1. VACCINES N7-2006 L. Duroux Slides assembled from diverse sources

2. Lecture Plan 1. Introduction 2. The Immune System 3. Subunit & Peptide Vaccines 4. Attenuated Vaccines 5. Vector Vaccines

3. 1. INTRODUCTION

4. Discovery of Vaccination  Discovered in 1796 by Dr. Edward Jenner  Tested empirical knowledge: mild cattle disease cowpox protects against deadly human disease smallpox  Inoculated 8-years-old boy with exudate from cowpox pustule: full protection against smallpox

5. Chicken ! BITCH !

6. 2. The Immune System

7. Function of the Immune System (Self/Non-self Discrimination)  To protect from pathogens Intracellular (e.g. viruses and some bacteria and parasites) Intracellular (e.g. viruses and some bacteria and parasites) Extracellular (e.g. most bacteria, fungi and parasites) Extracellular (e.g. most bacteria, fungi and parasites)  To eliminate modified or altered self

8. The Invaders...  Bacteria  Viruses  parasites such as fungi, protista, & worms

9. Our 1 st Line of Defense...  The Integumentary System… Skin Skin Mucous membranes Mucous membranes Mucous Mucous  provides a physical barrier preventing microbial access

10. Other mechanisms of Defense...  Physiological variables pH of our environment pH of our environment temperature of our environment temperature of our environment  chemical defenses nitric oxide, enzymes, proteins… nitric oxide, enzymes, proteins…  AND the IMMUNE SYSTEM…

11. Overview of the Immune System Immune System Innate (Nonspecific) Cellular ComponentsHumoral Components Adaptive (Specific) Protects/re-exposure Cellular ComponentsHumoral Components Interactions between the two systems

12. Innate Immunity Adaptive Immunity Comparison of Innate and Adaptive Immunity No memory No memory No time lag Not antigen specific A lag period Antigen specific Development of memory

13. Cells of the Immune System Immune System Myeloid Cells Granulocytic Neutrophils Basophils Eosinophils Monocytic Macrophages Kupffer cells Dendritic cells Lymphoid Cells T cells Helper cells Suppressor cells Cytotoxic cells B cells Plasma cells Natural Killer cells

14. Development of the Immune System myeloid Granulocyte lymphoid nk thymus CD8 + CD4 + CTL TH2 TH1 Monocyte B-Cells

15. What Happens during an infection?  Innate Immunity - the troops are called to battle… injury & infection injury & infection macrophages slip between cells [extravasation] to arrive macrophages slip between cells [extravasation] to arrive cytokine chemicals attract other “troops” [chemotaxis] cytokine chemicals attract other “troops” [chemotaxis] histamine chemicals dilate blood vessels for easier access to injury [vasodilatation] histamine chemicals dilate blood vessels for easier access to injury [vasodilatation]

16. What are macrophages ?  Phagocytic cells - able to ingest small foreign invaders neutrophils neutrophils monocyte monocyte  they release cytokines that enhance the immune response

17. Macrophages  Mast cells /basophils release histamine that dilates blood vessels release histamine that dilates blood vessels causes redness [erythrema], swelling [edema], and heat [fever] causes redness [erythrema], swelling [edema], and heat [fever]

18. Summary on Macrophages  Macrophages are able to launch the first strike…  more help is needed to overcome rapidly reproducing invaders…  Help from the ADAPTIVE IMMUNE System results in a coordinated successful defense !  Major players... the B lymphocytes

19. The Adaptative Immune System  There are 2 types of lymphocytes: T lymphocytes [ T - Helper cells ] - help signal immune cells into action T lymphocytes [ T - Helper cells ] - help signal immune cells into action B lymphocytes [ B cells ] - make special proteins called antibodies B lymphocytes [ B cells ] - make special proteins called antibodies

20. T-lymphocytes migrate to the thymus gland...  These Lymphocytes are sorted into 2 types  Identification tag is a protein called Major Histocompatability Complex [MHC] Self- IDForeign

21. & in the thymus gland...  All diversely varying MHC lymphocytes will wait for a call to action...  These Lymphocytes will mature into T-Helper cells  They function to stimulate B cells to activate their attack against the invaders Self- ID Foreign Saved to be educated… in body defense Dropped out!

22. Adaptive Immune System  T he 2nd type of lymphocyte is: B lymphocytes [ B cells] - start in the bone marrow and circulate through the body B lymphocytes [ B cells] - start in the bone marrow and circulate through the body they are called into action when stimulated by a foreign antigen... [ usually a protein from the invader] they are called into action when stimulated by a foreign antigen... [ usually a protein from the invader]

23. When an invader attacks...  An antigen is phagocytized by the B cell  is broken into non-infective pieces  & attached to the cell’s MHC when processed through the cell’s machinery  MHC-antigen complex is placed on the cell membrane surface  where it is recognized by the T Helper cell

24. When help arrives...  The T-helper cell receptor “docks” with the B cell’s MHComplex  B cells proliferate... Antigen & T-helper cell Proliferation of cell line Naïve cell

25. B cells differentiate into... Antibody producing cells [attack mode] Antibody producing cells [attack mode] Memory cells [remembers & future protection] Memory cells [remembers & future protection] Antigen & T-helper cell memory antibodies

26. The RESULT...  The Antibody producing B cells mounts a successful attack against the invader  the memory B cells save the “recognition ID” for many years in preparation for future invasion

27. 3. Principles of Vaccination

28. Principles of Vaccination  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.

29.  Old Technology: Grow in animals (vaccinia in calves for smallpox; rabbit brains for rabies) Grow in animals (vaccinia in calves for smallpox; rabbit brains for rabies) Simple bacterial culture (Cholera vibrio) then inactivation Simple bacterial culture (Cholera vibrio) then inactivation Grow in eggs (influenza, vaccinia) then inactivate Grow in eggs (influenza, vaccinia) then inactivate >100 million eggs used for influenza in the USA every year Vaccine Technology

30. Limitations To Traditional Vaccines 1. can’t grow all organisms in culture 2. safety to lab personnel 3. Expense 4. insufficient attentuation 5. reversion to infectious state 6. need refrigeration 7. do not work for all infectious agents 8. infants/children receive them – immature immunity

31. Recombinant Vaccines 1. Subunit Vaccines peptide vaccines Genetic immunization 3. Attenuated Vaccines 4. Vector Vaccines 5. Bacterial Antigen Delivery Systems

32. New Generation of Vaccines  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.

33. Recombinant Vaccines 1. Delete Virulence Genes (can not revert) V/B as Vaccine 2. Clone gene for pathogenic antigen into non-pathogenic virus or bacteria V/B as Vaccine 3. Clone pathogenic antigen gene into expression vector A. Vaccinate with ‘protein’ 1. Subunit 2. Peptide

34. New Generation of Vaccines  There are three types of vaccines we will be discussing:  Subunit (protein) vaccines  Attenuated vaccines  Vector vaccines

35. Vaccine Technology

36. 4. Subunit / Peptide Vaccines

37. 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

38. Structure of a Virus particle

39. Subunit vaccines born from following observation  It has been showed that the capsid or envelope proteins are enough to illicit 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.

40. A Subunit Vaccine for M. tuberculosis  Tuberculosis is caused by Mycobacterium tuberculosis.  The bacterium form lesions 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.

41. A Subunit Vaccine 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.

42. Selection & delivery of vaccine peptides  Antigenic determinants = epitopes on envelope proteins  Inert carrier: hemocyanin from keyhole limpet  Highly immunogenic carrier: Hepatitis B core prot.

43. 5. Attenuated Vaccines

44. 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.

45. A Live Cholera Vaccine  The causal agent of cholera is Vibrio cholerae and is transmitted through contaminated water.  V. cholerae produces a enterotoxin with an A subunit and 5 B subunits.  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.  In the sequence of the A peptide a tetracycline resistance gene is inserted.

46. A Live Cholera Vaccine  A plasmid with A peptide was digested with 2 restriction enzymes Cla1 and Xba1.  This removes 550 bases of A peptide.  A Xba1 linker was added and T4 ligase used to ligate the DNA. This plasmid was mixed with V. cholerae with tetracycline resistant gene.  By conjugation the plasmid was transferred to the strain with the tet R gene inserted into it’s chromosomal DNA.

47. Production of a Live Cholera Vaccine

48. A Live Cholera Vaccine  By recombination the A peptide with the tet R gene was replaced by the deleted A peptide.  The final result is V. cholerae with a 550 bp of the A peptide deleted.  If this can be used as a vaccine is being tested.

49. Production of a Live Cholera Vaccine

50. 6. Vector Vaccines

51. Vector Vaccine  A vector vaccine is a vaccine which is introduced by a vector e.g. vaccinia virus.  The vaccinia virus as a live vaccine led to the globally eradication of the smallpox virus.  The genome of the vaccinia virus has been completely sequenced.  The virus replicates in the cytoplasm rather than in the nucleus.  The vaccinia virus is generally nonpathogenic.

52. Vector Vaccine  These characteristics makes the vaccinia virus a good candidate for a virus vector to carry gene for antigenic determinants form other pathogens.  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.

53. Vector Vaccine  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.

54. Insertion of antigen gene into vaccinia virus genome by recombination

55. Vector Vaccine  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.