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