1. 7th SEMINAR VACCINATION, POLYCLONAL AND MONOCLINAL ANTIBODIES

2. THE FIRST VACCINE Edward 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 RABIES Louis 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) Emil Behring Shimbasaru Kitasato

5. EXTRACELLULAR PATHOGENS (ec. bacteria, multicellular parasites) INTRACELLULAR PATHOGENS (viruses, ic. bacteria, unicellular parasites) 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. replication outside host cells replication inside host cells WHAT HAPPENS WHEN YOU GET INFECTED WITH A PATHOGEN? …DEPENDS ON THE PATHOGEN

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

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

8. PRIMARY AND SECONDARY ADAPTIVE IMMUNE RESPONSES

9. PRINCIPLES OF VACCINATION II Immunological mechanism of protection: 1.Antibodies: neutralization – to block colonization precipitation/agglutination – to block spreading 2.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. Cultivated in conditions disabling their virulence (mostly viruses): MMR (Morbilli - Mumps - Rubella), OPV (oral polio vaccine = Sabin), BCG (tuberculosis), Rotavirus, Influenza (LAIV), Yellow fever Advantages  Mimic natural infection  Stimulate PRRs on innate cells  Induce CD4 and CD8 T cells  Effective CTL response Disadvantages  May cause disease in the immunocompromised Viral proteins synthesized inside the cells are efficiently presented on MHC I molecules (not characteristic for killed or subunit vaccines). TYPES OF VACCINES I Live-attenuated (weakened) pathogen containing vaccines

11. 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 TYPES OF VACCINES II Inactivated (dead) pathogen containing vaccines

12. Only the most characteristic parts (PAMPs) of the pathogen, usually conjugated to a carrier molecule Tetanus and diphteria toxoids (DT), Hepatitis B, Hib (Haemophilus influenzae 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) TYPES OF VACCINES III Subunit vaccines

13. 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 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 KEEP IN MIND… SAFETY EFFICACY

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 clones of many B cells POLYCLONAL ANTIBODIES binding to multiple epitopes

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

17.  Products of clones of one B-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) MONOCLONAL ANTIBODIES (MAb)

18. (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 STEPS OF MAb GENERATION *For more details see supplementary

19. Polyclonal antibodiesMonoclonal antibodies Number of recognized antigen determinants several (frequent cross-reactions) mostly one Specificitypolyspecificmonospecific AffinityVarying (diverse antibodies) high Concentration of non- specific immunoglobulins highlow Cost of preparationlowhigh StandardisabilityImpossible (or uneasy)easy Amountlimitedunlimited Applicabilitymethod-dependentexcellent FEATURES OF POLYCLONAL AND MONOCLONAL ANTIBODIES

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

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

22. PASSIVE IMMUNIZATION TypeApplication Intramuscular (less effective due to lower dose) HBV-Ig; Varicella-zoster-Ig; Diphtheria and tetanus antitoxins. Intravenous (IVIG)Bruton-agammaglobulinaemia; Variable and mixed immunodeficiencies with hypogammaglobulinaemia; Anti-venom antibody treatment; 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 1)Anti-TNF-α therapy 2)Anti-tumor therapy / targeted chemotherapy Monoclonal antibodies are specific to a cell-type specific it is hard to make them specific to malignant cells 3)Immunosuppression - prevention of organ rejection following transplantation Cell-type specific 4)Drug elimination e.g. anti-digoxin antibodies for treating digoxin-intoxication

25.  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) 1) ANTI-TNF- α THERAPY

26. 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. 2) ANTI-TUMOR THERAPY

27. Basiliximab Daclizumab 3) IMMUNOSUPPRESSION 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 NameTypeTargetIndications Alemtuzumab (Mabcampath) Daclizumab (Zenapax) Basiliximab (Simulect) Rituximab (Rituxan/Mabthera) Trastuzumab (Herceptin) Gemtuzumab Ibritumomab (Y 90 ) Edrecolomab Gefitinib Imatinib Monoclonal Ab, humanized Monoclonal IgG1, chimeric Monoclonal IgG1, humanized Monoclonal IgG4, humanized conjugated with calicheamicin Monoclonal IgG1, murine Monoclonal IgG2, murine EGFR-TKI KIT-TKI CD52 IL-2 R CD20 HER2/neu CD33 CD20 EpCAM EGFR TK TK CLL, CML transplantation lymphoma, RA breast cancer, NSC lung cancer leukemia lymphoma CRC NSCLC GIST, CML

29. SUPPLEMENTARY INFORMATION

31. CHECK FOR YOUR COUNTRY/REGION COMPLIANCE TO VACCINES

32. NOMENCLATURE OF MAbs PREFIXTARGETSOURCESUFFIX variable -ki(n)- interleukin-u- human mab -ci(r)- cardiovascular-o- mouse -co(l)- colonic tumor-xi- chimera -neu(r)- nervous system-zu- humanized

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

35. plasma cell repertoire of a health individual 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) plasma cell repertoire of a multiple myeloma patient