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
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
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
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
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
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
antigen concentration
%
05
10152025
0,01 0,1 1 10
antigen concentration
%
010203040506070
0,01 0,1 1 10
antigen concentration
%
05
1015202530
0,01 0,1 1 10
antigen concentration
%
0102030405060
0,01 0,1 1 10
antigen concentration
%
01020304050
0,01 0,1 1 10
antigen concentration
%
0
5
10
15
20
0,01 0,1 1 10
antigen concentration
%
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
EXP. 2B - Specific IgG1
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
EXP. 3B - Specific IgG1
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
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