hal allergy abstractbook eaaci 2015

Scientific Contribution 2015 34th Congress of the European Academy of Allergy and Clinical Immunology

EAACI, 6-10 June 2015, Barcelona, Spain

Dear Congress delegate,On behalf of HAL Allergy, we like to welcome you to the EAACI 2015 congress in Barcelona. The theme of this years congress is Allergy: new answers to old questions. This theme fits HAL Allergy very well. HAL Allergy, is an old (more than 55 years) and established company with a long and successful history in the field of allergen immunotherapy. Nevertheless, the company has commit-ted itself to continue to generate new answers to questions coming from the immunotherapy field: be it on the characterization and clinical efficacy of exis-ting products; or about the development of novel products and product claims.

In this abstract book you will find our abstracts dealing with various clinical studies and product development activities, including information on specific dates and times of the presentations.

HAL Allergys clinical development program, aimed at gaining registration for specific immunotherapy products, is progressing successfully. This year, we are presenting the design of the Phase III pivotal study for SUBLIVAC Birch. This study follows the completion of the Phase II dose range finding study in 2013. We also present clinical data on the possibility of accelerated up-dosing with our PURETHAL Birch product. This study resulted in the registration of the rush up-dosing scheme for our entire PURETHAL Pollen portfolio.

At this years meeting we also present two abstracts on the characterization and stability of our allergen products. Using antibody-based and physicoche-mical methods (i.e., mass spectrometry and circular dichroism) we compare and characterize wasp venom obtained from different sources, and perform extensive analysis on the stability of Phleum pratense extract.

Furthermore, we present data on the development of a manufacturing plat-form for recombinant Bet v1.0101 using CHO cells. This work was performed using the capability of HALIX, our subsidiary for contract manufacturing of clinical trial materials for third parties. It is shown that a recombinant form of Betula protein can be produced at an industrial scale.

We hope you will have a successful congress. If you require further information about our products, or our R&D program, please do not hesitate to visit our booth in the exhibition area.

Kind regards,

Dirk-Jan Opstelten, PhD Diderik Boot, PhDResearch & Development Director Medical Director

Diderik Boot, PhDMedical Director

Dirk-Jan Opstelten, PhDResearch & DevelopmentDirector

Scientific Contribution 2015 Contents

Clinical

Design of a phase III short-term efficacy study with allergen specific immunotherapy in patients with birch pollen induced allergic rhinitis/rhinoconjunctivitis ......................................................................... 6Poor correlation between symptom scores and absolute levels of allergen-specific serum IgG4 in allergic rhinitis .............................................................................................................................................. 8Accelerated up-dosing with a registered allergoid birch pollen SCIT preparation is non-inferior to conventional up-dosing ..................................................................................................................... 10Sublingual immunotherapy against alternaria allergens in patients with asthma ....................................................... 12Accelerated and conventional up-dosing with a registered allergoid birch pollen SCIT preparation: a comparison of tolerability between adults and adolescents ........................................................ 14Safety of a cluster-immunotherapy with a depot allergoid in patients with allergic rhinitis against grasses ...................................................................................................................................... 16

Development

Large Scale Manufacturing of Recombinant Bet v1.0101 in Chinese Hamster Ovary cells ......................................... 18Stress stability study on a Phleum pratense extract ...................................................................................................... 20Characterisation of wasp venom collected via electrostimulation or venom sac extraction ....................................... 22

Design of a phase III short-term efficacy study with allergen specific immunotherapy in patients with birch pollen induced allergic rhinitis/rhinoconjunctivitis.

O. Pfaar1, P. Kuna2, P. Panzner3, M. Dupinov4, M.J. van Nimwegen5, D. Boot5, C. Bachert6

1 Center for Rhinology and Allergology Wiesbaden, Germany; 2 Poradnia Alergologii i Chorb Puc Lodz, Poland; 3 Ustav imunologie a alergologie, Plzen,

Czech Republic; 4 ALIAN s.r.o. Ambulancia alergolgie a klinickej imunolgie, Bardejov, Slovakia; 5 HAL Allergy BV, Medical Department, Leiden, The

Netherlands; 6 University of Ghent, Clinical Trial Center, Upper Airways Research Laboratory, Ghent, Belgium.

BackgroundIn order to comply with the 2008 EMA guidelines on the development of SIT products, a clinical development program was

started to obtain full marketing authorization for a sublingual IT product for the treatment of birch pollen allergy. Previously

the optimal dose has been determined in a phase II dose finding study. The next step is, in patients with birch pollen induced

allergic rhinitis/rhinoconjunctivitis (AR), to determine the short-term efficacy. The recommended end-point is a combined

symptom and medication score (CSMS) during the pollen season. However, there is a wide variety in both the applied CSMS

scores and in the definition of the pollen season. This abstract highlights the design of the phase III study.

MethodThe current study is a randomized, double-blind, placebo-controlled, parallel-group study in 400 adult patients (200 per arm),

with moderate to severe birch pollen induced AR with or without mild to moderate persistent asthma (ClinicalTrials.gov

NCT02231307). Treatment is started at least 12 weeks before the pollen season whereby the birch pollen season is defined as

3 out of 5 consecutive days birch pollen counts with 80 grains/m3 per 24 hours and the birch peak pollen season is defined as

all days with birch pollen counts 500 grains/m3 per 24 hours. Recently, an EAACI task force has recommended the use of the

CSMS as the primary endpoint whereby 6 organ related symptoms (4 nasal, 2 ocular) and the need for anti-allergic medication

(stepwise) are balanced in an equally weighted manner. We have selected this score and, in accordance with guidelines, the

minimal clinically important difference in the primary endpoint between test and control population has been predefined

and justified upfront (23% decrease compared to placebo). The study is performed in 42 clinical study centers in 5 European

countries.

ResultsThe first patient was recruited in September 2014 and the screening target was met on 21st of October, resulting in 406

randomized patients. The results will be available in the second half of 2015.

ConclusionAccording to the EMA guidelines we designed a Phase III short-term efficacy study to identify the effect of the optimal

dose of SUBLIVAC FIX Birch SLIT in birch pollen allergic rhinitis/rhinoconjunctivitis patients. We implemented the recently

recommended EAACI CSMS score as the primary endpoint and the minimal clinically important difference has been defined

and justified upfront.

Abstract number: 1101 Session number, date and time: TPS 30, Monday 8 June 2015; 12:00 - 13:30

Session title: Sensitization, allergen extracts and efficacy of AIT

6 EAACI, 6-10 June 2015, Barcelona, Spain

abstract 2

TPS 30 - Sensitization, allergen extracts and efficacy of AIT


Poor correlation between symptom scores and absolute levels of allergen-specific serum IgG4 in allergic rhinitis.

O. Pfaar1,2, E. van Twuijver3, D. Boot3, R. El Galta3, L. Klimek1, R. van Ree4, P. Kuna5, P. Panzner6

1 Center for Rhinology and Allergology Wiesbaden, Germany, 2Department of Otorhinolaryngology, Head and Neck Surgery, Universittsmedizin

Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany, 3HAL Allergy BV, Leiden, The Netherlands, 4Department of

Experimental Immunology, Academic Medical Center, University of Amsterdam, The Netherlands, 5Barlicki University Hospital, Medical University of

Lodz, Poland, 6Dept. of Allergology and Immunology, Medical Faculty in Plzen, Charles University Prague, Czech Republic.

BackgroundThe efficacy of specific immunotherapy has been confirmed in numerous trials in patients with allergic rhinitis (AR) induced

by aeroallergens. Enhancement of clinical benefit might be feasible if biomarkers are available to identify (non-)responders,

to determine the optimal treatment period and to predict relapse. Therefore, surrogate endpoints might be crucial for the

development of new vaccines and to improve current treatment regimes.

MethodsIn a 5-month phase II study, 269 adult subjects (134 females;135 males) with birch pollen induced AR were treated with a

sublingual birch pollen preparation (3,333; 10,000; 20,000 or 40,000 AUN/ml) or with placebo. The absolute difference in

mean symptom score following a titrated nasal provocation test (TNPT) and the change in birch pollen specific serum IgG4

levels between 5 months of treatment and baseline were determined. Assessment of the correlation between the change from

baseline in symptom scores and log serum IgG4 levels was done using a bivariate normal model with and without adjustment for the treatment factor. The dependence was modelled using an unstructured covariance matrix.

ResultsFollowing 5 months of treatment a decrease in symptom scores was observed in all treatment groups, with active treatment

showing a larger decrease than placebo. Serum IgG4 levels significantly increased in all active treatment groups, but not in the placebo group. Assessing the correlation between symptom scores following TNPT and IgG4 levels with and without adjustment for the treatment effect indicated a lack of association between the two parameters.

ConclusionNo significant association could be demonstrated between the change in symptom score and birch pollen specific IgG4 levels. This confirms earlier reports that an increase in IgG4 levels per se is not predictive for clinical outcome. Testing for IgG4-

associated inhibitory activity rather than absolute IgG4 levels might have a better correlation with clinical outcome.

Abstract number: 1113Session number, date and time: TPS 30, Monday 8 June 2015; 12:00 - 13:30


8 EAACI, 6-10 June 2015, Barcelona, SpainEAACI, 6-10 June 2015, Barcelona, Spain 7

HAL Abstractbook 2015 A3 DEF.indd 2 20-05-15 15:56

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abstract 2




O. Pfaar1,2, E. van Twuijver3, D. Boot3, R. El Galta3, L. Klimek1, R. van Ree4, P. Kuna5, P. Panzner6

1 Center for Rhinology and Allergology Wiesbaden, Germany, 2Department of Otorhinolaryngology, Head and Neck Surgery, Universittsmedizin

Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany, 3HAL Allergy BV, Leiden, The Netherlands, 4Department of

Experimental Immunology, Academic Medical Center, University of Amsterdam, The Netherlands, 5Barlicki University Hospital, Medical University of

Lodz, Poland, 6Dept. of Allergology and Immunology, Medical Faculty in Plzen, Charles University Prague, Czech Republic.

BackgroundThe efficacy of specific immunotherapy has been confirmed in numerous trials in patients with allergic rhinitis (AR) induced

by aeroallergens. Enhancement of clinical benefit might be feasible if biomarkers are available to identify (non-)responders,

to determine the optimal treatment period and to predict relapse. Therefore, surrogate endpoints might be crucial for the

development of new vaccines and to improve current treatment regimes.

MethodsIn a 5-month phase II study, 269 adult subjects (134 females;135 males) with birch pollen induced AR were treated with a

sublingual birch pollen preparation (3,333; 10,000; 20,000 or 40,000 AUN/ml) or with placebo. The absolute difference in

mean symptom score following a titrated nasal provocation test (TNPT) and the change in birch pollen specific serum IgG4

levels between 5 months of treatment and baseline were determined. Assessment of the correlation between the change from

baseline in symptom scores and log serum IgG4 levels was done using a bivariate normal model with and without adjustment for the treatment factor. The dependence was modelled using an unstructured covariance matrix.

ResultsFollowing 5 months of treatment a decrease in symptom scores was observed in all treatment groups, with active treatment

showing a larger decrease than placebo. Serum IgG4 levels significantly increased in all active treatment groups, but not in the placebo group. Assessing the correlation between symptom scores following TNPT and IgG4 levels with and without adjustment for the treatment effect indicated a lack of association between the two parameters.

ConclusionNo significant association could be demonstrated between the change in symptom score and birch pollen specific IgG4 levels. This confirms earlier reports that an increase in IgG4 levels per se is not predictive for clinical outcome. Testing for IgG4-

associated inhibitory activity rather than absolute IgG4 levels might have a better correlation with clinical outcome.

Abstract number: 1113Session number, date and time: TPS 30, Monday 8 June 2015; 12:00 - 13:30




Accelerated up-dosing with a registered allergoid birch pollen SCIT preparation is non-inferior to conventional up-dosing.

Krzysztof Buczyko1, Jan F van der Werf2, Diderik Boot2, Ronald van Ree3, Bernadetta Majorek-Olechowska4.1NZOZ Centrum Alergologii, d, Poland, 2HAL Allergy B.V., Leiden, The Netherlands, 3Department of Experimental Immunology, Academic Medical

Center, University of Amsterdam, The Netherlands, 4Alergo-Med Specjalistyczna Przychodnia Lekarska Sp. z o.o. in Tarnow, Poland

BackgroundThe conventional registered up-dosing regimen (C-reg.) of the allergoid birch pollen preparation under evaluation requires

six injections over five weeks. It is recommended to reach the maintenance dose before the pollen season. Due to climatic

changes causing early pollen flight and new allergens prolonging the pollen season it is difficult, however, to up-dose outside

the pollen season. An accelerated up-dosing regimen (A-reg.) would also be helpful for patients with multiple pollen allergies,

patients coming near the beginning of pollen season, and patients who wish to up-dose faster. We performed a clinical study

to investigate if A-reg. is as safe as C-reg.

MethodsIn birch pollen allergic patients presenting with rhinitis or rhinoconjunctivitis with or without mild asthma (FEV1 > 70%) an

A-reg. (0.1-0.3-0.5 ml at weekly intervals) was compared to the C-reg. (0.05-0.1-0.2-0.3-0.4-0.5 ml at weekly intervals) using

a glutaraldehyde-modified birch pollen preparation adsorbed to aluminium hydroxide. After up-dosing the maintenance dose

(0.5 ml) was given 3 times at biweekly intervals. The multi-centre study was performed according to an open randomized,

parallel group design. Success rate was based on the (predefined) need of additional up-dosing steps because of the occurrence

of local and systemic reactions. Cases with more than 2 extra visits or a systemic reaction > grade I were considered as failures.

ResultsA total of 123 birch pollen allergic patients (81 adults, 42 adolescents) were randomized to either the C-reg. (62 patients) or

A-reg. (61 patients). A high proportion of patients successfully reached the maintenance dose without safety concerns in both

groups (98.4% and 96.7% for A-reg. and C-reg., respectively). The two-sided 95% confidence interval (CI) for the difference in

proportions between the two treatment groups was (3.8% - 7.1%), confirming non-inferiority of the A-reg. treatment.

No differences were observed between adult and adolescents, the latter age group having a 100% success rate in both A-reg.

and C-reg. At the end of study, significant increases in specific IgG and IgG4 were observed in both groups, independent from

age.

ConclusionThe accelerated SCIT regimen is as safe as the conventional and might be used to up-dose adult as well adolescent patients

within 2 weeks. Moreover, both up-dosing regimens resulted in similar immunological effects, as assessed after 3 injections

in the maintenance phase.

Abstract number: 1098

Session number, date and time: TPS 30, Monday 8 June 2015; 12:00 - 13:30





abstract 3


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BackgroundThe conventional registered up-dosing regimen (C-reg.) of the allergoid birch pollen preparation under evaluation requires

six injections over five weeks. It is recommended to reach the maintenance dose before the pollen season. Due to climatic

changes causing early pollen flight and new allergens prolonging the pollen season it is difficult, however, to up-dose outside

the pollen season. An accelerated up-dosing regimen (A-reg.) would also be helpful for patients with multiple pollen allergies,

patients coming near the beginning of pollen season, and patients who wish to up-dose faster. We performed a clinical study

to investigate if A-reg. is as safe as C-reg.

MethodsIn birch pollen allergic patients presenting with rhinitis or rhinoconjunctivitis with or without mild asthma (FEV1 > 70%) an

A-reg. (0.1-0.3-0.5 ml at weekly intervals) was compared to the C-reg. (0.05-0.1-0.2-0.3-0.4-0.5 ml at weekly intervals) using

a glutaraldehyde-modified birch pollen preparation adsorbed to aluminium hydroxide. After up-dosing the maintenance dose

(0.5 ml) was given 3 times at biweekly intervals. The multi-centre study was performed according to an open randomized,

parallel group design. Success rate was based on the (predefined) need of additional up-dosing steps because of the occurrence

of local and systemic reactions. Cases with more than 2 extra visits or a systemic reaction > grade I were considered as failures.

ResultsA total of 123 birch pollen allergic patients (81 adults, 42 adolescents) were randomized to either the C-reg. (62 patients) or

A-reg. (61 patients). A high proportion of patients successfully reached the maintenance dose without safety concerns in both

groups (98.4% and 96.7% for A-reg. and C-reg., respectively). The two-sided 95% confidence interval (CI) for the difference in

proportions between the two treatment groups was (3.8% - 7.1%), confirming non-inferiority of the A-reg. treatment.

No differences were observed between adult and adolescents, the latter age group having a 100% success rate in both A-reg.

and C-reg. At the end of study, significant increases in specific IgG and IgG4 were observed in both groups, independent from

age.

ConclusionThe accelerated SCIT regimen is as safe as the conventional and might be used to up-dose adult as well adolescent patients

within 2 weeks. Moreover, both up-dosing regimens resulted in similar immunological effects, as assessed after 3 injections

in the maintenance phase.







abstract 3


Sublingual immunotherapy against alternaria allergens in patients with asthma.

Antonio Carbonell Martnez1, Ana Escudero Pastor1, Juan Carlos Miralles Lpez1, Mara-Jess Cruz Carmona2.1 Hospital Reina Sofa, Murcia, 2 HAL Allergy, Spain

IntroductionSublingual immunotherapy (SLIT) is safe and effective, but data concerning this treatment against Alternaria allergy are

insufficient. The aim of this study was to evaluate the safety of SLIT against Alternaria and the possible changes in the need for

medication after one year of treatment in patients with asthma.

Material and methods44 patients (30 men), mean age 16 years, diagnosed with allergic asthma against Alternaria were included. Patients were

treated with SLIT against Alternaria alternata using a dosage schedule where the maintenance dose is reached after 5 days

of treatment (5 drops per day). All patients had primary treatment with inhaled corticosteroids, long acting beta agonist and

montelukast. During the study, control visits were performed during clinic visits at six months and after one year of treatment.

The doctor provided an assessment of asthma control and listed all medications prescribed.

ResultsIn all patients it was possible to achieve the maintenance dose, except in one case where the dose was reduced to three

drops after presenting oral itching and abdominal pain. No more side effects were observed during treatment. Regarding

the need for medication, after a year of monitoring, 17 patients (39%) did not require any additional treatment, 14 patients

(32%) needed rescue treatment only (Salbutamol or Ebastine 10 g) in 8 patients (18%) it was not possible to reduce the

daily dose of inhaled corticosteroids (budesonide 200 g / every 12 hours) and finally in 5 patients (11%) the SLIT failed to

modify the initial treatment (budesonide 160 g and formoterol 4.5 g, 1 dose every 12 hours and montelukast 10 mg). In the

latter group, 3 patients (7%) attended the emergency department during the study period and required treatment with oral

corticosteroids, no hospital admissions were observed.

ConclusionsSLIT is a safe treatment for allergic asthma to Alternaria. After a year of SLIT, it is possible to decrease the pharmacological

treatment in approximately 70% of patients.







abstract 4


1098

A

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erat

ed u

p-do

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gist

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Antonio Carbonell Martnez1, Ana Escudero Pastor1, Juan Carlos Miralles Lpez1, Mara-Jess Cruz Carmona2.1 Hospital Reina Sofa, Murcia, 2 HAL Allergy, Spain

IntroductionSublingual immunotherapy (SLIT) is safe and effective, but data concerning this treatment against Alternaria allergy are

insufficient. The aim of this study was to evaluate the safety of SLIT against Alternaria and the possible changes in the need for

medication after one year of treatment in patients with asthma.

Material and methods44 patients (30 men), mean age 16 years, diagnosed with allergic asthma against Alternaria were included. Patients were

treated with SLIT against Alternaria alternata using a dosage schedule where the maintenance dose is reached after 5 days

of treatment (5 drops per day). All patients had primary treatment with inhaled corticosteroids, long acting beta agonist and

montelukast. During the study, control visits were performed during clinic visits at six months and after one year of treatment.

The doctor provided an assessment of asthma control and listed all medications prescribed.

ResultsIn all patients it was possible to achieve the maintenance dose, except in one case where the dose was reduced to three

drops after presenting oral itching and abdominal pain. No more side effects were observed during treatment. Regarding

the need for medication, after a year of monitoring, 17 patients (39%) did not require any additional treatment, 14 patients

(32%) needed rescue treatment only (Salbutamol or Ebastine 10 g) in 8 patients (18%) it was not possible to reduce the

daily dose of inhaled corticosteroids (budesonide 200 g / every 12 hours) and finally in 5 patients (11%) the SLIT failed to

modify the initial treatment (budesonide 160 g and formoterol 4.5 g, 1 dose every 12 hours and montelukast 10 mg). In the

latter group, 3 patients (7%) attended the emergency department during the study period and required treatment with oral

corticosteroids, no hospital admissions were observed.

ConclusionsSLIT is a safe treatment for allergic asthma to Alternaria. After a year of SLIT, it is possible to decrease the pharmacological

treatment in approximately 70% of patients.







abstract 4


Accelerated and conventional up-dosing with a registered allergoid birch pollen SCIT preparation: a comparison of tolerability between adults and adolescents.



BackgroundRecently we compared accelerated (A-reg.) and conventional (C-reg.) up-dosing regimens with an allergoid birch pollen SCIT

preparation. The A-reg. was found to be non-inferior thus providing a safe option to up-dose patients within 2 weeks. In the

study both adults and adolescents were included, below an overview of all safety parameters will be given, comparing both populations.

MethodsIn birch pollen allergic rhinitis/rhinoconjunctivitis patients with or without mild asthma (FEV1 > 70%) an A-reg. (0.1-0.3-0.5

ml at weekly intervals) was compared to the C-reg. (0.05-0.1-0.2-0.3-0.4-0.5 ml at weekly intervals) using a glutaraldehyde-

modified birch pollen preparation adsorbed to aluminium hydroxide. After up-dosing 3 maintenance doses (0.5 ml) were

given. The multi-centre study was performed according to an open randomized, parallel group design. Early and late (within

30 min and within 24 hrs after injection) local (ELRs, LLRs) and systemic reactions (ESRs, LSRs) as well as general adverse events

(AEs) were recorded. Vital signs were also monitored.

ResultsA total of 123 birch pollen allergic patients were randomized to either the C-reg. (62 patients, 41 adults) or A-reg. (61 patients,

40 adults).

The majority of the subjects did not show ELRs, only wheals 5 cm diameter were found with similar frequencies for both age

groups. Regarding LLRs, 24.4% (n=30) of the subjects reported reactions with wheals 5 cm and 16.3% (n=20) with wheals > 5

cm 12 cm. One reaction with wheal > 12 cm was observed in the adult group (C-reg.). For the LLRs no consistent differences

between age groups were found.

Concerning ESRs the vast majority of subjects experienced no symptoms or non-specific symptoms (grade 0). None of the

adolescents and only one adult patient had an ESR grade I. LSRs of grade I occurred in 3.3 (C-reg.) to 6.5% (A-reg.) of the

subjects. No consistent differences between the adult and adolescent groups were found.

146 treatment-emergent (TE) AEs related to the medication were reported. The percentage of patients with a related TEAE

appears somewhat lower in the adolescent compared to the adult population.

ConclusionIn general a good tolerability was observed for both A-reg. and C-reg. and only small differences in the frequencies of local and

systemic reactions and general AEs between the two age groups were observed. In both treatment groups there is a tendency

towards better tolerance by the adolescent patients as compared to the adults.


Session number, date and time: PDS 25, Tuesday 9 June 2015; 10:30 - 12:00

Session title: Immunotherapy vaccines




abstract 5


1107

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= 44

Diag

nosis

, %

A

sthm

a

Asth

ma

+ Rh

initis

20

80

Adve

rse

reac

tions

afte

r SLI

T, n

* 1

Emer

genc

y dep

t. ad

miss

ions d

uring

SLI

T, n

3

0

39

20

32

40

18

40

11

051015202530354045

Base

line

After

1 yr

of tre

atmen

t

% patients

No tre

atmen

tRe

scue

med

icatio

n*Inh

aled c

ortico

steroi

ds**

Cortic

oster

oids +

LABA

+ Mo

nteluk

ast**

*

Tabl

e 2:

Alle

rgy

hist

ory

& m

ost

freq

uent

rel

ated

adv

erse

rea

ction

s. EAAC

I Con

gres

s 201

5In

rela

tion

to th

is p

rese

ntati

on, I

dec

lare

the

follo

win

g, re

al o

r per

ceiv

ed c

oni

ct o

f int

eres

t. M

J Cru

z is

an

empl

oyee

of H

AL A

llerg

y.





BackgroundRecently we compared accelerated (A-reg.) and conventional (C-reg.) up-dosing regimens with an allergoid birch pollen SCIT

preparation. The A-reg. was found to be non-inferior thus providing a safe option to up-dose patients within 2 weeks. In the

study both adults and adolescents were included, below an overview of all safety parameters will be given, comparing both populations.

MethodsIn birch pollen allergic rhinitis/rhinoconjunctivitis patients with or without mild asthma (FEV1 > 70%) an A-reg. (0.1-0.3-0.5

ml at weekly intervals) was compared to the C-reg. (0.05-0.1-0.2-0.3-0.4-0.5 ml at weekly intervals) using a glutaraldehyde-

modified birch pollen preparation adsorbed to aluminium hydroxide. After up-dosing 3 maintenance doses (0.5 ml) were

given. The multi-centre study was performed according to an open randomized, parallel group design. Early and late (within

30 min and within 24 hrs after injection) local (ELRs, LLRs) and systemic reactions (ESRs, LSRs) as well as general adverse events

(AEs) were recorded. Vital signs were also monitored.

ResultsA total of 123 birch pollen allergic patients were randomized to either the C-reg. (62 patients, 41 adults) or A-reg. (61 patients,

40 adults).

The majority of the subjects did not show ELRs, only wheals 5 cm diameter were found with similar frequencies for both age

groups. Regarding LLRs, 24.4% (n=30) of the subjects reported reactions with wheals 5 cm and 16.3% (n=20) with wheals > 5

cm 12 cm. One reaction with wheal > 12 cm was observed in the adult group (C-reg.). For the LLRs no consistent differences

between age groups were found.

Concerning ESRs the vast majority of subjects experienced no symptoms or non-specific symptoms (grade 0). None of the

adolescents and only one adult patient had an ESR grade I. LSRs of grade I occurred in 3.3 (C-reg.) to 6.5% (A-reg.) of the

subjects. No consistent differences between the adult and adolescent groups were found.

146 treatment-emergent (TE) AEs related to the medication were reported. The percentage of patients with a related TEAE

appears somewhat lower in the adolescent compared to the adult population.

ConclusionIn general a good tolerability was observed for both A-reg. and C-reg. and only small differences in the frequencies of local and

systemic reactions and general AEs between the two age groups were observed. In both treatment groups there is a tendency

towards better tolerance by the adolescent patients as compared to the adults.


Session number, date and time: PDS 25, Tuesday 9 June 2015; 10:30 - 12:00

Session title: Immunotherapy vaccines




abstract 5


Safety of a cluster-immunotherapy with a depot allergoid in patients with allergic rhinitis against grasses.

Susana Monzn Ballarn1, Nuria Prez Cinto2, Raquel Montijano Sanchez3, Mara-Jess Cruz Carmona4.1 Consulta de Alergia. Consorcio Aragones de Salud. Ejea-Tarazona. Zaragoza, Spain; 2 Consulta de Alergia. Consorcio Aragons de Salud. Centro Cinco

Villas. Ejea de los Caballeros. Zaragoza, Spain; 3 Consulta de Alergia. Consorcio Aragons de Salud. Centro Moncayo de Tarazona. Zaragoza; 4 HAL

Allergy, Spain.

IntroductionDespite the benefits of subcutaneous immunotherapy (SCIT), some patients refuse treatment, partly because of the associated

inconvenience. The cluster schemes can provide a more convenient treatment option, but there is a perception that they

produce a higher rate of adverse reactions. The aim of this study is to investigate the safety and efficacy of a cluster scheme

with a depot allergoid in patients with allergic rhinitis against grasses.

Material and methodsThirty patients (14 males), mean age 21 years, diagnosed with allergic rhinitis against grasses with or without mild persistent

asthma were included. Patients were treated with SIT. The treatment will be administered with an initial dose of 0.5 ml of

the maintenance vial (first injection 0.2 ml + 0.3 ml, at intervals of 30 min between them, and a waiting period of one hour

after the second dose), reaching a maximum dose of 0.5 ml two weeks later and repeating it each month. The safety of the

treatment will be evaluated with a patient questionnaire on the basis of side effects. The symptoms and need for additional

symptomatic medication during the season were also assessed before and after treatment.

ResultsThe proposed dose could be administered in all patients. No local reactions occurred during the clustered injections. In 3

patients (10%) late mild local reaction was observed at the 0.5 mL dose at two weeks (mean wheal size: 4.5 cm). No systemic

adverse reactions were observed. Regarding the need for medication, at baseline all patients were taking antihistaminics and

nasal steroids. Eleven patients also needed 2-agonists on demand. After a year of follow-up, six patients (20%) did not require

any additional treatment, 22 patients (73%) only needed antihistaminics and 7 of them (23%) plus 2-agonists on demand.

Finally, in two patients (7%) was not possible to modify the initial treatment.

ConclusionsFrequency and severity of adverse side effects in cluster-SIT correspond to those described on literature in conventional

dosage schedule. This SCIT treatment generated a significant improvement regarding the need of medication. The modified

extract used in the study appears to be a safe and well-tolerated treatment for allergy in patients with allergic rhinitis against

grasses.


Session number, date and time: TPS 49, Tuesday 9 June 2015; 12:00 - 13:30

Session title: Safety of immunotherapy and immunotherapy for food allergy


PDS 25 - Immunotherapy vaccines


abstract 6


529

Ac

cele

rate

d an

d co

nven

tiona

l up-

dosi

ng w

ith a

regi

ster

ed a

llerg

oid

birc

h po

llen

SCIT

pre

para

tion:

a c

ompa

rison

of t

oler

abili

ty b

etw

een

adul

ts a

nd a

dole

scen

ts

Con

vent

iona

l

No

Rea

ctio

n>

5 bu

t 1

2 cm

>12

cm

5 cm

All

Adu

ltA

dole

scen

t

(%)

020406080100

LLR

s

(%)

(%)

All

Adu

ltA

dole

scen

t

(%)

(%)

(%)

(%)

Acc

eler

ated

Tota

l

Con

vent

iona

l No

Rea

ctio

nG

rade

IG

rade

0

All

Adu

ltA

dole

scen

t

(%)

0%20%

40%

60%

80%

100%

LSR

s

(%)

(%)

All

Adu

ltA

dole

scen

t

(%)

(%)

(%)

(%)

Acc

eler

ated

Tota

l

EAAC

I Con

gres

s 20

15In

rela

tion

to th

is p

rese

ntati

on, I

dec

lare

the

follo

win

g, re

al o

r per

ceiv

ed c

oni

ct o

f int

eres

t. T

he p

rese

nter

is a

em

ploy

ee o

f HAL

Alle

rgy.



Susana Monzn Ballarn1, Nuria Prez Cinto2, Raquel Montijano Sanchez3, Mara-Jess Cruz Carmona4.1 Consulta de Alergia. Consorcio Aragones de Salud. Ejea-Tarazona. Zaragoza, Spain; 2 Consulta de Alergia. Consorcio Aragons de Salud. Centro Cinco

Villas. Ejea de los Caballeros. Zaragoza, Spain; 3 Consulta de Alergia. Consorcio Aragons de Salud. Centro Moncayo de Tarazona. Zaragoza; 4 HAL

Allergy, Spain.

IntroductionDespite the benefits of subcutaneous immunotherapy (SCIT), some patients refuse treatment, partly because of the associated

inconvenience. The cluster schemes can provide a more convenient treatment option, but there is a perception that they

produce a higher rate of adverse reactions. The aim of this study is to investigate the safety and efficacy of a cluster scheme

with a depot allergoid in patients with allergic rhinitis against grasses.

Material and methodsThirty patients (14 males), mean age 21 years, diagnosed with allergic rhinitis against grasses with or without mild persistent

asthma were included. Patients were treated with SIT. The treatment will be administered with an initial dose of 0.5 ml of

the maintenance vial (first injection 0.2 ml + 0.3 ml, at intervals of 30 min between them, and a waiting period of one hour

after the second dose), reaching a maximum dose of 0.5 ml two weeks later and repeating it each month. The safety of the

treatment will be evaluated with a patient questionnaire on the basis of side effects. The symptoms and need for additional

symptomatic medication during the season were also assessed before and after treatment.

ResultsThe proposed dose could be administered in all patients. No local reactions occurred during the clustered injections. In 3

patients (10%) late mild local reaction was observed at the 0.5 mL dose at two weeks (mean wheal size: 4.5 cm). No systemic

adverse reactions were observed. Regarding the need for medication, at baseline all patients were taking antihistaminics and

nasal steroids. Eleven patients also needed 2-agonists on demand. After a year of follow-up, six patients (20%) did not require

any additional treatment, 22 patients (73%) only needed antihistaminics and 7 of them (23%) plus 2-agonists on demand.

Finally, in two patients (7%) was not possible to modify the initial treatment.

ConclusionsFrequency and severity of adverse side effects in cluster-SIT correspond to those described on literature in conventional

dosage schedule. This SCIT treatment generated a significant improvement regarding the need of medication. The modified

extract used in the study appears to be a safe and well-tolerated treatment for allergy in patients with allergic rhinitis against

grasses.


Session number, date and time: TPS 49, Tuesday 9 June 2015; 12:00 - 13:30

Session title: Safety of immunotherapy and immunotherapy for food allergy


PDS 25 - Immunotherapy vaccines


abstract 6


Large Scale Manufacturing of Recombinant Bet v1.0101 in Chinese Hamster Ovary cells.

J. Rook1, H. Warmenhoven2, O. Brugman1, D. Verbart1, A. Huybens1 and H. van Schijndel2

1 HALIX BV, Leiden, The Netherlands; 2 HAL Allergy BV, Leiden, The Netherlands

BackgroundChinese Hamster Ovary (CHO) cells are the most widely used expression system for the production of therapeutic proteins.

Compared to bacterial expression systems, CHO cells possess human-like posttranslational modification machineries enabling

the production of properly folded recombinant proteins while no endotoxins are produced. Previously, a stable CHO cell line

(CHO-rBet v1.0101) was developed for expression of Bet v1.0101, the immunodominant isoform of the major birch pollen

allergen Bet v1. In this study, a large scale bioreactor manufacturing process was developed to optimize the yield.

MethodsTo assess the feasibility of a large scale manufacturing process, a single use 2 L shaker bag and traditional 10 L bioreactor setup

were inoculated with stably growing pre-cultures of CHO-rBet v1.0101 cells and cultured using a fed batch feeding strategy.

Cell density, viability and metabolites were monitored at regular time intervals. Expression of rBet v1.0101 was measured

using a in house developed Bet v1 ELISA kit. In addition, several commercially available CHO feeds were tested in a fed batch

shaker flask setup to optimize the cell growth and rBet v1.0101 expression.

ResultsCompared to a previously, glucose-fed only batch culture, the Bet v1.0101 titre obtained with the single use 2 L shaker bag and

the 10 L bioreactor were respectively 2.4 and 4 fold higher. Testing several commercially available CHO feeds showed higher

Bet v1.0101 concentrations, viable cell densities and prolonged viability. The best feed yielded 14 times more Bet v1.0101

compared to the glucose-fed only batch culture.

ConclusionIn this study, the feasibility of the CHO production platform for large scale manufacturing of recombinant Bet v1.0101 has

been demonstrated. Furthermore, optimizing the feeding strategy may increase the rBet v1.0101 production further. It is

expected that similar expression levels may be obtained by using an optimized feeding strategy in combination with a large

scale manufacturing process, thus generating sufficient yield per batch for economic viability.



Session title: Basic immunology


TPS 49 - Safety of immunotherapy and immunotherapy for food allergy


abstract 7


n

= 30

Age,

mea

n (S

D)

21 (1

1)

Sex

14 m

ale

Diag

nost

ics,

n (%

)

Rhi

nitis

Rhi

nitis

+ a

sthm

a

18 (6

0%)

12 (4

0%)

Base

line

tota

l IgE

kU/

L, m

ean

(SD)

* 43

5 (5

04)

Follo

w u

p to

tal I

gE k

U/L,

mea

n (S

D) *

431

(384

) Ba

selin

e sp

ecifi

c Ig

E, k

U/L,

mea

n (S

D) **

Phl

Phl

p1

P

hl p

5

58 (3

0)

50 (3

4)

45 (3

7)

Follo

w u

p sp

ecifi

c Ig

E, k

U/L,

mea

n (S

D) **

Phl

Phl

p1

P

hl p

5

62 (3

3)

43 (3

8)

42 (3

9)

Adve

rse

reac

tions

, n (%

)

Loc

al la

te re

actio

ns

S

yste

mic

reac

tions

3 (1

0%)

0

00

0

63

37

20

50

23

07

010203040506070

No tr

eatm

ent

Antih

istam

inics

Antih

istam

imics

+B2

ago

nist

Antih

istam

imics

+na

sal s

tero

idsAn

tihist

amim

ics +

nasa

l ste

roids

+B2

ago

nist

% Patients

Base

line

Follo

w-up

1429

S

afet

y of

a cl

uste

r-im

mun

othe

rapy

with

a d

epot

alle

rgoi

d in

pa

tient

s w

ith a

llerg

ic rh

initi

s ag

ains

t gra

sses

31

10,2

0,3

0,5

0,5

0,5

23

45

17

11W

eek

Initi

al tr

eatm

ent

Main

tena

nce t

reat

men

t

30 m

inut

es

Injec

tion

EAAC

I Con

gres

s 201

5In

rela

tion

to th

is p

rese

ntati

on, I

dec

lare

the

follo

win

g, re

al o

r per

ceiv

ed c

oni

ct o

f int

eres

t. M

J Cru

z is

an

empl

oyee

of H

AL A

llerg

y.



J. Rook1, H. Warmenhoven2, O. Brugman1, D. Verbart1, A. Huybens1 and H. van Schijndel2

1 HALIX BV, Leiden, The Netherlands; 2 HAL Allergy BV, Leiden, The Netherlands

BackgroundChinese Hamster Ovary (CHO) cells are the most widely used expression system for the production of therapeutic proteins.

Compared to bacterial expression systems, CHO cells possess human-like posttranslational modification machineries enabling

the production of properly folded recombinant proteins while no endotoxins are produced. Previously, a stable CHO cell line

(CHO-rBet v1.0101) was developed for expression of Bet v1.0101, the immunodominant isoform of the major birch pollen

allergen Bet v1. In this study, a large scale bioreactor manufacturing process was developed to optimize the yield.

MethodsTo assess the feasibility of a large scale manufacturing process, a single use 2 L shaker bag and traditional 10 L bioreactor setup

were inoculated with stably growing pre-cultures of CHO-rBet v1.0101 cells and cultured using a fed batch feeding strategy.

Cell density, viability and metabolites were monitored at regular time intervals. Expression of rBet v1.0101 was measured

using a in house developed Bet v1 ELISA kit. In addition, several commercially available CHO feeds were tested in a fed batch

shaker flask setup to optimize the cell growth and rBet v1.0101 expression.

ResultsCompared to a previously, glucose-fed only batch culture, the Bet v1.0101 titre obtained with the single use 2 L shaker bag and

the 10 L bioreactor were respectively 2.4 and 4 fold higher. Testing several commercially available CHO feeds showed higher

Bet v1.0101 concentrations, viable cell densities and prolonged viability. The best feed yielded 14 times more Bet v1.0101

compared to the glucose-fed only batch culture.

ConclusionIn this study, the feasibility of the CHO production platform for large scale manufacturing of recombinant Bet v1.0101 has

been demonstrated. Furthermore, optimizing the feeding strategy may increase the rBet v1.0101 production further. It is

expected that similar expression levels may be obtained by using an optimized feeding strategy in combination with a large

scale manufacturing process, thus generating sufficient yield per batch for economic viability.



Session title: Basic immunology


TPS 49 - Safety of immunotherapy and immunotherapy for food allergy


abstract 7


Stress stability study on a Phleum pratense extract.

Sandipta Acharya, Gert Jan Stam, Niels Sinnige, Rob van den Hout, Dion Luykx

HAL Allergy, Leiden, The Netherlands

BackgroundStressed and non-stressed Phleum pratense extracts were investigated with several analytical assays to monitor identity (SDS-

PAGE, immunoblot), content (protein, major allergen Phl p 5), potency (IgE potency) and structure (CD, Fluorescence, HP-SEC).

MethodsStressed conditions: Incubation at 45C (12 days), 60C (3 days) or 90C (1 hour), freeze-thawing (5 times) or shaking (15

min at 2600 rpm). SDS-PAGE: Reduced samples on 4-12% Bis-tris gels and stained with silver. Immunoblot: After SDS-PAGE,

proteins were transferred to PVDF membrane and stained using pooled sera from grass allergic patients, HRP-conjugated

antibody and CN/DAB substrate. Protein: Bradford assay using BSA as a standard. Major allergen content: An ELISA was used

to quantify Phl p 5 content. Potency: the allergenic activity was measured using an IgE-inhibition assay. CD: Spectra were

recorded from 260-190 nm. Fluorescence: Emission spectra were recorded from 290-400 nm, excitation at 280 nm. HP-SEC:

A Bio-Sec 3 size exclusion chromatography column was used with UV-detection.

ResultsSDS-PAGE showed disappearance of bands and appearance of higher molecular weight bands for the Phleum pratense extract upon thermal stressing. Immunoblot showed reduced intensity of major allergen bands for the thermal stressed samples. The protein content was not affected by stressing the Phleum pratense extract whereas the Phl p 5 content decreased for the 90C

sample. The IgE potency decreased by temperature stressing. Temperature stressing induced unfolding of proteins according to CD and fluorescence spectroscopy. HP-SEC showed aggregation of Phleum pratense proteins after thermal treatment.

Freeze-thawing and shaking did not affect any of the investigated properties of the Phleum pratense extract.

ConclusionTemperature stressing of a Phleum pratense allergen extract affected the protein profile and IgE potency. In parallel, protein unfolding and protein aggregation occurred.


Session title: Allergen standardization


TPS 26 - Basic immunology


abstract 8


1055

D

evel

opm

ent o

f a M

anuf

actu

ring

Platf

orm

for R

ecom

bina

nt

Bet v

1.01

01 u

sing

Chin

ese

Ham

ster

Ova

ry ce

lls

120

100 80 60 40 20 0

rBet v1.0101 (mg/L)

10L

Rea

ctor

Shak

er b

ag 2

LC

ontr

ol

0

Con

trol

100

200

300

400

rBet

v1.

0101

(m

g/L)

A

B

C

F

E D

0412162024

AB

CD

EF

Con

trol

8

VCD (10

6

cells/mL)

04

68

1012

1416

18Ti

me

(day

s)2

EAAC

I Con

gres

s 201

5In

rela

tion

to th

is p

rese

ntati

on, I

dec

lare

the

follo

win

g, re

al o

r per

ceiv

ed c

oni

ct o

f int

eres

t. T

he p

rese

nter

is a

n em

ploy

ee o

f HAL

IX/

the

HAL

Alle

rgy

grou

p.



Sandipta Acharya, Gert Jan Stam, Niels Sinnige, Rob van den Hout, Dion Luykx


BackgroundStressed and non-stressed Phleum pratense extracts were investigated with several analytical assays to monitor identity (SDS-

PAGE, immunoblot), content (protein, major allergen Phl p 5), potency (IgE potency) and structure (CD, Fluorescence, HP-SEC).

MethodsStressed conditions: Incubation at 45C (12 days), 60C (3 days) or 90C (1 hour), freeze-thawing (5 times) or shaking (15

min at 2600 rpm). SDS-PAGE: Reduced samples on 4-12% Bis-tris gels and stained with silver. Immunoblot: After SDS-PAGE,

proteins were transferred to PVDF membrane and stained using pooled sera from grass allergic patients, HRP-conjugated

antibody and CN/DAB substrate. Protein: Bradford assay using BSA as a standard. Major allergen content: An ELISA was used

to quantify Phl p 5 content. Potency: the allergenic activity was measured using an IgE-inhibition assay. CD: Spectra were

recorded from 260-190 nm. Fluorescence: Emission spectra were recorded from 290-400 nm, excitation at 280 nm. HP-SEC:

A Bio-Sec 3 size exclusion chromatography column was used with UV-detection.

ResultsSDS-PAGE showed disappearance of bands and appearance of higher molecular weight bands for the Phleum pratense extract upon thermal stressing. Immunoblot showed reduced intensity of major allergen bands for the thermal stressed samples. The protein content was not affected by stressing the Phleum pratense extract whereas the Phl p 5 content decreased for the 90C

sample. The IgE potency decreased by temperature stressing. Temperature stressing induced unfolding of proteins according to CD and fluorescence spectroscopy. HP-SEC showed aggregation of Phleum pratense proteins after thermal treatment.

Freeze-thawing and shaking did not affect any of the investigated properties of the Phleum pratense extract.

ConclusionTemperature stressing of a Phleum pratense allergen extract affected the protein profile and IgE potency. In parallel, protein unfolding and protein aggregation occurred.


Session title: Allergen standardization


TPS 26 - Basic immunology


abstract 8


Characterisation of wasp venom collected via electrostimulation or venom sac extraction.

Niels Sinnige, Susanne Quaak, Diederik van Deursen, Jolanda Meijlis, Rob van den Hout and Dion Luykx


BackgroundA characterisation study was performed on wasp venom obtained via either electrostimulation or venom sac extraction.

Different assays with respect to identity, content and protein structure were applied.

MethodsSDS-PAGE: Reduced samples were applied to 4-12% Bis-tris gels combined with silver staining. Immunoblot: After SDS-

PAGE, proteins were transferred to a PVDF membrane and stained using pooled sera of wasp venom allergic patients, HRP

conjugated antibodies and CN/DAB substrate. MS: Tryptic digests were prepared from wasp venom samples. Peptides were

separated via nano-HPLC before electrospray ionisation. Ionised peptides were fragmented revealing amino acid sequences.

Protein: Spectrophotometric method according to Lowry using BSA as a standard. Major allergen content: An ELISA was used

to quantify Ves v 5. CD: Far-UV CD spectra were recorded from 260-190 nm.

ResultsSDS-PAGE protein profiles revealed that wasp venom collected via sac extraction contains a high variety of proteins in

comparison to electrostimulation venom. Via immunoblot and MS allergens Ves v 1 (~35 kDa), Ves v 2 (~45 kDa), Ves v 3 (~

90 kDa) and Ves v 5 (~27 kDa) were identified in the wasp venom collected via the two different methods. The amount of

Ves v 5 in relation to total venom protein was higher in wasp venom collected via electrostimulation (4 %) compared to via

sac extraction (2 %). The CD-spectrum of sac extraction venom indicated a relative higher amount of helical proteins in

comparison to electrostimulation venom.

ConclusionThe SDS-PAGE profiles and Ves v 5 (%) in total protein indicate that wasp venom collected via venom sac extraction contains

more unidentified proteins compared to collection via electrostimulation. Ves v 1, 2, 3 and 5 were identified in both venoms.

Abstract number: 461Session number, date and time: PDS 20, Monday 8 June 2015; 15:45 - 17:15

Session title: Diagnosis and treatment of anaphylaxis and hymenoptera venom allergy


TPS 32 - Allergen standardization


abstract 9


1134

S

tress

stab

ility

stud

y on

a P

hleu

m p

rate

nse

extra

ct

ba

250

150

100

75 50 37 25 20 15 10

250

150

100 75 50 37 25 20 15 10

M

M

1

2

3

4

5

6

1

2

3

4

5

6

kDa

kDa

ab

10

0,1

0,2

0,3

0,4

0,5

0,6

0,7

Relative potency

Major Allergen (g/mL)

0.8

0,91

23

45

61

02468101214

23

45

6

mA

U

500

400

300

200

100 0

56

78

910

1112

13m

in

1.35

kDa

158k

Da

670k

Da

44kD

a17

kDa

mA

U 50 40 30 20 10 0

56

78

910

1112

13m

in

607080a bNo

n str

esse

d45

C60

C90

CFr

eeze

thaw

Shak

ing

EAAC

I Con

gres

s 201

5In

rela

tion

to th

is p

rese

ntati

on, I

dec

lare

the

follo

win

g, re

al o

r per

ceiv

ed c

oni

ct o

f int

eres

t. T

he p

rese

nter

is a

n em

ploy

ee o

f HAL

Alle

rgy.


Characterisation of wasp venom collected via electrostimulation or venom sac extraction.

Niels Sinnige, Susanne Quaak, Diederik van Deursen, Jolanda Meijlis, Rob van den Hout and Dion Luykx


BackgroundA characterisation study was performed on wasp venom obtained via either electrostimulation or venom sac extraction.

Different assays with respect to identity, content and protein structure were applied.

MethodsSDS-PAGE: Reduced samples were applied to 4-12% Bis-tris gels combined with silver staining. Immunoblot: After SDS-

PAGE, proteins were transferred to a PVDF membrane and stained using pooled sera of wasp venom allergic patients, HRP

conjugated antibodies and CN/DAB substrate. MS: Tryptic digests were prepared from wasp venom samples. Peptides were

separated via nano-HPLC before electrospray ionisation. Ionised peptides were fragmented revealing amino acid sequences.

Protein: Spectrophotometric method according to Lowry using BSA as a standard. Major allergen content: An ELISA was used

to quantify Ves v 5. CD: Far-UV CD spectra were recorded from 260-190 nm.

ResultsSDS-PAGE protein profiles revealed that wasp venom collected via sac extraction contains a high variety of proteins in

comparison to electrostimulation venom. Via immunoblot and MS allergens Ves v 1 (~35 kDa), Ves v 2 (~45 kDa), Ves v 3 (~

90 kDa) and Ves v 5 (~27 kDa) were identified in the wasp venom collected via the two different methods. The amount of

Ves v 5 in relation to total venom protein was higher in wasp venom collected via electrostimulation (4 %) compared to via

sac extraction (2 %). The CD-spectrum of sac extraction venom indicated a relative higher amount of helical proteins in

comparison to electrostimulation venom.

ConclusionThe SDS-PAGE profiles and Ves v 5 (%) in total protein indicate that wasp venom collected via venom sac extraction contains

more unidentified proteins compared to collection via electrostimulation. Ves v 1, 2, 3 and 5 were identified in both venoms.

Abstract number: 461Session number, date and time: PDS 20, Monday 8 June 2015; 15:45 - 17:15

Session title: Diagnosis and treatment of anaphylaxis and hymenoptera venom allergy


TPS 32 - Allergen standardization


abstract 9


Abbreviated leaflet texts.


PDS 20 - Diagnosis and tre

hal allergy abstractbook eaaci 2015

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