Assessment of Amb a 1 isoallergens as basis for

Assessment of Amb a 1 isoallergens as basis for development of a
recombinant ragweed SIT vaccine
Augustin S1; Wald M1; Asero R2; Reese G1; Klysner S1; Nandy A1.
1
Allergopharma GmbH & Co. KG, Reinbek, Germany and 2Clinica San Carlo, Paderno Dugnano, Italy
Background
Pollen from short ragweed (Ambrosia artemisiifolia) contains five isoallergens of the major allergen Amb a 1, with about 60-86% sequence identity. To provide rationale for a
potential isoallergen composition of a future recombinant immunotherapeutic vaccine for the treatment of ragweed allergy, recombinant and natural Amb a 1 isoallergens
were produced, purified, and analysed in detail.
Methods
Expression of the five Amb a 1 isoallergens was attempted in both, E. coli and P. pastoris, the products were subsequently purified and characterised by SDS-PAGE,
dynamic-light scattering (DLS) and size-exclusion chromatography with multiple-angle light scattering (SEC-MALS). The different purified recombinant isoallergens and
natural counterparts purified from1 short ragweed extract were compared immunologically by solid phase IgE-immunoblotting and IgE-inhibition experiments (EAST).
Results
Four isoallergens (Amb a 1.01, 1.02, 1.03 and 1.05) were successfully expressed in P. pastoris and purified as folded, soluble and monomeric proteins (Fig. 1 & Fig. 2). All
five isoallergens were expressible in E. coli as insoluble inclusion bodies and were subsequently purified as unfolded aggregated proteins (Fig. 1). In solid phase human IgEimmunoblots natural and recombinant Amb a 1.01, Amb a 1.03 and Amb a 1.02 showed the highest IgE-binding for most of the analysed sera of allergic individuals from Italy
and the US (Fig. 3). In contrast, Amb 1.05 (formerly known as Amb a 2) and Amb a 1.04 showed only low IgE-reactivity. Analysis of isoallergen reactivity by IgE-inhibition
experiments confirmed the importance of the isoallergens Amb a 1.01, Amb a 1.03 and Amb a 1.02 (Fig. 4).
2
100
75
50
50
35
35
25
rAmb a 1.0502
rAmb a 1.0402
rAmb a 1.0305
rAmb a 1.0202
rAmb a 1.0101
rAmb a 1.0502
rAmb a 1.0402
rAmb a 1.0305
25
7.0 x 10
4
6.0 x 10
4
5.0 x 10
4
rAmb a 1.0502
(100% monomer)
- DTT
0.5
rAmb a 1.0202
(94,65% monomer)
rAmb a 1.0101
(100% monomer)
+ DTT
20.0
24.0
26.0
28.0
rAmb a 1.0202
30
20
10
0
0.1
1.0
10
100
0.01
0.1
30.0
40
rAmb a 1.0305
30
1.0
10
100
Radius (nm)
monomer
60
Time (min)
20
10
monomer
90
80
70
60
50
rAmb a 1.0502
40
30
20
10
0
0
0.01
0.1
1.0
10
100
0.01
0.1
Radius (nm)
1.0
10
100
Radius (nm)
Fig. 2:. SEC-MALS and DLS analysis of purified recombinant Amb a 1 isoallergens
Amb a 1 isoallergens were characterised by analytical size-exclusion chromatography connected to a multiangle light scattering detector (SEC-MALS) (left) and by dynamic-light scattering (DLS) (right). Both methods
demonstrate that all four Amb a 1 isoallergens expressed heterologously in P. pastoris are largely monomeric
and have the expected molecular weight.
Fig. 1: SDS-PAGE analysis of purified recombinant Amb a 1 isoallergens
Amb a 1 isoallergens were expressed in P. pastoris (left) and in E. coli (right)
and subsequently purified. For comparison, nAmb a 1 purfied from short
ragweed pollen was applied. Amb a 1 isoallergens expressed in E. coli are
largely aggregated. - DTT, non-reduced; + DTT, reduced.
3
22.0
0.01
monomer
100
90
80
70
60
50
40
Radius (nm)
- DTT
Escherichia coli
Pichia pastoris
20
50
3.0 x 104
10
+ DTT
rAmb a 1.0101
30
0
0.0
10
40
10
4.0 x 104
15
15
1.0 x 105
9.0 x 104
8.0 x 104
50
% Mass
100
75
60
1.0
rAmb a 1.0305
(98,36% monomer)
DLS
monomer
% Mass
(kDa)
70
% Mass
(kDa)
SEC-MALS
Molar mass (g/mol)
MW
Amb a 1 isoallergens purified from P. pastoris are monomeric
UV (RU)
MW
rAmb a 1.0202
rAmb a 1.0101
rAmb a 1.0502
rAmb a 1.0305
rAmb a 1.0202
rAmb a 1.0101
nAmb a 1.01
rAmb a 1.0502
rAmb a 1.0305
rAmb a 1.0202
nAmb a 1.01
rAmb a 1.0101
Heterologous expression of Amb a 1 isoallergens
% Mass
1
Amb a 1.01 and Amb a 1.03 show the highest IgE-binding frequencies in immunoblots
US American ragweed allergic subjects
1
2 3 4
5 6 7
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 K 29 30 31
Italian ragweed allergic subjects
1 2 3
4 5
IgE-binding frequencies
7 8 9 10 11 12 1314 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 6162 63
6
Protein / Extract
Ragweed pollen extract
Deglycosylated ragweed pollen extract
Purified natural nAmb a 1.01
Recombinant rAmb a 1.0101 (P. pastoris)
Recombinant rAmb a 1.0202 (P. pastoris)
Recombinant rAmb a 1.0305 (P. pastoris)
Recombinant rAmb a 1.0502 (P. pastoris)
Recombinant rAmb a 1.0101 (E. coli)
Recombinant rAmb a 1.0202 (E. coli)
Recombinant rAmb a 1.0305 (E. coli)
Recombinant rAmb a 1.0402 (E. coli)
Recombinant rAmb a 1.0502 (E. coli)
Horseradish peroxidase
Bromelain
Ascorbate oxidase
USA
Italy
(n = 24)
(n = 61)
Ragweed extract
(deglycosylated)
100 %
100 %
nAmb a 1
92 %
100 %
rAmb a 1.0101 (P.p.)
71 %
100 %
rAmb a 1.0202 (P.p.)
71 %
90 %
rAmb a 1.0305 (P.p.)
83 %
98 %
rAmb a 1.0402 (E.c.)
4%
51 %
rAmb a 1.0502 (P.p.)
0%
43 %
P.p. = Pichia pastoris E.c. = Escherichia coli.
Fig. 3: IgE-reactivity of ragweed allergic subjects to purified allergens and extracts (immunoblot)
Reactivity of human IgE to proteins/extracts bound to membrane strips was investigated by colorimetric detection following binding of alkaline phosphatase coupled anti-human IgE secondary
antibody. Plasma from 28 individual ragweed allergic subjects and three plasma pools from the US (both obtained from Plasmalab International, Everett, WA) were analysed (left). In addition, 63 sera
of ragweed allergic individuals from Italy were investigated (middle). One serum obtained from a German allergic donor with a known high titre to cross-reactive carbohydrate determinants (CCDs)
was included (K). CCD-bearing proteins (horseradish peroxidase, bromelain, ascorbate oxidase) were used as positive CCD controls. Ragweed pollen extract and deglycosylated ragweed pollen
extract (periodate treated) were compared for exclusion of pure CCD-reactive individuals. Seven of 31 samples in the US American group and two of 63 Italien sera showed no reaction to
unglycosylated ragweed extract or to recombinant Amb a 1 and were therefore excluded from the final calculation of IgE binding frequencies (right).
4
Amb a 1.01, 1.02 and 1.03 apparently contain most of the relevant ragweed IgE-epitopes
Plasma 1 (US American ragweed allergic subject)
Extract
60
rAmb a 1.01 P.p.
40
nat. Amb a 1.01
20
0
rAmb a 1.01 E.c.
1:104
1:103
1:102
Dilution
Extract
60
rAmb a 1.02 P.p.
40
rAmb a 1.03 P.p.
20
0
1:10
rAmb a 1.01 P.p.
rAmb a 1.04 E.c.
rAmb a 1.05 P.p.
1:104
1:103
1:102
Dilution
1:10
100
80
80
Extract
60
rAmb a 1.01 P.p.
40
nat. Amb a 1.01
20
0
rAmb a 1.01 E.c.
1:104
1:103
1:102
Dilution
% Inhibition
80
100
% Inhibition
80
% Inhibition
100
% Inhibition
100
Plasma 6 (US American ragweed allergic subject)
Extract
60
rAmb a 1.02 P.p.
40
rAmb a 1.03 P.p.
20
0
1:10
rAmb a 1.01 P.p.
rAmb a 1.04 E.c.
rAmb a 1.05 P.p.
1:104
1:103 1:102
Dilution
1:10
Fig. 4: Analysis of IgE-reactivity by
IgE-inhibition (EAST)
Assays were conducted with plasma from
two US American ragweed allergic
subjects (see also Fig. 3). Natural
ragweed pollen extract was coated as
solid phase. For inhibition serial dilutions
of extract and purified Amb a 1 isoallergens (starting protein conc.: 50 µg/ml
and 20 µg/ml, respectively) were applied.
P.p., Pichia pastoris; E.c., Echerichia coli.
Conclusion
Amb a 1.01 expressed in P. pastoris is immunologically largely comparable with natural Amb 1.01. Based on IgE-immunoblots and IgE-inhibition Amb 1.01, Amb a 1.03 and
Amb a 1.02 are the most relevant Amb a 1 isoallergens. For final selection of the isoallergen/s suitable for immunotherapy more ragweed allergic patients' sera need to be
investigated with respect to isoallergen cross-reactivity and by analysing T-cell cross-reactivities of the different isoallergens.
Conflict of interest: SA, MW, GR, SK and AN are employees of Allergopharma GmbH & Co. KG. RA has nothing to declare.
EAACI & WAO World Allergy & Asthma Congress 2013, 22 - 26 June, Milan, Italy I Contact: Allergopharma GmbH & Co. KG I 21462 Reinbek, Germany I Phone ++49 40 72765-0 • Fax ++49 40 7227713 • www.allergopharma.com