1. 1 Vaccines Successes of the Past Possibilities for the Future www.freelivedoctor.com

2. Vaccines Immunity to viral infections usually depends on the development of an immune response to Antigens on the virus surface Antigens on the virus-infected cell In most cases response to internal proteins has little effect on humoral immunity to infection Humoral antibodies can be important diagnostically (HIV) www.freelivedoctor.com

3. 3 Vaccines Minor role for internal proteins can be seen in influenza pandemics New flu viral strain contains a novel glycoprotein Pandemic virus contains internal proteins to which the population has already been exposed Nevertheless the CTL response to internal proteins is important Surface glycoprotein = protective immunogen which must be identified for a logical vaccine www.freelivedoctor.com

4. 4 Vaccines Some viruses have more than one surface protein Influenza (Orthomyxovirus) Hemagglutinin - attaches virus to cell receptor Neuraminidase - involved in release of virus from cell Hemagglutinin is major target: stimulates neutralizing antibody www.freelivedoctor.com

5. 5 Vaccines Neutralization may result from: Binding of antibody to site on virus surface - block interaction with receptor Aggregation of virus by polyvalent antibody Complement-mediated lysis www.freelivedoctor.com

6. 6 Vaccines Addition points to note: Site in body at which virus replicates Three major sites for viral replication www.freelivedoctor.com

7. 7 Three major sites for viral replication Mucosal surfaces of respiratory tract and GI tract. Rhino; myxo; corona; parainfluenza; respiratory syncytial; rota Infection at mucosal surfaces followed by spread systemically via blood and/or neurones to target organs: picorna; measles; mumps; HSV; varicella; hepatitis A and B Direct infection of blood stream via needle or bites and then spread to target organs: hepatitis B; alpha; flavi; bunya; rhabdo Local immunity via IgA very important in 1 and 2. www.freelivedoctor.com

8. 8 There is little point in having a good neutralizing humoral antibody in the circulation when the virus replicates, for example, in the upper respiratory tract. Clearly, here secreted antibodies are important. Although in the case of influenza serum antibodies may be important www.freelivedoctor.com

9. 9 Vaccines - Problems Different viruses may cause similar disease--e.g. common cold Antigenic drift and shift -- especially true of RNA viruses and those with segmented genomes Shift: reassortment of segmented genomes (‘flu A but not rota or ‘flu B) Drift: rapid mutation - retroviruses Large animal reservoirs - Reinfection may occur www.freelivedoctor.com

10. 10 Vaccines - Problems Integration of viral DNA. Vaccines will not work on latent virions unless they express antigens on cell surface. In addition, if vaccine virus integrates it may cause problems Transmission from cell to cell via syncytia Recombination of the virulent strain or of the vaccine virus www.freelivedoctor.com

11. 11 Smallpox Mummies China/India Crusaders W Europe: fatality rate 25% History changed: Cortes Louis XIV www.freelivedoctor.com

12. 12 Smallpox Variolation 1% v. 25% mortality Life-long immunity: No drift or shift (proof reading) UK: 1700’s China 1950 Pakistan/Afghanistan/Ethiopia 1970 www.freelivedoctor.com

13. Smallpox Vaccination Jenner 1796 : Cowpox/Swinepox 1800’s Compulsory childhood vaccination 1930’s Last natural UK case 1940’s last natural US case 1958 WHO program October 1977: Last case (Somalia) www.freelivedoctor.com

14. Smallpox No animal reservoir Lifelong immunity Subclinical cases rare Infectivity does not precede overt symptoms One Variola serotype Effective vaccine Major commitment by governments www.freelivedoctor.com

15. 15 Small RNA virus Some drift…but not too far as non-viable US: Sabin attenuated vaccine ~ 10 cases vaccine-associated disease per year 50% vaccinees feces 50% contacts Vaccine-associated cases: revertants 1 in 4,000,000 vaccine infections paralytic polio 1 in 100 of wt infections Scandinavia: Salk dead vaccine No gut immunity Cannot wipe out wt virus Polio Vaccine www.freelivedoctor.com

16. 16 Sabin Polio Vaccine Attenuation by passage in foreign host More suited to foreign environment and less suited to original host Grows less well in original host Polio: Monkey kidney cells Grows in epithelial cells Does not grow in nerves No paralysis Local gut immunity (IgA) Pasteur rabies vaccine also attenuated www.freelivedoctor.com

17. 17 Salk Polio Vaccine Formaldehyde-fixed No reversion www.freelivedoctor.com

18. 18 Polio Vaccine Why use the Sabin vaccine?: Local immunity: Vaccine virus just like natural infection Stopping replication in G.I. Tract stops viral replication TOTALLY Dead Salk vaccine virus has no effect on gut replication No problem with selective inactivation Greater cross reaction as vaccine virus also has antigenic drift Life-long immunity www.freelivedoctor.com

19. 19 Polio Vaccine New CDC Guidelines Last US natural (non-vaccine associated) case was 15 years ago 2 does injectable (Salk) vaccine 2 doses oral Vaccine cases 1 in 3 million does New strategy will prevent about 5 of the 10 vaccine-associated cases (the five found in vaccinees) Cost $20 million Savings from eradication $230 million www.freelivedoctor.com

20. 20 New Recommendations To eliminate the risk for Vaccine- Associated Paralytic Poliomyelitis, the ACIP recommended an all-inactivated poliovirus vaccine (IPV) schedule for routine childhood polio vaccination in the United States. As of January 1, 2000, all children should receive four doses of IPV at ages 2 months, 4 months, 6-18 months, and 4-6 years. www.freelivedoctor.com

21. 21 Vaccines Activates all phases of immune system. Can get humoral IgG and local IgA Raises immune response to all protective antigens. Inactivation may alter antigenicity. More durable immunity; more cross- reactive Advantages of Attenuated Vaccines I www.freelivedoctor.com

22. 22 Vaccines Low cost Quick immunity in majority of vaccinees In case of polio and adeno vaccines, easy administration Easy transport in field Can lead to elimination of wild type virus from the community Advantages of Attenuated Vaccines II www.freelivedoctor.com

23. 23 Vaccines Disadvantages of Live Attenuated Vaccine Mutation; reversion to virulence (often frequent) Spread to contacts of vaccinee who have not consented to be vaccinated (could also be an advantage in communities where vaccination is not 100%) Spread vaccine not standardized--may be back-mutated Poor "take" in tropics Problem in immunodeficiency disease (may spread to these patients) www.freelivedoctor.com

24. 24 Vaccines Advantages of inactivated vaccines Gives sufficient humoral immunity if boosters given No mutation or reversion Can be used with immuno-deficient patients Sometimes better in tropics Disadvantages of inactivated vaccines Many vaccinees do not raise immunity Boosters needed No local immunity (important) Higher cost Shortage of monkeys (polio) Failure in inactivation and immunization with virulent virus www.freelivedoctor.com

25. 25 New Methods Selection of attenuated virus strain Varicella Hepatitis A Use monoclonal antibodies to select for virus with altered surface receptor Rabies Reo Use mutagen and grow virus at 32 degrees. Selects for temperature-sensitive virus. Grows in upper respiratory tract but not lower ‘flu (new vaccine) respiratory syncytial virus www.freelivedoctor.com

26. 26 New Methods Recent ‘flu vaccine from Aviron Passage progressively at cold temperatures TS mutant in internal proteins Can be re-assorted to so that coat is the strain that is this years flu strain www.freelivedoctor.com

27. 27 New Methods Deletion mutants Suppression unlikely (but caution in HIV) Viable but growth restrictions Problems Oncogenicity in some cases (adeno, retro) www.freelivedoctor.com

28. 28 New Methods Recombinant DNA Single gene (subunit) S-antigen mRNA cDNA Express plasmid S-antigen mRNA protein Hepatitis B vaccine raised in yeast www.freelivedoctor.com

29. 29 Single gene (subunit) - problems Surface glycoprotein poorly soluble - deletion? Poorly immunogenic Post-translational modifications Poor CTL response www.freelivedoctor.com

30. 30 Single gene (subunit) in expression vector Vaccinate with live virus Canary Pox Infects human cells but does not replicate Better presentation CTL response Vaccinia Attenuated Polio Being developed for anti-HIV vaccine www.freelivedoctor.com

31. 31 New Methods Chemically synthesized peptide malaria poorly immunogenic www.freelivedoctor.com

32. 32 antibody New methods Anti-idiotype vaccine epitope Antibody with epitope binding site Virus www.freelivedoctor.com

33. 33 antibody Anti-idiotype vaccine cont Make antibody against antibody idiotype Anti- idiotype antibody Anti-idiotype antibody mimics the epitope www.freelivedoctor.com

34. 34 Anti-anti-idiotype antibody Anti-idiotype antibody cont 2 Use anti-idiotype antibody as injectable vaccine Antibody to anti-idiotype antibody Binds and neutralizes virus Anti-idiotype antibody Anti-anti-idiotype antibody Use as vaccine www.freelivedoctor.com

35. 35 New Methods New “Jennerian Vaccines” Live vaccines derived from animal strains of similar viruses Naturally attenuated for humans Rotavirus: Monkey Rota 80% effective in some human populations Ineffective in others Due to differences in circulating viral serotypes www.freelivedoctor.com

36. 36 New Methods New Jennerian Vaccines Bovine parainfluenza Type 3 Bovine virus is: Infectious to humans Immunogenic (61% of children get good response) Poorly transmissable Phenotypicaly stable www.freelivedoctor.com

37. 37 New Methods Second Generation Jennerian Vaccines Rotavirus 11 segments of double strand RNA Two encode: VP4 (hemagglutinin) VP7 (glycoprotein) Co-infect tissue culture cells reassortment 10 segments from monkey rotavirus 1 segment outer capsid protein of each of four major rotavirus strains Efficacy >80% Elicit neutralizing antibodies www.freelivedoctor.com

38. 38 Vaccines 1796 Jenner: wild type animal-adapted virus 1800’s Pasteur: Attenuated virus 1996 DNA vaccines The third vaccine revolution www.freelivedoctor.com

39. 39 DNA Vaccines plasmid Muscle cell Gene for antigen Muscle cell expresses protein - antibody made CTL response www.freelivedoctor.com

40. 40 DNA Vaccines Plasmids are easily manufactured in large amounts DNA is very stable DNA resists temperature extremes so storage and transport are straight forward DNA sequence can be changed easily in the laboratory. This means that we can respond to changes in the infectious agent By using the plasmid in the vaccinee to code for antigen synthesis, the antigenic protein(s) that are produced are processed (post-translationally modified) in the same way as the proteins of the virus against which protection is to be produced. This makes a far better antigen than purifying that protein and using it as an immunogen. www.freelivedoctor.com

41. 41 DNA Vaccines Mixtures of plasmids could be used that encode many protein fragments from a virus/viruses so that a broad spectrum vaccine could be produced The plasmid does not replicate and encodes only the proteins of interest No protein component so there will be no immune response against the vector itself Because of the way the antigen is presented, there is a CTL response that may be directed against any antigen in the pathogen. A CTL response also offers protection against diseases caused by certain obligate intracellular pathogens (e.g. Mycobacterium tuberculosis) www.freelivedoctor.com

42. 42 DNA Vaccines Possible Problems Potential integration of plasmid into host genome leading to insertional mutagenesis Induction of autoimmune responses (e.g. pathogenic anti-DNA antibodies) Induction of immunologic tolerance (e.g. where the expression of the antigen in the host may lead to specific non-responsiveness to that antigen) www.freelivedoctor.com

43. 43 DNA Vaccines DNA vaccines produce a situation that reproduces a virally- infected cell Gives: Broad based immune response Long lasting CTL response Advantage of new DNA vaccine for flu: CTL response can be against internal protein In mice a nucleoprotein DNA vaccine is effective against a range of viruses with different hemagglutinins www.freelivedoctor.com

44. 44 Towards an anti-HIV Vaccine Questions: For a vaccine what are the measures of protection? Can we overcome polymorphism? What are the key antigens? Attenuated or killed or neither? Mucosal immunity critical? Prevent infection or prevent disease? Animal models How does HIV kill cells anyway? www.freelivedoctor.com

45. 45 Towards an anti-HIV Vaccine What should vaccine elicit? Humoral response neutralizing antibody kill free virus Cellular response kill infected cells problem of cell-cell infection www.freelivedoctor.com

46. 46 Towards an anti-HIV Vaccine Early faith in neutralizing antibodies backed by chimpanzee experiments HIV high levels of neutralizing antibody Can resist subsequent challenge by virus injected I.V. !!!! But not via rectum or vagina But chimps do not get AIDS www.freelivedoctor.com

47. 47 Towards an anti-HIV Vaccine Chimp studies designed for success Animals challenged with small doses of virus at moment that antibody levels high (virus --not infected cells!) Challenge virus same strain as that used to induce antibody No vaccine made from one virus strain has protected chimps from another virus strain Protection in man may not result from neutralizing antibodies at all Ability to raise neutralizing antibodies in monkeys does not correlate with protection Cell-mediated immunity is the key This is also key in humans HIV-exposed but not infected people shows signs of a cell-mediated response www.freelivedoctor.com

48. 48 Towards an anti-HIV Vaccine Since 1986: > 15 SUBUNIT VACCINES Based on gp160/gp120 All safe None effective Low levels of strain-specific antibodies that quickly disappear Only ephemeral effects of cell-mediated immunity All done with gp160/gp120 of syncytium-inducing virus None tested on large groups of high risk people www.freelivedoctor.com

49. 49 Towards an anti-HIV Vaccine A Classical Approach? December 1992: Live attenuated SIV vaccine protected all monkeys for 2 years against massive dose of virus All controls died cell mediated immunity was key www.freelivedoctor.com

50. 50 Towards an anti-HIV Vaccine Humans: NEF deletion mutant www.freelivedoctor.com

51. 51 Towards an anti-HIV Vaccine Live attenuated: Pro: SIV with NEF deletion protects after ONE immunization Long lived cell-mediated and humoral immunity Possible herd immunity Con: Safety in immunodeficient people LTR Reversion Need multiple strains: polymorphism www.freelivedoctor.com

52. 52 Towards an anti-HIV Vaccine Inactivated: Pro: Simple Mimics natural infection Protects against systemic and rectal challenge No reversion Con: Polymorphism LTR Inactivation failure www.freelivedoctor.com

53. 53 Towards an anti-HIV Vaccine Subunit vaccine: Pro: Safety Con: Ephemeral humoral response Little cell mediated response www.freelivedoctor.com

54. 54 Towards an anti-HIV Vaccine Subunit in vector Pro: Potent cell-mediated immunity www.freelivedoctor.com

55. 55 Towards an anti-HIV Vaccine Problems for all vaccines: Enhancing antibody Vaccine may be immunosuppressive (anti- MHC) www.freelivedoctor.com

56. Towards an anti-HIV Vaccine Summary of problems: Virus can hide in cells Cell-cell transmission Ethical problems Lack of animal models Immuno-silent sugars Polymorphism/hypervariability: DRIFT Activation of same cells that virus infects Useless if T4 cells are depleted Blood brain barrier Oncogenicity www.freelivedoctor.com