# Patent

## Title
Peptide carrier fusion proteins as allergy vaccines

## Abstract

The present invention relates to a polypeptide comprising at least three peptide fragments consisting of 10 to 50 consecutive amino acid residues of at least one wild-type allergen fused to the N- and C-terminus of a surface polypeptide of a virus of the hepadnaviridae family or at least one fragment of said surface polypeptide.

## Background
This application is a divisional of U.S. application Ser. No. 14/124,925 filed Dec. 9, 2013, now U.S. Pat. No. 9,308,251, which is a National Stage of PCT/EP2012/061040 filed Jun. 11, 2012 and claims the benefit of EP 11169365.1 filed Jun. 9, 2011.


The present invention relates to novel polypeptides and uses thereof.
Type I allergy is an IgE-mediated hypersensitivity disease affecting almost 25% of the population. It is based on the recognition of harmless airborne, insect, venom, food allergen and contact allergen antigens derived from per se harmless antigen sources such as pollen, insects, mold and animal proteins by specific immunoglobulin E. The crosslinking of effector cell-bound IgE antibodies leads to a release of inflammatory mediators (e.g., histamine, leucotrienes) and thus to the immediate symptoms of allergy (e.g., rhinoconjunctivitis, asthma, dermatitis, anaphylaxis). T-cell activation via IgE-dependent as well as IgE-independent mechanisms contributes to chronic allergic inflammation.
The probably only causative form of allergy treatment is allergen-specific immunotherapy, which is based on the repeated administration of increasing amounts of allergen extracts for most sources. Numerous clinical studies have documented the clinical efficacy of injection immunotherapy and there is evidence for several immunological mechanisms underlying this treatment. Due to the difficulty to prepare high quality allergen extracts for certain allergen sources and the fact that the administration of allergens to patients can cause severe side effects, allergen-specific immunotherapy can only be recommended for certain patients groups and disease manifestations. It is especially difficult to treat patients with co-sensitizations to several different allergen sources and patients suffering from severe disease manifestations such as allergic asthma. Allergic asthma is one of the most vigorous manifestations of allergy, because it severely affects the quality of daily life, causes a high rate of hospitality admissions and can manifest itself in serious, life-threatening forms requiring intensive care of the patient.
Allergen extracts prepared from natural allergen-sources are crude in nature, and it is impossible to influence the quality and amounts of individual allergens in such preparations by technical means. They also contain numerous undefined non-allergenic components, and several recent studies indicate the poor quality of such extracts and document their great heterogeneity.
In the last decade great progress has been made in the field of molecular allergen characterization using recombinant DNA technology. A large number of the most important disease-eliciting allergens has been characterized down to the molecular level, and recombinant allergens mimicking the epitope complexity of natural allergen extracts have been produced. Moreover, several research groups have used the knowledge regarding allergen structures to develop defined new allergy vaccines. Genetic engineering, synthetic peptide chemistry and conjugation of allergens with immunostimulatory DNA sequences have been used to reduce the allergenic activity of the new vaccines and thus the rate of therapy-induced side effects. First promising clinical studies were conducted with such allergen derivatives. Interestingly, it turned out that although IgE-reactivity of genetically engineered recombinant allergens and allergen-derived synthetic T-cell epitope-containing peptides could be strongly reduced or even abolished, these derivatives still could induce systemic side effects appearing several hours after injection. For example, it was reported that T-cell epitope peptides of the major cat allergen, Fel d 1, induced asthma and bronchial hyper reactivity several hours after intracutaneous injection, and there is strong evidence that this effect is T-cell mediated and MHC-restricted.
These results indicate that the removal of IgE-reactivity diminishes IgE-mediated side effects since no immediate reactions were recorded in the course of these immunotherapy studies. However, the allergen-specific T-cell epitopes which have been preserved in the recombinant allergen derivatives as well as in the peptide mixtures are responsible for the late side effects (e.g. very problematic or atopic dermatitis, chronic T-cell-mediated allergic skin manifestation). The side effects caused in the case of recombinant allergen-derivatives were relatively mild and in the case of the T-cell peptide vaccines may be overcome by adequate dosing. Both of the two new approaches therefore seem very promising for immunotherapy of allergic rhinoconjunctivitis but may have limitations when it comes to the treatment of severe forms of allergic asthma, where the induction of late side effects in the lung may be very problematic.
In order to administer and consequently to provoke an efficient immune response against peptides, polypeptides and proteins, adjuvants and/or carriers are regularly used. Complete Freund's adjuvant (CFA), for instance, is one of the most potent adjuvants available. There exists a need for vaccine compositions able to induce strong immune responses against peptides and polypeptides derived from allergens and of course of other antigens with or without the use of complete Freund's adjuvant. Further, while BSA has been used successfully as a carrier in animal models it may not be appropriate for use in human vaccine compositions because of the risk of adverse reactions such as the risk of transmitting prion disease (variant Creutzfeldt-Jakob disease). A further challenge to the development of an effective vaccine against allergens is the need for an immune response able to rapidly decrease allergens in an individual or animal. Therefore, high concentrations of allergen-specific antibodies in the blood, which are mainly of the IgG subtype, are needed. In mucosal surfaces IgA antibodies are also important.
Cholera toxin, a known carrier protein in the art, is also used regularly as an adjuvant. However, cholera toxin increases total and specific IgE antibody levels and leads to IgE-associated inflammatory reactions.
Due to the side effects provoked by most carrier proteins used for vaccination, there exists a need for carrier systems which are able to stimulate immune responses against allergens or other antigens, without using toxic adjuvants, without using poorly tolerated carrier proteins and, in certain situations, without stimulation of potentially pathologic immune responses. Novel carrier systems meeting these specifications can be used towards the formation of novel conjugates and compositions suitable for the treatment or prevention of diseases like allergic diseases.
In Bohle B. et al. (J. Immunol. 172 (11) (2004): 6642-6648) a recombinant fusion protein comprising an S-layer protein moiety and Bet v 1 moiety is described. This molecule comprises the native Bet v 1 allergen including Bet v 1-specific T cell epitopes.
WO 2004/004761 relates to virus like particles which are fused to an immunogen and which may be used for immunisation.
In WO 2004/003143 the use of fusion proteins comprising a virus like particle and an allergenic molecule as immunogen for vaccination is disclosed.
In WO 2007/140505 and Edlmayr et al. (J. Immunol. 182 (10) (2009) 6298-6306) the use of fusion proteins comprising various carrier molecules fused to allergen-derived peptides are described to induce allergen-specific IgG antibodies but these constructs do not exhibit an immunomodulatory effect which may be considered advantageous for allergic patients such as the induction of IL-10 or Th1 immunity. FIG. 4 of Edlmayr et al shows that KLH-fused peptides induce the Th2 cytokine IL-5 and VP1 fusion proteins do not induce IL-10 or IFN-gamma.
In Niespodziana et al (J. Allergy Clin. Immunol. 127 (6) (2011) 1562-1570) the use of fusion proteins each comprising Hepatitis B-derived PreS and two peptides derived from the major cat allergen Fel d 1 are described to induce allergen-specific IgG antibodies. However, no regimen suitable for vaccination of humans has been described and the peptides contained allergen-specific T cell epitopes.
It is an object of the present invention to provide medicaments and carriers which overcome the aforementioned drawbacks and allow an allergen vaccination with reduced side effects.
Therefore, the present invention relates to a polypeptide comprising at least three peptide fragments consisting of 10 to 50 consecutive amino acid residues of at least one wild-type allergen fused to the N- and C-terminus of a surface polypeptide of a virus of the hepadnaviridae family or at least one fragment of said surface polypeptide or comprising a surface polypeptide of a virus of the hepadnaviridae family or at least one fragment thereof fused N- and/or C-terminally to at least three peptides derived from at least one wild-type allergen.
In order to provoke an enhanced immune response against a molecule, in particular against an allergenic or hypoallergenic molecule according to the present invention, at least three peptide fragments derived from at least one wild-type allergen are fused (by genetic engineering) to a surface polypeptide of a virus of the hepadnaviridae family, preferably of a Hepatitis B virus, more preferably of a Hepatitis B PreS polypeptide, or at least one fragment thereof. It turned surprisingly out that in contrast to conventionally and regularly employed carrier proteins like KLH (Keyhole limpet hemocyanin) a surface polypeptide of a virus of the hepadnaviridae family, preferably of a Hepatitis B virus, more preferably of a Hepatitis B PreS polypeptide, or fragments thereof lead to an enhanced formation of antibodies directed to those peptides which are bound thereto.
Moreover, it turned out that allergen specific IgG antibodies induced by immunization with more than three properly selected allergen derived peptide fragments fused to the Hepatitis B PreS polypeptide are better focused to the IgE epitopes of the allergen while immunization with the wild-type allergen leads to IgG which are directed to all parts of the allergen—also those which are not IgE reactive. In an experiment normalized for IgG titers this leads to a better blocking capacity of PreS/peptide induced IgG compared to wild-type allergen induced (FIG. 12).
Also very surprisingly, it turned out that in cultures of human PBMCs fusion proteins of allergen derived peptide fragments to the Hepatitis B PreS polypeptide strongly induced the cytokines IL-10 and IFN-gamma, which have been attributed as positive indicators for a successful allergy immunotherapy. In contrast, induction of IL-10 and IFN-gamma was significantly lower with wild-type allergen, allergen derived peptide fragments alone or PreS alone (FIGS. 10 A-10 C).
“Fused to the N- and C-terminus”, as used herein, means that at least one peptide is fused to the N-terminus of a surface polypeptide of a virus of the hepadnaviridae family or at least one fragment of said surface polypeptide and at least one peptide is fused to the C-terminus of a surface polypeptide of a virus of the hepadnaviridae family or at least one fragment of said surface polypeptide. In a most simplest embodiment of the present invention a surface polypeptide of a virus of the hepadnaviridae family or at least one fragment of said surface polypeptide may comprise at the N-terminus one peptide and on the C-terminus two peptides or vice versa.
The polypeptide of the present invention preferably comprises at least four, more preferably at least five, even more preferably at least six, peptide fragments, preferably B cell binding peptides, derived from an allergen, whereby four peptides are most preferred.
According to a particularly preferred embodiment of the present invention the carrier protein is the Hepatitis B PreS polypeptide with the following amino acid sequence (SEQ ID No. 21):







GGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPIKDH


 


WPAANQVGVGAFGPGLTPPHGGILGWSPQAQGILTTVSTIPPPASTNRQS


 


GRQPTPISPPLRDSHPQAMQWNSTAFHQALQDPRVRGLYFPAGGSSSGTV


 


NPAPNIASHISSISARTGDPVTN






It is also possible to use fragments Hepatitis B PreS1 or Hepatitis B PreS2 of the Hepatitis B PreS polypeptide. A fragment of the Hepatitis B PreS polypeptide preferably comprises or consists of at least 30, preferably at least 40, more preferably at least 50, consecutive amino acid residues of SEQ ID No. 21.
“Hypoallergenic” as used herein, refers to molecules with reduced or no allergenic potential (i.e. IgE reactivity determined with IgE binding assays known in the art). Such molecules have a decreased capacity to provoke allergic reactions in an individual compared to the wild-type protein from which these molecules are derived.
The at least three, preferably at least four, more preferably at least five, even more preferably at least six, peptide fragments fused to the N- and C-terminus of a surface polypeptide of a virus of the hepadnaviridae family or at least one fragment of said surface polypeptide comprise or consist of 10 to 50 consecutive amino acids, more preferably 15 to 50 consecutive amino acids, in particular 20-50 consecutive amino acids, of at least one wild-type allergen and exhibit preferably reduced IgE reactivity compared to the wild-type allergen from which the peptide fragments are derived from. These peptide fragments are preferably designed to exclude allergen-specific T-cell epitopes which may cause T-cell-mediated side effects. T-cell epitopes and molecules exhibiting reduced T-cell response may be determined and identified by methods known by the person skilled in the art (e.g., Bercovici N. et al. Clin Diagn Lab Immunol. (2000) 7:859-864).
The at least three peptide fragments comprising or consisting of 10 to 50 consecutive amino acids, more preferably 15 to 50 consecutive amino acids, in particular 20-50 consecutive amino acids, of at least one wild-type allergen can be derived from one and the same allergen. If two or more fragments are derived from the same allergen these two or more fragments are not adjacently located in the wild type allergen and/or have an order in the polypeptide of the present invention which does not correspond to the order in the wild type allergen.
The term “peptide fragment” as used herein means a part/fragment of a hypoallergenic polypeptide or fusion protein of the invention which is derived from the primary structure of a wild-type allergen and comprise or consist of 10 to 50 consecutive amino acids, more preferably 15 to 50 consecutive amino acids, in particular 20-50 consecutive amino acids, of this wild-type allergen.
The terms “derived from an allergen” and “derived from at least one wild-type allergen”, as used herein, mean that the peptide fragments according to the present invention are obtained directly from an allergen by fragmentation or truncation. The amino acid sequence of these peptide fragments is preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical, in particular 100% identical, to the amino sequence stretch of the wild-type allergen, from which the peptide fragments are derived from. However, the peptides which are not 100% identical to the wild-type allergen fragments should be able to bind with at least 60%, preferably at least 70%, more preferably at least 80%, most preferably at least 90%, strength to an antibody or to antibodies, preferably to IgG antibodies, which are directed to said wild-type allergen fragments. “At least one wild-type allergen” means that the polypeptide of the present invention may comprise B-cell binding peptides of more than one, preferably two, more preferably three, different wild-type allergens (i.e. sources) (e.g. one peptide is derived from Bet v 1, one from Amb a 1 and one from Phl p 1 or two peptides are derived from Bet v 1 and one from Amb a 1).
The degree of identity of a first amino acid sequence to a second amino acid can be determined by a direct comparison between both amino acid sequences using certain algorithms. Such algorithms are, for instance, incorporated in various computer programs (e.g. “BLAST 2 SEQUENCES (blastp)” (Tatusova et al. (1999) FEMS Microbiol. Lett. 174:247-25; Corpet F, Nucl. Acids Res. (1988) 16:10881-10890).
The polypeptides of the present invention may be obtained by recombinant methods or chemical synthesis. Alternatively, it is, of course, also possible to obtain the molecules by enzymatic or chemical cleavage of the wild-type allergen or a polypeptide/protein harbouring the molecule of interest.
It was now surprisingly found that peptide carrier fusion proteins with improved properties can be obtained by employing surface proteins from viruses of the hepadnaviridae class, more specifically the human hepatitis B virus. One up to 20, preferably 3 or 4 up to 20, more preferably 3 or 4 up to 15, even more preferably 3 or 4 up to 10 (i.e. 3, 4, 5, 6, 7, 8, 9, 10), peptide fragments, preferably hypoallergenic peptide fragments, can be fused to the C-terminus and the N-terminus of a surface polypeptide of a virus of the hepadnaviridae family or at least one fragment of said surface polypeptide. A preferred embodiment of the current invention are therefore fusion proteins composed of at least 3 up to 6 hypoallergenic peptide fragments with a carrier protein derived from the surface antigens of human hepatitis B virus. According to a particularly preferred embodiment of the present invention such fusion proteins use the preS protein as carrier. A most preferred embodiment of this invention are fusion proteins where 4 hypoallergenic peptide fragments are fused to the preS carrier protein or a fragment thereof. The (hypoallergenic) peptide fragments can be the same or different and can derived from one or several allergenic proteins and the locus of the peptides within the fusion protein is the C-terminus and the N-terminus of the carrier protein. One up to three (hypoallergenic) peptide fragments can be fused to each of the C-terminus and the N-terminus in such a way that the sum of the (hypoallergenic) peptide fragments will be, for instance, three or four to six. The terms “fused” or “fusion protein”, refer to a preferred embodiment of the invention, meaning that the non-allergenic carrier protein and the (hypoallergenic) peptide fragments at the carrier's C- and N-terminus are expressed and prepared as one singular recombinant polypeptide chain
A most highly preferred embodiment of the current invention are fusion proteins of the hepatitis B virus preS protein, which carry (hypoallergenic) peptide fragments derived from a specific allergen, such that one or two, preferably two, peptide fragments each are fused to the C-terminus and the N-terminus of the carrier. For illustration, the preferred polypeptides of the current invention may have the general molecular structure represented by the following generic structures:












It is understood that peptides A, B, C and D can be the same or different and may be derived from the same allergen for each individual fusion protein or will be derived from different allergens.
The (hypoallergenic) peptides to be fused to the N- and C-terminus of the surface polypeptide of a virus of the hepadnaviridae family or at least one fragment of said surface polypeptide, preferably the preS protein or a fragment thereof, are preferably selected from the group consisting of major birch pollen allergens, in particular Bet v 1 and Bet v 4, major timothy grass pollen allergens, in particular Phl p 1, Phl p 2, Phl p 5, Phl p 6 and Phl p 7, major house dust mite allergens, in particular Der p 1, Der p 2, Der p 5, Der p 7, Der p 21 and Der p 23, major cat allergen Fel d 1, the major ragweed allergen Amb a 1, the major Japanese cedar allergens Cry j 1 and Cry j 2, major bee allergens, major wasp allergens, profilins, especially Phl p 7, Phl p 12.
Other suited allergens to be used according to the present invention can be derived from the following table 2 without being restricted to said table.







TABLE 2







Sources of hypoallergenic peptides













Allergen
Biochem. ID or

cDNA (C) or
Reference,


Species Name
Name
Obsolete name
MW
protein (P)
Acc. No.





Ambrosia artemisiifolia
Amb a 1
antigen E
 8
C
8, 20


short ragweed
Amb a 2
antigen K
38
C
8, 21



Amb a 3
Ra3
11
C
22



Amb a 5
Ra5
 5
C
11, 23



Amb a 6
Ra6
10
C
24, 25



Amb a 7
Ra7
12
P
26


Ambrosia trifida
Amb t 5
Ra5G
  4.4
C
9, 10, 27


giant ragweed


Artemisia vulgaris
Art v 1

27-29
C
28


mugwort
Art v 2

35
P
28A



Art v 3
lipid transfer protein
12
P
53



Art v 4
profilin
14
C
29


Helianthus annuus
Hel a 1

34

29A


sunflower
Hel a 2
profilin
  15.7
C
Y15210


Mercurialis annua
Mer a 1
profilin
14-15
C
Y13271


Caryophyllales
Che a 1

17
C
29B, AY049012


Chenopodium album
Che a 2
profilin
14
C
AY082337


lamb's-quarters, pigweed,
Che a 3
polcalcin
10
C
AY082338


white goosefoot


Salsola kali
Sal k 1

43
P
29C


Russian-thistle


Rosales
Hum j 4w


C
AY335187


Humulus japonicus


Japanese hop


Parietaria judaica
Par j 1
lipid transfer protein 1
15
C
see list of isoallergens



Par j 2
lipid transfer protein 2

C
see list of isoallergens



Par j 3
profilin

C
see list of isoallergens


Parietaria officinalis
Par o 1
lipid transfer protein
15

29D


B. Grasses
Cyn d 1

32
C
30, S83343


Poales
Cyn d 7


C
31, X91256


Cynodon dactylon
Cyn d 12
profilin
14
C
31a, Y08390


Bermuda grass
Cyn d 15

 9
C
AF517686












Cyn d 22w
enolase
data pending














Cyn d 23
Cyn d 14
 9
C
AF517685



Cyn d 24
Pathogenesis- related p.
21
P
pending


Dactylis glomerata
Dac g 1
AgDg1
32
P
32


orchard grass
Dac g 2

11
C
33, S45354



Dac g 3


C
33A, U25343



Dac g 5

31
P
34


Festuca pratensis
Fes p 4w

60
—


meadow fescue


Holcus lanatus
Hol l 1


C
Z27084


velvet grass


Lolium perenne
Lol p 1
group I
27
C
35, 36


rye grass
Lol p 2
group II
11
P
37, 37A, X73363



Lol p 3
group III
11
P
38



Lol p 5
Lol p IX, Lol p Ib
31/35
C
34, 39



Lol p 11
hom: trypsin inhibitor
16

39A


Phalaris aquatica
Pha a 1


C
40, S80654


canary grass


Phleum pratense
Phl p 1

27
C
X78813


timothy
Phl p 2


C
X75925, 41



Phl p 4


P
41A



Phl p 5
Ag25
32
C
42



Phl p 6


C
Z27082, 43



Phl p 11
trypsin inhibitor hom.
20
C
AF521563, 43A



Phl p 12
profilin

C
X77583, 44



Phl p 13
polygalacturonase
55-60
C
AJ238848


Poa pratensis
Poa p 1
group I
33
P
46


Kentucky blue grass
Poa p 5

31/34
C
34, 47


Sorghum halepense
Sor h 1


C
48


Johnson grass


C. Trees
Pho d 2
profilin
  14.3
C
Asturias p.c.


Arecales


Phoenix dactylifera


date palm


Fagales
Aln g 1

17
C
S50892


Alnus glutinosa


alder


Betula verrucosa
Bet v 1

17
C
see list of isoallergens


birch
Bet v 2
profilin
15
C
M65179



Bet v 3


C
X79267



Bet v 4

 8
C
X87153, S54819



Bet v 6
h: isoflavone reductase
  33.5
C
see list of isoallergens



Bet v 7
cyclophilin
18
P
P81531


Carpinus betulus
Car b 1

17
C
see list of isoallergens


hornbeam


Castanea sativa
Cas s 1

22
P
52


chestnut
Cas s 5
chitinase



Cas s 8
lipid transfer protein
  9.7
P
53


Corylus avellana
Cor a 1

17
C
see list of isoallergens


hazel
Cor a 2
profilin
14
C



Cor a 8
lipid transfer protein
9
C



Cor a 9
11S globulin-like protein
40/? 
C
Beyer p.c.



Cor a 10
luminal binding prot.
70
C
AJ295617



Cor a 11
7S vicilin-like prot.
48
C
AF441864


Quercus alba
Que a 1

17
P
54


White oak


Lamiales
Fra e 1

20
P
58A, AF526295


Oleaceae


Fraxinus excelsior


ash


Ligustrum vulgare
Lig v 1

20
P
58A


privet


Olea europea
Ole e 1

16
C
59, 60


olive
Ole e 2
profilin
15-18
C
60A



Ole e 3

  9.2

60B



Ole e 4

32
P
P80741



Ole e 5
superoxide dismutase
16
P
P80740



Ole e 6

10
C
60C, U86342



Ole e 7

?
P
60D, P81430



Ole e 8
Ca2+-binding protein
21
C
60E, AF078679



Ole e 9
beta-1,3-glucanase
46
C
AF249675



Ole e 10
glycosyl hydrolase hom.
11
C
60F, AY082335


Syringa vulgaris
Syr v 1

20
P
58A


lilac


Plantaginaceae
Pla l 1

18
P
P842242


Plantago lanceolata


English plantain


Pinales
Cry j 1

41-45
C
55, 56


Cryptomeria japonica
Cry j 2


C
57, D29772


sugi


Cupressus arisonica
Cup a 1

43
C
A1243570


cypress


Cupressus sempervirens
Cup s 1

43
C
see list of isoallergens


common cypress
Cup s 3w

34
C
ref pending


Juniperus ashei
Jun a 1

43
P
P81294


mountain cedar
Jun a 2


C
57A, AJ404653



Jun a 3

30
P
57B, P81295


Juniperus oxycedrus
Jun o 4
hom: calmodulin
29
C
57C, AF031471


prickly juniper


Juniperus sabinoides
Jun s 1

50
P
58


mountain cedar


Juniperus virginiana
Jun v 1

43
P
P81825, 58B


eastern red cedar


Platanaceae
Pla a 1

18
P
P82817


Platanus acerifolia
Pla a 2

43
P
P82967


London plane tree
Pla a 3
lipid transfer protein
10
P
Iris p.c.


D. Mites
Aca s 13
arthropod
 14*
C
AJ006774


Acarus siro

fatty acid binding prot.


mite


Blomia tropicalis
Blo t 1
cysteine protease
39
C
AF277840


mite
Blo t 3
trypsin
 24*
C
Cheong p.c.



Blo t 4
alpha amylase
56
C
Cheong p.c.



Blo t 5


C
U59102



Blo t 6
chymotrypsin
25
C
Cheong p.c.



Blo t 10
tropomyosin
33
C
61



Blo t 11
paramyosin
110 
C
AF525465, 61A



Blo t 12
Bt11a

C
U27479



Blo t 13
Bt6, fatty acid bind prot.

C
U58106



Blo t 19
anti-microbial pep. hom.
  7.2
C
Cheong p.c.


Dermatophagoides farinae
Der f 1
cysteine protease
25
C
69


American house dust mite
Der f 2

14
C
70, 70A, see list of isoallergens



Der f 3
trypsin
30
C
63



Der f 7

24-31
C
SW: Q26456, 71



Der f 10
tropomyosin

C
72



Der f 11
paramyosin
98
C
72A



Der f 14
mag3, apolipophorin

C
D17686



Der f 15
98k chitinase
98
C
AF178772



Der f 16
gelsolin/villin
53
C
71A



Der f 17
Ca binding EF protein
53
C
71A



Der f 18w
60k chitinase
60
C
Weber p.c.


Dermatophagoides microceras
Der m 1
cysteine protease
25
P
68


house dust mite


Dermatophagoides pteronyssinus
Der p 1
antigen P1, cysteine protease
25
C
62, see list of isoallergens


European house dust mite
Der p 2

14
C
62A-C, see list of isoallergens



Der p 3
trypsin
28/30
C
63



Der p 4
amylase
60
P
64



Der p 5

14
C
65



Der p 6
chymotrypsin
25
P
66



Der p 7

22/28
C
67



Der p 8
glutathione transferase

C
67A



Der p 9
collagenolytic serine pro.

P
67B



Der p 10
tropomyosin
36
C
Y14906



Der p 14
apolipophorin like prot.

C
Epton p.c.


Euroglyphus maynei
Eur m 2


C
see list of isoallergens


mite
Eur m 14
apolipophorin
177 
C
AF149827


Glycyphagus domesticus
Gly d 2


C
72B, see isoallergen list


storage mite


Lepidoglyphus destructor
Lep d 2

15
C
73, 74, 74A, see isoallergen list


storage mite
Lep d 1



Lep d 5


C
75, AJ250278



Lep d 7


C
75, AJ271058



Lep d 10
tropomyosin

C
75A, AJ250096



Lep d 13


C
75, AJ250279


Tyrophagus putrescentiae
Tyr p 2


C
75B, Y12690


storage mite


E. Animals
Bos d 2
Ag3, lipocalin
20
C
76, see isoallergen list


Bos domesticus
Bos d 3
Ca-binding S100 hom.
11
C
L39834


domestic cattle
Bos d 4
alpha-lactalbumin
  14.2
C
M18780


(see also foods)
Bos d 5
beta-lactoglobulin
  18.3
C
X14712



Bos d 6
serum albumin
67
C
M73993



Bos d 7
immunoglobulin
160 

77



Bos d 8
caseins
20-30

77


Canis familiaris
Can f 1

25
C
78, 79


(Canis domesticus)
Can f 2

27
C
78, 79


dog
Can f 3
albumin

C
S72946



Can f 4

18
P
A59491


Equus caballus
Equ c 1
lipocalin
25
C
U70823


domestic horse
Equ c 2
lipocalin
  18.5
P
79A, 79B



Equ c 3
Ag3 - albumin
67
C
79C, X74045



Equ c 4

17
P
79D



Equ c 5
AgX
17
P
Goubran Botros p.c.


Felis domesticus
Fel d 1
cat-1
38
C
15


cat (saliva)
Fel d 2
albumin

C
79E, X84842



Fel d 3
cystatin
11
C
79F, AF238996



Fel d 4
lipocalin
22
C
AY497902



Fel d 5w
immunoglobulin A
400 

Adedoyin p.c.



Fel d 6w
immunoglobulin M
 800-1000

Adedoyin p.c.



Fel d 7w
immunoglobulin G
150 

Adedoyin p.c.


Cavia porcellus
Cav p 1
lipocalin homologue
20
P
SW: P83507, 80


guinea pig
Cav p 2

17
P
SW: P83508


Mus musculus
Mus m 1
MUP
19
C
81, 81A


mouse (urine)


Rattus norvegius
Rat n 1

17
C
82, 83


rat (urine)


F. Fungi (moulds)
Alt a 1

28
C
U82633


1. Ascomycota
Alt a 2

25
C
83A, U62442


1.1 Dothideales
Alt a 3
heat shock prot.
70
C
U87807, U87808


Alternaria alternata
Alt a 4
prot. disulfideisomerase
57
C
X84217



Alt a 6 a
cid ribosomal prot. P2
11
C
X78222, U87806



Alt a 7
YCP4 protein
22
C
X78225



Alt a 10
aldehyde dehydrogenase
53
C
X78227, P42041



Alt a 11
enolase
45
C
U82437



Alt a 12
acid ribosomal prot. P1
11
C
X84216


Cladosporium herbarum
Cla h 1

13

83B, 83C



Cla h 2

23

83B, 83C



Cla h 3
aldehyde dehydrogenase
53
C
X78228



Cla h 4
acid ribosomal prot. P2
11
C
X78223



Cla h 5
YCP4 protein
22
C
X78224



Cla h 6
enolase
46
C
X78226



Cla h 12
acid ribosomal prot. P1
11
C
X85180


1.2 Eurotiales
Asp fl 13
alkaline serine protease
34

84


Aspergillus flavus


Aspergillus fumigatus
Asp f 1

18
C
M83781, S39330



Asp f 2

37
C
U56938



Asp f 3
peroxisomal protein
19
C
U20722



Asp f 4

30
C
AJ001732



Asp f 5
metalloprotease
40
C
Z30424



Asp f 6
Mn superoxide dismut.
  26.5
C
U53561



Asp f 7

12
C
AJ223315



Asp f 8
ribosomal prot. P2
11
C
AJ224333



Asp f 9

34
C
AJ223327



Asp f 10
aspartic protease
34
C
X85092



Asp f 11
peptidyl-prolyl isomerase
24

84A



Asp f 12
heat shock prot. P90 90

C
85



Asp f 13
alkaline serine protease
34

84B



Asp f 15

16
C
AJ002026



Asp f 16

43
C
g3643813



Asp f 17


C
AJ224865



Asp f 18
vacuolar serine protease
34

84C



Asp f 22w
enolase
46
C
AF284645



Asp f 23
L3 ribosomal protein
44
C
85A, AF464911


Aspergillus niger
Asp n 14
beta-xylosidase
105 
C
AF108944



Asp n 18
vacuolar serine protease
34
C
84B



Asp n 25
3-phytase B
 66-100
C
85B, P34754



Asp n ?

85
C
Z84377


Aspergillus oryzae
Asp o 13
alkaline serine protease
34
C
X17561



Asp o 21
TAKA-amylase A
53
C
D00434, M33218


Penicillium brevicompactum
Pen b 13
alkaline serine protease
33

86A


Penicillium chrysogenum
Pen ch 13
alkaline serine protease
34

87


(formerly P. notatum)
Pen ch 18
vacuolar serine protease
32

87



Pen ch 20
N-acetyl glucosaminidase
68

87A


Penicillium citrinum
Pen c 3
peroxisomal mem. prot.
18

86B



Pen c 13
alkaline serine protease
33

86A



Pen c 19
heat shock prot. P70
70
C
U64207



Pen c 22w
enolase
46
C
AF254643



Pen c 24
elongation factor 1 beta

C
AY363911


Penicillium oxalicum
Pen o 18
vacuolar serine protease
34

87B


1.3 Hypocreales
Fus c 1
ribosomal prot. P2
 11*
C
AY077706


Fusarium culmorum
Fus c 2
thioredoxin-like prot.
 13*
C
AY077707


1.4 Onygenales
Tri r 2


C
88


Trichophyton rubrum
Tri r 4
serine protease

C
88


Trichophyton tonsurans
Tri t 1

30
P
88A



Tri t 4
serine protease
83
C
88


1.5 Saccharomycetales
Cand a 1

40
C
89


Candida albicans
Cand a 3
peroxisomal protein
29
C
AY136739


Candida boidinii
Cand b 2

20
C
J04984, J04985


2. Basidiomycotina
Psi c 1


2.1 Hymenomycetes
Psi c 2
cyclophilin
16

89A


Psilocybe cubensis


Coprinus comatus
Cop c 1
leucine zipper protein
11
C
AJ132235


shaggy cap
Cop c 2



AJ242791



Cop c 3



AJ242792



Cop c 5



AJ242793



Cop c 7



AJ242794


2.2 Urediniomycetes
Rho m 1
enolase
47
C
89B


Rhodotorula mucilaginosa
Rho m 2
vacuolar serine protease
31
C
AY547285


2.3 Ustilaginomycetes
Mala f 2
MF1, peroxisomal
21
C
AB011804, 90


Malassezia furfur

membrane protein



Mala f 3
MF2, peroxisomal
20
C
AB011805, 90




membrane protein



Mala f 4
mitochondrial malate




dehydrogenase





35
C
AF084828, 90A


Malassezia sympodialis
Mala s 1


C
X96486, 91



Mala s 5

 18*
C
AJ011955



Mala s 6

 17*
C
AJ011956



Mala s 7


C
AJ011957, 91A



Mala s 8

 19*
C
AJ011958, 91A



Mala s 9

 37*
C
AJ011959, 91A



Mala s 10
heat shock prot. 70
86
C
AJ428052



Mala s 11
Mn superoxide dismut.
23
C
AJ548421


3. Deuteromycotina
Epi p 1
serine protease
30
P
SW: P83340, 91B


3.1 Tuberculariales


Epicoccum purpurascens


(formerly E. nigrum)


G. Insects
Aed a 1
apyrase
68
C
L12389


Aedes aegyptii
Aed a 2

37
C
M33157


mosquito


Apis mellifera
Api m 1
phospholipase A2
16
C
92


honey bee
Api m 2
hyaluronidase
44
C
93



Api m 4
melittin
 3
C
94



Api m 6

7-8
P
Kettner p.c.



Api m 7
CUB serine protease
39
C
AY127579


Bombus pennsylvanicus
Bom p 1
phospholipase
16
P
95


bumble bee
Bom p 4
protease

P
95


Blattella germanica
Bla g 1
Bd90k

C


German cockroach
Bla g 2
aspartic protease
36
C
96



Bla g 4
calycin
21
C
97



Bla g 5
glutathione transferase
22
C
98



Bla g 6
troponin C
27
C
98


Periplaneta americana
Per a 1
Cr-PII

C


American cockroach
Per a 3
Cr-PI
72-78
C
98A



Per a 7
tropomyosin
37
C
Y14854


Chironomus kiiensis
Chi k 10
tropomyosin
  32.5*
C
AJ012184


midge


Chironomus thummi thummi
Chi t 1-9
hemoglobin
16
C
99


midge
Chi t 1.01
component III
16
C
P02229



Chi t 1.02
component IV
16
C
P02230



Chi t 2.0101
component I
16
C
P02221



Chi t 2.0102
component IA
16
C
P02221



Chi t 3
component II-beta
16
C
P02222



Chi t 4
component IIIA
16
C
P02231



Chi t 5
component VI
16
C
P02224



Chi t 6.01
component VIIA
16
C
P02226



Chi t 6.02
component IX
16
C
P02223



Chi t 7
component VIIB
16
C
P02225



Chi t 8
component VIII
16
C
P02227



Chi t 9
component X
16
C
P02228


Ctenocephalides felis felis
Cte f 1


cat flea
Cte f 2
M1b
27
C
AF231352



Cte f 3

25
C


Thaumetopoea pityocampa
Tha p 1

15
P
PIR: A59396, 99A


pine processionary moth


Lepisma saccharina
Lep s 1
tropomyosin
36
C
AJ309202


silverfish


Dolichovespula maculata
Dol m 1
phospholipase A1
35
C
100


white face hornet
Dol m 2
hyaluronidase
44
C
101



Dol m 5
antigen 5
23
C
102, 103


Dolichovespula arenaria
Dol a 5
antigen 5
23
C
104


yellow hornet


Polistes annularies
Pol a 1
phospholipase A1
35
P
105


wasp
Pol a 2
hyaluronidase
44
P
105



Pol a 5
antigen 5
23
C
104


Polistes dominulus
Pol d 1



Hoffman p.c.


Mediterranean paper wasp
Pol d 4
serine protease
32-34
C
Hoffman p.c.



Pol d 5



P81656


Polistes exclamans
Pol e 1
phospholipase A1
34
P
107


wasp
Pol e 5
antigen 5
23
C
104


Polistes fuscatus
Pol f 5
antigen 5
23
C
106


wasp


Polistes gallicus
Pol g 5
antigen 5
24
C
P83377


wasp


Polistes metricus
Pol m 5
antigen 5
23
C
106


wasp


Vespa crabo
Vesp c 1
phospholipase
34
P
107


European hornet
Vesp c 5
antigen 5
23
C
106


Vespa mandarina
Vesp m 1



Hoffman p.c.


giant asian hornet
Vesp m 5



P81657


Vespula flavopilosa
Ves f 5
antigen 5
23
C
106


yellowjacket


Vespula germanica
Ves g 5
antigen 5
23
C
106


yellowjacket


Vespula maculifrons
Ves m 1
phospholipase A1
  33.5
C
108


yellowjacket
Ves m 2
hyaluronidase
44
P
109



Ves m 5
antigen 5
23
C
104


Vespula pennsylvanica
Ves p 5
antigen 5
23
C
106


yellowjacket


Vespula squamosa
Ves s 5
antigen 5
23
C
106


yellowjacket


Vespula vidua
Ves vi 5
antigen 5
23
C
106


wasp


Vespula vulgaris
Ves v 1
phospholipase A1
35
C
105A


yellowjacket
Ves v 2
hyaluronidase
44
P
105A



Ves v 5
antigen 5
23
C
104


Myrmecia pilosula
Myr p 1


C
X70256


Australian jumper ant
Myr p 2


C
S81785


Solenopsis geminata
Sol g 2



Hoffman p.c.


tropical fire ant
Sol g 4



Hoffman p.c.


Solenopsis invicta
Sol i 2

13
C
110, 111


fire ant
Sol i 3

24
C
110



Sol i 4

13
C
110


Solenopsis saevissima
Sol s 2



Hoffman p.c.


Brazilian fire ant


Triatoma protracta
Tria p 1
Procalin
20
C
AF179004, 111A.


California kissing bug


H. Foods
Gad c 1
allergen M
12
C
112, 113


Gadus callarias


cod


Salmo salar
Sal s 1
parvalbumin
12
C
X97824


Atlantic salmon


Bos domesticus
Bos d 4
alpha-lactalbumin
  14.2
C
M18780


domestic cattle
Bos d 5
beta-lactoglobulin
  18.3
C
X14712


(milk)
Bos d 6
serum albumin
67
C
M73993


see also animals
Bos d 7
immunoglobulin
160 

77



Bos d 8
caseins
20-30

77


Cyprinus carpio
Cyp c 1
parvalbumin
12
C
129


(Common carp)


Gallus domesticus
Gal d 1
ovomucoid
28
C
114, 115


chicken
Gal d 2
ovalbumin
44
C
114, 115



Gal d 3
Ag22, conalbumin
78
C
114, 115



Gal d 4
lysozyme
14
C
114, 115



Gal d 5
serum albumin
69
C
X60688


Metapenaeus ensis
Met e 1
tropomyosin

C
U08008


shrimp


Penaeus aztecus
Pen a 1
tropomyosin
36
P
116


shrimp


Penaeus indicus
Pen i 1
tropomyosin
34
C
116A


shrimp


Penaeus monodon
Pen m 1
tropomyosin
38
C


black tiger shrimp
Pen m 2
arginine kinase
40
C
AF479772, 117


Todarodes pacificus
Tod p 1
tropomyosin
38
P
117A


squid


Helix aspersa
Hel as 1
tropomyosin
36
C
Y14855, 117B


brown garden snail


Haliotis midae
Hal m 1

49

117C


abalone


Rana esculenta
Ran e 1
parvalbumin alpha
  11.9*
C
AJ315959


edible frog
Ran e 2
parvalbumin beta
  11.7*
C
AJ414730


Brassica juncea
Bra j 1
2S albumin
14
C
118


oriental mustard


Brassica napus
Bra n 1
2S albumin
15
P
118A, P80208


rapeseed


Brassica rapa
Bra r 2
hom: prohevein
25

P81729


turnip


Hordeum vulgare
Hor v 15
BMAI-1
15
C
119


barley
Hor v 16
alpha-amylase



Hor v 17
beta-amylase



Hor v 21
gamma-3 hordein
34
C
119A, SW: P80198


Secale cereale
Sec c 20
secalin


see isoall. list


rye


Triticum aestivum
Tri a 18
agglutinin


wheat
Tri a 19
omega-5 gliadin
65
P
PIR: A59156


Zea mays
Zea m 14
lipid transfer prot.
 9
P
P19656


maise, corn


Oryza sativa
Ory s 1


C
119B, U31771


rice


Apium graveolens
Api g 1
hom: Bet v 1
 16*
C
Z48967


celery
Api g 4
profilin


AF129423



Api g 5

55/58
P
P81943


Daucus carota
Dau c 1
hom: Bet v 1
16
C
117D, see isoallergen list


carrot
Dau c 4
profilin

C
AF456482


Corylus avellana
Cor a 1.04
hom: Bet v 1
17
C
see list of isoallergens


hazelnut
Cor a 2
profilin
14
C
AF327622



Cor a 8
lipid transfer protein
 9
C
AF329829


Malus domestica
Mal d 1
hom: Bet v 1

C
see list of isoallergens


apple
Mal d 2
hom: thaumatin

C
AJ243427



Mal d 3
lipid transfer protein
 9
C
Pastorello p.c.



Mal d 4
profilin
  14.4*
C
see list of isoallergens


Pyrus communis
Pyr c 1
hom: Bet v 1
18
C
AF05730


pear
Pyr c 4
profilin
14
C
AF129424



Pyr c 5
hom: isoflavone reductas
  33.5
C
AF071477


Persea americana
Pers a 1
endochitinase
32
C
Z78202


avocado


Prunus armeniaca
Pru ar 1
hom: Bet v 1

C
U93165


apricot
Pru ar 3
lipid transfer protein
 9
P


Prunus avium
Pru av 1
hom: Bet v 1

C
U66076


sweet cherry
Pru av 2
hom: thaumatin

C
U32440



Pru av 3
lipid transfer protein
10
C
AF221501



Pru av 4
profilin
15
C
AF129425


Prunus domestica
Pru d 3
lipid transfer protein
 9
P
119C


European plum


Prunus persica
Pru p 3
lipid transfer protein
10
P
P81402


peach
Pru p 4
profilin
14
C
see isoallergen list


Asparagus officinalis
Aspa o 1
lipid transfer protein
 9
P
119D


Asparagus


Crocus sativus
Cro s 1

21

Varasteh A-R p.c.


saffron crocus


Lactuca sativa
Lac s 1
lipid transfer protein
 9

Vieths p.c.


lettuce


Vitis vinifera
Vit v 1
lipid transfer protein
 9
P
P80274


grape


Musa × paradisiaca
Mus xp 1
profilin
15
C
AF377948


banana


Ananas comosus


pineapple
Ana c 1
profilin
15
C
AF377949



Ana c 2
bromelain
  22.8*
C
119E-G, D14059


Citrus limon
Cit l 3
lipid transfer protein
 9
P
Torrejon p.c.


lemon


Citrus sinensis
Cit s 1
germin-like protein
23
P
Torrejon p.c.


sweet orange
Cit s 2
profilin
14
P
Torrejon p.c.



Cit s 3
lipid transfer protein
 9
P
Torrejon p.c.


Litchi chinensis
Lit c 1
profilin
15
C
AY049013


litchi


Sinapis alba
Sin a 1
2S albumin
14
C
120


yellow mustard


Glycine max
Gly m 1
HPS
 7
P
120A


soybean
Gly m 2

 8
P
A57106



Gly m 3
profilin
14
C
see list of isoallergens



Gly m 4
(SAM22) PR-10 prot.
17
C
X60043, 120B


Vigna radiata
Vig r 1
PR-10 protein
15
C
AY792956


mung bean


Arachis hypogaea
Ara h 1
vicilin
  63.5
C
L34402


peanut
Ara h 2
conglutin
17
C
L77197



Ara h 3
glycinin
60
C
AF093541



Ara h 4
glycinin
37
C
AF086821



Ara h 5
profilin
15
C
AF059616



Ara h 6
hom: conglutin
15
C
AF092846



Ara h 7
hom: conglutin
15
C
AF091737



Ara h 8
PR-10 protein
17
C
AY328088


Lens culinaris
Len c 1
vicilin
47
C
see list of isoallergens


lentil
Len c 2
seed biotinylated prot.
66
P
120C


Pisum savitum
Pis s 1
vicilin
44
C
see list of isoallergens


pea
Pis s 2
convicilin
63
C
pending


Actinidia chinensis
Act c 1
cysteine protease
30
P
P00785


kiwi
Act c 2
thaumatin-like protein
24
P
SW: P81370, 121


Capsicum annuum
Cap a 1w
osmotin-like protein
23
C
AJ297410


bell pepper
Cap a 2
profilin
14
C
AJ417552


Lycopersicon esculentum
Lyc e 1
profilin
14
C
AJ417553


tomato
Lyc e 2
b-fructofuranosidase
50
C
see isoallergen list



Lyc e 3
lipid transfer prot.
6
C
U81996


Solanum tuberosum
Sola t 1
patatin
43
P
P15476


potato
Sola t 2
cathepsin D inhibitor
21
P
P16348



Sola t 3
cysteine protease inhibitor
21
P
P20347



Sola t 4
aspartic protease inhibitor
16 + 4
P
P30941


Bertholletia excelsa
Ber e 1
2S albumin
 9
C
P04403, M17146


Brazil nut
Ber e 2
11S globulin seed storage protein
29
C
AY221641


Juglans nigra
Jug n 1
2S albumin
 19*
C
AY102930


black walnut
Jug n 2
vicilin-like prot.
 56*
C
AY102931


Juglans regia
Jug r 1
2S albumin

C
U66866


English walnut
Jug r 2
vicilin
44
C
AF066055



Jug r 3
lipid transfer protein
 9
P
Pastorello


Anacardium occidentale
Ana o 1
vicilin-like protein
50
C
see isoallergen list


Cashew
Ana o 2
legumin-like protein
55
C
AF453947



Ana o 3
2S albumin
14
C
AY081853


Ricinus communis
Ric c 1
2S albumin

C
P01089


Castor bean


Sesamum indicum
Ses i 1
2S albumin
 9
C
121A, AF240005


sesame
Ses i 2
2S albumin
 7
C
AF091841



Ses i 3
7S vicilin-like globulin
45
C
AF240006



Ses i 4
oleosin
17
C
AAG23840



Ses i 5
oleosin
15
C
AAD42942


Cucumis melo
Cuc m 1
serine protease
66
C
D32206


muskmelon
Cuc m 2
profilin
14
C
AY271295



Cuc m 3
pathogenesis-rel p. PR-1
 16*
P
P83834


I. Others
Ani s 1

24
P
121B, A59069


Anisakis simplex
Ani s 2
paramyosin
97
C
AF173004


nematode
Ani s 3
tropomyosin
41
C
121C, Y19221



Ani s 4

 9
P
P83885


Argas reflexus
Arg r 1

17
C
AJ697694


piigeon tick


Ascaris suum
Asc s 1

10
P
122


worm


Carica papaya
Car p 3w
papain
  23.4*
C
122A, M15203


papaya


Dendronephthya nipponica
Den n 1

53
P
122B


soft coral


Hevea brasiliensis
Hev b 1
elongation factor
58
P
123, 124


rubber (latex)
Hev b 2
1,3-glucanase
34/36
C
125



Hev b 3

24
P
126, 127



Hev b 4
component of
100-115
P
128




microhelix complex



Hev b 5

16
C
U42640



Hev b 6.01
hevein precursor
20
C
M36986, p02877



Hev b 6.02
hevein
 5
C
M36986, p02877



Hev b 6.03
C-terminal fragment
14
C
M36986, p02877



Hev b 7.01
hom: patatin from B-serum
42
C
U80598



Hev b 7.02
hom: patatin from C-serum
44
C
AJ223038



Hev b 8
profilin
14
C
see list of isoallergens



Hev b 9
enolase
51
C
AJ132580



Hev b 10
Mn superoxide dismut.
26
C
see list of isoallergens



Hev b 11
class 1 chitinase

C
see list of isoallergens



Hev b 12
lipid transfer protein
  9.3
C
AY057860



Hev b 13
esterase
42
P
P83269


Homo sapiens
Hom s 1

 73*
C
Y14314


human autoallergens
Hom s 2

  10.3*
C
X80909



Hom s 3

  20.1*
C
X89985



Hom s 4

 36*
C
Y17711



Hom s 5

  42.6*
C
P02538


Triplochiton scleroxylon
Trip s 1
class 1 chitinase
  38.5
P
Kespohl p.c.


obeche









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According to a particularly preferred embodiment of the present invention at least one, preferably at least two, more preferably at least three, in particular all, of the at least three peptides derived from the at least one wild-type allergen is a B cell binding peptide.
“B cell binding peptides” to be used for allergy vaccination according to the invention are derived from or close to the IgE binding sites of allergens but per se show no or minimal IgE reactivity compared to the wild-type allergen (Focke M et al. Clinical & Experimental Allergy 40(2010):385-397). Requirements for their production and selection are the knowledge of the primary sequence of the allergen and regarding the IgE binding sites. Upon immunization, B cell binding peptides fused to a suitable immunogenic carrier, are capable of inducing the production of allergen-specific IgG which can block IgE binding to the allergen. Whether the IgG induced with the fusion protein can recognize the allergen can be determined, for instance, by testing the IgG for reactivity with the complete allergen. Suitable methods include ELISA, dot blot or Western blot assays. Those peptides are preferred which induce IgG that blocks patients IgE binding to the allergen.
The present invention shows that the use of suitable B cell binding peptides in particular when three or more are fused to a suitable carrier according to the present invention allows the induction of IgG responses which are better focused to the IgE epitopes than those induced by immunization even with a complete allergen. Furthermore, the invention shows that the combination of the appropriate peptides and their number with a suitable carrier can direct the allergen-specific immune response towards a favorable anti-allergic immune response (characterized by the induction of preferentially allergen-specific IgG and not IgE responses and tolerogenic (IL-10) and Th1 (Interferon gamma) cytokine responses.
Moreover, it surprisingly turned out that—despite the fact that they lack allergen-specific T-cell epitopes—polypeptides according to the invention containing 3 or more B cell binding peptides fused to an immunogenic carrier are able reduce allergen-specific T-cell reactions. This is shown by the fact that the presence of allergen-specific IgG induced by therapeutic vaccination with the hypoallergenic polypeptides of the present invention reduces allergen-specific T-cell activation caused by IgE facilitated antigen presentation in PBMCs from vaccinated human allergic individuals. (FIGS. 16 A and B).
According to a preferred embodiment of the present invention at least one of said at least three peptides exhibits no or reduced IgE-binding capacity compared to the wild-type allergen.
According to another preferred embodiment of the present invention at least one, preferably at least two, more preferably at least three, of said at least three B-cell binding peptides exhibits no or substantially no T-cell reactivity.
The presence of allergen-specific T cell epitopes may give rise to unwanted T cell mediated side effects. Therefore it is particularly preferred to use peptides exhibiting no or substantially no T-cell reactivity in order to obtain the polypeptides of the present invention.
However, also allergen fragments comprising at least one T-cell epitope may be used in the polypeptide according to the present invention.
“Exhibiting reduced IgE-binding capacity”, as used herein, means that the molecules according to the present invention show significantly reduced IgE-binding capacity or activity (at least 50% less, preferably at least 70% less, more preferably at least 80% less, even more preferably at least 90% less, most preferably at least 95% less, binding capacity compared to the wild-type allergen) or even lack IgE-binding at all.
IgE-binding activity/capacity of molecules like peptides and proteins can be determined by, for example, an enzyme linked immunosorbent assay (ELISA) using, for example, sera obtained from a subject, (i.e., an allergic subject) that has been previously exposed to the wild-type allergen. Briefly, a peptide to be tested is coated onto wells of a microtiter plate. After washing and blocking the wells, an antibody solution consisting of the plasma of an allergic subject, who has been exposed to the peptide being tested or the protein from which it was derived, is incubated in the wells. A labelled secondary antibody is added to the wells and incubated. The amount of IgE-binding is then quantified and compared to the amount of IgE bound by a purified wild-type allergen.
Alternatively, the binding activity of a peptide can be determined by Western blot analysis. For example, a peptide to be tested is run on a polyacrylamide gel using SDS-PAGE. The peptide is then transferred to nitrocellulose and subsequently incubated with serum from an allergic subject. After incubation with the labelled secondary antibody, the amount of IgE bound is determined and quantified.
Another assay which can be used to determine IgE-binding activity of a peptide is a competition ELISA assay. Briefly, an IgE-antibody pool is generated by combining plasma from allergic subjects who have been shown by direct ELISA to be IgE-reactive with wild-type allergen. This pool is used in ELISA competition assays to compare IgE-binding to wild-type allergen to the peptide tested. IgE-binding for the wild-type allergen and the peptide being tested is determined and quantified.
A “T-cell epitope” means a protein, peptide or polypeptide (e.g., allergen) or fragment thereof, for which a T-cell has an antigen specific binding site, the result of binding to said binding site activates the T-cell. The term “exhibiting reduced T-cell reactivity”, as used herein, refers to molecules which exhibit a T-cell reactivity which is significantly reduced compared to the stimulation induced by the wild-type allergen from which the hypoallergenic molecule is derived using equimolar amounts in standard assays known in the art (reduced T-cell reactivity means at least 30%, preferably at least 50%, more preferably at least 70%, most preferably at least 90%, less stimulation of hypoallergenic molecules compared to the wildtype allergen at equimolar amounts). In a particular preferred embodiment of this invention, the molecules may “lack” T-cell epitopes and thus molecule shows reduced T-cell reactivity in the individual(s) to be treated (i.e., who is to receive an epitope-presenting valency platform molecule). It is likely that, for example, an allergen-derived molecule may lack a T-cell epitope(s) with respect to an individual, or a group of individuals, while possessing a T-cell epitope(s) with respect to other individual(s). Methods for detecting the presence of a T-cell epitope are known in the art and include assays which detect T-cell proliferation (such as thymidine incorporation). Immunogens that fail to induce statistically significant incorporation of thymidine above background (i.e., generally p less than 0.05 using standard statistically methods) are generally considered to lack T-cell epitopes, although it will be appreciated that the quantitative amount of thymidine incorporation may vary, depending on the immunogen being tested (see, e.g., Zhen L. et al. (Infect Immun. (2003) 71:3920-3926)). Generally, a stimulation index below about 2-3, more preferably less than about 1, indicates lack of T-cell reactivity and epitopes. The presence of T-cell epitopes can also be determined by measuring secretion of T-cell-derived lymphokines according to standard methods. The stimulation index (SI) may be calculated by dividing the proliferation rate (Thymidine uptake) of stimulated cells through the proliferation rate of unstimulated cells in medium alone. SI=1 means no stimulation, and SI>1 indicates stimulation of cells. Location and content of T-cell epitopes, if present, can be determined empirically.
The cytokine secretion may be determined in addition to the stimulation of T cells. For example, IFN-gamma and IL-10 as biomarkers for increased activity of regulatory T cells have been recognized as cytokines accompanying a successful allergy immunotherapy.
The peptide fragments of the present invention are preferably composed or consist of amino acids 151 to 177, 87 to 117, 1 to 30, 43 to 70 or 212 to 241 of Phl p 1, amino acids 1 to 33, 8 to 39, 34 to 65 or 66 to 96 of Phl p 2, amino acids 93 to 128, 98 to 128, 26 to 53, 26 to 58, 132 to 162, 217 to 246, 252 to 283 or 176 to 212 of Phl p 5, amino acids 23 to 54, 56 to 90, 73 to 114 or 95 to 127 of Phl p 6, amino acids 1 to 34 or 35 to 70 of chain 1 of Fel d 1, amino acids 1 to 34, 35 to 63 or 64 to 92 of chain 2 of Fel d 1, amino acids 30 to 59, 50 to 79, 75 to 104, 30 to 74 or 60 to 104 of Bet v 1, amino acids 1 to 30, 52 to 84 or 188 to 222 of Der p 1, amino acids 1 to 33, 21 to 51, 42 to 73, 62 to 103 or 98 to 129 of Der p 2, amino acids 1 to 30, 20 to 50, 50 to 80, 90 to 125, 125 to 155 or 165 to 198 of Der p 7, amino acids 1-35, 36-70, 71-110, 111-145, 140-170, 175-205, 210-250 or 250-284 of Der p 10, amino acids 1 to 35, 35 to 72, 70 to 100 or 90 to 122 of Der p 21, amino acids 1 to 32, 15 to 48 or 32 to 70, 32 to 60, 52 to 84, 32 to 70 (Cys→Ser) of Der p 23, amino acids 19 to 58, 59 to 95, 91 to 120 or 121 to 157 of Alt a 1, amino acids 31 to 60, 45 to 80, 60 to 96 or 97 to 133 of Par j 2, amino acids 1 to 40, 36 to 66, 63 to 99, 86 to 120 or 107 to 145 of Ole e 1, amino acids 25 to 58, 99 to 133, 154 to 183, 277 to 307, 334 to 363, 373 to 402, 544 to 573, 579 to 608, 58 to 99, 125 to 165, 183 to 224, 224 to 261, 252 to 289, 303 to 340, 416 to 457, 460 to 500 or 501 to 542 of Fel d 2, amino acids 19 to 58, 52 to 91, 82 to 119, 106 to 144 or 139 to 180 of Can f 2, amino acids 19 to 56, 51 to 90, 78 to 118, 106 to 145 or 135-174 of Can f 1, amino acids 27 to 70, 70 to 100 or 92 to 132 of Art v 1, amino acids 31 to 70, 80 to 120, 125 to 155, 160 to 200, 225 to 263, 264 to 300 305 to 350 or 356 to 396 of Amb a 1, amino acids 1 to 34, 35 to 74, 74 to 115, 125 to 165, 174 to 213, 241 to 280, 294 to 333, 361 to 400 or 401 to 438 of Alt a 6, amino acids 1 to 40, 41 to 80, 81 to 120, 121 to 160 of Alt a 2 or fragments or sequence variations thereof.
The specific amino acid sequences of the above identified allergen-derived molecules are (peptides in the following table having an N- and/or C-terminal cysteine residue (C) being used in the polypeptide of the present invention may lack said cysteine residue):













Peptide
Position
Sequence
SEQ ID No.


















Pep Alt a 1.1
19-58
APLESRQDTASCPVTTEGDYVWKISEFYGRKPEGTYYN
23




SL



 


Pep Alt a 1.2
59-95
GFNIKATNGGTLDFTCSAQADKLEDHKWYSCGENSFM
24


 


Pep Alt a 1.3
 91-120
ENSFMDFSFDSDRSGLLLKQKVSDDITYVA
25


 


Pep Alt a 1.4
121-157 
TATLPNYCRAGGNGPKDFVCQGVADAYITLVTLPKSS
26


 


Pep Alt a 2.1
 1-40
MHSSNNFFKDNIFRSLSKEDPDYSRNIEGQVIRLHWDW
27




AQ



 


Pep Alt a 2.2
41-80
LLMLSAKRMKVAFKLDIEKDQRVWDRCTADDLKGRN
28




GFKR



 


Pep Alt a 2.3
 81-120
CLQFTLYRPRDLLSLLNEAFFSAFRENRETIINTDLEYAA
29


 


Pep Alt a 2.4
121-160
KSISMARLEDLWKEYQKIFPSIQVITSAFRSIEPELTVYT
30


 


Pep Alt a 2.5
161-190
CLKKIEASFELIEENGDPKITSEIQLLKAS
31


 


Pep Alt a 6.1
 1-34
MTITKIHARSVYDSRGNPTVEVDIVTETGLHRAI
32


 


Pep Alt a 6.2
35-74
VTETGLHRAIVPSGASTGSHEACELRDGDKSKWGGKGV
33




TK



 


Pep Alt a 6.3
 74-115
APALIKEKLDVKDQSAVDAFLNKLDGTTNKTNLGANAI
34




LGVS



 


Pep Alt a 6.4
125-165
EKGVPLYAHISDLAGTKKPYVLPVPF
35




QNVLNGGSHAGGRLA



 


Pep Alt a 6.5
174-213
CEAPTFSEAMRQGAEVYQKLKALAKKTYGQSAGNVGD
36




EGG



 


Pep Alt a 6.6
241-280
IKIAMDVASSEFYKADEKKYDLDFKNPDSDKSKWLTYE
37




QL



 


Pep Alt a 6.7
294-333
VSIEDPFAEDDWEAWSYFFKTYDGQIVGDDLTVTNPEFI
38




K



 


Pep Alt a 6.8
361-400
AKDAFGAGWGVMVSHRSGETEDVTIADIVVGLRSGQIK
39




TG



 


Pep Alt a 6.9
401-438
APARSERLAKLNQILRIEEELGDNAVYAGNNFRTAVNL
40


 


Pep Amb a 1.1
31-70
EILPVNETRRLTTSGAYNIIDGCWRGKADWAENRKALA
41




DC



 


Pep Amb a 1.2
 80-120
GGKDGDIYTVTSELDDDVANPKEGTLRFGAAQNRPLWI
42




IFE



 


Pep Amb a 1.3
125-155
IRLDKEMVVNSDKTIDGRGAKVEIINAGFTL
43


 


Pep Amb a 1.4
160-200
NVIIHNINMHDVKVNPGGLIKSNDGPAAPRAGSDGDAIS
44




IS



 


Pep Amb a 1.5
225-263
GTTRLTVSNSLFTQHQFVLLFGAGDENIEDRGMLATVA
45




F



 


Pep Amb a 1.6
264-300
NTFTDNVDQRMPRCRHGFFQVVNNNYDKWGSYAIGGS
46


 


Pep Amb a 1.7
305-350
ILSQGNRFCAPDERSKKNVLGRHGEAAAESMKWNWRT
47




NKDVLENGA



 


Pep Amb a 1.8
356-396
GVDPVLTPEQSAGMIPAEPGESALSLTSSAGVLSCQPGA
48




PC



 


Pep Art v 1.1
27-70
SKLCEKTSKTYSGKCDNKKCDKKCIEWEKAQHGACHK
49




REAGKES



 


Pep Art v 1.2
 70-100
SCFCYFDCSKSPPGATPAPPGAAPPPAAGGS
50


 


Pep Art v 1.3
 92-132
APPPAAGGSPSPPADGGSPPPPADGGSPPVDGGSPPPPST
51




H



 


Can f 1 Pep 1
19-56
QDTPALGKDTVAVSGKWYLKAMTADQEVPEKPDSVTP
52




M



 


Can f 1 Pep 2
51-90
DSVTPMILKAQKGGNLEAKITMLTNGQCQNITVVLHKT
53




SE



 


Can f 1 Pep 3
 78-118
CQNITVVLHKTSEPGKYTAYEGQRVVFIQPSPVRDHYIL
54




YC



 


Can f 1 Pep 4
106-145
QPSPVRDHYILYCEGELHGRQIRMAKLLGRDPEQSQEA
55




LE



 


Can f 1 Pep 5
135-174
RDPEQSQEALEDFREFSRAKGLNQEILELAQSETCSPGG
56




Q



 


Can f 2 Pep 1
19-58
QEGNHEEPQGGLEELSGRWHSVALASNKSDLIKPWGHF
57




RV



 


Can f 2 Pep 2
52-91
PWGHFRVFIHSMSAKDGNLHGDILIPQDGQCEKVSLTAF
58




K



 


Can f 2 Pep 3
 82-119
CEKVSLTAFKTATSNKFDLEYWGHNDLYLAEVDPKSYL
59


 


Can f 2 Pep 4
106-144
NDLYLAEVDPKSYLILYMINQYNDDTSLVAHLMVRDLS
60




R



 


Can f 2 Pep 5
139-180
VRDLSRQQDFLPAFESVCEDIGLHKDQIVVLSDDDRCQ
61




GSRD



 


Fel d 2 Pep 1
25-58
EAHQSEIAHRFNDLGEEHFRGLVLVAFSQYLQQC
62


 


Fel d 2 Pep 2
 99-133
CTVASLRDKYGEMADCCEKKEPERNECFLQHKDDN
63


 


Fel d 2 Pep 3
154-183
NEQRFLGKYLYEIARRHPYFYAPELLYYAE
64


 


Fel d 2 Pep 4
277-307
CADDRADLAKYICENQDSISTKLKECCGKPV
65


 


Fel d 2 Pep 5
334-363
VEDKEVCKNYQEAKDVFLGTFLYEYSRRHP
66


 


Fel d 2 Pep 6
373-402
LAKEYEATLEKCCATDDPPACYAHVFDEFK
67


 


Fel d 2 Pep 7
544-573
EKQIKKQSALVELLKHKPKATEEQLKTVMG
68


 


Fel d 2 Pep 8
579-608
VDKCCAAEDKEACFAEEGPKLVAAAQAALA
69


 


Fel d 2 Pep 9
58-99
CPFEDHVKLVNEVTEFAKGCVADQSAANCEKSLHELLG
70




DKLC



 


Fel d 2 Pep 10
125-165
CFLQHKDDNPGFGQLVTPEADAMCTAFHENEQRFLGK
71




YLYE



 


Fel d 2 Pep 11
183-224
EEYKGVFTECCEAADKAACLTPKVDALREKVLASSAKE
72




RLKC



 


Fel d 2 Pep 12
224-261
CASLQKFGERAFKAWSVARLSQKFPKAEFAEISKLVTD
73


 


Fel d 2 Pep 13
252-289
FAEISKLVTDLAKIHKECCHGDLLECADDRADLAKYIC
74


 


Fel d 2 Pep 14
303-340
CGKPVLEKSHCISEVERDELPADLPPLAVDFVEDKEVC
75


 


Fel d 2 Pep 15
416-457
CELFEKLGEYGFQNALLVRYTKKVPQVSTPTLVEVSRSL
76




GKV



 


Fel d 2 Pep 16
460-500 
CTHPEAERLSCAEDYLSVVLNRLCVLHEKTPVSERVTK
77




C



 


Fel d 2 Pep 17
501-542 
CTESLVNRRPCFSALQVDETYVPKEFSAETFTFHADLCT
78




LPE



 


Pep Ole e 1.1
 1-40
EDIPQPPVSQFHIQGQVYCDTCRAGFITELSEFIPGASLR
79


 


Pep Ole e 1.2
36-66
GASLRLQCKDKENGDVTFTEVGYTRAEGLYS
80


 


Pep Ole e 1.3
63-99
GLYSMLVERDHKNEFCEITLISSGRKDCNEIPTEGWA
81


 


Pep Ole e 1.4
 86-120 
GRKDCNEIPTEGWAKPSLKFKLNTVNGTTRTVNPL
82


 


Pep Ole e 1.5
107-145 
LNTVNGTTRTVNPLGFFKKEALPKCAQVYNKLGMYPP
83




NM



 


Pep Par j 2.1
31-60
GEEACGKVVQDIMPCLHFVKGEEKEPSKEC
84


 


Pep Par j 2.2
45-80
CLHFVKGEEKEPSKECCSGTKKLSEEVKTTEQKREA
85


 


Pep Par j 2.3
60-96
CCSGTKKLSEEVKTTEQKREACKCIVRATKGISGIKN
86


 


Pep Par j 2.4
 97-133
ELVAEVPKKCDIKTTLPPITADFDCSKIQSTIFRGYY
87


 


Der p 1 Pep 1
 1-30
TNACSINGNAPAEIDLRQMRTVTPIRMQGG
88


 


Der p 1 Pep 2
52-84
NQSLDLAEQELVDCASQHGCHGDTIPRGIEYIQ
89


 


Der p 1 Pep 3
 85-115
HNGVVQESYYRYVAREQSCRRPNAQRFGISN
90


 


Der p 1 Pep 4
 99-135
REQSCRRPNAQRFGISNYCQIYPPNVNKIREALAQTH
91


 


Der p 1 Pep 5
145-175
KDLDAFRHYDGRTIIQRDNGYQPNYHAVNIV
92


 


Der p 1 Pep 6
155-187
GRTIIQRDNGYQPNYHAVNIVGYSNAQGVDYWI
93


 


Der p 1 Pep 7
175-208
VGYSNAQGVDYWIVRNSWDTNWGDNGYGYFAANI
94


 


Der p 1 Pep 8
188-222
VRNSWDTNWGDNGYGYFAANIDLMMIEEYPYVVIL
95


 


Der p 1 Pep 1.2
 1-41
TNACSINGNAPAEIDLRQMRTVTPIRMQGGCGSCWAFS
143




GVA



 


Der p 1 Pep 2.2
42-82
ATESAYLAYRNQSLDLAEQELVDCASQHGCHGDTIPRG
144




IEYIQ



 


Der p 1 Pep 9
27-57
MQGGCGSCWAFSGVAATESAYLAYRNQSLD
145


 


Der p 2 Pep 1
 1-33
DQVDVKDCANHEIKKVLVPGCHGSEPCIIHRGK
96


 


Der p 2 Pep 2
21-51
CHGSEPCIIHRGKPFQLEAVFEANQNSKTAK
97


 


Der p 2 Pep 3
42-73
EANQNSKTAKIEIKASIEGLEVDVPGIDPNAC
98


 


Der p 2 Pep 4
 62-103
EVDVPGIDPNACHYMKCPLVKGQQYDIKYTWIVPKIAP
99




KSEN



 


Der p 2 Pep 5
 98-129
APKSENVVVTVKVMGDNGVLACAIATHAKIRD
100


 


Der p 5 Pep 1
 1-35
MEDKKHDYQNEFDFLLMERIHEQIKKGELALFYLQ
101


 


Der p 5 Pep 2
25-60
KKGELALFYLQEQINHFEEKPTKEMKDKIVAEMDTI
102


 


Der p 5 Pep 3
65-95
DGVRGVLDRLMQRKDLDIFEQYNLEMAKKSG
103


 


Der p 5 Pep 4
 78-114
DLDIFEQYNLEMAKKSGDILERDLKKEEARVKKIEV
104


 


Der p 7 Pep 1
 1-30
DPIHYDKITEEINKAVDEAVAAIEKSETFD
105


 


Der p 7 Pep 2
20-50
VAAIEKSETFDPMKVPDHSDKFERHIGIIDL
106


 


Der p 7 Pep 3
50-80
LKGELDMRNIQVRGLKQMKRVGDANVKSEDG
107


 


Der p 7 Pep 4
 90-125
VHDDVVSMEYDLAYKLGDLHPNTHVISDIQDFVVEL
108


 


Der p 7 Pep 5
125-155
LSLEVSEEGNMTLTSFEVRQFANVVNHIGGL
109


 


Der p 7 Pep 6
165-198
LSDVLTAIFQDTVRAEMTKVLAPAFKKELERNNQ
110


 


Der p 10 Pep 1
 1-35
MEAIKKKMQAMKLEKDNAIDRAEIAEQKARDANLR
111


 


Der p 10 Pep 2
36-70
AEKSEEEVRALQKKIQQIENELDQVQEQLSAANTK
112


 


Der p 10 Pep 3
 71-110
LEEKEKALQTAEGDVAALNRRIQLIEEDLERSEERLKIA
113




T



 


Der p 10 Pep 4
111-145
AKLEEASQSADESERMRKMLEHRSITDEERMEGLE
114


 


Der p 10 Pep 5
140-170
RMEGLENQLKEARMMAEDADRKYDEVARKLA
115


 


Der p 10 Pep 6
175-205
DLERAEERAETGESKIVELEEELRVVGNNLK
116


 


Der p 10 Pep 7
210-250
SEEKAQQREEAHEQQIRIMTTKLKEAEARAEFAERSVQ
117




KLQ



 


Der p 10 Pep 8
250-284
QKEVDRLEDELVHEKEKYKSISDELDQTFAELTGY
118


 


Der p 21 Pep 1
 1-35
MFIVGDKKEDEWRMAFDRLMMEELETKIDQVEKGL
119


 


Der p 21 Pep 2
35-72
LHLSEQYKELEKTKSKELKEQILRELTIGENFMKGAL
120


 


Der p 21 Pep 3
 70-100
GALKFFEMEAKRTDLNMFERYNYEFALESIK
121


 


Der p 21 Pep 4
 90-122
YNYEFALESIKLLIKKLDELAKKVKAVNPDEYY
122


 


Der p 23 Pep 1
 1-32
MANDNDDDPTTTVHPTTTEQPDDKFECPSRFG
123


 


Der p 23 Pep 2
15-48
PTTTEQPDDKFECPSRFGYFADPKDPHKFYICSN
124


 


Der p 23 Pep 3
32-70
GYFADPKDPHKFYICSNWEAVHKDCPGNTRWNEDEE
125




TCT



 


Der p 23 Pep 4
32-60
GYFADPKDPHKFYICSNWEAVHKDCPGNT
146


 


Der p 23 Pep 5
42-70
KFYICSNWEAVHKDCPGNTRWNEDEETCT
147


 


Der p 23 Pep 6
 32-70*
GYFADPKDPHKFYISSNWEAVHKDSPGNTRWNEDEETS
148



(Cys
T




->Ser)




 


Bet v 1 Pep 1
30-59
LFPKVAPQAISSVENIEGNGGPGTIKKISF
126


 


Bet v 1 Pep 2
50-79
GPGTIKKISFPEGFPFKYVKDRVDEVDHTN
127


 


Bet v 1 Pep 3
 75-104
VDHTNFKYNYSVIEGGPIGDTLEKISNEIK
128


 


Bet v 1 Pep A
30-74
LFPKVAPQAISSVENIEGNGGPGTIKKISFPEGFPFKYVK
143




DRVDE



 


Bet v 1 Pep B
 60-104
PEGFPFKYVKDRVDEVDHTNFKYNYSVIEGGPIGDTLEK
144




ISNEIKI



 


Fel d 1 chain 1
 1-34
EICPAVKRDVDLFLTGTPDEYVEQVAQYKALPVVC
129


Pep 1





 


Fel d 1 chain 1
35-70
LENARILKNCVDAKMTEEDKENALSLLDKIYTSPLC
130


Pep 2





 


Fel d 1 chain 2
 1-34
VKMAITCPIFYDVFFAVANGNELLLDLSLTKVNAC
131


Pep 1





 


Fel d 1 chain 2
35-63
TEPERTAMKKIQDCYVENGLISRVLDGLVC
132


Pep 2





 


Fel d 1 chain 2
64-92
CMTTISSSKDCMGEAVQNTVEDLKLNTLGR
133


Pep 3





 


Ph1 p 5 Pep 1
 98-128
CGAASNKAFAEGLSGEPKGAAESSSKAALTSK
134


 


Ph1 p 5 Pep 2
26-58
ADLGYGPATPAAPAAGYTPATPAAPAEAAPAGKC
135


 


Ph1 p 5 Pep 3
132-162
AYKLAYKTAEGATPEAKYDAYVATLSEALRIC
136


 


Ph1 p 5 Pep 4
217-246
CEAAFNDAIKASTGGAYESYKFIPALEAAVK
137


 


Phl p 5 Pep 5
252-283
TVATAPEVKYTVFETALKKAITAMSEAQKAAKC
138


 


Ph1 p 5 Pep 6
176-212
CAEEVKVIPAGELQVIEKVDAAFKVAATAANAAPAND
139




K



 


Ph1 p 5 Pep 1a
 93-128
CFVATFGAASNKAFAEGLSGEPKGAAESSSKAALTSK
141


 


Ph1 p 5 Pep 2b
26-53
ADLGYGPATPAAPAAGYTPATPAAPAEAC
142


 


Ph1 p 5 Pep 7
59-91
ATTEEQKLIEKINAGFKAALAAAAGVQPADKYR
22


 


Ph1 p 1 Pep 1
151-171
HVEKGSNPNYLALLVKYVNGDGDVVAVC
1


 


Ph1 p 1 Pep 2
 87-117
EPVVVHITDDNEEPIAPYHFDLSGHAFGAMAC
2


 


Ph1 p 1 Pep 3
 1-30
IPKVPPGPNITATYGDKWLDAKSTWYGKPTGC
3


 


Ph1 p 1 Pep 4
43-70
GYKDVDKPPFSGMTGCGNTPIFKSGRGC
4


 


Ph1 p 1 Pep 5
212-241
CVRYTTEGGTKTEAEDVIPEGWKADTSYESK
5


 


Ph1 p 2 Pep 1
 1-33
VPKVTFTVEKGSNEKHLAVLVKYEGDTMAEVELC
6


 


Ph1 p 2 Pep 2
28-39
CVEKGSNEKHLAVLVKYEGDTMAEVELREHGSD
7


 


Ph1 p 2 Pep 3
34-65
REHGSDEWVAMTKGEGGVWTFDSEEPLQGPFNC
8


 


Ph1 p 2 Pep 4
66-96
CFRFLTEKGMKNVFDDVVPEKYTIGATYAPEE
9


 


Ph1 p 6 Pep 1
23-54
GKATTEEQKLIEDVNASFRAAMATTANVPPAD
10


 


Ph1 p 6 Pep 2
56-90
YKTFEAAFTVSSKRNLADAVSKAPQLVPKLDEVYN
11


 


Ph1 p 6 Pep 3
 95-127
AADHAAPEDKYEAFVLHFSEALRIIAGTPEVHA
12


 


Ph1 p 6 Pep 4
 73-114
DAVSKAPQLVPKLDEVYNAAYNAADHAAPEDKY
13





*) Cysteins exchanged with serins (marked in bold)






The terms “fragments thereof” and “sequence variations thereof” refer to peptides which are deduced from the allergen-derived molecules disclosed herein and show biochemical properties (e.g. the capacity to prevent IgE binding to the allergen from which those molecules are derived from) which are comparable or identical to said allergen-derived molecules. The fragments of the present invention comprise at least 5, preferably at least 7, more preferably at least 10, successive and/or a maximum of 95%, preferably a maximum of 90%, more preferably a maximum of 80% amino acid residues of the allergen-derived molecule. The term “sequence variation” includes modifications of the peptides such as fragmentation (see above), amino acid substitutions (in particular cysteine or methionine residues may be exchanged with serine, alanine or other natural or non-natural amino acids or amino acid derivatives), deletions or additions. “Sequence variation” refers also to said allergen-derived molecules of the above table, wherein at least 1, preferably at least 2, more preferably at least 3, even more preferably at least 4 (5, 6, 7, 8, 9, 10, 15, 20) amino acid residues are added to the C- and/or N-terminus.
It is noted that the allergen referred to herein as “clone 30 allergen” is an allergen derived from the house dust mite Dermatophagoides pteronyssinus and consists of the following sequence: MANDNDDDPTTTVHPTTTEQPDDKFECPSRFGYFADPKDPHKFYICSNWEAVHKDCP GNTRWNEDEETCT (SEQ ID No. 140; see also WO 2007/124524). In the meantime, the allergen name Der p 23 has been assigned to clone 30 allergen. This means that Der p 23 and clone 30 allergen are synonyms.
According to the present invention also peptides are encompassed which are at least 80% identical, preferably 90% identical, to the amino sequences disclosed above.
According to a preferred embodiment of the present invention the surface polypeptide of the virus of the hepadnaviridae family or at least one fragment thereof comprises at least two B-cell binding peptide fragments derived from at least one wild-type allergen fused to its N-terminus and at least two B-cell binding peptide fragments derived from at least one wild-type allergen fused to its C-terminus.
In a particularly preferred embodiment of the present invention at least two of said at least three B-cell binding peptides are identical.
The polypeptide of the present invention can be used as vaccine in the treatment or prevention of an allergy in a human or animal.
The polypeptide is preferably administered to an individual in the amount of 0.01 microgram per kg body weight to 5 mg/kg body weight, pref-erably 0.1 microgram per kg body weight to 10 microgram per kg body weight.
According to a particularly preferred embodiment of the present invention the polypeptides of the present invention are administered to an individual in an amount of at least 10 μg, preferably at least 20 μg, per polypeptide. The maximum amount of polypeptides to be administered can be varied but is preferably below 100 μg, more preferably below 50 μg, even more preferably 40 μg or less, per polypeptide.
The amount of polypeptides that may be combined with excipients to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. The dose of the vaccine may vary according to factors such as the disease state, age, sex and weight of the individual, and the ability of antibody to elicit a desired response in the individual. Dosage regime may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. The dose of the vaccine may also be varied to provide optimum preventative dose response depending upon the circumstances. For instance, the polypeptides and vaccine of the present invention may be administered to an individual at intervals of several days, one or two weeks or even months depending always on the level of allergen-specific IgG induction.
In a preferred embodiment of the present invention the polypeptide/vaccine is applied between 2 and 10, preferably between 2 and 7, even more preferably up to 5 and most preferably up to 3 times. In a particularly preferred embodiment the time interval between the subsequent vaccinations is chosen to be between 2 weeks and 5 years, preferably between 1 month and up to 3 years, more preferably between 2 months and 1.5 years. The repeated administration of the peptide/vaccine of the present invention may maximize the final effect of a therapeutic vaccination.
According to a particularly preferred embodiment of the present invention three or more B-cell binding peptides selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 5, SEQ ID No. 9, SEQ ID No. 137, SEQ ID No. 139, SEQ ID No. 142 and SEQ ID No. 10 are bound N- and C-terminally to a surface polypeptide of the virus of the hepadnaviridae family, preferably the hepatitis PreS polypeptide or fragments thereof.
The polypeptides of the present invention comprising the at least three B-cell binding peptides derived from at least one wild-type allergen are preferably selected from the group consisting of SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18 and SEQ ID No. 19.
Another aspect of the present invention relates to a nucleic acid molecule encoding a polypeptide according to the present invention.
Another aspect of the present invention relates to a vector comprising a nucleic acid molecule according to the present invention.
Said vector is preferably an expression vector.
The vector harbouring the nucleic acid molecule of the present invention may be used for cloning purposes or for the production of expression vectors. Said vector can be a plasmid, cosmid, virus, bacteriophage or any other vector commonly used in genetic engineering, and can include, in addition to the nucleic acid molecule of the invention, eukaryotic or prokaryotic elements for the control of the expression, such as regulatory sequences for the initiation and the termination of the transcription and/or translation, enhancers, promoters, signal sequences and the like.
According to a preferred embodiment of the present invention the vector is a bacterial, fungal, insect, viral or mammalian vector.
The vector of the present invention may preferably be employed for cloning and expression purposes in various hosts like bacteria, yeasts, filamentous fungi, mammalian cells, insect cells, plant cells or any other prokaryotic or eukaryotic cells. Therefore, said vector comprises besides a nucleic acid encoding for a hypoallergenic molecule or fusion protein according to the present invention host specific regulatory sequences.
Another aspect of the present invention relates to a host comprising a nucleic acid molecule or a vector according to the present invention.
The nucleic acid molecule and the vector according to the present invention may be introduced into a suitable host. Said molecule may be incorporated into the genome of the host. The vector may exist extrachromosomally in the cytoplasm or incorporated into the chromosome of the host.
Yet another aspect of the present invention relates to an antibody directed against a hypoallergenic molecule, hypoallergenic fusion protein or a fusion protein according to the present invention.
Another aspect of the present invention relates to a vaccine formulation comprising at least one, preferably at least two, more preferably at least three, even more preferably at least 4, polypeptide according to the present invention.
In a particularly preferred embodiment of the present invention the vaccine comprises at least one, preferably at least two, preferably at least three, preferably at least four, preferably at least 5, polypeptides having an amino acid sequence selected from the group consisting of SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 149, SEQ ID No. 150, SEQ ID No. 151 and SEQ ID No. 152.
Depending on the composition such a vaccine can be used in the treatment and/or prevention of grass pollen allergies, birch pollen allergies, house dust mite allergies or a combination of those allergies in individuals suffering from such allergies or being at risk to suffer therefrom.
The term “preventing”, as used herein, covers measures not only to prevent the occurrence of disease, such as risk factor reduction, but also to arrest its progress and reduce its consequences once established. “Preventing” means also to prevent sensitization of an individual being at risk to get an allergy.
As used herein, the term “treatment” or grammatical equivalents encompasses the improvement and/or reversal of the symptoms of disease (e.g., allergy). A compound which causes an improvement in any parameter associated with disease when used in the screening methods of the instant invention may thereby be identified as a therapeutic compound. The term “treatment” refers to both therapeutic treatment and prophylactic or preventative measures.
According to one of the most preferred embodiment of the present invention the vaccine comprises polypeptides having amino acid sequence SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16 and SEQ ID No. 17.
According to another preferred embodiment of the present invention the vaccine comprises polypeptides having amino acid sequence SEQ ID No. 18 and/or SEQ ID No. 19.
According to a particularly preferred embodiment of the present invention the vaccine comprises polypeptides of the present invention which comprise allergen fragments derived from house dust mite allergens. Particularly preferred are amino acid residues 1 to 33, 21 to 51, 42 to 73, 62 to 103 or 98 to 129 of Der p 2, amino acid residues 1 to 30, 20 to 50, 50 to 80, 90 to 125, 125 to 155 or 165 to 198 of Der p 7, amino acid residues 1 to 35, 35 to 72, 70 to 100 or 90 to 122 of Der p 21, amino acids 1 to 32, 15 to 48 or 32 to 70, 32 to 60, 52 to 84, 32 to 70 (Cys→Ser) of Der p 23, amino acid residues 1 to 30, 1 to 41, 27 to 57, 42 to 82, 52 to 84, 85 to 115, 99 to 135, 145 to 175, 155 to 187, 175 to 208 or 188 to 222 of Der p 1. Most preferably, the vaccine comprises at least one of the polypeptides SEQ ID No. 149 to 152 (shown in FIGS. 18 A-18 D).
In a particularly preferred embodiment the polypeptide/vaccine of the present invention is administered 4 times per treatment year over a total treatment period of 1 to 5 years, preferably over 2 to 3 years. Of said 4 yearly administrations 3 are applied within a period of 6 to 12, preferably 8, weeks having intervals of 3 to 6 weeks, preferably 4 weeks, between administration 1 and 2 and another 3 to 6 weeks, preferably 4 weeks, between administration 2 and 3. The fourth administration is applied 3 to 7 months after the third administration. If the total treatment period exceeds 1 year, the same dosing regimen is applied in the following treatment years.
For the treatment of seasonal allergies (e.g. pollen allergies such as grass pollen allergy or birch pollen allergy) administration 1, 2, and 3 are preferably scheduled before the respective season with allergen exposure (pollen season), and the fourth administration is scheduled after the season.
The vaccine formulation according to the present invention may be formulated as known in the art and necessarily adapted to the way of administration of said vaccine formulation.
Preferred ways of administration of the vaccine formulation (of the present invention) include all standard administration regimes described and suggested for vaccination in general and allergy immunotherapy specifically (orally, transdermally, intraveneously, intranasally, via mucosa, rectally, etc). However, it is particularly preferred to administer the molecules and proteins according to the present invention subcutaneously or intramusculary.
The vaccine formulation according to the present invention may only comprise a viral capsid protein or fragments thereof of a member of the genus of hepadnaviridae.
Said formulation preferably further comprises at least one adjuvant, pharmaceutical acceptable excipient and/or preservative.
In order to increase the immunogenicity of the hypoallergenic molecules according to the present invention, adjuvants, for instance, may be used in a medicament according to the present invention. An adjuvant according to the present invention is an auxiliary agent which, when administered together or in parallel with an antigen, increases its immunogenicity and/or influences the quality of the immune response. Hence, the adjuvant can, e.g., considerably influence the extent of the humoral or cellular immune response. Customary adjuvants are, e.g., aluminum compounds, lipid-containing compounds or inactivated mycobacteria.
Generally, adjuvants can be of different forms, provided that they are suitable for administration to human beings. Further examples of such adjuvants are oil emulsions of mineral or vegetal origin, mineral compounds such as aluminium phosphate or hydroxide, or calcium phosphate, bacterial products and derivatives, such as P40 (derived from the cell wall of Corynebacterium granulosum), monophosphoryl lipid A (MPL, derivative of LPS) and muramyl peptide derivatives and conjugates thereof (derivatives from mycobacterium components), alum, incomplete Freund's adjuvant, liposyn, saponin, squalene, etc. (see, e.g., Gupta R. K. et al. (Vaccine 11:293-306 (1993)) and Johnson A. G. (Clin. Microbiol. Rev. 7:277-289). The medicament of the present invention comprises most preferably alum as adjuvant.
Another preferred embodiment of the present invention is a combination of more than one fusion protein containing hypoallergenic peptides and the hepatitis B pre S protein. These combinations may be derived from peptides from a single allergen or from different allergens of the same allergen source or from several different allergen source.
A preferred embodiment of the present invention relates to a mixture of four fusion proteins containing hypoallergenic peptides from Phl p 1, Phl p 2, Phl p 5, and Phl p 6 and the hepatitis B virus preS protein.
Another preferred embodiment of the present invention relates to a fusion protein or a mixture of 2 fusion proteins containing hypoallergenic peptides from Bet v 1 and the hepatitis B virus PreS protein.
Yet another preferred embodiment of the present invention relates to a mixture of at least 2 fusion proteins containing hypoallergenic peptides from house dust mite allergens, most preferably selected from Der p 1, Der p 2, Der p 5, Der p 7, Der p 21 and Der p 23 and the hepatitis B virus PreS protein. Most preferably, the mixture contains 3 fusion proteins containing hypoallergenic peptides derived from Der p 1, Der p 2, and Der p 23. It is particularly preferred that the mixture comprises at least one, preferably at least two, more preferably at least three, of the polypeptides shown in SEQ ID No. 149 to 152 (see also FIGS. 18 A-18 D).
Generally, specific vaccine formulations according to the present invention can be prepared for the treatment or prevention of different allergies by combination of hypoallergenic polypeptides of the invention representing the clinically relevant allergens of an allergen source. Methods to determine the clinically relevant allergens of an allergen source are known in the art and have been described before (Valenta and Niederberger, 2007, J Allergy Clin Immunol, 119 (4): 826-830). In a preferred embodiment, the hypoallergenic polypeptides of said specific vaccine formulation are adsorbed to an adjuvant which can be used in human (e.g. aluminium hydroxide), and the mixture is administered 3-4 times per year for 1-3 years applying more than 10 μg of each polypeptide present in the vaccine formulation per dose.
According to another preferred embodiment of the present invention said formulations comprise 10 ng to 1 g, preferably 100 ng to 10 mg, especially 0.5 μg to 200 μg of said hypoallergenic molecule or antibody.
Another aspect of the present invention relates to the use of a hypoallergenic protein or an antibody according to the present invention for manufacturing a medicament for the treatment or prevention of a viral infection and/or an allergy in a human or animal.
Said medicament preferably further comprises at least one adjuvant, pharmaceutical acceptable excipient and/or preservative.
The medicament according to the present invention may be used for active (administration of the hypoallergenic protein and/or molecules of the invention) as well as for passive immunization (antibodies directed to the hypoallergenic protein and/or molecules of the invention).
According to a preferred embodiment of the present invention said medicament comprises 10 ng to 1 g, preferably 100 ng to 10 mg, especially 0.5 μg to 200 μg of said hypoallergenic molecule, nucleic acid molecule, vector, host or antibody.
The medicament is preferably administered to an individual in amount of 0.01 μg/kg body weight to 5 mg/kg body weight, preferably 0.1 μg/kg body weight to 10 μg/kg body weight.
In a particularly preferred embodiment, the medicament is administered in a dose containing an absolute amount of 5-200 μg, more preferably 10-80 μg, most preferably 20-40 μg of each included hypoallergenic polypeptide
The particular dosage regimen, i.e., dose, timing and repetition, will depend on the particular individual and that individual's medical history. Empirical considerations, such as the half life, will generally contribute to determination of the dosage. Frequency of administration may be determined and adjusted over the course of therapy.
Most preferably, the dosing regimen for the medicament will consist of 4 yearly subcutaneous injections of one and the same dose over a total treatment period of 2 to 3 years. Of said 4 yearly subcutaneous injections 3 are applied within a period of 6 to 12, preferably 8, weeks having intervals of 3 to 6 weeks, preferably 4 weeks, between injection 1 and 2 and another 3 to 6 weeks, preferably 4 weeks, between injection 2 and 3. The fourth injection is applied 4 to 6 months after the third administration. The same dosing regimen is applied in the following treatment years.
For the treatment of seasonal allergies (e.g. pollen allergies such as grass pollen allergy or birch pollen allergy) administration 1, 2, and 3 are preferably scheduled before the respective season with allergen exposure (pollen season), and the fourth administration is scheduled after the season.
The individual to whom the medicament according to the present invention is administered is preferably an individual or animal which is having or is at risk of having an allergy.
Subjects having or at risk of having an allergy, allergic condition, allergic disorder or allergic disease include subjects with an existing allergic condition or a known or a suspected predisposition towards developing a symptom associated with or caused by an allergic condition. Thus, the subject can have an active chronic allergic condition, disorder or disease, an acute allergic episode, or a latent allergic condition, disorder or disease. Certain allergic conditions are associated with seasonal or geographical environmental factors. Thus, at risk subjects include those at risk from suffering from a condition based upon a prior personal or family history, and the season or physical location, but which the condition or a symptom associated with the condition may not presently manifest itself in the subject.
The administration of the medicament according to the present invention, which comprises at least one hypoallergenic molecule as described herein, to an individual may prevent sensitization of said individual or may induce an appropriate immune response to allergens. If the medicament of the present invention is used to prevent sensitization, it should be administered to an individual prior to the first contact with said allergen. Therefore, it is preferred to administer the medicament according to the present invention to neonates and children. It turned out that also the administration of the medicament according to the present invention to pregnant individuals will induce the formation of antibodies directed against allergens in the unborn child. It is especially beneficiary to use hypoallergenic molecules according to the present invention for such therapies, because due to the lack of allergen-specific T-cell epitopes side effects occurring in the course of allergen immunotherapy can significantly be reduced or even be completely avoided.
Another aspect of the present invention relates to the use of a viral capsid protein from a virus of the family of hepadnaviridae as a carrier in medicaments or vaccines.
One of the advantages of such a carrier is that not only the antigen fused or conjugated thereon may be exposed to the immune system, but also an immune response against the capsid protein of a hepadnavirus is induced. Consequently, such a vaccination may lead to the prevention and/or treatment of diseases caused by hepadnaviruses. The virus is preferably of the species of human hepatitis B virus.
Another aspect of the present invention relates to a hypoallergenic molecule derived from Phl p 5 (Genbank Nr. X7435) having a C- and/or N-terminal truncation and lacking substantially IgE-binding capacity.
Grass pollen is one of most potent outdoor seasonal sources of airborne allergens responsible for hay fever and allergic asthma.
More than 40% of allergic individuals display IgE-reactivity with grass pollen allergens, which are divided into more than 11 groups. More than 80% of the grass pollen allergic patients react with group 5 allergens.
Group 5 allergens are non-glycosylated, highly homologous proteins with a molecular mass range from 25-33 kD. Several group 5 allergens have been cloned and/or immunologically characterized.
The trial to reduce the allergenic activity by introducing point mutations, mutations of several amino acids in row or deletions showed no effect (Schramm G, et al. J Immunol 1999; 162: 2406-1435). IgE-binding regions of Phl p 5 (Flicker S, et al. J Immunol 2000; 165: 3849-3859) have already been described and the three-dimensional structure has been solved (Maglio O, et al. 2002. Protein Eng. 15:635-642).
It turned out that in particular the Phl p 5 peptides according to the present invention, which are C- and/or N-terminally truncated and lack IgE-binding capacity, may be employed for the active vaccination of individuals.
According to a preferred embodiment of the present invention the truncated molecule substantially lacks T-cell epitopes and, thus lacks Phl p 5-specific T-cell reactivity.
As already outlined above, late side effects of allergen immunotherapy can be significantly reduced or even be avoided if the hypoallergenic molecules substantially lack allergen-specific T-cell epitopes.
Truncated Phl p 5 molecules lacking T-cell epitopes are composed of amino acids 93 to 128, 98 to 128, 26 to 53, 26 to 58 or 252 to 283 of Phl p 5 or fragments or sequence variations thereof.
In particular these truncated molecules substantially show low or no allergen-specific T-cell reactivity and are, nevertheless, able to provoke an appropriate immune response directed against the wild-type allergen.
According to another preferred embodiment of the present invention the hypoallergenic truncated Phl p 5 is composed of amino acids 132 to 162, 217 to 246 or 176 to 212 of Phl p 5 or sequence variations thereof.
These hypoallergenic molecules comprise one or more T-cell epitopes but lack IgE-binding capacity.
Another preferred embodiment of the present invention are truncated Phl p 1 molecules lacking T-cell epitopes, which are composed of amino acids 1 to 30, 43 to 70, 87 to 117, 151 to 171 or 214 to 241 of Phl p 1 or sequence variations thereof fused to a viral carrier protein, preferable the Hep B pre S protein.
Another preferred embodiment of the present invention are truncated Phl p 2 molecules lacking T-cell epitopes, which are composed of amino acids 1 to 33, 8 to 39, 34 to 65 or 66 to 96 of Phl p 2 or sequence variations thereof fused to a viral carrier protein, preferrably the Hep B pre S protein.
Another preferred embodiment of the present invention are truncated Phl p 6 molecules lacking T-cell epitopes, which are composed of amino acids 23 to 54, 56 to 90, 73 to 114 or 95 to 127 of Phl p 6 or sequence variants thereof fused to a viral carrier protein, preferrably the Hep B pre S protein.
Another preferred embodiment of the present invention refers to truncated Bet v 1 molecules lacking T-cell epitopes, which are composed of amino acids 30 to 59, 50 to 79, 75 to 104, 30 to 74 or 60 to 104 of Bet v 1.
Another preferred embodiment of the present invention are combinations or mixtures of truncated Phleum pratense molecules lacking T-cell epitopes, fused to a viral carrier protein, preferrably the Hep B pre S protein, as described above.
A preferred embodiment of the present invention are combinations or mixtures of truncated Phleum pratense molecules lacking T-cell epitopes, which are composed of one each such fusion protein from truncated Phl p 1, Phl p 2, Phl p 5, and Phl p 6, as described above.
Another aspect of the present invention relates to a hypoallergenic molecule derived from Fel d 1 (Genbank Nr. X62477 and X62478) having a C- and/or N-terminal truncation and lacking IgE-binding capacity.
Allergies to animals affect up to 40% of allergic patients. In the domestic environment, allergies to the most popular pets, cats and dogs, are particularly prevalent and connected with perennial symptoms. Animal allergens are present in dander, epithelium, saliva, serum or urine. Exposure to the allergens can occur either by direct skin contact or by inhalation of particles carrying the allergens. The major cat and dog allergens were shown to be present widespread and could even be detected in non-pet owning households and in public places, e.g., schools. This can be attributed to the high and increasing number of households keeping pets in industrialized countries (about 50%) and the high stability of the allergens, which are carried off and distributed.
Fel d 1 was identified as the major cat allergen, which is recognized by more than 90% of cat allergic patients. Fel d 1 represents a 38 kDa acidic glycoprotein of unknown biological function. It consists of two identical non-covalently linked heterodimers, which, again, are composed of two polypeptide chains antiparallely linked by three disulfide bonds. Chain 1 and chain 2 are encoded on different genes, each consisting of 3 exons. Recombinant Fel d 1 (rFel d 1), expressed as a chain 2- to chain 1 fusion protein, has been generated in E. coli. This recombinant Fel d 1 is able to completely mimic the immunological properties of the wild-type allergen.
Peptides derived from the major cat allergen Fel d 1, and lacking IgE-binding capacity are suitable, e.g., for immunotherapy and prophylactic allergy vaccination. The Fel d 1-derived synthetic peptides—like the Phl p 5 and allergen-derived peptides disclosed herein—are capable of inducing an IgG response, i.e., the production of so called “blocking antibodies” or “protective antibodies”. These antibodies prevent IgE-binding to the allergen Fel d 1. A significant reduction in allergic symptoms may thus be achieved.
According to a preferred embodiment of the present invention the truncated molecule exhibits reduced T-cell reactivity.
In order to avoid or to significantly reduce late side effects the Fel d 1 derived hypoallergenic molecule exhibits reduced T-cell reactivity as defined in the present invention.
The truncated Fel d 1 is preferably composed of amino acids 1 to 34 or 35 to 70 of chain 1 of Fel d 1, amino acids 1 to 34, 35 to 63 or 64 to 92 of chain 2 of Fel d 1 or sequence variations thereof.
Another aspect of the present invention relates to hypoallergenic molecules being composed of or comprising amino acids 1 to 33, 21 to 51, 42 to 73, 62 to 103 or 98 to 129 of Der p 2, amino acids 1 to 30, 20 to 50, 50 to 80, 90 to 125, 125 to 155 or 165 to 198 of Der p 7, amino acids 1 to 35, 35 to 72, 70 to 100 or 90 to 122 of Der p 21, amino acids 1 to 32, 15 to 48 or 32 to 70, 32 to 60, 52 to 84, 32 to 70 (Cys→Ser) of Der p 23, amino acids 19 to 58, 59 to 95, 91 to 120 or 121 to 157 of Alt a 1, amino acids 31 to 60, 45 to 80, 60 to 96 or 97 to 133 of Par j 2, amino acids 1 to 40, 36 to 66, 63 to 99, 86 to 120 or 107 to 145 of Ole e 1, amino acids 25 to 58, 99 to 133, 154 to 183, 277 to 307, 334 to 363, 373 to 402, 544 to 573, 579 to 608, 58 to 99, 125 to 165, 183 to 224, 224 to 261, 252 to 289, 303 to 340, 416 to 457, 460 to 500 or 501 to 542 of Fel d 2, amino acids 19 to 58, 52 to 91, 82 to 119, 106 to 144 or 139 to 180 of Can f 2, amino acids 19 to 56, 51 to 90, 78 to 118, 106 to 145 or 135-174 of Can f 1, amino acids 27 to 70, 70 to 100 or 92 to 132 of Art v 1, amino acids 31 to 70, 80 to 120, 125 to 155, 160 to 200, 225 to 263, 264 to 300 305 to 350 or 356 to 396 of Amb a 1, amino acids 1 to 34, 35 to 74, 74 to 115, 125 to 165, 174 to 213, 241 to 280, 294 to 333, 361 to 400 or 401 to 438 of Alt a 6, amino acids 1 to 40, 41 to 80, 81 to 120, 121 to 160 of Alt a 2 or fragments or sequence variations thereof.
Methods for the production of fusion proteins are well known in the art and can be found in standard molecular biology references such as Sambrook et al. (Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory Press, 1989) and Ausubel et al. (Short Protocols in Molecular Biology, 3rd ed; Wiley and Sons, 1995). In general, a fusion protein is produced by first constructing a fusion gene which is inserted into a suitable expression vector, which is, in turn, used to transfect a suitable hosT-cell. In general, recombinant fusion constructs are produced by a series of restriction enzyme digestions and ligation reactions which result in the desired sequences being incorporated into a plasmid. If suitable restriction sites are not available, synthetic oligonucleotide adapters or linkers can be used as is known by those skilled in the art and described in the references cited above. The polynucleotide sequences encoding allergens and native proteins can be assembled prior to insertion into a suitable vector or the sequence encoding the allergen can be inserted adjacent to a sequence encoding a native sequence already present in a vector. Insertion of the sequence within the vector should be in frame so that the sequence can be transcribed into a protein. It will be apparent to those of ordinary skill in the art that the precise restriction enzymes, linkers and/or adaptors required as well as the precise reaction conditions will vary with the sequences and cloning vectors used. The assembly of DNA constructs, however, is routine in the art and can be readily accomplished by a person skilled in the art.
It is a specific and unexpected advantage, that the fusion proteins derived from truncated hypoallergenic allergen molecules and the human hepatitis B pre S protein can be reproducibly expressed in standard expression systems and easily be manufactured produced in high yield with processes and reproducibly in standard expression systems known to a person skilled in the art, most particularly by using in an Escherichia coli as expression system. Such manufacturing process typically comprise the expression of the molecules according to the invention by the cultivation of cells in a bioreactor (e.g. in a fermenter, shake flask), followed by cell harvest (e.g. by filtration, centrifugation, etc.) and cell disruption (e.g. by high-pressure homogenization, sonication, freeze/thaw cycles, enzymatic or chemical cell lysis, etc.), purification of the molecules (e.g. by chromatography, filtration, precipitation, ultra/diafiltration, etc.) and final product formulation. In order to obtain a high yield of the molecules according to the invention, preferably high-cell density cultivation processes are employed, by application of fed-batch fermentation.
Another aspect of the present invention relates to a nucleic acid molecule coding for a hypoallergenic molecule and a fusion protein according to the present invention.
The nucleic acid molecule of the present invention may be employed, e.g., for producing said molecules recombinantly.
Said nucleic acid molecule may—according to another aspect of the present invention—be comprised in a vector.
This vector is preferably an expression vector.



BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
The present invention is further illustrated by the following figures and examples, however, without being restricted thereto.
FIG. 1 A shows a schematic overview of vector HBV_Phlp1_4xP5
FIG. 1 B shows a schematic overview of vector HBV_Phlp2_4xP3
FIG. 1 C shows a schematic overview of vector HBV_Phlp5_V2
FIG. 1 D shows a schematic overview of vector HBV_Phlp6_4xP1
FIG. 2 A shows the primary sequence of fusion protein HBV_PhlP1_4xP5 (BM321, sequence ID Nr. 14)
FIG. 2 B shows the primary sequence of fusion protein HBV_Phlp2_4xP3 (BM322, sequence ID Nr. 15)
FIG. 2 C shows the primary sequence of fusion protein HBV_Phlp5_V2 (BM325, sequence ID Nr. 16)
FIG. 2 D shows the primary sequence of fusion protein HBV_Phlp6_4xP1 (B326, sequence ID Nr. 17)
FIG. 2 E shows the primary sequence of fusion protein HBV_Betv1_4PA (BM31a, sequence ID Nr. 18)
FIG. 2 F shows the primary sequence of fusion protein HBV_Betv1_2PA2PB (BM31, sequence ID Nr. 19)
FIG. 2 G shows the primary sequence of fusion protein HBV_Phlp5_V1 (sequence ID No. 20)
FIG. 3 A shows a Coomassie Blue stained 12% SDS Page gel containing purified fusion protein HBV_Phlp1_4xP5 (BM 321, lane 1 and 10: 5 ug molecular marker, lane 2, 3, 11 and 12 5 ug BM321, lane 4 and 13 2 ug BM321, lane 5 and 14 1 ug BM321, lane 6 and 15 0.5 ug BM321, lane 7 and 16 0.25 ug BM321, lane 8 and 17 0.1 ug BM 321, lane 9 and 18 0.05 ug BM321). Lanes 1 to 9 are under reducing and lanes 10-18 under non-reducing conditions.
FIG. 3 B shows a Coomassie Blue stained 12% SDS Page gel containing purified fusion protein HBV_Phlp2_4xP3 (BM 322, lane 1 and 10: 5 ug molecular marker, lane 2, 3, 11 and 12 5 ug BM322, lane 4 and 13 2 ug BM322, lane 5 and 14 1 ug BM322, lane 6 and 15 0.5 ug BM322, lane 7 and 16 0.25 ug BM322, lane 8 and 17 0.1 ug BM 322, lane 9 and 18 0.05 ug BM322). Lanes 1 to 9 are under reducing and lanes 10-18 under non-reducing conditions.
FIG. 3 C shows a Coomassie Blue stained 12% SDS Page gel containing purified fusion protein HBV_Phlp5_V2 (BM 325, lane 1 and 10: 5 ug molecular marker, lane 2, 3, 11 and 12 5 ug BM325, lane 4 and 13 2 ug BM325, lane 5 and 14 1 ug BM325, lane 6 and 15 0.5 ug BM325, lane 7 and 16 0.25 ug BM325, lane 8 and 17 0.1 ug BM 325, lane 9 and 18 0.05 ug BM325). Lanes 1 to 9 are under reducing and lanes 10-18 under non-reducing conditions.
FIG. 3 D shows a Coomassie Blue stained 12% SDS Page gel containing purified fusion protein HBV_Phlp6_4xP1 (BM 326, lane 1 and 10: 5 ug molecular marker, lane 2, 3, 11 and 12 5 ug BM326, lane 4 and 13 2 ug BM326, lane 5 and 14 1 ug BM326, lane 6 and 15 0.5 ug BM326, lane 7 and 16 0.25 ug BM326, lane 8 and 17 0.1 ug BM 326, lane 9 and 18 0.05 ug BM326). Lanes 1 to 9 are under reducing and lanes 10-18 under non-reducing conditions.
FIGS. 4 A-4 B demonstrate the lack of IgE reactivity of fusion peptides derived from grass pollen allergens. IgE binding of fusion proteins in comparison to the complete allergen was tested by IgE dot-blot assay. Sera from the indicated number of grass pollen allergic patients were incubated with dotted proteins and bound IgE was detected with 125I-labelled anti-human IgE. No IgE binding was detected for any of the four peptide-carrier fusion proteins.
FIG. 4 A shows the results from the dot blot assay using HBV_Phlp1_4XP5 (BM321);
FIG. 4 B shows the results from the dot blot assay using HBV_Phlp2_4xP3 (BM322);
FIG. 4 C shows the results from the blot assay using HBV_Phlp5_V2 (BM325);
FIG. 4 D shows the results from form the dot blot assay using HBV_Phlp6_4xP1 (BM326).
FIG. 5 shows the low allergenic activity of grass pollen allergen derived fusion protein HBV_Phlp1_4xP5 (BM321) as determined by CD203c expression on basophils of allergic patients. PBMCs from grass pollen allergic patients were incubated with serial dilutions of Phl p 1 (light grey bars) or BM321 (dark grey bars). Induction of CD203c was measured as mean florescense intensities, and calculated stimulation indices are shown on the y-axis.
FIG. 6 shows the low allergenic activity of grass pollen allergen derived fusion protein HBV_Phlp6_4xP1 (BM326) as determined by CD203c expression on basophils of allergic patients. PBMCs from grass pollen allergic patients were incubated with serial dilutions of Phl p 6 (light grey bars) or BM326 (dark grey bars). Induction of CD203c was measured as mean florescence intensities, and calculated stimulation indices are shown on the y-axis.
FIGS. 7 A-7 D show Timothy grass pollen allergen-specific IgG1 responses in mice. Groups of 4 mice were immunized with 20 ug of fusion proteins (single fusion proteins and combination of 4 fusion proteins) and 10 μg each (Phl p1 and 5) or 5 μg each (Phl p2 and 6) of wild-type allergen at study week 0 and 3 followed by a boost immunization at study week 17. Antigens were administered subcutaneously in the back region of the animals. Blood was collected at study week 0, 3, 6, 9, 12, 17, 20 and 22 from the tail vein of the mice. In study weeks with immunizations blood was collected one day before the immunization. Immune sera of mice were investigated for the presence of allergen-specific IgG1 by ELISA. Pre-Immune sera before the first immunization were negative in all animals. Individual fusion proteins were compared to the application of a mixture of fusion proteins.

    
    
        a) FIG. 7 A shows the immune response against rPhl p 1 antigen for HBV_Phlp1_4xP5 (BM321 as single component), BM321 in a mixture with BM322, BM325 and BM326, and rPhl p 1 immunized mice.
        b) FIG. 7 B shows the immune response against rPhl p 2 antigen for HBV_Phlp2_4xP3 (BM321 as single component), BM322 in a mixture with BM321, BM325 and BM326, and rPhl p 2 immunized mice.
        c) FIG. 7 C shows the immune response against rPhl p 5 antigen for HBV_Phlp5_V2 (BM325 as single component), BM325 in a mixture with BM321, BM322 and BM326, and rPhl p 5 immunized mice.
        d) FIG. 7 D shows the immune response against rPhl p 6 antigen for HBV_Phlp6_4xP1 (BM326 as single component), BM326 in a mixture with BM321, BM322 and BM325, and rPhl p 6 immunized mice.
    
    


FIGS. 8 A and 8B show the molecular and immunological characterization of recombinant fusion proteins.
FIG. 8 A. Coomassie-stained SDS-PAGE showing four PreS fusion proteins with Bet v1 derived peptides (lane 1: 2xPA-PreS, lane 2: 2xPB-PreS, lane 3: 4xPA-PreS, lane 4: 2xPA2xPB-PreS) and the carrier PreS (lane 5).
FIG. 8 B. Nitrocellulose dotted recombinant fusion proteins and PreS are probed with a rabbit anti-PreS serum (lane 1), rabbit preimmune-serum (lane 3) buffer control for rabbit antibodies (lane 3) and monoclonal antibodies directed against Bet v 1-derived peptide P2′ (mAb2) (lane 4) and P4′ (mAb12) (lane 5) and buffer control for monoclonal mouse antibodies (lane 6).
FIG. 9 A shows IgE reactivity of rBet v 1 and recombinant fusion proteins of PreS with Bet v 1 derived peptides. Sera from birch pollen allergic patients, from non-allergic controls and only buffer were tested for their reactivity to dot-blotted rBet v 1, the four recombinant fusion proteins (2PA-PreS, 2PB-PreS, 4PA-PreS, 2PA2PB-PreS) and PreS alone. Bound human IgE was detected with 125I-labeled anti-human IgE antibodies. Counts per minute (cpm) corresponding to bound IgE are measured with a γ-counter and indicated at Y-axis. Box plots show the results of 50 birch pollen allergic patients.
FIG. 9 B shows the basophil activation by rBet v1 and the four PreS fusion proteins as measured by CD 203c upregulation. Blood samples of birch pollen allergic patients were exposed to increasing concentrations (0.001-1 μg/ml) of antigens, anti-IgE of buffer control (Co). Results of one representative patient are shown. CD 203c expression was determined by FACS analysis and is displayed as stimulation index (SI (y-axis). Means of triplicate measurements are shown and standard deviations are indicated.
FIGS. 10 A-10 C show lymphoproliferative responses and cytikine production of PBMC of birch pollen allergic patients. PBMCs of birch pollen allergic patients have been stimulated with equimolar amounts of rBet v 1, the Bet v 1 derived peptides PA and PB, PreS alone, and PreS fusion proteins (i.e. 2PA-PreS, 2PB-PreS, 4PA-PreS, 2PAPB-PreS). Stimulation indices (SI) (y-axes) are displayed.
(A) In FIG. 10 A, SI for the highest concentration (5 μg/well of Bet v 1 and equimolar amounts of the peptides, PreS and PreS fusion proteins) of 6 birch pollen allergic patients are shown as box blots, where 50% of the values are within the boxes and non-outliers are between the bars. The lines within the boxes indicate the median values.

(B) In FIG. 10 B, SI for four concentrations (1=5 μg/well, 2=2.5 μg/well, 3=1.25 μg/ml, 4=0.63 μg/well of rBet v1 and equimolar amounts of the peptides, PreS and PreS fusion proteins) are shown for one representative patient.

(C) In FIG. 10 C, Cytokine production in supernatants of PBMCs of 6 birch pollen allergic patients, stimulated with with 2.5 μg/mL of rBet v 1 and equimolar amounts of peptides PA and PB, PreS and four PreS fusion proteins, have been measured. Observed concentrations (pg/mL) (y-axes) after stimulation with antigens are shown in box blots, where 50% of the values are within the boxes and non-outliers are between the bars. The lines within the boxes indicate the median values.

FIGS. 11 A and 11B show the induction of IgG antibodies specific for rBet v 1 and Bet v 1 homologous allergens after subcutaneous immunization by PreS fusion proteins in rabbits.
(A) In FIG. 11 A, rabbits have been immunized with Alumhydroxide-adsorbed (Alum) (top) or complete Freund's adjuvant (CFA)-adsorbed (bottom) fusion proteins (2PA-PreS, 2PB-PreS, 4PA-PreS, 2PAPB-PreS) and rBet v 1. Rabbit IgG specific for rBet v 1 has been measured and mean optical density (OD) values for duplicate measurements are displayed (y-axes) for different dilutions of rabbit anti-sera (x-axes).

(B1) FIG. 11 B shows a multiple sequence alignment of Bet v 1 and Bet v 1-homologous allergens in alder (Aln g 1), hazel (Cor a 1) and apple (Mal d 1). Same amino acids are indicated as dots, gaps are indicated as dashes. Percentage identity of Bet v 1 homologous allergens to Bet v 1 is shown at the right side. Bet v 1-derived peptide A (PA, dashed line) and peptide B (PB, full line) are framed.

(B2) In FIG. 11 C, IgG antibodies of anti-rabbit sera (rab α-2PA-PreS, rab α-2PB-PreS, rab α-4PA-PreS, rab α-2PAPB-PreS) directed against rBet v 1, rAln g 1, rCor a 1 and rMal d 1 (x-axis) have been measured by ELISA. Means of duplicate measurements are shown. Optical density (OD) corresponding to allergen-specific IgG in rabbit sera (post) is displayed in comparison with corresponding preimmune sera (pre) (y-axes).

(C) In FIG. 11 D, IgG antibodies of rabbit immunized with rBet v 1 and recombinant fusion proteins (2PA-PreS, 2PB-PreS, 4PA-PreS, 2PAPB-PreS) directed against six Bet v 1-derived peptides (P1′-P6′) (x-axis) have been measured by ELISA. Means of optical densitiy (OD) values for duplicate measurements (y-axis) are displayed.

FIG. 12 shows the inhibition of Anti-2xPA2xPB-PreS rabbit serum against allergic patients' IgE compared to rabbit serum against complete rBet v 1. The percentage inhibition of IgE binding to rBet v 1 (y-axes) obtained with anti-2xPA2xPB-PreS and anti-rBet v 1 rabbit sera were determined by means of inhibition ELISA and are displayed as box blots, where 50% of the values are within the boxes and nonoutliers are between the bars. The lines within the boxes indicate the median values. Results of 21 birch pollen allergic patients are shown.
FIG. 13 shows a titration of rabbit IgG raised after immunisation with PreS-fusion proteins containing either 2 or 4 copies of a Phl p 6 derived peptide. For the immunogenicity testing rabbits (New Zealand White rabbits) were immunized with the different fusion proteins using aluminium hydroxide as adjuvant. The induction of specific antibodies was monitored in ELISA assays. Results show that the fusion proteins containing 4 peptides are more immunogenic than the fusion proteins containing 2 peptides.
FIGS. 14 A-14 D show the induction of a robust IgG response directed to the grass pollen allergens Phl p 1 (FIG. 14 A), Phl p2 (FIG. 14 B), Phl p 5 (FIG. 14 C), and Phl p 6 (FIG. 14 D) following in human grass pollen allergics following subcutaneous immunization with a vaccine formulation (BM32) comprising a mixture of the 4 hypoallergenic fusion proteins with SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, and SEQ ID NO. 17. The determination of IgG was carried out by ELISA. IgG levels before treatment (pre) are compared to IgG levels post-treatment (post).
FIG. 15 shows the results of T-cell proliferation assays performed on T-cells from grass pollen allergic individuals after immunization with a vaccine formulation consisting of a mixture of the 4 hypoallergenic fusion proteins with SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, and SEQ ID NO. 17. The T-cell reactivity is strongly reduced or absent if compared to grass pollen. The y-axis of the graph reflects the stimulation index.
FIGS. 16 A and 16 B show that IgG induced by therapy with a vaccine formulation (BM32) comprising a mixture of the 4 hypoallergenic fusion proteins with SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, and SEQ ID NO. 17 reduces lymphoproliferative responses to grass pollen allergens in human PBMCs.
FIG. 16 A shows the experimental set-up.
FIG. 16 B shows results from T-cell proliferation assays performed in the absence (+serum before) and presence (+serum after) of treatment-induced IgG. The y-axis of the graph reflects the stimulation index. P1-P5 indicate results from different study participants.
FIG. 17 shows the set-up of a clinical study carried out in 69 grass pollen allergic individuals using the vaccine formulation BM32 comprising a mixture of the 4 hypoallergenic fusion proteins with SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, and SEQ ID NO. 17
FIG. 18 A shows the primary sequence of fusion protein HBV Der p2-2xP2-2xP4 (sequence ID Nr. 149)
FIG. 18 B shows the primary sequence of fusion protein HBV Der p2-3xP2-3xP4 (sequence ID Nr. 150)
FIG. 18 C shows the primary sequence of fusion protein HBV Der p23-2xP4-2xP5 (sequence ID Nr. 151)
FIG. 18 D shows the primary sequence of fusion protein HBV Der p23-4xP6 (sequence ID Nr. 152)
FIG. 19 A shows the change in nasal symptoms induced by treatment with 3 subcutaneous injections of the vaccine formulation BM32 comprising a mixture of the 4 hypoallergenic fusion proteins with SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, and SEQ ID NO. 17. Black bars: before treatment, grey bars: after treatment.
FIG. 19 B shows the change in the mean wheal area between titrated skin prick test before and after treatment with the vaccine formulation BM32. The titrated skin prick test was carried out using 8 serial dilutions of grass pollen extract (undiluted to 1:128).
FIG. 20 shows IgE binding of the Der p 2 derived peptides in comparison to the complete allergen tested by an IgE dot-blot assay. Sera from 26 house dust mite allergic patients were incubated with dotted KLH-conjugated peptides and bound IgE was detected with 125I-labelled anti-human IgE. No IgE binding was detected for any of the 5 peptides as in example 26.



EXAMPLES
Example 1: Construction of Expression Plasmid for HBV_Phlp1_4xP5 (BM321)
The synthetic BM321 gene were assembled from synthetic oligo-nucleotides and/or PCR products and was cloned into an appropriate standard vector (pMK-RQkanR). The plasmid was purified from a transformed E. coli K12 strain (DH10B-T1R) and concentration was determined by UV spectroscopy. The final synthetic and codon-optimized BM321 DNA-sequence was further cloned into the expression vector pET28b(+) using appropriate restriction sites (NcoI site at the 5′-end and EcoRI at the 3′-end). The plasmid DNA was purified from transformed E. coli K12 DH10B (dam+dcm+) and concentration determined by UV spectroscopy. The final construct was verified by sequencing of the insert. A summary of plasmid data and a plasmid map of final expression vector “pBM-321” is shown below.
Summary of BM321 sequence cloned into final expression vector pET-28b(+).
















Alias name
Gene
Plasmid
Plasmid
Restriction


Sequence
sequence
size
size
name
sites







BM321
HBV_Phlp1_4xP5
882 bp
6153 bp
pBM-
NcoI/






321
EcoRI









Example 2: Transformation of Expression Plasmid into Expression Host for HBV_Phlp1_4xP5 (BM321)
Chemically competent E. coli BL21(DE3) cells were transformed with the expression plasmid by heat shock method. Transformed cells were plated on LB-agar-plates consisting of 0.5% sodium chloride 1% soy peptone, 0.5% yeast extract, 1.5% agar and 50 μg/mL kanamycin for selection. Cells on LB plates were grown by over-night cultivation at 37° C. Single colonies of transformed BL21(DE3) E. coli cells were isolated, cultured in LB-medium and screened for growth and expression of BM321. The best performing clone was selected for the further establishment of a Master Cell Bank.
Example 3: Preparation of a Master Cell Bank for HBV_Phlp1_4xP5 (BM321)
An aliquot of the selected clone was used for inoculation of 150 mL culture medium (composition: 0.5% sodium chloride, 1% soy peptone, 0.5% yeast extract, 50 μg/mL kanamycin). The Master Cell Bank (MCB) culture was incubated at 37° C. under constant agitation at 200 rpm until the culture reached an optical density of OD600=1-2. Glycerol was added in order to obtain a final glycerol concentration of 15% v/v and the MCB was aliquoted into 1 mL vials and stored in an ultra deep freezer at −75±10° C.
Example 4: High Cell Density Fed-Batch Fermentation of HBV_PhlP1_4xP5 (BM321)
Synthetic culture medium (100 mL, pH=6.8, salts and trace elements, 10 g/L glucose as carbon source) was inoculated with 1 mL of Master Cell Bank (E. coli BL21(DE3)/pBM321) and cultured in a shake flask (37° C., 200 rpm) until an optical density target value of OD=1 was reached. A 22 L stainless steel fermenter was used to perform the fed-batch fermentation. For automatic and reproducible feed control, a recipe was programmed allowing to pre-define specific growth-rate, feed rate, duration of batch-phase and duration of exponential feed-phase. In order to increase the oxygen transfer rate of the fermenter, back-pressure was controlled and set to 1 bar. The fermenter was in-situ sterilized with the synthetic culture medium as mentioned above and the fermentation was started by inoculation with preculture. After depletion of glucose, the exponential feeding phase was started in order to maintain a specific growth rate of μ=0.25 h−1. At an OD=45, the expression of recombinant BM321 was induced by the bolus addition of IPTG (0.8 mM final concentration). The culture was harvested at OD600=73. BM321 product titer obtained from the fed-batch fermentation was 1.2 g per L culture broth. Afterwards, the bacterial culture broth was cooled down to ≦20° C. and centrifuged at 7,000 rpm (5,500 g) at 4° C. for 15 min. Wet cell biomass was aliquoted and stored at −75° C.
Example 5: Cell Disruption and Clarification
For cell disruption, 748 gram biomass from Example 6 were thawed and subdivided into aliquots á 125 gram and resuspended in a homogenization buffer (20 mM Tris, 1 mM EDTA, 0.1% Triton X-100, pH 11.0) under mechanical agitation at room temperature for 30 min. For cell disruption, a freeze/thaw procedure was applied by freezing −75° C. and subsequent thawing, followed by mechanical homogenisation. The pH of the homogenate was adjusted to pH=10.0. The crude cell homogenate was subjected to a centrifugation step at 7,000 rpm (5,500 g) at 4° C. for 30 min. The supernatants were subjected to precipitation with PEI (polyethyleneimine) under mechanical agitation. Insoluble matters were separated by a subsequent centrifugation step. The clarified supernatants were subjected to the following chromatography step.
Example 6: Chromatographic Purification of HBV_Phlp1_4xP5 (BM321)
A total of 1840 mL of the PEI precipitation supernatant from the clarification step as described in Example 7 were loaded on a 5×30 cm Q-Sepharose FF column and equilibrated with buffer A (TrisHCl, EDTA). Unbound material was removed by washing with buffer A, followed by a wash with buffer C (1 sodium phosphate, EDTA, pH 7.0). Elution of the product fraction was accomplished by a linear gradient elution with 0-100% BM32 buffer E (sodium phosphate, EDTA, NaCl pH 7.0) in BM32 buffer C. Selection of product-containing fractions for pooling was performed according to SDS-PAGE analysis, by densitometric evaluation of fraction purity and by product band intensity.
The pooled fractions from the capture step were adjusted to a conductivity of 115 mS/cm by the addition of 2.5 M sodium chloride, and this feedstock was loaded on a Phenyl Sepharose HP column equilibrated with buffer D (sodium phosphate, EDTA, NaCl pH 7.0). Unbound material was removed by washing with buffer D. Elution of the product fraction was accomplished by a gradient elution from 40-100% buffer C (sodium phosphate, EDTA, pH 7.0) in buffer D. Selection of product-containing fractions for pooling was performed according to SDS-PAGE analysis, by densitometric evaluation of fraction purity and by product band intensity.

The pooled fractions from the intermediate step were adjusted to a conductivity of 80 mS/cm by the addition of 2.5 M sodium chloride, and this feedstock was loaded on a Toyopearl Butyl 650-S column equilibrated with a mixture buffer F (sodium phosphate, EDTA, NaCl pH 7.0). Unbound material was removed by a gradient wash with 80-0% BM32 buffer F in buffer C (sodium phosphate, EDTA, pH 7.0). Elution of the fraction was accomplished by a gradient elution from 0-1 buffer G (sodium phosphate, EDTA, isopropanol, pH 7.0) in buffer C. Selection of product-containing fractions for pooling was performed according to SDS-PAGE analysis, by densitometric evaluation of fraction purity and by product band intensity.

Example 7: Manufacturing of HBV_Phlp2_4xP3 (BM322), HBV_Phlp5_V2 (BM325), and HBV_Phlp6_4xP1 (BM326)
For expression and manufacturing of the recombinant molecules according to the invention, namely HBV_Phlp2_4xP3 (BM322), HBV_Phlp5_V2 (BM325), and HBV_Phlp6_4xP1 (BM326), the same, similar or comparable methods and procedures as described in Example 1, Example 2, Example 3, Example 4, Example 5 and Example 6 were applied.
Example 8: Preparation of an Injectable Formulation Consisting of a Mixture of HBV_PhlP1_4xP5 (BM321); HBV_PhlP2_4xP3 (BM322), HBV_PhlP5_V2 (BM325), and HBV_PhlP6_4xP1 (BM326)
Each of the recombinant purified proteins was dissolved in an isotonic buffer containing 0.9% sodium chloride and 2 mM sodium phosphate and to each protein solution an appropriate amount of aluminium hydroxide was added. A mixture containing equal parts of the four resulting suspensions was prepared and aliquoted under sterile condition into sealed vials. The injectable formulation obtained by this procedure contained 0.4 mg/mL of each HBV_PhlP1_4xP5; HBV_PhlP2_4xP3, HBV_PhlP5_V2 and HBV_PhlP6_4xP1.
Example 9: Preparation of his-Tagged HBV_Betv1_4xPA
The gene coding for fusion proteins consisting of PreS fused with Bet v 1-derived peptide PA twice at the N- and C-terminus (i.e. 4PA-PreS) was synthesized by ATG:biosynthetics, Merzhausen, Germany and inserted into the NdeI/XhoI sites of the vector pET-17b (Novagen, Germany). The DNA sequences were confirmed by means of automated sequencing of both DNA strands (Microsynth, Balgach, Switzerland).
The fusion protein was expressed in E coli strain BL21 (DE3; Stratagene, La Jolla, Calif.). Cells were grown in Luria Bertani-medium containing 50 μg/mL kanamycin to an OD of 0.6. Protein expression was induced by adding isopropyl-β-D-thiogalactopyranoside to a final concentration of 1 mmol/L over night at 37° C. Cells were harvested by centrifugation at 3500 rpm for 10 minutes. The protein product was mainly detected in the inclusion body fraction. It was solubilized in 6M GuHCl, 100 mM NaH2PO4, 10 mM TRIS, pH 8.0 over night. The homogenate was centrifuged at 14,000 g for 18 minutes. Supernatants of were incubated with 2 mL of a previously equilibrated Ni-NTA resin for 4 hours (Qiagen, Hilden, Germany) and the suspensions were subsequently loaded onto a column, washed with 2 column volumes of washing buffer (8 mol/L urea, 100 mmol/L NaH2PO4, and 10 mmol/LTris-HCl [pH=6.1]), and eluted with the same buffer (pH=3.5). The purified protein was dialyzed against water.
The purity of recombinant proteins was analyzed by Coomassie-stained SDS-PAGE (12.5%) under reducing conditions.
The identity of the fusion protein was confirmed by the means of dot blot using monoclonal antibodies, specific for Bet v 1-derived peptides P2′ (mAb2) and P4′ (mAb12) and PreS-specific rabbit antibodies as well as corresponding rabbit preimmune IgGs. One μg of PreS fusion proteins, PreS and HSA (control) have been immobilized on nitrocellulose and were incubated with monoclonal as well as rabbit sera diluted 1:1000 have at 4° C. Bound antibodies were detected with iodine 125-labelled rabbit anti-mouse IgG (mAb2, mAb12) or 125I-goat anti-rabbit IgG (rabbit anti-PreS, rabbit preimmune) (Perkin-Elmer, Waltham, Mass.) diluted 1:500 for 2 hours and visualized by autoradiography. Furthermore ELISA plates (Maxisorp, Nunc, Denmark) were coated with 2 μg of PreS fusion protein and PreS, diluted in 0.1 mol/L carbonate buffer, pH 9.6 washed with PBS containing 0.05% vol/vol Tween 20 (PBST) 3 times and blocked for 2 hours with 1% BSA-PBST. Subsequently plates were incubated with mAb2, mAb12, anti-PreS rabbit serum and rabbit anti-Bet v 1 antibodies in a dilution of 1:5000 (dilution buffer: 0.5% wt/vol BSA in PBST) overnight at 4° C. After washing 5 times, bound IgG antibodies have been detected with a HRP-labelled sheep anti-mouse antibody (for mAb2, mAb12) or HRP-labelled donkey anti-rabbit antibody (rabbit sera) (both GE Healthcare, Uppsala, Sweden) and colour reaction was developed.
Example 10: Preparation of his-Tagged HBV_Betv1_2 xPA2xPB (BM31)
Genes coding for fusion protein consisting of PreS fused twice with Bet v 1-derived peptides at the N- and C-terminus 2xPA2xPB-PreS) was synthesized by GenScript Piscataway, N.J., USA, 2PAPB-Pres) and inserted into the NdeI/XhoI sites of the vector pET-17b (Novagen, Germany). The DNA sequences were confirmed by means of automated sequencing of both DNA strands (Microsynth, Switzerland).
The recombinant PreS fusion proteins was expressed in E coli strain BL21 (DE3; Stratagene, Calif.). Cells were grown in Luria Bertani-medium containing 50 μg/mL kanamycin to an OD of 0.6. Protein expression was induced by adding isopropyl-β-D-thiogalactopyranoside to a final concentration of 1 mmol/L over night at 37° C. Cells were harvested by centrifugation at 3500 rpm for 10 minutes. Proteins were mainly detected in the inclusion body fraction. The resulting protein was solubilized in 6M GuHCl, 100 mM NaH2PO4, 10 mM TRIS, pH 8.0 over night. The homogenate was centrifuged at 14,000 g for 18 minutes. Supernatants of were incubated with 2 mL of a previously equilibrated Ni-NTA resin for 4 hours (Qiagen, Hilden, Germany) and the suspensions were subsequently loaded onto a column, washed with 2 column volumes of washing buffer (8 mol/L urea, 100 mmol/L NaH2PO4, and 10 mmol/LTris-HCl [pH=6.1]), and eluted with the same buffer (pH=3.5). Protein was dialyzed against 10 mM NaH2PO4.
The purity of recombinant proteins was analyzed by Coomassie-stained SDS-PAGE (12.5%) under reducing conditions. The identity of the fusion proteins was confirmed by the means of dot blot using monoclonal antibodies, specific for Bet v 1-derived peptides P2′ (mAb2) and P4′ (mAb12) and PreS-specific rabbit antibodies as well as corresponding rabbit preimmune IgGs. One μg of PreS fusion protein, PreS and HSA (control) have been immobilized on nitrocellulose and were incubated with monoclonal as well as rabbit sera diluted 1:1000 have at 4° C. Bound antibodies were detected with iodine 125-labelled rabbit anti-mouse IgG (mAb2, mAb12) or 125I-goat anti-rabbit IgG (rabbit anti-PreS, rabbit preimmune) (Perkin-Elmer, Waltham, Mass.) diluted 1:500 for 2 hours and visualized by autoradiography. Furthermore ELISA plates (Maxisorp, Nunc, Rosklide, Denmark) were coated with 2 μg of PreS fusion protein and PreS, diluted in 0.1 mol/L carbonate buffer, pH 9.6 washed with PBS containing 0.05% vol/vol Tween 20 (PBST) 3 times and blocked for 2 hours with 1% BSA-PBST. Subsequently plates were incubated with mAb2, mAb12, anti-PreS rabbit serum and rabbit anti-Bet v 1 antibodies in a dilution of 1:5000 (dilution buffer: 0.5% wt/vol BSA in PBST) overnight at 4° C. After washing 5 times, bound IgG antibodies have been detected with a HRP-labelled sheep anti-mouse antibody (for mAb2, mAb12) or HRP-labelled donkey anti-rabbit antibody (rabbit sera) (both GE Healthcare, Uppsala, Sweden) and colour reaction was developed.
Example 11: Detection of IgE Reactivity of Fusion Protein HBV_Phlp1_4xP5 (BM3212
IgE binding in comparison to the complete allergen was tested by IgE dot-blot assay. Sera from grass pollen allergic patients were incubated with dotted proteins and bound IgE was detected with 125I-labelled anti-human IgE. No IgE binding was detected for HBV_Phlp1_4xP5 (BM321) as shown in FIG. 4A.
Example 12: Detection of IgE Reactivity of Fusion Protein HBV_Phlp2_4xP3 (BM322)
IgE binding in comparison to the complete allergen was tested by IgE dot-blot assay. Sera from grass pollen allergic patients were incubated with dotted proteins and bound IgE was detected with 125I-labelled anti-human IgE. No IgE binding was detected for HBV_Phlp2_4xP3 (BM321) as shown in FIG. 4B.
Example 13: Detection of IgE Reactivity of Fusion Protein HBV_Phlp5_V2 (BM3252
IgE binding in comparison to the complete allergen was tested by IgE dot-blot assay. Sera from grass pollen allergic patients were incubated with dotted proteins and bound IgE was detected with 125I-labelled anti-human IgE. No IgE binding was detected for HBV_Phlp5_V2 (BM325) as shown in FIG. 4C.
Example 14: Detection of IgE Reactivity of Fusion Protein HBV_Phlp6_4xP1 (BM326)
IgE binding in comparison to the complete allergen was tested by IgE dot-blot assay. Sera from grass pollen allergic patients were incubated with dotted proteins and bound IgE was detected with 125I-labelled anti-human IgE. No IgE binding was detected for HBV_Phlp1_4xP1 (BM326) as shown in FIG. 4D.
Example 15: Detection of IgE Reactivity of Fusion Protein HBV_etV1_4xPA und HBV_Betv1_2 xPA2xPB (BM31)
IgE binding in comparison to the complete allergen was tested by IgE dot-blot assay. Sera from grass pollen allergic patients were incubated with dotted proteins and bound IgE was detected with 125I-labelled anti-human IgE. No IgE binding was detected for both fusion proteins as shown in FIG. 5
Example 16: Rabbit Anti-r89P5 Antibodies Block Patient's IgE-Binding to rPhl p 1
To determine the ability of peptide-induced rabbit Ig to inhibit the binding of allergic patients' IgE antibodies to rPhl p 1, ELISA plates were coated with 1 μg/ml rPhl p 1, washed and blocked. The plates were preincubated with 1:100-diluted rabbit anti-peptide (HBV_Phlp1_4xP5, KLHP5), a rabbit anti rPhl p 1 and, for control purposes, with the corresponding preimmune sera. After washing, plates were incubated with human sera from Phl p 1-allergic patients (1:3 diluted) and bound IgE was detected with mouse anti-human IgE (Pharmingen 1:1000) and then with sheep anti-mouse IgG POX-coupled (Amersham Bioscience) 1:2000. The percentage of inhibition of IgE-binding achieved by preincubation with the anti-peptide antisera was calculated as follows: 100-ODi/ODP×100.
ODi and ODP represent the extinctions after preincubation with the rabbit immune and preimmune serum, respectively. Table 1 shows the capacity of anti-Phl p 1 peptide antibodies to inhibit the binding of 13 allergic patients' IgE to complete rPhl p 1. Anti-fusion protein sera blocked the IgE-binding to the same extent as sera against rPhl p 1 and KLHP5. Table 2 shows the inhibition (in %) of all 13 patients.







TABLE 1







% inhibition of 13 patients' IgE-binding to rPhl p 1 after


incubation with rabbit anti-rPhl p 1, anti-HBV_Phlp1_4xP5


and anti-KLHP5 antisera









% inhibition












patient
rPhl p 1
HBV_Phlp1_4xP5
KLHP5
















1
83.63
86.11
85.17



2
88.74
95.69
93.85



3
95.66
96.80
98.42



4
97.43
97.72
96.29



5
92.77
90.84
88.45



6
93.56
91.93
90.07



7
95.00
94.56
96.84



8
85.25
89.10
90.05



9
97.07
104.72
93.73



10 
91.55
103.02
95.47



11 
98.85
102.43
100.49



12 
94.01
92.12
93.91



13 
87.75
59.62
42.98



Mean
92.41
92.59
89.67










Example 17: Rabbit Anti-HBV_Phlp2_4xP3 Antibodies Block Patient's IgE-Binding to rPhl p 2
To determine the ability of peptide-induced rabbit Ig to inhibit the binding of allergic patients' IgE antibodies to rPhl p 2, ELISA plates were coated with 1 μg/ml rPhl p 2, washed and blocked. The plates were preincubated with 1:100-diluted rabbit anti-peptide (HBV_Phlp2_4xP3, KLHP3), a rabbit anti rPhl p 2 and, for control purposes, with the corresponding preimmune sera. After washing, plates were incubated with human sera from Phl p 2-allergic patients (1:3 diluted) and bound IgE was detected with mouse anti-human IgE (Pharmingen 1:1000) and then with sheep anti-mouse IgG POX-coupled (Amersham Bioscience) 1:2000. The percentage of inhibition of IgE-binding achieved by preincubation with the anti-peptide antisera was calculated as follows: 100−ODi/ODP×100.
ODi and ODP represent the extinctions after preincubation with the rabbit immune and preimmune serum, respectively. Table 2 shows the capacity of anti-Phl p 2 peptide antibodies to inhibit the binding of 19 allergic patients' IgE to complete rPhl p 2. Anti-fusion protein sera blocked the IgE-binding to the same extent as sera against rPhl p 2 and KLHP3. Table 2 shows the inhibition (in %) of all 19 patients.







TABLE 2







% inhibition of 19 patients' IgE-binding to rPhl p 2 after


incubation with rabbit anti-rPhl p 1, anti-HBV_Phlp2_4xP3


and anti-KLHP3 antisera









% inhibition












patient
rPhl p 2
HBV_Phlp2_4xP3
KLHP3







1

98.24
81.36



2

97.50
83.90



3
96.46
98.57
90.58



4

98.31
86.77



5

96.46
81.17



6

99.43
72.45



9
91.25
91.38
90.44



8

95.78
54.49



9

98.60
87.55



10 

95.45
82.68



11 
91.36
96.70
78.21



12 

98.47
90.21



13 

97.67
93.20



14 

96.57
85.64



15 

97.00
91.35



16 
93.73
98.06
83.62



17 

95.55
76.27



18 

95.91
86.49



19 

95.90
83.99



Mean
93.20
97.19
83.18










Example 18: Rabbit Anti-HBV_Phlp5_V2 Antibodies Block Patient's IgE-Binding to rPhl p 5
To determine the ability of peptide-induced rabbit Ig to inhibit the binding of allergic patients' IgE antibodies to rPhl p 5, ELISA plates were coated with 1 μg/ml rPhl p 5, washed and blocked. The plates were preincubated with 1:100-diluted rabbit anti-peptide (HBV_Phl p2_V2), a rabbit anti rPhl p 5 and, for control purposes, with the corresponding preimmune sera. After washing, plates were incubated with human sera from Phl p 5-allergic patients (1:3 diluted) and bound IgE was detected with mouse anti-human IgE (Pharmingen 1:1000) and then with sheep anti-mouse IgG POX-coupled (Amersham Bioscience) 1:2000. The percentage of inhibition of IgE-binding achieved by preincubation with the anti-peptide antisera was calculated as follows: 100−ODi/ODP×100.
ODi and ODP represent the extinctions after preincubation with the rabbit immune and preimmune serum, respectively. Table 3 shows the capacity of anti-Phl p 5 peptide antibodies to inhibit the binding of 16 allergic patients' IgE to complete rPhl p 5. Anti-fusion protein sera blocked the IgE-binding to the same extent as sera against rPhl p 5 and better than KLH peptide mix. Table 3 shows the inhibition (in %) of all 16 patients.







TABLE 3







% inhibition of 13 patients' IgE-binding to rPhl p 5 after


incubation with rabbit anti-rPhl p 1, anti-HBV_Phlp5_V2


and anti-KLH peptide mix antisera









% inhibition












patient
rPhl p 5
HBV_Phlp5_V2
KLHPmix







1
99.00
96.69
91.74



2
94.57
94.15
68.42



3
98.98
95.88
85.74



4
97.39
88.38
80.23



5
98.95
93.74
62.33



6
98.52
93.36
78.82



9
97.22
91.35
79.94



8
96.02
89.70
80.14



9
97.09
88.48
61.11



10 
99.30
84.03
92.92



11 
99.50
94.09
86.46



12 
95.45
88.97
81.31



13 
96.22
93.34
60.87



14 
90.86
94.80
83.02



15 
98.45
94.15
83.60



16 
94.68
92.46
91.77



Mean
97.01
92.10
79.28










Example 19: Rabbit Anti-HBV_Phlp6_4xP1 Antibodies Block Patient's IgE-Binding to rPhl p 6
To determine the ability of peptide-induced rabbit Ig to inhibit the binding of allergic patients' IgE antibodies to rPhl p 6, ELISA plates were coated with 1 μg/ml rPhl p 6, washed and blocked. The plates were preincubated with diluted rabbit anti-peptide (HBV_Phlp6_4xP1, KLHP1), a rabbit anti rPhl p 6 and, for control purposes, with the corresponding preimmune sera. After washing, plates were incubated with human sera from Phl p 6-allergic patients (1:3 diluted) and bound IgE was detected with mouse anti-human IgE (Pharmingen 1:1000) and then with sheep anti-mouse IgG POX-coupled (Amersham Bioscience) 1:2000. The percentage of inhibition of IgE-binding achieved by preincubation with the anti-peptide antisera was calculated as follows: 100−ODi/ODP×100. ODi and ODP represent the extinctions after preincubation with the rabbit immune and preimmune serum, respectively. Table 4 shows the capacity of anti-Phl p 6 peptide antibodies to inhibit the binding of 21 allergic patients' IgE to complete rPhl p 6. Anti-fusion protein sera blocked the IgE-binding to the same extent as sera against rPhl p 6 and KLHP1. Table 4 shows the inhibition (in %) of all 21 patients.







TABLE 4







% inhibition of 21 patients' IgE-binding to rPhl p 6 after


incubation with rabbit anti-rPhl p 6, anti-HBV_Phlp6_4xP1


and anti-KLHP1 antisera









% inhibition












patient
rPhl p 6
HBV_Phlp6_4xP1
KLHP1
















 1
96.52
95.96
95.64



 2
88.26
91.20
88.06



 3
95.07
95.39
94.10



 4
82.77
83.74
81.98



 5
96.71
96.35
95.20



 6
95.46
93.38
92.83



 7
90.52
88.07
86.06



 8
86.69
85.14
83.08



 9
89.09
91.56
89.00



10
97.05
96.48
97.42



11
86.97
89.19
84.95



12
37.22
49.14
44.90



13
75.97
79.19
75.85



14
91.05
92.13
87.93



15
89.01
88.25
85.82



16
92.46
91.82
91.30



17
78.99
84.13
77.93



18
47.25
67.02
67.825



19
93.84
86.62
79.841



20
58.42
56.69
71.388



21
39.92
56.69
67.797



Mean
81.39
83.36
82.81










Example 20: IgE Reactivity of PreS Fusion Proteins Determined by Dot Blot and ELISA
Purified rBet v 1, recombinant fusion proteins 4xPA-PreS, 2xPA2xPB-PreS were tested for their IgE reactivity by RAST-based, non-denaturing dot blot assays. Two μg of the purified proteins and, for control purposes, HSA were dotted onto nitrocellulose membrane strips (Schleicher & Schuell, Dassel, Germany).
Nitrocellulose strips were blocked in buffer A (Vrtala, J Clin Invest, 1997) and incubated with sera from birch pollen allergic patients (n=50), sera from non-allergic persons (n=3) diluted 1:10, buffer control and positive control (1:1000 diluted rabbit anti-rBet v 1 antiserum). Bound IgE antibodies were detected with 125I-labelled anti-human IgE antibodies (BSM Diagnostica, Vienna, Austria), bound rabbit antibodies with a 125I-labeled goat anti-rabbit antiserum (Perkin-Elmer) and visualized by autoradiography (Valenta et al., 1992). Additionally, ELISA plates were coated with rBet v 1 and the purified PreS fusion proteins (5 μg/mL). After washing and blocking as described above, plates were incubated with sera of birch pollen allergic patients (n=21) and three non-allergic control sera diluted 1:5. Bound IgE was detected by purified mouse anti human IgE (BD Pharmingen) diluted 1:1000 overnight and visualized with HRP-labelled sheep anti mouse IgG (GE Healthcare) diluted 1:2000. After washing, colour reaction was determined as described above.
Example 21: Allergen-Induced Upregulation of CD203c of Allergic Patients' Basophils
Heparinized blood samples were obtained from birch allergic patients after informed consent was given and were incubated with increasing concentrations of rBet v 1, 4PA-PreS, 2PAPB-PreS ranging from 0.001 to 1 mg/mL, a monoclonal anti-IgE antibody (Immunotech, Marseille, France) as positive control, or PBS (negative control) for 15 min (37° C.). CD 203c expression was determined as previously described.
Example 22: Lymphoproliferative Responses and Cytokine Induction in PBMC from Birch Pollen Allergic Patients
PBMCs from birch pollen allergic patients (n=6) have been isolated by Ficoll (Amersham Biosciences, Uppsala, Sweden) density gradient centrifugation. Subsequently PBMCs were resuspended in AIM V medium (Life Technologies, Grand Island, N.Y.) to a final concentration of 2×105 cells/well and stimulated with decreasing antigen doses (equimolar amounts of 5 μg/well rBet v 1, PA, PB, PreS, 2PA-PreS, 2PB-PreS, 4PA-PreS, 2PAPB-PreS), with medium alone (negative control) or with IL-2 (4 IE/well) (positive control). After 6 days, proliferative responses were measured by [3H] thymidine incorporation and are expressed as stimulation indices (SI).
Furthermore cytokine production of 17 different cytokines (i.e. IL-113, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, IFN-γ, TNF-α, G-CSF, GM-CSF, MIP-1β, MCP-1) has been measured after 6 days of stimulation with Bio-plex Pro Human Cytokine 17-Plex Panel (Bio-Rad Laboratories) according the manufacturer's instructions. Briefly, the undiluted supernatants were mixed with anti-cytokine/chemokine mouse monoclonal antibodies coupled to different beads as capture antibodies (Bio-Rad). An 8-point standard curve was used to achieve low-end sensitivity. After washing, anti-cytokine biotinylated detection antibody was added. The reaction was visualized by adding Streptavidin-labelled Phycoerythrin (PE) and assay buffer. The samples were analyzed on a Luminex 100 instrument (Biosource, Nivelles, Belgium) and the data were acquired using the Bio-Plex Manager 6.0 software. All samples were analyzed in one run. Results are shown in FIGS. 10 A-10 C.
Example 23: Analysis of Rabbit Sera Immunized with rBet v 1 and PreS Fusion Proteins for their Recognition of rBet v 1, Bet v 1 Homologous Allergens and Bet v 1-Derived Peptides by ELISA
ELISA plates (Maxisorp, Nunc) were coated either with 1 μg/ml rBet v 1 or homologous allergens in alder (rAln g 1), hazel (rCor a 1), apple (rMal dl) and additionally with several Bet v 1-derived peptides in a concentration of 1 μg/ml overnight at 4° C. After washing and blocking as described above sera from rabbits immunized with rBet v 1 and the PreS fusion proteins conjugated to alum or CFA, were incubated in serial 1:2 dilutions ranging from 1:500 to 1:1 280 000 and in a concentration of 1:1000. Bound rabbit IgG was detected with HRP-labelled donkey anti-rabbit antibodies (GE Healthcare) and colour reaction was determined as described above.
Example 24: Inhibition of Allergic Patients' IgE Binding to rBet v 1
An inhibition ELISA was used to study the inhibition of the binding of birch pollen allergic patients' IgE to rBet v 1. ELISA plates were coated with rBet v 1 in a concentration of 1 μg/m at 4° C. overnight. After washing and blocking plates were pre-incubated with rabbit sera directed against the PreS fusion protein 2PAPB-PreS and anti-Bet v 1 rabbit serum in a dilution of 1:80 and 1:160 in comparison with rabbit preimmune sera overnight at 4° C. After an additional washing step sera of birch pollen allergic patients diluted 1:5 were added overnight at 4° C. and bound human IgE were detected with a 1:1000 diluted alkaline phosphatase-conjugated mouse monoclonal anti human IgE antibody (BD Pharmingen). The percentage of inhibition of IgE binding to rBet v 1 after pre-incubation with 2PAPB-PreS rabbit antisera and Bet v 1 rabbit antisera was calculated as follows: percent inhibition=100−(ODi×100/ODp). ODp and ODi represent the extinctions after pre-incubation with specific rabbit IgG (ODi) or preimmune sera (ODp), respectively. (FIG. 12)
Example 25: Use of a Vaccine Formulation Comprising a Mixture of 4 Hypoallergenic Fusion Proteins for the Treatment of Grass Pollen Allergy in Grass Pollen Allergic Human Individuals
An injectable formulation of hypoallergenic fusion proteins SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, and SEQ ID No. 17 with aluminum hydroxide was prepared as described in example 8. In the course of a clinical study, the vaccine was administered 3 times subcutaneously to 69 grass pollen allergic human subjects. (FIG. 17)
Vaccination with the vaccine formulation led to a robust IgG immune response. Induction of allergen-specific IgG following s.c. injection of the 3 different dose levels of the vaccine and placebo was determined by ELISA in the sera collected from the study participants before and after treatment with 3 s.c. injections of the vaccine formulation. (FIGS. 14 A-14 D).
For this purpose, ELISA plates (Nunc Maxisorp, Roskilde, Denmark) were coated with 5 μg/ml of the antigens Phl p 1, Phl p 2, Phl p 5, and Phl p 6 or human serum albumin (HSA) as control over night at 4° C. After washing with PBS containing 0.5% Tween 20 (PT) and blocking with 2% w/v BSA in PT, plates were subsequently incubated with 1:10 to 1:100 diluted sera from patients, serum from a non-atopic individual or buffer alone in triplicates overnight at 4° C. Bound IgE antibodies were detected with HRP-coupled anti-human IgE antibodies diluted in PT, 0.5% w/v BSA. The colour development was performed by addition of staining solution ABTS (2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt; Sigma-Aldrich, St. Louis, Mo., USA) (100 μl/well). The optical density was measured using an ELISA Reader at 405 nm. The results of IgG assessments are shown in FIGS. 14 A-14 D.
The vaccine did not provoke any relevant T-cell reactivity towards the hypoallergenic fusion proteins present in the vaccine formulation as determined by in-vitro T-cell proliferation assay (FIG. 15), thus demonstrating the lack of T-cell reactivity of the hypoallergenic fusion proteins.
T-cell proliferation assays were performed using the following procedure: Peripheral blood mononuclear cells (PBMC) were isolated from heparinised blood samples of the grass pollen allergic patients by Ficoll (Amersham Pharmacia Biotech, Little Chalfont, UK) density gradient centrifugation. PBMC (2×105) were then cultured in triplicates in 96-well plates (Nunclone; Nalge Nunc International, Roskilde, Denmark) in 200 μl serum-free Ultra Culture medium (BioWhittaker, Rockland, Me.) supplemented with 2 mM L-glutamin (SIGMA, St. Louis, Mo.), 50 μM b-mercaptoethanol (SIGMA) and 0.1 mg gentamicin per ml (SIGMA) at 37° C. and 5% CO2 in a humidified atmosphere. Cells were stimulated with a mixture containing 0.25 μg of each polypeptide component of the vaccine and for comparison an equimolar concentrations of grasspollen extract or for control purposes with 4 U Interleukin-2 per well (Boehringer Mannheim, Germany) or medium alone. After 6 d culture 0.5 μCi per well [3H]thymidine (Amersham Pharmacia Biotech) was added and 16 h thereafter incorporated radioactivity was measured by liquid scintillation counting using a microbeta scintilllation counter (Wallac ADL, Freiburg, Germany). Mean cpm were calculated from the triplicates and stimulation indices (SI) were calculated as the quotient of the cpm obtained by antigen or interleukin-2 stimulation and the unstimulated control. Results of proliferation assays are shown in FIG. 15.
Treatment with the vaccine induced IgG antibodies with the capability to modulate the allergen-specific T-cell response as demonstrated by a reduced proliferative response upon stimulation with grass pollen allergens in the presence of treatment-induced IgG. (FIGS. 16 A and 16 B).
For this purpose, T-cell proliferation assays were performed with PBMCs isolated from study participants after treatment as described above with the exception that the stimulation was done with a mixture of the 4 grass pollen allergens Phl p 1, Phl p 2, Phl p5, and Phl p 6 (0.25 μg per allergen) together with serum collected from the same participant before and after the treatment. The experimental set-up and results are shown in FIGS. 16 A and 16 B.
Reduction of nasal allergy symptoms induced by provocation in a pollen chamber and reduction of skin reactivity as determined by titrated skin prick testing was observed in patients having received 3 injections containing either 20 μg or 40 μg of each of the 4 polypeptides while there was no reduction in those parameters after treatment with doses of 10 μg of each polypeptide. (see FIGS. 19 A and 19 B).
Example 26: Selection of Peptides Derived from House Dust Mite Allergen Der p 2 and Design of PreS Fusion Proteins Using Those Peptides
The 5 non IgE binding Der p 2 derived peptides—Der p2 Pep1 (SEQ ID No. 96), Der p2 Pep2 (SEQ ID No. 97), Der p2 Pep3 (SEQ ID No. 98), Der p2 Pep4 (SEQ ID No. 99), and Der p2 Pep5 (SEQ ID No. 100)—were screened with respect to

    
    
        their IgE binding properties (dot blot assay)
        their potential to induce Der p 2 specific T-cell reactions, and (T-cell proliferation assay)
        their ability to induce Der p 2-specific antibodies with the capacity to block human patient's IgE to Der p 2. (inhibition ELISA using rabbit anti-peptide IgG)
    
    


For that purpose, each of the peptides was chemically coupled to KLH. KLH and chemical coupling of the peptides was used in this screening experiment because it is an easy-to-use and well established and straight forward model system allowing initial comparison of the different peptides.
IgE binding of the Der p 2 derived peptides in comparison to the complete allergen was tested by IgE dot-blot assay. Sera from 26 house dust mite allergic patients were incubated with dotted KLH-conjugated peptides and bound IgE was detected with 125I-labelled anti-human IgE. No IgE binding was detected for any of the 5 peptides as shown below.
To identify peptides which induce a low lymphoproliferative response in PBMC from house dust mite allergic patients PBMCs isolated from 10 patients were stimulated with the 5 Der p 2 derived peptides alone, the KLH-conjugated peptides, and wild-type Der p 2 for comparison.
PBMCs from all 10 patient were stimulated by the wild-type Der p 2, and there was no or only very low proliferation upon stimulation with Der p2 Pep1, Der p2 Pep2, and Der p2 Pep4. Stimulation with Der p2 Pep3 and Der p2 Pep5 however, resulted in significant proliferation of the PBMCs in 4 out of 10 and 3 out of 10 cases, respectively, indicating that peptides 3 and 5 contain important T-cell epitopes.
To identify the ability of the peptides to induce blocking IgG, rabbits were immunized with the 5 individual KLH-peptide conjugates. Subsequently, the ability of peptide-induced rabbit IgG to inhibit the binding of allergic patients' IgE antibodies to rDer p 2 was investigated by ELISA. ELISA plates were coated with 1 μg/ml rDer p 2, washed and blocked. The plates were preincubated with 1:100-diluted rabbit anti-peptide (KLH-P1, KLH-P2, KLH-P3, KLH-P4, and KLH-P5), a rabbit anti rDer p 2 and, for control purposes, with the corresponding preimmune sera. After washing, plates were incubated with human sera from house dust mite allergic, Der p 2 sensitized patients (1:3 diluted) and bound IgE was detected with mouse anti-human IgE (Pharmingen 1:1000) and then with sheep anti-mouse IgG POX-coupled (Amersham Bioscience) 1:2000. The percentage of inhibition of IgE-binding achieved by preincubation with the anti-peptide antisera was calculated as follows: 100−ODi/ODP×100.







TABLE 5







Inhibition capacity of anti-Der p 2-peptide antibodies to inhibit the 


binding of 20 allergic patients' IgE to complete rDer p 2. Anti- 


KLH-peptide sera induced by peptides 2, 3, and 4 blocked the 


IgE-binding to the same extent as sera against wild-type Der p 2. 


Table 5 shows the inhibition (in %) of all 20 patients.













Patient 
Peptide 
Peptide 
Peptide 
Peptide 
Peptide 
Der 


#
1
2
3
4
5
p 2
















1
50.63
74.41
78.36
75.50
1.07
78.26


2
49.61
77.15
82.95
77.85
4.16
82.74


3
64.73
87.41
92.13
89.25
0.00
93.34


4
37.98
72.24
81.08
75.60
2.48
84.25


5
0.00
43.56
50.52
47.28
0.00
56.70


6
54.12
80.63
82.64
80.94
1.10
83.21


7
51.43
79.64
92.08
83.25
16.16
93.51


8
42.93
71.02
79.55
75.44
0.83
78.35


9
30.33
58.36
50.94
56.49
7.76
57.03


10
38.46
66.79
71.20
71.25
0.00
69.06


11
48.15
74.60
83.13
78.97
5.59
83.56


12
46.06
68.54
74.05
71.32
10.05
76.46


13
44.71
73.62
87.29
77.19
4.97
84.34


14
39.20
63.55
53.94
65.30
0.00
66.20


15
43.62
71.82
89.94
74.54
0.51
94.39


16
38.09
69.94
84.08
72.45
1.29
86.83


17
43.63
74.16
87.12
78.50
2.98
89.10


18
29.09
73.75
89.97
77.59
1.38
90.66


19
40.44
56.77
62.09
62.30
0.00
66.16


20
20.89
60.85
70.76
63.16
2.69
74.98


mean
40.71
69.94
77.69
72.71
3.15
79.46
















TABLE 6







Decision matrix for selection of peptides. Peptides 2 and 4


meet all requirements of peptide fragments of the present


invention.














peptide induces






IgG which



peptide is
peptide induces
inhibit binding



non-IgE
no or only low
of human IgE
Peptide



binding
T-cell reactivity
to Der p 2
suitable?















Der p2 Pep1
✓
✓
X
no


Der p2 Pep2
✓
✓
✓
yes


Der p2 Pep3
✓
X
✓
no


Der p2 Pep4
✓
✓
✓
yes


Der p2 Pep5
✓
X
X
no









Example 27: Selection of Der p 1 Derived Hypoallergenic Peptides
The ability of Der p 1 derived peptides to induce IgE-blocking IgG antibodies was determined using rabbit-anti-peptide KLH antisera and sera from 6 house dust mite allergic patients in an inhibition ELISA as described in example 26 with the exception that the ELISA plates were coated with wild-type Der p 1 instead of Der p 2.







TABLE 7







Inhibition capacity of anti-Der p 1- peptide antibodies to 


inhibit the binding of 6 allergic patients' IgE to complete


Der p 1. Anti-KLH-peptide sera induced by peptides 1, 2, and 8 


were found to block the IgE-binding to a similar extent as sera against 


wild-type Der p 1. Table 7 shows the inhibition (in %) of 6 patients.















Patient I
Patient II
Patient III
Patient IV
Patient V
Patient VI
mean

















der p 1
72.9
91.3
80
90.8
87.5
89.7
85.4


peptide 1
50
68.4
65.5
87.7
77.4
85.1
72.4


peptide 2
47.8
73.4
66.1
83.2
72.6
82.5
70.9


peptide 3
22.5
28.2
22.1
35.5
26.4
27.6
27.1


peptide 4
24.4
42.4
33.4
46.5
33.2
42
37.0


peptide 5
22.7
31.4
23.3
38.4
30.4
31.5
29.6


peptide 6
1.9
12.8
3.6
5.6
4.2
5.4
5.6


peptide 7
30
51.8
43.5
67.4
52.1
59.6
50.7


peplide 8
41.1
65.8
52.8
76
66.2
73.9
62.6

## Claims

1. A nucleic acid molecule encoding a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16 and SEQ ID No. 17.

2. A vector, comprising the nucleic acid molecule of claim 1.

3. The vector of claim 2, which is an expression vector.

4. The vector of claim 2, which is a bacterial, fungal, insect, viral or mammalian vector.

5. A host cell, comprising the nucleic acid molecule of claim 1.

6. A host cell, comprising the vector of claim 2.

7. A method for treating a grass pollen allergy in a human or animal subject in need thereof, the method comprising administering an effective amount of the nucleic acid molecule of claim 1 to the subject.