U.S. patent application number 15/126069 was filed with the patent office on 2017-03-16 for toll-like receptor 2 agonists and vaccines and uses thereof.
This patent application is currently assigned to The Provost, Fellows, Foundation Scholars & the Other Members of Board, of the College of the Holy. The applicant listed for this patent is The Provost, Fellows, Foundation Scholars & the Other Members of Board, of the College of the Holy. Invention is credited to Aisling DUNNE, Kingston MILLS.
Application Number | 20170072044 15/126069 |
Document ID | / |
Family ID | 50342204 |
Filed Date | 2017-03-16 |
United States Patent
Application |
20170072044 |
Kind Code |
A1 |
MILLS; Kingston ; et
al. |
March 16, 2017 |
TOLL-LIKE RECEPTOR 2 AGONISTS AND VACCINES AND USES THEREOF
Abstract
The present invention relates to Toll-Like Receptor 2 (TLR2)
agonists, in particular, to TLR2-activating lipoproteins, and more
particularly to TLR2-activating lipopeptides derived from the
bacteria Bordetella pertussis. The invention further extends to the
use of said TLR2-activating lipoproteins as a therapeutic or as
part of a vaccine composition in the treatment and prevention of
infectious diseases, cancer or allergic diseases.
Inventors: |
MILLS; Kingston; (Dublin,
IE) ; DUNNE; Aisling; (Dublin, IE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Provost, Fellows, Foundation Scholars & the Other Members
of Board, of the College of the Holy |
Dublin |
|
IE |
|
|
Assignee: |
The Provost, Fellows, Foundation
Scholars & the Other Members of Board, of the College of the
Holy
Dublin
IE
|
Family ID: |
50342204 |
Appl. No.: |
15/126069 |
Filed: |
March 18, 2015 |
PCT Filed: |
March 18, 2015 |
PCT NO: |
PCT/EP2015/055735 |
371 Date: |
September 14, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/6018 20130101;
A61K 39/099 20130101; A61K 39/39 20130101; A61K 2039/55516
20130101; A61K 2039/57 20130101; A61K 2039/55511 20130101; A61K
2039/585 20130101; C07K 14/705 20130101; A61K 2039/572
20130101 |
International
Class: |
A61K 39/02 20060101
A61K039/02 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 19, 2014 |
EP |
14160791.1 |
Claims
1. A lipoprotein obtainable from Bordetella pertussis for enhancing
a Th1 response, the lipoprotein having an N terminal signal peptide
of less than 40 amino acids in length wherein the N terminal signal
peptide comprises a lipobox comprising an amino acid sequence X1,
X2, X3, X4, wherein X1 can be selected from Leucine, Valine and
Isoleucine; X2 can be selected from Alanine, Serine, Threonine,
Valine and Isoleucine; X3 can be selected from Glycine, Alanine,
and Serine; and X4 is Cysteine, wherein X4 is capable of being
acylated, or a fragment or derivative thereof wherein the
lipoprotein or the fragment or derivative thereof is a Toll-like
receptor 2 agonist.
2. The lipoprotein as claimed in claim 1, wherein the lipoprotein
comprises an amino acid sequence selected from SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 or a
derivative or fragment thereof.
3. The lipoprotein as claimed in claim 1, wherein the lipoprotein
is a lipopeptide comprising an amino acid sequence of SEQ ID NO:13
or SEQ ID NO:14.
4. The lipoprotein of claim 1 for use in medicine.
5. The lipoprotein of claim 1 for use in the treatment or
prevention of a condition caused by a pathogen, cancer or an
allergic disease.
6. Use of a lipoprotein obtainable from Bordetella pertussis for
enhancing a Th1 response, the lipoprotein having an N terminal
signal peptide of less than 40 amino acids in length wherein the N
terminal signal peptide comprises a lipobox comprising an amino
acid sequence X1, X2, X3, X4, wherein X1 can be selected from
Leucine, Valine and Isoleucine; X2 can be selected from Alanine,
Serine, Threonine, Valine and Isoleucine; X3 can be selected from
Glycine, Alanine, and Serine; and X4 is Cysteine, wherein X4 is
capable of being acylated, or a fragment or derivative thereof
wherein the lipoprotein or the fragment or derivative thereof is a
Toll-like receptor 2 agonist in the preparation of a medicament for
the prevention or treatment of a condition caused by a pathogen or
tumourigenesis.
7. The lipoprotein for use as claimed in claim 5, wherein the
pathogen is a bacterium, a virus, a fungus or a parasite.
8. The lipoprotein for use or the use as claimed in claim 7,
wherein the bacterium is B. pertussis.
9. The use as claimed in claim 6, wherein the lipoprotein comprises
an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 or a derivative
or fragment thereof.
10. The use as claimed in claim 6, wherein the lipoprotein is a
lipopeptide comprising an amino acid sequence of SEQ ID NO:13 or
SEQ ID NO:14.
11. A method for the administration of at least one lipoprotein
obtainable from Bordetella pertussis for enhancing a Th1 response,
the lipoprotein having an N terminal signal peptide of less than 40
amino acids in length wherein the N terminal signal peptide
comprises a lipobox comprising an amino acid sequence X1, X2, X3,
X4, wherein X1 can be selected from Leucine, Valine and Isoleucine;
X2 can be selected from Alanine, Serine, Threonine, Valine and
Isoleucine; X3 can be selected from Glycine, Alanine, and Serine;
and X4 is Cysteine, wherein X4 is capable of being acylated, or a
fragment or derivative thereof wherein the lipoprotein or the
fragment or derivative thereof is a Toll-like receptor 2 agonist as
part of a vaccine composition.
12. The method of claim 11, wherein the vaccine composition is for
the treatment of or prophylaxis of a condition caused by a
pathogen, cancer or an allergic disease.
13. The method as claimed in claim 11, wherein the at least one
lipoprotein is an adjuvant.
14. The method as claimed in claim 11, wherein the at least one
TLR2-activating lipoprotein is an antigen.
15. The method of claim 11, wherein the vaccine composition
comprises at least one antigen derived from an infectious disease
or a tumour antigen.
16. The method as claimed in claim 11, wherein the lipoprotein
comprises an amino acid sequence selected from SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 or a
derivative or fragment thereof.
17. The method as claimed in claim 11, wherein the lipoprotein is a
lipopeptide comprising an amino acid sequence of SEQ ID NO:13 or
SEQ ID NO:14.
18. A composition to mediate an immune response in a subject
comprising at least one lipoprotein obtainable from Bordetella
pertussis for enhancing a Th1 response, the lipoprotein having an N
terminal signal peptide of less than 40 amino acids in length
wherein the N terminal signal peptide comprises a lipobox
comprising an amino acid sequence X1, X2, X3, X4, wherein X1 can be
selected from Leucine, Valine and Isoleucine; X2 can be selected
from Alanine, Serine, Threonine, Valine and Isoleucine; X3 can be
selected from Glycine, Alanine, and Serine; and X4 is Cysteine,
wherein X4 is capable of being acylated, or a fragment or
derivative thereof wherein the lipoprotein or the fragment or
derivative thereof is a Toll-like receptor 2 agonist, optionally
comprising an antigen from a pathogen, from a tumour cell or an
allergen.
19. The composition of claim 18, wherein the at least one
lipoprotein is an immunogenic determinant.
20. The composition of claim 18, wherein the at least one
lipoprotein is an adjuvant.
21. The composition of claim 18, wherein the composition is for use
in mediating an immune response against a pathogen.
22. The composition of claim 18, wherein the composition is an
acellular pertussis vaccine composition.
23. The composition as claimed in claim 18, wherein the lipoprotein
comprises an amino acid sequence selected from SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 or a
derivative or fragment thereof.
24. The composition as claimed in claim 18, wherein the lipoprotein
is a lipopeptide comprising an amino acid sequence of SEQ ID NO:13
or SEQ ID NO:14.
25. The composition of claim 18 for use in medicine.
26. Use of the composition of claim 18 in the preparation of a
medicament for the prevention or treatment of a condition caused by
a pathogen or cancer or an allergic disease.
27. The composition of claim 18 for use in the treatment or
prevention of a condition caused by a pathogen or
tumourigenesis.
28. Use of a composition of claim 18 in a method of vaccinating a
subject to induce immunity against a pathogen.
29. The use as claimed in claim 26 wherein the pathogen is an
infectious agent.
30. The use as claimed in claim 29, wherein the infectious agent is
a bacterium, a virus, a fungus or a parasite.
31. The use as claimed in claim 30, wherein the bacterium is B.
pertussis.
32. A composition comprising at least one lipoprotein obtainable
from Bordetella pertussis for enhancing a Th1 response, the
lipoprotein having an N terminal signal peptide of less than 40
amino acids in length wherein the N terminal signal peptide
comprises a lipobox comprising an amino acid sequence X1, X2, X3,
X4, wherein X1 can be selected from Leucine, Valine and Isoleucine;
X2 can be selected from Alanine, Serine, Threonine, Valine and
Isoleucine; X3 can be selected from Glycine, Alanine, and Serine;
and X4 is Cysteine, wherein X4 is capable of being acylated, or a
fragment or derivative thereof wherein the lipoprotein or the
fragment or derivative thereof is a Toll-like receptor 2 agonist
for use as a booster to enhance the immune response generated in a
host against a pathogen to which the subject has previously been
exposed.
33. The composition as claimed in claim 32, wherein the lipoprotein
comprises an amino acid sequence selected from SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 or a
derivative or fragment thereof.
34. The composition as claimed in claim 32, wherein the lipoprotein
is a lipopeptide comprising an amino acid sequence of SEQ ID NO:13
or SEQ ID NO:14.
35. The use as claimed in claim 32 wherein the pathogen is an
infectious agent.
36. The use as claimed in claim 35, wherein the infectious agent is
a bacterium, a virus, a fungus or a parasite.
37. The use as claimed in claim 36, wherein the bacterium is B.
pertussis.
38. A method of mediating an immune response in a subject against
an infectious disease, said method comprising the steps of:
providing a composition comprising at least one lipoprotein
obtainable from Bordetella pertussis for enhancing a Th1 response,
the lipoprotein having an N terminal signal peptide of less than 40
amino acids in length wherein the N terminal signal peptide
comprises a lipobox comprising an amino acid sequence X1, X2, X3,
X4, wherein X1 can be selected from Leucine, Valine and Isoleucine;
X2 can be selected from Alanine, Serine, Threonine, Valine and
Isoleucine; X3 can be selected from Glycine, Alanine, and Serine;
and X4 is Cysteine, wherein X4 is capable of being acylated, or a
fragment or derivative thereof wherein the lipoprotein or the
fragment or derivative thereof is a Toll-like receptor 2 agonist;
and administering the composition to the subject in a
therapeutically effective or prophylactically effective amount
sufficient to elicit an immune response in the subject against the
infectious disease.
39. The method as claimed in claim 38, wherein the lipoprotein
comprises an amino acid sequence selected from SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 or a
derivative or fragment thereof.
40. The method as claimed in claim 38, wherein the lipoprotein is a
lipopeptide comprising an amino acid sequence of SEQ ID NO:13 or
SEQ ID NO:14.
41. The method as claimed in claim 38, wherein the composition
includes an antigen from a pathogen that causes the infectious
disease.
42. The method as claimed in claim 38 wherein the pathogen is an
infectious agent.
43. The method as claimed in claim 42, wherein the infectious agent
is a bacterium, a virus, a fungus or a parasite.
44. The method as claimed in claim 43, wherein the bacterium is B.
pertussis.
45. The method of claim 38, wherein the composition is a vaccine
composition.
46. A method of mediating an immune response in a subject with
cancer, said method comprising the steps of: providing a
composition comprising at least one lipoprotein obtainable from
Bordetella pertussis for enhancing a Th1 response, the lipoprotein
having an N terminal signal peptide of less than 40 amino acids in
length wherein the N terminal signal peptide comprises a lipobox
comprising an amino acid sequence X1, X2, X3, X4, wherein X1 can be
selected from Leucine, Valine and Isoleucine; X2 can be selected
from Alanine, Serine, Threonine, Valine and Isoleucine; X3 can be
selected from Glycine, Alanine, and Serine; and X4 is Cysteine,
wherein X4 is capable of being acylated, or a fragment or
derivative thereof wherein the lipoprotein or the fragment or
derivative thereof is a Toll-like receptor 2 agonist; and
administering the composition to the subject in a therapeutically
effective or prophylactically effective amount.
47. The method as claimed in claim 46, wherein the lipoprotein
comprises an amino acid sequence selected from SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 or a
derivative or fragment thereof.
48. The method as claimed in claim 46, wherein the lipoprotein is a
lipopeptide comprising an amino acid sequence of SEQ ID NO:13 or
SEQ ID NO:14.
49. The method of claim 46, wherein the composition further
comprises a tumour-specific antigen, tumour associated antigen
tumour, a heat shock protein and antigenic peptide or a synthetic
peptide antigen.
50. The method of claim 49, wherein the antigen is killed whole
tumor cells or extracts.
51. The method as claimed in claim 46, wherein the cancer is
hepatic cancer, lung cancer, in particular non-small cell lung
cancer, prostate cancer, ovarian cancer, breast cancer, melanoma,
basal cell carcinoma, or haematological malignancies.
52. The method of claim 46, wherein the composition is a vaccine
composition.
53. A method of mediating an immune response in a subject with an
allergic disease, said method comprising the steps of: providing a
composition comprising at least one lipoprotein obtainable from
Bordetella pertussis for enhancing a Th1 response, the lipoprotein
having an N terminal signal peptide of less than 40 amino acids in
length wherein the N terminal signal peptide comprises a lipobox
comprising an amino acid sequence X1, X2, X3, X4, wherein X1 can be
selected from Leucine, Valine and Isoleucine; X2 can be selected
from Alanine, Serine, Threonine, Valine and Isoleucine; X3 can be
selected from Glycine, Alanine, and Serine; and X4 is Cysteine,
wherein X4 is capable of being acylated, or a fragment or
derivative thereof wherein the lipoprotein or the fragment or
derivative thereof is a Toll-like receptor 2 agonist; and
administering the composition to the subject in a therapeutically
effective or prophylactically effective amount.
54. The method as claimed in claim 53, wherein the allergic disease
is asthma.
55. The method of claim 53, wherein the composition is a vaccine
composition.
56. The method as claimed in claim 53, wherein the lipoprotein
comprises an amino acid sequence selected from SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 or a
derivative or fragment thereof.
57. The method as claimed in claim 53, wherein the lipoprotein is a
lipopeptide comprising an amino acid sequence of SEQ ID NO:13 or
SEQ ID NO:14.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to Toll-Like Receptor 2 (TLR2)
agonists, in particular, to TLR2-activating lipoproteins, and more
particularly to TLR2-activating lipopeptides derived from the
bacteria Bordetella pertussis. The invention further extends to the
use of said TLR2-activating lipoproteins as a therapeutic or as
part of a vaccine composition in the treatment and prevention of
infectious diseases, cancer or allergic diseases.
BACKGROUND TO THE INVENTION
[0002] The bacterium Bordetella pertussis is the causative agent of
whooping cough, a severe and debilitating respiratory tract
infection affecting infants and young children. Whooping cough
(pertussis) still accounts for over 300,000 infant deaths annually,
mostly in developing countries. The disease was largely controlled
in developed countries through vaccination with whole cell
pertussis vaccines (Pw), which were introduced in the 1950s.
However, these vaccines were associated with unacceptable side
effects and were replaced in many countries in the 1990s by
acellular pertussis vaccines (Pa), composed of individual B.
pertussis antigens absorbed to alum as the adjuvant. More recently,
studies in children and mice have demonstrated that Pa promote the
induction of Th2 and Th17 cells and this has been attributed to the
use of alum as the adjuvant. In contrast, Pw induce Th1 and Th17
responses and confer a higher level of protection against infection
in mice and in children and this is thought to reflect the presence
of B. pertussis-derived pathogen associated molecular patterns
(PAMPs), including agonists for Toll-like receptors (TLRs) (Higgs
et al).
[0003] Th1 cells are characterized by the production of
pro-inflammatory cytokines such as IFN-.gamma., IL-2, and
TNF-.beta.. Th1 cells are involved in cell-mediated immunity (CMI),
this being the immune response typically mounted against viruses
and intracellular pathogens.
[0004] Th17 cells secrete IL-17 and are involved in immune
responses to infection and tumours. Functionally, Th17 cells play a
role in host defence against extracellular pathogens by mediating
the recruitment of neutrophils and macrophages to infected tissues.
They are, therefore, largely part of the cellular immune response
together with Th1 cells. The IL-17 cytokine family is a group of
cytokines including IL-17A, B, C, D, IL-17E (IL-25) and IL-17F. It
is increasingly recognized that besides T cells, other cells such
as NK cells and neutrophils might also be an important source of
IL-17. Besides IL-17A, the major cytokine produced by Th17 cells,
these cells also release IL-17F, IL-21 and IL-22.
[0005] Toll-like Receptors (TLRs) are part of a family of pattern
recognition receptors (PRRs) which have evolved for innate immune
recognition of conserved microbial products. TLRs have a key role
in modulating the innate immune response; they are also involved in
tissue repair, maintenance of tissue integrity and tumorigenesis.
Eleven Toll-like Receptors have been identified in humans to date.
The binding of pattern-associated molecular patterns (PAMPs), such
as TLR ligands to pathogen recognition receptors on cells of the
innate immune system, such as macrophages and dendritic cells
(DCs), activates signalling pathways leading to pro-inflammatory
gene expression and the induction of innate immune responses. This
in turn helps to drive adaptive immunity. Consequently TLR agonists
have been exploited commercially as adjuvants in vaccines to boost
immune responses to antigens and as direct immunotherapeutics for
cancer. The members of the TLR family are highly conserved, with
most mammalian species having between 10 to 15 Toll-like Receptors.
Toll-like Receptor 2 (TLR2, CD282, TLR-2) can be activated by
peptidoglycan, lipoproteins, lipoteichoic acid and endogenous
ligands. Lipoproteins are biochemical assemblies comprising both
proteins and lipids. The consensus view is that TLR2 activation is
more anti-inflammatory than other TLRs; for example it has been
reported that TLR2 induced regulatory antigen presenting cells and
immunological tolerance (Dillon et al).
[0006] Although the number of cases of pertussis continued to
decline following introduction of acellular pertussis vaccines
(Pa), in recent years there has been alarming increases in the
incidence of disease not only in infants but also in adolescents
and young adults. This has been attributed to antigenic variation
in protective antigens or waning and ineffective immunity induced
with current Pa. The resurgence in pertussis has consequently
called into question the level of protection provided by current
vaccines and highlighted the need for a better vaccine.
SUMMARY OF THE INVENTION
[0007] The inventors of the present invention have surprisingly
discovered that novel Toll-like Receptor 2 lipoprotein ligands from
B. pertussis are capable of activating innate inflammatory immune
responses that drive the induction of protective adaptive cellular
immunity to B. pertussis. The present inventors have identified
lipopeptides that enhance Th1 and Th17 responses and in particular
provide more effective and longer lasting protective immunity to B.
pertussis.
[0008] Following extensive experimentation, the present inventors
have identified and characterised novel TLR2-activating
lipopeptides derived from B. pertussis. The present inventors have
demonstrated that these novel lipopeptides specifically activate
TLR2 and surprisingly drive potent pro-inflammatory cytokine
production. Furthermore, the inventors have demonstrated that
corresponding synthetic lipopeptides have potent adjuvant
properties, promoting protective Th1 and Th17 responses against B.
pertussis infection in vivo when co-administered with pertussis
antigens. These findings demonstrate that combining protective
antigens with an adjuvant based on an endogenous TLR2 ligand from
B. pertussis has considerable potential for the development of a
more effective vaccine capable of generating protective cellular
immunity against pathogens and in generating Th1/Th17 immune
responses in relation to other conditions such as cancer or
allergic diseases and in particular against the re-emerging B.
pertussis pathogen.
[0009] According to a first aspect of the present invention, there
is provided at least one lipoprotein obtainable from Bordetella
pertussis (B. pertussis) for enhancing a Th1 response, the
lipoprotein having an N terminal signal peptide of less than 40
amino acids in length wherein the N terminal signal peptide
comprises a lipobox comprising an amino acid sequence X1, X2, X3,
X4, wherein X1 can be selected from Leucine, Valine and Isoleucine;
X2 can be selected from Alanine, Serine, Threonine, Valine and
Isoleucine; X3 can be selected from Glycine, Alanine, and Serine;
and X4 is Cysteine, wherein X4 is capable of being acylated, or a
fragment or derivative thereof wherein the lipoprotein or the
fragment or derivative thereof is a Toll-like receptor 2 (TLR-2)
agonist.
[0010] As will be understood by those of skill in the art, whilst
the lipoproteins of the present invention were identified from
Bordetella pertussis, such lipoproteins or a fragment or derivative
thereof may also be obtained by synthetic routes.
[0011] Suitably, a lipoprotein or a fragment or derivative thereof
may be obtained from B. pertussis.
[0012] Suitably a lipoprotein or a fragment or derivative thereof
may be obtained from a soluble secreted fraction of the B.
pertussis.
[0013] In embodiments a lipoprotein of the present invention can
comprise an amino acid sequence selected from SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 or can
be a derivative or fragment thereof.
[0014] In embodiments a lipoprotein of the present invention can
comprise or consist of an amino acid sequence selected from SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ
ID NO:6 or can be a derivative or fragment thereof.
[0015] In embodiments, a lipoprotein of the invention or a
fragment, or derivative thereof can promote a Th1 response and/or a
Th17 response or suppress a Th2 response against a pathogen, cancer
or an allergic disease.
[0016] In embodiments a derivative can be a Toll-like receptor 2
(TLR-2) agonist lipoprotein of SEQ ID NO:1, SEQ ID NO:2, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 from a gram
negative bacterium which is not B. pertussis.
[0017] In embodiments a derivative of a lipoprotein of the first
aspect of the invention can be a lipopeptides comprising an exposed
acyl coupled N-terminus. Suitably the exposed acyl coupled
N-terminus may be formed from cleavage of a lipoprotein of the
invention between X3 and X4 of the lipobox comprising an amino acid
sequence X1, X2, X3, X4, wherein X1 can be selected from Leucine,
Valine and Isoleucine; X2 can be selected from Alanine, Serine,
Threonine, Valine and Isoleucine; X3 can be selected from Glycine,
Alanine, and Serine; and X4 is Cysteine.
[0018] In embodiments a derivative or fragment of a lipoprotein of
the first aspect of the invention can be from the soluble fraction
from B. Pertussis. In alternative embodiments, a derivative or
fragment of a lipoprotein of the first aspect of the invention can
be synthetically or recombinantly produced.
[0019] In embodiments a fragment of a lipoprotein of the first
aspect of the invention can be a lipopeptide fragment selected from
SEQ ID NO: 13 or SEQ ID NO: 14.
TABLE-US-00001 (Lipopeptide 1569) SEQ ID NO: 13 CSDVNQLLGNEESVD
(Lipopeptide 2992) SEQ ID NO: 14 CANPSASSGVYTYGQ
[0020] In embodiments, a lipopeptide comprising or consisting of
SEQ ID NO: 13 or SEQ ID NO: 14 comprises an exposed acyl coupled
N-terminus, for example a diacylated or triacylated N-terminal
cysteine, in particular the cysteine can be palmitoylated.
[0021] A fragment can comprise at least 10, suitably 20, 50,
suitably 100 and suitably 150 or greater contiguous amino acids
from the relevant sequence and which is functionally active.
[0022] A derivative can comprise an amino acid sequence which is at
least 70% homologous to the relevant sequence, more preferably at
least 80% homologous, more preferably at least 90% homologous, even
more preferably at least 95% homologous, more preferably at least
97%, most preferably at least 99% homologous. A derivative
encompasses a polypeptide sequence of SEQ ID NO: 1 which includes
substitution of amino acids, especially a substitution(s) which
is/are known for having a high probability of not leading to any
significant modification of the biological activity or
configuration, or folding, of the protein. These substitutions,
typically known as conserved substitutions, are known in the art.
For example the group of arginine, lysine and histidine are known
interchangeable basic amino acids. Suitably, in embodiments amino
acids of the same charge, size or hydrophobicity may be substituted
with each other. Suitably, any substitution may be selected based
on analysis of amino acid sequence alignments of lipoproteins to
provide amino acid substitutions to amino acids which are present
in other lipoproteins at similar or identical positions when the
sequences are aligned. Hybrids, and derivatives and fragments
thereof may be generated using suitable molecular biology methods
as known in the art.
[0023] Suitably, the N-terminal signal peptide including the
lipobox further comprises a region of positively charged amino
acids and a region of hydrophobic amino acids. Suitably, the
N-terminal signal peptide can thus comprise the structure, a region
of positively charged amino acids towards the N-terminus of the
N-terminal signal peptide, a lipobox at the C-terminus of the
N-terminal signal peptide and a, region of hydrophobic amino acids
interposed between the positively charged amino acid region and the
lipobox.
[0024] By positively charged region it is meant about 5-7 residues,
comprising at least 2 positively charged amino acid residues, for
example Lys or Arg residues. By hydrophobic region it is meant an
intervening stretch of uncharged amino acid residues, for example
phenylalanine, leucine, alanine, isoleucine, valine, methionine,
tryptophan, proline, between the positively charged region and the
lipobox.
[0025] Typically the positively charged region and hydrophobic
region can span 7-22 amino acids in length. By lipobox, it is meant
a distinct sequence at the C-terminal end of the signal peptide.
The lipobox contains the invariant Cys that is lipid-modified.
[0026] In embodiments, the at least one TLR2-activating lipoprotein
or lipopeptide can be obtained from the bacteria Bordetella
pertussis.
[0027] In embodiments, the at least one TLR2-activating lipoprotein
or lipopeptide can be provided by recombinant means.
[0028] In embodiments, the exposed acyl of a lipopeptide of the
invention can be a diacylated group or a triacylated group and
preferably a triacylated group.
[0029] Suitably, a lipoprotein or fragment or derivative thereof
enhances a Th1 response against a pathogen or cancer or in an
allergic disease or condition.
[0030] Suitably a pathogen may be any infectious agent, in
particular a bacterium, a virus, a fungus or a parasite.
[0031] In certain embodiments, the pathogen can be B.
pertussis.
[0032] In embodiments, cancer can be hepatic cancer, lung cancer,
in particular non-small cell lung cancer, prostate cancer, ovarian
cancer, breast cancer, melanoma, basal cell carcinoma, or
haematological malignancies.
[0033] In certain embodiments, the allergic disease can be
asthma.
[0034] In certain embodiments, a TLR2-activating lipopeptide can be
a fragment of a lipoprotein comprising the amino acid sequence of
SEQ ID NO:1 or SEQ ID NO:2.
[0035] SEQ ID NO:1 is the full length sequence of B. pertussis
lipoprotein BP1569; SEQ ID NO:2 is the full length sequence of B.
pertussis lipoprotein BP2992; SEQ ID NO:3 is the full length
sequence of B. pertussis lipoprotein BP0205; SEQ ID NO:4 is the
full length sequence of B. pertussis lipoprotein BP3342; SEQ ID
NO:5 is the full length sequence of B. pertussis lipoprotein BP3819
and SEQ ID NO:6 is the full length sequence of B. pertussis
lipoprotein BP2508.
[0036] In the below sequences positively charged residues and
putative lipobox are highlighted in bold and the invariant cysteine
residue is underlined.
TABLE-US-00002 (BP1569) SEQ ID NO: 1
MRMNKRHAGASALMALALLAGCSDVNQLLGNEESVDYKSTRRGDPLSIPP
DLTQANNDPRYKAPASGTATYSQFQQQGLQQQASAGQNTNVLPERADMRV
ERDGDLRWLVIERPPEQLFSKVVDFWTDTGFTVSVNNPQAGIIETDWAEN
RAKIPESWLRQVLGSVLETAWDSGEREKFRTRVERVNGHTEIYITHNQML
EKRVGSDGGQVQWTHGKEDPGLNAAMLARLMVYLGTDVDAARKLVAQAEA
APQAPKVQSVRAEGAMLVVDESFDRAWRRVGVALDSGGFAVDDRDRSAGE
YFVRYVDTDTGAQNEQPGFFSRLFSSDKKAQAPQYRIRLTGSGTQTQVTV
LDANGQRDSSATAQRMLSVLKDKMV (BP2992) SEQ ID NO: 2
NYMHSPSVVAGRARRLLAVAAVAGSVAVLAGCANPSASSGVYTYGQAQRE
QIVRTGTVTGVRPITIQNDKSSGVGLVAGGALGGVAGNAVGGGTGRTIATV
GGVILGALAGNAIENRAGKSSGYEITVRLDNGETRVVAQEADVPISVGQRV QVISGAGPTRVTPY
(BP0205) SEQ ID NO: 3
MQLTIRKLAYTLAFSTLVLAGCTTASKKTDGQAATPADQASSQQASAASV
EFYVAQAKAGDGLMEVKVPDGSLYMQRQPVLTRADLTEAAALVDRQGQNF
VGLRFTEAGARKLNDISSKNIGNMLALVIDRELVAAPRIAEPLNRGVLAF
GVPSAKAASEIAAKIRGDAGAPAAGVPAAPAPKPAPKP (BP3342) SEQ ID NO: 4
MKSRIAKSLTIAALAATLAACSSVPLDDKAGQAGGSGQGSASGQILDPFN
PQSILAQQRSVYFDFDSYTVSEQYRGLVETHARYLASNNQQRIKIEGNTD
ERGGAEYNLALGQRRADAVRRMMTLLGVSDNQIETISFGKEKPKATGSSE ADFAENRRADIVYQR
(BP3819) SEQ ID NO: 5
MSAPLDTPALRLNTRFATGIVLAGTLALAGCAQQRSAGYYDPPGASTITD
AQYQGQAAGYRTVVHAPSQLQIELKPNQPARQQNAQAQAGQQSTEDGTAV
PEGQAAPQPQPETASPGAQAIIPQAQTYQGTFPCFAAGLACEAQRVTLTL
APNGRWRSRTNYLDKQPQASAPVAEQGCWDATQERPPRVLLLDGSGNMRA
ELVMTANNVLRVRSVGGRTPNLNYNLTRQPDLDAIAELDKQAAPKCP (BP2508) SEQ ID NO:
6 MIARISLRPLKGLAVAVLAASALTACSSGKWGFPYKAGVQQGNWITKEQV
ALLQQGMSREQVRFALGSPTLTSVLHADRWDYPYYFKPGYGKAQERQFTV
WFENDHLVRWSGDEQPDLQPFQIEKVNAKQEEKADAQVDTAEKRQEGIDK
AEKVRPHVDVTTPDNPTLDYPGEPGQTFEPLK
[0037] Without wishing to be bound by theory, the inventors submit
that the lipoproteins identified in the present invention contain a
unique N-terminal signal peptide characteristic of bacterial
lipoproteins from Gram negative bacteria. In embodiments, the
lipoproteins of the present invention can be triacylated with
palmytic acid or can be a diacylated lipid.
[0038] According to a second aspect of the present invention there
is provided a method for the simultaneous, separate or sequential
administration of at least one TLR2-activating lipoprotein, or
fragment or derivative thereof, for example a lipopeptide
comprising the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:14
as part of a vaccine composition for the treatment of or
prophylaxis of a condition caused by a pathogen or tumourigenesis.
In certain embodiments, the at least one TLR2-activating
lipoprotein, or fragment or derivative thereof, for example a
lipopeptide comprising the amino acid sequence of SEQ ID NO:13 or
SEQ ID NO:14 is an adjuvant. In certain embodiments, the pathogen
is the bacteria B. pertussis. In certain embodiments, the vaccine
composition comprises at least one antigen derived from an
infectious disease or a tumour for example a tumour specific or
tumour-associated antigen. In certain embodiments, the at least one
antigen is derived from B. pertussis. In certain embodiments, the
vaccine composition comprises an allergen.
[0039] According to a further aspect of the present invention,
there is provided a composition to mediate an immune response in a
subject the composition comprising the at least one TLR2-activating
lipoprotein, or fragment or derivative thereof of the present
invention, for example a lipopeptide comprising the amino acid
sequence of SEQ ID NO:13 or SEQ ID NO:14. In embodiments, the
lipoprotein, or fragment or derivative thereof, for example the
lipopeptide comprising the amino acid sequence of SEQ ID NO:13 or
SEQ ID NO:14 is an immunogenic determinant in such a composition.
In embodiments, the lipoprotein, or fragment or derivative thereof,
for example the lipopeptide comprising the amino acid sequence of
SEQ ID NO:13 or SEQ ID NO:14 can provide adjuvant activity in such
a composition.
[0040] In certain embodiments, the lipoprotein, or fragment or
derivative thereof of the present invention, for example
compositions comprising a lipopeptide comprising the amino acid
sequence of SEQ ID NO:13 or SEQ ID NO:14 are for use in mediating
an immune response against a pathogen, against tumour cells or
against an allergen. Said lipoprotein, or fragment or derivative
thereof, for example vaccine compositions including the
lipoprotein, or fragment or derivative thereof, are typically
administered to mammals, in particular humans, in order to confer
protective immunity against the pathogen/infectious agent.
[0041] In certain embodiments, the composition comprises the at
least one TLR2-activating lipoprotein, or fragment or derivative
thereof, for example a lipopeptide comprising the amino acid
sequence of SEQ ID NO:13 or SEQ ID NO:14 as the adjuvant. In
certain embodiments the vaccine or vaccine composition comprises
the at least one TLR2-activating lipoprotein, or fragment or
derivative thereof, for example lipopeptide as the antigen.
[0042] In embodiments, there is provided a composition comprising
at least one TLR2-activating lipoprotein, or fragment or derivative
thereof of the invention, for example a lipopeptide comprising the
amino acid sequence of SEQ ID NO:13 or SEQ ID NO:14 which provides
protective immunity to a subject.
[0043] In certain embodiments, the composition can comprise the at
least one TLR2-activating lipoprotein, or fragment or derivative
thereof, for example a lipopeptide comprising the amino acid
sequence of SEQ ID NO:13 or SEQ ID NO:14 as the adjuvant and an
antigen from the bacteria B. pertussis.
[0044] In certain embodiments, the composition can comprise a
tumour antigen for example a tumour specific or tumour-associated
antigen.
[0045] In certain embodiments, the composition can be an acellular
pertussis vaccine.
[0046] In certain further aspects the present invention provides a
composition according to the invention for use in medicine.
[0047] In certain further aspects the present invention provides
the use of at least one TLR2-activating lipoprotein, or fragment or
derivative thereof of the invention, for example a lipopeptide
comprising the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:14
in the preparation of a medicament for the prevention or treatment
of a condition caused by a pathogen, cancer or an allergic
disease.
[0048] In certain further aspects, the present invention provides
at least one TLR2-activating lipoprotein, or fragment or derivative
thereof of the invention, for example comprising a lipopeptide
comprising the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:14
for use in the treatment or prevention of a condition caused by a
pathogen, cancer or an allergic disease.
[0049] In certain embodiments, a pathogen can be any infectious
agent, in particular a bacterium, a virus, a fungus or a
parasite.
[0050] In certain embodiments, the pathogen can be B.
pertussis.
[0051] In embodiments, tumourigenesis may be hepatic cancer, lung
cancer, in particular non-small cell lung cancer, prostate cancer,
ovarian cancer, breast cancer, melanoma, basal cell carcinoma, or
haematological malignancies.
[0052] In certain embodiments, the allergic disease is asthma.
[0053] In certain embodiments, the at least one TLR2-activating
lipoprotein, or fragment or derivative thereof of the invention,
for example a lipopeptide comprising the amino acid sequence of SEQ
ID NO:13 or SEQ ID NO:14 or the compositions containing the same
are administered prophylactically to a subject. In certain further
embodiments, the at least one TLR2-activating lipoprotein, or
fragment or derivative thereof, for example a lipopeptide
comprising the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:14
or the compositions comprising the same are administered
therapeutically. Prophylactic and therapeutic compositions may be
administered to subjects in need thereof as required.
[0054] In various further aspects, the present invention extends to
the at least one TLR2-activating lipoprotein, or fragment or
derivative thereof of the invention, for example comprising a
lipopeptide comprising the amino acid sequence of SEQ ID NO:13 or
SEQ ID NO:14 or to preparations or mixtures comprising the same, or
to compositions containing the same, for use as a booster to
enhance the immune response generated in a host against a pathogen
to which the subject has previously been exposed, typically by way
of infection or due to the previous administration of a primary
vaccine.
[0055] The invention further provides for the use of the at least
one TLR2-activating lipoprotein, or fragment or derivative thereof,
for example a lipopeptide comprising the amino acid sequence of SEQ
ID NO:13 or SEQ ID NO:14 of the invention in a method of
vaccinating a subject to induce immunity against infectious disease
and in particular against B. pertussis derived infectious
disease.
[0056] Accordingly a further aspect of the invention provides for a
method of mediating an immune response in a subject against an
infectious disease, said method comprising the steps of: [0057]
providing a composition comprising at least one TLR2-activating
lipoprotein, or fragment or derivative thereof of the invention,
for example comprising a lipopeptide comprising the amino acid
sequence of SEQ ID NO:13 or SEQ ID NO:14, and [0058] administering
the composition to the subject in a therapeutically effective or
prophylactically effective amount sufficient to elicit an immune
response in the subject against the infectious disease.
[0059] According to a further aspect of the invention, there is
provided a lipoprotein, or fragment or derivative thereof, of a
first aspect of the invention for use in the preparation of a
medicament for the treatment of a condition requiring enhancement
of a Th1 and/or Th17 response in particular a pathogen or a
malignant condition/cancer.
[0060] In certain embodiments of the aspects of the present
invention, the composition can include an antigen from pathogen
that causes an infectious disease. In certain embodiments the
composition can include an antigen from a pathogen that causes
tetanus, diphtheria, hepatitis B virus, polio, haemophilus
influenza B, influenza, meningococcal disease Mycobacterium
tuberculosis. In certain embodiments the composition can include an
antigen from a pathogen that causes a respiratory tract infection.
In certain embodiments, the respiratory tract infection is from B.
pertussis and causes whooping cough.
[0061] Accordingly a further aspect of the invention provides for a
method of mediating an immune response in a subject with cancer,
said method comprising the steps of: [0062] providing a composition
comprising at least one TLR2-activating lipoprotein, or fragment or
derivative thereof, for example comprising a lipopeptide comprising
the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:14, and [0063]
administering the composition to the subject in a therapeutically
effective or prophylactically effective amount.
[0064] In certain embodiments, the antigen can be a tumour-specific
protein or peptide or killed whole tumor cells or extracts. In
certain embodiments, the antigen can be a tumour associated
antigen, a tumour specific antigen, a heat shock protein and
antigenic peptide or a synthetic peptide antigen derived from or
corresponding to that in a cancerous cell.
[0065] In embodiments of the aspects of the invention, the cancer
can be melanoma or non-Small cell Lung Carcinoma, prostate cancer,
ovarian cancer, breast cancer. melanoma, basal cell carcinoma, or
haematological malignancies.
[0066] Accordingly a further aspect of the invention provides for a
method of mediating an immune response in a subject with an
allergic disease, said method comprising the steps of: [0067]
providing a composition comprising at least one TLR2-activating
lipoprotein, or fragment or derivative thereof, for example
comprising a lipopeptide comprising the amino acid sequence of SEQ
ID NO:13 or SEQ ID NO:14, and [0068] administering the composition
to the subject in a therapeutically effective or prophylactically
effective amount.
[0069] In certain embodiments, the allergic disease is asthma.
[0070] In embodiments of the aspects of the present invention, the
composition can be a vaccine composition.
[0071] According to a further aspect, there is provided a
lipoprotein, or fragment or derivative thereof, of the first aspect
of the invention for use in mediating an immune system in a subject
in particular for use in the treatment or conditions requiring
enhancement of Th1 and/or Th17 responses, in particular a pathogen
or a malignant condition/cancer.
[0072] As used herein, the term "vaccine composition" means any
composition containing an immunogenic determinant which stimulates
the immune system in a manner such that it can better respond to
subsequent challenges or pathogenic infections or a tumour. It will
be appreciated that a vaccine usually contains an immunogenic
determinant and optionally an adjuvant, the adjuvant serving to
non-specifically enhance the immune response to the immunogenic
determinant.
[0073] In embodiments, the composition can be provided in
combination with an immune modulator. In embodiments the immune
modulator can be a Phosphoinositide kinase-3 (PI3K) inhibitor, a
mitogen activated (MAP) kinase inhibitor or immune checkpoint
inhibitors. In embodiments, the immune checkpoint inhibitors can be
anti-Cytotoxic T-Lymphocyte Antigen 4 (CTLA4) or anti-Programme
Death 1 (PD1)/Programmed Death Ligand-1 (PDL1).
[0074] Phosphoinositide kinase-3 (PI3K) is a proto-oncogene which
regulates cell longevity. In one embodiment the pI3K inhibitor is
LY294002, a pharmaceutically acceptable salt or solvate thereof, or
an analogue thereof, wherein the analogue has pI3K inhibitory
activity. Alternatively the pI3K inhibitor may be wortmannin (WMN)
or a pharmaceutically acceptable salt or solvate thereof, or an
analogue thereof.
[0075] MAP kinases are proteins which are involved with cellular
responses, inflammation and growth. P38 kinase (p38) is a member of
the stress activated protein kinase (SAPK) group of MAP kinases. In
certain embodiments, the inhibitor is a p38 kinase inhibitor.
Preferably the p38 kinase inhibitor is SB203580 or a
pharmaceutically acceptable salt or solvate thereof, or an analogue
thereof, wherein the analogue has p38 inhibitory activity.
Alternatively the inhibitor may be SB220025 or SB239063 or a
pharmaceutically acceptable salt or solvate thereof, or an analogue
thereof, wherein the analogue has p38 kinase inhibitory
activity.
BRIEF DESCRIPTION OF THE FIGURES
[0076] Embodiments of the present invention will now be described
by way of example only with reference to the figures described
below.
[0077] FIG. 1A shows SEQ ID NOs:1-6 which are full length sequences
of B. pertussis lipoproteins BP1569, BP2992, BP0205, BP3342, BP3819
and BP2508. FIG. 1B shows SEQ ID NOs:7-12 which are N-terminal
signal peptide sequences from putative B. pertussis lipoproteins
(positively charged residues and putative lipobox are highlighted
in bold, invariant cysteine residue is underlined). FIG. 1C shows
SEQ ID NO:13 and SEQ ID NO:14 which are the sequences of the
lipopeptides from B. pertussis.
[0078] FIG. 2A shows an SDS-PAGE analysis of Bordetella pertussis
lipoprotein BP1569 (lane 1: 10 ng BP1569, Lane 2: molecular weight
markers) following nickel affinity and ion-exchange chromatography.
FIG. 2B shows cytokine production (TNF-alpha, IL-6, IL-12p70,
IL-23) measured by ELISA when Dendritic Cells from C3H/HeJ mice
were cultured with BP1569 (100 and 1000 ng/ml) for 24 h. FIG. 2C
shows surface expression of MHCII, CD80 and CD86 determined by flow
cytometry following treatment of C3H/HeJ Dendritic Cells with
BP1569 (100 ng/ml; bold line) or medium only (grey line) for 24 h.
FIG. 2D shows TNF-.alpha. production measured by ELISA when
Dendritic Cells from C3H/HeJ mice were treated with BP1569 (100
ng/ml) for 24 h with or without addition of anti-TLR2 antibody
(T2.5; 2.5 .mu.g/ml). FIG. 2E shows concentrations of IL-6 in
supernatants quantified by ELISA when BP1569 was treated with
lipase from Aspergillus (As) or Pseudomonas (Ps) at the indicated
concentrations for 18 h at 37.degree. C. Lipase-treated or
untreated BP1569 (100 ng/ml) was used to stimulate BMDC from
C3H/HeJ mice.
[0079] FIG. 3 demonstrates the activation of NF-.kappa.B and MAP
kinase pathways by BP1569. FIG. 3A shows luciferase activity of
HEK-293T cells stably expressing human TLR2 which were transfected
with an NF-.kappa.B luciferase reporter construct prior to
stimulation with increasing doses of BP1569. Luciferase activity
was quantified after 24 h. FIG. 3B shows IL-8 production of
HEK-293T cells stably expressing human TLR2 which were transfected
with an NF-.kappa.B luciferase reporter construct prior to
stimulation with increasing doses of BP1569. IL-8 production was
quantified after 24 h. FIG. 3C shows phosphorylation of p38 which
was assessed following stimulation of spleen cells from C3H/HeJ
mice with BP1569 with or without addition anti-TLR2 antibody of
(T2.5 2.5 .mu.g/ml) or an isotype control. FIG. 3D shows IL-6
concentrations in supernatants of human PBMC which were treated
with BP1569 (100 ng/ml) in the presence and absence of anti-TLR1 or
anti-TLR6 neutralising antibodies. After 24 h, the concentrations
of IL-6 in supernatants were determined by ELISA.
[0080] FIG. 4 demonstrates how BP1569 induces innate cytokine and
antigen-specific IL-17 and IFN-.gamma. production in vivo. FIG. 4A
shows IL-12 and IL-6 concentrations when C3H/HeJ mice were injected
intraperitoneal (i.p.) with BP1569 in PBS (70 .mu.g) or PBS only.
After 3 h serum IL-12 and IL-6 concentrations were quantified by
ELISA. FIG. 4B shows concentration of antigen-specific IFN-.gamma.
quantified by ELISA when C3H/HeJ mice were injected in the footpad
with PBS or 10 .mu.g of BP1569. After seven days the draining lymph
node was harvested and cells were stimulated with BP1569 (2
.mu.g/ml). FIG. 4C shows concentration of antigen-specific
IFN-.gamma. quantified by ELISA when C3H/HeJ mice were injected in
the footpad with PBS or 10 .mu.g of BP1569. After seven days the
draining lymph node was harvested and cells were stimulated with
total heat killed B. pertussis 1-100.times.10.sup.6/ml).
[0081] FIG. 5 demonstrates how synthetic lipopeptide LP1569 induces
cytokine production by mouse Dendritic Cells and macrophage and
human PBMC. FIG. 5A shows TNF-.alpha., IL-12p40, IL-12p70, IL-6 and
IL1.alpha. production from dendritic cells from C57BL/6 mice which
were stimulated with increasing concentrations of lipopeptides
LP1569. Anti-TLR2 (T2.5 2.5 .mu.g/ml) was co-incubated with the
highest dose of peptide. After 24 hr culture, cytokine production
was quantified by ELISA. FIG. 5B shows TNF-.alpha. production from
dendritic cells from C57BL/6 mice stimulated with LP1569 or LP1569
with anti-TLR2 (.alpha.TLR2; T2.5: 2.5 .mu.g/ml). After 24 hr,
TNF-.alpha. production was quantified by ELISA. FIG. 5C shows
TNF.alpha. production from human PBMC which were treated with
increasing concentrations of LP1569 for 24 hr and TNF.alpha. and
IL-6 production was quantified by ELISA. FIG. 5D shows serum
cytokines from C57BL/6 mice which were injected i.p. with 50 or 100
.mu.g of LP1569. After 3 hr serum IL-6 and IL-12p40 were quantified
by ELISA.
[0082] FIG. 6 demonstrates how LP1569 enhances activation of
.gamma..delta. T cells and CD4 T cells. FIG. 6A shows spleen cells
from C57BL/6 mice stimulated with LP1569 (100 ng/ml) or medium with
or without anti-IL-12p40 (.alpha.p40) or anti-IL-23p19 (.alpha.p19)
blocking antibodies. After 72 h supernatants were tested for
IFN-.gamma. by ELISA. FIG. 6B shows spleen cells from C57BL/6 mice
stimulated with LP1569 (100 ng/ml) or medium with or without
IL-1.beta. or IL-23. After 72 h supernatants were tested for IL-17
by ELISA. FIG. 6C shows intracellular cytokine staining for
IFN-.gamma. gated on CD4 T cells from spleen cell cultures
stimulated with LP1569 (100 ng/ml), IL-1.beta. or both. FIG. 6D
shows intracellular cytokine staining for IFN-.gamma. gated on CD8
T cells from spleen cell cultures stimulated with LP1569 (100
ng/ml), IL-1.beta. or both. FIG. 6E shows intracellular cytokine
staining for IL-17 gated on .gamma..delta. T cells from spleen cell
cultures stimulated with LP1569, IL-23 or both.
[0083] FIG. 7 demonstrates how LP1569 acts as an adjuvant for an
experimental acellular pertussis vaccine and promotes protective
cellular immunity against B. pertussis. C57BL/6 mice were immunized
twice (0 and 4 weeks) with PTd, FHA and pertactin alone or
formulated with LP1569 or PBS as control. Two weeks after the
second immunization, mice were challenged by aerosol exposure to B.
pertussis. FIG. 7A shows CFU counts which were performed on lung
homogenates, 3, 7 and 10 days post challenge. FIG. 7B shows serum
FHA-specific IGg1 and IgG2a antibody titres which were determined
by ELISA on serum prepared on day of challenge. FIG. 7C shows
IL-17, IFN-.gamma. and IL-5 concentrations in supernatants from
spleen cells from immunized mice on day of challenge stimulated
with FHA and after 3 days IL-17, IFN-.gamma. and IL-5
concentrations in supernatants were quantified by ELISA.
[0084] FIG. 8 demonstrates how LP1569 as an adjuvant enhances Th1
responses and protective efficacy of acellular pertussis vaccine.
Mice were immunized i.p. twice (0 and 28 days) with and acellular
pertussis vaccine absorbed to alum alone (Pa) or with LP1569 added
to Pa with alum. 14 days after the second immunization, mice were
challenged by exposure to an aerosol of live B. pertussis. The
number of CFU in the lungs were quantified at intervals after
challenge. The results of two independent experiments are shown in
FIG. 8A and FIG. 8B. FIG. 8C shows B. pertussis-FHA-specific
IFN-.gamma. production by spleen cells on day of challenge.
[0085] FIG. 9 demonstrates how the lipoprotein from Bordetella
pertussis BP2992 induces maturation and pro-inflammatory cytokine
production by dendritic cells (DCs). FIG. 9A shows SDS-PAGE
analysis of BP2992 following nickel affinity and ion-exchange
chromatography. Lane 1: 5 ng BP2992; lane 2: 10 ng BP2992. FIG. 9B
shows cytokine production when dendritic cells from C3H/HeJ mice
which were cultured with BP2992 (50, 100 and 1000 ng/ml) for 24 h
and cytokine production was measured by ELISA. FIG. 9C shows
surface expression of MHC class II, CD80 and CD86 which was
determined by flow cytometry following treatment of C3H/HeJ DC with
BP2992 (100 ng/ml; bold line) or medium only (grey line) for 24
h.
[0086] FIG. 10 demonstrates how BP2992 induces pro-inflammatory
cytokine production by dendritic cells (DCs) in a TLR2 dependent
manner. FIG. 10A shows IL-6 production when dendritic cells from
C3H/HeJ mice were treated with BP2992 (100 ng/ml) or
PAM.sub.3Cys.sub.4 with or without addition of anti-TLR2 antibody
(T2.5; 2.5 .mu.g/ml). After 24 hours IL-6 production was measured
by ELISA. FIG. 10B shows TNF-.alpha. production when dendritic
cells from C3H/HeJ mice were treated with BP2992 (2.5-500 ng/ml) or
500 ng/ml BP2992 with an anti-TLR2 antibody (T2.5; 2.5 .mu.g/ml).
After 24 hours TNF-.alpha. production was measured by ELISA.
[0087] FIG. 11 demonstrates how BP2992 induces pro-inflammatory
cytokine production from human peripheral blood mononuclear cells
(PBMC). PBMC from two human donors (A and B) were treated with
BP2992 (0.01-1 .mu.g/ml), After 24 h, the concentrations of
TNF-.alpha. in supernatants were determined by ELISA.
[0088] FIG. 12 demonstrates how the synthetics lipopetides LP1569
and LP2992 enhance activation of CD4 T cells and .gamma..delta. T
cells. FIG. 11A shows ELISA for IL-17 and IFN.gamma. on
supernatants from spleen cell stimulated with LP1569, LP2992 or
Pam.sub.3Cys.sub.4 with or without IL-1.beta. or IL-23 for 3 days.
FIG. 12B shows intracellular cytokine staining for IL-17 gated on
.gamma..delta. T cells from spleen cell cultures stimulated with
LP1569, LP2992 or Pam3Cys in the presence or absence of IL-23. FIG.
12C shows CFSE staining of CD4.sup.+ T cells stimulated with
anti-CD3 alone (shaded histogram) or in the presence of LP1569,
LP292 or Pam3Cys (solid line).
[0089] FIG. 13 demonstrates how LP1569 and LP2992 act as adjuvants
in vivo, promoting Th1 and Th17 responses to co-injected antigens.
C57BL/6 mice were immunized (day 0, and 21) i.p with KLH (10 .mu.g)
mixed with LP1569 or LP2992 (10 .mu.g). Seven days following the
second immunization, spleen cells were harvested and re-stimulated
with increasing concentrations of KLH for 3 days. IL-17 and
IFN.gamma. were detected in the spleen cell supernatants by
ELISA.
[0090] FIG. 14 demonstrates how therapeutic administration of
LP1569 slows tumour growth and enhances survival of mice challenged
with CT26 colon carcinoma cells. BALB/c mice were injected
subcutaneously (s.c.) with 3.times.10.sup.5 CT26 colon carcinoma
cells on day 0. FIG. 14A shows tumor volume and FIG. 14B shows
survival which were monitored in mice injected in site of the
tumour with LP1569 (50 .mu.g) in DMSO and PBS or DMSO and PBS alone
on days 3, 10 and 17. Results in FIG. 14A are representative of two
experiments (n=6) and results for FIG. 14B are pooled data from 2
experiments (n=12).
DETAILED DESCRIPTION OF THE INVENTION
Administration of Vaccine Compositions
[0091] In certain embodiments, the vaccine compositions of the
invention may comprise a further adjuvant. In certain embodiments,
the adjuvant is selected from the group consisting of, but not
limited to, Freund's complete adjuvant, Freund's incomplete
adjuvant, Quil A, Detox, ISCOMs and squalene. Further suitable
adjuvants include mineral gels or an aluminium salt such as
aluminium hydroxide or aluminium phosphate, but may also be a salt
of calcium, iron or zinc, or may be an insoluble suspension of
acylated tyrosine, or acylated sugars, or may be cationically or
anionically derivatised saccharides, polyphosphazenes,
biodegradable microspheres, monophosphoryl lipid A (MPL), lipid A
derivatives (e.g. of reduced toxicity), 3-0-deacylated MPL, quil A,
Saponin, QS21, Freund's Incomplete Adjuvant (Difco Laboratories,
Detroit, Mich.), Merck Adjuvant 65 (Merck and Company, Inc., USA),
AS-2, AS01, AS03, AS04, AS15 (GSK, USA), MF59 (Novartis, Sienna,
Italy), CpG oligonucleotides, bioadhesives and mucoadhesives,
microparticles, liposomes, outer membrane vesicles, polyoxyethylene
ether formulations, polyoxyethylene ester formulations, muramyl
peptides or imidazoquinolone compounds.
[0092] The TLR2-activating lipoprotein of the present invention may
be administered to a patient in need of treatment via any suitable
route. Typically, a composition of the invention can be
administered parenterally by injection or infusion. Examples of
preferred routes for parenteral administration include, but are not
limited to; intravenous, intracardial, intraarterial,
intraperitoneal, intramuscular, intracavity, subcutaneous,
transmucosal, inhalation or transdermal. Routes of administration
may further include topical and enteral, for example, mucosal
(including pulmonary), oral, nasal, rectal.
[0093] In embodiments where the composition is delivered as an
injectable composition, for example in intravenous, intradermal or
subcutaneous application, the active ingredient can be in the form
of a parenterally acceptable aqueous solution which is pyrogen-free
and has suitable pH, isotonicity and stability. Those of relevant
skill in the art are well able to prepare suitable solutions using,
for example, isotonic vehicles such as sodium chloride injection,
Ringer's injection or, Lactated Ringer's injection. Preservatives,
stabilisers, buffers, antioxidants and/or other additives may be
included, as required.
[0094] The composition may also be administered via microspheres,
liposomes, other microparticulate delivery systems or sustained
release formulations placed in certain tissues including blood.
[0095] Examples of the techniques and protocols mentioned above and
other techniques and protocols which may be used in accordance with
the invention can be found in Remington's Pharmaceutical Sciences,
18th edition, Gennaro, A. R., Lippincott Williams & Wilkins;
20th edition ISBN 0-912734-04-3 and Pharmaceutical Dosage Forms and
Drug Delivery Systems; Ansel, H. C. et al. 7th Edition ISBN
0-683305-72-7, the entire disclosures of which is herein
incorporated by reference.
[0096] The composition of the invention is typically administered
to a subject in a "therapeutically effective amount", this being an
amount sufficient to show benefit to the subject to whom the
composition is administered. The actual dose administered, and rate
and time-course of administration, will depend on, and can be
determined with due reference to, the nature and severity of the
condition which is being treated, as well as factors such as the
age, sex and weight of the subject being treated, as well as the
route of administration. Further due consideration should be given
to the properties of the composition, for example, its binding
activity and in-vivo plasma life, the concentration of the antibody
or binding member in the formulation, as well as the route, site
and rate of delivery.
[0097] Dosage regimens can include a single administration of the
composition, or multiple administrative doses of the composition.
The compositions can further be administered sequentially or
separately with other therapeutics and medicaments which are used
for the treatment of the condition for which the TLR2-activating
lipoprotein of the present invention is being administered to
treat.
[0098] Examples of dosage regimens which can be administered to a
subject can be selected from the group comprising, but not limited
to; 1 .mu.g/kg/day through to 20 mg/kg/day, 1 .mu.g/kg/day through
to 10 mg/kg/day, 10 .mu.g/kg/day through to 1 mg/kg/day. In certain
embodiments, the dosage will be such that a plasma concentration of
from 1 .mu.g/ml to 100 .mu.g/ml of the lipoprotein is obtained.
However, the actual dose of the composition administered, and rate
and time-course of administration, will depend on the nature and
severity of the condition being treated. Prescription of treatment,
e.g. decisions on dosage etc, is ultimately within the
responsibility and at the discretion of general practitioners and
other medical doctors, and typically takes account of the disorder
to be treated, the condition of the individual patient, the site of
delivery, the method of administration and other factors known to
practitioners.
DEFINITIONS
[0099] Unless otherwise defined, all technical and scientific terms
used herein have the meaning commonly understood by a person who is
skilled in the art in the field of the present invention.
[0100] Throughout the specification, unless the context demands
otherwise, the terms `comprise` or `include`, or variations such as
`comprises` or `comprising`, `includes` or `including` will be
understood to imply the inclusion of a stated integer or group of
integers, but not the exclusion of any other integer or group of
integers.
[0101] As used herein, terms such as "a", "an" and "the" include
singular and plural referents unless the context clearly demands
otherwise. Thus, for example, reference to "an active agent" or "a
pharmacologically active agent" includes a single active agent as
well as two or more different active agents in combination, while
references to "a carrier" includes mixtures of two or more carriers
as well as a single carrier, and the like.
[0102] As used herein, the term "treatment" and associated terms
such as "treat" and "treating" means the reduction of the
progression, severity and/or duration of Whooping Cough or at least
one symptom thereof, wherein said reduction or amelioration results
from the administration of a TLR2-activating lipoprotein of B.
pertussis. The term `treatment` therefore refers to any regimen
that can benefit a subject. The treatment may be in respect of an
existing condition or may be prophylactic (preventative treatment).
Treatment may include curative, alleviative or prophylactic
effects. References herein to "therapeutic" and "prophylactic"
treatments are to be considered in their broadest context. The term
"therapeutic" does not necessarily imply that a subject is treated
until total recovery. Similarly, "prophylactic" does not
necessarily mean that the subject will not eventually contract a
disease condition.
[0103] As used herein, the term "subject" refers to an animal,
preferably a mammal and in particular a human. In a particular
embodiment, the subject is a mammal, in particular a human. The
term "subject" is interchangeable with the term "patient" as used
herein.
[0104] As used herein, the terms "mount", "mounted", "elicit" or
"elicited" when used in relation to an immune response mean an
immune response which is raised against the immunogenic determinant
of a vaccine composition which is administered to a subject.
Typically the immunogenic determinant of the vaccine composition
comprises the at least one lipoprotein of the present
invention.
[0105] As used herein, the term "immune response" includes T cell
mediated and/or B cell mediated immune responses that are
influenced by modulation of T cell co-stimulation. The term immune
response further includes immune responses that are indirectly
effected by T cell activation such as antibody production (humoral
responses) and the activation of cytokine responsive cells such as
macrophages.
[0106] The inventors of the present invention have identified novel
TLR2 lipoprotein ligands from B. pertussis capable of activating
innate immune responses that drive the induction of protective
adaptive cellular immunity. Additionally, the present inventors
have demonstrated that these novel proteins specifically activate
TLR2 and drive potent pro-inflammatory cytokine production.
[0107] A number of Gram negative bacteria have been shown to
express ligands for TLR2. TLR2 forms a heterodimer with either TLR1
or TLR6 to recognize triacylated or diacylated lipoproteins,
respectively. These lipoproteins contain a common N-terminal signal
sequence comprising a positively charged region followed by a
hydrophobic region of 7-22 residues and finally a lipobox within
the first 40 residues from the N-terminus with the consensus
sequence [LVI][ASTVI][ASG][C]. Without being bound by theory, the
inventors submit that during biosynthesis of the lipoprotein of the
present invention, an acyl group such as palmytic acid or a
diacylated lipid is covalently attached to the conserved cysteine
residue in the lipobox and the signal sequence is enzymatically
cleaved, leaving an exposed acyl coupled N-terminus. The acyl group
on a lipopeptide physically interacts with TLR2 to activate the
receptor and subsequent downstream signalling pathways. Preferably
the lipoproteins of the present invention can be triacylated
because activity can be blocked with neutralising antibodies to
TLR1 and TLR2 but not TLR6. This is because TLR1/2 heterodimers
recognize triacylated lipoproteins whereas TLR2/6 heterodimers
recognize diacylated lipoproteins.
[0108] The proteins identified in the present invention contain
unique N-terminal signal peptide characteristic of bacterial
lipoproteins from Gram negative bacteria. It is demonstrated that
BP1569 and BP2992 have potent immunostimulatory activity, driving
DC maturation and pro-inflammatory cytokine production.
Furthermore, the present inventors have demonstrated that the
corresponding synthetic lipopeptide agonist of TLR2, LP1569, is an
effective adjuvant for an experimental acellular pertussis vaccine
(Pa) that induced Th1 and Th17 responses and conferred a high level
of protection against B. pertussis infection of the respiratory
tract in mice.
[0109] Host immunity to B. pertussis involves a combination of
innate and adaptive immune responses. The induction of Th1 and Th17
cells is dependent on dendritic cell maturation and production of
innate cytokines, including IL-12, and IL-1, IL-6 and IL-23
respectively. The induction of DC maturation and cytokine
production is driven by activation through PRR, including TLRs and
NLRs. Indeed it has been demonstrated that TLR4 plays a critical
role in natural and vaccine-induced protective cellular immunity to
B. pertussis. Surprisingly, during B. pertussis infection Th1
responses were stronger in TLR4-defective mice when compared with
wildtype mice and this was attributed to weaker Treg cells
responses due to the loss of TLR-4-induced innate IL-10 (Higgins S
C, et al.). It has been suggested that B. pertussis must contain
other IL-12-inducing PAMPs that promote Th1 responses. The present
study demonstrates that TLR2 ligands including BP1569 induce potent
IL-12 production by DC and macrophages and promote Th1 responses to
an experimental Pa and may therefore impart drivers of protective
Th1 responses in natural host immunity to B. pertussis.
[0110] In addition to the well established function of Th1 cells in
protective immunity to B. pertussis, by virtue of their in role in
macrophage activation and opsonising antibody production, evidence
is emerging that Th17 cells also play a role in immunity to B.
pertussis through recruitment and activation of neutrophils in the
respiratory tract (Ross P J, et al.). The present inventors have
demonstrated that Adenylate Cyclase Toxin (ACT) from B. pertussis
promotes Th17 responses by inducing IL-1.beta. production via
activation of the NLRP3 inflammasome and caspase-1 which is
required for cleavage of pro-IL-1.beta. (Dunne A, et al.). The
induction of pro-IL-1.beta. is driven through a TLR-induced
NF.kappa.B pathway. IL-1.beta. synergises with IL-23 to induce
expansion of Th17 cells but also promotes innate IL-17 production
by .gamma..delta. T cells. TLR agonists have also been shown to
induce IL-17 production by .gamma..delta. T cells. IL-17-secreting
.gamma..delta. T cells play an important role early in infection
and help to drive IL-17 production by CD4 T cells. The present
inventors have demonstrated that the TLR2 lipopeptide LP1569
induces IL-12 which promotes IFN-.gamma. production by CD4 and CD8
T cells. LP1569 induced significant IL-17 in combination with
IL-23. IL-1 and IL-23 are required to induce IL-17 production by
.gamma..delta. T cells, and the lack of IL-17 production in vitro
without exogenous IL-23 may reflect the fact that LP1569 is more
effective at inducing IL-1 than IL-23. Furthermore, it promoted
IL-17 production by CD4 T cells in vivo when used as an adjuvant
for an experimental Pa.
[0111] Immunity to infection by B. pertussis conferred by
vaccination with Pa wanes significantly over a relatively short
period; the efficacy of the vaccines has been shown to be as low as
24% in children aged 8 to 12 years (and this may explain the recent
resurgence of whopping cough). Current Pa administered with alum as
the adjuvant and studies in mice and humans have shown that these
vaccines preferentially induce Th2-type responses. However, recent
studies in mice and baboons have suggested that a failure of Pa to
induce Th1 or Th17 responses may explain their limited ability to
prevent infection with B. pertussis (Ross P J, et al.). The present
inventors demonstrate that the use of a Th1/Th17 promoting adjuvant
such the TLR2 agonists BP1569 or BP2992 or their corresponding
synthetic lipopeptides LP1569 and LP2992 have the capacity to
improve the efficacy of current Pa by promoting the induction of
protective cellular immunity. Furthermore, since these lipoproteins
are B. pertussis antigens as well as adjuvant TLR2 agonists they
have considerable potential for inclusion in a more effective
vaccine against B. pertussis.
[0112] The inventors of the present invention have used mass
spectroscopy and bioinformatic approaches to identify six putative
TLR-activating lipoproteins from B. pertussis (Table 1).
TABLE-US-00003 TABLE 1 Putative lipoproteins from Bordetella
pertussis. Primary Electronic Name Accession Annotation Size
Similar to BP0205 Q7W0D8 Putative ~19 kDa Hypothetical protein
lipoprotein Q56428 From Thermus thermophilis BP1569 Q7VXZ9 Putative
~40 kDa Lipoprotein NMB0928 from lipoprotein Neisseria menigitidis
BP3342 Q7VU04 Putative ~16 kDa Lipoprotein Omp P6 from lipoprotein
Haemophilus influenzae (Verified TLR2 agonist (14); 39% sequence
identity) BP3819 Q7VSV3 Uncharacterized ~25 kDa Poor matches BP2508
Q9X6Z0 Putative ~19 kDa Lipoprotein OmlA from lipoprotein
Burkholderia pseudomallei Synonym: OmlA BP2992 Q7VUT2 Putative ~16
kDa Outer membrane lipoprotein lipoprotein PCP from H. influenzae
(Verified TLR2 agonist (14); 40% sequence identity)
[0113] The inventors demonstrate that at least two of these novel
proteins specifically activate TLR2 and drive potent
pro-inflammatory cytokine production (BP1569 and BP2992). These
proteins contain a characteristic N-terminal signal peptide that is
unique to Gram negative bacteria. Table 2 shows SEQ ID NOs:7-12
which are the N-terminal signal peptides of these putative
lipoproteins from B. pertussis.
TABLE-US-00004 (LP1569) SEQ ID NO: 7 MRMNK R HAGASALMALAL LAGC
(LP2992) SEQ ID NO: 8 MNYMHSPSVVAGRARRLLAVAAVAGSVAVLAGC (LP0205)
SEQ ID NO: 9 MQLTIR K LAYTLAFSTLV LAGC (LP3342) SEQ ID NO: 10
MKSRIA K SLTIAALAAT LAAC (LP3819) SEQ ID NO: 11
MSAPLDTPALRLNTRFATGIVLAGTLA LAGC (LP2508) SEQ ID NO: 12 MIARISLRPL
K GLAVAVLAASA LTAC
TABLE-US-00005 TABLE 2 N-terminal signal peptide of putative
lipopetides from B. pertussis Name N-terminal signal peptide LP1569
MRMNK R HAGASALMALAL LAGC (SEQ ID NO: 7) LP2992
MNYMHSPSVVAGRARRLLAVAAVAGSVAV LAGC (SEQ ID NO: 8) LP0205 MQLTIR K
LAYTLAFSTLV LAGC (SEQ ID NO: 9) LP3342 MKSRIA K SLTIAALAAT LAAC
(SEQ ID NO: 10) LP3819 MSAPLDTPALRLNTRFATGIVLAGTLA LAGC (SEQ ID NO:
11) LP2508 MIARISLRPL K GLAVAVLAASA LTAC (SEQ ID NO: 12)
[0114] The present inventors have demonstrated that BP1569 and
BP2992 activate murine dendritic cells and macrophages and human
mononuclear cells via TLR2. Furthermore, the inventors have
demonstrated that the corresponding synthetic lipopeptides LP1569
and LP2992 have potent immunostimulatory and adjuvant properties,
capable of enhancing Th1, Th17 and IgG2a antibody responses induced
in mice with an experimental acellular pertussis vaccine and
conferred protective immunity against respiratory infection with B.
pertussis.
[0115] Furthermore, the inventors consider that the lipoproteins of
the present invention can be utilised with kinase inhibitors and/or
tumour associated antigens to provide a Th1/Th17 mediated response
against tumours.
[0116] The present inventors have demonstrated that therapeutic
administration of the synthetic lipopeptide LP1569 slows tumour
growth and enhances survival in a murine colon cancer model. The
lipoproteins of the present invention have considerable potential
as therapeutics alone or in combination with PI3K kinase inhibitors
or other inhibitors of regulatory responses or for inclusion in
prophylactic and/or therapeutic vaccines for the treatment and
prevention of cancer.
[0117] The present inventors predict that the lipopeptides of the
present invention can be used as a therapeutic or as a vaccine
composition in the treatment and/or prevention of allergic diseases
such as asthma. This may relate to IFN-gamma mediated suppression
of the Th2/Th17 response or IL-10-mediated suppression. In
experiments carried out by the inventors, IFN-gamma induction and
some IL-10 production are detected by the TLR2-activated dendritic
cells. There are also reports that TLR2 agonists can
induce/activate Treg cells.
[0118] The present invention will now be described with reference
to the following examples which are provided for the purpose of
illustration and are not intended to be construed as being limiting
on the present invention.
EXAMPLES
Materials and Methods
Mice
[0119] C57BL/6 mice and C3H/HeJ mice containing a mutation in the
tlr4 gene were obtained from Harlan UK and maintained at Trinity
College Dublin in a specific pathogen-free facility.
Reagents
[0120] Lipases from Aspergillus and Pseudomonas were obtained from
Sigma. ELISA for TNF, IL-23, IL-10 and IL-17 were obtained from
R&D. ELISA for IFN-.gamma., IL-6 and IL-12p40 were obtained
from BD Biosciences. Lipopeptide LP1569 were synthesized by EMC
Microcollections.
Cloning and Purification of BP1569 and BP2992
[0121] DNA encoding BP1569 and BP2992 was amplified from B.
pertussis genomic DNA and cloned into the pET21a bacterial
expression vector (Invitrogen) following sequence verification.
C-terminal histidine versions were generated in E. coli BL21 pLysS
cells following 0.2 mM IPTG induction for 18 hr at 30.degree. C.
Cells were lysed using Bugbuster (Novagen) and proteins were
subsequently purified by nickel affinity followed by DEAE
ion-exchange chromatography. Following desalting on PD10 columns
(GE healthcare), protein purity was determined by SDS-PAGE and
coomassie staining or western blotting for the histidine tagged
proteins.
Cell Preparation and Stimulation
[0122] Bone marrow derived dendritic cells (DCs) were prepared by
culturing bone marrow cells obtained from the femur and tibia of
mice in complete RPMI (cRPMI, RPMI containing 10% fetal calf serum,
100 U/ml penicillin, 100 .mu.g/ml streptomycin, 2 mM 1-glutamine
(Invitrogen), and 50 .mu.M 2-ME (Sigma-Aldrich) supplemented with
GM-CSF 40 ng/ml. Cells were re-cultured with fresh medium
containing 40 ng/ml GM-CSF every 3 d for a period of 8 d. DC were
seeded at 1.times.10.sup.6 cells per ml 24 hr prior to stimulation.
ELISA assays were performed using R&D kits according to the
manufacturer's instructions. Spleen cells from C3H/HeJ mice were
seeded at 2.times.10.sup.6 cells per ml and stimulated with
Pam.sub.3Cys.sub.4 (120 nM) or BP1569 (120 nM) for the indicated
times, with or without addition of T2.5-anti-TLR2 antibody or an
isotype control (Hycult Biotech). For p38 activation assays,
samples were lysed with RIPA buffer and SDS-PAGE was performed
followed by Western blotting with anti-phospho-p38 and .beta.-actin
antibodies (Cell Signaling).
Luciferase Assay
[0123] HEK 293T cells stably expressing human TLR2 were transfected
with an NF-.kappa.B luciferase construct as described previously
(21) and stimulated overnight with the indicated concentrations of
BP1569.
Flow Cytometry
[0124] Following stimulation, DC were stained with CD11c (clone
N418; eBioscience), MHCII (clone M5/114.15.2 ebioscience), CD80
(clone 16-10A1 eBioscience) and CD86 (clone 16-10A1 eBioscience).
Samples were analyzed with a FACS DIVA and FloJo software.
Cytokine Induction In Vivo
[0125] C3H/HeJ mice were treated intraperitoneally with BP1569 (70
.mu.g) or PBS control and serum cytokines were measured by ELISA
after 3 h. Significant concentrations of IL-6 and IL-12 were
detected in the serum of mice treated with the BP1569 versus PBS
controls (FIG. 4A).
Antigenicity of BP1569
[0126] C3H/HeJ mice were injected into the footpad with BP1569 (10
.mu.g) diluted in PBS or with PBS only. After seven days the
draining lymph node was harvested and the lymph node cells were
stimulated with either BP1569 (2 .mu.g/ml) or heat killed B.
pertussis pertussis (1-100.times.10.sup.6/ml). After 3 days of
culture, the concentration of IFN-.gamma. in supernatants was
quantified by ELISA. BP1569-specific IFN-.gamma. was induced at
significant levels in mice immunized with BP1569 but not in mice
immunized with PBS (FIG. 4B). Furthermore, cells from immunized
mice produced IFN-.gamma. upon re-stimulation with heat killed B.
pertussis, thus providing evidence that BP1569 is an antigenic
component of the bacteria (FIG. 4C).
Adjuvant Activity of BP1569
[0127] Mice were immunized i.p. twice (wk 0 and 4) with an
experimental laboratory prepared Pa using two purified antigens,
detoxified PT and FHA (1 and 2.5 .mu.g/mouse respectively). PT was
detoxified with formaldehyde as described (Sutherland et al). FHA
was purchased from Kaketsuken, Kumamoto, Japan. Both preparation
were highly purified, as determined by SDS gel chromatography and
were free of detectable LPS. Mice were challenged with B. pertussis
by aerosol inoculation or sacrificed 2 wks after second
immunization.
B. pertussis Respiratory Challenge
[0128] Mice were infected with B. pertussis by exposure to an
aerosol of live B. pertussis as previously described (22). The
course of B. pertussis infection was followed by performing CFU
counts on lungs from groups of 4 mice at intervals after challenge.
The lungs were aseptically removed and homogenised in 1 ml of
sterile physiological saline with 1% casein on ice. Undiluted and
serially diluted homogenate (100 .mu.l) from individual lungs was
spotted in triplicate onto Bordet-Gengou agar plates, and the
number of CFU was calculated after 5 days incubation at 37.degree.
C. The limit of detection was approximately 0.3 log.sub.10 CFU per
lung for groups of 4 mice at each time point.
T Cell Cytokine Production
[0129] Spleen cells (2.times.10.sup.6/ml) from immunized mice were
cultured at 37.degree. C. and 5% CO.sub.2 with heat killed B.
pertussis or purified FHA. Stimulation with PMA (250 ng/ml; Sigma)
and anti-mouse CD3 (1 .mu.g/ml; Pharmingen, San Diego, USA) or
medium only was used as positive and negative controls
respectively. Supernatants were removed after 72 h and IL-4, IL-13,
IL-17 and IFN-.gamma. concentrations determined by two-site
ELISA.
FHA-Specific Antibody Production
[0130] Serum antibody responses to B. pertussis were quantified by
ELISA using plate-bound FHA (5 .mu.g/ml). Bound antibodies were
detected using biotin-conjugated anti-mouse IgG1 or IgG2a (Caltag)
and peroxidase-conjugated streptavidin (BD Pharmingen). Antibody
levels are expressed as the mean endpoint titre (.+-.SE),
determined by extrapolation of the linear part of the titration
curve to 2 SE above the background value obtained with non-immune
mouse serum.
Example 1
Identification, Cloning, Expression and Purification of
TLR2-Activating Lipoproteins from B. pertussis
[0131] Putative B. pertussis lipoproteins were identified using the
DOLOP database (http://www.mrc-lmb.cam.ac.uk/genomes/dolop) which
searches for the presence of the N-terminal signal peptide found in
lipoproteins from Gram-negative bacteria. The sequences of
uncharacterised proteins identified in a mass spectroscopy analysis
of secreted proteins from B. pertussis were used as a source for
this screen in order to ensure that the proteins identified are
indeed expressed proteins. The highest scoring proteins are listed
in Table 1 alongside putative homologs from other bacterial
species.
[0132] All six proteins contain the characteristic positively
charged region followed by a stretch of hydrophobic amino acids and
the lipobox containing the invariant cysteine residue to which the
acyl group is attached during biosynthesis (FIG. 1). BR1569, BP2509
and BP2992 share some sequence similarity with lipoproteins from
Neisseria Meningitidis, Burkholderia pseudomallei and Haemophilis
Influenzae respectively.
[0133] C-terminal histidine versions of the putative lipoproteins
were constructed and expressed in E. coli by IPTG induction. BP1569
and BP2992 were successfully expressed and purified by nickel
affinity followed by ion-exchange chromatography. Although BP2992
was found to be an immunologically active ligand for TLR2 (data not
shown), the present inventors decided to focus on BP1569 because
its expression levels were higher and they could generate
significant quantities of the lipoprotein for more extensive in
vitro and in vivo studies. Analysis of the purity of the BP1569
preparation revealed a strong band at 40 kDa, with a weaker band at
about 35 kDa (FIG. 2A). Western blotting suggested that the second
band is a breakdown product of the full length protein as a result
of proteolysis that was not prevented by the presence of protease
inhibitors. The lipoproteins were co-purified with LPS, some of
which we could remove using polymyxin B columns, but because of the
sticky nature of the lipopeptides, it proved impossible to obtain a
preparation completely free of LPS. Therefore, the inventors
adopted a strategy of carrying out all initial studies with
lipoproteins in TLR4 defective cells or mice and then synthesized
lipopeptide versions of the lipoproteins for more thorough
immunological analysis.
Example 2
BP1569 Induces DC Maturation and Cytokine Production in a TLR2
Dependent Manner
[0134] The present inventors examined the capacity of pertussis
lipoprotein to activate innate immune cells in vitro using bone
marrow-derived DCs from TLR4-defective C3H/HeJ mice. BP1569 induced
robust IL-6, IL-12, IL-23 and TNF-.alpha. production by DC from
C3H/HeJ mice (FIG. 2B). Furthermore, stimulation of DCs with BP1569
for 24 hours enhanced surface expression of MHC class II, CD80 and
CD86 detectable by flow cytometry (FIG. 2C).
[0135] Blocking antibodies were used against TLR2 to determine if
the proteins can activate TLR2 specifically. BP1569-induced
TNF-.alpha. production from DC C3H/HeJ was completely abrogated in
the presence of the TLR2 blocking antibody (FIG. 2D). Lipase
treatment was used to confirm that the immunostimulatory effects of
BP1569 were due the presence of the characteristic acyl side chain
of the lipoprotein. The recombinant lipoprotein was incubated with
two separate lipases for 18 h prior to stimulation of DC. Lipase
treatment abolished BP1569-induced IL-12p40 and IL-6 production
(FIG. 2E), but had no effect on cytokine production induced by the
TLR9 agonist CpG (data not shown), confirming that BP1569 contains
lipid side chains capable of triggering TLR2-induced inflammatory
cytokine production. These data demonstrate that BP1569 is a
lipoprotein agonist for TLR2 and activates maturation and
inflammatory cytokine production by murine DC.
Example 3
BP1569 Activates NF-.kappa.B and MAP Kinase Pathways Downstream of
TLR2
[0136] Cytokine production by TLR2 requires activation of the
transcription factors NF-.kappa.B and p38 MAP kinase. To determine
if BP1569 activates NF-.kappa.B, HEK 293T cells stably expressing
TLR2, but devoid of TLR4, were transfected with an NF-.kappa.B
luciferase reporter construct. Stimulation of these cells with
BP1569 (100 ng/ml) resulted in a significant increase in luciferase
activity (FIG. 3A). IL-8 production by TLR2 transfected HEK 293T
cells was also increased following stimulation with BP1569, linking
activation of the NF-.kappa.B pathway with cytokine production
(FIG. 3B). To assess activation of the MAP kinase pathway, spleen
cells from C3H/HeJ mice were stimulated with BP1569. This treatment
enhanced p38 phosphorylation 15 minutes following stimulation,
which was inhibited by addition of anti-TLR2 blocking antibody
(FIG. 3C). These results demonstrate that BP1569 induces
TLR2-dependent activation of NF-.kappa.B and p38, two pathways
shown to be required for TLR2-induced cytokine production. TLR
agonists can bind to TLR1/2 or TLR2/6 heterodimers, therefore, the
role of these TLRs using specific blocking antibodies to human TLR1
and TLR6 was examined. Incubation of human PBMC with a TLR1
blocking antibody significantly reduced BP1569-induced IL-6
production, whereas an anti-TLR6 antibody had little effect,
suggesting that BP1569 is triacylated rather than diacylated (FIG.
3D).
Example 4
BP1569 Induces Innate Inflammatory Cytokines and is Immunogenic In
Vivo
[0137] Having demonstrated that BP1569 is capable of activating
TLR2 in vitro, whether or not the lipoprotein can induce
pro-inflammatory cytokine responses in vivo was determined.
[0138] C3H/HeJ mice were treated intraperitoneally with BP1569 (70
.mu.g) and serum cytokines were measured after 3 h. Significant
concentrations of IL-6 and IL-12 were detected in the serum of mice
treated with the BP1569 versus PBS controls (FIG. 3A).
[0139] The possibility that BP1569 was immunogenic in vivo and
capable of inducing B. pertussis-specific immune responses was
examined. C3H/HeJ mice were injected into the footpad with BP1569
(10 .mu.g) diluted in PBS or with PBS only. After seven days the
draining lymph node was harvested and the lymph node cells were
stimulated with either BP1569 (2 .mu.g/ml) or heat killed B.
pertussis. BP1569-specific IFN-.gamma. was induced at significant
levels in mice immunized with BP1569 but not in mice immunized with
PBS (FIG. 3B). Furthermore, cells from immunized mice produced
IFN-.gamma. upon re-stimulation with heat killed B. pertussis, thus
providing evidence that BP1569 is an antigenic component of the
bacteria (FIG. 3C).
Example 5
Synthetic Lipopeptide LP1569 Induces Inflammatory Cytokines by
Human and Murine Innate Immune Cells
[0140] The above results demonstrate that BP1569 has
immunomodulatory as well as antigenic properties and the former is
due to its ability to activate TLR2. In order to provide evidence
of TLR2-mediated immunomodulatory activity, and to examine the
adjuvant properties of the B. pertussis lipoproteins in vivo in
conventional mice, a synthetic lipopeptide version of BP1569 was
generated. The lipopeptide, named LP1569 to distinguish it from the
full length protein, has the conserved cysteine residue
palmitylated and followed by 11 amino acids of the protein sequence
of BP1569. This represents the mature N-terminus of the lipoprotein
following removal of the signal peptide during biosynthesis.
[0141] It was first demonstrated that this lipopeptide specifically
activates TLR2 by stimulating murine DC with LP1569 in the presence
and absence of a TLR2 blocking antibody. LP1569 induced TNF-.alpha.
production, which was blocked by anti-TLR2 (FIG. 5A). Furthermore,
LP1569 induced robust expression of TNF-.alpha., IL-10 and IL-6 by
murine macrophages (FIG. 5B). LP1569 also stimulated TNF.alpha.
production by human PBMC (FIG. 5C). Finally, it was demonstrated
that LP1569 induced pro-inflammatory cytokine production in vivo.
Injection of mice with LP1569 resulted in a significant enhancement
of serum concentrations of IL-12 and IL-6 over that observed in
mice injected with PBS (FIG. 5D). These findings demonstrate that
the synthetic peptide LP1569 is a TLR2 agonist and activates innate
immune responses in vitro and in vivo.
Example 6
LP1569 Enhances Activation of T Cells
[0142] The present inventors have shown that BP1569 and LP1569
promote inflammatory cytokine production by DCs, including IL-12,
IL-6, and IL-23 that promote the induction or expansion of Th1 and
Th17 cells.
[0143] Stimulation of spleen cells with LP1569 induced the
production of IFN-.gamma. detected by ELISA, which was inhibited
upon co-incubation with and anti-IL-12p40 but not an anti-p19
antibody, indicating a role for LP1569 driven IL-12p70 in
IFN-.gamma. production (FIG. 6A). Furthermore LP1569 induced IL-17
production by spleen cells following addition of exogenous IL-23
(FIG. 6B). Intracellular cytokine staining and FACS analysis
revealed that CD4.sup.+ and CD8.sup.+ T cells produce IFN-.gamma.
following stimulation of the spleen cells with LP1569, which was
slightly augmented by addition of IL-1.beta. (FIG. 6C and FIG. 6D).
Furthermore, intracellular cytokine staining showed that the
combination of LP1569 and IL-23, but LP12569 or IL-23 alone,
induced a clear population of IL-17-secreting .gamma..delta. T
cells (FIG. 6E).
Example 7
LP1569 is an Effective Adjuvant for Promoting Protective Cellular
Immunity Against B. pertussis
[0144] Having shown that LP1569 has immunostimulatory activity,
promoting innate cytokines that drive T cell responses, the present
inventors assessed its adjuvant activity in vivo using protective
antigens from B. pertussis and established respiratory infection
model. The inventors' previous studies using this model have
demonstrated a critical role for Th1 and Th17 cells in natural and
vaccine-induced immunity to B. pertussis (Ross P J et al). Mice
were immunized with the B. pertussis antigens, FHA, PTd and
pertactin alone or with LP1569 or with PBS only and boosted 4 weeks
later. Mice were challenged by aerosol exposure to live B.
pertussis 2 weeks after the second immunization. Assessment of CFU
counts in the lungs revealed that immunization with B. pertussis
antigens without an adjuvant conferred limited protection against
B. pertussis challenge (FIG. 6A). In contrast, immunization with
the experimental acellular pertussis vaccines (Pa) formulated with
LP1569 conferred a high level of protection against B. pertussis;
bacteria were undetectable in immunized mice 3, 7 and 10 days after
challenge (FIG. 7A).
[0145] Antibody and T cell responses specific for one of the B.
pertussis antigens, FHA, in immunized mice on the day of challenge
were assessed. Immunization with B. pertussis antigens alone
induced weak FHA-specific serum IgG1 and undetectable IgG2a. In
contrast, immunization with B. pertussis antigens in combination
with LP1569 generate high FHA-specific IgG2a titres in serum (FIG.
7B). Furthermore, the experimental acellular pertussis vaccines
(Pa) with LP1569 generated potent Th1 and Th17 responses, with high
concentrations of IFN-.gamma. and IL-17 detected in supernatants of
FHA-stimulated spleen cells from immunized mice (FIG. 7C). In
contrast, immunization with the B. pertussis antigens in the
absence of the lipopeptide generated Th2 type responses, with high
IL-5, but substantially lower concentrations of IL-17 and
IFN-.gamma. than that generated with the experimental vaccine
formulated with LP1569. These findings demonstrate that LP1569 is a
potent adjuvant for induction of Th1/Th17 type responses and
protection against B. pertussis.
Example 8
Therapeutic Administration of LP1569 Slows Tumour Growth and
Enhances Survival
[0146] The present inventors demonstrated how therapeutic
administration of LP1569 slows tumour growth and enhances survival
of mice challenged with CT26 colon carcinoma cells. Mice were
injected subcutaneously (s.c.) with CT26 colon carcinoma cells and
then treated on days 3, 10 and 17 with LP1569 or vehicle only. The
data show that the rate of tumor growth is slower in mice treated
with LP1569. Furthermore, the survival of tumor-bearing mice is
enhanced following treatment with LP1569. These data demonstrated
that LP1569 has anti-tumour properties, most likely due to
induction of innate and adaptive immune responses against the
tumor. Furthermore, this could be enhanced by blocking the
anti-inflammatory or regulatory responses also induced with the
TLR2 agonists, using inhibitors of Pi3 kinase or p38 MAP kinase or
immune checkpoint inhibitors e.g anti-Cytotoxic T-Lymphocyte
Antigen 4 (CTLA4) or anti-Programme Death 1 (PD1)/Programmed Death
Ligand-1 (PDL1).
[0147] All documents referred to in this specification are herein
incorporated by reference. Various modifications and variations to
the described embodiments of the inventions will be apparent to
those skilled in the art without departing from the scope of the
invention. Although the invention has been described in connection
with specific preferred embodiments, it should be understood that
the invention as claimed should not be unduly limited to such
specific embodiments. Indeed, various modifications of the
described modes of carrying out the invention which are obvious to
those skilled in the art are intended to be covered by the present
invention.
REFERENCES
[0148] Dillon et al (2006) Yeast zymosan, a stimulus for TLR2 and
dectin-1, induces regulatory antigen-presenting cells and
immunological tolerance. J Clin Invest; 116(4):916-28. [0149] Dunne
A, et al. (2010) Inflammasome activation by adenylate cyclase toxin
directs Th17 responses and protection against Bordetella pertussis.
J Immunol 185(3):1711-1719. [0150] Higgs R, Higgins S C, Ross P J,
& Mills K H (2012) Immunity to the respiratory pathogen
Bordetella pertussis. Mucosal Immunol 5(5):485-500. [0151] Higgins
S C, et al. (2003) Toll-like receptor 4-mediated innate IL-10
activates antigen-specific regulatory T cells and confers
resistance to Bordetella pertussis by inhibiting inflammatory
pathology. J Immunol 171(6):3119-3127. [0152] Ross P J, et al.
(2013) Relative contribution of Th1 and Th17 cells in adaptive
immunity to Bordetella pertussis: towards the rational design of an
improved acellular pertussis vaccine. PLoS Pathog 9(4):e1003264.
[0153] Sutherland et al. (2011) Antibodies Recognizing Protective
Pertussis Toxin Epitopes Are Preferentially Elicited by Natural
Infection versus Acellular Immunization Clin Vaccine Immunol;
18(6): 954-962.
Sequence CWU 1
1
141375PRTBordetella pertussis 1Met Arg Met Asn Lys Arg His Ala Gly
Ala Ser Ala Leu Met Ala Leu 1 5 10 15 Ala Leu Leu Ala Gly Cys Ser
Asp Val Asn Gln Leu Leu Gly Asn Glu 20 25 30 Glu Ser Val Asp Tyr
Lys Ser Thr Arg Arg Gly Asp Pro Leu Ser Ile 35 40 45 Pro Pro Asp
Leu Thr Gln Ala Asn Asn Asp Pro Arg Tyr Lys Ala Pro 50 55 60 Ala
Ser Gly Thr Ala Thr Tyr Ser Gln Phe Gln Gln Gln Gly Leu Gln 65 70
75 80 Gln Gln Ala Ser Ala Gly Gln Asn Thr Asn Val Leu Pro Glu Arg
Ala 85 90 95 Asp Met Arg Val Glu Arg Asp Gly Asp Leu Arg Trp Leu
Val Ile Glu 100 105 110 Arg Pro Pro Glu Gln Leu Phe Ser Lys Val Val
Asp Phe Trp Thr Asp 115 120 125 Thr Gly Phe Thr Val Ser Val Asn Asn
Pro Gln Ala Gly Ile Ile Glu 130 135 140 Thr Asp Trp Ala Glu Asn Arg
Ala Lys Ile Pro Glu Ser Trp Leu Arg 145 150 155 160 Gln Val Leu Gly
Ser Val Leu Glu Thr Ala Trp Asp Ser Gly Glu Arg 165 170 175 Glu Lys
Phe Arg Thr Arg Val Glu Arg Val Asn Gly His Thr Glu Ile 180 185 190
Tyr Ile Thr His Asn Gln Met Leu Glu Lys Arg Val Gly Ser Asp Gly 195
200 205 Gly Gln Val Gln Trp Thr His Gly Lys Glu Asp Pro Gly Leu Asn
Ala 210 215 220 Ala Met Leu Ala Arg Leu Met Val Tyr Leu Gly Thr Asp
Val Asp Ala 225 230 235 240 Ala Arg Lys Leu Val Ala Gln Ala Glu Ala
Ala Pro Gln Ala Pro Lys 245 250 255 Val Gln Ser Val Arg Ala Glu Gly
Ala Met Leu Val Val Asp Glu Ser 260 265 270 Phe Asp Arg Ala Trp Arg
Arg Val Gly Val Ala Leu Asp Ser Gly Gly 275 280 285 Phe Ala Val Asp
Asp Arg Asp Arg Ser Ala Gly Glu Tyr Phe Val Arg 290 295 300 Tyr Val
Asp Thr Asp Thr Gly Ala Gln Asn Glu Gln Pro Gly Phe Phe 305 310 315
320 Ser Arg Leu Phe Ser Ser Asp Lys Lys Ala Gln Ala Pro Gln Tyr Arg
325 330 335 Ile Arg Leu Thr Gly Ser Gly Thr Gln Thr Gln Val Thr Val
Leu Asp 340 345 350 Ala Asn Gly Gln Arg Asp Ser Ser Ala Thr Ala Gln
Arg Met Leu Ser 355 360 365 Val Leu Lys Asp Lys Met Val 370 375
2166PRTBordetella pertussis 2Asn Tyr Met His Ser Pro Ser Val Val
Ala Gly Arg Ala Arg Arg Leu 1 5 10 15 Leu Ala Val Ala Ala Val Ala
Gly Ser Val Ala Val Leu Ala Gly Cys 20 25 30 Ala Asn Pro Ser Ala
Ser Ser Gly Val Tyr Thr Tyr Gly Gln Ala Gln 35 40 45 Arg Glu Gln
Ile Val Arg Thr Gly Thr Val Thr Gly Val Arg Pro Ile 50 55 60 Thr
Ile Gln Asn Asp Lys Ser Ser Gly Val Gly Leu Val Ala Gly Gly 65 70
75 80 Ala Leu Gly Gly Val Ala Gly Asn Ala Val Gly Gly Gly Thr Gly
Arg 85 90 95 Thr Ile Ala Thr Val Gly Gly Val Ile Leu Gly Ala Leu
Ala Gly Asn 100 105 110 Ala Ile Glu Asn Arg Ala Gly Lys Ser Ser Gly
Tyr Glu Ile Thr Val 115 120 125 Arg Leu Asp Asn Gly Glu Thr Arg Val
Val Ala Gln Glu Ala Asp Val 130 135 140 Pro Ile Ser Val Gly Gln Arg
Val Gln Val Ile Ser Gly Ala Gly Pro 145 150 155 160 Thr Arg Val Thr
Pro Tyr 165 3188PRTBordetella pertussis 3Met Gln Leu Thr Ile Arg
Lys Leu Ala Tyr Thr Leu Ala Phe Ser Thr 1 5 10 15 Leu Val Leu Ala
Gly Cys Thr Thr Ala Ser Lys Lys Thr Asp Gly Gln 20 25 30 Ala Ala
Thr Pro Ala Asp Gln Ala Ser Ser Gln Gln Ala Ser Ala Ala 35 40 45
Ser Val Glu Phe Tyr Val Ala Gln Ala Lys Ala Gly Asp Gly Leu Met 50
55 60 Glu Val Lys Val Pro Asp Gly Ser Leu Tyr Met Gln Arg Gln Pro
Val 65 70 75 80 Leu Thr Arg Ala Asp Leu Thr Glu Ala Ala Ala Leu Val
Asp Arg Gln 85 90 95 Gly Gln Asn Phe Val Gly Leu Arg Phe Thr Glu
Ala Gly Ala Arg Lys 100 105 110 Leu Asn Asp Ile Ser Ser Lys Asn Ile
Gly Asn Met Leu Ala Leu Val 115 120 125 Ile Asp Arg Glu Leu Val Ala
Ala Pro Arg Ile Ala Glu Pro Leu Asn 130 135 140 Arg Gly Val Leu Ala
Phe Gly Val Pro Ser Ala Lys Ala Ala Ser Glu 145 150 155 160 Ile Ala
Ala Lys Ile Arg Gly Asp Ala Gly Ala Pro Ala Ala Gly Val 165 170 175
Pro Ala Ala Pro Ala Pro Lys Pro Ala Pro Lys Pro 180 185
4165PRTBordetella pertussis 4Met Lys Ser Arg Ile Ala Lys Ser Leu
Thr Ile Ala Ala Leu Ala Ala 1 5 10 15 Thr Leu Ala Ala Cys Ser Ser
Val Pro Leu Asp Asp Lys Ala Gly Gln 20 25 30 Ala Gly Gly Ser Gly
Gln Gly Ser Ala Ser Gly Gln Ile Leu Asp Pro 35 40 45 Phe Asn Pro
Gln Ser Ile Leu Ala Gln Gln Arg Ser Val Tyr Phe Asp 50 55 60 Phe
Asp Ser Tyr Thr Val Ser Glu Gln Tyr Arg Gly Leu Val Glu Thr 65 70
75 80 His Ala Arg Tyr Leu Ala Ser Asn Asn Gln Gln Arg Ile Lys Ile
Glu 85 90 95 Gly Asn Thr Asp Glu Arg Gly Gly Ala Glu Tyr Asn Leu
Ala Leu Gly 100 105 110 Gln Arg Arg Ala Asp Ala Val Arg Arg Met Met
Thr Leu Leu Gly Val 115 120 125 Ser Asp Asn Gln Ile Glu Thr Ile Ser
Phe Gly Lys Glu Lys Pro Lys 130 135 140 Ala Thr Gly Ser Ser Glu Ala
Asp Phe Ala Glu Asn Arg Arg Ala Asp 145 150 155 160 Ile Val Tyr Gln
Arg 165 5247PRTBordetella pertussis 5Met Ser Ala Pro Leu Asp Thr
Pro Ala Leu Arg Leu Asn Thr Arg Phe 1 5 10 15 Ala Thr Gly Ile Val
Leu Ala Gly Thr Leu Ala Leu Ala Gly Cys Ala 20 25 30 Gln Gln Arg
Ser Ala Gly Tyr Tyr Asp Pro Pro Gly Ala Ser Thr Ile 35 40 45 Thr
Asp Ala Gln Tyr Gln Gly Gln Ala Ala Gly Tyr Arg Thr Val Val 50 55
60 His Ala Pro Ser Gln Leu Gln Ile Glu Leu Lys Pro Asn Gln Pro Ala
65 70 75 80 Arg Gln Gln Asn Ala Gln Ala Gln Ala Gly Gln Gln Ser Thr
Glu Asp 85 90 95 Gly Thr Ala Val Pro Glu Gly Gln Ala Ala Pro Gln
Pro Gln Pro Glu 100 105 110 Thr Ala Ser Pro Gly Ala Gln Ala Ile Ile
Pro Gln Ala Gln Thr Tyr 115 120 125 Gln Gly Thr Phe Pro Cys Phe Ala
Ala Gly Leu Ala Cys Glu Ala Gln 130 135 140 Arg Val Thr Leu Thr Leu
Ala Pro Asn Gly Arg Trp Arg Ser Arg Thr 145 150 155 160 Asn Tyr Leu
Asp Lys Gln Pro Gln Ala Ser Ala Pro Val Ala Glu Gln 165 170 175 Gly
Cys Trp Asp Ala Thr Gln Glu Arg Pro Pro Arg Val Leu Leu Leu 180 185
190 Asp Gly Ser Gly Asn Met Arg Ala Glu Leu Val Met Thr Ala Asn Asn
195 200 205 Val Leu Arg Val Arg Ser Val Gly Gly Arg Thr Pro Asn Leu
Asn Tyr 210 215 220 Asn Leu Thr Arg Gln Pro Asp Leu Asp Ala Ile Ala
Glu Leu Asp Lys 225 230 235 240 Gln Ala Ala Pro Lys Cys Pro 245
6182PRTBordetella pertussis 6Met Ile Ala Arg Ile Ser Leu Arg Pro
Leu Lys Gly Leu Ala Val Ala 1 5 10 15 Val Leu Ala Ala Ser Ala Leu
Thr Ala Cys Ser Ser Gly Lys Trp Gly 20 25 30 Phe Pro Tyr Lys Ala
Gly Val Gln Gln Gly Asn Trp Ile Thr Lys Glu 35 40 45 Gln Val Ala
Leu Leu Gln Gln Gly Met Ser Arg Glu Gln Val Arg Phe 50 55 60 Ala
Leu Gly Ser Pro Thr Leu Thr Ser Val Leu His Ala Asp Arg Trp 65 70
75 80 Asp Tyr Pro Tyr Tyr Phe Lys Pro Gly Tyr Gly Lys Ala Gln Glu
Arg 85 90 95 Gln Phe Thr Val Trp Phe Glu Asn Asp His Leu Val Arg
Trp Ser Gly 100 105 110 Asp Glu Gln Pro Asp Leu Gln Pro Phe Gln Ile
Glu Lys Val Asn Ala 115 120 125 Lys Gln Glu Glu Lys Ala Asp Ala Gln
Val Asp Thr Ala Glu Lys Arg 130 135 140 Gln Glu Gly Ile Asp Lys Ala
Glu Lys Val Arg Pro His Val Asp Val 145 150 155 160 Thr Thr Pro Asp
Asn Pro Thr Leu Asp Tyr Pro Gly Glu Pro Gly Gln 165 170 175 Thr Phe
Glu Pro Leu Lys 180 722PRTBordetella pertussis 7Met Arg Met Asn Lys
Arg His Ala Gly Ala Ser Ala Leu Met Ala Leu 1 5 10 15 Ala Leu Leu
Ala Gly Cys 20 833PRTBordetella pertussis 8Met Asn Tyr Met His Ser
Pro Ser Val Val Ala Gly Arg Ala Arg Arg 1 5 10 15 Leu Leu Ala Val
Ala Ala Val Ala Gly Ser Val Ala Val Leu Ala Gly 20 25 30 Cys
922PRTBordetella pertussis 9Met Gln Leu Thr Ile Arg Lys Leu Ala Tyr
Thr Leu Ala Phe Ser Thr 1 5 10 15 Leu Val Leu Ala Gly Cys 20
1021PRTBordetella pertussis 10Met Lys Ser Arg Ile Ala Lys Ser Leu
Thr Ile Ala Ala Leu Ala Ala 1 5 10 15 Thr Leu Ala Ala Cys 20
1131PRTBordetella pertussis 11Met Ser Ala Pro Leu Asp Thr Pro Ala
Leu Arg Leu Asn Thr Arg Phe 1 5 10 15 Ala Thr Gly Ile Val Leu Ala
Gly Thr Leu Ala Leu Ala Gly Cys 20 25 30 1226PRTBordetella
pertussis 12Met Ile Ala Arg Ile Ser Leu Arg Pro Leu Lys Gly Leu Ala
Val Ala 1 5 10 15 Val Leu Ala Ala Ser Ala Leu Thr Ala Cys 20 25
1315PRTBordetella pertussis 13Cys Ser Asp Val Asn Gln Leu Leu Gly
Asn Glu Glu Ser Val Asp 1 5 10 15 1415PRTBordetella pertussis 14Cys
Ala Asn Pro Ser Ala Ser Ser Gly Val Tyr Thr Tyr Gly Gln 1 5 10
15
* * * * *
References