccine These domains are ‘epitopes’ antigenic determinants are recognized by antibodies

26. FMDV peptide vaccine Problem: Large quantities of peptide needed to be used to get immunological response Solution: Use highly immunogenic carrier molecule HBcAg was a suitable carrier (Hepatitis Core Protein) Fused peptide DNA with gene for HBcAg This fusion protein used as Vaccine

27. Peptide Vaccines • Peptides generally not good antigens by themselves • Improved antigenicity when attached to larger carrier protein • Highly antigenic protein such as hepatitis B core protein works very well as a carrier • Up to 500X better than peptide alone

28. Peptide Limitations • Epitopes must be continuous run of amino acids • Peptide must assume same conformation as it would as part of complete protein • Sometimes single epitopes do no elicit sufficient immunological response • Posttranslational modifications???

29. Yeast Retroposon Ty p1 Protein • Another potentially useful carrier • Studied using Plasmodium falciparum (malaria) • Sometimes subunit vaccines with different carriers used in concert provide best immunity

30. Genetic Immunization Delivery of a gene for the antigen to a host organism Use vector containing cDNA from viral protein/ eukaryotic promoter Inject into muscle/microprojectile system POTENTIAL eliminates purification of antigen protein is modified post-translationally

31. DNA Vaccines • Genetic immunization • Gold projectiles coated with influenza A gene biolistically delivered into mouse ears • No response to vector • Only expressed gene • Can therefore be reused repeatedly

32. Shigella-based Delivery Systems • Shigella can enter cells without phagocytosis • Directs plasmids to cytoplasm (with inserted genes) • Provides immunity to expressed antigens

33. Microparticle DNA Delivery • DNA adheres to cationic surface • Can be injected into tissue and DNA slowly released over several days • Gives prolonged though transient expression by cells • Provides up to 250-fold better effect as compared to “naked” DNA

34. Advantages of Genetic Immunization Over Conventional Vaccines

35. Rhesus Monkey Immunization Against Simian Immunodeficiency Virus • DNA constructs for expression of SIV proteins injected at 0 and 8 weeks • Booster of recombinant vaccinia virus expressing same proteins at 24 weeks • Immunity to SIV infection through mucosal tissues at 7 months post booster

36. Attenuated vaccines • Genetic manipulation of carrier (nonpathogenic) or pathogenic one • Cholera • caused by bacterium • lives in intestine causing diarrhea, dehydration • poor sanitation (water supply, sewage) • secretes an enterotoxin (A1) which causes disease • killed vaccine not effective long-term • subunit not effective • Phenol-killed cholera used as vaccine currently • Typically double deletions are preferred • can not multiple in host

37. Cholera Toxin • B subunit for binding to membrane receptor site • A2 peptide acts as connector between B and A1 subunits • A1 peptide is the enzymatically active component which modifies target G protein, locking adenylate cyclase in on position in intestinal mucosal cells

38. Cholera Vaccines A. Insert tetracycline gene into bacterium’s host chromosome This gene interrupts A1 peptide gene (toxic portion of the enterotoxin) NOT ACCEPTABLE Reversion by spontaneous excision B. Deleted A1 peptide sequence created

39. Deletion-attenuated Toxin Gene • Clone toxin gene • Cut out internal • segment • Ligate using linker • Conjugate into wt • cell • Recombination • Chromosomal gene • now attenuated by • deletion

40. 550 bp removed Plasmid will eventually be lost Bacterium will be tet sensitive

41. Vector Vaccines:Virus as Antigen Gene Delivery System Antigen Gene Virus Patient Antigen Protein is Made

42. Vector Vaccines • Use one organism to express antigens from another pathogenic organism • Vaccinia virus a good candidate vector • Broad host range • Well-characterized and benign • But spreads like… • Large ds DNA genome (187 kb) • Replicates and expressed in cytoplasm • brings own DNAP, RNAP, etc. • New genes introduced by in vivo recombination

43. Vector vaccines development • Insert cloned gene encoding antigen • B) Interrupt thymidine kinase (non-essential gene) • C. Infect host cell with native virus • D) Transform these cells with recombinant plasmid • E) HOMOLOGOUS RECOMBINATION • F) Select cells which are resistant to • BROMODEOXYURIDINE • **MODIFIED VIRUS USED AS VACCINE**

44. Recombinant Vaccinia Virus • Can insert more than one antigen gene

45. Vaccines Against Bacteria • Not all bacterial diseases treatable by antibiotics • Resistance is proliferating to many antibiotics • Antibiotics usually require refrigeration • Patients don’t always complete antibiotic regimen

46. Tuberculosis • 2 billion persons currently infected • 2-3 million deaths/yr • Antibiotics work but resistant strains becoming more common • Attenuated BCG strain of Mycobacterium bovis developed in early 1900’s used for immunization • But infects immunocompromised individuals • Gives false positive for standard tuberculosis test

47. Subunit Vaccines to Tuberculosis • Isolate proteins secreted into culture medium • Identify which induce protection • Construct subunit vaccine(s) based on promising antigens

48. Improving BCG Strain • E. coli/Mycobacterium shuttle vector • Introduce identified antigen gene into expression module • Vaccine was more potent than traditional one

49. Bacterial Antigen Delivery Systems:Bacterial Vectors Antigen Gene Bacterium Antigen Proteins made on Bacterial cell Vaccinate Patient

50. Bacterial Antigen Delivery Systems • Use live nonpathogenic bacterium which contains antigen • Insert antigen gene into flagellin gene • Epitope is expressed on the flagellum surface • ***Flagellin-engineered bacteria is VACCINE** • Advantage - Oral Administration