From Allergen Back to Antigen:. a Rational Approach to New Forms of Immunotherapy

From allergen back to antigen: a rational approach to new forms of immunotherapy

Paolo Colombo

Istituto di Biomedicina e di Immunologia Molecolare“Alberto Monroy”

del Consiglio Nazionale delle RicerchePalermo,Italy

DAA Data Analysis in Astronomy Erice 15-22/04/2007

Allergic reaction

from Holt and al. Nature,vol402,supp,1999

Parietaria pollen

Parj1.01 m.w. 14986

profilin m.w. 13945Parj2 m.w. 11345

Parj1.02 m.w. 10677CBP m.w. 9182

CBP+

profilin

Parj1+

Parj2

Immunological relevance

ELISA inhibition to natural Parietaria extract

P.judaica EX rParj1 rParj2 rParj1

+ rParj2

PjEDcys

Patient 1 15% 75% 80% 7%

Patient 2 40% 70% 75% 3,5%

Patient 3 35% 37% 80% 10%

Patient 4 59% 80% 86% 8%

Patient 5 55% 30% 68% 10%

Patient 6 30% 65% 69% 30%

Patient 7 38% 58% 79% 28%

Mean value 38,85% 59,29% 76,71% 13,78%

90% 5%

Par j 1.0101 139 aa 14400 DaPar j 1.0201 102 aa 10677 Da

Par j 2.0101 102 aa 11344 Da

C4 C14 C29C30 C50 C52 C75 C91

C4 C14 C29C30 C50 C52 C75 C91

C4 C14 C29C30 C50 C52 C75 C91

non-specific Lipid Transfer Proteins

Colombo et al. J.Immunol 1998

Parj 1 QETCGTMRV ALMPCLPFVQ GKEKE PSKGCCSGAKR LDGETKTGP QRVHACECIQTParj 2 EEACGKVVQ DIMPCLHFVK GEEKE PSKECCSGTKK LSEEVKTTE QKREACKCIVR

alpha 1 loop1 alpha 2 loop2 alpha 3

Parj 1 AMKTYS DIDGKLVSE VPKHCGIVVSKLPPIDV NMDCK TL-------------Parj 2 ATKGISGI KNELVAEVP KKCDIKTTLPPITA DFDCS KIQSTIFRGYY----

Loop3 alpha 4 loop4 beta

helix 1helix 2

helix 3

helix 4

A rational approach to site-specific mutagenesis

Parj1 3D model

The symptoms of the IgE-mediated allergic reactions can be transiently ameliorated by pharmacological treatments which do not interfere with the ethiogenesis and progression of the disease.

According to WHO: “The only treatment able to modify the natural outcome of the disease restoring a normal immunity against allergens is specific immunotherapy (SIT)”

(ANERGY, TOLERANCE, IMMUNE DEVIATION)

Therapy of allergic reaction

Therapy of allergic reaction

So far, from 1911 immunotherapy is performed by administration of gradually increasing doses of the extract to whom the patient is allergic by s.c. injection or mucosal administration without takingcare of the individual sensitisation profile of the patient

Despite its usage in clinical practise for almost one century, the molecular mechanisms involved in successful SIT are not fully understood.Several factors seem to influence the immune response such as:

1) the concentration of the allergen;2) the type of antigen presenting cell;3) the structure of the allergen;4) the type of adjuvant used for the formulation of the vaccine.

Natural extracts vs recombinant allergens

# “Natural” # Standardization of product# Pure protein# Tailored therapy# Genetic engineering of the molecules

From an extract-based Specific Immunotherapy to an allergen-

specific Immunotherapy

Par j 1 w.t. Pj A Pj B Pj C Pj D

C4 C14 C29C30 C50C52 C75 C91

C4 C14 SS C50C52 C75 C91

S C4 C14 C29C30 S C75 C91

S C14 SS C50C52 C75 C91

C4 C14 SS S S C75 C91

A B C D E

IgE binding activity

00,10,20,30,40,50,60,7

Parj1 Parj1.1 Parj1.2 Parj1.3 Parj1.4

O.D

.non allergic serum allergic sera

Parj1

PjA PjB PjC PjD

Parj1 PjA PjB PjC PjD

IgE binding activity

00,10,20,30,40,50,60,70,80,9

Parj2 Parj2cys

O.D

.

allergic patients non allergic subject

ELISA inhibition to natural Parietaria extract.

P.judaica EX rParj1 rParj2 rParj1

+ rParj2

PjEDcys

Patient 1 15% 75% 80% 7%

Patient 2 40% 70% 75% 3,5%

Patient 3 35% 37% 80% 10%

Patient 4 59% 80% 86% 8%

Patient 5 55% 30% 68% 10%

Patient 6 30% 65% 69% 30%

Patient 7 38% 58% 79% 28%

Mean value 38,85% 59,29% 76,71% 13,78%

Recombinant allergens

Anaphilactic activity

in vitro and in vivo assays

SPT with a mixture containing rParj1 + rParj2 allergens and with the PjEDcys hybrid

on 5 Pj allergic patients

Parj1 + Parj2 mix PjEDcys histamine PBS

5 μg/ml 50 μg/ml 5 μg/ml 50 μg/ml

Patient 1 47,1 108,33 4,71 15,7 19,62 -

Patient 2 43,96 58,87 6.25 6.28 56.52 -

Patient 3 12,56 43.96 - - 32.97 -

Patient 4 43.96 62,8 7.06 11.77 54.95 -

Patient 5 37,68 94,2 12.56 21.98 62.8 -

The concentration of the molecules used in the test are displayed, the wheal diameters are expressed as mm2.

CFDA-SE staining

PjEDcys

1 μg/ml 10 μg/ml

1 μg/ml 10 μg/ml

rParj1+rParj2

CSFE

CSFE

CD3+

CD3+

CD3+

CSFE

unstimulated

Parj1+Parj2 mix PjEDcys

1μg/ml 10μg/ml 1μg/ml 10μg/ml

Patient 1 1,6 3,1 3,4 7,5 Patient 2 1,4 17.7 35,3 27,2 Patient 3 0,5 1,5 1 1 Patient 4 5,4 5,8 5,7 7,3 Patient 5 3,4 1,5 1,6 1,8 Patient 6 2,4 2,1 3,6 0,9

Blocking antibodies induced by specific allergy vaccination prevent the activation of CD4+ T cells by inhibiting serum-IgE-facilitated allergen presentation

R.J.J. van Neerven et al. J. Immunol 1999, 163: 2944-2952

immune serum preimmune serum

rPar

j1rP

arj2

PjEkDa

51.734.128.020.0

6.4

rPjE

Dcys

rPar

j1rP

arj2

PjErPjE

Dcys

8

A rational approach to site-specific mutagenesis

Parj1 3D model

1 13 256

Parj2 G-S Parj1

1 13 256

A23A24 A27 G-S A23A24 A27PjEDmut

PjED

rPar

j1rP

arj2

rPjE

DrP

jEDm

ut

IgE bindingactivity

His-specific probe

Recombinant allergens

0

20

40

60

80

0,01 0,1 1 10

antigen concentration

%

0102030405060

0,01 0,1 1 10


%

05

10152025

0,01 0,1 1 10


%

010203040506070

0,01 0,1 1 10


%

05

1015202530

0,01 0,1 1 10


%

0102030405060

0,01 0,1 1 10


%

01020304050

0,01 0,1 1 10


%

0

5

10

15

20

0,01 0,1 1 10


%

rPj1+rPj2 Mix rPjED rPjEDmut

05

101520253035

0,01 0,1 1 10

antigen concentration %

Histamine release assay

CFDA-SE staining

unstimulated rParj1+rParj2

PjED PjEDmut

1 μg/ml 10 μg/ml

1 μg/ml 10 μg/ml

1 μg/ml 10 μg/ml

CSFE

CSFE CSFE

CD3+CD3+

CD3+CD3+

CSFE

EXP. 1B - Specific IgG1

A B C0

250000

500000

750000

1000000

Groups

AU

/ml

EXP 1B - Specific IgG2a

A B C0

500

1000

1500

2000

Groups

AU

/ml

1st bleeding


D E F0

250000

500000

750000

1000000

Groups

AU

/ml

EXP 2B - Specific IgG2a

D E F0

500

1000

1500

2000

Groups

AU

/ml

1st bleeding


G H I0

250000

500000

750000

1000000

Groups

AU

/ml

1st bleeding

Mice immunized with: A,D,G mix rParj1+rParj2; B,E.H PjED; C,F,I PjEDmut

Specific IgG1

p<0.02 p<0.03A B

C

p<0.06

0

20

40

60

80

100

1/ 25 1/ 100 1/ 400 1/ 1600 1/ 6400

mouse sera dilution

% in

hibi

tion

% Inhibition of IgE binding

0

2 0

4 0

6 0

8 0

10 0

1/ 2 5 1/ 10 0 1/ 4 0 0 1/ 16 0 0 1/ 6 4 0 0

mouse sera dilution

% in

hibi

tion

% Inhibition of IgE binding

A

PjED

B

PjEDmut

Conclusion

- Parietaria allergic subjects produce IgE mainly against the Parj1 and Parj2 proteins.

- The use of the most remarkable allergenic components of the pollen instead of complete extracts excludes the possibility that patients became sensitive to other components of the extract and inducinga new immune reaction against the most remarkable allergenic components of the pollen.

- In addition, the use of hybrid polypeptides, produced by engineering the major Parietaria allergens, presents a few advantages respect to the natural extracts with:

1)reduced anaphylactic activity;2)higher antibody production in mice and retained T cell

proliferative response in human;3)the production of protective antibodies against the most

relevant allergenic components of the pollen.

ANGELA BONURAMIMMO GERACIIstituto di Biomedicina e di Immunologia Molecolaredel Consiglio Nazionale delle RicerchePalermo, Italy

SAVERIO AMOROSOU.O. Allergologia delloOspedale Civico-Palermo, Italy

RUDOLF VALENTAKERSTIN WESTRITSCHNIGUniversity of Vienna. Austria

GABRIELLA DI FELICESILVIA CORINTIIstituto Superiore di Sanità,Roma, Italy

Moverare et al Allergy 2002

Parj1

Parj2

Profilin

Ca2+ binding protein

Cross-reactive and species-specific Parietaria allergens

Pj CBP Cherry CBP Olea CBP

From Allergen Back to Antigen:. a Rational Approach to New Forms of Immunotherapy

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