bewerbung für den dissertationspreis der Ögai 2018 · curriculum vitae dr. aglas lorenz gregor,...
TRANSCRIPT
Bewerbung für den Dissertationspreis der ÖGAI 2018
Bewerber: Lorenz Aglas
Inhalt:
• Short scientific CV • Approbation der Dissertation und der Institution, an der die Arbeit durchgeführt wurde • Dissertation
Curriculum Vitae Dr. AGLAS Lorenz Gregor, MSc, BSc Personal Data Name Aglas Lorenz Address Hellbrunnerstr. 34, 5020 Salzburg Date of Birth April, 12th 1989 Nationality Austrian Marital Status Single Education and Training 01.10.2007 – 12.09.2011 Bachelor Genetics at the University of Salzburg
14.09.2011 – 23.10.2013 Master Genetics and Biotechnology at the University of Salzburg
Master Thesis at the Department of Molecular Biology in the Working Group of Fatíma Ferreira; Title: Recombinant production and protein purification of the major allergen of cupressus arizonica (cypress) Cup a 1 and development of a hypoallergenic variant of Cup a 1
04.11.2013 – 18.08.2017 PhD. Student Molecular Biology at the University of Salzburg Department of Molecular Biology of the University of Salzburg; Working Group of Fatíma Ferreira; Title: Intrinsic properties of the Bet v 1 fold: impact on immunogenicity and allergenicity
Since 21.08.2017 Senior Scientist in the department of Molecular Biology at the University of Salzburg
Scientific Publications - Context matters: Th2 polarization resulting from pollen composition and not from protein-intrinsic
allergenicity. Aglas L, Gilles S, Bauer R, Huber S, Araujo GR, Mueller G, Scheiblhofer S, Amisi M, Dang HH, Briza P, Bohle B, Horejs-Hoeck J, Traidl-Hoffmann C, Ferreira F. Journal of Allergy and Clinical Immunology 2018 May
- Endolysosomal protease susceptibility of Amb a 1 as a determinant of allergenicity Wolf M, Aglas L, Twaroch T, Steiner M, Hauser M, Hofer H, Parigiani A, Ebner C, Bohle B, Briza P, Neubauer A, Stolz F, Wallner M, Ferreira F Journal of Allergy and Clinical Immunology 2017 October
- Two Distinct Conformations in Bet v 2 Determine Its Proteolytic Resistance to Cathepsin S. Soh W, Briza P, Dall E, Asam C, Schubert M, Huber S, Aglas L, Bohle B, Ferreira F, Brandstetter H Journal of Molecular Sciences 2017 October
- Amb a 1 isoforms: unequal siblings with distinct immunological features Authors: Wolf M, Twaroch T, Huber S, Steiner M, Aglas L, Hauser M, Aloisi I, Hofer H, Parigiani A, Ebner C, Bohle B, Briza P, Neubauer N, Stolz F, Wallner M, Ferreira F Allergy 2017 May
- Tree pollen allergens-an update from a molecular perspective. Asam C, Hofer H, Wolf M, Aglas L, Wallner M. Allergy 2015 October
- Bet v 1 – a Trojan horse for small ligands boosting allergic sensitization? Asam C, Batista A, Moraes A, de Paula V, Almeida F, Aglas L, Kitzmüller C, Bohle B, Ebner C, Ferreira F, Wallner M, Valente A. Clinical & Experimental Allergy 2014 April
Book Chapters
- The Pollen-Food Syndrome: a molecular perspective Asam C, Aglas L, Huber S, Ferreira F, Roulias A Food Allergy – Methods for detection and clinical studies 2016
- The Pollen-Food Syndrome: An update on diagnostic and therapeutic approaches Huber S, Asam C, Roulias A, Ferreira F, Aglas L Food Allergy – Methods for detection and clinical studies 2016
Congress Participation - EAACI annual congress 2018 - ISMA 2017 - EAACI annual congress 2016:
Vienna, Austria, 11 – 15 June 2016; Posters: “Development of an aptamer-based tool for quality control of a birch pollen immunotherapy vaccine“ and “Immunologic evaluation of the hypoallergenic birch pollen AIT vaccine candidate BM4 during toxicity testing”
- 3rd Oxford Symposium on Aptamers: Oxford, united Kingdom, 4 – 5 April 2016; Poster: “Development of an enzyme-linked apta-sorbent assay (ELASA) for the quality control of a birch pollen immunotherapy vaccine”
- ÖGMBT 2015: 7th ÖGMBT (Austrian Association of Molecular Life Sciences and Biotechnology) Annual Meeting “Salzburg goes Science” Salzburg, Austria, 9 – 11 September 2015; Poster: “The influence of ligand-binding on the stablity of the major birch pollen allergen Bet v 1”
- EAACI annual congress 2015: Barcelona, Spain, 6 – 10 June 2015; Poster: “Ligand-binding influences the physico-chemical properties of the major birch pollen allergen Bet v 1“
- EAACI Summer school 2014: Allergy School on An Insight into Allergy and Allergen Immunotherapy Athens, Greece, 11 – 13 September 2014; Poster: “The Major Birch Pollen Allergen Bet v 1 and Its Immunological Behavior Concerning Ligand Binding”
- ÖGAI 2014: General meeting Salzburg, Austria, 06 – 08 November 2014; Poster: “Analysis of interactions of the major birch pollen allergen Bet v 1 with naturally occurring and synthetic ligands”
Scientific Associations: Memberships
- Junior member of the European Academy of Allergy and Clinical Immunology (EAACI) - Member of the Austrian Society for Allergology and Immunology (ÖGAI) - Member of the Austrian Association of Molecular Life Sciences and Biotechnology (ÖGMBT)
Intrinsic properties of the Bet v 1 fold: impact on immunogenicity and allergenicity
Dissertation
Zur Erlangung des Doktorgrades
Doktor rerum naturalium (Dr.rer.nat)
an der naturwissenschaftlichen Fakultät
der Paris-Lodron-Universität Salzburg
Eingereicht von
Lorenz Aglas, MSc
Betreuer:
Univ. Prof. Dr. Fátima Ferreira
Fachbereich: Molekulare Biologie
Salzburg, Mai, 2017
“We mortals have many weaknesses; we feel too much, hurt too much and too soon we die, but we do have the chance of love.”
Elizabeth: The Golden Age (2007)
“What man is a man who does not make the world better.”
Kingdom of Heaven (2005)
And the reason why I have studied molecular biology…
“Their brains weren't large enough to harvest sufficient amounts of the protein complex. So we violated the Harvard Compact. Jim and I used gene therapies to increase their brain mass. A larger
brain means more protein. As a side effect the sharks got smarter.”
Deep Blue Sea (1999)
ii
Acknowledgments
Sooooo... It seems like, I have finally completed my doctoral thesis. Although it was a long, long, long
and rugged way to go, I have to admit that I am very thankful for every single moment I have
invested in this project (PhD). Of course, it was an intense time and sometimes almost unbearable,
but I am still grateful because it were these experiences that made me stronger in so many ways and
gave me the chance to grow. Therefore, I want thank all my colleagues and friends who supported
me on this journey, especially Martin, Chris, Marco, Steffi, Teresa, Ricardo, Xin, Carlos, Isi and Yoan.
Sara, you deserve your own sentence(s). I am not sure, if you know how much you helped me during
my PhD, no matter if it was scientific assistance or emotional support. Thank you so much for that.
Olivia and Galber, the both of you are so wonderful persons. I cannot even mention how much I
appreciate your friendship. You two mean the world to me. Thank you for everything! Beijos.
Special thanks also go to the USA, in particular to Geoffrey Mueller from the National Institute of
Environmental Health Sciences (NIEHS) who provided the NMR data for the ligand binding study and
plenty of helpful advices.
I would also like to thank Peter Briza for the MS analyses. He is spending so much time in this small
room without windows and this really deserves special appreciation.
Fátima, thank you so much for supporting me, supervising me, guiding me, inspiring me, correcting
me, motivating me, believing in me… for everything! You are an amazing leader, and were there for
us when we needed you the most. Muito obrigado!
Finally, my family: I want to thank my parents, Ines and Ferdinand, as well as my brothers and my
amazing sister for their support and, of course, for loving me and accepting me for who I am.
At the end, I just want to say: No LOGRETS!
iii
Table of contents
Abbreviations .......................................................................................................................................... 4
Abstract ................................................................................................................................................... 6
Introduction ............................................................................................................................................. 7
Type-I hypersensitivity reaction (allergy) ............................................................................................ 7
Allergic sensitization ............................................................................................................................ 7
The effector phase – the actual allergic immune response ................................................................ 8
Respiratory allergies, inhalant allergens and diagnosis of birch pollen allergy .................................. 8
Allergen immunotherapy (AIT) and hypoallergens ............................................................................. 9
Aim of the thesis ................................................................................................................................ 10
Chapter 1: Ligand binding of Bet v 1 ..................................................................................................... 11
Introduction ....................................................................................................................................... 11
The major birch pollen allergen Bet v 1 ........................................................................................ 11
Identified and discussed ligands of Bet v 1 ................................................................................... 12
Aims ............................................................................................................................................... 13
Material and methods ....................................................................................................................... 14
Birch pollen allergic patients ......................................................................................................... 14
Protein expression, purification and characterization of recombinant Bet v 1.0101 (rBet v 1) ... 14
Soluble fraction of aqueous birch pollen extract .......................................................................... 15
Purification of natural Bet v 1 (nBet v 1) ....................................................................................... 15
Identification of bacterial colonies on pollen grains ..................................................................... 15
Quantification of LPS and LTA present in the birch pollen extract ............................................... 15
Used ligands .................................................................................................................................. 16
ANS displacement assay ................................................................................................................ 16
Protein-ligand interaction studies used to determine the affinity constant KD............................ 16
Pull-down assay using biotinylated LPS ......................................................................................... 17
Analysis of secondary structural elements and thermal stability ................................................. 17
Production of 15N-1H labeled Bet v 1 for NMR analysis ................................................................. 18
NMR spectroscopy ........................................................................................................................ 18
In vitro endo-/lysosomal degradation ........................................................................................... 19
Mediator release assay.................................................................................................................. 19
Stimulation and antigen uptake of murine bone marrow-derived dendritic cells (mBMDCs) ..... 20
1
Maturation of human monocyte-derived dendritic cells (moDCs) and analysis of cytokine secretion ........................................................................................................................................ 20
Immunization of IL-4/GFP-enhanced transcript (4get) mice......................................................... 21
Results ............................................................................................................................................... 21
Identification of bacterial colonies on pollen grains ..................................................................... 21
Quantification of LPS and LTA present in the birch pollen extract ............................................... 22
Production and purification of aqueous birch pollen extract, nBet v 1, rBet v 1 and BM4 .......... 22
ANS displacement assay ................................................................................................................ 23
Protein-ligand interaction studies used to determine the affinity constant KD............................ 24
NMR spectroscopy ........................................................................................................................ 26
Pull-down assay using biotinylated LPS ......................................................................................... 27
Analysis of secondary structural elements and thermal stability ................................................. 28
In vitro endo-/lysosomal degradation ........................................................................................... 31
Mediator release assay.................................................................................................................. 32
Stimulation and antigen uptake of murine bone marrow-derived dendritic cells (mBMDCs) ..... 35
Maturation of human monocyte-derived dendritic cells (moDCs) and analysis of cytokine secretion ........................................................................................................................................ 36
Immunization of IL-4/GFP-enhanced transcript (4get) mice......................................................... 38
Discussion .......................................................................................................................................... 39
Chapter 2: BM4SIT ................................................................................................................................. 43
Introduction ....................................................................................................................................... 43
The BM4 molecule ......................................................................................................................... 43
The role of vitamin D3 as novel adjuvants .................................................................................... 44
The BM4SIT project ....................................................................................................................... 44
Aims ............................................................................................................................................... 44
Material and methods ....................................................................................................................... 45
Mass spectrometry analysis .......................................................................................................... 45
CD and FTIR ................................................................................................................................... 45
Dynamic light scattering (DLS) ....................................................................................................... 45
Time-dependent endo-/lysosomal degradation ........................................................................... 46
Monitoring stability of the protein under different storage conditions (aging control) ............... 46
Development of a BM4-specific ELISA to monitor the integrity of immune epitopes .................. 46
Development of a potency assay for quality control .................................................................... 48
Development of a sandwich enzyme-linked apta-sorbent assay (ELASA) for quality control of the formulated BM4 drug product ...................................................................................................... 50
2
Immunological evaluation of BM4 drug product within the BM4SIT acute toxicity study ........... 53
Results ............................................................................................................................................... 55
Characterization of the BM4 molecule ......................................................................................... 55
Development of a potency assay for quality control of the formulated BM4 drug product ........ 61
Development of a sandwich ELASA for quality control of the BM4 drug product ........................ 68
Immunological evaluation of the BM4 drug product within the BM4SIT acute toxicity study ..... 72
Immunological evaluation of the BM4 drug product within the BM4SIT repeated toxicity study 75
Discussion .......................................................................................................................................... 77
Chapter 3: General discussion and conclusion ...................................................................................... 83
Bibliography ........................................................................................................................................... 85
Appendix ................................................................................................................................................ 90
Scientific curriculum vitae ................................................................................................................. 90
3
Abbreviations
3D three-dimensional 4get IL-4/GFP-enhanced transcript ANS 8-Anilinonaphthalene-1-sulfonic acid AP alkaline phosphatase APC antigen-presenting cells BM4 Bet v 1 mutant 4 Bp base pairs CD circular dichroism Cfu colony forming units DAMP damage-associated molecular pattern DC dendritic cell DLS dynamic light scattering DOC sodium deoxycholate dsDNA double-stranded DNA DTT dithiothreitol ELASA enzyme-linked apta-sorbent assay ELISA enzyme-linked immunosorbent assay FCSi fetal calf serum inactivated FcεRI high-affinity IgE receptor FTIR Fourier transform infrared GM-CSF granulocyte-macrophage colony-stimulating factor GMP good manufacturing practices h hour/hours HCl hydrochloric acid hRBL humanized rat basophilic leukemia cells HRP horseradish peroxidase IC50 inhibition concentration IgE immunoglobulin E Kdo2 Kdo2-Lipid A LOD limit of detection LPS lipopolysaccharide LTA lipoteichoic acid mBMDC murine bone marrow-derived dendritic cell min minutes moDCs monocyte-derived dendritic cells MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide NBT/BCIP nitroblue tetrazolium/5-Bromo-4-chloro-3-indolyl phosphate NMR nuclear magnetic resonance NZW New Zealand White rabbit o/n over night OVA ovalbumin from chicken 97% PALMs pollen-associated lipid mediators PAMP pathogen-associated molecular pattern PBS phosphate-buffered saline PCR polymerase chain reaction PDB protein data bank PLUS Paris-Lodron-University Salzburg PP phytoprostane PPAR-γ peroxisome proliferator-activated receptor γ
4
PR-10 pathogenesis-related proteins of class 10 PRR pattern recognition receptor Q3OS quercetin 3-O-sophoroside RH hydrodynamic radius RT room temperature (usually 22°C) SAW surface acoustic wave SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SELEX systematic evolution of ligands by exponential enrichment SOP standard operating procedure SPT skin prick test ssDNA single-stranded DNA TBS tris-buffered saline TBST tris-buffered saline plus tween TLR toll-like receptor
5
Abstract
Allergic reactions to birch (Betula verrucosa) pollen are the most prevalent tree pollen allergies in
Europe. Over 100 million allergic patients worldwide suffer from birch pollen allergy, and more than
95% of them are sensitized to a protein designated Bet v 1, thus rendering it the major birch pollen
allergen. So far, the allergic sensitization-driving property of Bet v 1, which is linked to the induction
of a strong Th2 immune response, remains elusive.
The intrinsic properties of the Bet v 1 fold have been investigated in detail and hereby one special
feature in particular has attracted attention: the potential of Bet v 1 to act as a promiscuous acceptor
for various ligands via binding to its hydrophobic cavity, which comprises the core of the allergen.
The objective of this doctoral thesis was to investigate the influence of ligand binding on the
allergenicity of Bet v 1, thereby considering physicochemical and immunological properties. The
ligands we chose for this study were, on the one hand, natural pollen-derived compounds, and on
the other hand, the microbial-derived compounds lipopolysaccharide (LPS) and lipoteichoic acid,
which were selected for their presence as contaminants in aqueous pollen extracts and for their
potential to activate toll-like receptors (TLR). We could demonstrate that Bet v 1 is able to bind the
pollen-derived compounds but not the TLR-2 and TLR-4 agonists. Ligand binding increased the
proteolytic and thermal stability of the protein, whereas the non-binding microbial-derived
compounds were shown to destabilize the protein. However, neither one of the investigated
compounds did endow Bet v 1 with the capacity to induce Th2 polarization in vivo. In contrast, birch
pollen extracts were shown to promote Th2 polarization. From these findings we can conclude that
TLR-co-stimulation alone is not a decisive aspect in birch pollen sensitization and that Bet v 1
sensitization is rather determined by the complex pollen environment. Hence, a superior role of the
intrinsic properties of this allergen on the induction of a Th2-favoured immune response most likely
can be excluded. Future studies focusing on the pollen matrix could shed light on the substance(s)
contributing to its allergenicity.
Besides the ligand binding study of Bet v 1, we investigated the reduced allergenicity of BM4, a
hypoallergenic variant of the Bet v 1 molecule displaying a completely different fold than the wild-
type allergen. Here, the objective was to characterize the hypoallergen in detail and also to compare
it with the wild-type protein. We found that BM4 is a potent immunogen ready to be evaluated in a
first-in-man allergen immunotherapy (AIT) clinical trial and having the potential to provide a safer
and more efficacious option to the natural birch pollen extracts that are presently used in AIT.
6
Introduction
Type-I hypersensitivity reaction (allergy)
The human immune system is able to react towards pathogens such as, bacteria, parasites, infectious
microbes, and viruses with two kinds of protective responses, the innate and the adaptive immune
response. The innate immune response is fast, but unspecific and its main function is to cause an
immediate defense mechanism to protect the human body against pathogens. On the other hand,
the adaptive immunity, whilst occurring later, results in a more efficient pathogen elimination. It
specifically adapts to the pathogen exposure and also generates an immunological memory in order
to be prepared for a consecutive encounter. The immune system recognizes specific pathogen- or
damage-associated molecular patterns (PAMPs, DAMPs) such as, proteins, lipids, and
polysaccharides, and small chemicals via pattern recognition receptors (PRRs) expressed on antigen-
presenting cells (APCs). The immune system of atopic individuals, i.e those with a genetic
predisposition to develop allergic hypersensitivity reactions, recognize, per se, harmless
environmental antigens (allergens) and develop a response against it. In non-atopic human beings,
those allergens are usually tolerated by the immune system. This often described clinical
phenomenon is called a hypersensitivity reaction and those classified as type-I hypersensitivity
reaction are better known as allergic reactions (allergy). Type-I hypersensitivity reactions are defined
as immunoglobulin E (IgE)-mediated immune reactions towards the allergen and are typically divided
into two phases: the sensitization phase and the effector phase[1].
Allergic sensitization
Upon first encounter of the allergen with APCs, an immunologic response is induced by allergen-
specific Th2 and B cells, this mechanism is called allergic sensitization. In detail, the allergen is
internalized and processed by dendritic cells (DCs), the most potent kind of APCs, into short, linear
peptides that are presented to naïve CD4+ T cells via MHC-II molecules expressed on the surface of
the DCs. After the internalization of the allergen, the antigen-loaded DC migrates to the lymph nodes
where the presentation to the naïve T cells occurs. The activation of T cells is facilitated by the
expression of co-stimulatory molecules such as, CD40, CD80 and CD86 that are up-regulated by PRR
stimulation[2]. Mentionable PRRs are the Toll like receptors (TLRs), which are able to recognize
bacterial compounds such as, lipopolysaccharides (LPS) and lipoteichoic acid (LTA), C-type lectin
receptors (CLRs), NOD-like receptors (NLRs) and protease-activated receptors (PARs). In case of
allergens, the recognition of PAMPs by DCs is inducing the secretion of Th2-polarizing cytokines,
including IL-4, IL-5, and IL-13. Together with antigen-presentation and the expression of co-
7
stimulatory molecules, these cytokines are responsible for the priming of naïve CD4+ T cells to
effector Th2 cells. In turn, these cytokines promote a class switch in B cells to become allergen-
specific IgE-producing plasma cells. The produced IgE antibodies then bind to the high-affinity IgE
receptor (FcεRI) expressed on mast cells and basophils[3, 4]. Unbound IgE circulating in the blood
rarely exceeds a half-life of two days, whereas bound to FcεRI the half-life is prolonged for at least 30
days. Once allergen-specific IgE has bound to mast cells or basophils the sensitization process is
completed. A co-stimulatory signal represented by the CD40 ligand, which is expressed on the
surface of T cells and interacts with CD40 on B cells, is necessary to mediate the class switch of B
cells[5]. B cell-activation and class switching happens in so-called germinal centers (GCs), which are
assemblies of diverse kinds of immune cells, including B cells, Th2 cells, and DCs and generated in the
lymph nodes[6]. In these germinal centers, not only the activation of the B cells is happening, but
also other B cell-associated molecular events. Events include, an excessive proliferation of B cells, as
well as affinity maturation, production of memory B cells and long-living plasma cells.
The effector phase – the actual allergic immune response
Upon a repeated encounter of the allergen with the pre-primed immune system, two or more
allergen-specific IgE antibodies are immobilized on the FcεRI expressed on basophils and mast cells
and become cross-linked with the allergen. This mechanism is called the effector phase and leads to
an immediate release of certain mediators such as, tryptase and chymase, serine esterases and the
short-lived vasoactive amine histamine. Further to this the so-called late-phase reaction described as
a consecutive inflammatory immune response is initiated and further mediated by the secretion of
inflammatory chemokines, cytokines, and lipid mediators such as prostaglandins, leukotrienes,
thromboxanes and platelet-activating factor. In course of the late-phase reaction, which usually
occurs three to nine hours after allergen exposure, other immune cells including eosinophils,
neutrophils, basophils, TH2 lymphocytes, and B cells are being recruited. The typical described
symptoms arising from an allergic immune response are allergen-dependent and include vascular
leakage, allergic rhinoconjunctivitis, tissue inflammation, tissue remodeling, allergic asthma, atopic
dermatitis, bronchoconstriction, intestinal hypermotility, but also life-threatening anaphylaxis[7, 8].
Respiratory allergies, inhalant allergens and diagnosis of birch pollen allergy
Allergic diseases are affecting 30 to 40% of the world´s population and there is a clear trend that the
prevalence of developing an allergic condition is increasing steadily[9]. In general, allergens can be
clustered into four groups depending on their site of exposure: inhalants, ingestants, contactants and
8
injectants. Herein, we focus on inhalant allergens, with special emphasis on pollen-derived
allergens[10, 11].
The main reason for the development of seasonal respiratory allergies is pollen released from trees
during their pollination period. About 10% of the European population suffer from flu-mimicking
symptoms such as, rhinitis and conjunctivitis caused by allergic reactions towards pollen. In Europe,
76% of allergic patients are allergic to pollen and 35% are solely sensitized to pollen[12]. In vast parts
of Europe, trees of the Betulaceae family are the most relevant pollen-releasing source. In particular,
birch (Betula verrucosa) is the main cause for winter and spring pollinosis.
A patient suffering from an allergic condition triggered by birch pollen is usually diagnosed by
methods such as, Skin Prick Test (SPT), serological analysis to measure the level of allergen-specific
IgE antibodies, cell-based techniques, provocation tests, etc., and supported by the patient´s clinical
history[13-15].
The current available diagnostic approaches are only able to determine patients’ sensitization profile,
but can usually not explicitly detect the allergen that is responsible for the onset of symptoms. In this
respect, there are also other methods like the provocation test, which links the exposure of a certain
allergen source directly to the occurrence of symptoms. But these tests are rarely done, not just
because performing of such can be complex and expensive, but also because it is extremely
uncomfortable for the patient. However, the lack of diagnostic methods that addresses directly an
allergic manifestation is an important missing aspect in current allergy diagnosis and further
approaches are required to improve the present situation.
Allergen immunotherapy (AIT) and hypoallergens
The vast majority of therapeutic approaches in the treatment of respiratory allergy are only
addressing the symptoms caused by the disease. That is why most clinicians prescribe symptom-
suppressive substances such as, antihistamine. At present, the only long-lasting curative treatment
method targeting the disease at a molecular level is allergen immunotherapy (AIT), and thus has the
potential to increase the patient´s quality of life. AIT is able to modify the allergen-specific immune
response by initiating a state of immunologic tolerance. This is facilitated via the induction of
allergen-specific regulatory T-cells (Treg), as well as allergen-specific blocking antibodies, such as IgG4,
IgG1 and IgA[16-20].
Besides all these positive effects of AIT, the possibility remains that side-effects can occur in course
of the treatment. In contrast to weak side-effects such as small local inflammations and swellings,
also severe life-threatening systemic reactions can occur. Usually this happens because the therapy is 9
performed with natural allergen extracts or IgE-reactive purified allergens. To overcome this
potential risk of side-effects it has been suggested to substitute “active” allergens by their generated
hypoallergenic variants, also called hypoallergens. These variants possess a diminished allergenic
potential and reduced IgE reactivity compared to the wild-type molecule, but are still immunogenic
and thus able to induce immunologic tolerance without causing unexpected side-effects. There are
several possibilities to generate such hypoallergens. Mentionable techniques achieved via genetic
modification are recombinant allergens, with destroyed conformational IgE epitopes, or generated
recombinant fragments, oligomers and mosaics[21, 22].
Aim of the thesis
This doctoral thesis is dealing with the intrinsic properties of Bet v 1, the major birch pollen allergen,
and the impact of its fold on immunogenicity and allergenicity. So far, there are many open questions
regarding this topic. For example, what is the intrinsic property of Bet v 1 that makes it the major
birch pollen allergen? Other allergens usually possess certain intrinsic features such as protease
activity or defined glycosylation patterns that can be recognized by the immune system and
therefore are able to provoke allergic reactions. For the particular case of Bet v 1 such an intrinsic
property has not yet been clearly defined. The current idea regarding Bet v 1 allergenicity lies in its
ability to bind to a huge variety of low-molecular weight ligands facilitated by its unique fold.
Although this feature has been described excessively in the literature the question remains how this
property can be translated into an allergic sensitization-inducing immune response. Chapter I of this
thesis will provide new insights into this topic and offer explanations why ligand binding is probably
not the answer for the allergenicity of Bet v 1. Chapter II is dealing with the creation of
hypoallergenic molecules and their efficacy in the treatment of birch pollen allergy. In this respect,
the reduced allergenic potential of a Bet v 1-derived hypoallergic variant, called BM4, is discussed.
10
Chapter 1: Ligand binding of Bet v 1
Introduction
The major birch pollen allergen Bet v 1
Birch (Betula verrucosa) pollen has been described as the main cause for winter and spring pollinosis
within the temperate climate zone of the northern hemisphere. Besides the classical symptoms
affecting the respiratory tract such as, sneezing, coughing, scratchy throat, allergic asthma, etc., but
also other symptoms, such as gastrointestinal disturbances, swollen eyes, urticaria and other
inflammation reactions can occur if an allergic patient is exposed to birch pollen[23]. Betula
verrucosa is a member of the Betulaceae family, which in fact represents the main source of pollen
allergens in Europe.
The major allergen found in birch pollen is called Bet v 1 and sensitization towards this allergen
occurs in over 95% of all birch pollen allergic patients[24, 25]. Bet v 1 has many isoforms with divers
immunological properties[26]. In this thesis, if not otherwise explicitly mentioned, we are always
referring to the Bet v 1.0101 isoform.
Bet v 1 is a member of the pathogenesis-related proteins of class 10 (PR-10). These proteins play a
role within the general immune defense and stress management of plants and are expressed upon
induction by bacterial, fungal and/or viral invasion, or by abiotic environmental factors such as, cold,
drought, oxidative stress and physical damage[27]. Beside the suggested protective role of PR-10
proteins, less is known about the physiological function of Bet v 1. It is supposed that Bet v 1 has an
impact on plant growth, reproduction and development since it is expressed in large amounts in the
pollen grain and released quickly upon hydration[27, 28]. Information regarding the physicochemical
properties and the molecular structure of Bet v 1 is more readily available, as revealed by X-ray
crystallographic and NMR spectroscopic methods. With a molecular weight of 17.5 kDa the globular
protein has a defined 3D fold consisting of a seven-stranded anti-parallel β-sheet structure wrapping
around a relatively long C-terminal α-helix. A solvent-accessible cavity in the shape of a “Y” is covered
by this antiparallel β-sheet. This cavity inside the protein represents the center of the molecule and is
responsible for the characteristic function of Bet v 1 to act as a carrier or storage protein for a huge
diversity of different, naturally occurring ligands[29]. The ligands that are discussed to interact with
Bet v 1, can be classified into the three major groups of flavonoids (i), phytohormones (ii), both
derived from the pollen interior, and microbial-derived compounds (iii).
11
Identified and discussed ligands of Bet v 1
Pollen-derived compounds
Q3OS – the natural ligand
In 2014, Seutter von Loetzen et al. reported the identification of the first natural, physiological ligand
of Bet v 1 through extraction of the glycosylated flavonoid quercetin 3-O-sophoroside (Q3OS; 626.5
Da) from purified Bet v 1 of a birch pollen extract[30]. They further raised the question about the
potential function of Bet v 1 as a carrier of flavonoids like Q3OS in birch pollen. In general, flavonoids
are secondary plant metabolites that play a role in protection from UV radiation, plant development
and defense, communication with microorganisms, pollen viability and pigment formation to attract
pollinators by providing appealing flower colors[30, 31]. In plants, the expression of Q3OS is
upregulated upon UV-B radiation stress. In a follow-up-study, Seutter von Loetzen et al. suggested
that the complex formation of Bet v 1 with the physiological ligand prevents the glycosylated
flavonoid from degradation, thus providing chemical stability to maintain its UV-B screening,
protective function[32]. In addition, also binding of Bet v 1 to other members of the flavonoid family
has been reported, including flavone and naringenin. These two possess structural similarities to
kaempferol, which is a flavanol and was described to have features that are essential for pollen
fertility. Here, it was also proposed that Bet v 1 acts as a carrier protein to transport flavonoids to the
stigmatic surface, where pollen germination takes place[28].
PPE1 and DOC – phytohormones
Another group of chemical compounds that are studied intensively with regard to interaction with
Bet v 1 aside from flavonoids are, phytohormones, including: pollen-associated lipid mediators
(PALMs), cytokinins and brassinosteroids. Phytohormones are co-delivered as non-allergenic, pollen-
delivered, low molecular weight compounds together with the allergens and are supposed to have a
leading impact upon Th2- polarization. Among those the group of phytoprostanes E1 (PPE1; 356.5
Da), which are a type of eicosanoid-like, monohydroxylated derivative of linoleic and linolenic acid,
represent molecules highly homologous to leukotrienes and prostaglandin E2, that are both
associated with the induction of inflammation, and are found in plant cells in amounts ranging from
4.5 -61 ng/g of dry weight[33]. Recently it was shown that this group of phytoprostanes is involved in
processes inhibiting the Th1-polarizing production of IL-12p70 in human dendritic cells (DCs) via
activation of the nuclear peroxisome proliferator-activated receptor γ (PPAR-γ) and thus favoring a
Th2-dominated allergic immune response. In vitro, they potently attract and activate human
12
neutrophils and eosinophils, and promote the production of allergen-specific IgE in Th2-primed B
cells[34].
Other phytohormones that have been studied in detail with regard to interactions with Bet v 1 are
cytokinins, for example kinetin, and plant steroid hormones of the brassinosteroid class, such as
brassinoloids, brassinolide and 24-epicastasterone[35]. In other topic-related scientific publications
the anionic detergent sodium deoxycholate (DOC; 414.6 Da), which possesses structural similarities
to brassinosteroids, is frequently used as a surrogate to address the influence of ligand binding of Bet
v 1 to plant steroid hormones[36]. Furthermore, it has been reported that the isoform Bet v 1a is
able to bind two molecules of DOC within its hydrophobic cavity as it was confirmed by x-ray
crystallography[37].
LTA and LPS – Microbial-derived compounds
Besides the mentioned pollen-derived compounds, Bet v 1 was discussed to interact with
immunomodulatory microbial compounds, which in turn can contribute to the process of allergic
sensitization. In previous studies, the interaction of the antigen with microbial components was
shown to mimic a pathogen-associated microbial pattern that activates Toll-like receptors (TLRs), a
conserved family of PRRs, to shift the immune response to an allergy-inducing Th2 response[38]. In
this respect, TLR-2 and TLR-4 seem to play an important role in the promotion of allergic
sensitization. The endotoxin lipopolysaccharide (LPS; 10,000-20,000 Da), a cell wall constituent of
Gram-negative bacteria, activates TLR-4, whereas lipoteichoic acid (LTA; 4,000-8,000 Da) as major
structural element of the cell wall composition of Gram-positive bacteria represents a significant
agonist of TLR-2[39]. The pollen is not sterile and actually provides a biotope for a huge diversity of
microbiota. Therefore, high amounts of microbial lipids can be found in the pollen cover[40]. Until
now, several allergens have been investigated towards their lipid-or LPS-binding affinity, which has
been shown to be the responsible mechanism to trigger sensitization in cases such as, Der p 2
(house-dust mite), Fel d 1 (cat), Bla g 1 (cockroach) and Can f 6 (dog)[41].
Aims
For birch pollen extracts, a proteolytic activity able to degrade tight junctions was described, but not
for the particular case of Bet v 1[42]. The allergen also possesses no glycosylation profile that
represents the driving force of allergic sensitization. The potential association of ligand binding of Bet
v 1 and allergic sensitization has been discussed extensively within the literature and is expected to
further elucidate key questions surrounding the topic of allergy, including how an individual becomes
13
sensitized to Bet v 1[28]. So far, the property that links Bet v 1 to Th2 polarization, and thus
rendering it the major birch allergen still remains elusive. The aim of this study is to investigate the
interaction of Bet v 1 with several pollen-derived and -associated compounds, and also the influence
of ligand binding on its intrinsic, physicochemical, as well as immune-modulatory properties. In turn,
we will be able to evaluate how far ligand binding is contributing to the allergenicity of the major
birch pollen allergen, and to clarify present misunderstandings regarding this topic. For this purpose,
we have selected the three pollen-derived molecules (Q3OS, PPE1 and DOC) and two microbial-
derived compounds (LPS and LTA). In addition, the LPS-substructure Kdo2-Lipid A (Kdo2; 2,306.8 Da)
has been used to provide a defined, nearly homogenous composition to LPS with similar endotoxin
activity, in order to achieve an improved reproducibility.
Material and methods
Birch pollen allergic patients
The sera used within this study were obtained from birch pollen-allergic patients (n = 6) that were
selected by case history, positive skin prick test and Bet v 1-specific IgE reactivity. Detection of the
latter was performed by Immuno-CAP (Thermo Fisher Scientific, Uppsala, Sweden). The study was
approved by the Ethic Committee of the Medical University and General Hospital of Vienna (no.
EK028/2006).
Protein expression, purification and characterization of recombinant Bet v 1.0101 (rBet v 1)
Recombinant Bet v 1.0101, called rBet v 1, was expressed and purified as previously described[24]. In
short, for the expression of rBet v 1 the Escherichia coli strain BL21 StarTM (DE3) (Invitrogen, Carlsbad,
CA, USA) was used. Protein purification was performed using protein precipitation with 200 mM
sodium chloride, and low-pressure chromatography using a 10 ml phenyl Sepharose as well as a
DEAE Sepharose column (GE Healthcare Biosciences, Little Chalfont, UK). Endotoxin contamination
was determined by EndoZyme® recombinant Factor C (rFC) assay (Hyglos GmbH, Bernried am
Starnberger See, Deutschland), Limulus amoebocyte lysate (LAL) assay (Associates of Cape Cod, Inc.,
East Faulmouth, MA, USA) and a HEK-Blue™ mTLR-4 reporter cell line (Invivogen, San Diego,
California, USA). Detected endotoxin levels were below the threshold of 0.3 ng/ml. After proper
physicochemical characterization of rBet v 1, the protein was stored lyophilized at -20°C.
14
Soluble fraction of aqueous birch pollen extract
An amount of 5 mg of Betula pendula pollen (Allergon AB, Ängelholm, Sweden) were weighed and
dissolved in PBS. After shaking the suspension for 24 h at 4°C, a centrifugation step (three times for 5
min at 12,000 x g at 4°C) was used to collect the supernatant. The obtained extract was filtered
through a 0.2 µm pore-size sterile filter (Merck Millipore, Merck KGaA, Darmstadt, Germany).
Purification of natural Bet v 1 (nBet v 1)
Natural Bet v 1 (nBet v 1) was purified from an birch pollen extract of 5 mg Betula pendula (Allergon
AB, Ängelholm, Sweden) pollen. The extract was prepared in 50 ml endotoxin-free water
supplemented with 0.5 M NaCl. The pollen-buffer suspension was shaking at 1,400 rpm for 5 min at
RT and then centrifuged for 5 min at 12,000 x g at 4°C. The obtained supernatant was filtered
through a 0.45-µm filter (GE Healthcare Biosciences, Little Chalfont, UK). The purification of nBet v 1
was performed using a combination of hydrophobic chromatography size exclusion chromatography.
In this respect a 10-ml Phenylsepharose column and a Superdex 75 10/300 GL column (both from GE
Healthcare Biosciences) were used. The purified nBet v 1 was stored in solution at -20°C. Mass
spectrometric analysis revealed that the nBet v 1 preparation is a heterogeneous mixture of several
Bet v 1 isoforms: Bet v 1a (MS-score 492.42, coverage 93.13), Bet v 1f (MS-score 507.42, coverage
73.75), Bet v 1g (MS-score 401.48, coverage 70.63), Bet v 1m (MS-score 454.27, coverage 67.50) and
some Bet v 1-derived fragments.
Identification of bacterial colonies on pollen grains
Birch pollen (Betula pendula) was harvested during the flowering season found on trees (n=5) at
different sites of Salzburg, Austria. Collected pollen grains (10 mg) were resolved in 1 ml of a PBS
buffer. A total of 100 µl of pollen suspension was plated either on a GC-agar plates (5% FCS, 1 µg/ml
Nystatin) or on nutrient agar plates (1 µg/ml Nystatin). The plates of both agar types were incubated
at 20°C or 37°C. Bacterial colonies were selected and identified via gram staining and 16S rRNA
sequencing.
Quantification of LPS and LTA present in the birch pollen extract
The amounts of LPS and LTA in birch pollen extracts were determined by titration of LTA and LPS
(from 10 pg/ml to 100 ng/ml) with a luciferase reporter assay using hTLR-2 or hTLR-4 transfected 15
HEK293 cells (as previously described[43]. The results obtained by the titration experiment were
then compared with the values of birch pollen extract.
Used ligands
The three compounds, sodium deoxycholate (DOC), lipoteichoic acid (LTA) from Staphylococcus
aureus, and lipopolysaccharides from Escherichia coli O111:B4 were ordered from Sigma-Aldrich, Inc.
(St. Louis, MO, USA). Kdo2-Lipid A (Kdo2) was purchased from both, Adipogen, Inc. (Songdo-dong,
Yeonsu-gu, Incheon, South Korea) and Avanti Polar Lipids, Inc. (Alabaster, Alabama, USA); and the
flavonoid quercetin 3-O-sophoroside (Q3OS) from Haihang Industry Co., Ltd. (Jinan City, China). All
phytoprostane isotypes, including E1-phytoprostanes (PPE1), B1-phytoprostanes (PPB1), F1-
phytoprostanes (PPF1) and the isomeric mixture consisting of all these three phytoprostanes (PPmix)
were synthetized by autoxidation of α-linolenic acid, as previously described[44]. In most of the
experiments, the molar ration that was used for rBet v 1 plus ligand was 1:10. If not, it was
mentioned explicitly in the text. The protein-ligand mixture was incubated either o/n at 4°C or for 2 h
at RT. PPA1 was purchased by Cayman Chemical (Ann Arbor, Michigan, USA).
ANS displacement assay
An amount of 50 µl of rBet v 1 (10 µM) was pre-incubated with the ligand in the above-mentioned
molar ratio. The experiment was performed for each ligand. The protein-ligand solution was
incubated for 5 min at RT with 50 µl of a 8-Anilinonaphthalene-1-sulfonic acid (ANS) reagent (50 µM)
and then measured within a transparent 96-well F-bottom plate (Greiner Bio-One, Kremsmünster,
Austria). The ANS displacement assay data were generated by recording an emission scan starting
from 400 up to 600 nm (in 2 nm steps). The excitation wavelength of the measurement was 370 nm.
For each ligand the background signal (ligand plus ANS plus sodium phosphate buffer, pH 7.4) was
subtracted.
Protein-ligand interaction studies used to determine the affinity constant KD
The affinity constant KD was determined via using the surface acoustic waves (SAW) technology.
With the sam® 5BLUE biosensor equipment (nanotemper, Munich, Germany) the interaction capacity
of rBet v 1 with each one of the six ligands was analyzed. In this regard, each ligand was titrated
towards immobilized rBet v 1. The protein was immobilized on a SAW CM-Dextran 3D sensor chip by
activating the surface of the chip with a freshly prepared mixture of NHS and EDC in a concentration
16
of 100 mM and 400 mM, respectively. Free, uncoupled binding sites were blocked with 1 M
ethanolamine, pH 8.5. To determine the affinity constant KD, different concentrations of each ligand
were applied on the coupled chip. The buffer used for this titration experiment was a 5 mM sodium
phosphate buffer, pH 7.4. According to which ligand was used, the concentration range of the
titration experiment differed slightly and ranged between 5 µM and 500 µM. After each samole
injection, residual ligand was removed by washing the chip with a 10 mM citric acid regeneration
buffer, pH 2.8. For the analysis of the recorded SAW phase changes and the calculation of the affinity
constant KD the software TraceDrawer 1.7 (Ridgeview Instruments, Uppsala, Sweden) was used. The
KD was determined by both, kinetic and affinity/EC50 evaluation. All interaction studies were
performed using two individually coated chips. To ensure coupling efficiency, the chip was analyzed
using a mouse monoclonal anti-Bet v 1 antibody.
Pull-down assay using biotinylated LPS
The LPS pull-down was performed using an adaptation of a previous reported protocol[45]. In brief,
10 µl of biotinyled LPS from E. Coli 011:B4 (Invivogen, San Diego, California, USA) in a concentration
of 1 mg/ml immobilized on 20 µl Strep-Tactin® Sepharose® (IBA Lifesciences, Goettingen, Germany)
was incubated with 10 µl of rBet v 1 (1 mg/ml) under shaking conditions for 20 min at RT. The
solution was centrifuged for 5 min at 14,000 x g at RT and followed by three washing steps using 100
µl of PBS with 0.05% Tween 20. SDS sample buffer with 5 mM DTT was added to the beads (volume
approximately 20 µl) and analyzed using SDS-PAGE. The controls for this assay were: rBet v 1 only,
non LSP-removed rBet v 1, biotinyled LPS only, beads only, and beads incubated with the protein in
order to exclude unspecific binding to the beads.
Analysis of secondary structural elements and thermal stability
Circular dichroism (CD) spectroscopy and Fourier transform infrared (FTIR) spectroscopy was used in
order to address the influence of ligand binding on the secondary structural elements and the
thermal stability of rBet v 1. CD spectra were recorded from 190 to 260 nm using a JASCO J-815
spectropolarimeter fitted with a PTC-423S Peltier-type single position cell holder (Jasco, Tokyo,
Japan). Each sample was diluted in a 10 mM potassium phosphate buffer, pH 7.4. The final
concentration was 0.1 mg/ml. For monitoring the thermal stability, the signal in millidegrees (mdeg)
was recorded from 20 to 95°C (temperature slope: 1°C/min) at a wavelength of 222 nm.
The FTIR amide 1 and amide 2 spectra were recorded using an AquaSpec transmission cell adapted to
a Tensor II FTIR system (Bruker Optics Inc., Billerica, MA, USA). The sample concentration ranged 17
between 1.0-2.0 mg/ml. Each analyzed sample was filter prior to the analysis through an amicon
ultra-0.5 centrifugal filter unit, 3 kDa cutoff (Merck Millipore, Darmstadt, Germany) in order to
concentrate the sample and to obtain the accurate buffer for background subtraction. The spectra
were recorded at 25°C, thermostatted by a temperature-controlled Haake F8 thermostat (Thermo
Electron, Germany). The acquired data were analyzed with the OPUS spectroscopy software 6.0
(Bruker Optics Inc., Billerica, MA, USA). All spectra were vector normalized over the amide 1 band.
The second derivative values were calculated of smoothed data (Savitzky-Golay algorithm, 25
smoothing points). Secondary structural elements were analyzed with a Quant2 method provided by
the OPUS software. The spectra used for thermal stability determination were recorded from 25 to
95°C (dT=2.5K) using a BioATR II unit (Bruker Optics Inc., Billerica, MA, USA). Difference spectra were
generated by subtracting the original spectrum measured at 25°C from the spectra recorded at the
other temperatures. From the thereof resulting relative signal change of the amide I band the
melting point (Tm) was calculated.
Production of 15N-1H labeled Bet v 1 for NMR analysis 15N-1H labeled Bet v 1 was expressed as previously described[46]. In brief, an amount of two liters of
bacteria culture grown in LB medium for 24 h, was harvested. The obtained bacteria pellet was
resolved in 0.5 L M9 salts lacking any carbon or nitrogen source. After feeding the bacteria with
glucose, 15N ammonium chloride, and 15N Celltone (1/25 g) (Cambridge Isotope Labs), the suspension
was incubated for 35 min. For the induction of protein expression IPTG was used. 15N-1H labeled Bet
v 1was purified with the same protocol as described above (Protein expression, purification and
characterization of rBet v 1).
NMR spectroscopy
NMR spectroscopic measurements were performed with both, a 600 or 800 MHz Agilent DD2
spectrometer with a cryogenically cooled probe using the pulse sequence gNhsqc. According to the
ligand the different buffer conditions and protein concentrations were used. 45 mM Bet v 1 and 300
mM LTA was analyzed in PBS, 10% 2H2O, 60 mM DSS. The interaction with PPE1 was investigated
using 25 mM Bet v 1, 2 mM PPE1 and a PBS buffer containing 5% 2H-(d6) DMSO, 10% 2H2O, 60 mM
DSS. For PPA1 100 mM Bet v 1 and 302 mM PPA1 was used in PBS, 8.7% 2H-(d6) DMSO, 10% 2H2O, 60
mM DSS. Similar conditions were used for LPS (100 mM Bet v 1, 400 mM LPS, PBS, 10% 2H2O, 60 mM
DSS). The interaction of 100 mM Bet v 1 with 422 mM Kdo2 was analyzed in a PBS buffer, pH 7.0,
10% 2H2O, 8.6% 2H(d6) DMSO, 60 mM DSS.
18
In vitro endo-/lysosomal degradation
The endo-/lysosomal degradation of rBet v 1 with and without pre-incubation with each one of the
six ligands was simulated as previously described[47]. In this respect, cells from the JAWS II cell line
(American Type Culture Collection, Manassas, VA, USA) were centrifuged using ultracentrifugation in
order to obtain the microsomal fraction. The 7 µg microsomes were incubated with 5 µg of protein in
complex with or without ligand in 100 mM citrate buffer, pH 4.8 with 2 mM dithiothreitol (DTT). The
degradation was performed at 37°C over 48 h and analyzed at defined time points (0, 0.5, 1, 3, 6, 12,
24 and 48 h). After the degradation reaction of the last sample was stopped, all degradation samples
were analyzed using SDS-PAGE. The gels were quantitatively analyzed with the Image Lab 4.0.1
Software (Bio-Rad). For mass spectrometric analysis of the degradation only the samples obtained
after 12 h of incubation were used. The mass spectrometric analysis was performed using a Q-
Exactive Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) with
nanoelectrospray and nano-HPLC (Dionex Ultimate 3000, Thermo Fisher Scientific). Acquired data
were analyzed with the web-based application MSTools[48].
Mediator release assay
The influence of protein-ligand interaction on the allergenic potential of rBet v 1 to induce the
release of mediators was investigated using a rat basophil (RBL-2H3) cells, transfected with the
human high-affinity IgE receptor (FceRI)[49]. The rat basophil cells were passively sensitized with sera
of birch pollen allergic patients (1:10). Degranulation was stimulated via a titration of the antigen in
1:10 dilution steps from 1 µg/ml to 0.01 pg/ml. Fluorescence data were normalized towards the total
enzyme release, as identified by cells lysed with Triton X-100, and presented as percentage of
release. From the resulting titration curves it was able to determine the half maximal release (in ng).
Statistics were performed using a repeated ANOVA followed by a Dunnett post-hoc test. All half
maximal release values were logarithmically transformed prior to the statistical analysis. A p-value
that was higher than 0.05 was considered as not significant. In parallel, a cell viability assay was
performed with MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide; Sigma-Aldrich,
Inc., St. Louis, MO, USA) in order to determine if there was a cytotoxic effect caused by the ligands
used in this experiment.
19
Stimulation and antigen uptake of murine bone marrow-derived dendritic cells (mBMDCs)
For the stimulation, BMDCs isolated from C57BL/6 mouse bone marrow were incubated with the
antigen over a certain period of time (24, 14.5, 6, 3, 1 and 0 h). The cell isolation was performed as
previously described[50]. Bone marrow cells, extracted from female mouse femora, were cultured in
RPMI 1640 medium (5% fetal calf serum, 2 mM L-glutamine, 1% Penicillin-Streptomycin, 20% GM-CSF
supernatant and 200 µM ß-mercaptoethanol) for 10 days, and then either immediately used for the
stimulation/uptake experiment or frozen. 2x105 cells of mBMDCs were stimulated with 0.5 µg of rBet
v 1. In contrast to the antigen stimulation/maturation experiment where unlabeled rBet v 1 was
used, the antigen uptake experiment was performed using rBet v 1 labeled with pHrodo™ Red,
succinimidyl ester (ThermoFisher Scientific). As control, the cells were stimulated with medium alone
and ligand without protein. Cell treated with 100 ng/ml LPS represented the positive control. For
comparison with birch pollen extract, the amount of Bet v 1 in the extract was determined in order
to adjust the Bet v 1 content of the extract towards rBet v 1 (0.5 µg/2x105 cells). LPS was used in a
ratio of 40 pg of LPS per µg rBet v 1 (nLPS samples); PPE1 in a 1:1 molar ratio. For the flow cytometric
analysis, the cells were labeled with allophycocyanin (APC)-conjugated anti-mouse CD11c antibody
(clone N418; eBioscience, Inc., San Diego, CA, USA), Fluorescein isothiocyanate (FITC)-conjugated
anti-mouse CD86 antibody (clone GL-1; BioLegend, San Diego, CA, USA), PerCP/Cy5.5 anti-mouse
CD40 antibody (clone 3/23; BioLegend, San Diego, CA, USA). Cell viability was investigated using a
fixable viability stain 450 (BD Biosciences). The V450 Rat anti-Mouse LY-6G and LY-6C (BD
Biosciences) antibody was used to determine the amount of granulocytes and monocytes within the
cell suspension. The cells were measured with a FACSCanto II instrument (BD Biosciences, San Jose,
CA, USA). The measurement was compensated and the acquired data were analyzed using the
BDFACSDiva software (BD Biosciences).
Maturation of human monocyte-derived dendritic cells (moDCs) and analysis of cytokine secretion
The stimulation of moDCs was performed using the ligands in a 1:10 molar ratio. The moDCs were
isolated from PBMCs according to a previous described protocol[44]. MoDCs were generated from
PBMCs of healthy, non-atopic as well as atopic donors. Cell viability was investigated using Aqua® dye
(Invitrogen, Carlsbad, CA, USA). Expression of CD1a (eBioscience, Inc., San Diego, CA, USA) and loss of
CD14 (BD Biosciences) was analyzed by flow cytometry using a Navios flow cytometer (Beckman
Coulter, Brea, CA, USA). The concentration of rBet v 1 alone or in complex with one of the six ligands
used to stimulate 1*106 cells/ml moDCs was 1000 ng/ml. Stimulation was performed for 24 h. The
same controls were used as for the mBMDC stimulation (unstimulated moDCs and ligands only). The 20
cells were not co-stimulated using LPS. The investigated maturation markers were: CD40
(eBioscience), HLA-DR, CD80, CD83 and CD86 (BD Biosciences). After the stimulation, different levels
of cytokine expression of the supernatant were determined. The analyzed cytokines were: CCL17, IL-
1β, IL-10 (BD Biosciences), IL-6, IL-12 (eBioscience), and TNFα (R&D Systems, Inc., Minneapolis, MN,
USA).
Immunization of IL-4/GFP-enhanced transcript (4get) mice
A total of 13 4get mice (Jackson laboratory, Bar Harbor, Maine, USA) were immunized by applying
two intradermal injections of 25 µl at each side of the abdominal region. The mice were grouped into
animals that received rBet v 1-combination (n=5), birch pollen extract (n=5) or rBet v 1 (n=3). 65 µg
of birch pollen extract were injected. A corresponding amount of rBet v 1, as quantified from the
extract, was injected. For those animals that received rBet v 1-combination, a mixture of the
determined amount of rBet v 1 and Q3OS, PPE1 (both in a 1:1 molar ratio to rBet v 1), the natural
amount of LPS and LTA (0.04 ng/0.5 ng per 1 µg, respectively) was prepared. For each animal, sample
administration was on the right lateral, whereas on the left site PBS was injected as control. After
sacrificing the mice on day 5 post-immunization, the excision of skin-draining inguinal lymph nodes
was performed. The extracted lymph node cells were labeled using an APC-conjugated anti-mouse
CD4 antibody (BD Biosciences, San Jose, CA, USA). IL-4/eGFP expressing CD4+ T cells were counted
with flow cytometry. The obtained data were background subtracted and statistical analysis was
performed. In order to compare all groups with each other a one-way ANOVA with a Bonferroni post-
test was used. The in vivo experiment were approved by the Austrian Federal Ministry of Science,
Research and Economy (BMWF-66.012/0010-II/3b/2013) and performed according to their
guidelines.
Results
Identification of bacterial colonies on pollen grains
Pollen grains are known to provide a biotope for a huge variety of bacteria[40]. It is possible to detect
high amounts of microorganisms-derived endotoxins in pollen coats. In this respect, we analyzed the
microbiome found on pollen, which was collected from different birch trees around the area of
Salzburg, Austria. The analysis of the microbiome resulted in 51 identified microorganism isolates.
Thereof, 10 hits were Gram-negative and 41 were Gram-positive microorganisms (Table 1). The
identified Gram-negative bacteria were part of the families of Pseudomonadaceae (3 hits),
Moraxellaceae (2 hits), Caulobacteraceae, Xanthomonadaceae, Sphingomonadaceae,
21
Noctuoideaceae and Rhodobacteraceae (1 hit each). In contrast to that, the amount of
representative species of Gram-positive bacteria was much higher, and the identified isolates could
be classified into the families of Bacillaceae (32 hits), Microbacteriaceae (3 hits), Micrococcaceae (2
hits), Staphylococcaceae, Nocardioidaceae, Streptomycetaceae and Gordoniaceae (1 hit each).
Table 1. Identification of Gram-negative and Gram-positive bacteria strains found on birch pollen grains.
Order Family Count1 Gram-negative Pseudomonadales Pseudomonadaceae 3 Moraxellaceae 2 Caulobacterales Caulobacteraceae 1 Xanthomonadales Xanthomonadaceae 1 Sphingomonadales Sphingomonadaceae 1 Enterobacteriales Noctuoideaceae 1 Rhodobacterales Rhodobacteraceae 1 Gram-positive Bacillales Bacillaceae 32 Staphylococcaceae 1 Actinomycetales Micrococcaceae 2 Nocardioidaceae 1 Microbacteriaceae 3 Streptomycetaceae 1 Gordoniaceae 1 1total number of identified clones in colony forming units (cfu)
Quantification of LPS and LTA present in the birch pollen extract
Since we were able to identify such a comprehensive diversity of both, Gram-positive and Gram-
negative bacteria in the pollen coat, we wanted to know if a pollen extract has the capability to
initiate the activation of human TLR-4 and TLR-2. Therefore, a titration experiment using a HEK293
luciferase reporter cell line was performed with the respective agonists, LPS and LTA, and compared
with the extract (data not shown). From the resulting data it was possible to quantify the amount of
LTA and LPS within an aqueous birch pollen extract necessary to generate the same level of TLR-2 or
TLR-4 activation, respectively. In this respect, an amount 0.04 ng LPS and 0.5 ng LTA per 1 µg of total
protein of the extract was determined. In addition, we determined the capacity of PPE1 and Q3OS to
activate both, TLR-4 and TLR-2. Both molecules did not activate one of these Toll-like receptors (data
not shown).
Production and purification of aqueous birch pollen extract, nBet v 1, rBet v 1 and BM4
The experiments presented in this thesis were performed with an aqueous birch pollen extract or
with the purified proteins, nBet v 1, rBet v 1 or BM4, which is a hypoallergenic variant of Bet v 1 and
22
will be described in detail in chapter 2. All three purified proteins as well as the birch pollen extract
have been analyzed with SDS-PAGE (Fig. 1). A sandwich ELISA using a mouse monoclonal Bet v 1-
specific antibody and polyclonal affinity-purified anti-Bet v 1 antibody was performed to determine
the exact amount of Bet v 1 within the birch pollen extract (data not shown). The percentage of Bet v
1 content within the analyzed birch pollen extract batch was around 25%.
Figure 1. SDS-PAGE analysis of an aqueous birch extract (birch Ex.), natural purified Bet v 1 (nBet v 1), recombinantly produced Bet v 1 (rBet v 1) and the hypoallergenic variant of Bet v 1, BM4.
ANS displacement assay The ANS displacement assay was performed using a 1:10 ratio of rBet v 1 towards the
known/potential ligands (10-fold molar excess of known/potential ligand). This assay is used to get a
first idea about the capacity of Bet v 1 to bind Q3OS, DOC, PPE1, LTA, and LPS. A substructure of LPS,
called Kdo2-Lipid A (Kdo2; 2,306.8 Da) was used in addition to the other five molecules to be analyzed
within these interaction studies. In contrast to LPS, Kdo2 possess a defined, homogenous structure,
but is supposed to have a strong endotoxin activity similar to LPS. For the ANS displacement assay,
rBet v 1 pre-incubated with each ligand was incubated with ANS and then the resulting fluorescence
signal was compared with rBet v 1 without ligand pre-incubation. A decrease of signal caused by the
protein-ligand interaction was shown for each of the six ligands, meaning they were able to replace
ANS binding (Fig. 2). Furthermore, the molecular structure and the molecular weight of the six
ligands are displayed.
23
Figure 2. ANS displacement assay indicating protein-ligand interaction. Fluorescence intensities were measured from 400 to 600 nm. The molar ratio of protein to ligand was 1:10. Continuous line represents the fluorescence intensity of the rBet v 1-ANS complex, whereas the dashed line shows the signal reduction caused by the displacement of ANS by one of the six ligands. Chemical structures and molecular weight of the potential ligands is displayed on top of each graph.
Protein-ligand interaction studies used to determine the affinity constant KD
Since a certain potential of all six selected ligands to interact with Bet v 1 was identified with the ANS
displacement assay, the subsequent, logical step was to measure the exact binding affinity (KD) of
each interaction. This was achieved by performing interaction studies using the surface acoustic
wave (SAW) technology. For these measurements, rBet v 1 was immobilized on a SAW chip and then
titrated towards each ligand (Fig. 3). Whenever the ligand interacts with the protein, this results in a
phase shift of the acoustic wave. The thereof generated phase shifts can be used to determine the
binding-affinity (KD) via kinetic or affinity/EC50 evaluation (Table 2). The protein was found to
interact with a high binding affinity with the natural, pollen-derived components, Q3OS and PPE1
24
(1.47 and 0.48 µM, respectively). In contrast to that, the determined KD values of LTA (199.81 µM),
LPS (160.1 µM), and the synthetic substances, DOC (95.02 µM) and Kdo2 (379.76 µM), were much
higher, and thus the binding affinity much lower. In general, the KD value is reversibly proportional to
the binding affinity[51]. The high KD values determined for LPS, LTA and Kdo2 were found close to the
limit of detection of the SAW technology, which is indicating that the ligand is not binding under
physiological conditions. Moreover, we determined the KD of other phytoprostane isoforms, B1-
phytoprostane (PPB1, 1.03 µM) and F1-phytoprostane (PPF1, 2.39 µM), and also an isomeric mixture
of B1-, E1- and F1-phytoprostanes (PPmix, 1.22 µM). The isomeric mixture is relevant in respect to
naturally occurring phytoprostanes identified within birch pollen extract[52]. In addition, as a control
ligand and in order to point out the relevance of the ANS displacement assay, the binding affinity of
ANS was determined to be 32.72 µM.
Table 2. Binding affinity (KD) of rBet v 1 to ligands determined by SAW interaction studies and binding confirmation by NMR spectroscopy.
Ligand KD [µM] SD [µM] NMR
Q3OS 1.47 ± 0.12 [32]
DOC 95.02 ± 32.40 [37]
PPmix PPB1 PPF1 PPE1
1.22 1.03 2.39 0.48
± 0.15 ± 0.42 ± 0.48 ± 0.14
-2 - - interaction confirmed (KD low to sub µM)
LTA 199.81 ± 55.72 No significant interactions
LPS 160.10 ± 123.32 No significant interactions
Kdo2 379.76 ± 62.82 No significant interactions
ANS 32.72 ± 0.28 [37] 2 not measured (-)
25
Figure 3. Surface acoustic wave (SAW) interaction studies. The SAW phase changes were recorded over time at different concentrations of the investigated ligands in order to calculate the binding affinity.
NMR spectroscopy
In order to definitely proof or exclude that rBet v 1 is not specifically binding to or interacting with
one of the selected ligands, nuclear magnetic resonance (NMR) spectroscopic measurements were
performed. In this respect, only the interactions of those molecules were investigated, which until
now has not been described in previous studies using NMR spectroscopic techniques, including PPE1,
LTA, LPS and Kdo2 (Table 2, Fig. 4). The interaction of rBet v 1 with Q3OS and DOC was already
demonstrated. Of all four analyzed molecules, specific binding by 15N-labelled Bet v 1 was only
confirmed for PPE1, since significant differences between the PPE1-bound and ligand-free 1H-15N
HSQC spectra were observable (Fig. 4, PPE1 full and zoomed spectra). The KD was found in the low to
sub µM range, and thus similar to the value determined by the SAW measurement. No significant
interactions were detected for LTA, LPS and Kdo2, indicating that these endotoxins and endotoxin-
like compounds do not bind rBet v 1 in a specific way.
26
Figure 4. NMR 1H-15N HSQC spectra of rBet v 1 and the possible ligands. Overlay of two spectra of 15N-labelled Bet v 1 in the absence (black) and presence of PPE1 (violet), Kdo2 (green), LTA (red), LPS (light blue) or PPA1 (dark blue).
Pull-down assay using biotinylated LPS
Because of the discrepancies found in the data recorded by the two interaction study techniques,
SAW and NMR, it became evident to take an in-depth look at the subject of LPS-binding to rBet v 1. In
this respect, a pull-down assay was performed using biotinylated LPS immobilized on streptavidin 27
beads (Fig. 5). In case the protein binds LPS it is pulled-down with the LPS-bead-complex and
afterwards can be analyzed with SDS-PAGE. In this assay, rBet v 1 was not binding LPS, and the faint
band that appeared on the SDS-PAGE only resulted from unspecific binding of rBet v 1 to the beads
(lane without biotin-LPS). Finally, we could verify that rBet v 1 is definitely not binding LPS.
Figure 5. LPS pull-down assay is revealing that Bet v 1 is not interacting with LPS. Biotinylated LPS was immobilized on streptavidin-conjugated beads and incubated with rBet v 1. The complex was pulled-down and washed three times. After the washing of the beads only a faint protein band at the height of rBet v 1 was visible and associated with unspecific binding.
Analysis of secondary structural elements and thermal stability
Although it was already confirmed that LTA, LPS and Kdo2 are not binding rBet v 1, we aimed to
investigate whether or not the selected molecules used in this study have the capability to influence
the secondary structure of rBet v 1. Therefore, the protein complex preincubated with each one of
the six molecules was analyzed in separate using circular dichroism (CD) spectroscopy and Fourier
transform infrared (FTIR) spectroscopy. None of the selected molecules possessed the ability to alter
the secondary structural content of the allergen (Fig. 6).
Interestingly, we found that the presence of the ligands and non-ligands has an influence on the
thermal stability of rBet v 1. To determine the melting point (Tm) of rBet v 1, its denaturation in the
different preparations was record starting from 20/25°C up to 95°C. From the resulting melting curve
(data not shown) it was possible to calculate the Tm (Table 3). In contrast to LPS (mean ΔTm -3.81)
and Kdo2 (mean ΔTm -2.4), all analyzed ligands and LTA were able to increase in the thermal stability
of the allergen. The binding of rBet v 1 to the pollen-derived PPE1 even increased the thermal
stability by approximately 6.5°C. The increase caused by DOC, LTA and Q3OS on the other hand was
28
rather modest (ΔTm about 1-3.5°C). The data recorded by the both methods, CD and FTIR, were
comparable.
Table 3. Influence on the thermal stability of rBet v 1*
Ligand Tm CD SD CD Tm FTIR SD FTIR Δ CD Δ FTIR
- 63.68 ± 0.06 63.38 ± 2.24
Q3OS 64.04 ± 0.10 65.26 ± 1.77 + 0.36 + 1.88
DOC 67.44 ± 0.58 66.6 ± 4.36 + 3.81 + 3.22
PPE1 70.62 ± 0.15 69.31 ± 0.05 + 6.94 + 5.93
LTA 65.35 ± 0.08 65.16 ± 2.21 + 1.67 + 1.78
LPS 60.27 ± 0.15 59.17 ± 3.05 - 3.41 - 4.21
Kdo2 62.95 ± 0.05 59.31 ± 4.47 - 0.73 - 4.07
CD, circular dichroism; FTIR, Fourier transform infrared spectroscopy; Tm, melting point; SD, Standard deviation *All values are in °C. Light highlighting refers to increase of thermal stability in comparison to rBet v 1 without a ligand, whereas dark highlighting to a decrease.
29
Figure 6. Interaction does not affect the secondary structural elements of rBet v 1. Secondary structure elements were analyzed by CD (a) and FTIR (b) measurements.
30
In vitro endo-/lysosomal degradation
At this point, we were already able to show that some of the selected molecules used within this
study bind to Bet v 1 (PPE1, Q3OS and DOC), (as has been previously shown[32, 37]. Other molecules
(LPS, LTA and Kdo2), whilst not directly interacting with the allergen, possess important co-
stimulatory functions on the immune system and, with the exception of Kdo2, were identified within
pollen extracts. Therefore, it proved logical to also investigate those molecules in the presence of Bet
v 1 in the following experiments.
To investigate if the ligands/non-ligands have an impact on the proteolytic stability of rBet v 1, an
endo-/lysosomal degradation simulation assay was performed over 48 h. All three ligands of Bet v 1
(PPE1, Q3OS and DOC) enhanced the proteolytic stability of the allergen, as displayed by the
densitometric analysis of SDS-PAGE (Fig. 7a, b). In complex with PPE1 or DOC, even after 48 h the
majority of rBet v 1 still was intact and unaffected by proteolytic degradation. On the contrary, the
presence of the three molecules that do not bind to Bet v 1 (LPS, LTA and Kdo2) either did not affect
proteolytic stability of the protein (LPS) or even resulted in a decreased stability towards endo-
/lysosomal degradation. With the exception of LTA, all these identified effects on the proteolytic
stability of rBet v 1 correlated with the results obtained for the thermal stability.
In previous studies, a very likely correlation between the proteolytic stability of allergens and the
quantity as well as quality of the resulting immune response was discussed[53]. With regard to
peptide presentation by MHC class 2 molecules to naïve CD4+ T cells, the sequences of generated
peptides after 12 hours of endo-/lysosomal degradation were analyzed by mass spectrometry (Fig.
7c). The peptide clusters of rBet v 1 generated in presence with LTA or Kdo2 were found to have
increased variety of different peptides. In contrast, the LPS preparation displayed peptide clusters
comparable to rBet v 1 alone. Although the binding of Q3OS resulted in an increased stability
towards endo-/lysosomal degradation, the allergen bound to Q3OS showed a higher frequency of
generated peptides in comparison to unbound rBet v 1. On the contrary, binding to PPE1 and DOC
decreased the frequency of peptides, and thus these data are in good correlation with the induced
increased proteolytic stability (Fig. 7a, b) facilitated by ligand binding. However, the generation of
peptides in the region of the immunodominant T-cell epitope of Bet v 1 (Fig. 7c, grey box) in general
was scarce. Interestingly, when comparing all seven clusters a higher frequency of peptides in this
region was observable for those samples where rBet v 1 was bound either to the physiologic ligand
Q3OS or to DOC.
31
Figure 7. Ligand interaction alters the proteolytic susceptibility of rBet v 1. (a) SDS-PAGE analysis of in vitro endo-/lysosomal degradation of rBet v 1 with and without ligands recorded at different time points from 0 to 48 hours. (b) Relative quantification of SDS-PAGE results, interpreted by the Image Lab 4.0.1 Software (Bio-Rad). (c) Generated peptide clusters obtained after 12 hours of proteolytic degradation analyzed by mass spectrometry. The immunodominant T-cell epitope of Bet v 1 is highlighted in grey.
Mediator release assay
Next, the effect on the IgE-mediated mediator release was examined using rat basophilic leukemia
cells transfected with the human FcεRI IgE receptor (hRBL). Therefore, hRBL cells were passively
sensitized with serum from six patients allergic to birch pollen. The cells were stimulated with the
antigen and the resulting cross-linking of the allergen by immobilized IgEs caused a release of β-
32
hexosaminidase. The release was monitored in a concentration-dependent way (Fig. 8). Using this
assay, no significant influence of the six ligands/non-ligands was observed. Since none of the six
investigated molecules altered the secondary structural elements of rBet v 1, and consequently the
IgE epitopes remained intact and unaffected, it was quite predictable that there would be no
influence on the IgE-mediated release of inflammatory mediators. The following immunological
experiments are focusing on the influence of the ligands and non-interacting molecules on allergic
sensitization towards Bet v 1 in a naïve model. Whereas here (mediator release assay), we
investigated the influence on a pre-established immune response towards Bet v 1, since the hRBL
cells were stimulated with serum of allergic patients.
33
Figure 8. Protein-ligand interaction does not alter the IgE binding and cross-linking activities of rBet v 1. (a) Mediator release assay; amount of rBet v 1 with and without ligand (in ng) necessary to induce a half maximal β-hexosaminidase release using serum of six birch pollen allergic donors. P-values were calculated with ANOVA of prior transformed data [y= log(y)]. There was no statistically significant difference observed between the seven groups (P < 0.05). (b) Individual mediator release assay titration curves from each patient.
34
Stimulation and antigen uptake of murine bone marrow-derived dendritic cells (mBMDCs)
The importance of the involvement of APCs in the process of allergic sensitization, needed for the
priming of naïve T cells, is incontestable. In fact, APCs are necessary to take-up, process and present
the allergen in order to promote Th2 polarization. In this context, dendritic cells represent a very
relevant group of APCs. Therefore, we wanted to investigate if the maturation and the antigen
uptake of CD11c+ mBMDCs upon stimulation with different preparations of rBet v 1 plus ligands/non-
ligands differ from un-stimulated or extract-treated cells. The cells were stimulated over 24 h with
the antigen to determine if BMDC maturation markers such as, CD86 and CD40 are up-regulated (Fig.
9a). The different mixtures of rBet v 1 plus ligand/non-ligand used in this experiment were chosen
upon their attributes to mimic natural levels of exposure. PPE1 was chosen as representative ligand
of Bet v 1, since it has been described to possess a Th2 polarizing capacity[34]. LPS was chosen as a
representative of non-interacting bacterial compound and used in the same concentration as
measured in aqueous birch pollen extracts (nLPS). Also a mixture containing rBet v 1 and both
molecules was prepared (rBet v 1+PPE1+nLPS). The controls were PPE1/nLPS only, an aqueous birch
pollen extract and of course rBet v 1 alone. Moreover, LPS in a concentration of 100 ng/ml was used
as a positive control. The extract induced a statistically significant, time-dependent activation of
both, CD86 and CD40, compared to the unstimulated cells (medium control), and the MFI was even
higher than the positive control. With the exception of the birch pollen extract and the positive
control, none of the analyzed samples were able to induce a significantly upregulated expression of
CD86. In comparison, CD40 was also slightly upregulated upon stimulation with rBet v 1+nLPS and
rBet v 1+PPE1+nLPS, although no maturation-stimulating effect was shown for nLPS alone.
In order to exclude that the lack of maturation induction was influenced by the missing of other Bet v
1-isoforms (not rBet v 1), or by the recombinant production procedure of rBet v 1, we used a batch of
natural Bet v 1 purified from birch pollen extract and compared it with the recombinant variant (Fig.
9b). The expression of CD86 was no significantly altered in both natural and recombinant Bet v 1
preparations when compared to medium-treated cells.
As already mentioned above, also the effects of the selected six molecules on antigen uptake of
murine BMDCs were investigated. In this case, the cells were incubated with pHrodo™ Red-labeled
rBet v 1, pre-incubated with one of the molecules and compared with apo-rBet v 1 (Fig. 9c). When
treating the cells with rBet v 1 in the presence of LTA or DOC, an increase in the percentage of
antigen-processing cells was observable, indicating fast and efficient antigen-internalization.
Significant alterations between the different samples were not detected after 1 h and 6 h of
incubation. After treating the cells for 6 h with the different antigen preparations, all samples except
35
apo-rBet v 1 reached an uptake-plateau. In contrast, rBet v 1 alone had been internalized by the cells
until 24 h. In general, we can conclude that although all rBet v 1-containing samples are processed
fast (approximately 40% of CD11c+ BMDCs after 1 h of treatment), as determined by the increase of
fluorescence of pHrodo in endo-/lysosomal acidic environment, this antigen uptake is not translated
into maturation of CD11c+ murine BMDCs.
Figure 9. Activation and uptake of Bet v 1 by murine BMDCs observed over time. Time-dependent maturation and uptake experiments were measured by flow cytometry. BMDC activation was performed with birch pollen extract, rBet v 1 +/- the naturally occurring amount of LPS (nLPS), or PPE1 phytoprostanes in a 1:1 molar ratio (according to the NMR results) or a combination of both (rBet v 1+PPE1+nLPS). Murine BMDCs (CD11c+) were stained for the maturation markers CD86 and CD40. Dashed line represents the mean of basal activation of uninduced cells. Cells treated with 100 ng/ml of LPS served as a positive control for BMDC activation (a). Comparison of CD86 activation using rBet v 1 or nBet v 1 (purified from a birch pollen extract (b). Percentage of BMDCs that are taking up pHrodo™ Red, succinimidyl ester-labeled rBet v 1 in complex with or without ligand are shown (c). Data represent means of duplicate values *P<0.05, **P<0.01, ***P<0.001 significantly different versus the medium control (a) or rBet v 1 without ligand (b, c) as calculated by using repeated ANOVA. Data derived from at least two independently performed experiments.
Maturation of human monocyte-derived dendritic cells (moDCs) and analysis of cytokine secretion
Next, we addressed the effect of the six molecules (1:10 molar ratio) on the activation of human
dendritic cells stimulated by rBet v 1. In this respect, the expression of maturation markers CD40, 36
HLA-DR, CD83, CD80 and CD86 of moDCs was analyzed by flow cytometry (Fig. 10a). In addition, the
supernatant of the stimulated moDCs was collected and the secreted cytokines were analyzed (Fig .
10b). For this experiment, moDCs generated from PBMCs of both, atopic and non-atopic donors were
used. Compared to unstimulated cells, unbound rBet v 1, as well as in complex with the ligands,
Q3OS, PPE1 and DOC did not alter the maturation profile or cytokine production of moDCs
significantly. As a control the stimulating effect of the six molecules in absence of rBet v 1 was
investigated, but in this case also did neither induce statically significant expression of maturation
markers, nor secretion of cytokines.
On the contrary, the bacterial-derived compounds (LPS, LTA, Kdo2) had a stimulatory effect on
moDCs, which was observed for the expression of maturation markers as well as for the secretion of
cytokines. However, with the exception HLA-DR and CD86 expression stimulated by LTA alone, none
of the effects on the expression of maturation markers was statistically significant. No additive effect
caused by rBet v 1 was found when analyzing both, the expression of maturations markers and the
cytokine production. In general, significant differences between moDCs derived from atopic and non-
atopic donors were not observable.
37
Figure 10. Expression of maturation markers (CD40, HLA-DR, CD83, CD80 and CD86) and cytokines (IL-6, IL-12, CCL17, IL-10, IL-1β and TNF-α) of moDCs derived from atopic and non-allergic donors upon stimulation with Bet v 1 in combination with either pollen- or bacterial-derived compounds. Maturation experiments were performed by flow cytometry and results were presented as median fluorescence. MoDCs were treated with 1000 ng/ml of rBet v 1 with or without ligand pre-incubation in a 1:10 molar ratio. No influence of DOC, Q3OS or PPE1 on the expression of maturation markers of moDCs was found (data not shown). Data represented as mean with SEM. P-values were calculated with ANOVA. All statistical calculations were performed using the GraphPad Prism 5 software; Ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
Immunization of IL-4/GFP-enhanced transcript (4get) mice
With both experiments, the stimulation of murine BMDCs and of human moDCs, we could
demonstrate that Bet v 1 without the pollen context is not sufficient to induce activation of dendritic
cells. This fact prompted us to investigate the contribution of those molecules that are naturally 38
occurring in the pollen (LPS, LTA, Q3OS and PPE1) on allergic sensitization towards Bet v 1 and
promotion of a Th2-biased immune response in more detail using an in vivo approach. For this
purpose, IL-4/GFP-enhanced transcript (4get) mice were immunized either with an aqueous birch
pollen extract, rBet v 1 alone or the above-mentioned combination. After immunization, the IL-4
gene activation of CD4+ T cells of the skin-draining inguinal lymph nodes was monitored by flow
cytometric analysis. Interestingly, the physiologically relevant cocktail of rBet v 1 plus ligands (Q3OS,
PPE1) and bacterial compounds (LPS, LTA) was not able to induce a Th2 polarization, and neither did
the control group where rBet v 1 alone was injected. On the contrary, the immunization with the
birch pollen extract resulted in a massive activation of IL-4 gene expression (Fig. 11).
Figure 11. In vivo mouse model demonstrating the lack of Th2-polarising properties of rBet v 1 alone or in combination with pollen- and bacterial-derived compounds. Birch pollen extract clearly shows Th2-polarization activity. Gating strategy used to quantify CD4+ IL-4 GFP+ T cells (a). CD4+ lymphocytes were selected and analyzed with reference to Th2 differentiation. Percentage of IL-4 expressing CD4+ T cells of 4get mice immunized with rBet v 1, rBet v 1 in combination with pollen-derived compounds or aqueous birch pollen extract (b). Statistical analysis of 4get in vivo data was performed using one-way ANOVA with a Bonferroni post-test; Ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
Discussion
Recently, the majority of scientific publications directly associate the term “allergenicity” with the
intrinsic molecular properties of the allergens themselves. Mentionable examples are the protease
activity of mite Der p 1, the glycosylation patterns of mite Der p 1 and Der p 2, cat Fel d 1, peanut Ara
h 1, cockroach Bla g 2, and dog Can f 1, as well as the LPS-binding activity of Der p 2[45, 54-57]. In
case of Bet v 1 the situation is different, since it lacks protease activity and a glycosylation profile. 39
Therefore, the intrinsic properties of Bet v 1 that explains its allergenic potential remain elusive. With
regard to this, the ligand binding capacity of Bet v 1 was postulated to be responsible for its
allergenicity, as it was demonstrated already for a couple of food and respiratory allergens. Within
this chapter, ligand binding of Bet v 1 was investigated excessively[28]. We found that the allergen is
able to bind many low molecular weight, pollen-derived compounds, but cannot interact with
microbial-derived compounds possessing a molecular weight higher than 2000 Da. However, since
these microbial-derived compounds are present in aqueous pollen extracts, and there might
represent a source for co-stimulatory signals to influence the immune system, their contribution on
allergic sensitization was investigated. Neither the examination of pollen-derived ligands, nor of the
microbial-derived molecules revealed a significant influence on the allergenicity of Bet v 1.
Both, the TLR-2 and TLR-4 agonists, LTA and LPS, were identified within birch pollen extracts and
most likely derive from microorganisms inhabiting the pollen grains. The ratio of Gram-positive to
Gram-negative microorganisms determined on birch pollen was in imbalance; actually the number of
Gram-positive exceeded the number of Gram-negative bacteria by far. In this respect, much higher
amounts of LTA than LPS were quantified within the aqueous pollen extract. Although we identified a
couple of other members of the Bacillaceae family, the data we provided within this study on the
bacterial microbiota of birch pollen grains collected in around the area of Salzburg, Austria is
comparable to the reported microbiota data of German pollen[40]. The levels of LPS (40 pg /µg total
protein) and LTA (500 pg /µg total protein) we quantified in the extract were also similar to reported
values that usually range between 16.31 to 2300 pg/µg of protein[58, 59]. In general, pollen grains
do not represent a sterile environment. In this context, an interaction of PR-10 proteins like Bet v 1
with such microbial-derived compounds is possible[41]. In the performed experiments, either a
binding affinity of Bet v 1 to LTA, LPS and Kdo2 close to the detection limit was determined (SAW
interaction study)[60, 61] or even completely excluded (NMR spectroscopy). According to the data
presented in this study, an interaction of Bet v 1 to bacterial compounds (>2000 Da) is rather
questionable. Considering the size of the cavity of Bet v 1 and its even smaller, solvent-accessible
entrances, it becomes obvious that molecules bigger than 1400 Da are unlikely to represent
reasonably sized ligand candidates[62, 63]. Only a partial interaction of Bet v 1 with the diacyl chain
of LTA fitting into the cavity, would be possible, whereas it is rather unlikely that a multi-acyl
molecule like LPS would fit. In addition, the immunological data generated in this study, confirmed
that the immune-modulatory effects induced by the microbial molecules was not enhanced in the
presence of Bet v 1. Further, we were able to demonstrate that the amount of LPS detectable in birch
pollen extracts is not capable of causing DC maturation. In contrast to the determined level of LPS
(nLPS), the birch pollen extract was triggering the expression of CD86 and CD40 maturation markers.
Only a much higher LPS concentration (100 ng/ml) is able to induce a comparable response in DCs. In 40
how far LPS is necessary to facilitate birch pollen-induced DC maturation has been discussed
intensively in the literature. For example, Gutermuth et al. could only demonstrate DC maturation
induced by birch pollen extracts if the DCs were co-stimulated with LPS[64]. On the contrary, also
pollen extract were capable of promoting DC maturation without LPS co-stimulation[58]. However,
we found that the endotoxin levels detected in pollen extracts were not sufficient to induce
maturation of both, human and murine DCs. This finding, that allergic sensitization appears to be
independent of co-stimulatory signals from bacterial compounds, is also supported by a previous
study that was able to show that pollen extracts are capable of inducing maturation of TLR-4-
deficient DCs[58]. This lack of contribution of TLR-2 and TLR-4 agonists to the allergenicity of Bet v 1,
is further supported by the hypothesis that allergen recognition is almost completely PRR
independent[65].
Bacterial-derived stimuli like LPS are known to activate TLRs, although at classical sites where allergic
sensitization in the human body takes place such as, the mucosa or the respiratory tract, bacteria are
omnipresent, which might lead to a constant activation of TLRs. In this respect, the human immune
system has developed a control mechanism to differentiate between pathogenic and commensal
bacteria and is coordinated by TLR co-receptors expressed on epithelial cells. Hence, it is important
to identify TLR co-receptor mechanisms involved in the process of allergic sensitization rather than
TLR activation per se[66].
In previous studies it was demonstrated that PPE1 has the capacity to downregulate the IL-12p70
cytokine secretion in human DCs via signaling cascades involving the nuclear peroxisome proliferator-
activated receptor γ (PPAR-γ). In turn, this inhibiting effect resulted in an immune response
characterized by Th2-polarization[44]. Taking this Th2-favoring potential of PPE1 into account, we
analyzed the effect of Bet v 1-bound PPE1, since we were able to show that PPE1 has a relatively high
binding affinity to the Bet v 1 molecule (KD in low micromolar range), towards allergen uptake and
stimulation of DCs. Although a marginally increased expression of CD40 upon stimulation with PPE1-
bound and unbound rBet v 1 in a mixture with nLPS compared to apo-rBet v 1 or nLPS alone was
identified, we were not able to reproduce the high activation of DCs triggered by the birch pollen
extract. Notably, also a mixture of natural Bet v 1 isoforms purified from birch pollen extracts (nBet v
1) was not able to stimulate CD40 expression of DCs. Thus, an essential contribution in respect of DC
maturation facilitated by the pollen context is very likely.
In several previous studies, the accessibility of the cavity of Bet v 1 to potential ligands was dissected
using the fluorescence signal of 1-anilino-8-naphthalene sulfonate (ANS). In this case ligand binding
was described by the substitution of ANS by a ligand, as demonstrated by the decrease of signal
intensity caused by the binding of ANS to Bet v 1. Using these indirect measurements, a huge variety
41
of ligands was identified[24, 28]. Subsequently, this resulted many scientific publications dealing with
the influence of Bet v 1 ligand binding on protein dynamic and fold stability, as well as antigen
processing and presentation[53, 67]. In respect of Bet v 1, a direct link between proteolytic
processing of the allergen and class II MHC loading was demonstrated, and thus these findings were
used to explain its Th2 polarizing potential and allergenicity. However, the authors did not consider
that Bet v 1 per se is not capable of inducing a Th2 response[68].
From our results, we can conclude that ligand binding to the pollen-derived ligands has an overall
stabilizing effect on the protein, whereas the non-interacting bacterial molecules obviously provided
a destabilizing environment, as was observed for the thermal and proteolytic stability. We could
show that the effects on the thermal stability correlated with the proteolytic stability, which
subsequently has the potential to influence the allergenicity of the Bet v 1[69]. However, we could
not confirm that the identified effects on the thermal and proteolytic stability of the protein neither
influenced the IgE-binding affinity of Bet v 1 (mediator release assay) nor DC maturation and Th2
polarization (mBMDCs, moDCs, 4get). This fact leads us to the conclusion that the intrinsic properties
of Bet v 1 are not sufficient to explain its allergenicity, although the possibility remains that the ligand
responsible for a Th2 polarization towards Bet v 1 might still be unidentified. On the other hand, it is
probably more likely that allergic sensitization to Bet v 1 arises from Bet v 1-unrelated, co-stimulatory
molecules from pollen able to interact with the immune system and to promote Th2 phenotype. If
this is the case, the question still remains, why Bet v 1 represents the birch pollen major allergen. Is it
just a quantitative effect? With a percentage ranging between 10 to 25 % of soluble protein, Bet v 1
represents the most abundant protein found in birch pollen[70], and thus it is relevant to investigate
further in this direction.
Whatever the answer is, the pollen matrix seems to contribute to allergic sensitization, as supported
by the fact that in vivo Bet v 1 even in complex with pollen-derived ligands and microbial-derived
compounds but otherwise unaffected by the pollen content fails to induce a Th2-favoured immune
response, as observed using the 4get Th2 polarization reporter mouse strain. Remarkably, the
immunization with an aqueous birch pollen extract resulted in a statistically significant activation of
the IL-4 gene in CD4+ T-cells. Considering that Bet v 1 itself intrinsically possesses a very weak
sensitizing potential, but represents the major allergen is somehow quite contradictory. Finally, we
can conclude that the allergenicity of Bet v 1 results from several factors that also includes the
involvement of the pollen context, and cannot just be traced-back on its intrinsic protein properties.
In this respect, we suggest to always look at allergens as a part of a bigger picture, no matter from
which source they derive. For future investigations, it is important to focus on the identification of
42
specific molecules able to promote Th2 polarization, but also to find the reason why atopic people
are prone to allergenic sources and non-atopics are not.
Chapter 2: BM4SIT
Introduction
The BM4 molecule
The therapeutic approaches to treat allergic diseases are being revolutionized by the development of
molecule-based vaccines. In birch pollen allergy, allergen-immunotherapy (AIT) – whether it is
administered subcutaneously (SCIT) or sublingual (SLIT) – is usually performed using natural pollen
extracts. The composition of such extracts differs extensively due to factors such as, manufacture´s
production procedures and pollen origins. These differences are reflected in the quantity of extract-
containing proteins, glycoproteins, polysaccharides, lipids and other low-molecular-weight
compounds. Thus, standardization of these heterogeneous mixtures, to be used as AIT extracts, is
challenging and practically unattainable. Therefore, in diagnosis and treatment of allergies it
becomes increasingly routine to replace current natural extracts by recombinant proteins. Aside from
the standardization issue, the treatment with natural extracts is often accompanied by the
occurrence of side effects such as, local inflammation, rhinoconjunctivitis and oral-pharyngeal
itching. In this respect, it is necessary to increase the antigen dose carefully during the treatment. In
order to reduce such side effects, innovative alternatives to extracts are developed. An option is to
use genetically engineered hypoallergens possessing a reduced IgE-reactivity. The overall goal of the
therapeutic approach to replace extracts by hypoallergenic molecules is to make AIT more efficient,
less invasive and safer, and thus will lead to an improvement of the patients´ life quality[71, 72].
The BM4 molecule is a hypoallergenic variant of Bet v 1 (Bet v 1.0101) and was invented by grafting
an 8 amino acid long IgE epitope sequence from Mal d 1.0108, the PR-10 homologue of Bet v 1 in
apple, onto the Bet v 1 sequence[73]. The result of this grafting procedure was a genetically
engineered monomeric molecule comprising an inaccessible cavity and a completely different fold to
the Bet v 1 allergen. The hypoallergen exhibits a reduced IgE reactivity and capacity to cause
mediator release because of the loss of conformational IgE-binding epitopes. However, the T-cell
activation capacity of the molecule remains and is even increased[74, 75]. Furthermore, BM4 is able
to skew the allergic Th2-immune response towards a mixed Th1/Th2/Treg-immune response[76].
The reduced allergenicity, but retained immunogenicity of the designed mutant represents the ideal
properties of a birch pollen AIT vaccine to promote the induction of Tregs in an efficient way. These
properties make BM4 an excellent candidate to be investigated in a first-in-man clinical trial.
43
The role of vitamin D3 as novel adjuvants
The idea behind using vitamin D3 as an adjuvant alternative to the otherwise frequently used
aluminum hydroxide, is to effectively skew the immune response away from Th2 towards an anti-
inflammatory response mediated by allergen-specific Tregs. Calcitriol (1,25 dihydroxy vitamin D3),
the physiologically active version of vitamin D3, is enhancing the beneficial effects necessary for an
effective immunotherapeutic outcome such as, downregulation of Th2 cytokines, diminishing of
allergen-specific IgE antibody levels and the priming of DCs to induce the development of Treg
cells[77, 78]. In a mouse model for cat allergy, it has been demonstrated that vitamin D3 coupled to
the major cat allergen Fel d 1 decreases the number of inflammation-associated cells and of Th2
cytokines in the bronchoalveolar lavage, and suppresses airway hyperresponsiveness[79]. Similar
effects have been shown for another mouse model, where the co-administration of calcitriol with the
antigen, in this case ovalbumin, resulted in an effective induction of Tregs and the release of the
associated cytokines IL-10 and TGF-β[80]. Thus, the aforementioned anti-inflammatory,
immunosuppressive attributes of vitamin D3 make it reasonable to be investigated as novel adjuvant
in order to improve AIT.
The BM4SIT project
BM4SIT is an abbreviation for “Bet v 1 Mutant for [4] Specific Immuno Therapy”. The goal of the
project is to combine the hypoallergenic but hyperimmunogenic molecule BM4 with the anti-
inflammatory properties of vitamin D3. This combinatory vaccine, consisting of the hypoallergen and
the novel adjuvant will be evaluated upon AIT-beneficial attributes such as, rapid induction of an
anti-inflammatory immune response, treatment efficacy and reduction of allergic side effects, within
a clinical trial. This concept should make AIT safer, more effective and above all a more attractive
treatment for patients. BM4SIT is funded by the European Union within the 7th Framework program
under the call identifier FP7-HEALTH-2013-INNOVATION-1. It is a co-operation of 7 partner
organizations from the 6 different European countries; Austria, Denmark, Finland, Germany, The
Netherlands and Poland.
Further project-related information can be found on the projects´ website www.BM4SIT.eu.
Aims
Within the BM4SIT project the tasks of the Paris-Lodron-Universität Salzburg (PLUS) are clearly
defined and categorized in work packages containing certain aims, deliverables and milestones. 44
Besides the coordination of dissemination and exploitation activities, which also included the
development and maintenance of the projects´ website (www.BM4SIT.eu), our tasks were to provide
a detailed description of analytical methods that are observing structural and immunogenic
properties of the BM4 molecule. In this respect, our first aim (i) was to physicochemically and
immunologically characterize the hyperallergenic molecule in detail. In order to assess the potency of
the formulated BM4 drug substance we developed a potency assay (ii) for quality control, which in
principal is based on an inhibition ELISA set-up. As a part of quality control, we developed aptamers
that specifically recognize the 3D shape of the BM4 hypoallergen (iii). Aptamers are short DNA or
RNA oligonucleotides comprising 20 to 100 nucleotides that can adopt a target-specific 3D shape and
thus, are able to bind molecules with a high affinity and specificity[81]. Therefore, this novel
technique can be used to address folding, as well as the immunogenic structure of the hypoallergenic
molecule. The last aim was the evaluation of the humoral immune response induced by the BM4
drug product during the BM4SIT acute (iv) and repeated (v) toxicity study. To assess toxicological
safety of a drug product, it is a mandatory aspect of vaccine development and prerequisite for
human clinical trials to perform a preclinical toxicity study[82]. To sum up, the overall objective of
this thesis regarding the BM4SIT project was to evaluate the efficacy of the drug product on a
molecular level.
Material and methods
Mass spectrometry analysis
Protein identity was determined by mass spectrometry using a Quadrupole time-of-flight mass
spectrometer with electrospray ionization (Waters Ges.m.b.H., Vienna, Austria).
CD and FTIR
Determination of secondary structural elements was performed by CD and FTIR spectroscopy as
descried in chapter I.
Dynamic light scattering (DLS)
DLS was used to determine the aggregation behavior of BM4 in solution. 20 μl of protein sample in a
concentration of 0.5 to 1 µg/ml were measured within the DLS 802 system (Viscotek Corp., Houston,
TX, US). For data evaluation, the OmniSize™ software (Viscotek Corp., Houston, TX, US) was used to
analyze the combined data curve of at least 15 individual measurements to calculate intensity and 45
mass distribution, as well as the derived hydrodynamic radius, RH in nm. The obtained results were
compared with those of Bet v 1 (always rBet v 1.0101 was used but in terms of simplicity will be
called “Bet v 1” in the whole chapter).
Time-dependent endo-/lysosomal degradation
Proteolytic degradation was monitored using the endo-/lysosomal degradation assay as described in
chapter I.
Monitoring stability of the protein under different storage conditions (aging control)
The BM4 was stored in solution under different temperature conditions (-20°C, 4°C, RT and 37°C)
over a period of seven months. After each time point, starting with 6 h, the samples were analyzed
using SDS-PAGE.
Development of a BM4-specific ELISA to monitor the integrity of immune epitopes
Indirect ELISA using anti-BM4 antibodies
The three mouse monoclonal anti-BM4 antibodies, A1, F8 and I6, which were generated by
hybridoma technology and purified using Protein G sepharose (GE healthcare, Little Chalfont, UK),
were tested in an indirect ELISA regarding their efficiency to react with the BM4 molecule. In this
respect, 50 µl of BM4 was coated in a concentration of 2 µg/ml onto Maxisorp plates (NUNC™
Thermo Fisher, Waltham, MA, USA) and incubated o/n at 4°C. The coating buffer for all ELISA
experiments, if not mentioned explicitly, was PBS. The in-between washing steps were performed
with TBST buffer (TBS, pH 7.4, 0.05% Tween). For the blocking buffer and the dilution of primary and
secondary antibodies a TBST buffer containing 0.5% BSA was used. The different antibodies were
titrated ten times 1:2 starting with a concentration of 1 µg/ml. As secondary antibody an AP-
conjugated rabbit anti-mouse IgG + IgM antibody (Jackson ImmunoResearch Europe Ltd., Oaks Drive
Newmarket, Suffolk, UK) was used at a concentration of 1 µg/ml. For detection, 100 µl of 4-
nitrophenyl phosphate disodium salt was used as substrate for the alkaline phosphatase and the
resulting colorimetric shift was measured after 30 min of incubation at 405 nm with a reference
wavelength of 492 nm with a Tecan Sunrise plate reader (Tecan Austria GmbH., Grödig, Austria). All
ELISA experiments were either performed in duplicates or triplicates.
46
Identification of matching anti-BM4 antibody pairs in sandwich ELISA
Each one of the three antibodies (A1, F8 or I6) was coated in a concentration of 2 µg/ml in PBS. After
washing and blocking, where the same buffers were used as described above, the plates were
incubated with 1 µg/ml of BM4, and thereafter with one of the complementary antibodies of the
three anti-BM4 antibodies at a concentration of 1 µg/ml, which were biotinylated using Biotin-X-NHS
(Merck Millipore, Darmstadt, Germany) prior to this experiment. As detection antibody a HRP-
conjugated streptavidin (Jackson ImmunoResearch, West Grove, PA, USA) was used in combination
with the substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid). The plate was also
measured after 30 min at 405 nm (reference wavelength: 492 nm).
Sandwich ELISA using the anti-BM4 antibodies (A1, F8 or I6) and an affinity-purified polyclonal rabbit
anti-Bet v 1.0101 antibody
Since no matching antibody pairs were found, a sandwich ELISA using the monoclonal anti-BM4
antibodies in combination with a polyclonal rabbit anti-Bet v 1.0101 antibody, was established.
Therefore, each one of the anti-BM4 antibodies was coated separately (2 µg/ml) and after the
blocking step incubated with a 1:2 serial dilution of the antigen (either BM4 or Bet v 1), starting with
a concentration of 1 µg/ml. As secondary antibody an affinity-purified polyclonal rabbit anti-Bet v
1.0101 antibody was chosen in a concentration of 1 µg/ml. The detection antibody was an AP-
conjugated goat anti-rabbit antibody (1 µg/ml). The absorbance was measured after 30 min as
described above.
Indirect ELISA using polyclonal anti-BM4 rabbit sera
In order to refine the BM4-specific Sandwich ELISA, we wanted to replace the affinity-purified
polyclonal rabbit anti-Bet v 1.0101 antibody by a polyclonal antibody specific for the BM4 molecule.
Therefore, we had to produce and titrate a polyclonal anti-BM4 rabbit serum. For that reason, two
NZW rabbits (Charles River Laboratories, Sulzfeld, Germany) were immunized with 1 mg BM4
formulated with Alu-Gel-S (Serva, Heidelberg, Germany) in a 1/1 vol/vol dilution to a final
concentration of 1 mg/ml. The obtained rabbit sera were tested in an indirect ELISA coating
unformulated BM4 (2 µg/ml). The plate was incubated with a serial dilution of the individual or
pooled (ratio 1/1 vol/vol) rabbit serum (called anti-BM4 rabbit sera mix). For detection, a HRP-
conjugated goat anti-rabbit IgG, Fc Fragment antibody was used (Jackson ImmunoResearch, West
Grove, PA, USA) in a concentration of 1:5000. As substrate the 1-component SureBlue™ TMB
microwell peroxidase substrate (KPL, Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD, USA)
47
was used. After 5 min of incubation, the colorimetric reaction was stopped by the addition of 1 M HCl
and the plate was measured at 450 nm.
Sandwich ELISA using the anti-BM4 antibody A1 and the polyclonal anti-BM4 rabbit sera mix
For the final BM4-specific Sandwich ELISA, which should be used for quality control to monitor the
integrity of immune epitopes, we combined the monoclonal anti-BM4 antibody A1 with the
polyclonal anti-BM4 rabbit sera mix. The anti-BM4 antibody A1 was chosen because it displayed the
best reactivity in all performed ELISAs. In this sandwich ELISA, the anti-BM4 antibody A1 was coated
(50 µl/well, 2 µg/ml) and after the blocking step incubated with the antigen (BM4 or Bet v 1).
Therefore, a serial dilution with 1:5 steps starting with at a concentration of 5 µg/ml was prepared.
For detection, the polyclonal anti-BM4 rabbit sera mix was used in a dilution of 1:5000. The detection
step was performed with the HRP-conjugated goat anti-rabbit IgG, Fc Fragment antibody and
SureBlue™ TMB microwell peroxidase substrate as described in the previous paragraph.
Development of a potency assay for quality control
Inhibition ELISA matrix
For a first screening to determine the inhibition capacity of the anti-BM4 rabbit sera mix (Charles
River Laboratories, Sulzfeld, Germany), we performed an inhibition ELISA analyzing at the same time
different dilutions of the sera mix and different concentrations of the unformulated BM4. At first, 50
µl of the protein solution is coated in a concentration of 2 µg/ml onto a Maxisorp 96-well
transparent flat-bottom plate (NUNC™ Thermo Fisher, Waltham, MA, USA) and incubated o/n at 4°C.
The anti-BM4 rabbit sera mix was titrated in a range from 1:3200 to 1:204800, and the BM4 used for
inhibition was titrated from 4 µg/ml to 0.007813 µg/ml. For the inhibition, BM4 and anti-BM4 rabbit
sera mix were incubated for 2 h at RT in a reaction tube (50 µl volume), transferred onto the coated,
washed and blocked plate and incubated for another hour at RT. As secondary antibody the HRP-
conjugated goat anti-rabbit IgG, Fc Fragment antibody (Jackson ImmunoResearch, West Grove, PA,
USA) was used 1:5000 and incubated for 1 h. The plates were washed and only uninhibited plate-
bound rabbit anti-BM4 IgG was detected. The detection is performed after a 2 min incubation with
the SureBlue™ TMB microwell peroxidase substrate (KPL, Kirkegaard & Perry Laboratories Inc.,
Gaithersburg, MD, USA). Absorbance was measured after stopping the colorimetric reaction with 1 M
HCl at 450 nm. All measurements were performed in triplicates.
48
Original protocol of the potency assay
For the quality control of the BM4 drug substance, a potency assay has been developed. This potency
assay functions like an inhibition ELISA. In this assay, a “gold standard” BM4 is coated on a 96-well
plate and afterwards incubated with the anti-BM4 rabbit sera mix. The sera mix was pre-incubated
with different analysis samples (e.g. aluminum hydroxide-formulated BM4 or Bet v 1) and compared
with a “gold standard” of BM4. The “gold standard”, which has been fully characterized and
represents the unformulated drug substance, was provided by our cooperation partner Biomay AG,
aliquoted and stored at -70°C. For this original potency assay protocol, a dilution of 1:25,000 of the
anti-BM4 rabbit sera mix was chosen. For the inhibition mix first 3 dilutions (3 µg, 1 µg, and 0.5 µg)
and later 5 dilutions (5 µg, 2.5 µg, 1.25 µg, 0.625 µg, and 0.3125 µg) of inhibitor were chosen. In all
other respects, the inhibition ELISA was performed as described in the previous paragraph. For the
maximum control we used uninhibited wells. For the background values, the wells were incubated
without primary antibody (anti-BM4 rabbit sera mix). For the data analysis, the background was
subtracted from all obtained values. To determine the inhibition values [%], at first the values were
multiplied with 100 and divided by the mean of the maximum control values (represents normalized
absorbance value in %), and afterwards this value was subtracted from 100.
Potency assay refinement and final protocol
The original potency assay protocol was later optimized. Using 12 dilutions of the inhibition mix we
obtained a sigmoidal curve, which is necessary for the half maximal inhibitory concentration (IC50)
determination and to describe the potency of the drug product. The IC50 was determined using
GraphPad Prism 6 software (GraphPad Software, Inc., CA, USA). The original five 1:2 dilution steps (5
µg, 2.5 µg, 1.25 µg, 0.625 µg, and 0.3125 µg) were adjusted to 1:3 dilution steps, and the starting
concentration of inhibitor was increased to 160 µg/ml (the highest possible concentration for the
formulated BM4 drug product). The anti-BM4 rabbit sera mix dilution remained the same (1:25,000).
For a precise IC50 determination, the sigmoidal curve has to comprise at least two values in the top
plateau and two values in the bottom plateau. In case of the formulated drug substance this was not
achieved. Therefore it was necessary to set constraints to define the plateau values, characterized by
the uninhibited values (100% signal) and the background values. The reproducibility of the potency
assay was validated since it was repeated on three consecutive days and performed by at least three
different people. The analyzed samples comprised a BM4 skin prick test (SPT), BM4 stability (stored
under different conditions), BM4 toxicity (used for the toxicity study), aluminum hydroxide-
formulated BM4, placebo (same amount of aluminum hydroxide, used as a control) and of course a
“gold standard” BM4 sample. A SOP has been generated and is available at the PLUS.
49
Development of a sandwich enzyme-linked apta-sorbent assay (ELASA) for quality control of the
formulated BM4 drug product
Selection of anti-BM4-specific aptamers
We aimed to establish a sandwich ELASA experimental set-up for quality control to address folding,
as well as the immunogenic structure of the hypoallergenic molecule. Aptamers are developed by
the so-called systematic evolution of ligands by exponential enrichment (SELEX) process. We used a
slightly adapted version of the protocol from Stoltenburg et al.[81]. A pool of a random ssDNA
oligonucleotide library “BANK”, containing 60 nucleotides each, was heated up 94°C for 8 min and
cooled afterwards on ice for 15 min to produce defined three-dimensional shaped DNA aptamers,
which have the chance to bind to a target molecule. For the folding procedure an aptamer-binding
buffer was used (20 mM Tris, 100 mM NaCl, 2 mM MgCl2, 5 mM KCl, 1 mM CaCl, 0.02% Tween 20,
pH 7.6). For the development of anti-BM4-specific aptamers, BM4, provided by Biomay AG, was
immobilized on PierceTM NHS-activated magnetic beads (Pierce Biotechnology, Rockford, USA). The
ssDNA oligonucleotide library “BANK” (IBA GmbH, Goettingen, Germany) was incubated with the
BM4-coupled magnetic beads. After several washing steps, where unbound aptamers were removed,
the bound DNA was eluted from the target molecule and amplified using a biotinylated primer. Then
the amplified dsDNA was separated by heating to become ssDNA. For the separation step PierceTM
streptavidin magnetic beads (Pierce Biotechnology, Rockford, USA), able to bind the biotinylated
lagging strand and thus separating them from the leading strands of interest, are used. The cycle was
repeated 7 times, followed by a counter selection cycle. During the SELEX cycles those aptamers with
a higher specificity were increased whereas those with less specificity were sorted out. After the last
cycle, the double-stranded PCR product was ligated into a pGEM-T easy vector (Promega
Corporation, Madison, USA) and then cloned into a bacterial host. The aptamer sequence-containing
plasmids were sent for sequencing to Eurofins (Eurofins Genomics GmbH, Ebersberg, Germany).
50
Figure 12. Schematic overview of the SELEX procedure to identify anti-BM4-specific aptamers.
Dot blot using biotinylated aptamers
Five identified aptamer sequences with a biotinylation on the 5´end were purchased (Eurofins
Genomics GmbH, Ebersberg, Germany) and used in a dot blot to determine their specificity. In this
respect, either 2 µl of 1 mg/ml BM4 or Bet v 1 was coated on a nitrocellulose membrane (Whatman
plc, part of GE Healthcare Life Sciences, Maidstone, United Kingdom) for 15 min at RT and then
blocked for 2 h at RT (0.5% BSA, 150 mM NaCl, 25 mM Tris/HCl pH 7.5, 0.05% NaN3, 0.5% Tween 20).
The membrane was incubated with 50 nM of the biotinylated aptamers diluted in blocking buffer at
RT in the dark o/n. After incubation the incubation with the biotinylated aptamers, the membrane
was washed 3 times with blocking buffer. As detection antibody an AP-conjugated streptavidin
antibody (Caltag Laboratories, CA, USA) was used in a dilution 1:1000, diluted in blocking buffer and
incubated with the membrane for 1 h at RT. After washing the membrane three times with blocking
buffer, the membrane was equilibrated with AP-detection buffer (100 mM Tris/HCl pH 9.5, 100 mM
NaCl, 5 mM MgCl2) and then incubated with NBT/BCIP. After 5 to 10 min the membrane should
develop a dark staining. The staining reaction was stopped by washing the membrane with distilled
water.
51
Indirect ELASA using biotinylated aptamers
For the folding of the aptamer sequences, 500 nM of the five biotinylated aptamers were diluted in
aptamer-binding buffer and folded as described in the SELEX procedure. Either BM4, Bet v 1 or OVA
(Sigma-Aldrich, St. Louis, USA) was coated in a concentration of 2 µg/ml onto a Maxisorp 96-well
transparent flat-bottom plate (NUNC™ Thermo Fisher, Waltham, MA, USA) and incubated o/n at 4°C.
After blocking of the plate, the aptamers were applied in aptamer-binding buffer supplemented with
0.5% BSA. The aptamer-binding reaction was performed for 1 h at RT in the dark. After washing, a
HRP-conjugated streptavidin detection antibody (Jackson Immuno Research Inc., West Grove, PA,
USA) was used diluted 1:2000 in ELISA blocking buffer. Detection was performed using the SureBlue™
TMB microwell peroxidase substrate as described above.
Sandwich ELASA
The biotinylated anti-BM4 aptamers 1 to 5 were immobilized in a concentration of 500 nM on
streptavidin-coated ELISA plates (2 µg/ml, Streptavidin from Streptomyces avidinii, Sigma-Aldrich, St.
Louis, USA) and incubated with the target protein, either BM4 or Bet v 1, in a concentration of 1
µg/ml. As capture antibody, the mouse monoclonal anti-BM4 antibody was used. For the detection
an HRP-conjugated goat anti-mouse IgG (H+L) antibody (Bio-Rad Laboratories, Inc., Hercules, CA,
USA) was diluted 1:1000. For the sandwich ELASA titration experiment, the two anti-BM4 aptamers 1
and 2 were titrated from 500 to 3.9 nM in 1:2 dilution steps in aptamer-binding buffer supplemented
with 0.5% BSA.
52
Figure 13. Schematic representation of the developed sandwich ELASA.
Immunological evaluation of BM4 drug product within the BM4SIT acute toxicity study
Study design
Figure 14. Study design of both, the acute and repeated toxicity study.
53
Samples of acute toxicity study
The acute toxicity studies were performed in New Zealand White rabbits and Wistar rats. 50% of the
animals were immunized with a single dose of BM4 (320 μg per injection, adsorption to alum
hydroxide), which is 4-times the intended human clinical dose. The other 50% underwent
immunization with placebo. The placebo samples contained the same buffer and amount of alum
hydroxide as the BM4 sample. The 96 serum samples (2 x 28 rat sera, 2 x 20 rabbit sera) were
received on 18th of January 2016 frozen on dry ice and stored at -20°C until the analysis was
performed. Pre- and post-immunization sera of 14 rats per group (7 female,7 male) and 10 rabbits
per group (5 female, 5 male) were analyzed.
Sample of repeated toxicity study
The repeated toxicity study was performed in Wistar rats. The animals were bi-weekly immunized
over a period of six months with either a human clinical dose of BM4 (80 µg BM4/1 mg alum/0.9%
NaCl in 500 µl), a high dose of the drug product (160 µg BM4/1 mg alum/0.9% NaCl in 500 µl) or
placebo. Animals of the main group were sacrificed one week after the last injection. Animals of the
recovery group were sacrificed after a 6-week observation period. The 90 serum samples (3 x 20 rats
per main group, 3 x 10 rats per recovery group) arrived either on the 8th of September 2016 or on the
22nd of September 2016 frozen on dry ice.
Indirect ELISA using rat serum
The indirect ELISA to determine BM4-specific IgE, IgG1, IgG2a and IgG2b levels within the rat sera
was performed according to a normal indirect ELISA protocol. As primary antibody a HRP-conjugated
monoclonal rat anti-mouse antibody was used with a specificity either for IgE (clone MARE-1, isotype
IgG1, product# MA516813, ThermoFisherScientific, Rockford, USA), IgG1 (SB 3060-05, G1 7E7,
SouthernBiotech,Birmingham, USA), IgG2b (SB 3065-05, 2A 8F4, SouthernBiotech,Birmingham, USA)
or IgG2b (SB 3070-05, 2B 10A8, SouthernBiotech,Birmingham, USA). Each serum was titrated to
determine the endpoint titer and each measurement was performed in duplicates. For the endpoint
titer analysis the LOD was used.
54
Indirect ELISA using rabbit serum
To evaluate BM4-specific IgG levels, each rabbit serum was titrated and analyzed using a HRP-
conjugated goat anti-rabbit IgG, Fc Fragment antibody (Jackson ImmunoResearch Inc., Suffolk, United
Kingdom). For data analysis and endpoint titer determination again the LOD was used. Since there
was no anti-rabbit IgE antibody commercially available for the evaluation of BM4-specific IgE titers,
an ELISA kit from BlueGene Biotech (Putuo District, Shanghai, China) was used for the quantitative
determination of rabbit total IgE.
Results
Characterization of the BM4 molecule
Within the BM4SIT project a major task was to characterize the hypoallergenic BM4 molecule, which
was provided by our cooperation partner Biomay AG, in detail and to compare it with the major bich
pollen allergen, Bet v 1. We addressed the integrity of the molecule on a physicochemical as well as
on an immunological level. Further, the proteolytic and also the storage stability of the protein was
investigated under certain defined conditions.
Physicochemical characterization
For the physicochemical analysis of the unformulated BM4 molecule, produced by Biomay AG,
different analytic methods were used. Mass spectrometry was used to confirm the protein identity.
The determined molecular weight was 17418.9730 Da, and thus almost equal to the calculated
molecular weight (17418.91 Da) that was determined by using the PeptideMass application from
ExPASy according to the sequence of BM4[83].
The secondary structural elements were analyzed by CD and FTIR spectroscopy. According to the CD
spectrum of Bet v 1 at 20°C (Fig. 15a, left), the protein exhibited a mixed secondary structure
phenotype consisting of α-helices and β-sheets, while the BM4 molecule at the same temperature
was lacking the specific Bet v 1-fold. The CD spectrum of BM4 was mostly characterized by a
pronounced signal minimum at approximately 200 nm, indicating a mostly unordered structure. At
95°C both proteins showed the same unordered folding (Fig. 15a, right).
The analysis of the FTIR spectra revealed similar results. The BM4 amide I band had a maximum at
the wavenumber 1655 cm-1 and a shape clearly indicative of a protein with a higher α-helical content
in relation to the number of β-sheet elements (Fig. 15b, right). In contrast to that, the amide I band
of Bet v 1 was characterized by a broad peak at 1637 cm-1, which is representative for higher β-sheet
55
content. The second derivative of the amide I and amide II bands demonstrated the relative amounts
of secondary structural elements at both wavelengths, 1655 cm-1 and 1637 cm-1 (Fig. 15b, left).
Compared to Bet v 1, where a higher percentage of β-sheets was found, the second derivative
spectrum of BM4 showed a higher amount of α-helical elements in relation to its β-sheet content.
When quantifying the secondary structural elements (Table 4), with a content of 28,054% α-helices
and 38,226% β-sheets the data obtained for Bet v 1 reflect precisely the theoretical value found in
the Protein Data Bank (PDB). In comparison, the β-sheet content of BM4 was massively reduced
(12,447%), while the percentage of α-helical elements only decreased slightly (22,012%).
The aggregation behavior of BM4 and Bet v 1 in solution was determined by DLS (Fig. 15c). Both
proteins were almost 100% monomeric in solution. The measured hydrodynamic radius of BM4 was
2.1 nm. In contrast, the hydrodynamic radius of Bet v 1 was slightly smaller (1.8 nm).
56
Figure 15. CD spectra of BM4 and Bet v 1 at 20 and 95°C (a), recorded from a wavelength of 190 to 260 nm and presented as mean residue molar ellipticity. The FTIR spectra (b) of Bet v 1 and BM4 are presented as amide I and II bands, as well as the second derivatives thereof. The two typical minima at 1655 and 1637 cm-1 are representatives for α-helical and intramolecular β-sheet structures, respectively. Analysis of the aggregation behavior of Bet v 1 and BM4 in solution analyzed by DLS (c). The hydrodynamic radius (RH) of both molecules was calculated using a mass distribution model for proteins.
theoretical value for Bet v 1 (PDB)
Bet v 1 BM4
α-helical elements 25% 28.054% 22.012% β-sheets 39% 38.226% 12.447%
Table 4. Determination of secondary structure elements were performed by FTIR spectroscopy.
57
Time-dependent endo-/lysosomal degradation
The proteolytic stability of BM4 was monitored over 48 h using an endo-/lysosomal degradation
assay. In figure 16, the relative quantification of the SDS-PAGE results (not shown) of the degradation
of BM4 is shown. In contrast to the proteolytic stability of Bet v 1, BM4 was quickly degraded and
was gone after 6 h. Moreover, BM4 was used as a negative control when we investigated the
influence of ligand binding on the proteolytic stability of Bet v 1, as presented in chapter 1. Five of
the six graphs showing a ligand co-incubation displayed a similar degradation pattern like apo BM4,
indicating no influence of ligand binding on the proteolytic stability of BM4. Surprisingly, the
combination of BM4 and PPE1 resulted in an enhanced proteolytic stability.
Figure 16. Time-dependent endo-/lysosomal degradation of BM4 was monitored over 48 h. The data are presented values determined by relative quantification of SDS-PAGE results, interpreted by the Image Lab 4.0.1 Software (Bio-Rad). In addition, BM4 was used as a negative control for ligand binding (chapter 1).
58
Monitoring protein stability under different storage conditions
To assess the stability of BM4 when storing it over a period of six months either at -20°C, 4°C, 37°C or
at room temperature, the protein was analyzed at defined time points using SDS-PAGE (Fig. 17).
When storing the protein at -20°C, it was stable over the whole six months. The protein solution was
clear and showed no sign of precipitation. At 4°C, the protein remained relative stable as well. Only
after five months a faint band appeared underneath the BM4 band, indicating that the protein was
slightly degraded. Surprisingly, when the protein was stored at 37°C or at room temperature, it was
stable for at least one month. After two months, the gel displayed strong degradation bands.
59
Figure 17. The stability of BM4 under different storage conditions (at 4°C, -20°C, 37°C and RT) was monitored using SDS-PAGE, h: hours; d: days; m: months.
Immunological characterization
We established a BM4-specific sandwich ELISA to monitor the integrity of the immune epitopes of
the unformulated drug product. Therefore, three mouse monoclonal anti-BM4 antibodies (A1, F8 and
I6), generated by hybridoma technology prior to the start of this project, were analyzed using an
indirect ELISA upon their reactivity towards the BM4 molecule (Fig. 18a). The antibodies A1 and F8
exhibited a high reactivity, whereas the third antibody (I6) showed a reduced reactivity and only
generated an acceptable signal at a much higher concentration. In order to identify matching anti-
BM4 antibody pairs, the three mouse monoclonal antibodies were biotinylated and used in a
sandwich ELISA set-up (Fig. 18b). Although the efficiency of the biotinylated mouse monoclonal
antibodies was previously determined by an indirect ELISA using a streptavidin-conjugated secondary
antibody (data not shown), no matching antibody pairs were found, indicating all three anti-BM4
antibodies are recognizing similar epitopes. An alternative solution for the secondary antibody of the
sandwich ELISA was to use a polyclonal antibody instead. First, an affinity-purified polyclonal rabbit
anti-Bet v 1 antibody was used, which is recognizing BM4 as well (Fig. 18c). Only the capture
antibodies A1 and F8 worked in combination with the polyclonal antibody. When substituting BM4
by Bet v 1, no signal was observed at all. These promising results convinced us to use the monoclonal
anti-BM4 antibody A1 in combination with a polyclonal rabbit anti-BM4 serum. For that reason, BM4
formulated with Alu-Gel-S was provided to Charles River Laboratories in order to immunize two
rabbits. The obtained rabbit sera were analyzed and titrated using unformulated BM4 in an indirect
ELISA (Fig. 18e). Final sandwich ELISA was performed using the anti-BM4 A1 capture antibody in
combination with the polyclonal anti-BM4 rabbit sera mix (Fig. 18d). The results of the final assay
displayed a high specificity and reactivity towards BM4, whereas for Bet v 1 the signal was rather
weak.
60
Figure 18. The reactivity of the anti-BM4 antibodies A1, F8 and I6 towards BM4 was investigated using an indirect ELISA (a). Identification of matching anti-BM4 antibody pairs in a sandwich ELISA (b). The first antibody mentioned in the legend represents the coated capture antibody, whereas the second represents the biotinylated matching antibody. Sandwich ELISA using the anti-BM4 antibodies (A1, F8 or I6) and an affinity-purified polyclonal rabbit anti-Bet v 1.0101 antibody (c). Sandwich ELISA using the anti-BM4 antibody A1 and the polyclonal anti-BM4 rabbit sera mix (d). In these both sandwich ELISA experiments (c + d) the quality of the assays towards their specificity to the BM4 molecule was determined and compared with Bet v 1. An indirect ELISA was performed to titrate the polyclonal anti-BM4 rabbit sera in order to determine a suitable dilution (e).
Development of a potency assay for quality control of the formulated BM4 drug product
Original potency assay protocol
To assess the potency of the BM4 drug product in a formulated state, a potency assay has been
established. The assay principle of the potency assay is set up in a similar way as an inhibition ELISA.
A BM4 standard, which has been fully characterized and represents the unformulated drug
substance, was aliquoted and stored at -70°C. To determine the potency of different BM4 batches,
61
standard BM4 was coated on ELISA plates and then incubated with the inhibition mixture consisting
of the polyclonal anti-BM4 rabbit sera mix (described above) and either an analysis sample or the
standard. To determine the optimal serum dilution in relation to the optimal amount of BM4 used as
inhibitor, an inhibition ELISA matrix experiment was performed (Fig. 19a, left and right). According to
the data from this experiment the BM4 inhibition range of 100 to 6.25 µg/ml and the rabbit anti-BM4
sera mix dilution 1:25,000 were defined. To determine the performance of the potency assay with
BM4 formulated with aluminum hydroxide a test assay was performed (Fig. 19b, left and right).
Therefore, two different batches of BM4 (one produced by Biomay and the other one by PLUS) and
one batch of BM4 adsorbed to Alu-gel-S (formulated BM4) were analyzed. Further, Bet v 1 was used
as a reference protein. Both unformulated BM4 batches showed similar inhibition efficiency,
whereas the inhibition signal of formulated BM4 was slightly lower. Bet v 1 almost did not inhibit in
this assay. For the first potency assay test, three different concentrations of inhibition antigen were
used (3 µg, 1 µg, and 0.5 µg, respectively). Later on two more dilutions were included (Fig. 19c, d).
This first potency assay protocol was used to determine the potency of formulated BM4 provided by
Biomay at day 0 (t=0) and one month after formulation (Fig. 19c). The inhibition signal of the BM4
standard was consistent and reproducible during six independently performed assays (including once
by another person). The measurement of t=0 samples was repeated four times in order to address
the reproducibility of the assay. There was no relevant difference between the signals of t=0 and t=1
samples. Similar results were obtained when performing the assay with t=3 and t=6 samples (Fig.
19d).
62
Figure 19. Inhibition ELISA matrix to determine a suitable dilution of the anti-BM4 rabbit sera mix presented as absorbance values and the resultant normalized inhibition values (a). The determined dilution was tested using 3, 1 and 0.5 µg of inhibitor (b). The analyzed samples were three different batches of BM4, one produced at the PLUS, the other two (formulated and unformulated with alum hydroxide) produced at Biomay. The first potency assay protocol was later on adjusted and therefore 5 concentrations of inhibitor (5 µg, 2.5 µg, 1.25 µg, 0.625 µg, and 0.3125 µg) were used instead of three. The reproducibility of the original potency assay protocol was investigated, also when performed by another person (c). The assay was used to assess stability during different storage conditions (d), t: time in months.
63
Potency assay refinement and final protocol
The original potency assay protocol has been optimized in order to determine the relative inhibition
concentration (IC50). The IC50 is an important value in pharmacology and vaccine development
describing the effectiveness of a drug product. The protocol was extended to a total of 12 dilutions of
the inhibition mix in order to obtain a sigmoidal curve, which is necessary for the IC50 analysis of the
drug product. Using the original 1:2 dilution series (Fig. 20a) did neither comprise the right
concentration range for a sigmoidal curve of the standard nor of formulated BM4. Adjusting the 1:2
dilution to a 1:3 dilution resulted in a sigmoidal curve behavior for the gold standard containing the
characteristic top and bottom signal plateau (Fig. 20b). However, for the formulated drug substance
no inhibition plateau was reached. Also when titrating the rabbit anti-BM4 sera mix 1:10,000,
1:25,000, 1:50,000 or 1:100,000, it was not possible to reach the top plateau (Fig. 20c, d). Therefore,
we decided to keep the 1:25,000 dilution for the final potency assay protocol. Also when increasing
the starting concentration of formulated BM4 to the highest possible concentration without
concentrating the sample (160 µg/ml) a top inhibition plateau was not reached (Fig. 20e). For a
precise IC50 determination, the sigmoidal curve has to comprise at least two values in the top
plateau and two values in the bottom plateau. In case of the formulated drug substance this was not
achieved. Therefore, it was necessary to set constraints to define the plateau values, characterized
by the uninhibited values (100% signal) and the background values. The final potency assay protocol
was established considering all these relevant factors (Fig. 21a, b). The assay was performed on two
different days revealing similar results, pointing out that the data were reproducible. Furthermore,
both, inhibition curves and the associated curve fitting of the standard were parallel to the sample.
64
Figure 20. The original potency assay protocol was optimized in order to obtain a sigmoidal curve of the inhibition signal. Therefore, instead of the previously used five dilutions, we now used 11 dilutions starting with 5 µg (a). Since this was not enough to reach a top and bottom signal plateau, the curve was enlarged to 12 dilution steps and using a 1:3 instead of a 1:2 dilution series (b). Titration of the anti-BM4 rabbit sera mix (1:10,000; 1:25,000; 1:50,000 and 1:100,000) using the highest possible concentration of the formulated BM4 drug product (c and d). The final potency assay protocol (e) was performed using the highest possible concentration of the formulated drug product.
65
Figure 21. The final potency assay protocol was performed on to different days in order to determine its reproducibility. The curve fitting (faint line) to find the relative IC50 was performed by using the GraphPad Prism 7 software (GraphPad Software, Inc., La Jolla, CA, USA). Measurements were performed in triplicates.
Potency assay in use
The final BM4 potency assay was used to evaluate the potency of the formulated drug product and a
skin prick test (SPT) sample during advanced stability testing at time point 0 (Biomay production
release date). The two samples were compared with the physicochemically characterized standard,
stored at -70°C, and a so-called “placebo control” sample, which contained the same amount of
aluminum hydroxide and the same buffer as the formulated BM4. The potency assay was performed
with regular rabbit anti-BM4 serum mix (PLUS) and used in the same dilution (1:25000) as used
during the assay development period. In addition and as requested by Biomay, the potency assay
was performed with another polyclonal rabbit anti-BM4 serum (origin: Biomay) in a concentration
1:35000. The potency assay was performed on three different days using the rabbit anti-BM4 serum
from Biomay and on two different days using the conventional rabbit anti-BM4 serum mix (origin:
PLUS). When performing the assay with the serum from Biomay (Fig. 22a), the two sample curves
(formulated BM4 and SPT BM4) exhibited a relatively parallel curve progression and were
comparable with the standard curve. In contrast, the placebo control was negative and followed a
constant fluctuation around zero percent of inhibition. At the highest concentration, the placebo
control was slightly increased. The control displayed a basically linear curve behavior and was not
described by a sigmoidal curve behavior, which is required to obtain a meaningful curve fitting. The
IC50 calculation of the fitted generated data was not possible. Interestingly, the SPT BM4 sample that
was analyzed the first time using the potency assay reached a top inhibition plateau in contrast to
the formulated sample. However, the SPT BM4 sample exhibited an overall reduced inhibition
potential. All three potency assays were performed on different days and were reproducible,
reflected also by the calculated IC50 values (Table 5). In comparison, the results of the two potency
66
assays using the sera mix from PLUS were similar (Fig. 22b). In this particular case, the standard curve
and the formulated BM4 sample and also the thereof derived curve fittings showed an almost ideal
overlay. Although there was only enough sample left to repeat the potency assay once using the
PLUS sera mix, the generated data of both assays were reproducible. Also here, the calculation of the
IC50 resulted in values that lay in a comparable range (Fig. 23, Table 5).
Figure 22. In respect of advanced Non-Good Manufacturing Practices (Non-GMP) stability testing, the final potency assay protocol was used to determine the potency of the formulated drug product and the BM4 SPT sample GMP batch. Both
67
samples were compared with the standard and the placebo control. One the one hand an anti-BM4 rabbit serum from Biomay was used in a dilution of 1:30,000 (a) and on the other hand the anti-BM4 rabbit sera mix from PLUS in a dilution of 1:25,000 dilution (b). The potency assay was repeated on three different days in a row using the Biomay serum, and on two different days using the PLUS sera mix.
Figure 23. Generated IC50 values and median displayed in a vertical scatter plot, left Biomay serum, right PLUS serum.
SPT BM4 formulated BM4 Standard t0d1 Biomay Serum 0.60 80.65 12.52 t0d2 Biomay Serum 1.03 85.58 7.70 t0d3 Biomay Serum 0.63 42.60 9.77 Average Biomay Serum 0.75 69.61 9.99 t0d1 PLUS Serum 12.68 42.60 33.63 t0d2 PLUS Serum 1.79 23.43 11.21 Average PLUS Serum 7.23 33.01 22.42
Table 5. IC50 values (presented as µg/ml) as determined by the potency assay.
Development of a sandwich ELASA for quality control of the BM4 drug product
A major aspect of the project BM4SIT was quality control of the BM4 drug substance. Therefore, we
established a method to produce BM4-specific aptamers and to use them in an ELASA experimental
set-up in order to investigate folding and stability of the drug product. The identification of the BM4-
specific aptamers was performed using the SELEX selection process. In this respect, a pre-GMP batch
of BM4 was used and coupled to NHS-activated magnetic beads. The coupling efficiency was
analyzed with a dot blot assay using the previously described anti-BM4 antibody A1 (Fig. 24a). A
positive signal is indicated by a dark blue color caused by the enzymatically processing of the alkaline
phosphatase substrate. A strong signal of the BM4-coupled beads as well as of the positive control
BM4 was observable. Uncoupled beads and the second antibody control were negative. The brown
color resulted from the magnetic beads themselves. In figure 24b all double stranded polymerase
68
chain reaction (dsPCR) products of the SELEX cycles 1 to 8 were listed. The dsPCR products of all
cycles were found at a molecular weight of approximately 150 bp. After the last cycle, non-modified
primers were used for the amplification (Fig. 24c). The resulting final dsPCR product was ligated into
a pGEM-T easy vector and then cloned into a bacterial host. The identification of positive clones was
performed using the colony-screening PCR method (Fig. 24d). Five duplicates of anti-BM4 aptamer
sequences that had a sequence identity of 100% were identified (Fig. 24e). The potential 3D structure
of the anti-BM4 aptamer sequences A1, A2, A3, A4 and A5 was generated using the online structure
prediction tool mfold Web server[84] (Fig. 24f). In case of aptamer A3, two possible 3D structures
were generated by the program.
69
Figure 24. Evaluation of coupling efficiency of the NHS-activated magnetic beads coupled to BM4 in a dot blot assay using the mouse monoclonal anti-BM4 antibody A1 (a). The dsPCR products of the SELEX cycles 1 to 8 were found at a molecular weight of around 150 bp (b). Final dsPCR product using non-modified primers (c) was used for ligation into the pGEM-T easy vector and then cloned into a bacterial host. Positive bacterial clones were identified by colony-screening PCR (d). Five duplicate anti-BM4 aptamer sequences were identified (e) and called aptamer sequence A1 to A5. Potential structure of anti-BM4 aptamers using the mfold Web server (f).
In order to assess efficiency and specificity of the identified anti-BM4 aptamers, different approaches
were used. At first, the anti-BM4 aptamers were biotinylated and then applied instead of a primary
antibody in a dot blot experimental set-up in a concentration of 50 nM (Fig. 25a). Aptamers A1 and
A2 displayed a high reactivity to BM4, whereas the other three (A3, A4 and A5) were less reactive
and only a slight signal shadow was observable. Interestingly, all five aptamers exhibited a strong
reaction when the BM4-couple beads were coated on the membrane. None one of the aptamers
70
recognized Bet v 1 in this assay. The detection antibody control was negative. The specificity of the
biotinylated anti-BM4 aptamers was further analyzed within an indirect ELASA (Fig. 25b). In this
experiment, aptamers A1 to A4 showed a high and specific reactivity towards the coated target
molecule, whereas the reactivity of aptamer A5 was rather weak and unspecific. With respect to
quality control of the BM4 drug product, the goal was to implement a sandwich ELASA (Fig. 25c).
Therefore, the biotinylated aptamers were immobilized in a concentration of 500 nM on streptavidin
coated ELISA plates and then incubated with the target protein. The mouse monoclonal anti-BM4
antibody A1 complemented the sandwich ELASA. As target proteins both, BM4 and Bet v 1 were
analyzed. The aptamer A1 showed the best reactivity towards the BM4 molecule, whereas reactivity
to Bet v 1 in this case was rather low. In the other four cases (A2 to A5) only moderate signal
difference was observable between BM4 and Bet v 1. In order to determine the most efficient
aptamer concentration for the sandwich ELASA, a titration experiment using only the aptamers A1
and A2 was performed based on the previous experiment (Fig. 25d). The titration experiment was
only done for BM4. The absorbance values were found in a plateau until 10 nM and then seemed to
drop rapidly.
Figure 25. Dot blot assay using biotinylated anti-BM4 aptamers A1 to A5 in a concentration of 50 nM (a). Indirect ELASA using the biotinylated anti-BM4 aptamers A1 to A5 in a concentration of 50 nM (b), coated was either BM4, Bet v 1 or Ova. The aptamers were tested in a sandwich ELASA set-up in a concentration of 500 nM (c). A titration experiment from 500 to 3.9 nM was performed to determine the ideal concentration of the aptamers A1 and A2 within the sandwich ELASA (d).
71
Immunological evaluation of the BM4 drug product within the BM4SIT acute toxicity study
A major aspect of vaccine development is to evaluate the safety level of the drug product, which is
accomplished by toxicology studies. The toxicology studies are performed prior to first-in-man clinical
trials. In these studies, accurate animal models are used and the obtained data can be translated into
potential risks for participants of the clinical trial. Within the acute toxicity study the effects of a
single subcutaneous injection of formulated BM4 was investigated within two different mammalian
species, New Zealand White rabbits and Wistar rats. Our task within the acute toxicity study was to
evaluate the influence of the drug product on the humoral immune responses (IgG, IgE) of the two
species. In the rabbits, the serological BM4-specific IgG and the total IgE titers were quantified (Fig.
26a). The specific IgG titers were significantly increased compared to the pre-immunization but also
to the placebo group. No effect on the total IgE response could be shown. However, a significant
difference of the IgG response between the pre-placebo group and the pre-immunization group was
found, although both cohorts should be similar. The analysis of both groups was repeated, but the
result stayed consistent. The sera of the rats were analyzed upon the BM4-specific IgE, IgG1, IgG2a
and IgG2b levels (Fig. 26b). A single injection of the drug product resulted in a significant increase of
IgG1, IgG2a and IgG2b, whereas BM4-specific IgE titers remained unaffected. In contrast to the
results obtained for the rabbits, no statistical difference between the pre-placebo group and the pre-
immunization group was found in the rats.
72
Figure 26. Determination of BM4-specific IgG and total IgE levels in rabbit sera within the acute toxicity study (a). BM4-specific IgE, IgG1, IgG2a and IgG2b levels were determined within rat sera of the acute toxicity study (b). For each animal, pre- and post-treatment serum samples were assayed by ELISA. The animals were grouped into P (animals treated with placebo) and Test (animals treated with formulated BM4 sample). Statistics were calculated using either a t-test or a paired t-test. All statistical analyses were performed using the Graphpad Prism 5 software; Ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
An important aspect was to examine if the induced BM4-specific antibodies are cross-reacting with
Bet v 1 (Fig. 27a, b). In this experiment there was no need to analyze all placebo samples individually,
therefore we pooled the serum samples of each group in order to have some background indication
73
(dashed line). The same pattern of Bet v 1-specific IgG induction was found in both animal species, as
reported for BM4. All in all, the titers were slightly lower than found for BM4. There was again no
induction of Bet v 1-specific IgE titers within the rat serum samples.
Figure 27. Determination of Bet v 1-specific IgG levels in rabbit sera (a) and of Bet v 1-specific IgE, IgG1, IgG2a and IgG2b in rat sera of the acute toxicity study (b). For each animal, pre- and post-treatment serum samples were assayed by ELISA. The sera of the placebo group were pooled (dashed line). Statistics were calculated using a paired t-test. All statistical analyses were performed using the Graphpad Prism 5 software; Ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
74
Immunological evaluation of the BM4 drug product within the BM4SIT repeated toxicity study
Here, the evaluation of the humoral immune response (IgG and IgE) induced by a bi-weekly
immunization of Wistar rats over a period of six months with either the human clinical dose of BM4
or a high dose (double the intended clinical dose) of the drug product is presented (Fig. 28). A
placebo control group was also included in the repeated toxicity study. IgG1, IgG2a and IgG2b level of
both, the clinical and the high group were intensively increased compared to the placebo group.
Interestingly, when analyzing IgG2a a slight statistical difference between the clinical and the high
cohort was found (P ≤ 0.05), indicating that immunizations using the double clinical dose induced an
even higher IgG2a immune response. In contrast to the results of the acute toxicity study, the
analysis of the humoral immune response induced by repeated injections of the drug product,
resulted in elevated IgE titers in both clinical and the high groups. Considering the magnitudes of the
endpoint titers, the induction of IgE titers was way lower in relation to the IgG titers.
Figure 28. BM4-specific IgE, IgG1, IgG2a and IgG2b levels in rat sera of the main group were determined by ELISA. The animals were grouped into Placebo, Clinical (animals treated with human clinical dose; 80 µg BM4/1 mg alum/0.9% NaCl in 500 µl) and High (animals treated with 160 µg BM4/1 mg alum/0.9% NaCl in 500 µl). The median of each data group is shown in the scatter plot. Statistics were calculated on transformed data (Y=Log[Y]) using a one-way ANOVA. A Bonferroni post test was used to compare all groups with each other. All statistical calculations were performed using the GraphPad Prism 5 software; Ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
75
In the repeated toxicity study there was another group of animals that was sacrificed after a 6-week
observation period and called the “recovery group”. In toxicity studies, the purpose of a recovery
group is to investigate whether the induced toxicological effects observed when completing the
dosing phase remain or are reversible. Here, the recovery group was analyzed upon the ability to
maintain a specific immune response towards the drug product (Fig. 29). Although, the levels of all
three IgG subtypes of the clinical and the high group decreased a little bit compared to the main
group (Fig. 28), the immune response was still remarkably high. In contrast, the IgE titers drop
completely and there was no statistically significant difference between all three groups observable.
Within the recovery group, the difference between the placebo and the clinical group was not
statistically significant in none of the four cases.
Figure 29. BM4-specific IgE, IgG1, IgG2a and IgG2b levels in rat sera of the recovery group were determined by ELISA. The animals were grouped into the same groups as described for the main group of the repeated toxicity study (Placebo, Clinical and High. The median of each data group is shown on the scatter plot. Statistics were calculated on transformed data (Y=Log[Y]) using a one-way ANOVA. A Bonferroni post test was used to compare all groups with each other. All statistical calculations were performed using the GraphPad Prism 5 software; Ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
76
Discussion
The all-embracing objective of the project BM4SIT is to find a safer, more efficient and patient-
friendlier alternative to current AIT therapeutic approaches. With regard to a first-in-man clinical
trial, a detailed characterization of the BM4 drug product is required. With respect to the
physicochemically determination of the BM4 molecule, the generated results were in accordance
with previously published data. The purity of BM4 was determined by SDS-PAGE. Protein identity was
confirmed by mass spectrometry. The secondary structure elements of recombinant Bet v 1 and its
hypoallergenic mutant were compared using CD and FTIR spectroscopy. Analyzing the recorded CD
spectra at 20°C revealed two distinctly different protein configurations. In contrast, when measuring
the proteins at 95°C both spectra were similar. Both, the Bet v 1 and the BM4 CD spectrum display
comparable pattern like the published ones[74, 85, 86]. However, minor discrepancies to the
reported CD data by Wallner et al. are found[74]. In our case, the spectrum minima of both proteins
align when measured at 20°C. This difference can occur due to concentration issues.
The FTIR data obtained for BM4 and Bet v 1 are in good accordance with the result of the CD spectra
both proteins. The amide I band is the most sensitive region of a FTIR spectrum and characterized by
the C = O stretch vibrations of the peptide bonds[87, 88]. The signal generated in this region reflects
the secondary structure elements of a protein. The FTIR spectroscopic analysis of the amide I band
and the thereof calculated second derivative of both proteins at 25°C revealed that the β-sheet
content of BM4 is diminished massively compared to Bet v 1. This distinct loss of the typical Bet v 1
fold has been described in the literature[74]. Also here we found slight deviations to the published
results. For Bet v 1, our determined 28.054% of α-helices and 38.226% of β-sheets is in good
accordance with the theoretical value as well as the published data. Concerning BM4, we found
22.012% of α-helices and 12.447% of β-sheets. According to the data of BM4 published by Wallner et
al.[74], the protein possess a fold containing 9.9% of α-helices and 17.7% of β-sheets, but is
contradictory to their second derivative data, which explicitly display a higher α-helical than β-sheet
content as specified by the higher characteristic peak at 1655 cm-1. Since the data of both
experiments were vector-normalized and thus became concentration-independent, there are only
two possible explanations for the differing results. Either the background signal of the published
result was not accurately subtracted, which is indicated by the deviation of the signal at positions like
1690, 1595 and 1550 cm-1 that in our case perfectly align, or it is a matter of batch-to-batch
variability. Furthermore, FTIR spectroscopy is more reliable and sensitive for β-sheet that for α-
helical structures [89].
The same discrepancies between our and the published data are found in the determined
hydrodynamic radiuses of Bet v 1 and BM4, 1.8 and 2.1 nm in our experiment and 2.1 and 3.0 nm in
77
the published experiment, respectively[74]. Even in terms of proteolytic stability we were able to
show that BM4 is slightly less stable than Bet v 1, which is also on the contrary to the publication by
Wallner et al[74]. This difference most likely results from the activity and quality of the isolated
microsomes that were used within the experiment. We found out that storage of isolated
microsomes for more than one night at -70°C can decrease their proteolytic activity; therefore, the
herein presented experiments were performed with the same batch of microsomes. Also slight
differences in the buffer pH were shown to have this potential[53]. Besides that, it was planned to
use the BM4 molecule as a reference for non-ligand binding for the ligand binding study presented in
chapter 1, since the hydrophobic cavity of Bet v 1 is not supposed to be accessible for ligands [74].
Nevertheless, the combination of BM4 and PPE1 increased the stability of BM4 towards proteolytic
degradation; although not as much as it was the case for Bet v 1. There are two possible explanations
for that. Either PPE1 is able to bind BM4 somewhere else as in the cavity, or it possesses an inhibiting
effect on endo- or exoproteases. On the other hand, phytoprostanes are described as non-enzymatic
lipid peroxidation products[90, 91]. However, nothing related to this topic can be found in the
literature.
Within the project BM4SIT, the BM4 hypoallergen will be investigated in a first-in-man clinical trial
and in this case will be used adsorbed to aluminum hydroxide. Therapeutic vaccines that are
formulated with aluminum hydroxide are not supposed to be stored below 0°C since freezing of the
preparation provokes adjuvant particle aggregation[92]. According to the patient information leaflet
of Alutard SQ®, AIT vaccines based on pollen extracts are usually stored at 4°C for maximal six
months[93]. Although the BM4 that was used in this study to monitor the effects of storage
conditions on the protein was not formulated, storing the protein at 4°C for six months should also
be feasible.
A BM4-specific sandwich ELISA was established in order to monitor the integrity of the immune
epitopes of the hypoallergenic BM4 in course of quality control during drug development (Fig. 18d).
Considering that it takes a 100,000 fold higher amount of Bet v 1 than BM4 to reach an absorbance
signal of 0.5 (OD405) underlines the specificity and sensitivity of the established assay. So far, the
assay was only performed with unformulated BM4. Next step would be to perform the assay with the
formulated drug product and to compare it with unformulated BM4. On the contrary, the sandwich
ELISA performed using an anti-Bet v 1.0101 antibody was less sensitive (Fig. 18c), which can be
traced back mostly onto the specificity of the antibody towards Bet v 1.0101 and not the BM4
molecule.
In addition to this immunological characterization used for quality control of BM4, a potency assay
was established to assess the potency of the drug product. The purpose of the BM4-potency assay is
78
to provide detailed information about the impact of temperature over a period of time on the
potency of the drug product. The potency determined by IC50 values of each, formulated BM4 and
the BM4 SPT solution is used in order to evaluate the suitability of the drug product for the clinical
study of BM4SIT. For an accurate IC50 determination a sigmoidal curve behavior is necessary, and
the standard (unformulated BM4) and the sample curves have to be parallel to each other in order to
compare the potency of the drug product with the potency of the standard[94]. To achieve a
sigmoidal curve behavior the original potency assay protocol (as presented in Fig. 19) was refined
(Fig. 20) and finally the resulting potency assay met all the requirements for a correct IC50
determination (Fig. 21).
To verify the efficacy of the established potency assay, the assay was performed in course of Non-
GMP stability testing using either a polyclonal anti-BM4 rabbit serum from Biomay or from PLUS for
the inhibition (Fig. 22). The three GMP samples, formulated BM4, formulated placebo and a BM4 SPT
sample were used and compared with the unformulated BM4 standard. When analyzing the three
potency assays that were performed using the Biomay sera, it seems obvious that the BM4 SPT
sample possesses a higher inhibition potential in terms of IC50 concentration than the aluminum
hydroxide-formulated BM4, and in two of the three cases the inhibition capacity was even higher
than the standard. However, the BM4 SPT curve reaches a maximal inhibition plateau at
approximately 75%, and thus it is lower than the maximal inhibition of the standard (100%). The
comparison with the maximum inhibition value of the aluminum hydroxide-formulated BM4 sample
is not meaningful since the derived curve does not reach the top plateau, but still it appears that it
will jut out the SPT curve. The BM4 SPT sample, which contains glycerol in the final formulation, was
measured for the first time within this potency assay set-up. Therefore no reference data are
available for comparison yet. In future, the influence/interference of glycerol upon the assay has to
be determined, although nothing regarding this topic can be found in the literature. In case of the
standard, for the inhibition curves a noticeable bottom and top plateau were reached. Furthermore,
we found negligible effects of interference caused by aluminum hydroxide only at the highest
concentration of the placebo sample. The rest of the placebo curve exhibited only a background
inhibition signal fluctuating around 0% of inhibition. Therefore, it was not possible to analyze the
curve and to generate the IC50 of the placebo sample. When analyzing the GMP samples using the
PLUS sera mix, the same pattern occurred. However, it has to be mentioned that in this case the
standard curve matched the curve of the formulated drug product. This almost perfect curve overlay
between the sample curve and the standard was not found when the assay was established using the
PLUS sera mix (Fig. 21). This can be due to batch-to-batch variation and the decreased potency of the
formulated BM4 that was used for establishing the assay. We received the formulated sample from
Biomay at the 28th of September, 2016. The final experiments to set-up the potency assay were 79
performed by end of November, 2016. Therefore, it can be concluded that the formulated sample
used to establish the assay was stored for two months at 4°C, which can result in decreased potency
compared to analyzed stability batch that was immediately analyzed upon receipt. In general, the
potency assay showed a high reproducibility that is also reflected by the generated IC50 values (Table
5, Fig. 23). As expected from the curve behavior, the lowest IC50 values were found for SPT BM4,
followed by the BM4 standard. The formulated BM4 exhibited the highest IC50. The IC50 is a
measure of the effectiveness of a substance and thus is inversely proportional to its potency,
meaning a low IC50 is an indicator for a potency of the drug product[95, 96]. Hence, it can be
concluded that the potency and the inhibition potential of formulated BM4 is decreased compared
to unformulated BM4. The phenomenon that adsorption to aluminum hydroxide may affect the
potency of a substance is also discussed in the literature[92, 97].
A technique was established to address the 3D structure integrity of the BM4 molecule in course of
quality control using an ELASA experimental set-up with identified BM4-specific aptamers. In total,
five duplicates of anti-BM4 aptamer sequences, which displayed a sequence identity of 100%, were
identified with the SELEX method (Fig. 24). Sequences that are occurring more frequently than others
are supposed to bind more efficiently the target than other aptamer sequences[98-100]. Different
assays were used to determine the specificity of the identified aptamers, including dot blot assays
and indirect ELASAs (Fig. 25). Biotinylated aptamers were used in this respect. In the dot blot
experiment, the reactivity of the aptamers was stronger towards BM4-coupled beads than towards
the single BM4 molecule. Usually, reported aptamer sequences range between 15 to 70 nucleotides
in length, of course dependent on the target size[100]. The dot blot results and the relatively big size
of the aptamer sequences indicate that identified aptamers are probably more specifically
recognizing the BM4-bead complex than the single molecule. Within the indirect ELASA experiment,
the BM4-bead complex was not investigated, although performing the assay with such should
definitely be considered in future. In the indirect ELASA, all five aptamer sequences were neither
recognizing Bet v 1 nor ovalbumin, demonstrating that the aptamers are BM4-specific. The same
result was observed with the dot blot assay. However, it has to be mentioned that the ELASA signal in
respect of the BM4 molecule was highly fluctuating. Hence, the assay stability has to be more refined
regarding reproducibility. When performing a sandwich ELASA with the BM4-specific aptamers in
combination with the mouse monoclonal anti-BM4 antibody A1, in general a high reactivity towards
Bet v 1 was observed. This either resulted from unspecific background signal caused by the coated
streptavidin or from the mouse monoclonal anti-BM4 A1 antibody that was shown to react also with
Bet v 1. Although this is very unlikely since the aptamers-binding is BM4 specific. Anyways, the
reason for the unspecific binding within this sandwich ELASA experiment needs to be clarified.
Therefore, it would be worth trying to replace the antibody by the polyclonal anti-BM4 rabbit sera 80
mix. On the other hand, when BM4 was titrated in the sandwich ELASA experiment, the absorbance
signal was found in a plateau until 10 nM and then dropped. This range of aptamer specificity is
similar to what is reported in the literature[81]. Another important thing that needs to be
investigated is the influence of biotinylation on BM4 accessibility and binding reactivity, but with a
different experimental approach. Moreover, it is recommended to identify shorter aptamers not only
with respect to generate more specific aptamers but also to have a control of the SELEX method per
se.
Within the BM4SIT acute toxicity study the humoral immune response (IgE and IgG) in NZW rabbits
and Wistar rats caused by a single subcutaneous injection of the BM4 drug product was evaluated. In
course of the following repeated toxicity study, then the IgG and IgE immune response induced by a
bi-weekly immunization of Wistar rats over a period of six months was investigated. A single
subcutaneous injection was able to induce a robust BM4-specific IgG immune response in both
investigated animals, whereas the IgE antibody titers remained unaffected (Fig. 26). An unpaired t-
test revealed that there is a highly significant difference (P ≤ 0.001) between the two groups, pre-
placebo and pre-immunization when analyzing BM4-specific rabbit IgG (not shown in figure 26a, IgG).
Although the measurement and the titer determination were repeated for both groups, there is no
proper explanation for this increased basal BM4-specific IgG level. Next, the cross-reactivity of the
induced BM4-specific antibodies regarding Bet v 1 was investigated (Fig. 27). Albeit the Bet v 1-
specific titers were slightly lower compared to the BM4-specific response, the same pattern of IgG
and not IgE induction is apparent. This indicates that BM4 is a potent immunogen, able to channel
the immune response towards Th1, and capable of inducing a high, Bet v 1-crossreacting IgG immune
response that potentially possesses blocking activity in humans. In humans, there are four subtypes
of IgG: IgG1, IgG2, IgG3 and IgG4[101]. In contrast, rats express IgG1, IgG2a, IgG2b and Ig2c[102].
Although the subtypes of Ig classes between human and rodents are not directly correlating, it is
clear that the relevant Th2 cytokine IL-4 induces IgG1 and IgE in mice, whereas in humans, a class-
switch towards IgG4 and IgE is induced[103]. This fact also strengthens the hypothesis that the strong
IgG response found in the two investigated species can be translated into a blocking IgG response in
human. Sequence comparison revealed that the constant region of rat IgG2b is most homologous to
mouse IgG2a and IgG2b, whereas the constant regions of rat IgG1 and IgG2a are most homologous to
the constant region of mouse IgG1[104]. Therefore, we have focused on IgG1, IgG2a and IgG2b when
analyzing the rat sera. IgG2c was omitted. In contrast, rabbits only have one IgG subclass.
The immune response induced during the repeated toxicity study only differed a little bit from the
acute toxicity one. With regard to IgE, two distinct populations of the clinical and the high group
were found. On the one hand those who had marginally increased IgE titers, and on the other hand a
81
population where the production of BM4-specifc IgE was not induced. Although between the clinical
and the high group a few significant differences were identified, the clinical group is inducing a
robust IgG immune response (Fig. 28). In comparison, the slightly elevated IgE titers dropped
completely in the recovery group, whereas the relevant IgG responses remained (Fig. 29). In mice,
repeated subcutaneous immunizations with BM4 resulted in high levels of Bet v 1-specific IgG1 and
IgG2a, but also Bet v 1-specific IgE antibodies. In this experiment, BM4 was able to induce murine IgG
antibodies that possessed the ability to block the binding of human serum IgE to Bet v 1[74]. In
another mouse model, the animals were sensitized subcutaneously with Bet v 1 adsorbed to
aluminum hydroxide and afterwards treated either with Bet v 1, BM4 or PBS, followed by an aerosol
challenges. The resulting immune response was analyzed and revealed that BM4 induced a high level
of Bet v 1 cross-reactive IgG antibodies. Compared to Bet v 1, the treatment with BM4 in this in vivo
model significantly reduced Bet v 1-specific IgE levels[76]. Hence, it can be concluded that the
immune response induced by BM4 is species-specific, dependent on the antigen dose as well as the
numbers of antigen applications.
82
Chapter 3: General discussion and conclusion
This thesis provides useful insights on allergenicity of proteins and the characteristics that are
responsible for it such as, induction of allergic sensitization (Th2 immune response), IgE reactivity,
protein stability, and processing. Since the major purpose of this thesis was to investigate the reason
why Bet v 1 is the major allergen found within birch pollen, the clinical relevance of this research and
its impact on following studies is not deniable. This kind of basic research is necessary in order to find
the villain that responsible for allergic sensitization towards Bet v 1 and, in allergic patients, the
thereof resulting clinical manifestations. Whenever the real cause will be discovered, prophylactic
and/or protective strategies can be developed in order to treat birch pollen allergy or even to
eliminate the onset of such.
Here we found that besides the influences of ligand binding on the stability of the protein no
significant impacts on its immunogenicity and allergenicity occurred. However, we could
demonstrate that pollen extracts have the potential to induce Th2 polarization, whereas Bet v 1 lacks
this property, even in a mixture with immune-modulatory ligands and TLR-2 and TLR-4 agonists.
Therefore, we want to state that it would probably make more sense to focus on the identification of
a co-stimulatory, sensitization-inducing substance found in the pollen and the next reasonable step
would be to investigate the interaction of such with Bet v 1. However, so far we cannot exclude that
this substance is a potential ligand of Bet v 1. These findings will prompt researches to consider the
pollen context as a relevant source for co-stimuli that induce sensitization, and not just focus on the
allergens themselves.
Additionally, we investigated the efficacy of the Bet v 1 homologues hypoallergen, BM4. The
objective of the project BM4SIT is to use the hypoallergenic molecule in a first-in-man clinical trial.
BM4 has the potential to become a safer and more efficient alternative to the birch pollen extracts
that are currently used in AIT. As part of this doctoral thesis, we provided detailed information on the
characterization of the BM4 molecule, and developed different analytical methods for quality control
of the drug product in order to guarantee that the product is safe and effective before its application
in the clinical trial. In this context, quality control is a major aspect of drug development. Further, the
induction of a humoral immune response (IgE and IgG) by the subcutaneous administration of the
BM4 drug product was investigated in two different mammalian species. This has been done in order
to investigate efficacy, but also evaluate the safety level of the drug product. In general, we could
demonstrate that BM4 is an effective immunogen and able to induce a strong and potentially
protective IgG immune response, which is cross-reactive to Bet v 1. From the data we generated so
83
far and that are described in detail in this thesis, we can conclude that BM4 possess the potential to
revolutionize current AIT therapeutic approaches by offering a safer and more effective alternative.
84
Bibliography
1. Abbas, A.K., A.H. Lichtman, and S. Pillai, Cellular and molecular immunology. 2012, Philadelphia: Elsevier/Saunders.
2. Takeuchi, O. and S. Akira, Pattern recognition receptors and inflammation. Cell, 2010. 140(6): p. 805-20.
3. Jabara, H.H., et al., TRAF2 and TRAF3 independently mediate Ig class switching driven by CD40. Int Immunol, 2009. 21(4): p. 477-88.
4. Kracker, S. and A. Durandy, Insights into the B cell specific process of immunoglobulin class switch recombination. Immunol Lett, 2011. 138(2): p. 97-103.
5. Ozcan, E., et al., Toll-like receptor 9, transmembrane activator and calcium-modulating cyclophilin ligand interactor, and CD40 synergize in causing B-cell activation. J Allergy Clin Immunol, 2011. 128(3): p. 601-9 e1-4.
6. Giltiay, N.V., C.P. Chappell, and E.A. Clark, B-cell selection and the development of autoantibodies. Arthritis Res Ther, 2012. 14 Suppl 4: p. S1.
7. Bloemen, K., et al., The allergic cascade: review of the most important molecules in the asthmatic lung. Immunol Lett, 2007. 113(1): p. 6-18.
8. Hirose, K., K. Takahashi, and H. Nakajima, Roles of IL-22 in Allergic Airway Inflammation. J Allergy (Cairo), 2013. 2013: p. 260518.
9. Ruby Pawankar, G.W.C., Stephen T. Holgate, Richard F. Lockey, WAO White Book on Allergy 2011-2012: Executive Summary., in WAO World Allergy Organization - A World Federation of Allergy, Asthma and Clinical Immunology Societies. 2011-2012.
10. Platts-Mills, T.A. and J.A. Woodfolk, Allergens and their role in the allergic immune response. Immunol Rev, 2011. 242(1): p. 51-68.
11. Karp, C.L., Guilt by intimate association: what makes an allergen an allergen? J Allergy Clin Immunol, 2010. 125(5): p. 955-60; quiz 961-2.
12. Chivato, T., et al., Allergy, living and learning: diagnosis and treatment of allergic respiratory diseases in Europe. J Investig Allergol Clin Immunol, 2012. 22(3): p. 168-79.
13. Kim, Y.H., et al., Correlation between skin prick test and MAST-immunoblot results in patients with chronic rhinitis. Asian Pac J Allergy Immunol, 2013. 31(1): p. 20-5.
14. Cross, S., S. Buck, and J. Hubbard, Allergy in general practice. BMJ, 1998. 316(7144): p. 1584-7.
15. Sastre, J., Molecular diagnosis in allergy. Clin Exp Allergy, 2010. 40(10): p. 1442-60. 16. Ceuppens, J.L., et al., Immunotherapy with a modified birch pollen extract in allergic
rhinoconjunctivitis: clinical and immunological effects. Clin Exp Allergy, 2009. 39(12): p. 1903-9.
17. Akdis, C.A. and M. Akdis, Mechanisms of allergen-specific immunotherapy and immune tolerance to allergens. World Allergy Organ J, 2015. 8(1): p. 17.
18. Kappen, J.H., et al., Applications and mechanisms of immunotherapy in allergic rhinitis and asthma. Ther Adv Respir Dis, 2017. 11(1): p. 73-86.
19. Soyka, M.B., D. Holzmann, and C.A. Akdis, Regulatory cells in allergen-specific immunotherapy. Immunotherapy, 2012. 4(4): p. 389-96.
20. Valenta, R., et al., Recombinant allergens for allergen-specific immunotherapy: 10 years anniversary of immunotherapy with recombinant allergens. Allergy, 2011. 66(6): p. 775-83.
21. Luger, E.O., et al., Allergy for a lifetime? Allergol Int, 2010. 59(1): p. 1-8. 22. Pelaia, G., A. Vatrella, and R. Maselli, The potential of biologics for the treatment of asthma.
Nat Rev Drug Discov, 2012. 11(12): p. 958-72. 23. Rentzos, G., et al., Intestinal allergic inflammation in birch pollen allergic patients in relation
to pollen season, IgE sensitization profile and gastrointestinal symptoms. Clin Transl Allergy, 2014. 4: p. 19.
85
24. Asam, C., et al., Bet v 1--a Trojan horse for small ligands boosting allergic sensitization? Clin Exp Allergy, 2014. 44(8): p. 1083-93.
25. Smith, M., et al., Geographic and temporal variations in pollen exposure across Europe. Allergy, 2014. 69(7): p. 913-23.
26. Breitenbach, M., et al., Biological and immunological importance of Bet v 1 isoforms. Adv Exp Med Biol, 1996. 409: p. 117-26.
27. Fernandes, H., et al., Structural and functional aspects of PR-10 proteins. FEBS J, 2013. 280(5): p. 1169-99.
28. Mogensen, J.E., et al., The major birch allergen, Bet v 1, shows affinity for a broad spectrum of physiological ligands. J Biol Chem, 2002. 277(26): p. 23684-92.
29. Radauer, C., P. Lackner, and H. Breiteneder, The Bet v 1 fold: an ancient, versatile scaffold for binding of large, hydrophobic ligands. BMC Evol Biol, 2008. 8: p. 286.
30. Seutter von Loetzen, C., et al., Secret of the major birch pollen allergen Bet v 1: identification of the physiological ligand. Biochem J, 2014. 457(3): p. 379-90.
31. Trapero, A., et al., Characterization of a glucosyltransferase enzyme involved in the formation of kaempferol and quercetin sophorosides in Crocus sativus. Plant Physiol, 2012. 159(4): p. 1335-54.
32. Seutter von Loetzen, C., et al., Ligand Recognition of the Major Birch Pollen Allergen Bet v 1 is Isoform Dependent. PLoS One, 2015. 10(6): p. e0128677.
33. Parchmann, S. and M.J. Mueller, Evidence for the formation of dinor isoprostanes E1 from alpha-linolenic acid in plants. J Biol Chem, 1998. 273(49): p. 32650-5.
34. Oeder, S., et al., Pollen-derived nonallergenic substances enhance Th2-induced IgE production in B cells. Allergy, 2015. 70(11): p. 1450-60.
35. Chwastyk, M., M. Jaskolski, and M. Cieplak, Structure-based analysis of thermodynamic and mechanical properties of cavity-containing proteins--case study of plant pathogenesis-related proteins of class 10. FEBS J, 2014. 281(1): p. 416-29.
36. Markovic-Housley, Z., et al., Crystal structure of a hypoallergenic isoform of the major birch pollen allergen Bet v 1 and its likely biological function as a plant steroid carrier. J Mol Biol, 2003. 325(1): p. 123-33.
37. Kofler, S., et al., Crystallographically mapped ligand binding differs in high and low IgE binding isoforms of birch pollen allergen bet v 1. J Mol Biol, 2012. 422(1): p. 109-23.
38. Thomas, W.R., Innate affairs of allergens. Clin Exp Allergy, 2013. 43(2): p. 152-63. 39. Schroder, N.W., et al., Immune responses induced by spirochetal outer membrane
lipoproteins and glycolipids. Immunobiology, 2008. 213(3-4): p. 329-40. 40. Ambika Manirajan, B., et al., Bacterial microbiota associated with flower pollen is influenced
by pollination type, and shows a high degree of diversity and species-specificity. Environ Microbiol, 2016. 18(12): p. 5161-5174.
41. Bublin, M., T. Eiwegger, and H. Breiteneder, Do lipids influence the allergic sensitization process? J Allergy Clin Immunol, 2014. 134(3): p. 521-9.
42. Runswick, S., et al., Pollen proteolytic enzymes degrade tight junctions. Respirology, 2007. 12(6): p. 834-42.
43. Schwarz, H., et al., Residual endotoxin contaminations in recombinant proteins are sufficient to activate human CD1c+ dendritic cells. PLoS One, 2014. 9(12): p. e113840.
44. Gilles, S., et al., Pollen-derived E1-phytoprostanes signal via PPAR-gamma and NF-kappaB-dependent mechanisms. J Immunol, 2009. 182(11): p. 6653-8.
45. Herre, J., et al., Allergens as immunomodulatory proteins: the cat dander protein Fel d 1 enhances TLR activation by lipid ligands. J Immunol, 2013. 191(4): p. 1529-35.
46. Marley, J., M. Lu, and C. Bracken, A method for efficient isotopic labeling of recombinant proteins. J Biomol NMR, 2001. 20(1): p. 71-5.
47. Egger, M., et al., Assessing protein immunogenicity with a dendritic cell line-derived endolysosomal degradome. PLoS One, 2011. 6(2): p. e17278.
86
48. Kavan, D. and P. Man, MSTools—Web based application for visualization and presentation of HXMS data. International Journal of Mass Spectrometry, 2011. 302(1–3): p. 53-58.
49. Nowak-Wegrzyn, A.H., et al., Mediator release assay for assessment of biological potency of German cockroach allergen extracts. J Allergy Clin Immunol, 2009. 123(4): p. 949-955 e1.
50. Behboudi, S., et al., The effects of DNA containing CpG motif on dendritic cells. Immunology, 2000. 99(3): p. 361-6.
51. Williamson, M.P., Using chemical shift perturbation to characterise ligand binding. Prog Nucl Magn Reson Spectrosc, 2013. 73: p. 1-16.
52. Traidl-Hoffmann, C., et al., Pollen-associated phytoprostanes inhibit dendritic cell interleukin-12 production and augment T helper type 2 cell polarization. J Exp Med, 2005. 201(4): p. 627-36.
53. Machado, Y., et al., Fold stability during endolysosomal acidification is a key factor for allergenicity and immunogenicity of the major birch pollen allergen. J Allergy Clin Immunol, 2016. 137(5): p. 1525-34.
54. Wan, H., et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions. J Clin Invest, 1999. 104(1): p. 123-33.
55. Zhang, J., et al., Comparative enzymology of native and recombinant house dust mite allergen Der p 1. Allergy, 2009. 64(3): p. 469-77.
56. Royer, P.J., et al., The mannose receptor mediates the uptake of diverse native allergens by dendritic cells and determines allergen-induced T cell polarization through modulation of IDO activity. J Immunol, 2010. 185(3): p. 1522-31.
57. Trompette, A., et al., Allergenicity resulting from functional mimicry of a Toll-like receptor complex protein. Nature, 2009. 457(7229): p. 585-8.
58. Kamijo, S., et al., Cupressaceae pollen grains modulate dendritic cell response and exhibit IgE-inducing adjuvant activity in vivo. J Immunol, 2009. 183(10): p. 6087-94.
59. Varga, A., et al., Ragweed pollen extract intensifies lipopolysaccharide-induced priming of NLRP3 inflammasome in human macrophages. Immunology, 2013. 138(4): p. 392-401.
60. Bracke, N., et al., Surface acoustic wave biosensor as a functional quality method in pharmaceutics. Sensors and Actuators B: Chemical, 2015. 210: p. 103-112.
61. Patching, S.G., Surface plasmon resonance spectroscopy for characterisation of membrane protein-ligand interactions and its potential for drug discovery. Biochim Biophys Acta, 2014. 1838(1 Pt A): p. 43-55.
62. Chwastyk, M., M. Jaskolski, and M. Cieplak, The volume of cavities in proteins and virus capsids. Proteins, 2016. 84(9): p. 1275-86.
63. Erickson, H.P., Size and shape of protein molecules at the nanometer level determined by sedimentation, gel filtration, and electron microscopy. Biol Proced Online, 2009. 11: p. 32-51.
64. Gutermuth, J., et al., Immunomodulatory effects of aqueous birch pollen extracts and phytoprostanes on primary immune responses in vivo. J Allergy Clin Immunol, 2007. 120(2): p. 293-9.
65. Iwasaki, A. and R. Medzhitov, Control of adaptive immunity by the innate immune system. Nat Immunol, 2015. 16(4): p. 343-53.
66. Fischer, H., et al., Mechanism of pathogen-specific TLR4 activation in the mucosa: fimbriae, recognition receptors and adaptor protein selection. Eur J Immunol, 2006. 36(2): p. 267-77.
67. Grutsch, S., et al., Ligand binding modulates the structural dynamics and compactness of the major birch pollen allergen. Biophys J, 2014. 107(12): p. 2972-81.
68. Freier, R., E. Dall, and H. Brandstetter, Protease recognition sites in Bet v 1a are cryptic, explaining its slow processing relevant to its allergenicity. Sci Rep, 2015. 5: p. 12707.
69. Ohkuri, T., et al., A protein's conformational stability is an immunologically dominant factor: evidence that free-energy barriers for protein unfolding limit the immunogenicity of foreign proteins. J Immunol, 2010. 185(7): p. 4199-205.
70. Schappi, G.F., et al., Concentrations of the major birch tree allergen Bet v 1 in pollen and respirable fine particles in the atmosphere. J Allergy Clin Immunol, 1997. 100(5): p. 656-61.
87
71. Ferreira, F., M. Wolf, and M. Wallner, Molecular approach to allergy diagnosis and therapy. Yonsei Med J, 2014. 55(4): p. 839-52.
72. Winther, L., H.J. Malling, and H. Mosbech, Allergen-specific immunotherapy in birch- and grass-pollen-allergic rhinitis. II. Side-effects. Allergy, 2000. 55(9): p. 827-35.
73. Klinglmayr, E., et al., Identification of B-cell epitopes of Bet v 1 involved in cross-reactivity with food allergens. Allergy, 2009. 64(4): p. 647-51.
74. Wallner, M., et al., Reshaping the Bet v 1 fold modulates T(H) polarization. J Allergy Clin Immunol, 2011. 127(6): p. 1571-8 e9.
75. Kitzmuller, C., et al., A hypoallergenic variant of the major birch pollen allergen shows distinct characteristics in antigen processing and T-cell activation. Allergy, 2012. 67(11): p. 1375-82.
76. Pichler, U., et al., The fold variant BM4 is beneficial in a therapeutic Bet v 1 mouse model. Biomed Res Int, 2013. 2013: p. 832404.
77. van der Aar, A.M., et al., Vitamin D3 targets epidermal and dermal dendritic cells for induction of distinct regulatory T cells. J Allergy Clin Immunol, 2011. 127(6): p. 1532-40 e7.
78. Bakdash, G., et al., Vitamin D3 metabolite calcidiol primes human dendritic cells to promote the development of immunomodulatory IL-10-producing T cells. Vaccine, 2014. 32(47): p. 6294-302.
79. Grundstrom, J., et al., Covalent coupling of vitamin D3 to the major cat allergen Fel d 1 improves the effects of allergen-specific immunotherapy in a mouse model for cat allergy. Int Arch Allergy Immunol, 2012. 157(2): p. 136-46.
80. Taher, Y.A., et al., 1alpha,25-dihydroxyvitamin D3 potentiates the beneficial effects of allergen immunotherapy in a mouse model of allergic asthma: role for IL-10 and TGF-beta. J Immunol, 2008. 180(8): p. 5211-21.
81. Stoltenburg, R., C. Reinemann, and B. Strehlitz, FluMag-SELEX as an advantageous method for DNA aptamer selection. Anal Bioanal Chem, 2005. 383(1): p. 83-91.
82. Forster, R., Study designs for the nonclinical safety testing of new vaccine products. J Pharmacol Toxicol Methods, 2012. 66(1): p. 1-7.
83. Artimo, P., et al., ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res, 2012. 40(Web Server issue): p. W597-603.
84. Zuker, M., Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res, 2003. 31(13): p. 3406-15.
85. Husslik, F., et al., Folded or Not? Tracking Bet v 1 Conformation in Recombinant Allergen Preparations. PLoS One, 2015. 10(7): p. e0132956.
86. Groh, N., et al., IgE and allergen-specific immunotherapy-induced IgG4 recognize similar epitopes of Bet v 1, the major allergen of birch pollen. Clin Exp Allergy, 2017. 47(5): p. 693-703.
87. Kong, J. and S. Yu, Fourier transform infrared spectroscopic analysis of protein secondary structures. Acta Biochim Biophys Sin (Shanghai), 2007. 39(8): p. 549-59.
88. Hu, X., D. Kaplan, and P. Cebe, Determining Beta-Sheet Crystallinity in Fibrous Proteins by Thermal Analysis and Infrared Spectroscopy. Macromolecules, 2006. 39(18): p. 6161-6170.
89. Demirdoven, N., et al., Two-dimensional infrared spectroscopy of antiparallel beta-sheet secondary structure. J Am Chem Soc, 2004. 126(25): p. 7981-90.
90. Collado-Gonzalez, J., et al., New UHPLC-QqQ-MS/MS method for quantitative and qualitative determination of free phytoprostanes in foodstuffs of commercial olive and sunflower oils. Food Chem, 2015. 178: p. 212-20.
91. Collado-Gonzalez, J., et al., Water deficit during pit hardening enhances phytoprostanes content, a plant biomarker of oxidative stress, in extra virgin olive oil. J Agric Food Chem, 2015. 63(14): p. 3784-92.
92. Clapp, T., et al., Vaccines with aluminum-containing adjuvants: optimizing vaccine efficacy and thermal stability. J Pharm Sci, 2011. 100(2): p. 388-401.
93. ABELLÓ, A., Informationen zu Alutard SQ®, in package insert. 2010. 94. Held, P., Determination of Relative Potency Using Parallel Line Analysis
88
with Gen5™ Data Analysis Software. Parallelism Analysis, BioTek Instruments, 2008. 95. GraphPad Curve Fitting Guide, in GraphPad Prism 6., GraphPad Software Inc. 96. Beck B, C.Y., Dere W, et al., Assay Operations for SAR Support. Sittampalam GS, Coussens NP,
Brimacombe K, et al., editors. Assay Guidance Manual, 2012(Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences).
97. Wei Wang, M.S., Selection of Adjuvants for Enhanced Vaccine Potency. World Journal of Vaccines, 2011. 1(33-78).
98. Tombelli, S., M. Minunni, and M. Mascini, Analytical applications of aptamers. Biosens Bioelectron, 2005. 20(12): p. 2424-34.
99. Zichel, R., et al., Aptamers as a sensitive tool to detect subtle modifications in therapeutic proteins. PLoS One, 2012. 7(2): p. e31948.
100. Kadioglu, O., et al., Aptamers as a novel tool for diagnostics and therapy. Invest New Drugs, 2015. 33(2): p. 513-20.
101. Vidarsson, G., G. Dekkers, and T. Rispens, IgG subclasses and allotypes: from structure to effector functions. Front Immunol, 2014. 5: p. 520.
102. Nilsson, R., et al., Fractionation of rat IgG subclasses and screening for IgG Fc-binding to bacteria. Mol Immunol, 1982. 19(1): p. 119-26.
103. Mestas, J. and C.C. Hughes, Of mice and not men: differences between mouse and human immunology. J Immunol, 2004. 172(5): p. 2731-8.
104. Bruggemann, M., et al., Immunoglobulin heavy chain locus of the rat: striking homology to mouse antibody genes. Proc Natl Acad Sci U S A, 1986. 83(16): p. 6075-9.
89