7 th SEMINAR

1. 7th SEMINAR VACCINATION, POLYCLONAL AND MONOCLINAL ANTIBODIES

2. THE FIRST VACCINEEdward Jenner - 1796 He proved that a vaccination with a weak pathogen isolated from another specie (cowpox) can grant immunity against a similar but dangerous human pathogen (smallpox) Thanks to the vaccination campaign of the WHO smallpox infection rate reached zero in 1976. Then, in 1979, years after the last registered case smallpox was declared eradicated.

3. IMMUNIZATION WITH ATTENUATED (WEAKENED) PATHOGEN AGAINST RABIESLouis Pasteur - 1880 He infected hares with the virus and isolated their nerves. Then the pathogens were weakened by drying and used for vaccination.

4. ANTIBODIES AGAINST DIPHTERIA AND TETANUS TOXINS (ANTITOXINS)Koch Laboratory, Germany, 1890. Investigations: Protective humoral factors (pathogen-specific antibodies) in the blood • Many diseases occur only once (natural protection) • Some diseases can be prevented by vaccination • The blood contains anti-bacterial activity (anti-toxins  serum therapy) ShimbasaruKitasato Emil Behring

5. WHAT HAPPENS WHEN YOU GET INFECTED WITH A PATHOGEN? …DEPENDS ON THE PATHOGEN EXTRACELLULAR PATHOGENS (ec. bacteria, multicellular parasites) INTRACELLULAR PATHOGENS (viruses, ic. bacteria, unicellular parasites) replication outside hostcells replication inside hostcells Many effector mechanisms of the innate and adaptive responses are used against them. Antibodies play an important role. NK/CTL and Th1/macrophage dominance. Neutralizing antibodies help.

6. PRINCIPLES OF VACCINATION I • Goals: Prevent infection, transmission and/or disease • Specificity: Generating an immune response againsta specific pathogen • Memory:Maintaining that response over time in order to prevent re-infection with thesameora similar pathogen

7. THE AIM OF VACCINATION IS TO PROVOKE THE PRIMARY RESPONSE First exposure to antigen (primary adaptive response) Second exposure to antigen (secondary adaptive response) Effector cells Effector cells Naïve cell Memory cells Memory cells

8. PRIMARY AND SECONDARY ADAPTIVE IMMUNE RESPONSES

9. PRINCIPLES OF VACCINATION II Immunological mechanism of protection: • Antibodies: • neutralization – to block colonization • precipitation/agglutination – to block spreading • T cell responses: helper T cells enhance antibody production + formation of CTL memory cells (viral vaccines - better anti-viral immunity if antigens are presented on MHC I class molecules  CTL activation)

10. TYPES OF VACCINES ILive-attenuated (weakened) pathogen containing vaccines Cultivated in conditions disabling their virulence (mostly viruses): MMR (Morbilli - Mumps - Rubella), OPV (oral polio vaccine = Sabin), BCG (tuberculosis), Rotavirus, Influenza (LAIV), Yellow fever • Disadvantages • May cause disease in the immunocompromised • Advantages • Mimic natural infection • Stimulate PRRs on innate cells • Induce CD4 and CD8 T cells • Effective CTL response Viral proteins synthesized inside the cells are efficiently presented on MHC I molecules (not characteristic for killed or subunit vaccines).

11. TYPES OF VACCINES IIInactivated (dead) pathogen containing vaccines Previously virulent microorganism killed by chemicals, heat or radioactivity Influenza, Pertussis, Hepatitis A, IPV (inactivated polio vaccine = Salk) • Advantages • Contain the microbial pattern that stimulates an innate immune response • Disadvantages • Don’t induce CD8 CTL response • Inactivation may lower immunogenicity

12. TYPES OF VACCINES IIISubunit vaccines Only the most characteristic parts (PAMPs) of the pathogen, usually conjugated to a carrier molecule Tetanus and diphteria toxoids (DT), Hepatitis B, Hib (Haemophilusinfluenzae type B), Meningococcus C antigen Toxoids are inactivated exotoxins. Hib: capsular polysaccharide Hep B: surface antigen produced by yeast cells Men C: a polysaccharide coupled to a carrier protein (complex antigen) • Advantages • Purified microbial antigens • May be simpler to produce • Reduced risk of adverse effects • Disadvantages • Don’t induce CD8 CTL response • Require addition of adjuvant(s)

13. KEEP IN MIND… • Safety standards are much higher for preventive treatments compared to therapeutic treatments • Live-attenuated vaccines can be more effective than non-replicating vaccines but pose more risks EFFICACY SAFETY • Immunity that is induced must be robust and durable in order to be clinically relevant • Risk vs. benefits of the individual and the society (Relative and changes with time) • Ethical issues, mandated vs. recommended vaccination

14. SOME CONTRAINDICATIONS FOR VACCINATING • Do not give vaccines to actually ill patients • Do not give live vaccines to immunosuppressed patients • Avoid giving live vaccines to pregnant women • Avoid all types of vaccines in the first trimester of pregnancy • In spite of immune suppression in HIV infected, we can give MMR but not BCG

15. MONOCLONAL ANTIBODIES monoclonal antibodies clones of a single B cell binding to a single epitope POLYCLONAL ANTIBODIES polyclonal antibodies clones of many B cells binding to multiple epitopes

16. Ag Ag Ag POLYCLONAL ANTIBODIES Polyclonal antibody Immunserum • Products of a set of B-lymphocyte clones • Heterogeneous in antigenspecificity, affinity, and isotype Set of B-cells Activated B-cells Antibody-producing plasma-cells Antigen-specific antibodies

17. MONOCLONAL ANTIBODIES (MAb) • Products of clones of oneB-lymphocyte • Homogeneous in specificity, affinity, and isotype • Can be found in humans in a pathologic condition called multiple myeloma, which is a malignant proliferation of a plasma cell (see supplementary)

18. STEPS OF MAbGENERATION (1)Immunization of a mouse(2) Isolation of B cells from the spleen(3) Cultivation of myeloma cells(4) Fusion of myeloma and B cells(5) Separation of cell lines(6) Screening of suitable cell lines(7) in vitro (a) or in vivo (b) multiplication(8) Harvesting *For more details see supplementary

19. FEATURES OF POLYCLONAL AND MONOCLONAL ANTIBODIES

20. humanized mouse monoclonal antibodies Human immunoglobulin transgenic mouse ENDANGERED SUBJECT (Immunodeficiency - e.g. hypogammaglobulinemia) PASSIVE IMMUNIZATION immunization mouse monoclonal antibodies immunization • PROTECTED SUBJECT • serum antibody human monoclonal antibodies • The immune system of recipient is not activated • Prompt but temporary protection/effect  Immunoglobulin degradation (3-6 months)

21. Human Mouse Humanized DIFFERENT TYPES AND IMMUNOGENICITIES OF ANTIBODIES USED IN THERAPY Chimeric *Humanized antibodiesare from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans (except CDR loops)!

22. PASSIVE IMMUNIZATION These are artificially acquired while maternal IgG transfer are usually termed naturally aqcuired

23. DIAGNOSTIC USES OF MAbs Identifying cell types Immunohistochemistry Characterization of lymphomas with CD (cluster of differentiation) markers Isolation of cells Isolation of CD34+ stem cells for autologous/allogeneic transplantation (from peripheral blood) Blood group determination(with anti-A, anti-B, and anti-D monoclonal antibodies) Identification of cell surface and intracellular antigens Cell activation state

24. THERAPEUTIC USE OF MAbs • Anti-TNF-α therapy • Anti-tumor therapy / targeted chemotherapyMonoclonal antibodies are specific to a cell-type specific it is hard to make them specific to malignant cells • Immunosuppression - prevention of organ rejection following transplantationCell-type specific • Drug eliminatione.g. anti-digoxin antibodies for treating digoxin-intoxication

25. 1) ANTI-TNF- α THERAPY • Infliximab(Remicade): since 1998, chimeric • Adalimumab(Humira): since 2002, recombinant human • Etanercept (Enbrel) – dimer fusion protein (TNF-α receptor + Ig Fc-part) Not a real monoclonal antibody, no Fab end, the specificity is given by TNF-receptor! Indications of anti-TNF-α therapy: • Rheumatoid arthritis • Spondylitis ankylopoetica • Psoriasis vulgaris, arthritis psoriatica • Crohns’ disease, colitis ulcerosa (usually - still – not as first line therapy)

26. 2) ANTI-TUMOR THERAPY Unconjugated MAb(on figure: Naked MAb) Anti-CD20 (rituximab – Mabthera/Rituxan, chimeric): B-cell Non-Hodgkin lymphoma Anti-CD52 (campath – Mabcampath, humanized): chronic lymphoid leukemia Anti-ErbB2 (trastuzumab – Herceptin, humanized): breast cancer Anti-VEGF (bevacizumab – Avastin, humanized): colorectal tu. (+ Lucentis!) Anti-EGFR (cetuximab – Erbitux, chimeric): colorectal tu. (+ Vectibix, recomb. human!) Killing of tumor cells via opsonized phagocytosis, ADCC or CDC. Conjugated MAb(on figure: Immunoconjugates) Anti-CD20 + yttrium-90 isotope (ibritumomab- Zevalin) Anti-CD20 + iodine-131 (tositumomab – Bexxar) The conjugates act right next to the tumor.

27. 3) IMMUNOSUPPRESSION Basiliximab Daclizumab Targeting IL-2Rs on T cells  prevention of transplantation rejection • OTHERS : • Omalizumab (Xolair): anti-IgE for moderate to severe allergic asthma • (binds mIgE-expressing B cells, not those already bound to the high affinity FcεRI) • Efalizumab(Raptiva): anti-CD11a, humanized, used in psoriasis

28. TARGETED MAb THERAPIES

29. SUPPLEMENTARY INFORMATION

31. CHECK FOR YOUR COUNTRY/REGION COMPLIANCE TO VACCINES

32. NOMENCLATURE OF MAbs

34. Spleen Immunization B cells, HGPRT+ Myeloma cell HGPRT- PEG fusion HAT selection Testing supernatants for specific antibody production Selection of hybridoma cells aminopterine * HAT= hypoxanthine, aminopterine, thymidine *Hypoxantine-guanine phosphoribosyltransferase

35. plasma cell repertoire of a health individual plasma cell repertoire of a multiple myeloma patient Myeloma multiplex = malignant tumor of plasma cells uncontrolled replication of clones  production of monoclonal antibodies (same type of heavy and light chains, same subclass if IgG or IgA)