U.S. patent application number 14/026436 was filed with the patent office on 2014-05-15 for rna vaccines.
This patent application is currently assigned to Biomay AG. The applicant listed for this patent is Biomay AG. Invention is credited to Angelika Fruhwirth, Elisabeth Rosler, Sandra Scheiblhofer, Josef Thalhamer, Richard Weiss.
Application Number | 20140134129 14/026436 |
Document ID | / |
Family ID | 38847002 |
Filed Date | 2014-05-15 |
United States Patent
Application |
20140134129 |
Kind Code |
A1 |
Thalhamer; Josef ; et
al. |
May 15, 2014 |
RNA Vaccines
Abstract
A RNA vaccine containing a RNA molecule encoding an allergen or
derivative thereof, in which the allergen is an allergen of Alnus
glutinosa, Alternaria alternata, Ambrosia artemisiifolia, Apium
graveolens, Arachis hypogaea, Betula verrucosa, Carpinus betulus,
Castanea sativa, Cladosporium herbarum, Corylus avellana,
Cryptomeria japonica, Cyprinus carpio, Daucus carota,
Dermatophagoides pteronyssinus, Fagus sylvatica, Felis domesticus,
Hevea brasiliensis, Juniperus ashei, Malus domestica, Quercus alba
or Phleum pratense.
Inventors: |
Thalhamer; Josef;
(Lamprechtshausen, AT) ; Weiss; Richard;
(Salzburg, AT) ; Rosler; Elisabeth; (Salzburg,
AT) ; Scheiblhofer; Sandra; (Salzburg, AT) ;
Fruhwirth; Angelika; (Innsbruck, AT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Biomay AG |
Vienna |
|
AT |
|
|
Assignee: |
Biomay AG
Vienna
AT
|
Family ID: |
38847002 |
Appl. No.: |
14/026436 |
Filed: |
September 13, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12680354 |
Nov 1, 2010 |
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PCT/EP08/63035 |
Sep 29, 2008 |
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14026436 |
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Current U.S.
Class: |
424/85.2 ;
424/275.1; 536/23.1 |
Current CPC
Class: |
C12N 2770/36143
20130101; A61K 39/36 20130101; C12N 7/00 20130101; A61K 2039/57
20130101; A61K 2039/55522 20130101; A61P 37/04 20180101; A61K
2039/575 20130101; A61K 38/2086 20130101; A61K 2039/53 20130101;
A61K 39/35 20130101; A61P 37/08 20180101; A61K 2039/55561 20130101;
A61K 38/208 20130101 |
Class at
Publication: |
424/85.2 ;
424/275.1; 536/23.1 |
International
Class: |
A61K 39/35 20060101
A61K039/35; A61K 38/20 20060101 A61K038/20 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 28, 2007 |
EP |
07450169.3 |
Claims
1. A RNA vaccine, comprising a RNA molecule encoding for an
allergen or derivative thereof, wherein the allergen is at least
one allergen selected from the group consisting of Alnus glutinosa,
Alternaria alternata, Ambrosia artemisiifolia, Apium graveolens,
Arachis hypogaea, Betula verrucosa, Carpinus betulus, Castanea
sativa, Cladosporium herbarum, Corylus avellana, Cryptomeria
japonica, Cyprinus carpio, Daucus carota, Dermatophagoides
pteronyssinus, Fagus sylvatica, Felis domesticus, Hevea
brasiliensis, Juniperus ashei, Malus domestica, Quercus alba and
Phleum pratense.
2. The vaccine according to claim 1, wherein the allergen is
selected from the group consisting of Aln g 1, Alt a 1, Alt a 3,
Alt a 4, Alt a 5, Alt a 6, Alt a 7, Alt a 8, Alt a 10, Alt a 12,
Alt a 13, Amb a 1, Amb a 2, Amb a 3, Amb a 5, Amb a 6, Amb a 7, Amb
a 8, Amb a 9, Amb a 10, Api g 1, Api g 4, Api g 5, Ara h 1, Ara h
2, Ara h 3, Ara h 4, Ara h 5, Ara h 6, Ara h 7, Ara h 8, Bet v 1,
Bet v 2, Bet v 3, Bet v 4, Bet v 6, Bet v 7, Car b 1, Cas s 1, Cas
s 5, Cas s 8, Cla h 2, Cla h 5, Cla h 6, Cla h 7, Cla h 8, Cla h 9,
Cla h 10, Cla h 12, Cor a 1, Cor a 2, Cor a 8, Cor a 9, Cor a 10,
Cor a 11, Cry j 1, Cry j 2, Cyp c 1, Dau c 1, Dau c 4, Der p 1, Der
p 2, Der p 3, Der p 4, Der p 5, Der p 6, Der p 7, Der p 8, Der p 9,
Der p 10, Der p 11, Der p 14, Der p 20, Der p 21, Clone 30
allergen, Fag s 1, Fel d 1, Fel d 2, Fel d 3, Fel d 4, Fel d 5w,
Fel d 6w, Fel d 7w, Hev b 1, Hev b 2, Hev b 3, Hev b 4, Hev b 5,
Hev b 6.01, Hev b 6.02, Hev b 6.03, Hev b 7.01, Hev b 7.02, Hev b
8, Hev b 9, Hev b 10, Hev b 11, Hev b 12, Hev b 13, Jun a 1, Jun a
2, Jun a 3, Mal d 1, Mal d 2, Mal d 3, Mal d 4, Que a 1, Phl p 1,
Phl p 2, Phl p 4, Phl p 5, Phl p 6, Phl p 7, Phl p 11, Phl p 12 and
Phl p 13.
3. The vaccine according to claim 1, characterised in that wherein
the allergen is selected from the group consisting of Aln g 1, Alt
a 1, Amb a 1, Api g 1, Ara h 2, Bet v 1, beta-casein, Car b 1, Cas
s 1, Cla h 8, Cor a 1, Cry j 1, Cyp c 1, Dau c 1, Der p 2, Fag s 1,
Fel d 1, Hev b 6, Jun a 1, Mal d 1, ovalbumin (OVA), Phl p 1, Phl p
2, Phl p 5, Phl p 6 and Phl p 7.
4. The vaccine according to claim 1, wherein the allergen is
selected from the group consisting of Phl p 1, Phl p 2, Phl p 5 Phl
p6, Aln g 1, Cor a 1, Que a 1, Car b 1 and Bet v 1.
5. The vaccine according to claim 1, wherein the allergen
derivative is hypoallergenic.
6. The vaccine according to claim 5, wherein the hypoallergenic
allergen derivative exhibits an IgE reactivity which is at least
10% lower than the IgE reactivity of the wild-type allergen.
7. The vaccine according to claim 1, wherein the RNA molecule
encoding the allergen or derivative thereof is fused to a further
molecule encoding a peptide, polypeptide or protein.
8. The vaccine according to claim 1, wherein the RNA molecule
comprises at least one further element selected from the group
consisting of replicase, 1-globin leader sequence, cap0, cap1 and
poly A tail.
9. The vaccine according to claim 1, further comprising a CpG-DNA
adjuvant.
10. The vaccine according to claim 1, wherein the vaccine is
adapted for intramuscular, intradermal, intravenous, transdermal,
topical or biolistic administration.
11. A method for treating or preventing allergy, comprising
administering the RNA vaccine according to claim 1 into a body or
into cells outside of the body.
12. A method for manufacturing a protective and therapeutic vaccine
for hyposensitising an individual to an allergen, comprising
employing a RNA molecule encoding for an allergen or derivative
thereof, to obtain the RNA vaccine according to claim 1.
13. The method according to claim 11, wherein the vaccine further
comprises an adjuvant selected from the group consisting of CpG-DNA
and cytokines.
14. The method according to claim 11, wherein the vaccine is
adapted for intramuscular, intradermal, intravenous, transdermal,
topical or biolistic administration.
15. An isolated RNA molecule comprising a nucleotide sequence
encoding an allergen or derivative thereof as defined in claim
1.
16. The method according to claim 12, wherein the vaccine further
comprises an adjuvant selected from the group consisting of CpG-dNA
and cytokines.
17. The method according to claim 12, wherein the vaccine is
adapted for intramuscular, intradermal, intravenous, transdermal,
topical or biolistic administration.
18. The vaccine according to claim 1, further comprising a cytokine
adjuvant.
19. The vaccine according to claim 18, wherein the cytokine
adjuvant is at least one selected from the group consisting of
IL-12 and IL-15.
Description
CROSS REFERENCE TO RELATED APPLICATION(S)
[0001] This application is a continuation of U.S. application Ser.
No. 12/680,354, filed on Nov. 1, 2010, the entire content of which
is incorporated herein by reference.
[0002] The present invention relates to RNA vaccines.
[0003] During the last decades, type I allergic diseases have
emerged as a major public health problem in Western industrialised
countries with about 25% of the population being affected by
now.
[0004] In addition to family predisposition, conditions of growing
up--including early childhood infections--and dietary habits, but
also environmental factors such as passive smoking or exposure to
air pollutants have been demonstrated to be of great relevance for
the development of atopic diseases.
[0005] Specific immunotherapy, which is performed by injections of
escalating doses of allergen(s) over years, currently represents
the only available therapeutic intervention. However, due to the
high doses administered, the risk of anaphylactic side effects is
evident and the use of crude, barely characterised allergen
extracts implies the possibility for sensitisation of the patient
against previously unrecognised components.
[0006] Additionally, there is no preventive vaccination against
type I allergy available, although prevention of young children
with increased hereditary risk to develop allergic disease may be
the most feasible approach. Training of the naive immune system is
easier to accomplish than balancing an already manifested allergic
immune phenotype.
[0007] In Ying et al. (Nature Med (1999) 5:823-827)
self-replicating RNA vaccines are disclosed whose RNA encodes for
.beta.-galactosidase, which is often used as a model molecule for
studying immunological processes. In Ying et al. the anti-tumour
reaction was studied and the induction of CD8 positive cells was
observed. However, CD4 positive cells which were not investigated
in Ying et al. mediate in contrast to CD8 positive cells
immunological protection against allergies and prevent a class
switch towards IgE in B-cells.
[0008] Recently, nucleic acid based vaccines have become a
promising approach to bias immune mechanisms underlying allergic
diseases. It has been shown in numerous animal studies, that DNA
vaccines can prevent from the induction of type I allergic
responses and even reverse an already established allergic TH2
immune status (Weiss, R. et al. (2006) Int Arch Allergy Immunol
139:332-345).
[0009] Nevertheless, general concerns have been raised regarding
the safety of DNA based vaccines: The introduced DNA molecules
could potentially integrate into the host genome or, due to their
distribution to various tissues, could lead to sustained delivery
of allergen, thus inducing uncontrollable anaphylactic reactions
within patients with pre-existing allergen-specific IgE molecules.
Furthermore, vaccination of healthy children requires the highest
safety standards for any anti-allergy vaccine.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 shows in vitro transfection of BHK-21 cells with RNA
(.beta.Gal-RNA) or self-replicating RNA (.beta.Gal-repRNA)
transcripts encoding .beta.-galactosidase.
[0011] FIG. 2A shows Phl p 5 specific IgG1 and IgG2a levels after
nucleic acid vaccination.
[0012] FIG. 2B shows Phl p 5 specific IgG1 and IgG2a levels after
subsequent sensitisation with recombinant allergen in alum.
[0013] FIG. 3 shows Phl p 5 specific IgE measured via RBL release
assay.
[0014] FIG. 4A shows the number of IFN-gamma secreting splenocytes
after in vitro re-stimulation with recombinant Phl p 5 as
determined by ELISPOT.
[0015] FIG. 4B shows the number of IL-4 secreting splenocytes after
in vitro re-stimulation with recombinant Phl p 5 as determined by
ELISPOT.
[0016] FIG. 4C shows the number of IL-5 secreting splenocytes after
in vitro re-stimulation with recombinant Phl p 5 as determined by
ELISPOT.
[0017] FIG. 5A shows the number of total leukocyte in BALF of
sensitised mice after i.n. application of allergen.
[0018] FIG. 5B shows the number of total eosinophils in BALF of
sensitised mice after i.n. application of allergen.
[0019] FIG. 6A shows the levels of IL-5 in BALF of sensitised mice
after i.n. application of allergen.
[0020] FIG. 6B shows the levels of IFN-.gamma. in BALF of
sensitised mice after i.n. application of allergen.
[0021] FIGS. 7A, 7B, and 7C show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Bet v 1.
[0022] FIGS. 8A, 8B, and 8C show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Car b 1.
[0023] FIGS. 9A, 9B, and 9C show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Cas s 1.
[0024] FIGS. 10A and 10B show the induction of Th 1 memory by RNA
pTNT-Phl p 1.
[0025] FIGS. 11A, 11B, and 11C show the induction of Th 1 memory
and suppression of IgE responses by RNA pTNT-Phl p 6.
[0026] FIG. 12 shows the induction of Th 1 memory by RNA pTNT-Cor a
1.
[0027] FIG. 13 shows the induction of Th 1 memory by RNA pTNT-Aln g
1.
[0028] FIG. 14A, 14B, and 14C show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Fag s 1.
[0029] FIGS. 15A and 15B show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Phl p 2.
[0030] FIGS. 16A and 16B show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Phl p 7.
[0031] FIG. 17A, 17B, and 17C show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-hybrid (Phl p
1-2-5-6).
[0032] FIGS. 18A and 18B show the induction of Th 1 memory by RNA
pTNT-Cry j 1.
[0033] FIG. 19 shows the induction of Th 1 memory by RNA pTNT-Jun a
1.
[0034] FIG. 20 shows the induction of Th 1 memory by RNA pTNT-Amb a
1.
[0035] FIG. 21A, 21B, and 21C show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Api g 1.
[0036] FIGS. 22A and 22B show the induction of Th 1 memory by RNA
pTNT-Dau c 1.
[0037] FIG. 23A, 23B, and 23C show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Mal d 1.
[0038] FIG. 24A, 24B, and 24C show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Ova.
[0039] FIGS. 25A and 25B show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Beta-Casein.
[0040] FIG. 26 shows the induction of Th 1 memory responses by RNA
pTNT-Cyp c 1.
[0041] FIGS. 27A and 27B show the induction of Th 1 memory
responses by RNA pTNT-Fel d 1.
[0042] FIGS. 28A and 28B show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Der p 2.
[0043] FIG. 29A, 29B, and 29C shows the induction of Th 1 memory
and suppression of IgE responses by RNA pTNT-Alt a 1.
[0044] FIGS. 30A and 30B show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Cla h 8.
[0045] FIGS. 31A and 31B show the induction of Th 1 memory by RNA
pTNT-Hev b 6.
[0046] FIG. 32 shows the induction of Th 1 memory by RNA
pTNT-hybrid (allergen).
[0047] FIGS. 33A and 33B show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Ara h 2.
[0048] FIGS. 34A and 34B show the induction of Th 1 memory by RNA
pTNT-Que a 1.
[0049] FIGS. 35A and 35B show no induction of Th 1 memory by RNA
pTNT-Art v 1.
[0050] FIGS. 36A and 36B show no induction of Th 1 memory or
suppression of IgE responses by RNA pTNT-Ole e 1.
[0051] It is therefore an object of the present invention to
provide an allergen vaccine which overcomes the drawbacks of DNA
vaccines and still allows for an effective treatment of allergies
or successfully prevents from sensitisation against an
allergen.
[0052] Therefore the present invention relates to an RNA vaccine
comprising at least one RNA molecule encoding for at least one
allergen or derivative thereof, wherein said allergen is an
allergen of Alnus glutinosa, Alternaria alternata, Ambrosia
artemisiifolia, Apium graveolens, Arachis hypogaea, Betula
verrucosa, Carpinus betulus, Castanea sativa, Cladosporium
herbarum, Corylus avellana, Cryptomeria japonica, Cyprinus carpio,
Daucus carota, Dermatophagoides pteronyssinus, Fagus sylvatica,
Felis domesticus, Hevea brasiliensis, Juniperus ashei, Malus
domestica, Quercus alba and Phleum pratense.
[0053] It turned out that RNA molecules encoding an allergen or
derivative thereof may also be used efficiently as RNA vaccines.
RNA vaccines exhibit the features attributed to DNA vaccines for
the treatment of allergic diseases: They provide the allergen in
its purest form, i.e. its genetic information, and, similar to DNA
vaccines, they induce TH1-biased immune reactions. Furthermore,
similar methods as developed for DNA vaccines to create
hypoallergenic gene products, can be implemented with RNA vaccines,
as well.
[0054] Besides, RNA vaccines offer striking advantages over DNA
vaccines: (i) The vaccine contains the pure genetic information of
the allergen but no additional foreign sequences, such as viral
promoters, antibiotic resistance genes, or viral/bacterial
regulatory sequences that are usually present in the backbone of
plasmids used for DNA vaccines. (ii) RNA cannot integrate into the
host genome thus abolishing the risk of malignancies. (iii) RNA is
translated in the cytoplasm of the cell, hence the transcription
machinery of the cell nucleus is not required, rendering RNA
vaccines independent of transport into and out of the nucleus as
well as of nuclear stages. (iv) Due to the rapid degradation of
RNA, expression of the foreign transgene is short-lived, avoiding
uncontrollable long term expression of the antigen.
[0055] The RNA vaccine of the present invention may comprise more
than one RNA molecule encoding an allergen, preferably two, three,
five, ten, etc. However, one RNA molecule may also encode for at
least one allergen, which means that one RNA molecule comprises a
nucleotide sequence encoding for at least one, two, three, five,
ten, etc. different or identical allergens. The allergens to be
encoded by one or more RNA molecules may be selected from the list
below in any combination.
[0056] As used herein, the term "RNA vaccine" refers to a vaccine
comprising an RNA molecule as defined herein. Said vaccine may
comprise, however, of course other substances and molecules which
are required or which are advantageous when said vaccine is
administered to an individual (e.g. pharmaceutical excipients).
[0057] The term "allergen of" is used interchangeable with the
terms "allergen derived from" and "allergen obtained from". This
means that the allergen is naturally expressed in said organisms
and the DNA/RNA encoding said allergens is isolated in order to
produce the RNA molecules of the present invention.
[0058] It turned out that not all RNA molecules encoding an
allergen can induce the formation of allergen-specific antibodies
when administered to a mammal or human being. RNA molecules
encoding for Artemisia vulgaris allergen Art v 1 and Olea europea
allergen Ole e 1, for instance, are not able to induce Th 1 memory
and to suppress the allergen specific IgE response. However, RNA
molecules encoding the allergen of the above mentioned sources are
capable to do so.
[0059] According to a preferred embodiment of the present invention
the allergen of Alnus glutinosa is Aln g 1, the allergen of
Alternaria alternata is selected from the group consisting of Alt a
1, Alt a 3, Alt a 4, Alt a 5, Alt a 6, Alt a 7, Alt a 8, Alt a 10,
Alt a 12 and Alt a 13, the allergen of Ambrosia artemisiifolia is
selected from the group consisting of Amb a 1, Amb a 2, Amb a 3,
Amb a 5, Amb a 6, Amb a 7, Amb a 8, Amb a 9 and Amb a 10, the
allergen of Apium graveolens is selected from the group consisting
of Api g 1, Api g 4 and Api g 5, the allergen of Arachis hypogaea
is selected from the group consisting of Ara h 1, Ara h 2, Ara h 3,
Ara h 4, Ara h 5, Ara h 6, Ara h 7 and Ara h 8, the allergen of
Betula verrucosa is selected from the group consisting of Bet v 1,
Bet v 2, Bet v 3, Bet v 4, Bet v 6 and Bet v 7, the allergen of
Carpinus betulus is Car b 1, the allergen of Castanea sativa is
selected from the group consisting of Cas s 1, Cas s 5 and Cas s 8,
the allergen of Cladosporium herbarum is selected from the group
consisting of Cla h 2, Cla h 5, Cla h 6, Cla h 7, Cla h 8, Cla h 9,
Cla h 10 and Cla h 12, the allergen of Corylus avellana is selected
from the group consisting of Cor a 1, Cor a 2, Cor a 8, Cor a 9,
Cor a 10 and Cor a 11, the allergen of Cryptomeria japonica is
selected from the group consisting of Cry j 1 and Cry j 2, the
allergen of Cyprinus carpio is Cyp c 1, the allergen of Daucus
carota is selected from the group consisting of Dau c 1 and Dau c
4, the allergen of Dermatophagoides pteronyssinus is selected from
the group consisting of Der p 1, Der p 2, Der p 3, Der p 4, Der p
5, Der p 6, Der p 7, Der p 8, Der p 9, Der p 10, Der p 11, Der p
14, Der p 20, Der p 21 and Clone 30 allergen, the allergen of Fagus
sylvatica is Fag s 1, the allergen of Felis domesticus is selected
from the group consisting of Fel d 1, Fel d 2, Fel d 3, Fel d 4,
Fel d 5w, Fel d 6w and Fel d 7w, the allergen of Hevea brasiliensis
is selected from the group consisting of Hey b 1, Hey b 2, Hey b 3,
Hey b 4, Hey b 5, Hey b 6.01, Hey b 6.02, Hev b 6.03, Hey b 7.01,
Hey b 7.02, Hey b 8, Hey b 9, Hey b 10, Hey b 11, Hey b 12 and Hey
b 13, the allergen of Juniperus ashei is selected from the group
consisting of Jun a 1, Jun a 2 and Jun a 3, the allergen of Malus
domestica is selected from the group consisting of Mal d 1, Mal d
2, Mal d 3 and Mal d 4, the allergen of Quercus alba is Que a 1 and
the allergen of Phleum pratense is selected from the group
consisting of Phl p 1, Phl p 2, Phl p 4, Phl p 5, Phl p 6, Phl p 7,
Phl p 11, Phl p 12 and Phl p 13.
[0060] According to a preferred embodiment of the present invention
the allergen is selected from the group consisting of:
TABLE-US-00001 Grass Pollen: Phl p 1, Phl p 2, Phl p 5, Phl p 6,
Phl p 7, Phl p 12 House Dust Mite: Der p 1, Der p 2, Der p 7, Der p
21, Clone 30 allergen (PCT- application AT2007/000201, Austrian
patent application AT 503530: MKFNIIIVFI SLAILVHSSY AANDNDDDPT
TTVHPTTTEQ PDDKFECPSR FGYFADPKDP HKFYICSNWE AVHKDCPGNT RWNEDEETCT,
SEQ ID No. 1) Birch Pollen: Bet v 1 and its homologous tree (Aln g
1, Cor a 1, Fag s 1) or food allergens) Mal d 1, Api g 1, Pru p 1)
Cat: Fel d 1, Fel d 2 Weeds (Ragweed, Amb a 1 Mugwort):
Cypress/Juniper/ Cry j 1, Cry j 2, Jun a 1, Jun a 3, Cha o 1, Cha o
2, Cup a 1, Cedar: Cup a 3, Jun a 1, Jun a 3, Pla a 3 Peanut: Ara h
1, Ara h 2, Ara h 4 Hazelnut: Cor a 8, Cor a 9 Fish/Shrimps: Gad c
1, Cyp c 1, Pen a 1
[0061] Especially preferred allergens to be used in an RNA vaccine
of the present invention are selected from the group consisting of
Aln g 1, Alt a 1, Amb a 1, Api g 1, Ara h 2, Bet v 1, beta-casein,
Car b 1, Cas s 1, Cla h 8, Cor a 1, Cry j 1, Cyp c 1, Dau c 1, Der
p 2, Fag s 1, Fel d 1, Hev b 6, Jun a 1, Mal d 1, ovalbumin (OVA),
Phl p 1, Phl p 2, Phl p 5, Phl p 6 and Phl p 7.
[0062] It turned out that the above identified allergens are
particularly suited to be used in RNA vaccines. However, it is of
course also possible to use the present invention for other
allergens, such as Amb a 1, Amb a 2, Amb a 3, Amb a 5, Amb a 6, Amb
a 7, Amb a 8, Amb a 9, Amb a 10, Amb t 5, Hel a 1, Hel a 2, Hel a
3, Mer a 1, Che a 1, Che a 2, Che a 3, Sal k 1, Cat r 1, Pla l 1,
Hum j 1, Par j 1, Par j 2, Par j 3, Par o 1, Cyn d 1, Cyn d 7, Cyn
d 12, Cyn d 15, Cyn d 22w, Cyn d 23, Cyn d 24, Dac g 1, Dac g 2,
Dac g 3, Dac g 5, Fes p 4w, Hol l 1, Lol p 1, Lol p 2, Lol p 3, Lol
p 5, Lol p 11, Pha a 1, Phl p 1, Phl p 2, Phl p 4, Phl p 5, Phl p
6, Phl p 11, Phl p 12, Phl p 13, Poa p 1, Poa p 5, Sor h 1, Pho d
2, Aln g 1, Bet v 1, Bet v 2, Bet v 3, Bet v 4, Bet v 6, Bet v 7,
Car b 1, Cas s 1, Cas s 5, Cas s 8, Cor a 1, Cor a 2, Cor a 8, Cor
a 9, Cor a 10, Cor a 11, Que a 1, Fra e 1, Lig v 1, Syr v 1, Cry j
1, Cry j 2, Cup a 1, Cup s 1, Cup s 3w, Jun a 1, Jun a 2, Jun a 3,
Jun o 4, Jun s 1, Jun v 1, Pla a 1, Pla a 2, Pla a 3, Aca s 13,
Blot 1, Blo t 3, Blo t 4, Blo t 5, Blo t 6, Blo t 10, Blo t 11, Blo
t 12, Blo t 13, Blo t 19, Der f 1, Der f 2, Der f 3, Der f 7, Der f
10, Der f 11, Der f 14, Der f 15, Der f 16, Der f 17, Der f 18w,
Der m 1, Der p 1, Der p 2, Der p 3, Der p 4, Der p 5, Der p 6, Der
p 7, Der p 8, Der p 9, Der p 10, Der p 11, Der p 14, Der p 20, Der
p 21, Eur m 2, Eur m 14, Gly d 2, Lep d 1, Lep d 2, Lep d 5, Lep d
7, Lep d 10, Lep d 13, Tyr p 2, Tyr p 13, Bos d 2, Bos d 3, Bos d
4, Bos d 5, Bos d 6, Bos d 7, Bos d 8, Can f 1, Can f 2, Can f 3,
Can f 4, Equ c 1, Equ c 2, Equ c 3, Equ c 4, Equ c 5, Fel d 1, Fel
d 2, Fel d 3, Fel d 4, Fel d 5w, Fel d 6w, Fel d 7w, Cav p 1, Cav p
2, Mus m 1, Rat n 1, Alt a 1, Alt a 3, Alt a 4, Alt a 5, Alt a 6,
Alt a 7, Alt a 8, Alt a 10, Alt a 12, Alt a 13, Cla h 2, Cla h 5,
Cla h 6, Cla h 7, Cla h 8, Cla h 9, Cla h 10, Cla h 12, Asp fl 13,
Asp f 1, Asp f 2, Asp f 3, Asp f 4, Asp f 5, Asp f 6, Asp f 7, Asp
f 8, Asp f 9, Asp f 10, Asp f 11, Asp f 12, Asp f 13, Asp f 15, Asp
f 16, Asp f 17, Asp f 18, Asp f 22w, Asp f 23, Asp f 27, Asp f 28,
Asp f 29, Asp n 14, Asp n 18, Asp n 25, Asp o 13, Asp o 21, Pen b
13, Pen b 26, Pen ch 13, Pen ch 18, Pen ch 20, Pen c 3, Pen c 13,
Pen c 19, Pen c 22w, Pen c 24, Pen o 18, Fus c 1, Fus c 2, Tri r 2,
Tri r 4, Tri t 1, Tri t 4, Cand a 1, Cand a 3, Cand b 2, Psi c 1,
Psi c 2, Cop c 1, Cop c 2, Cop c 3, Cop c 5, Cop c 7, Rho m 1, Rho
m 2, Mala f 2, Mala f 3, Mala f 4, Mala s 1, Mala s 5, Mala s 6,
Mala s 7, Mala s 8, Mala s 9, Mala s 10, Mala s 11, Mala s 12, Mala
s 13, Epi p 1, Aed a 1, Aed a 2, Api m 1, Api m 2, Api m 4, Api m
6, Api m 7, Born p 1, Bom p 4, Bla g 1, Bla g 2, Bla g 4, Bla g 5,
Bla g 6, Bla g 7, Bla g 8, Per a 1, Per a 3, Per a 6, Per a 7, Chi
k 10, Chi t 1-9, Chi t 1.01, Chi t 1.02, Chi t 2.0101, Chi t
2.0102, Chi t 3, Chi t 4, Chi t 5, Chi t 6.01, Chi t 6.02, Chi t 7,
Chi t 8, Chi t 9, Cte f 1, Cte f 2, Cte f 3, Tha p 1, Lep s 1, Dol
m 1, Dol m 2, Dol m 5, Dol a 5, Pol a 1, Pol a 2, Pol a 5, Pol d 1,
Pol d 4, Pol d 5, Pol e 1, Pol e 5, Pol f 5, Pol g 5, Pol m 5, Vesp
c 1, Vesp c 5, Vesp m 1, Vesp m 5, Ves f 5, Ves g 5, Ves m 1, Ves m
2, Ves m 5, Ves p 5, Ves s 5, Ves vi 5, Ves v 1, Ves v 2, Ves v 5,
Myr p 1, Myr p 2, Sol g 2, Sol g 4, Sol i 2, Sol i 3, Sol i 4, Sol
s 2, Tria p 1, Gad c 1, Sal s 1, Bos d 4, Bos d 5, Bos d 6, Bos d
7, Bos d 8, Gal d 1, Gal d 2, Gal d 3, Gal d 4, Gal d 5, Met e 1,
Pen a 1, Pen i 1, Pen m 1, Pen m 2, Tod p 1, Hel as 1, Hal m 1, Ran
e 1, Ran e 2, Bra j 1, Bra n 1, Bra o 3, Bra r 1, Bra r 2, Hor v
15, Hor v 16, Hor v 17, Hor v 21, Sec c 20, Tri a 18, Tri a 19, Tri
a 25, Tri a 26, Zea m 14, Zea m 25, Ory s 1, Api g 1, Api g 4, Api
g 5, Dau c 1, Dau c 4, Cor a 1.04, Cor a 2, Cor a 8, Fra a 3, Fra a
4, Mal d 1, Mal d 2, Mal d 3, Mal d 4, Pyr c 1, Pyr c 4, Pyr c 5,
Pers a 1, Pru ar 1, Pru ar 3, Pru av 1, Pru av 2, Pru av 3, Pru av
4, Pru d 3, Pru du 4, Pru p 3, Pru p 4, Aspa o 1, Cro s 1, Cro s 2,
Lac s 1, Vit v 1, Mus xp 1, Ana c 1, Ana c 2, Cit 13, Cit s 1, Cit
s 2, Cit s 3, Lit c 1, Sin a 1, Gly m 1, Gly m 2, Gly m 3, Gly m 4,
Vig r 1, Ara h 1, Ara h 2, Ara h 3, Ara h 4, Ara h 5, Ara h 6, Ara
h 7, Ara h 8, Len c 1, Len c 2, Pis s 1, Pis s 2, Act c 1, Act c 2,
Cap a lw, Cap a 2, Lyc e 1, Lyc e 2, Lyc e 3, Sola t 1, Sola t 2,
Sola t 3, Sola t 4, Ber e 1, Ber e 2, Jug n 1, Jug n 2, Jug r 1,
Jug r 2, Jug r 3, Ana o 1, Ana o 2, Ana o 3, Ric c 1, Ses i 1, Ses
i 2, Ses i 3, Ses i 4, Ses i 5, Ses i 6, Cuc m 1, Cuc m 2, Cuc m 3,
Ziz m 1, Ani s 1, Ani s 2, Ani s 3, Ani s 4, Arg r, Asc s 1, Car p
1, Den n 1, Hev b 1, Hev b 2, Hev b 3, Hev b 4, Hev b 5, Hev b
6.01, Hev b 6.02, Hev b 6.03, Hev b 7.01, Hev b 7.02, Hev b 8, Hev
b 9, Hev b 10, Hev b 11, Hev b 12, Hev b 13, Homs 1, Hom 2, Horn s
3, Horn s 4, Horn s 5 and Trip s 1.
[0063] According to a preferred embodiment of the present invention
the allergen derivative is hypoallergenic.
[0064] In order to induce a specific immune response in a mammal,
in particular in a human, without provoking an allergenic reaction
or by provoking a significantly reduced allergenic reaction, it is
preferred that the allergen or derivative thereof exhibits
hypoallergenic properties, i.e. the hypoallergenic molecule shows
no or significantly reduced IgE reactivity.
[0065] As used herein, the term "hypoallergenic" refers to the
ability of a peptide, polypeptide or protein derived from an
allergen with allergenic properties to induce the induction of T
cells specific for said allergen and exhibiting reduced or no
allergic reactions when administered to an individual. The reduced
or missing ability of "hypoallergenic" derivatives of an allergen
to induce an allergic reaction in an individual is obtained by
removing or destroying the IgE binding epitopes from said
allergens, however, by conserving the T cell epitopes present on
said allergens. This can be achieved, for instance, by splitting
the allergen into fragments with reduced or no IgE binding capacity
and optionally fusing some or all of said fragments in an order
together which does not correspond to the order of the fragments in
the wild-type allergen (see e.g. EP 1 440 979). Another method for
producing "hypoallergenic" molecules from allergens involves C-
and/or N-terminal deletions of the wild-type allergen (see e.g. EP
1 224 215). Of course it is also possible to generate
hypoallergenic molecules by introducing specific mutations
affecting one or more amino acid residues of the wild-type
allergen, whereby said modifications result in a loss of the
three-dimensional structure.
[0066] RNA vaccines are rendered hypoallergenic by targeting the
resulting protein into the ubiquitination pathway of the cell,
where the respective protein is degraded into hypoallergenic
peptides. This is achieved by fusing the sequence encoding
ubiquitin to the 5' end of the allergen encoding RNA.
Ubiquitination efficacy can be enhanced by mutating amino acid
residue 76 from glycine to alanine (G76.fwdarw.A76). Ubiquitination
efficacy can be further enhanced by mutating the first amino acid
of the allergen (methionine) to a destabilizing amino acid
(Arginine) (M77.fwdarw.R77). Alternatively, ubiquitination of the
resulting gene product can be achieved by adding a carboxyterminal
destabilizing sequence known as PEST sequence.
[0067] According to a preferred embodiment of the present invention
the hypoallergenic allergen derivative encoded by the RNA in the
vaccine exhibits an IgE reactivity which is at least 10%,
preferably at least 20%, more preferably at least 30%, in
particular at least 50%, lower than the IgE reactivity of the
wild-type allergen.
[0068] Hypoallergenicity of RNA vaccines can be routinely tested by
translating the RNA in vitro in a rabbit reticulocyte lysate
system. The resulting gene product will be analyzed by IgE western
blots using pools of appropriate patients' sera. Reduction of IgE
binding capacity of the respective hypoallergen will be assessed
compared to the IgE binding capacity of the wild-type molecule,
translated in said reticulocyte lysate system.
[0069] According to a particularly preferred embodiment of the
present invention the RNA molecule of the invention may encode for
more than one, preferably more than two, more preferably more than
three, even more preferably more than four, allergens or
derivatives thereof. In particular, the RNA molecule may encode for
Phl p 1, Phl p 2, Phl p 5 and Phl p6, or for Aln g 1, Cor a 1, Que
a 1, Car b 1 and Bet v 1.
[0070] The RNA molecule encoding the allergen or derivative thereof
is fused to at least one further peptide, polypeptide or
protein.
[0071] The allergen encoding RNA sequence can by fused to RNA
sequences encoding peptides, polypeptides, or proteins. These
peptides can be signal peptides that target the allergen into the
endoplasmic reticulum and thereby enhance protein secretion from
the cell, for example the human tissue plasminogen activator signal
peptide (hTPA). Said peptide or protein can be the
lysosome-associated membrane protein (LAMP) or the 20-amino acid
C-terminal tail of the lysosomal integral membrane protein-II
(LIMP-II). The LAMP/LIMP-II sequences are used to direct the
antigen protein to the major histocompatibility class II (MHC II)
vesicular compartment of transfected professional
antigen-presenting cells (APCs) thereby enhancing activation of T
helper cells which increases vaccine efficacy. Said proteins or
polypeptides can also be proteins that enhance the TH1 bias of the
vaccine, e.g. the heat shock protein 70 (HSP70), or bacterial
toxins like cholera toxin (CT) or related toxins such as heat
labile enterotoxin (LT) of Escherichia coli.
[0072] According to a preferred embodiment of the present invention
the RNA molecule comprises at least one further element selected
from the group consisting of replicase, .beta.-globin leader
sequence, cap0, cap 1 and poly A tail.
[0073] The RNA vaccine consists of the RNA sequence encoding the
respective allergen.
[0074] This RNA sequence can be the wild-type sequence of the
allergen or can be adapted with respect to its codon usage.
Adaption of codon usage can increase translation efficacy and
half-life of the RNA. A poly A tail consisting of at least 30
adenosine residues is attached to the 3' end of the RNA to increase
the half-life of the RNA. The 5' end of the RNA is capped with a
modified ribonucleotide with the structure m7G(5')ppp(5')N (cap 0
structure) or a derivative thereof which can be incorporated during
RNA synthesis or can be enzymatically engineered after RNA
transcription by using Vaccinia Virus Capping Enzyme (VCE,
consisting of mRNA triphosphatase, guanylyl-transferase and
guanine-7-methylransferase), which catalyzes the construction of
N7-monomethylated cap 0 structures. Cap 0 structure plays a crucial
role in maintaining the stability and translational efficacy of the
RNA vaccine. The 5' cap of the RNA vaccine can be further modified
by a 2'-O-Methyltransferase which results in the generation of a
cap 1 structure (m7 Gppp[m2'-O]N), which further increases
translation efficacy.
[0075] RNA vaccines can be further optimised by converting them
into self-replicating vaccines. Such vectors include replication
elements derived from alphaviruses and the substitution of the
structural virus proteins with the gene of interest.
Replicase-based RNA vaccines have been demonstrated to induce
antibody as well as cytotoxic responses at extremely low doses due
to immune activation mediated by virus-derived danger signals
(Ying, H. et al. (1999) Nat Med 5:823-827).
[0076] The RNA vaccine can also be a self-replicating RNA vaccine.
Self-replicating RNA vaccines consisting of a replicase RNA
molecule derived from semliki forest virus (SFV), sindbis virus
(SIN), venezuelan equine encephalitis virus (VEE), Ross-River virus
(RRV), or other viruses belonging to the alphavirus family.
Downstream of the replicase lies a subgenomic promoter that
controls replication of the allergen RNA followed by an artificial
poly A tail consisting of at least 30 adenosine residues.
[0077] According to another preferred embodiment of the present
invention the vaccine comprises further CpG-DNA and cytokines,
preferably interleukin (IL)-12 and IL-15.
[0078] The vaccine or vaccine formulation according to the present
invention can further include an adjuvant. "Adjuvant", according to
the present invention, refers to a compound or mixture that
enhances the immune response to an antigen. An adjuvant may also
serve as a tissue depot that slowly releases the antigen. Adjuvants
include among others complete Freund's adjuvant, incomplete
Freund's adjuvant, saponin, mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, Levamisol, CpG-DNA, oil or
hydrocarbon emulsions, and potentially useful adjuvants such as BCG
(bacille Calmette-Guerin) and Corynebacterium parvum.
[0079] Alternatively, or in addition, also immunostimulatory
proteins can be provided as an adjuvant or to increase the immune
response to a vaccine. Vaccination effectiveness may be enhanced by
co-administration of an immunostimulatory molecule (Salgaller and
Lodge, J. Surg. Oncol. (1988) 68:122), such as an
immunostimulatory, immunopotentiating or pro-inflammatory cytokine,
lymphokine, or chemokine with the vaccine, particularly with a
vector vaccine. For example, cytokines or cytokine genes such as
IL-2, IL-3, IL-12, IL-15, IL-18, IFN-gamma, IL-10, TGF-beta,
granulocyte-macrophage (GM)-colony stimulating factor (CSF) and
other colony stimulating factors, macrophage inflammatory factor,
Flt3 ligand (Lyman, Curr. Opin. Hematol., 1998, 5:192), CD40
ligand, as well as some key costimulatory molecules or their genes
(e.g., B7.1, B7.2) can be used. These immunostimulatory molecules
can be delivered systemically or locally as proteins or be encoded
by the RNA molecule or a further RNA molecule in the RNA vaccine of
the present invention. As immunostimulatory molecules also
polycationic peptides such as polyarginine may be employed.
[0080] According to a further preferred embodiment of the present
invention the vaccine is adapted for intramuscular, intradermal,
intravenous, transdermal, topical, or biolistic administration.
[0081] The RNA vaccine of the present invention may be administered
in various ways. One way, for instance, is to transfer in vivo the
RNA vaccine directly into a body (e.g. intramuscular, intradermal,
intravenous, intranasal etc.). Alternatively it is possible to
place RNA into cells (e.g. epidermal cells) outside of the body,
e.g. epidermal cells are transfected with the RNA vaccine in vitro
and then administered (transplanted) to a body. The cells can be
transfected by exogenous or heterologous RNA when such RNA has been
introduced inside the cell. The RNA can be introduced into the
cells by pulsing, i.e. incubating the cells with the RNA molecules
of the invention. Alternatively, the RNA can be introduced in vivo
by lipofection, as naked RNA, or with other transfection
facilitating agents (peptides, polymers, etc.). Synthetic cationic
lipids can be used to prepare liposomes for in vivo transfection.
Useful lipid compounds and compositions for transfer of nucleic
acids are, e.g. DODC, DOPE, CHOL, DMEDA, DDAB, DODAC, DOTAP and
DOTMA. Other molecules are also useful for facilitating
transfection of a nucleic acid in vivo, such as cationic
oligopeptides (e.g. WO 95/21931), peptides derived from DNA binding
proteins (e.g. WO96/25508), or cationic polymers (e.g. WO
95/21931). Also polyethylenimine and its derivatives,
polylactide-polyglycolide, and chitosan may be used. Alternatively,
RNA molecules can be introduced into the desired host cells by
methods known in the art, e.g. electroporation, microinjection,
cell fusion, DEAE dextran, calcium phosphate precipitation, or use
of a gene gun (biolistic transfection, see e.g. Tang et al., Nature
(1992) 356: 152-154).
[0082] Another aspect of the present invention relates to the use
of at least one RNA molecule as defined herein for the manufacture
of a vaccine for treating or preventing allergy.
[0083] A further aspect of the present invention relates to the use
of at least one RNA molecule as defined herein for the manufacture
of a vaccine for hyposensitising an individual to an allergen.
[0084] According to another preferred embodiment of the present
invention the vaccine is adapted for intramuscular, intradermal,
intravenous, transdermal, topical or biolistic administration.
[0085] Another aspect of the present invention relates to an
isolated RNA molecule comprising at least one nucleotide sequence
encoding at least one allergen or derivative thereof. Said RNA
molecule preferably comprises at least one nucleotide sequence
selected from the group consisting of cap0, cap1, 5' .beta.-globin
leader sequence, self-replicating RNA, recoded allergen sequence
and artificial poly-A tail, whereby Cap0--allergen sequence--poly A
tail is an especially preferred RNA molecule. Cap0 is useful for
the in vivo production of antibodies and with respect to
self-replicating RNA vaccines for the induction of allergen
specific T cells and IFN-gamma secretion.
[0086] The present invention is further illustrated by the
following figures and examples without being restricted
thereto.
[0087] FIG. 1 shows in vitro transfection of BHK-21 cells with RNA
(.beta.Gal-RNA) or self-replicating RNA (.beta.Gal-repRNA)
transcripts encoding .beta.-galactosidase. RNA transcripts with
(cap) or without (no cap) addition of a m7G(5')ppp(5')G cap
structure were tested. Untransfected cells served as background
control (untransfected). Data are shown as means.+-.SEM of three
independent transfection experiments.
[0088] FIGS. 2A and 2B show Phl p 5 specific IgG1 and IgG2a levels
after nucleic acid vaccination (FIG. 2A) and subsequent
sensitisation with recombinant allergen in alum (B). Sera were
diluted 1:1000 (A) and 1:100000 (FIG. 2B). Numbers on top of bars
represent average IgG1:IgG2a ratios for the respective group. Data
are shown as means.+-.SEM (n=4).
[0089] FIG. 3 shows Phl p 5 specific IgE measured via RBL release
assay. IgE levels were measured after vaccination with the
respective nucleic acid vaccines (grey bars) and after subsequent
sensitisation with recombinant allergen in alum (black bars).
Values are shown as means of % specific hexosaminidase
release.+-.SEM (n=4). ***: P<0.001.
[0090] FIGS. 4A and 4B show the number of IFN-gamma (FIG. 4A), IL-4
(FIG. 4B), and IL-5 (FIG. 4C) secreting splenocytes after in vitro
re-stimulation with recombinant Phl p 5 as determined by ELISPOT.
Data are shown as means.+-.SEM (n=4) of numbers of cytokine
secreting cells per 10.sup.6 splenocytes.
[0091] FIGS. 5A and 5B show the number of total leukocytes (FIG.
5A) and eosinophils (FIG. 5B) in BALF of sensitised mice after i.n.
application of allergen. Values are shown as means.+-.SEM (n=4). *:
P<0.05; **: P<0.01.
[0092] FIGS. 6A and 6B show the levels of IL-5 (FIG. 6A) and
IFN-.gamma. (FIG. 6B) in BALF of sensitised mice after i.n.
application of allergen. Values are shown as means.+-.SEM (n=4). *:
P<0.05; **: P<0.01; ***: P<0.001.
[0093] FIGS. 7A, 7B, and 7C show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Bet v 1.
[0094] FIGS. 8A, 8B, and 8C show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Car b 1.
[0095] FIGS. 9A, 9B, and 9C show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Cas s 1.
[0096] FIGS. 10A and 10B show the induction of Th 1 memory by RNA
pTNT-Phl p 1.
[0097] FIG. 11A, 11B, and 11C show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Phl p 6.
[0098] FIG. 12 shows the induction of Th 1 memory by RNA pTNT-Cor a
1.
[0099] FIG. 13 shows the induction of Th 1 memory by RNA pTNT-Aln g
1.
[0100] FIG. 14A, 14B, and 14C show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Fag s 1.
[0101] FIGS. 15A and 15B show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Phl p 2.
[0102] FIGS. 16A and 16B show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Phl p 7.
[0103] FIG. 17A, 17B, and 17C show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-hybrid (Phl p
1-2-5-6).
[0104] FIGS. 18A and 18B show the induction of Th 1 memory by RNA
pTNT-Cry j 1.
[0105] FIG. 19 shows the induction of Th 1 memory by RNA pTNT-Jun a
1.
[0106] FIG. 20 shows the induction of Th 1 memory by RNA pTNT-Amb a
1.
[0107] FIG. 21A, 21B, and 21C show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Api g 1.
[0108] FIGS. 22A and 22B show the induction of Th 1 memory by RNA
pTNT-Dau c 1.
[0109] FIG. 23A, 23B, and 23C show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Mal d 1.
[0110] FIG. 24A, 24B, and 24C show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Ova.
[0111] FIGS. 25A and 25B show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Beta-Casein.
[0112] FIG. 26 shows the induction of Th 1 memory responses by RNA
pTNT-Cyp c 1.
[0113] FIGS. 27A and 27B show the induction of Th 1 memory
responses by RNA pTNT-Fel d 1.
[0114] FIGS. 28A and 28B show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Der p 2.
[0115] FIG. 29A, 29B, and 29C show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Alt a 1.
[0116] FIGS. 30A and 30B show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Cla h 8.
[0117] FIGS. 31A and 31B show the induction of Th 1 memory by RNA
pTNT-Hev b 6.
[0118] FIG. 32 shows the induction of Th 1 memory by RNA
pTNT-hybrid (allergen).
[0119] FIGS. 33A and 3B show the induction of Th 1 memory and
suppression of IgE responses by RNA pTNT-Ara h 2.
[0120] FIGS. 34A and 34B show the induction of Th 1 memory by RNA
pTNT-Que a 1.
[0121] FIGS. 35A and 35 B show no induction of Th 1 memory by RNA
pTNT-Art v 1.
[0122] FIGS. 36A and 36B show no induction of Th 1 memory or
suppression of IgE responses by RNA pTNT-Ole e 1.
EXAMPLES
Example 1
[0123] In the present example it is shown, that RNA as well as
replicase-based RNA vaccines encoding the clinically relevant
timothy grass pollen allergen Phl p 5 can effectively prevent from
allergic responses.
[0124] Materials and Methods
[0125] Plasmids Used for RNA Transcription
[0126] Vector pTNT was purchased from Promega (Mannheim, Germany)
and includes some special features providing advantages over other
vectors. Two promoters, one for the SP6 and the other for the T7
polymerase, are present to allow SP6--as well as T7-based in vitro
transcription. They lie in tandem adjacent to the multiple cloning
site (MCS). A 5' .beta.-globin leader sequence helps to increase
the translation of several genes for a more rapid initiation of
translation. Another feature to enhance gene expression is its
synthetic poly(A)30 tail.
[0127] Vector pSin-Rep5 (Invitrogen, Austria) is derived from
sindbis alphavirus, which is an enveloped, positive-stranded RNA
virus. Alphavirus based replicon vectors lack viral structural
proteins, but maintain the replication elements (replicase)
necessary for cytoplasmic RNA self-amplification and expression of
the inserted genes via an alphaviral promoter.
[0128] The Phl p 5 gene was excised from vector pCMV-PhlpS via
NheUXbaI (Gabler et al. (2006), J Allergy Clin Immunol 118:734-741)
and ligated into the XbaI restriction site of pTNT and pSin-Rep5
resulting in pTNT-P5 and pSin-Rep5-P5 respectively.
[0129] RNA Transcription
[0130] Plasmids pTNT-P5 and pSin-Rep5-P5 were linearised with the
corresponding restriction enzymes; templates were purified via
Phenol-Chloroform-Isoamylalcohol extraction, followed by a single
Chloroform-Isoamylalcohol extraction. After addition of 1/10 volume
of 3M Na-acetate pH 5.2 plasmids were precipitated with 2 volumes
of 100% EtOH and washed 3 times with 70% EtOH.
[0131] All transcription reactions were performed with a T7 or SP6
RiboMAX.TM. Large Scale RNA Production Systems (Promega) according
to the manufacturer's protocol. Briefly, for a 100 .mu.l reaction,
20 .mu.l Transcription buffer, 30 .mu.l rNTPs, 5-10 .mu.g template,
and 10 .mu.l Enzyme mix were filled up to 100 ml with Nuclease-free
H2O and incubated for 2-3 h at 37.degree. C. When using the SP6
RiboMax kit, 20 .mu.l instead of 30 .mu.l rNTPs were used.
[0132] To mimic the capped structure of mRNA, a 5' 7-methyl
guanosine nucleotide (m7G(5')ppp(5')G) or cap analog (EPICENTRE,
USA) was incorporated during RNA synthesis. The rNTP mix was
prepared as a 25:25:25:22.5:2.5 mM mix of rATP, rCTP, rUTP, rGTP
and m7G(5')ppp(5')G.
[0133] Following transcription, RNA was precipitated by adding 1
volume of 5M ammonium acetate to the reaction tube and incubating
the mixture for 10-15 minutes on ice. After a centrifugation period
of 15 minutes (13000 rpm) at 4.degree. C. or room temperature, the
pellet was washed with 70% ethanol and resuspended in nuclease-free
H.sub.2O.
[0134] Results
[0135] In Vitro Transfection with RNA and Self-Replicating RNA
[0136] BHK-21 cells were transfected in vitro with two different
RNA transcripts encoding .beta.-galactosidase, either as
conventional RNA vaccine transcribed from vector pTNT-.beta.Gal
(.beta.Gal-RNA) or as self-replicating RNA transcribed from vector
pRep5-.beta.Gal (.beta.Gal-repRNA).
[0137] RNA transcripts were tested with or without addition of a
m7G(5')ppp(5')G cap structure. FIG. 1 shows that transfection with
equal amounts of self replicating RNA induces a 7.5-fold higher
expression of the transgene compared to conventional RNA.
Additionally, stabilising RNA with a cap structure is essential for
in vitro transfection/translation of RNA.
[0138] RNA-Based Vaccines Encoding the Allergen Phlp 5 are
Immuno-Genic and Prevent from IgE Induction
[0139] To investigate the potential of RNA-based vaccines to
prevent from induction of allergy, female BALB/c mice were
immunised with either conventional RNA endcoding Phl p 5 or
self-replicating RNA encoding Phl p 5. To estimate the potency of
the RNA vaccines also corresponding groups were immunised with the
same doses of a conventional DNA vaccine (pCMV-P5) and a
self-replicating DNA vaccine (pSin-P5) encoding Phl p 5. Mice were
immunised three times in weekly intervals and two weeks later
sensitised via two injections of recombinant Phl p 5 complexed with
alum, a protocol known to induce an allergic phenotype,
characterised by high levels of IgE and a TH2 biased cytokine
profile of T cells.
[0140] FIG. 2A shows, that both RNA vaccines induce similar humoral
immune responses compared to the self-replicating DNA vaccine
pSin-P5. In contrast, the humoral immune response induced by the
conventional DNA vaccine pCMV-P5 was approximately one order of
magnitude higher compared to the other vaccines. All vaccine types
displayed a clearly TH1 biased serological profile characterised by
low IgG1/IgG2a ratios and no induction of functional IgE as
measured by RBL release assay (FIG. 3, grey bars).
[0141] After sensitisation, the control group, that had not been
pre-immunised, showed a strictly TH2 biased serology with high IgG1
levels and a high IgG1/IgG2a ratio, indicative of an allergic
sensitisation. In contrast, all vaccinated groups maintained a TH1
balanced immunophenotype (FIG. 2B). Pre-vaccination with both types
of RNA vaccines induced similar or better suppression of IgE
induction compared to control animals as their DNA counterparts
(FIG. 3, black bars). Overall, pre-vaccination with both types of
RNA vaccines resulted in a 93% suppression of IgE induction upon
allergic sensitisation.
[0142] RNA-Based Vaccines Induce a TH1 Biased T Cell Memory
[0143] Two weeks after the final sensitisation, splenocytes were
re-stimulated in vitro with recombinant Phl p 5 protein to assess
their TH1/TH2 profile. Therefore, the number of IFN-.gamma., IL-4,
and IL-5 secreting cells was determined via ELISPOT.
[0144] All groups pre-vaccinated with nucleic acid vaccines showed
significant induction of IFN-.gamma. secreting cells (FIG. 4A)
compared to the control group. Simultaneously, the amount of cells
secreting the TH2 type cytokines IL-4 (FIG. 4B) and IL-5 (FIG. 4C)
were suppressed, indicating that similar to DNA vaccines, RNA
vaccines could establish a TH1 biased antigen specific memory, that
could be reactivated upon subsequent allergen exposure.
[0145] RNA-Based Vaccines Alleviate Allergen Induced Lung
Inflammation
[0146] To investigate the effect of RNA-vaccination on the
induction of lung pathology, two weeks after the last
sensitisation, lung inflammation was induced by two daily i.n.
applications of 1 .mu.g recombinant Phl p 5. This protocol induced
strong infiltration of leukocytes into the broncho alveolar lavage
fluid (BALF) of sensitised mice (FIG. 5A, control). Approximately
80% of the infiltrating leukocytes were eosinophils (FIG. 5B). In
contrast, pre-vaccinated mice showed significantly reduced numbers
of total leukocyte infiltrate, and an even greater reduction with
respect to eosinophils.
[0147] The reduction of inflammatory infiltrate was also reflected
by a strong suppression of IL-5 in the BALF (FIG. 6A). The
suppression of IL-5 was inversely correlated with an induction of
IFN-.gamma. FIG. 6B).
CONCLUSION
[0148] DNA vaccines hold great promise for prevention and treatment
of allergic diseases. However, hypothetical risks associated with
DNA vaccines question the use of this novel type of vaccine for
clinical use in healthy adults or even children.
[0149] In this example it could be demonstrated for the first time,
that naked RNA vaccination with a clinically relevant allergen can
prevent from induction of allergy to the same extent as a
comparable DNA vaccine applied at the same dosage.
[0150] To address the problem of producing larger quantities of
RNA, conventional RNA was compared to self-replicating RNA derived
from a Sindbis virus replicon. In vitro transfection with both
types of RNA demonstrated that antigen expression depends among
other factors on the addition of a m7G(5')ppp(5')G cap analogon.
The majority of eukaryotic mRNAs is known to possess such a
m7G(5')ppp(5')G cap structure at the 5'-end, which is important for
binding translation initiation factors and contributes to mRNA
stability. Additionally, it could be shown, that similar amounts of
self-replicating RNA translate into 7-fold higher levels of
proteins (FIG. 1), which can easily be attributed to the
self-amplification of subgenomic RNA encoding the respective
antigen. This is in contrast to self-replicating DNA vaccines,
where protein expression is low compared to conventional DNA
vaccines, an effect that has been attributed to the induction of
apoptosis in transfected cells. Yet, the expression of RNA vaccines
is only transient and therefore comparable to cells that undergo
apoptosis shortly after transfection with self-replicating
vaccines. Indeed self-replicating RNA vaccines induce similar
humoral immune responses compared to self-replicating DNA vaccines
(FIG. 2A), whereas the conventional DNA vaccine--with its
continuous expression of antigen--displays the highest humoral
immune response.
[0151] Although in the present example the self-replicating nucleic
acid vaccines were applied at a five-fold reduced dose compared to
conventional RNA/DNA vaccines, a similar induction of TH1
memory--indicated by a boost of IgG2a after subsequent
sensitisation with recombinant allergen in alum (FIG. 2B) and a TH1
cytokine profile of re-stimulated splenocytes--as well as a high
protective capability (FIG. 3)--were observed. Here, both RNA
vaccines, and the self-replicating DNA vaccine show an even higher
protective capacity than the conventional DNA vaccine, albeit the
latter induces higher levels of intact antigen and higher humoral
immune responses. This indicates that a vaccine induced long
lasting secretion of the allergen may be counter-productive
compared to short-term vaccine expression as seen with RNA and
self-replicating vaccines.
[0152] RNA vaccination also resulted in a similar reduction of lung
infiltration after i.n. provocation with allergen compared to DNA
vaccines (FIG. 5A), which was mainly due to a drastic decrease in
the amount of eosinophils in BALF (FIG. 5B). This correlated with a
reduction of IL-5 (FIG. 6A) and induction of moderate levels of
IFN-.gamma. (FIG. 2B) in the lung, indicating that the
vaccine-induced generation of TH1 cells also affects the TH1/TH2
cytokine balance in the lung. Although in viral models IFN-.gamma.
in the lung can have detrimental effects on asthma and lung
pathology, this seems to be an indirect effect as IFN-.gamma. can
activate lung epithelial cells to recruit more TH2 cells into the
tissue. Indeed, in allergy models, it could be shown, that
redirecting TH2 immunity towards a more balanced TH1 milieu has a
beneficial effect on lung inflammation and airway hyperreactivity,
mainly by counterregulating IL-5 and IL-13 (Ford, J. G. et al.
(2001) J Immunol 167:1769-1777).
[0153] Taken together, it could be demonstrated, that RNA-based
vaccines can induce significant protection from allergic
sensitisation, and that by using self-replicating RNA-vaccines,
this effect can be achieved at low doses. Given the excellent
safety profile of RNA vaccines, this opens the door to clinical
application of RNA vaccines not only in a therapeutic setting but
also in healthy individuals with a high risk for development of
allergic disorders.
Example 2
Materials and Methods
[0154] Plasmids and RNA Transcription
[0155] As described for example 1, the cDNA encoding Bet v 1 was
cloned into vector pTNT. RNA transcripts were prepared as described
and capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0156] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0157] Immunization and Sensitization
[0158] Mice were immunized with RNA pTNT-Bet v 1 three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 .mu.g recombinant Bet v 1 complexed with alum to
induce an allergic phenotype. Control animals were only sensitized
and did not receive pre-vaccination with the RNA vaccine.
[0159] Measurement of Th 1 Memory Induction and Protection
[0160] One week after the last sensitization, allergen specific
serum IgG2a and IgE were measured by ELISA and RBL as described for
experiment 1. Ten days after the final sensitization, splenocytes
were re-stimulated in vitro with recombinant Bet v 1 for 72 h and
cell culture supernatants were analyzed for IFN-.gamma. as an
indicator of allergen-specific Th 1 cell activation
[0161] Results
[0162] Pre-vaccination with RNA pTNT-Bet v 1 (hatched bars)
resulted in recruitment of allergen-specific Th1 cells as indicated
by the increased induction of IgG2a (FIG. 7A) and secretion of
IFN-.gamma. (FIG. 7B) in contrast to sensitization controls (black
bars) or naive mice (white bars). This Th 1 priming was able to
suppress the induction of allergen specific IgE responses (FIG.
7C)
Example 3
Materials and Methods
[0163] Plasmids and RNA Transcription
[0164] As described for example 1, the cDNA encoding Car b 1 was
cloned into vector pTNT. RNA transcripts were prepared as described
and capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0165] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0166] Immunization and Sensitization
[0167] Mice were immunized with RNA pTNT-Car b 1 three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 .mu.g recombinant Car b 1 complexed with alum to
induce an allergic phenotype. Control animals were only sensitized
and did not receive pre-vaccination with the RNA vaccine.
[0168] Measurement of Th 1 Memory Induction and Protection
[0169] One week after the last sensitization, allergen specific
serum IgG2a and IgE were measured by ELISA and RBL as described for
experiment 1. Ten days after the final sensitization, splenocytes
were re-stimulated in vitro with recombinant Car b 1 for 72 h and
cell culture supernatants were analyzed for IFN-.gamma. as an
indicator of allergen-specific Th 1 cell activation
[0170] Results
[0171] Pre-vaccination with RNA pTNT-Car b 1 (hatched bars)
resulted in recruitment of allergen-specific Th1 cells as indicated
by the increased induction of IgG2a (FIG. 8A) and secretion of
IFN-.gamma. (FIG. 8B) in contrast to sensitization controls (black
bars) or naive mice (white bars). This Th 1 priming was able to
suppress the induction of allergen specific IgE responses (FIG.
8C)
Example 4
Materials and Methods
[0172] Plasmids and RNA Transcription
[0173] As described for example 1, the cDNA encoding Cas s 1 was
cloned into vector pTNT. RNA transcripts were prepared as described
and capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0174] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0175] Immunization and Sensitization
[0176] Mice were immunized with RNA pTNT-Cas s 1 three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 .mu.g recombinant Cas s 1 complexed with alum to
induce an allergic phenotype. Control animals were only sensitized
and did not receive pre-vaccination with the RNA vaccine.
[0177] Measurement of Th 1 Memory Induction and Protection
[0178] One week after the last sensitization, allergen specific
serum IgG2a and IgE were measured by ELISA and RBL as described for
experiment 1. Ten days after the final sensitization, splenocytes
were re-stimulated in vitro with recombinant Cas s 1 for 72 h and
cell culture supernatants were analyzed for IFN-.gamma. as an
indicator of allergen-specific Th 1 cell activation.
[0179] Results
[0180] Pre-vaccination with RNA pTNT-Cas s 1 (hatched bars)
resulted in recruitment of allergen-specific Th 1 cells as
indicated by the increased induction of IgG2a (FIG. 9A) and
secretion of IFN-.gamma. (FIG. 9B) in contrast to sensitization
controls (black bars) or naive mice (white bars). This Th 1 priming
was able to suppress the induction of allergen specific IgE
responses (FIG. 9C)
Example 5
Materials and Methods
[0181] Plasmids and RNA Transcription
[0182] As described for example 1, the cDNA encoding Phl p 1 was
cloned into vector pTNT. RNA transcripts were prepared as described
and capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0183] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0184] Immunization and Sensitization
[0185] Mice were immunized with RNA pTNT-Phl p 1 three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 .mu.g recombinant Phl p 1 complexed with alum to
induce an allergic phenotype. Control animals were only sensitized
and did not receive pre-vaccination with the RNA vaccine.
[0186] Measurement of Th 1 Memory Induction and Protection
[0187] One week after the last sensitization, allergen specific
serum IgG2a was measured by ELISA as described for experiment 1.
Ten days after the final sensitization, splenocytes were
re-stimulated in vitro with recombinant Phl p 1 for 72 h and cell
culture supernatants were analyzed for IFN-.gamma. as an indicator
of allergen-specific Th 1 cell activation.
[0188] Results
[0189] Pre-vaccination with RNA pTNT-Phl p 1 (hatched bars)
resulted in recruitment of allergen-specific Th 1 cells as
indicated by the increased induction of IgG2a (FIG. 10A) and
secretion of IFN-.gamma. (FIG. 10B) in contrast to sensitization
controls (black bars) or naive mice (white bars).
Example 6
Materials and Methods
[0190] Plasmids and RNA Transcription
[0191] As described for example 1, the cDNA encoding Phl p 6 was
cloned into vector pTNT. RNA transcripts were prepared as described
and capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0192] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0193] Immunization and Sensitization
[0194] Mice were immunized with RNA pTNT-Phl p 6 three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 .mu.g recombinant Phl p 6 complexed with alum to
induce an allergic phenotype. Control animals were only sensitized
and did not receive pre-vaccination with the RNA vaccine.
[0195] Measurement of Th 1 Memory Induction and Protection
[0196] One week after the last sensitization, allergen specific
serum IgG2a and IgE were measured by ELISA and RBL as described for
experiment 1. Ten days after the final sensitization, splenocytes
were re-stimulated in vitro with recombinant Phl p 6 for 72 h and
cell culture supernatants were analyzed for IFN-.gamma. as an
indicator of allergen-specific Th 1 cell activation.
[0197] Results
[0198] Pre-vaccination with RNA pTNT-Phl p 6 (hatched bars)
resulted in recruitment of allergen-specific Th1 cells as indicated
by the increased induction of IgG2a (FIG. 11A) and secretion of
IFN-.gamma. (FIG. 11B) in contrast to sensitization controls (black
bars) or naive mice (white bars). This Th 1 priming was able to
suppress the induction of allergen specific IgE responses (FIG.
11C).
Example 7
Materials and Methods
[0199] Plasmids and RNA Transcription
[0200] As described for example 1, the cDNA encoding Cor a 1 was
cloned into vector pTNT. RNA transcripts were prepared as described
and capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0201] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0202] Immunization and Sensitization
[0203] Mice were immunized with RNA pTNT-Cor a 1 three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 .mu.g recombinant Cor a 1 complexed with alum to
induce an allergic phenotype. Control animals were only sensitized
and did not receive pre-vaccination with the RNA vaccine.
[0204] Measurement of Th 1 Memory Induction and Protection
[0205] One week after the last sensitization, allergen specific
serum IgG2a was measured by ELISA.
[0206] Results
[0207] Pre-vaccination with RNA pTNT-Cor a 1 (hatched bars)
resulted in recruitment of allergen-specific Th 1 cells as
indicated by the increased induction of IgG2a (FIG. 12) in contrast
to sensitization controls (black bars).
Example 8
Materials and Methods
[0208] Plasmids and RNA Transcription
[0209] As described for example 1, the cDNA encoding Aln g 1 was
cloned into vector pTNT. RNA transcripts were prepared as described
and capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0210] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0211] Immunization and Sensitization
[0212] Mice were immunized with RNA pTNT-Aln g 1 three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 .mu.g recombinant Aln g 1 complexed with alum to
induce an allergic phenotype. Control animals were only sensitized
and did not receive pre-vaccination with the RNA vaccine.
[0213] Measurement of Th 1 Memory Induction and Protection
[0214] Ten days after the final sensitization, splenocytes were
re-stimulated in vitro with recombinant Aln g 1 for 72 h and cell
culture supernatants were analyzed for IFN-.gamma. as an indicator
of allergen-specific Th 1 cell activation.
[0215] Results
[0216] Pre-vaccination with RNA pTNT-Aln g 1 (hatched bars)
resulted in recruitment of allergen-specific Th 1 cells as
indicated by the increased secretion of IFN-.gamma. (FIG. 13) in
contrast to sensitization controls (black bars) or naive mice
(white bars).
Example 9
Materials and Methods
[0217] Plasmids and RNA Transcription
[0218] As described for example 1, the cDNA encoding Fag s 1 was
cloned into vector pTNT. RNA transcripts were prepared as described
and capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0219] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0220] Immunization and Sensitization
[0221] Mice were immunized with RNA pTNT-Fag s 1 three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 .mu.g recombinant Fag s 1 complexed with alum to
induce an allergic phenotype. Control animals were only sensitized
and did not receive pre-vaccination with the RNA vaccine.
[0222] Measurement of Th 1 Memory Induction and Protection
[0223] One week after the last sensitization, allergen specific
serum IgG2a and IgE were measured by ELISA and RBL as described for
experiment 1. Ten days after the final sensitization, splenocytes
were re-stimulated in vitro with recombinant Fag s 1 for 72 h and
cell culture supernatants were analyzed for IFN-.gamma. as an
indicator of allergen-specific Th 1 cell activation.
[0224] Results
[0225] Pre-vaccination with RNA pTNT-Fag s 1 (hatched bars)
resulted in recruitment of allergen-specific Th 1 cells as
indicated by the increased induction of IgG2a (FIG. 14A) and
secretion of IFN-.gamma. (FIG. 14B) in contrast to sensitization
controls (black bars) or naive mice (white bars). This Th 1 priming
was able to suppress the induction of allergen specific IgE
responses (FIG. 14C).
Example 10
Materials and Methods
[0226] Plasmids and RNA Transcription
[0227] As described for example 1, the cDNA encoding Phl p 2 was
cloned into vector pTNT. RNA transcripts were prepared as described
and capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0228] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0229] Immunization and Sensitization
[0230] Mice were immunized with RNA pTNT-Phl p 2 three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 .mu.g recombinant Phl p 2 complexed with alum to
induce an allergic phenotype. Control animals were only sensitized
and did not receive pre-vaccination with the RNA vaccine.
[0231] Measurement of Th 1 Memory Induction and Protection
[0232] One week after the last sensitization, allergen specific
serum IgE was measured by RBL as described for experiment 1. Ten
days after the final sensitization, splenocytes were re-stimulated
in vitro with recombinant Phl p 2 for 72 h and cell culture
supernatants were analyzed for IFN-.gamma. as an indicator of
allergen-specific Th 1 cell activation
[0233] Results
[0234] Pre-vaccination with RNA pTNT-Phl p 2 (hatched bars)
resulted in recruitment of allergen-specific Th 1 cells as
indicated by the increased secretion of IFN-.gamma. (FIG. 15A) in
contrast to sensitization controls (black bars) or naive mice
(white bars). This Th1 priming was able to suppress the induction
of allergen specific IgE responses (FIG. 15B).
Example 11
Materials and Methods
[0235] Plasmids and RNA Transcription
[0236] As described for example 1, the cDNA encoding Phl p 7 was
cloned into vector pTNT. RNA transcripts were prepared as described
and capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0237] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0238] Immunization and Sensitization
[0239] Mice were immunized with RNA pTNT-Phl p 7 three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 .mu.g recombinant Phl p 7 complexed with alum to
induce an allergic phenotype. Control animals were only sensitized
and did not receive pre-vaccination with the RNA vaccine.
[0240] Measurement of Th 1 Memory Induction and Protection
[0241] One week after the last sensitization, allergen specific
serum IgE was measured by RBL as described for experiment 1.
[0242] Results
[0243] Pre-vaccination with RNA pTNT-Phl p 7 (hatched bars)
resulted in recruitment of allergen-specific Th 1 cells as
indicated by the increased induction of IFN-.gamma. (FIG. 16A) in
contrast to sensitization controls (black bars) or naive mice
(white bars). This Th 1 priming was able to suppress the induction
of allergen specific IgE responses (FIG. 16B).
Example 12
Materials and Methods
[0244] Plasmids and RNA Transcription
[0245] As described for example 1, a hybrid cDNA encoding Phl p 1,
Phl p 2, Phl p 5, and Phl p 6 (Linhart B. and Valenta R., Int Arch
Allergy Immunol (2004) 134:324-331) was cloned into vector pTNT.
RNA transcripts were prepared as described and capped using a
ScriptCap kit (Ambion) according to the manufacturer's
protocol.
[0246] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0247] Immunization and Sensitization
[0248] Mice were immunized with RNA pTNT-hybrid (Phl p 1-2-5-6)
three times in weekly intervals and were sensitized one week later
via two weekly injections of 1 .mu.g recombinant Phl p 1, Phl p 2,
Phl p 5, and Phl p 6 complexed with alum to induce an allergic
phenotype. Control animals were only sensitized and did not receive
pre-vaccination with the RNA vaccine.
[0249] Measurement of Th 1 Memory Induction and Protection
[0250] One week after the last sensitization, allergen specific
serum IgG2a and IgE were measured by ELISA and RBL as described for
experiment 1. Ten days after the final sensitization, splenocytes
were re-stimulated in vitro with recombinant allergens for 72 h and
cell culture supernatants were analyzed for IFN-.gamma. as an
indicator of allergen-specific Th 1 cell activation.
[0251] Results
[0252] Pre-vaccination with RNA pTNT-hybrid (Phl p 1-2-5-6)
(hatched bars) resulted in recruitment of allergen-specific Th1
cells as indicated by the increased induction of IgG2a (FIG. 17A)
and secretion of IFN-.gamma. (FIG. 17B) in contrast to
sensitization controls (black bars) or naive mice (white bars).
This Th1 priming was able to suppress the induction of allergen
specific IgE responses (FIG. 17C).
Example 13
Materials and Methods
[0253] Plasmids and RNA Transcription
[0254] As described for example 1, the cDNA encoding Cry j 1 was
cloned into vector pTNT. RNA transcripts were prepared as described
and capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0255] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0256] Immunization and Sensitization
[0257] Mice were immunized with RNA pTNT-Cry j 1 three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 .mu.g recombinant Cry j 1 complexed with alum to
induce an allergic phenotype. Control animals were only sensitized
and did not receive pre-vaccination with the RNA vaccine.
[0258] Measurement of Th 1 Memory Induction and Protection
[0259] One week after the last sensitization, allergen specific
serum IgG2a were measured by ELISA as described for experiment 1.
Ten days after the final sensitization, splenocytes were
re-stimulated in vitro with recombinant Cry j 1 for 72 h and cell
culture supernatants were analyzed for IFN-.gamma. as an indicator
of allergen-specific Th 1 cell activation.
[0260] Results
[0261] Pre-vaccination with RNA pTNT-Cry j 1 (hatched bars)
resulted in recruitment of allergen-specific Th1 cells as indicated
by the increased induction of IgG2a (FIG. 18A) and secretion of
IFN-.gamma. (FIG. 18B) in contrast to sensitization controls (black
bars) or naive mice (white bars).
Example 14
Materials and Methods
[0262] Plasmids and RNA Transcription
[0263] As described for example 1, the cDNA encoding Jun a 1 was
cloned into vector pTNT. RNA transcripts were prepared as described
and capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0264] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0265] Immunization and Sensitization
[0266] Mice were immunized with RNA pTNT-Jun a 1 three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 .mu.g recombinant Jun a 1 complexed with alum to
induce an allergic phenotype. Control animals were only sensitized
and did not receive pre-vaccination with the RNA vaccine.
[0267] Measurement of Th 1 Memory Induction and Protection
[0268] Ten days after the final sensitization, splenocytes were
re-stimulated in vitro with recombinant Jun a 1 for 72 h and cell
culture supernatants were analyzed for IFN-.gamma. as an indicator
of allergen-specific Th 1 cell activation
[0269] Results
[0270] Pre-vaccination with RNA pTNT-Jun a 1 (hatched bars)
resulted in recruitment of allergen-specific Th1 cells as indicated
by the increased induction of IFN-.gamma. (FIG. 19) in contrast to
sensitization controls (black bars) or naive mice (white bars).
Example 15
Materials and Methods
[0271] Plasmids and RNA Transcription
[0272] As described for example 1, the cDNA encoding Amb a 1 was
cloned into vector pTNT. RNA transcripts were prepared as described
and capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0273] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0274] Immunization and Sensitization
[0275] Mice were immunized with RNA pTNT-Amb a 1 three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 .mu.g purified Amb a 1 complexed with alum to
induce an allergic phenotype. Control animals were only sensitized
and did not receive pre-vaccination with the RNA vaccine.
[0276] Measurement of Th 1 Memory Induction and Protection
[0277] Ten days after the final sensitization, splenocytes were
re-stimulated in vitro with purified Amb a 1 for 72 h and cell
culture supernatants were analyzed for IFN-.gamma. as an indicator
of allergen-specific Th 1 cell activation.
[0278] Results
[0279] Pre-vaccination with RNA pTNT-Amb a 1 (hatched bars)
resulted in recruitment of allergen-specific Th1 cells as indicated
by the increased secretion of IFN-.gamma. (FIG. 20) in contrast to
sensitization controls (black bars) or naive mice (white bars).
Example 16
Materials and Methods
[0280] Plasmids and RNA Transcription
[0281] As described for example 1, the cDNA encoding Api g 1 was
cloned into vector pTNT. RNA transcripts were prepared as described
and capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0282] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0283] Immunization and Sensitization
[0284] Mice were immunized with RNA pTNT-Api g 1 three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 .mu.g recombinant Api g 1 complexed with alum to
induce an allergic phenotype. Control animals were only sensitized
and did not receive pre-vaccination with the RNA vaccine.
[0285] Measurement of Th 1 Memory Induction and Protection
[0286] One week after the last sensitization, allergen specific
serum IgG2a and IgE were measured by ELISA and RBL as described for
experiment 1. Ten days after the final sensitization, splenocytes
were re-stimulated in vitro with recombinant Api g 1 for 72 h and
cell culture supernatants were analyzed for IFN-.gamma. as an
indicator of allergen-specific Th 1 cell activation.
[0287] Results
[0288] Pre-vaccination with RNA pTNT-Api g 1 (hatched bars)
resulted in recruitment of allergen-specific Th 1 cells as
indicated by the increased induction of IgG2a (FIG. 21A) and
secretion of IFN-.gamma. (FIG. 21B) in contrast to sensitization
controls (black bars) or naive mice (white bars). This Th 1 priming
was able to suppress the induction of allergen specific IgE
responses (FIG. 21C)
Example 17
Materials and Methods
[0289] Plasmids and RNA Transcription
[0290] As described for example 1, the cDNA encoding Dau c 1 was
cloned into vector pTNT. RNA transcripts were prepared as described
and capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0291] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0292] Immunization and Sensitization
[0293] Mice were immunized with RNA pTNT-Dau c 1 three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 .mu.g recombinant Dau c 1 complexed with alum to
induce an allergic phenotype. Control animals were only sensitized
and did not receive pre-vaccination with the RNA vaccine.
[0294] Measurement of Th 1 Memory Induction and Protection
[0295] One week after the last sensitization, allergen specific
serum IgG2a was measured by ELISA as described for experiment 1.
Ten days after the final sensitization, splenocytes were
re-stimulated in vitro with recombinant Dau c 1 for 72 h and cell
culture supernatants were analyzed for IFN-.gamma. as an indicator
of allergen-specific Th 1 cell activation.
[0296] Results
[0297] Pre-vaccination with RNA pTNT-Dau c 1 (hatched bars)
resulted in recruitment of allergen-specific Th 1 cells as
indicated by the increased induction of IgG2a (FIG. 22A) and
secretion of IFN-.gamma. (FIG. 22B) in contrast to sensitization
controls (black bars) or naive mice (white bars).
Example 18
Materials and Methods
[0298] Plasmids and RNA Transcription
[0299] As described for example 1, the cDNA encoding Mal d 1 was
cloned into vector pTNT. RNA transcripts were prepared as described
and capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0300] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0301] Immunization and Sensitization
[0302] Mice were immunized with RNA pTNT-Mal d 1 three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 .mu.g recombinant Mal d 1 complexed with alum to
induce an allergic phenotype. Control animals were only sensitized
and did not receive pre-vaccination with the RNA vaccine.
[0303] Measurement of Th 1 Memory Induction and Protection
[0304] One week after the last sensitization, allergen specific
serum IgG2a and IgE were measured by ELISA and RBL as described for
experiment 1. Ten days after the final sensitization, splenocytes
were re-stimulated in vitro with recombinant Mal d 1 for 72 h and
cell culture supernatants were analyzed for IFN-.gamma. as an
indicator of allergen-specific Th 1 cell activation.
[0305] Results
[0306] Pre-vaccination with RNA pTNT-Mal d 1 (hatched bars)
resulted in recruitment of allergen-specific Th 1 cells as
indicated by the increased induction of IgG2a (FIG. 23A) and
secretion of IFN-.gamma. (FIG. 23B) in contrast to sensitization
controls (black bars) or naive mice (white bars). This Th1 priming
was able to suppress the induction of allergen specific IgE
responses (FIG. 23C).
Example 19
Materials and Methods
[0307] Plasmids and RNA Transcription
[0308] As described for example 1, the cDNA encoding Ova was cloned
into vector pTNT. RNA transcripts were prepared as described and
capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0309] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0310] Immunization and Sensitization
[0311] Mice were immunized with RNA pTNT-Ova three times in weekly
intervals and were sensitized one week later via two weekly
injections of 1 .mu.g recombinant Ova complexed with alum to induce
an allergic phenotype. Control animals were only sensitized and did
not receive pre-vaccination with the RNA vaccine.
[0312] Measurement of Th 1 Memory Induction and Protection
[0313] One week after the last sensitization, allergen specific
serum IgG2a and IgE were measured by ELISA and RBL as described for
experiment 1. Ten days after the final sensitization, splenocytes
were re-stimulated in vitro with recombinant Ova for 72 h and cell
culture supernatants were analyzed for IFN-.gamma. as an indicator
of allergen-specific Th 1 cell activation.
[0314] Results
[0315] Pre-vaccination with RNA pTNT-Ova (hatched bars) resulted in
recruitment of allergen-specific Th1 cells as indicated by the
increased induction of IgG2a (FIG. 24A) and secretion of
IFN-.gamma. (FIG. 24B) in contrast to sensitization controls (black
bars) or naive mice (white bars). This Th 1 priming was able to
suppress the induction of allergen specific IgE responses (FIG.
24C).
Example 20
Materials and Methods
[0316] Plasmids and RNA Transcription
[0317] As described for example 1, the cDNA encoding Beta-Casein
was cloned into vector pTNT. RNA transcripts were prepared as
described and capped using a ScriptCap kit (Ambion) according to
the manufacturer's protocol.
[0318] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0319] Immunization and Sensitization
[0320] Mice were immunized with RNA pTNT-Beta-Casein three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 .mu.g recombinant Beta-Casein complexed with alum
to induce an allergic phenotype. Control animals were only
sensitized and did not receive pre-vaccination with the RNA
vaccine.
[0321] Measurement of Th 1 Memory Induction and Protection
[0322] One week after the last sensitization, allergen specific
serum IgE was measured by RBL as described for experiment 1. Ten
days after the final sensitization, splenocytes were re-stimulated
in vitro with recombinant Beta-Casein for 72 h and cell culture
supernatants were analyzed for IFN-.gamma. as an indicator of
allergen-specific Th 1 cell activation
[0323] Results
[0324] Pre-vaccination with RNA pTNT-Beta-Casein (hatched bars)
resulted in recruitment of allergen-specific Th 1 cells as
indicated by the increased secretion of IFN-.gamma. (FIG. 25A) in
contrast to sensitization controls (black bars) or naive mice
(white bars). This Th 1 priming was able to suppress the induction
of allergen specific IgE responses (FIG. 25B).
Example 21
Materials and Methods
[0325] Plasmids and RNA Transcription
[0326] As described for example 1, the cDNA encoding Cyp c 1 was
cloned into vector pTNT. RNA transcripts were prepared as described
and capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0327] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0328] Immunization and Sensitization
[0329] Mice were immunized with RNA pTNT-Cyp c 1 three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 .mu.g recombinant Cyp c 1 complexed with alum to
induce an allergic phenotype. Control animals were only sensitized
and did not receive pre-vaccination with the RNA vaccine.
[0330] Measurement of Th 1 Memory Induction and Protection
[0331] One week after the last sensitization, allergen specific
serum IgG2a was measured by ELISA as described for experiment
1.
[0332] Results
[0333] Pre-vaccination with RNA pTNT-Cyp c 1 (hatched bars)
resulted in recruitment of allergen-specific Th 1 cells as
indicated by the increased induction of IgG2a (FIG. 26).
Example 22
Materials and Methods
[0334] Plasmids and RNA Transcription
[0335] As described for example 1, the cDNA encoding Fel d 1 was
cloned into vector pTNT. RNA transcripts were prepared as described
and capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0336] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0337] Immunization and Sensitization
[0338] Mice were immunized with RNA pTNT-Fel d 1 three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 .mu.g recombinant Fel d 1 complexed with alum to
induce an allergic phenotype. Control animals were only sensitized
and did not receive pre-vaccination with the RNA vaccine.
[0339] Measurement of Th 1 Memory Induction and Protection
[0340] One week after the last sensitization, allergen specific
serum IgG2a was measured by ELISA as described for experiment 1.
Ten days after the final sensitization, splenocytes were
re-stimulated in vitro with recombinant Fel d 1 for 72 h and cell
culture supernatants were analyzed for IFN-.gamma. as an indicator
of allergen-specific Th 1 cell activation.
[0341] Results
[0342] Pre-vaccination with RNA pTNT-Fel d 1 (hatched bars)
resulted in recruitment of allergen-specific Th 1 cells as
indicated by the increased induction of IgG2a (FIG. 27A) and
secretion of IFN-.gamma. (FIG. 27B) in contrast to sensitization
controls (black bars) or naive mice (white bars).
Example 23
Materials and Methods
[0343] Plasmids and RNA Transcription
[0344] As described for example 1, the cDNA encoding Der p 2 was
cloned into vector pTNT. RNA transcripts were prepared as described
and capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0345] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0346] Immunization and Sensitization
[0347] Mice were immunized with RNA pTNT-Der p 2 three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 .mu.g recombinant Der p 2 complexed with alum to
induce an allergic phenotype. Control animals were only sensitized
and did not receive pre-vaccination with the RNA vaccine.
[0348] Measurement of Th 1 Memory Induction and Protection
[0349] One week after the last sensitization, allergen specific
serum IgG2a and IgE were measured by ELISA and RBL as described for
experiment 1.
[0350] Results
[0351] Pre-vaccination with RNA pTNT-Der p 2 (hatched bars)
resulted in recruitment of allergen-specific Th 1 cells as
indicated by the increased induction of IgG2a (FIG. 28A). This Th 1
priming was able to suppress the induction of allergen specific IgE
responses (FIG. 28B).
Example 24
Materials and Methods
[0352] Plasmids and RNA Transcription
[0353] As described for example 1, the cDNA encoding Alt a 1 was
cloned into vector pTNT. RNA transcripts were prepared as described
and capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0354] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0355] Immunization and Sensitization
[0356] Mice were immunized with RNA pTNT-Alt a 1 three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 .mu.g recombinant Alt a 1 complexed with alum to
induce an allergic phenotype. Control animals were only sensitized
and did not receive pre-vaccination with the RNA vaccine.
[0357] Measurement of Th 1 Memory Induction and Protection
[0358] One week after the last sensitization, allergen specific
serum IgG2a and IgE were measured by ELISA and RBL as described for
experiment 1. Ten days after the final sensitization, splenocytes
were re-stimulated in vitro with recombinant Alt a 1 for 72 h and
cell culture supernatants were analyzed for IFN-.gamma. as an
indicator of allergen-specific Th 1 cell activation.
[0359] Results
[0360] Pre-vaccination with RNA pTNT-Alt a 1 (hatched bars)
resulted in recruitment of allergen-specific Th 1 cells as
indicated by the increased induction of IgG2a (FIG. 29A) and
secretion of IFN-.gamma. (FIG. 29B) in contrast to sensitization
controls (black bars) or naive mice (white bars). This Th 1 priming
was able to suppress the induction of allergen specific IgE
responses (FIG. 29C).
Example 25
Materials and Methods
[0361] Plasmids and RNA Transcription
[0362] As described for example 1, the cDNA encoding Cla h 8 was
cloned into vector pTNT. RNA transcripts were prepared as described
and capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0363] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0364] Immunization and Sensitization
[0365] Mice were immunized with RNA pTNT-Cla h 8 three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 .mu.g recombinant Cla h 8 complexed with alum to
induce an allergic phenotype. Control animals were only sensitized
and did not receive pre-vaccination with the RNA vaccine.
[0366] Measurement of Th 1 Memory Induction and Protection
[0367] One week after the last sensitization, allergen specific
serum IgE was measured RBL as described for experiment 1. Ten days
after the final sensitization, splenocytes were re-stimulated in
vitro with recombinant Cla h 8 for 72 h and cell culture
supernatants were analyzed for IFN-.gamma. as an indicator of
allergen-specific Th 1 cell activation.
[0368] Results
[0369] Pre-vaccination with RNA pTNT-Cla h 8 (hatched bars)
resulted in recruitment of allergen-specific Th 1 cells as
indicated by the secretion of IFN-.gamma. (FIG. 30A) in contrast to
sensitization controls (black bars) or naive mice (white bars).
This Th 1 priming was able to suppress the induction of allergen
specific IgE responses (FIG. 30B).
Example 26
Materials and Methods
[0370] Plasmids and RNA Transcription
[0371] As described for example 1, the cDNA encoding Hev b 6 was
cloned into vector pTNT. RNA transcripts were prepared as described
and capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0372] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0373] Immunization and Sensitization
[0374] Mice were immunized with RNA pTNT-Hev b 6 three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 .mu.g recombinant Hev b 6 complexed with alum to
induce an allergic phenotype. Control animals were only sensitized
and did not receive pre-vaccination with the RNA vaccine.
[0375] Measurement of Th 1 Memory Induction and Protection
[0376] One week after the last sensitization, allergen specific
serum IgG2a was measured by ELISA as described for experiment 1.
Ten days after the final sensitization, splenocytes were
re-stimulated in vitro with recombinant Hev b 6 for 72 h and cell
culture supernatants were analyzed for IFN-.gamma. as an indicator
of allergen-specific Th 1 cell activation
[0377] Results
[0378] Pre-vaccination with RNA pTNT-Hev b 6 (hatched bars)
resulted in recruitment of allergen-specific Th 1 cells as
indicated by the increased induction of IgG2a (FIG. 31A) and
secretion of IFN-.gamma. (FIG. 31B) in contrast to sensitization
controls (black bars) or naive mice (white bars).
Example 27
Materials and Methods
[0379] Plasmids and RNA Transcription
[0380] As described for example 1, a hybrid cDNA encoding parts of
5 different allergens was cloned into vector pTNT. RNA transcripts
were prepared as described and capped using a ScriptCap kit
(Ambion) according to the manufacturer's protocol.
[0381] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0382] Immunization and Sensitization
[0383] Mice were immunized with RNA pTNT-hybrid
(Aln-Cor-Que-Car-Bet) three times in weekly intervals and were
sensitized one week later via two weekly injections of 1 .mu.g
recombinant whole allergens complexed with alum to induce an
allergic phenotype. Control animals were only sensitized and did
not receive pre-vaccination with the RNA vaccine.
[0384] Measurement of Th 1 Memory Induction and Protection
[0385] One week after the last sensitization, allergen specific
serum IgG2a was measured by ELISA as described for experiment
1.
[0386] Results
[0387] Pre-vaccination with RNA pTNT-hybrid (allergen) (hatched
bars) resulted in recruitment of allergen-specific Th 1 cells as
indicated by the increased induction of IgG2a (FIG. 32).
Example 28
Materials and Methods
[0388] Plasmids and RNA Transcription
[0389] As described for example 1, the cDNA encoding Ara h 2 was
cloned into vector pTNT. RNA transcripts were prepared as described
and capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0390] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0391] Immunization and Sensitization
[0392] Mice were immunized with RNA pTNT-Ara h 2 three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 mg recombinant Ara h 2 complexed with alum to
induce an allergic phenotype. Control animals were only sensitized
and did not receive pre-vaccination with the RNA vaccine.
[0393] Measurement of Th 1 Memory Induction and Protection
[0394] One week after the last sensitization, allergen specific
serum IgE was measured by ELISA and RBL as described for experiment
1. Ten days after the final sensitization, splenocytes were
re-stimulated in vitro with recombinant Ara h 2 for 72 h and cell
culture supernatants were analyzed for IFN-.gamma. as an indicator
of allergen-specific Th 1 cell activation.
[0395] Results
[0396] Pre-vaccination with RNA pTNT-Ara h 2 (hatched bars)
resulted in recruitment of allergen-specific Th 1 cells as
indicated by the secretion of IFN-.gamma. (FIG. 33A). This Th 1
priming was able to suppress the induction of allergen specific IgE
responses (FIG. 33B).
Example 29
Materials and Methods
[0397] Plasmids and RNA Transcription
[0398] As described for example 1, the cDNA encoding Que a 1 was
cloned into vector pTNT. RNA transcripts were prepared as described
and capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0399] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0400] Immunization and Sensitization
[0401] Mice were immunized with RNA pTNT-Que a 1 three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 .mu.g recombinant Que a 1 complexed with alum to
induce an allergic phenotype. Control animals were only sensitized
and did not receive pre-vaccination with the RNA vaccine.
[0402] Measurement of Th 1 Memory Induction and Protection
[0403] One week after the last sensitization, allergen specific
serum IgG2a was measured by ELISA as described for experiment 1.
Ten days after the final sensitization, splenocytes were
re-stimulated in vitro with recombinant Que a 1 for 72 h and cell
culture supernatants were analyzed for IFN-.gamma. as an indicator
of allergen-specific Th 1 cell activation
[0404] Results
[0405] Pre-vaccination with RNA pTNT-Que a 1 (hatched bars)
resulted in recruitment of allergen-specific Th 1 cells as
indicated by the increased induction of IgG2a (FIG. 34A) and
secretion of IFN-.gamma. (FIG. 34B) in contrast to sensitization
controls (black bars) or naive mice (white bars).
Example 30
Materials and Methods
[0406] Plasmids and RNA Transcription
[0407] As described for example 1, the cDNA encoding Art v 1 was
cloned into vector pTNT. RNA transcripts were prepared as described
and capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0408] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0409] Immunization and Sensitization
[0410] Mice were immunized with RNA pTNT-Art v 1 three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 us recombinant Art v 1 complexed with alum to
induce an allergic phenotype. Control animals were only sensitized
and did not receive pre-vaccination with the RNA vaccine.
[0411] Measurement of Th 1 Memory Induction and Protection
[0412] One week after the last sensitization, allergen specific
serum IgG2a was measured by ELISA and RBL as described for
experiment 1. Ten days after the final sensitization, splenocytes
were re-stimulated in vitro with recombinant Art v 1 for 72 h and
cell culture supernatants were analyzed for IFN-.gamma. as an
indicator of allergen-specific Th 1 cell activation
[0413] Results
[0414] Pre-vaccination with RNA pTNT-Art v 1 (hatched bars)
resulted in no recruitment of allergen-specific Th 1 cells as
indicated by no increased induction of IgG2a (FIG. 35A) or
secretion of IFN-.gamma. (FIG. 35B).
Example 31
Materials and Methods
[0415] Plasmids and RNA Transcription
[0416] As described for example 1, the cDNA encoding Ole e 1 was
cloned into vector pTNT. RNA transcripts were prepared as described
and capped using a ScriptCap kit (Ambion) according to the
manufacturer's protocol.
[0417] Capped transcripts were incubated with RNAse free DNAse
(Promega) for 15 min at 37.degree. C. to remove template DNA.
Subsequently, RNA was precipitated by adding 1 volume of 5M
ammonium acetate to the reaction tube and incubating the mixture
for 10-15 minutes on ice. After a centrifugation period of 15
minutes (13000 rpm) at 4.degree. C. or room temperature, the pellet
was washed with 70% ethanol and re-suspended in nuclease free
H.sub.2O.
[0418] Immunization and Sensitization
[0419] Mice were immunized with RNA pTNT-Ole e 1 three times in
weekly intervals and were sensitized one week later via two weekly
injections of 1 .mu.g recombinant Ole e 1 complexed with alum to
induce an allergic phenotype. Control animals were only sensitized
and did not receive pre-vaccination with the RNA vaccine.
[0420] Measurement of Th 1 Memory Induction and Protection
[0421] One week after the last sensitization, allergen specific
serum IgG2a and IgE were measured by ELISA and RBL as described for
example 1.
[0422] Results
[0423] Pre-vaccination with RNA pTNT-Ole e 1 (hatched bars)
resulted in no recruitment of allergen-specific Th 1 cells as
indicated by no increased induction of IgG2a (FIG. 36A).
Furthermore, no suppression of the induction of allergen specific
IgE responses could be measured (FIG. 36B).
Sequence CWU 1
1
1190PRTDermatophagoides pteronyssinus 1Met Lys Phe Asn Ile Ile Ile
Val Phe Ile Ser Leu Ala Ile Leu Val 1 5 10 15 His Ser Ser Tyr Ala
Ala Asn Asp Asn Asp Asp Asp Pro Thr Thr Thr 20 25 30 Val His Pro
Thr Thr Thr Glu Gln Pro Asp Asp Lys Phe Glu Cys Pro 35 40 45 Ser
Arg Phe Gly Tyr Phe Ala Asp Pro Lys Asp Pro His Lys Phe Tyr 50 55
60 Ile Cys Ser Asn Trp Glu Ala Val His Lys Asp Cys Pro Gly Asn Thr
65 70 75 80 Arg Trp Asn Glu Asp Glu Glu Thr Cys Thr 85 90
* * * * *