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.

Claims

1. A method for enhancing a Th1 and Th17 response in a subject in need thereof comprising: administering to the subject a lipoprotein obtainable from Bordetella pertussis, wherein the lipoprotein comprises 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, and X4; wherein X1 is selected from the group consisting of Leucine, Valine and Isoleucine X2 is selected from the group consisting of Alanine, Serine, Threonine, Valine and Isoleucine X3 is selected from the group consisting of Glycine, Alanine, and Serine and X4 is Cysteine; wherein X4 is capable of being acylated: wherein the lipoprotein is a Toll-like receptor 2 agonist and an adjuvant; and enhancing the Th1 and Th17 response, by the lipoprotein, in the subject, wherein the lipoprotein comprises the amino acid sequence of SEQ ID NO:13.

2. The method as claimed in claim 1, wherein the method is a method of treatment or prevention of a condition caused by B. pertussis.

3. The method as claimed in 2, wherein the method is a method of vaccinating the subject to induce immunity against B. pertussis.

4. The method as claimed in claim 2, wherein the method further comprises administering to the subject an antigen from B. pertussis.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) Embodiments of the present invention will now be described by way of example only with reference to the figures described below.

(2) 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.

(3) 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 24h. FIG. 2D shows TNF-α 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 μg/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° C. Lipase-treated or untreated BP1569 (100 ng/ml) was used to stimulate BMDC from C3H/HeJ mice.

(4) FIGS. 3A-3D demonstrate the activation of NF-κB 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-κB 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-κB 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 μg/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.

(5) FIGS. 4A-4C demonstrate how BP1569 induces innate cytokine and antigen-specific IL-17 and IFN-γ 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 μg) 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-γ quantified by ELISA when C3H/HeJ mice were injected in the footpad with PBS or 10 μg of BP1569. After seven days the draining lymph node was harvested and cells were stimulated with BP1569 (2 μg/ml). FIG. 4C shows concentration of antigen-specific IFN-γ quantified by ELISA when C3H/HeJ mice were injected in the footpad with PBS or 10 μg of BP1569. After seven days the draining lymph node was harvested and cells were stimulated with total heat killed B. pertussis 1-100×10.sup.6/ml).

(6) FIGS. 5A-5D demonstrate how synthetic lipopeptide LP1569 induces cytokine production by mouse Dendritic Cells and macrophage and human PBMC. FIG. 5A shows TNF-α, IL-12p40, IL-12p70, IL-6 and IL1α production from dendritic cells from C57BL/6 mice which were stimulated with increasing concentrations of lipopeptides LP1569. Anti-TLR2 (T2.5 2.5 μg/ml) was co-incubated with the highest dose of peptide. After 24 hr culture, cytokine production was quantified by ELISA. FIG. 5B shows TNF-α production from dendritic cells from C57BL/6 mice stimulated with LP1569 or LP1569 with anti-TLR2 (αTLR2; T2.5: 2.5 μg/ml). After 24 hr, TNF-α production was quantified by ELISA. FIG. 5C shows TNFα production from human PBMC which were treated with increasing concentrations of LP1569 for 24 hr and TNFα 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 μg of LP1569. After 3 hr serum IL-6 and IL-12p40 were quantified by ELISA.

(7) FIGS. 6A-6C demonstrate how LP1569 enhances activation of γδ 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 (αp40) or anti-IL-23p19 (αp19) blocking antibodies. After 72 h supernatants were tested for IFN-γ by ELISA. FIG. 6B shows spleen cells from C57BL/6 mice stimulated with LP1569 (100 ng/ml) or medium with or without IL-1β or IL-23. After 72 h supernatants were tested for IL-17 by ELISA. FIG. 6C shows intracellular cytokine staining for IFN-γ gated on CD4 T cells from spleen cell cultures stimulated with LP1569 (100 ng/ml), IL-1β or both. FIG. 6D shows intracellular cytokine staining for IFN-γ gated on CD8 T cells from spleen cell cultures stimulated with LP1569 (100 ng/ml), IL-1β or both. FIG. 6E shows intracellular cytokine staining for IL-17 gated on γδ T cells from spleen cell cultures stimulated with LP1569, IL-23 or both.

(8) FIGS. 7A-7C demonstrate 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-γ 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-γ and IL-5 concentrations in supernatants were quantified by ELISA.

(9) FIGS. 8A-8C demonstrate 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-γ production by spleen cells on day of challenge.

(10) FIGS. 9A-9C demonstrate 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.

(11) FIGS. 10A-10B demonstrate 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 μg/ml). After 24 hours IL-6 production was measured by ELISA. FIG. 10B shows TNF-α 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 μg/ml). After 24 hours TNF-α production was measured by ELISA.

(12) 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 μg/ml), After 24 h, the concentrations of TNF-α in supernatants were determined by ELISA.

(13) FIGS. 12A1, 12A2, 12B, and 12C demonstrate how the synthetics lipopetides LP1569 and LP2992 enhance activation of CD4 T cells and γδ T cells. FIG. 12A1 and FIG. 12A2 show ELISA for IL-17 (FIG. 12A1) and IFNγ (FIG. 12A2) on supernatants from spleen cell stimulated with LP1569, LP2992 or Pam.sub.3Cys.sub.4 with or without IL-1β or IL-23 for 3 days. FIG. 12B shows intracellular cytokine staining for IL-17 gated on γδ 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+ T cells stimulated with anti-CD3 alone (shaded histogram) or in the presence of LP1569, LP292 or Pam3Cys (solid line).

(14) 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 μg) mixed with LP1569 or LP2992 (10 μg). Seven days following the second immunization, spleen cells were harvested and re-stimulated with increasing concentrations of KLH for 3 days. IL-17 and IFNγ were detected in the spleen cell supernatants by ELISA.

(15) FIGS. 14A-14B demonstrate 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 3×10.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 μg) 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 EMBODIMENTS OF THE INVENTION

Administration of Vaccine Compositions

(16) 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, MI), Merck Adjuvant 65 (Merck and Company, Inc., USA), AS-2, AS01, AS03, A504, 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.

(17) 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.

(18) 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.

(19) The composition may also be administered via microspheres, liposomes, other microparticulate delivery systems or sustained release formulations placed in certain tissues including blood.

(20) 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.

(21) 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.

(22) 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.

(23) Examples of dosage regimens which can be administered to a subject can be selected from the group comprising, but not limited to; 1 μg/kg/day through to 20 mg/kg/day, 1 μg/kg/day through to 10 mg/kg/day, 10 μg/kg/day through to 1 mg/kg/day. In certain embodiments, the dosage will be such that a plasma concentration of from 1 μg/ml to 100 m/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

(24) 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.

(25) 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.

(26) 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.

(27) 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.

(28) 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.

(29) 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.

(30) 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.

(31) 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.

(32) 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.

(33) 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.

(34) 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.

(35) 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-1β production via activation of the NLRP3 inflammasome and caspase-1 which is required for cleavage of pro-IL-1β (Dunne A, et al.). The induction of pro-IL-1β is driven through a TLR-induced NFκB pathway. IL-1β synergises with IL-23 to induce expansion of Th17 cells but also promotes innate IL-17 production by γδ T cells. TLR agonists have also been shown to induce IL-17 production by γδ T cells. IL-17-secreting γδ 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 lipoptide LP1569 induces IL-12 which promotes IFN-γ 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 γδ 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.

(36) 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.

(37) 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).

(38) 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)

(39) 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.

(40) TABLE-US-00004 (LP1569) SEQ ID NO: 7 MRMNK R HAGASALMALAL LAGC (LP2992) SEQ ID NO: 8 MNYMHSPSVVAGRARRLLAVAAVAGSVAV LAGC (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

(41) 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)

(42) 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.

(43) 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.

(44) 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.

(45) 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.

(46) 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

(47) 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

(48) 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-γ, IL-6 and IL-12p40 were obtained from BD Biosciences. Lipopeptide LP1569 were synthesized by EMC Microcollections.

Cloning and Purification of BP1569 and BP2992

(49) 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 18hr at 30° 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

(50) 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 μg/ml streptomycin, 2 mM 1-glutamine (Invitrogen), and 50 μM 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 1×10.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 2×10.sup.6 cells per ml and stimulated with Pam.sub.3Cys.sub.4 (120nM) 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 β-actin antibodies (Cell Signaling).

Luciferase Assay

(51) HEK 293T cells stably expressing human TLR2 were transfected with an NF-κB luciferase construct as described previously (21) and stimulated overnight with the indicated concentrations of BP1569.

Flow Cytometry

(52) 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

(53) C3H/HeJ mice were treated intraperitoneally with BP1569 (70 μg) or PBS control and serum cytokines were measured by ELISA after 3h. 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

(54) C3H/HeJ mice were injected into the footpad with BP1569 (10 μg) 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 μg/ml) or heat killed B. pertussis pertussis (1-100×10.sup.6/ml). After 3 days of culture, the concentration of IFN-γ in supernatants was quantified by ELISA. BP1569-specific IFN-γ 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-γ 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

(55) 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 μg/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

(56) 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 μl) 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° 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

(57) Spleen cells (2×10.sup.6/ml) from immunized mice were cultured at 37° 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 μg/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-γ concentrations determined by two-site ELISA.

FHA-Specific Antibody Production

(58) Serum antibody responses to B. pertussis were quantified by ELISA using plate-bound FHA (5 μg/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

(59) 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.

(60) 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.

(61) 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

(62) 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-α 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).

(63) Blocking antibodies were used against TLR2 to determine if the proteins can activate TLR2 specifically. BP1569-induced TNF-α 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 immunostimulatry 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-κB and MAP Kinase Pathways Downstream of TLR2

(64) Cytokine production by TLR2 requires activation of the transcription factors NF-κB and p38 MAP kinase. To determine if BP1569 activates NF-κB, HEK 293T cells stably expressing TLR2, but devoid of TLR4, were transfected with an NF-κB 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-κB 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-κB 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

(65) 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.

(66) C3H/HeJ mice were treated intraperitoneally with BP1569 (70 μg) 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).

(67) 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 μg) 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 μg/ml) or heat killed B. pertussis. BP1569-specific IFN-γ 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-γ 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

(68) 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.

(69) 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-α production, which was blocked by anti-TLR2 (FIG. 5A). Furthermore, LP1569 induced robust expression of TNF-α, IL-10 and IL-6 by murine macrophages (FIG. 5B). LP1569 also stimulated TNFα 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

(70) 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.

(71) Stimulation of spleen cells with LP1569 induced the production of IFN-γ 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-γ 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-γ following stimulation of the spleen cells with LP1569, which was slightly augmented by addition of IL-1β (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 γδ T cells (FIG. 6E).

Example 7

LP1569 is an Effective Adjuvant for Promoting Protective Cellular Immunity Against B. pertussis

(72) 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).

(73) 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-γ 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 lipopetide generated Th2 type responses, with high IL-5, but substantially lower concentrations of IL-17 and IFN-γ 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

(74) 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).

(75) 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.

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