Manipulation of the immune response:

1. 2. Manipulation of the immune response: • Antigen specific • Immunostimulation • Immunosuppression • Non-antigen specific -Immunostimulation • -Immunosuppression • Immunomodulation

2. Targets of immunotherapies: elements of the immune response: cytokines, adhesion molecules, cell membrane molecules, receptors, antibodies Antibodies as drugs: specificity  antigenicity humanized antibodies, „targeting”

3. Immunotherapies Immunostimulation • Induction of immune response against • pathogenic microbes • Inducing immunreponse against tumor cells Immunosuppression -inhibition of autoimmun processes -inhibition of allergy -inducing transplantion tolerance -fight against newborn haemolytic aenemia Immunomodulation • shifting TH1 / TH2 balance in autoimmune diseases or allergy • -modulation of antibody isotype in allergy

4. Fighting against infections After infection  • Traditional drugs (antibiotics, anti-viral drugs) • Passive immunization (antibodies, cells) • Aktive vaccination Before infection  vaccination  1) increase the ratio of antigen specific cells 2) inducing specific immunological memory

5. IMMUNOSTIMULATION -vaccination Infectious diseases, epidemics Edward Jenner, 1796, smallpox „A landmark was discovery of the germ theory, which included small parasites, bacteria and viruses. This theory was mainly based on the studies of RobertKoch (1843-1910), Louis Pasteur(1822-1895) and many others.”

6. What will happen next.......

8. Vaccine approaches

9. Increase of the bodys’ specific protecting capability • Antigen specific immunostimulation: • Active immunization: • killed microbes • attenuated microbes • crossreactive microbe • non-pathogenic live microbes • non-toxic modified form • modified microbial toxin • Results: • antibody production + effector T cells • Adjuvants: support immunostimulation • concentrating ag • prolonged contact with ag • stimulation ofAPC

10. Passive immunization:rapid treatment of potentially fatal disease antigen specific IgG from hyperimmunized animal (or human) • advantage: immediate protection, but transient, disadvantage: no memory, • elimination of IgG, hypersensitivity, neutralization, • species specific immune response • application: antitetanic sera, snakebite • human IgG: antibody defficiency • Adaptive immunization: • therapy with immunocompetent cells • - immunodeficiency: • congenital cellular immunodeficieny • bone marrow (MHC compatibility!) or immunocompetent cells from fetal liver, thymus -enzyme deficiency: adenosin deaminase, • nucleosid phosphorylase • somatic gene therapy: bone marrow stem cells transfected with a viral vector containing the desired gene

11. Vaccination – antigen specific immunostimulation Vaccine: against the microbe, or toxin produced by the microbe Live, attennuated virus are more efficient compared to killed virus (effector mechanisms, CD8+T cells higher) – but: risk! New techniques: Recombinant DNA technologies Immunization with dendritic cells – new type vaccines • Attennuation of pathogenic microbes: • Culturing virus in monkey cells  mutations virus growth in monkey cells, butdoes not growth in human cells vaccine • In vitro mutagenesis:irreversible modification of virus gene • influensa- changes every year - antigén shift • directed mutagenezis

12. Attenuation of the pathogenic virus by culturing in non-human cells

14. Mechanisms of the changes of surface antigens on influensa virus – antigen-drift and antigen-shift human virus antigen drift lung epithel cells lung epithel cells human virus bird virus antigen shift lung epithel cells lung epithel cells Antigen drift: continous small changes in viral genes Antigen shift: genes from two different virus strains are mixed -> new virus

15. Antigenic Drift Each year’s flu vaccine contains three flu strains -- two A strains and one B strain -- that can change from year to year. After vaccination, your body produces infection-fighting antibodies against the three flu strains in the vaccine. If you are exposed to any of the three flu strains during the flu season, the antibodies will latch onto the virus’s HA antigens, preventing the flu virus from attaching to healthy cells and infecting them. Influenza virus genes, made of RNA, are more prone to mutations than genes made of DNA. If the HA gene changes, so can the antigen that it encodes, causing it to change shape If the HA antigen changes shape, antibodies that normally would match up to it no longer can, allowing the newly mutated virus to infect the body’s cells. This type of genetic mutation is called “antigenic drift.”

16. Pathogenic virus Mutation or deletion of virulence gene Immunogenic but avirulent virus -> vaccine

17. Development of non-pathogenic mutants: • Virus: polio, mumps, rubella, measles, etc. – deletion or mutation of gene (s) necessery for virulence • Bacterium: Salmonella typhy: non virulent mutants were selected • - UDP galactose epimerase enzyme mutation--LPS synthesis LPS low in mutants • -Targeting:enzyme Tyr, Phe syinthesis,  • slow proliferation - vaccination • chiken salmonella – vaccination important • Conjugate vaccine: B and helper T cells recognize different epitopes in the same molecular complex • Haemophilus influenzae B: • T cell-independent B cell response, to polysacharide chain of bacteria • tetanusz toxoid + polysacharide conjugate - > T dependent, efficient response even below two years age • Tetanusz toxoid specific T cells produce cytokines • B cell: antibody against bactaerial polysacharide

18. Haemophilus influenzae type B vaccine • Conjugate vaccine : • B and helper T cells recognize different epitopes in the same molecular complex

21. Reverse immunogenetics • Determination of T cell epitopes HLAB53 protects against fatal cerebral malaria. HLAB53–binding peptides are identified: nonapeptide with proline at position 2 From pathogen infected cells -> identification of the bound peptide with X Pro sequence Plasmodium falciparum Strong T cell proliferation Peptide -> terapy

22. Attennuated live microorganisms, as vector • combined vaccine: • Salmonella: tetanus toxoid ag, +Listeria monocytogenezis • Leishmania • Yersinia pestis • Schistosoma mansoni genes • virus: non-pathogenic (plant), – many genes in one • „microbe” as carrier: antigenebuilt in, • cannot be repeated • Synthetic peptides • identification of T cell epitopes,  peptidesynthesis • Disadvantage: variability • ISCOM: immune stimulatory complex • liposomes with peptides – sejtbe bejut

23. Immunstimuláló komplex peptiddel Fúzió Peptid transzport az ER -be Peptid bemutatása az MHCI-en keresztül a T sejtek számára

27. Succesful vaccinations SSPE stands for subacute sclerosing panencephalitis, a brain disease that is a late consequence of measles infection in a few patients.

33. Diseases for which effective vaccines are still needed. *The number of people infected is estimated at ~200 million, of which 20 million have severe disease. †Current measles vaccines are effective but heat-sensitive, which makes their use difficult in tropical countries. Estimated mortality data for 1999 from World Health Report 2000 (World Health Organization).

34. Types of virus infection polio, influenza, mumps, Yellow fever herpes, varicella, EBV HIV, hepatitis B, hepatitis C

35. Immune response after infection

36. Kinetics of antibody response

37. Targets of virus specific antibodies

38. PROBLEMS with vaccines: Specificity, isotype, localization of antibody response is not correct Antibody response does not provide protection Adaptation mechanisms of pathogens inhibit the immune reponse

39. Antibody production + citotoxic T cells activation –protection against the virus • DNA targeting: • ·      the right ligand, • ·      internalizationand direction to endosome • ·      fusion withlysosomes • ·      lysosomal enzymes degrade enzimek • Synthetic virus

40. Intranasal, intrarectal, intravaginal immunization mucosal immunization TL induction in Peyer plaques, lamina propria Adjuvants : pl. cholera toxin B: cAMP induction, IL-12 production suitable for mucosal immunization DNA based vaccination: Delivery: in vivo electroporation, gene gun  whole protein gene, or peptide MHCI, MHCII presentation Viral vector (vaccinia, poxvirus) strengthen the efficiency of rec DNA Nuclear transzlocation signál DNA plasmid ligand Endosomal lyzis, or bypass ? Tissue specific regulated promoter Therapic gene

42. epitope adjuvant Vaccine design delivery, site of delivery

43. Selection of the epitope APC T B

44. conformation N linear limited proteolysis C denaturation hidden linear determinant N N C C N C new determinant Selection of the epitope

45. Strategies to develop new generation of vaccines • Virus – do not express suitable T cell epitopes (selection during evolution) • Tumor – suitable T cells are deleted • „Epitope enhancement” • increase peptide- MHC binding affinity - MHCII –TH cells repair of anchoring aa. • combinatorial peptide libraries • peptide-MHC complex – increase TCR- binding affinity : activation of both small and large affinity T cells  • increase the number of T cells that recognize tumor epitope • increase TCR crossreactivity: peptid chimera – recognition of different virus strains

46. Design of new peptides Efficiency can be increased by modifying peptide sequence

47. To increase immunogenicity of antigen /carrier and adjuvant particularisation (Al-hidroxid/phosphate, liposome, virosome polimerisation ( mannose polimerek, MAP) emulsion (oil / water) microcapsula ( polymers degrading with different kinetics) bacterial products chemical adjuvants ( polinucleotides, CpG ) cytokines ( IL-2, IL-4, IL-6, IL-10, IL-12, GM-CSF, TNF-, IFN-) Targeting ( CR , FcR , MR, TLR ) VACCINE DESIGN

48. VACCINE TYPES live, attennuated killed / subunit DNA / RNA TYPE OF IMMUNE RESPONSE Antibody mediated B- cells +++ +++ +++ cellular CD4+ T-cells +/- TH1 +/-TH1* +++ TH1* CD8+ T-sejtek +++ - ++ antigen presentation MHC I / II MHC II MHC I / II memory humoral +++ +++ +++ sejtes +++ +/- ++ production Difficulty of development + ++ ++++ price + + +++ Transportation storage + +++ +++ safety ++ ++++ +++ COMPARISON OF VARIOUS TYPES OF VACCINES

49. AGE memory naive age naive memória TOXICITY Effect of adjuvant (TH1/TH2) autoimmuny (molecular mimicri) impurities (virus, prion) build into genom / activation of oncogenes

50. The efficient vaccine: - safe –can be applied to everyone including children - efficient to protect against infection or disease (less efficient) - protection for life long – memory - induce neutralizing antibodies - induce T cell response - stabile, cheap, no/few side effect - easy applicable (oralis vaccine, e.g. Sabin dropp) - acceptable and applicable everywhere (developing world) Adjuvants:non-specific signal, stimulation of APC cytokine induction, antigén-depo: slow felszívódás aluminium hidroxid, or oil emulsion. mixed vaccines - one can activate the other ( e.g. Bordatella Pertussis+ tetanus+difteria) cytokines : IL-12 - TH1 response The way of immunization Important: the site of infection/ immunizations Oral vaccines – the role of mucosa (MALT)