Week 2 Antigens and Immunization

8/12/2019 Week 2 Antigens and Immunization

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Some terminology"Antigenicity": the ability to interact with an antibody"Immunogenicity": the ability to induce an immuneresponse

"Hapten": an antigen that is not immunogenic

HaptensImmunogens

Note : Immunogenicity does depend one os as we ...


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Epitopes

"Epitope" = "Antigenic determinant" =

- -

form a bimolecular complex

form a trimolecular complex

n so u e ant gen

no MHC involvement

Anti en can be rotein

una e to in so u e antigenMHC restricted

polysaccharide or lipid

Epitope is accessible, often Epitope is internal, denaturedconformational, hydrophilic

and mobile

linear peptide, hydrophobic and

MHC-bound


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Two kinds of B-cell epitopes

"Discontinuous" or "conformational" epitopes

" " " "

denature

denature


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Factors that influence immunogenicity

Part I : Contribution of the immunogen

Foreignness : a molecule must be recognized asnon-se .

Molecular size : 5 - 10 kDa is the minimum

Chemical composition and heterogeneity : themore complex, the better.

processing by APCs is an essential step.


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antibody Nopara

hapten

metacarrier binding

Weak

binding

conjugate

ort o

cross-react on


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Increase susce tibilit to ha oc tosis 1

Couple the antigen to beads

The protein can be covalently linked to commonly

available activated e. .CNBr-activated a arosebeads.

ternative y, re oo ce scan e use .Coupling agents include tannic acid, chromic chlorideor g utera e y e.

Anti ens can also be adsorbed to activated r norto colloidal goldparticles.


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Increase susceptibility to phagocytosis (2)

se e ec rop ore ca y pur e an genDirect injectionof a gel fragment (rabbits).

- Freeze the gel slize (liquid N2) and grind it in an eppendorf tube. Suspend in buffer.

Excise the blotted antigenfrom a nitrocellulose (NC) membrane- Finely grind the NC slice in a mortar and suspend in buffer, OR...

- Dissolve the NC slice (1-2 cm2) in 0.3 ml DMSO, slowly add to 3 ml 15 mMNaHCO3 bu er H 9.6 under vortexin . NC reci itates as ine artricles.

Sediment at 10,000g, wash twice with PBS and resuspend in 1 ml PBS, OR...- Directly implant the NC slice subcutaneously.

Use e ectro-e ut on o t e ant gen rom t e ge s ice. T e"trick" is to localize the target on the gel ...This can be achieved by:

- stainin a side stri- by using reversible gel staining methods (e.g.: Copper, Zinc, Nile Red, Sypro

Orange stain)


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Part II : Contribution of the host

Genot e of the reci ient animal : enetic control ofimmune responsiveness is largely confined to MHC

e use o a uvan s : reun s a uvan , a um e c.Immuno en dosa e and fre uenc : avoid both lowand high zone tolerance; use repeated injections.

oute o a ministration : i.v., i.m., s.c., i. ., i.p.etc.


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How do adjuvants work?

Antigen depot formation: a protected reservoir ofanti en or slow release to drainin l m h nodes.This hel s the

formation of memory cells and prolonged antibody responses.

Injection-site depots of antigen and adjuvant can induce theormat on o granu omas.

Delivery vehicle or inert carrier: help direct antigento perip era immune organs an serve to concentrate oraggregate the antigen for efficient delivery to the immunes stem. E. .: sur actants concentrate rotein molecules on alarge surface area.

of the immune system, resulting in cell proliferation and/ordifferentiation. Included are: mineral salts, surfactants, peptidesand cytokines.


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Ad uvants hel

Antibody t iters

virus alone virus and adjuvant

100% 100%0%

1 year

postvaccination postvaccination


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Adjuvants help

Postulated mode of action :

Freund's incom lete ad uvant

epo

formation

granu oma

formation

non-spec c

stimulation

+ + -

Freund's complete adjuvant (#)

Alum

+

+

+ +

+

+

-

Mycobacterium tuberculosis(*)Bordetella pertussis(**)

++--

--

y

Synthetic polynucleotides +

-

-

-

-

Active adjuvant components : (*) muramyl dipeptide (MDP);

(**) LPS and pertussis toxin (whooping cough); (***) lipid A

,

"Granuloma" : tumour-like mass, nodule of granulation tissue containing lymphocytes,

macrophages and fibroblasts; result of chronic inflammatory response.


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Some valuable alternatives to Freund's

Replace crude preparations (whole bacteria) by refined products.Refined immunostimulators include: Trehalose Dimycolate (TDM)

Monophosphoryl Lipid A (MPL), Muramyl Dipeptide (MDP), Lipoteichoid

Acids (LTA).

Replace mineral oil (paraffin) by metabolizable oils (squalene).Numerous commercial products are now available:

- Ribi Adjuvant System : TDM and or MPL, squalene

- TiterMax: co ol mer CRL-8941 s ualene

- Montanide ISA Adjuvants- Syntex Adjuvant Formulation (SAF): thr-MDP, squalene

- etc.

Cheap home-made alternatives:

- Aluminum Salt Adjuvants- Nitrocellulose-adsorbed Protein

- Entrapped antigen (acrylamide, liposomes = lipide bilayer vesicles )

- ISCOMS (immune-stimulating complexes = saponin/cholesterol

micelles)

- etc.


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'

... e sure to use syr nges

... be sure to thoroughly MIX Complete adjuvant

... make a "stiff mayonaise"

... in ect small volumes er site es eciall i.d.


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Immunization routes. . -

- no adjuvant, except for liposomes or iscoms

- no large particles

- anger o anap y ax s or so u ze an gens

- helps "spreading" the immune response over different

lymphoid tissues

I.d. - limited to small volumes

- severe hypersensitivity reactions may result in combination

'

S.c. - preferred route for booster

- slow Ag release minimizes the risk for anaphylaxis

- use e our corners o e an ma

I.p.: - best for large volumes

- ood access of the A to the l m hatics

I.m.: - preferred route for poultry

- relatively large volumes are possible

Other(incl. intrasplenic, intranodal, intrafootpad):

- only if the amount of available Ag is very limited.


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Immunogen dose : don't overshoot ...

There is a huge workable range.

Much depends on the immunogenicity of the molecule,

including foreignness and immunogen presentation.

Rules of thumb:- Mouse: 1 - 100 g

- Rabbit: 10 1000 g

- Chicken: 1 100 g

- Goat (Sheep): 20 - 2000 g

Use enough for the primary immunization but decrease the

dose for the boost injections!

- This helps selection towards antibodies with high affinity(affinity

" "

dose of antigen. Binding of its antigen is the first step in activation of

a memory cell.

-

contaminants start being produced. Thus: better specificity.


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A routine immunization schedule

Rabbit Mouse Goat

Dose u 50-1000 5-100 500-2000

Formulation

CFA - IFA (*)

CFA - IFA CFA - IFA

n ec on vo .Route

m 200 u 2 m

i.d. - s.c. s.c. i.d. - s.c.

Frequency

Sam le vol.

at least 3 weeks interval

20 ml 200 ul 200 ml

(*) 50-50 emulsion of adjuvant and antigen solution; first injection

with Com lete Freund's Ad the followin boosts with Incom lete

Freund's Adjuvant. ! SMALL volumes per site, esp. i.d.!!!!


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Troubleshooting ...

Antigen property Classificationof defect Result RemedyDefect

Lacks an epitope No Ag binding toB-cells

B-cell tolerance No response None

Lacks Class II

protein binding site

No Ag-fragmentpresentation

Non-responsiveness

No response orprimaryresponse only

Switch animalsor attach class IIbinding site

Lacks T-cell

recepter binding site

No - e per ce

involvement

-ce to erance No response or

primaryresponse only

w tc an ma s

or attach TCRbinding site

Nondegradable

Small sizeno T-helper cell

o g- ragmenpresentation

-responsiveness

Non-No response

primary resp.

Conjugate to

Nonparticulate

Form

Poor phagocytosis Weakerresponse

Self-polymerizeCouple to beads

-


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How about a leap into Phylogeny?

Use chickens...

million years): excellent for conserved antigens

on- nvas ve, easy an o y co ec on: eggs

Very productive: "1 chicken = 10 rabbits" (15% of

t e oo vo ume per 4 wee s) or 1 c ic en wee= 3 rabbit months"

Easy to keep

No interaction with rheumatoid factors, com lementsystem nor with Fc receptors

No bindin to rotein A or rotein G


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Serum can be used in crude form, egg yolk

nee s pur cat onStep 1: Separation of egg white and yolk- Freeze and thaw the eggs

- Roll the semi-solid yolk ball on absorbent paperStep 2: Delipidation of the yolk- Dilute yolk with 9 vol. of dH2O and mix thoroughly, pH to 7.0 with 0.1 M NaOH

- reeze e emu s on a -

- Spin down the lipids (20 min x 2,000-3,000g)- pH the supernatant to 7.6

Step 3: Purification of IgG (Y) by salting out

- Add 18.5 g powdered (NH4)2SO4 to 100 ml of delipidated yolk solutionunder constant stirring and leave stirring for 1 hour- Spin down IgG (20 min x 2,000-3,000g), dissolve in 10 ml TBS and

spin again. Collect the supernatant.- Repeat precipitation by addition of 1 vol. of 3.6 M (NH4)2SO4 if necessary


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Purification of chicken IgG made easy...

Promega "Eggstract"Separate yolk from egg

white using EggSeparator

"A 60 (75) minute protocol yields

PrecipitateLipids

- mg o pure g

per egg."

A PrecipitationSolution A, mix,

centrifuge

Add PrecipitationSolution B, mix,

centrifuge

PrecipitateIgY

Solubilize IgY to desiredconcentration


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Some applications need minor adaption

Immune complexes with chicken IgG are moresoluble. Gel diffusion results can be improved by addition

of PEG 6000 (2-6%) or 1.5M NaCl.Chicken IgG has a lower isoelectric point (pI) thanmammalian IgGs.

E.g.: rocket electrophoresis needs to be run at pH 5.5-6,not at pH 8.6.

Pepsin does not work for digestion into (Fab)'2 (but

is enerally not needed either).Chicken IgG is more susceptible to aggregation onfreezin and thawin . Stora e at 4? C is re erable.


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Immunization of chickens

Emulsify 0.5 ml of antigen solution (1 to 100 g)and 0.55 ml of Complete Freund's Adjuvant or RibiAdjuvant System.

In ect 0.5 ml of the emulsion into each breastmuscle.

'

Adjuvant or Ribi.

'pointless, can yield severe hypersensitivity reactions

and ma result in cessation of e roduction ! .

A fast and strong antibody response is routinely.

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Week 2 Antigens and Immunization