Transcript 7th seminar - vaccination, mono- and polyclonal antibodies

7th SEMINAR
VACCINATION,
POLYCLONAL AND MONOCLONAL
ANTIBODIES
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.
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 (antitoxins  serum therapy)
Emil Behring
Shimbasaru Kitasato
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 host cells
replication inside host cells
Many effector mechanisms of the innate and
adaptive responses are used against them.
NK/CTL and Th1/macrophage dominance.
Neutralizing antibodies help.
Antibodies play an important role.
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
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
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)
TYPES OF VACCINES I
Live-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
Advantages




Mimic natural infection
Stimulate PRRs on innate cells
Induce CD4 and CD8 T cells
Effective CTL response
 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 II
Inactivated (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
TYPES OF VACCINES III
Subunit 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 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)
KEEP IN MIND…
• Safety standards are much higher for
preventive treatments compared to
therapeutic treatments
EFFICACY
SAFETY
• 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
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
(measles, mumps, rubella) but not BCG (Bacillus Calmette–Guérin)
POLYCLONAL ANTIBODIES
clones of many B cells
polyclonal
antibodies
binding to multiple epitopes
MONOCLONAL ANTIBODIES
clones of a single B cell
monoclonal
antibodies
binding to a
single epitope
POLYCLONAL ANTIBODIES
Ag
Immunserum
Polyclonal
antibody
- Products of a set of B-lymphocyte clones
- Heterogeneous in antigen specificity, affinity, and isotype
Set of B-cells
Ag
Activated B-cells
Antibodyproducing
plasma-cells
Antigen-specific
antibodies
MONOCLONAL ANTIBODIES (MAb)
 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)
STEPS OF MAb
GENERATION
(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
FEATURES OF POLYCLONAL AND
MONOCLONAL ANTIBODIES
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 nonspecific immunoglobulins
high
low
Cost of preparation
low
high
Standardisability
Impossible (or uneasy)
easy
Amount
limited
unlimited
Applicability
method-dependent
excellent
PASSIVE IMMUNIZATION
immunization
mouse monoclonal
antibodies
humanized mouse monoclonal
antibodies
Human immunoglobulin
transgenic mouse
PROTECTED
SUBJECT
serum antibody
 The immune system of recipient is not activated
 Prompt but temporary protection/effect
 Immunoglobulin degradation (3-6 months)
human monoclonal
antibodies
ENDANGERED SUBJECT
(Immunodeficiency - e.g. hypogammaglobulinemia)
DIFFERENT TYPES AND IMMUNOGENICITIES
OF ANTIBODIES USED IN THERAPY
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)!
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, 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
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)
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.
3) IMMUNOSUPPRESSION
Daclizumab
Basiliximab
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
Monoclonal Ab, humanized
CD52
CLL, CML
Monoclonal IgG1, chimeric
IL-2 R
transplantation
Monoclonal IgG1, chimeric
IL-2 R
transplantation
Monoclonal IgG1, chimeric
CD20
lymphoma, RA
Monoclonal IgG1, humanized
HER2/neu
Monoclonal IgG4, humanized
CD33
breast cancer, NSC
lung cancer
leukemia
Monoclonal IgG1, murine
CD20
lymphoma
Monoclonal IgG2, murine
EGFR-TKI
KIT-TKI
EpCAM
EGFR TK
TK
CRC
NSCLC
GIST, CML
(Mabcampath)
Daclizumab
(Zenapax)
Basiliximab
(Simulect)
Rituximab
(Rituxan/Mabthera)
Trastuzumab
(Herceptin)
Gemtuzumab
conjugated with calicheamicin
Ibritumomab
(Y90)
Edrecolomab
Gefitinib
Imatinib
SUPPLEMENTARY
INFORMATION
CHECK FOR YOUR COUNTRY/REGION
COMPLIANCE TO VACCINES
NOMENCLATURE OF MAbs
PREFIX
TARGET
SOURCE
-ki(n)- interleukin
-u- human
-ci(r)- cardiovascular
-o- mouse
SUFFIX
mab
variable
-co(l)- colonic tumor
-xi- chimera
-neu(r)- nervous system
-zu- humanized
Immunization
Selection of hybridoma cells
Spleen
B cells,
*HGPRT+
Myeloma cell
HGPRT-
aminopterine
PEG fusion
HAT selection
Testing supernatants for specific antibody production
HAT= hypoxanthine, aminopterine, thymidine
*Hypoxantine-guanine phosphoribosyltransferase
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)