7th seminar vaccination, polyclonal and monoclinal antibodies
TRANSCRIPT
7th SEMINARVACCINATION,
POLYCLONAL AND MONOCLINAL ANTIBODIES
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.
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.
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
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
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
Naïve cell
Effector cells
First exposure to antigen (primary
adaptive response)
Second exposure to antigen (secondary adaptive response)
Memory cellsMemory cells
Effector cells
THE AIM OF VACCINATION IS TO PROVOKE THE PRIMARY RESPONSE
PRIMARY AND SECONDARY ADAPTIVE IMMUNE RESPONSES
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)
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 ILive-attenuated (weakened) 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
TYPES OF VACCINES IIInactivated (dead) pathogen containing vaccines
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 polysaccharideHep B: surface antigen produced by yeast cellsMen 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 IIISubunit vaccines
• 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
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
MONOCLONAL ANTIBODIES
monoclonal antibodies
clones of a single B cell binding to a single epitope
polyclonal antibodiesclones of many B cells
POLYCLONAL ANTIBODIES
binding to multiple epitopes
POLYCLONAL ANTIBODIESAg
ImmunserumPolyclonal antibody
Ag
Ag
Set of B-cells
Activated B-cells Antibody-producing
plasma-cellsAntigen-specific
antibodies
- Products of a set of B-lymphocyte clones- Heterogeneous in antigen specificity, affinity, and isotype
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)
(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
Polyclonal antibodies Monoclonal antibodies
Number of recognized antigen determinants
several (frequent cross-reactions)
mostly one
Specificity polyspecific monospecific
Affinity Varying (diverse antibodies)
high
Concentration of non-specific immunoglobulins
high low
Cost of preparation low high
Standardisability Impossible (or uneasy) easy
Amount limited unlimited
Applicability method-dependent excellent
FEATURES OF POLYCLONAL AND MONOCLONAL ANTIBODIES
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 antibodiesimmunization
PASSIVE IMMUNIZATION
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
PASSIVE IMMUNIZATION
Type Application
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
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
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
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
Unconjugated MAb (on figure: Naked MAb)Anti-CD20 (rituximab – Mabthera/Rituxan, chimeric): B-cell Non-Hodgkin lymphomaAnti-CD52 (campath – Mabcampath, humanized): chronic lymphoid leukemiaAnti-ErbB2 (trastuzumab – Herceptin, humanized): breast cancerAnti-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
BasiliximabDaclizumab
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
TARGETED MAb THERAPIES
Name Type Target Indications
Alemtuzumab (Mabcampath)
Daclizumab(Zenapax)
Basiliximab(Simulect)
Rituximab(Rituxan/Mabthera)
Trastuzumab(Herceptin)
Gemtuzumab
Ibritumomab (Y90)EdrecolomabGefitinib Imatinib
Monoclonal Ab, humanized
Monoclonal IgG1, chimeric
Monoclonal IgG1, chimeric
Monoclonal IgG1, chimeric
Monoclonal IgG1, humanized
Monoclonal IgG4, humanized conjugated with calicheamicin
Monoclonal IgG1, murine
Monoclonal IgG2, murineEGFR-TKIKIT-TKI
CD52
IL-2 R
IL-2 R
CD20
HER2/neu
CD33
CD20
EpCAMEGFR TKTK
CLL, CML
transplantation
transplantation
lymphoma, RA
breast cancer, NSC lung cancerleukemia
lymphoma
CRCNSCLCGIST, CML
SUPPLEMENTARY INFORMATION
CHECK FOR YOUR COUNTRY/REGION COMPLIANCE TO VACCINES
NOMENCLATURE OF MAbs
PREFIX TARGET SOURCE SUFFIX
variable
-ki(n)- interleukin -u- human
mab-ci(r)- cardiovascular -o- mouse
-co(l)- colonic tumor -xi- chimera
-neu(r)- nervous system -zu- humanized
SpleenImmunization
Myeloma cellHGPRT-
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
plasma cell repertoire of a health individual
Myeloma multiplex = malignant tumor of plasma cellsuncontrolled 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