FIELD OF THE INVENTION
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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.
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TO THE INVENTION
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).
Th1 cells are characterized by the production of pro-inflammatory cytokines such as IFN-γ, IL-2, and TNF-β. Th1 cells are involved in cell-mediated immunity (CMI), this being the immune response typically mounted against viruses and intracellular pathogens.
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.
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).
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.
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OF THE INVENTION
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.
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.
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.
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.
Suitably, a lipoprotein or a fragment or derivative thereof may be obtained from B. pertussis.
Suitably a lipoprotein or a fragment or derivative thereof may be obtained from a soluble secreted fraction of the B. pertussis.
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.
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.
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.
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.
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.
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.
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.
SEQ ID NO: 13