Novel immunostimulation method

Abstract

To provide compositions for treating or preventing respiratory conditions associated with one or more infectious factors in a subject.SOLUTION: Provided is a composition comprising a TLR2 agonist conjugated to a solubilizing agent, the solubilizing agent increasing the solubility of the TLR2 agonist in a polar or aqueous solvent, the composition not containing an antigen that enhances an antigen-specific immune response to the one or more infectious factors, the TLR2 agonist being a lipopeptide or containing a lipid moiety, the lipid moiety being palmitoyl, the solubilizing agent containing polyethylene glycol (PEG), the composition not containing a TLR9 agonist, and the composition not containing a peptide antigen containing a T-helper epitope.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to novel methods of eliciting an innate immune response in a subject that involve the use of TLR2 moieties, including TLR2 agonists. [Background technology]

[0002] Influenza A virus (IAV) infections cause up to 1 billion infections and 300,000 to 500,000 deaths annually, and the 2009 global outbreak of swine H1N1 influenza A virus highlighted the limited antiviral options available to combat pandemic influenza. While vaccines are available for seasonal IAV epidemics, these vaccines induce antibodies against the constantly evolving neuraminidase and hemagglutinin surface proteins of IAV and therefore require annual reformulation and administration. Furthermore, these vaccines are generally ineffective against pandemic outbreaks caused by emerging viruses. An alternative is to target conserved internal regions of IAV. However, the recent pandemic outbreak of swine H1N1 influenza A virus has led to a quest to discover broad-spectrum prophylactic vaccines and antiviral options against pandemic influenza.

[0003] The present invention relates to the development of novel approaches to treating influenza and other infectious diseases and cancer. Summary of the Invention

[0004] In a first aspect of the present invention, there is provided a method of treating or preventing a disease by enhancing an innate immune response in a subject, the method comprising the step of administering to the subject an effective amount of a composition comprising a TLR2 moiety in solution, wherein the TLR2 moiety comprises a TLR2 agonist, and wherein the disease is not treated or prevented by a humoral or cellular immune response against the TLR2 moiety.

[0005] In a second aspect of the present invention, there is provided a method of treating or preventing a disease caused by an infectious agent, the method comprising the step of administering to a subject in need thereof an effective amount of a composition comprising a TLR2 moiety in solution, wherein the TLR2 moiety comprises a TLR2 agonist, and wherein the TLR2 moiety does not induce a specific cellular or humoral immune response against the infectious agent.

[0006] In a third aspect of the present invention, there is provided a method of treating or preventing cancer by enhancing an innate immune response in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of a composition comprising a TLR2 moiety in solution, wherein the TLR2 moiety comprises a TLR2 agonist, and wherein the TLR2 moiety does not induce a specific cellular or humoral immune response against the cancer.

[0007] In a fourth aspect of the present invention, there is provided a pharmaceutical composition for treating or preventing a disease by enhancing an innate immune response in a subject, comprising an effective amount of a TLR2 moiety in solution together with a pharmaceutically acceptable carrier or excipient, wherein the TLR2 moiety comprises a TLR2 agonist, and wherein the disease is not treated or prevented by a humoral or cellular immune response against the TLR2 moiety.

[0008] In a fifth aspect of the present invention, there is provided the use of an effective amount of a TLR2 moiety in solution for the manufacture of a medicament for treating or preventing a disease in a subject, wherein the TLR2 moiety comprises a TLR2 agonist, and the TLR2 agonist enhances an innate immune response in the subject, and the disease is not treated or prevented by a humoral or cellular immune response against the TLR2 moiety. [Brief explanation of the drawings]

[0009] [Figure 1]Schematic diagram of lipopeptide vaccine candidates. Schematic diagram of Pam2Cys-based constructs. (A) Pegylated Pam2Cys (Pam2Cys-PEG11) consists of one Pam2Cys molecule coupled to undecaethylene glycol (polyethylene glycol, PEG) via two serines. (B) Pam2Cys-based lipopeptide vaccine candidates consist of a target CD8+ T cell epitope and a helper T epitope linked via a single lysine (K) residue. The Pam2Cys lipid moieties are attached via two serine residues (Ser) to form a branched peptide structure. [Figure 2] Figure 1 shows that intranasal administration of lipopeptides expands lung cell populations. C57BL / 6 mice were intranasally inoculated with 25 nmol of OT2-P2C-gB498-505 lipopeptide or 25 nmol of the OT2-gB498-505 peptide (without Pam2Cys), and lung cell populations were characterized at the indicated time points (n = 3 / group / time point). (A) The total number of lung cells is shown. Symbols represent the mean cell number, and error bars indicate SEM. # = P < 0.01 vs. day 0. (B) The composition of lung cells is shown. Each bar represents the mean cell number (n = 3), and error bars indicate SD. * = P < 0.05 vs. the cell population on day 0. Statistical analysis was performed using one-way ANOVA and post-hoc Dunnett's multiple comparison test. Repeated experiments yielded similar results. [Figure 3]Figure 1 shows that Pam2Cys-based lipopeptides enhance IAV clearance. (A) Mice were inoculated with saline, IAV-LP (containing IAV-derived epitopes), or non-IAV-LP and challenged with 10 pfu of Mem71 (H3N1) influenza virus 3 days (upper panel) or 7 days (lower panel) after lipopeptide inoculation (n = 3-5 / group). Lung virus titers were assessed on day 5 of infection and are shown for BALB / c (◇), C57BL / 6 (□), and HHD (◯) mice. Symbols represent titers obtained from individual mice, and lines indicate the mean virus titers for the groups. (B) Percentages of virus clearance relative to saline controls are shown above the lipopeptide groups. IAV-specific CD8+ T cell responses were detected in C57BL / 6 mice challenged with Mem71 on day 7 after LP inoculation. On day 5 after challenge, PA224-233-specific CD8+ T cells were detected in the spleen using intracellular staining assays for IFN-γ and TNF-α. Bars represent the mean cytokine-specific response in each group, and error bars indicate SD. *=P<0.05, **P<0.01 vs. saline (one-way ANOVA analysis and post-hoc Dunnett's multiple comparison test). [Figure 4] Figure 1 shows that Pam2Cys-based lipopeptides reduce the effects of highly pathogenic IAV infection. Upper panel: Kaplan-Meier plot showing survival of C57BL / 6 mice (left) and HHD mice (right) intranasally inoculated with saline, IAV-LP, or non-IAV-LP (n=5 / group) and challenged with H1N1 PR8 virus 7 days later. Lower panel: Body weight change after infection. Symbols indicate mean, error bars indicate SEM. These results were reflected in independent replicate experiments in both mouse strains. [Figure 5]Figure 1. Effect of Pam2Cys on the lung cytokine environment. C57BL / 6 mice were administered 20 nmol of Pam2Cys-PEG11 (P2C-PEG11) or 50 μl of saline (in). Cytokine concentrations in bronchoalveolar lavage (BAL) fluid were determined on days 3 (D3) or 7 (D7) post-administration using a BD™ Cytometric Bead Array. Bars represent the mean response for each group (n = 3); error bars indicate SD. * = P < 0.05; ** = P < 0.01; *** = P < 0.001 vs. saline and naive groups (one-way ANOVA and post-hoc Tukey's multiple comparison test). [Figure 6] Figure 1 shows that Pam2Cys mediates viral clearance. Mice were inoculated with saline or 20 nmol of Pam2Cys-PEG11 and challenged with 10 pfu of Mem71 (H3N1) virus (n = 3-5 / group) 1 (upper panel), 3 (middle panel), or 7 (lower panel) days later. Viral titers were assessed in BALB / c (◇), C57BL / 6 (□), and HHD (◯) mice on day 5 postinfection. Symbols represent titers obtained from individual mice, and lines indicate the mean viral titer for the group. * = P < 0.05 vs. saline (unpaired Student's t test). Percentage of viral clearance relative to the saline group is shown above the Pam2Cys response. [Figure 7] Figure 1 shows that intranasal Pam2Cys administration expands cell subpopulations in the lung. C57BL / 6 mice were administered 20 nmol of pegylated Pam2Cys (P2C-PEG11) or 50 μl of saline (in), and lung cell populations were characterized 72 hours after administration. Bars represent the mean response for each group (n = 3), and error bars indicate SD. * = P < 0.05; vs. saline group (unpaired Student's t-test). [Figure 8]Figure 1 shows that Pam2Cys protects against virulent IAV infection. C57BL / 6 mice were inoculated intranasally with saline or 20 nmol of PEGylated Pam2Cys (P2C-PEG11) (n=5 / group) and challenged with 200 pfu of H1N1 PR8 virus 7 days later. One group of mice received 20 nmol of P2C-PEG11 and were challenged 72 hours later. Post-infection body weight changes are shown as percentages of original body weight. Symbols represent means, and error bars indicate SEM. These results reflect independent replicate experiments. [Figure 9] This figure shows that prophylaxis with Pam2Cys reduces viral load and contact transmission of influenza. BALB / c mice were inoculated with saline (white bars) or 20 nmol of PEGylated Pam2Cys (P2C-PEG11) (gray bars) and challenged with 10 pfu of Udorn (H3N2) virus (n = 2 per group) 7 days, 5 days, 72 hours, or 24 hours later. These mice were designated spreader mice. 24 hours after challenge, the spreader mice were paired with naive recipient mice. After 24 hours of pairing, the spreader mice were removed, and their nasal turbinates, trachea, and lungs were removed to determine viral titers (upper panel). 3.5 days after exposure to the spreader mice, organs were harvested from recipient mice, and viral titers were assessed (lower panel). White or gray arrows indicate successful contact-mediated transmission from the respective treatment groups to paired recipients. [Figure 10]

[0023] Figure 1 shows that a single dose of PEG-Pam2Cys protects against IAV when delivered intranasally. Mice were administered prophylactic PEG-Pam2Cys (20 nmol) via the intranasal (in), subcutaneous (sc), or intravenous (iv) routes and challenged 3 days later with a lethal dose of PR8 virus. Mice were then monitored for weight and survival for 8 days post-PR8 and euthanized at the endpoint. [Figure 11]Figure 1 shows that some Pam2Cys mutants also confer protection against IAV challenge. Mice were administered 20 nmol of various Pam2Cys-containing constructs via the intranasal route as prophylaxis and challenged with a lethal dose of PR8 virus 3 days later. Mice were then monitored for body weight (A) and survival (B) over 8 days post-PR8. The weight loss observed in the saline group was statistically significant (P<0.01) when compared with each of the other treatment groups. [Figure 12] Figure 1 shows that PEG-Pam2Cys is effective when given in repeated doses. Balb / c mice were administered a single dose of PEG-Pam2Cys or two doses of PEG-Pam2Cys, separated by three weeks, and then challenged with PR8 three days after the second dose. Mice were then monitored for weight and survival for eight days after challenge and sacrificed on day seven after PR8 for assessment of lung viral load. The weight loss observed in the saline group was statistically significant (P<0.01) when compared with each of the other treatment groups (A). Viral loads in mice treated with PEG-Pam2Cys were substantially lower than those in mice given saline alone (B). [Figure 13] Figure 1 shows that lower doses of PEG-Pam2Cys are also effective. Mice were prophylactically treated with lower doses of PEG-Pam2Cys: 2, 5, and 10 nmol (compare 20 nmol) and challenged with a lethal dose of PR8 virus 3 days later. Mice were monitored for weight change and survival for 18 days post-challenge and euthanized at the endpoint. No mice in the saline group survived more than 8 days post-PR8 challenge. [Figure 14]Protection against IAV challenge is independent of IFN-γ or type 1 interferon (i.e., IFN-α). IFN-γ-deficient mice (B6.IFN-γ- / -) (A) or type 1 interferon receptor-deficient mice (IFNAR- / -) (B) were prophylactically administered 20 nmol of PEG-Pam2Cys(in) and challenged with a lethal dose of PR8 virus 3 days later. These mice were protected against weight loss and lethality associated with PR8 infection. The B6.IFN-γ- / - cohort was euthanized on day 5, the endpoint at which the saline group was euthanized. [Figure 15] (Figure 1) PEG-Pam2Cys is effective as a therapeutic agent. Balb / c mice were challenged in with 104.5 PFU of Udorn virus and, 4 hours later, administered 20 nmol of PEG-Pam2Cys in. Two days later, the animals were sacrificed, and viral loads were determined in the nose, oropharynx, trachea, and lungs. [Figure 16] Figure 1 shows that PEG-Pam2Cys is effective as an antibacterial agent. C57BL / 6 mice were pretreated (in) with 20 nmol of PEG-Pam2Cys and challenged 3 days later with 1 x 10 CFU of L. pneumophila (JR32Δfla strain). Mice were monitored daily after intranasal challenge with L. pneumophila, and bacterial burden in the lungs of mice was assessed 1, 2, and 3 days after challenge (A). Each symbol represents the mean bacterial burden obtained at each time point, and error bars represent the standard deviation (SD). Statistical significance is indicated by * (p < 0.05), obtained using a Student's t-test comparing saline and PEG-Pam2Cys-treated groups. The mean percentage of bacterial reduction relative to the saline group is indicated above each symbol. To demonstrate the duration of protection provided by PEG-Pam2Cys, mice were pretreated with PEG-Pam2Cys 3 days (B) or 7 days (C) before challenge with L. pneumophila. Bacterial burdens at 3 days post-infection are shown in panels B and C. DETAILED DESCRIPTION OF THE INVENTION

[0010] [Detailed explanation] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" shall be understood to imply the inclusion of a stated element or integer or group of elements or integers and not the exclusion of any other element or integer or group of elements or integers.

[0011] Reference herein to any prior publication (or information derived therefrom) or any matter that is publicly known is not, and should not be construed as, an admission or acknowledgment or any form of suggestion that the prior publication (or information derived therefrom) or matter forms part of the common general knowledge in the field of endeavor to which this specification pertains.

[0012] All publications mentioned herein are incorporated by reference in their entirety.

[0013] It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes one agent as well as two or more agents, and reference to "the composition" includes one composition as well as two or more compositions.

[0014] As used herein, the term "TLR2" is intended to mean Toll-like receptor 2 protein. TLR2 is a membrane receptor protein family of Toll-like receptors (i.e., "TLRs") that includes TLR1, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In humans, TLR2 is encoded by the TLR2 gene. TLR2 is expressed on the surface of certain cells and plays a fundamental role in pathogen recognition and innate immune activation.

[0015] A TLR2 agonist is an agent that binds to Toll-like receptor 2. A TLR2 agonist can also bind to TLR2 as a homodimer or heterodimer.

[0016] The present invention is premised on the observation that TLR2 agonists, such as S-[2,3-bis(palmitoyloxy)propyl]cysteine ​​(Pam2Cys), exhibit the ability to enhance innate immune responses in subjects, in particular inducing prophylactic and therapeutic effects against infectious agents such as viruses (e.g., influenza A) and bacteria (e.g., L. pneumophila) in a non-antigen-specific manner.

[0017] Accordingly, in a first aspect of the present invention, there is provided a method of treating or preventing a disease by enhancing an innate immune response in a subject, the method comprising the step of administering to the subject an effective amount of a composition comprising a TLR2 moiety in solution, wherein the TLR2 moiety comprises a TLR2 agonist, and wherein the disease is not treated or prevented by a humoral or cellular immune response against the TLR2 moiety.

[0018] In some embodiments, the TLR2 agonist is a lipopeptide or comprises a lipid moiety.

[0019] An exemplary lipopeptide according to this embodiment of the invention is the lipopeptide "Pam2Cys." Those skilled in the art will understand that the term "lipopeptide" refers to any composition comprising one or more lipid moieties and one or more conjugated amino acid sequences. "Pam2Cys" (also known as dipalmitoyl-S-glyceryl-cysteine ​​or S-[2,3bis(palmitoyloxy)propyl]cysteine) has been synthesized and corresponds to the lipid moiety of MALP-2, a macrophage-activating lipopeptide isolated from Mycoplasma fermentans. Pam2Cys is known to be a ligand for TLR2.

[0020] Pam2Cys has the following structure: [ka]

[0021] Another exemplary lipopeptide is the lipoamino acid N-palmitoyl-S-[2,3-bis(palmitoyloxy)propyl]cysteine, also known as Pam3Cys or Pam3Cys-OH, which is a synthetic form of the N-terminal portion of the Braun lipoprotein that spans the inner and outer membranes of Gram-negative bacteria. Pam3Cys has the following structure: [ka]

[0022] U.S. Patent No. 5,700,910 describes several N-acyl-S-(2-hydroxyalkyl)cysteines for use as intermediates in the preparation of lipopeptides used as synthetic adjuvants, B lymphocyte stimulators, macrophage stimulators, or synthetic vaccines. U.S. Patent No. 5,700,910 also teaches the use of such compounds as intermediates in the synthesis of Pam3Cys-OH and lipopeptides containing this lipoamino acid or its analogs at the N-terminus.

[0023] Other lipid moieties that can be used to target cell surface TLRs include palmitoyl, myristoyl, stearoyl, lauroyl, octanoyl, or decanoyl.

[0024] In addition to Pam2Cys and Pam3Cys, the present invention also contemplates the use of Set2Cys, Lau2Cys, and Oct2Cys in accordance with the present invention. Those skilled in the art will be aware that Set2Cys is also known as S-[2,3-bis(stearoyloxy)propyl]cysteine ​​or distearoyl-S-glyceryl-cysteine, Lau2Cys is also known as S-[2,3-bis(lauroyloxy)propyl]cysteine ​​or dilauroyl-S-glyceryl-cysteine, and Oct2Cys is also known as S-[2,3-bis(octanoyloxy)propyl]cysteine ​​or dioctanoyl-S-glyceryl-cysteine.

[0025] Other suitable TLR2 agonists include, but are not limited to, synthetic triacylated and diacylated lipopeptides, FSL-I (a synthetic lipoprotein derived from Mycoplasma salivarium 1), Pam3Cys (tripalmitoyl-S-glyceryl cysteine) and S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-cysteine, where "Pam3" is "tripalmitoyl-S-glyceryl." Derivatives of Pam3Cys are also suitable TLR2 agonists, including, but not limited to, S-[2,3-bis(palmitoyloxy)-(2-R,S)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-(Lys)-hydroxytrihydrochloride, Pam3Cys-Ser-Ser-Asn-Ala; PaM3Cys-Ser-(Lys)4, Pam3Cys-Ala-Gly, Pam3Cys-Ser-Gly; Pam3Cys-Ser, PaM3CyS-OMe, Pam3Cys-OH, PamCAG, palmitoyl-Cys((RS)-2,3-di(palmitoyloxy)-propyl)-Ala-Gly-OH, and the like. Another non-limiting example of a suitable TLR2 agonist is Pam2CSK4, PaM2CSK4 (dipalmitoyl-S-glycerylcysteine-serine-(lysine)4 or Pam2Cys-Ser-(Lys)4), a synthetic diacylated lipopeptide. Other synthetic TLR agonists include those described, for example, in Kellner et al. (1992, Biol. Chem. 373:1:51-5); Seifer et al. (1990, Biochem. J. 26:795-802); and Lee et al. (2003, J. Lipid Res. 44:479-486).

[0026] As will be appreciated by those skilled in the art, TLR2 agonists are typically non-polar and therefore soluble in non-polar solvents but only sparingly soluble in polar and aqueous solvents. If it is desired to use the TLR2 agonist in a polar or aqueous solvent, the TLR2 agonist may be conjugated to a solubilizing agent.

[0027] The solubilizing agent may comprise one or more solubilizing agents that may be conjugated to the TLR2 agonist to improve the solubility of the TLR2 moiety. Solubilizing agents are generally polar moieties that increase the solubility of the TLR2 moiety in polar or aqueous solvents.

[0028] In other further embodiments of the present invention, the solubilizing agent comprises one or more of the group consisting of "PEG" (or polyethylene glycol) and a polar polypeptide, such as "R4", a hyperbranched tetra-arginine complex; "H4", a hyperbranched tetra-histidine complex; "H8", a linear peptide containing a histidine residue; and "E8", a linear peptide containing a glutamic acid residue. Other linear and branched lipid solubilizing agents, including hyperbranched peptides containing glutamic acid residues, are also contemplated (see, for example, "Branched E8" below). In other further embodiments of the present invention, the solubilizing agent comprises PEG and one or more of the group consisting of R4, H4, H8, and E8 (linear or branched). R4, H4, H8, and E8 have been previously described in PCT / AU2009 / 000469 (WO / 2010 / 115230) and have the following structures:

[0029] [ka]

[0030] [ka]

[0031] [ka]

[0032] [ka]

[0033] [ka]

[0034] Those skilled in the art will appreciate that the present invention is not limited to the particular solubilizing agents exemplified, and that other suitable solubilizing agents known in the art, such as carbohydrates, can be used in accordance with the present invention.

[0035] The methods by which one or more solubilizing agents can be conjugated to the lipids according to the invention are well known to those skilled in the art. For example, conjugation via Fmoc chemistry, via disulfide or diether bridges, or via oxime chemistry is envisaged. In a particular embodiment of the invention, a soluble form of Pam2Cys is prepared by conjugating it to O-(N-Fmoc-2-aminoethyl)-O'-(2-carboxyethyl)-undecaethyleneglycol (Fmoc-PEG). 11 -OH, Merck Ltd) to Pam2Cys, resulting in a PEGylated form of the lipid, Pam2Cys-PEG. 11 which is then suitable for administration to a subject.

[0036] In a particularly preferred form according to the invention, the TLR2 moiety comprises a conjugate comprising Pam2Cys conjugated to PEG.

[0037] As previously shown, the present inventors have made the surprising observation that Pam2Cys exhibits prophylactic and therapeutic activity against infections with infectious agents, such as viruses (e.g., influenza A) or bacteria (e.g., L. pneumophila), in a non-antigen-specific manner. For example, when delivered intranasally, a single dose of soluble Pam2Cys provided immediate and significant protection against heterologous subtypes of influenza A infection in C57BL / 6, BALB / c, and HHD mice, as manifested by up to a 99% reduction in viral load after mild H3N1 infection and a significant reduction in morbidity and mortality associated with highly pathogenic H1N1 infection.

[0038] The inventors have also found that administration of a TLR2 agonist according to the methods of the invention elicits an innate immune response in a subject in the absence of any co-administered TLR agonist, including a TLR9 agonist. Thus, in some embodiments, the compositions according to the invention do not include a TLR9 agonist.

[0039] The inventors have shown that TLR2 agonists according to the present invention can enhance a non-antigen-specific, innate immune response in a subject. This is demonstrated by experiments involving the administration of a TLR2 moiety comprising one or more peptide antigens, which peptide antigens are "unrelated" to the disease being treated or prevented. As used herein, the term "unrelated" is intended to mean incapable of enhancing a humoral or cellular response to a specific antigen or antigens, and in the context of the present invention, does not enhance a humoral or cellular immune response to the TLR2 moiety.

[0040] Thus, a TLR2 agonist according to the present invention can further comprise one or more peptide antigens unrelated to the treatment or prevention of a disease, including, but not limited to, a helper T epitope and / or a cytotoxic T-lymphocyte (CTL) epitope. It is important to keep in mind that because a TLR2 moiety according to the present invention can elicit an innate immune response in a subject in a non-antigen-specific manner, one skilled in the art will understand that the TLR2 moiety can comprise one or more peptide antigens that will be unrelated to the disease being prevented or treated, or that the TLR2 moiety can be used in the absence of one or more peptide antigens.

[0041] By way of example, the present invention demonstrates that in the treatment of IAV, a TLR2 moiety comprising one or more "irrelevant" peptide antigens demonstrates the same ability to enhance non-antigen-specific / innate immune responses following administration of the moiety as an identical TLR2 agonist without the attached peptide antigen. In these experiments, the inventors used compositions comprising a TLR2 moiety, which included a TLR2 agonist (e.g., Pam2Cys), a helper T epitope (OT2), and / or a cytotoxic T lymphocyte epitope, a herpes simplex virus 1-derived CD8+ T cell epitope (see Table 1). Both epitopes are unrelated to IAV. Thus, the inventors have demonstrated that a TLR2 moiety according to the present invention can enhance an antigen-independent innate immune response in a subject to which it is administered.

[0042] Accordingly, in another aspect of the present invention, there is provided a method of treating or preventing a disease caused by an infectious agent, the method comprising the step of administering to a subject in need thereof an effective amount of a composition comprising a TLR2 moiety in solution, wherein the TLR2 moiety comprises a TLR2 agonist, and wherein the TLR2 moiety does not induce a specific cellular or humoral immune response against the infectious agent.

[0043] In some embodiments, the present invention provides methods for enhancing an innate immune response in a subject after infection, which has an immediate antiviral effect against the virus. Specifically, this means that administration of a TLR2 moiety according to the present invention can have a prophylactic effect in a subject after viral infection, particularly influenza A infection. Thus, in a further embodiment according to the present invention, administration of a TLR2 receptor agonist can be used to prevent disease caused by an infectious agent in a subject. In this way, the methods according to the present invention can be used to elicit an innate immune response in the prevention of infection by infectious agents, including, but not limited to, influenza A virus (IAV), hepatitis C virus (HCV), Mycobacterium tuberculosis, L. pneumophila, and infectious agents known to cause cancer.

[0044] The present invention also contemplates a method of enhancing the innate immune response in a subject already infected or established with an infectious agent. In particular, this means that administration of a composition according to the present invention can have a therapeutic effect after infection or establishment by an infectious agent in a subject. Thus, in a further embodiment, administration of a composition according to the present invention can be used to treat a disease caused by an infectious agent in a subject.

[0045] The inventors have further demonstrated that pre-treatment of subjects with a TLR2 moiety according to the present invention can significantly reduce bacterial burden in the lungs and trachea after intranasal bacterial challenge, even when the bacterial infection occurs 7 days after administration of the TLR2 moiety. Thus, in some embodiments, the infectious agent is a bacterium. The bacterium can be an intracellular gram-positive or gram-negative bacterium. In one embodiment, the bacterium includes, but is not limited to, Staphylococcus, Bacillus, Francisella, Yersinia, Legionella pneumophila, and Mycobacterium tuberculosis. In one embodiment, the infectious agent is Mycobacterium tuberculosis. In another embodiment, the infectious agent is Legionella pneumophila.

[0046] In some embodiments, the infectious agent is the cause of a secondary infection (e.g., pneumonia) in a subject. Accordingly, the present invention also provides a method of treating or preventing a secondary infection in a subject by enhancing an innate immune response, the method comprising administering to the subject an effective amount of a composition comprising a TLR2 moiety in solution, wherein the secondary infection is not treated or prevented by a humoral or cellular immune response to the soluble TLR2 moiety.

[0047] The inventors further demonstrate that when administered prophylactically, compositions comprising a TLR2 moiety according to the present invention can provide immediate protection against mild pathogenic infections with infectious agents, such as influenza A, and that this protection is associated with the influx of innate immune mediators into the lungs. This antiviral activity is not antigen-specific.

[0048] The present invention also contemplates the use of a TLR2 moiety as defined herein for the treatment of cancer in a subject. Accordingly, in another aspect of the present invention, there is provided a method of treating or preventing cancer by enhancing an innate immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a TLR2 moiety in solution, wherein the TLR2 moiety comprises a TLR2 agonist, and the TLR2 moiety does not directly induce a specific cellular or humoral immune response against the cancer. In some embodiments, administration of the TLR2 moiety inhibits the growth or spread of the cancer.

[0049] Those skilled in the art will recognize that cancer may or may not be caused by an infectious agent. Thus, established cancers not caused by an infectious agent can also be treated according to the methods of the present invention. For example, a TLR2 moiety can be administered directly to the site of a cancer-causing tumor in a subject to induce an innate immune response in the subject. Direct administration of a TLR2 agonist to the site of a cancer-causing tumor can be accompanied by recruitment of cells of the innate immune system (e.g., neutrophils, macrophages, and cytokines) to the tumor site. Thus, in some embodiments, a composition is administered directly to the site of a cancer-causing tumor. The term "tumor" is intended to mean a neoplasm or solid lesion (sometimes referred to as "neoplastic") formed by abnormal cell growth. It is important to keep in mind that the term tumor is not necessarily synonymous with cancer. Tumors can be benign, premalignant, or malignant, while cancer is by definition malignant, although tumors are often associated with cancer. As used herein, the term "cancer" refers to a group of diseases or disorders characterized by uncontrolled cell growth (e.g., tumor formation) without any differentiation of those cells into specialized, distinct cells.

[0050] As used herein, the term "subject" refers to animals, particularly mammals and more particularly primates, including lower primates, and even more particularly humans, who can benefit from the medical protocols of the present invention. Whether a human or non-human animal or embryo, the subject may be referred to as an individual, subject, animal, patient, host, or recipient. The present invention has both human and veterinary applications. For convenience, "animal" particularly includes livestock animals, such as cattle, horses, sheep, pigs, camels, goats, and donkeys. With respect to horses, these include horses used in the racing industry and horses used for recreation or animal husbandry. Examples of laboratory test animals include mice, rats, rabbits, guinea pigs, and hamsters. Rabbits and rodents, such as rats and mice, provide convenient test systems or animal models similar to primates and lower primates. In some embodiments, the subject is a human.

[0051] The compositions of the present invention are administered in effective amounts. As used herein, the terms "effective amount" and "therapeutically effective amount" of a TLR2 moiety refer to an amount sufficient to provide the desired therapeutic or physiological effect in at least a statistically significant number of subjects in the process. Undesirable effects, such as side effects, sometimes occur along with the desired therapeutic effect; therefore, practitioners must balance the potential benefits against the potential risks in determining what an appropriate "effective amount" is. The exact amount required will vary from subject to subject and will depend on the subject's species, age, and general condition, mode of administration, and the like. Thus, it may not be possible to specify an exact "effective amount." However, an appropriate "effective amount" in any individual case can be determined by one of ordinary skill in the art using only routine experimentation. In some embodiments, the effective amount for a human subject is in the range of about 0.1 nmol / kg body weight / dose to 1 mol / kg body weight / dose. In some embodiments, the range is about 1 nmol to 1 mol, about 1 μmol to 1 mol, 1 μmol to 500 μmol, 1 μmol to 250 μmol, 1 μmol to 50 μmol, or 1 nmol to 1 μmol per kg body weight per dose. In some embodiments, the range is about 0.08 μmol to 0.11 μmol per kg body weight per dose of the TLR2 moiety. Dosage regimens can be tailored to the exigencies of the situation and adjusted to provide an optimal therapeutic dose. For example, doses can be provided daily, weekly, monthly, or at other appropriate time intervals.

[0052] The term "treatment" or "treating" includes, but is not limited to, (i) slowing or halting the progression of the disease, (ii) partially ameliorating the progression of the disease, and (iii) completely ameliorating the progression of the disease (i.e., curing the disease). The term "prevent" or "preventing" should not be construed as being limited to complete prevention of the disease (i.e., not developing the disease), but may also include minimizing the progression of the disease, e.g., the disease occurring with less intensity or progressing at a slower rate in a subject as a result of the prophylactic administration of a composition according to the invention.

[0053] The composition according to the present invention may be administered in a single dose or in a series of doses. Although it is possible to administer the conjugate alone, it is preferable to present it as a composition, preferably a pharmaceutical composition. The preparation of such compositions is well known to those skilled in the art. The composition may contain any pharmaceutically acceptable carrier, diluent or excipient. The appropriate dosage and dosage regimen can be determined by the attending physician and may depend on the specific condition being treated, the severity of the condition, and the overall age, health, and weight of the subject.

[0054] A "pharmaceutically acceptable" carrier, excipient, or diluent refers to a pharmaceutical vehicle consisting of a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject together with the selected conjugate without causing any or substantial adverse reactions. Carriers can include excipients and other additives, such as diluents, detergents, colorants, wetting or emulsifying agents, pH buffers, preservatives, and the like. Carriers can further include all conventional solvents, dispersion media, fillers, solid carriers, coating agents, antifungal and antibacterial agents, skin penetration agents, surfactants, isotonic and absorption agents, and the like. It will be understood that the compositions of the present invention can further include other supplementary physiologically active agents.

[0055] Accordingly, the present invention also provides a pharmaceutical composition for treating or preventing a disease by enhancing an innate immune response in a subject, comprising an effective amount of a TLR2 moiety in solution, together with a pharmaceutically acceptable carrier or excipient, wherein the TLR2 moiety comprises a TLR2 agonist, and the disease is not treated or prevented by a humoral or cellular immune response against the TLR2 moiety.

[0056] The compositions of the present invention can be administered by any means known to those of skill in the art, including, but not limited to, intranasal, oral, and intravenous. In some embodiments, the compositions are administered intranasally.

[0057] The present invention also contemplates the use of an effective amount of a TLR2 moiety in solution for the manufacture of a medicament for treating or preventing a disease in a subject, wherein the TLR2 moiety comprises a TLR2 agonist, and the TLR2 agonist enhances an innate immune response in the subject, and the disease is not treated or prevented by a humoral or cellular immune response to the TLR2 moiety.

[0058] Those skilled in the art will understand that the invention described herein is susceptible to variations and modifications other than those specifically described. It will be understood that the invention includes all such variations and modifications that fall within the spirit and scope of the invention. The invention also includes all steps, features, compositions, and compounds referred to or shown in this specification, individually or collectively, and any combination of any two or more of said steps or features.

[0059] Specific embodiments of the present invention will now be described with reference to the following examples, which are intended for illustrative purposes only and are not intended to limit the scope of the generality of the foregoing. [Example]

[0060] Materials and Methods Synthesis, Purification, and Validation of Peptides and Lipopeptides. Lipopeptide and peptide synthesis was carried out by conventional solid-phase synthesis, using Fmoc (9-fluorenylmethoxycarbonyl) chemistry throughout. Peptides were assembled automatically using a Symphony Multiplex synthesizer (Protein Technologies Inc., Arizona, USA) or a Liberty synthesizer (CEM, North Carolina, USA), which uses microwave technology to facilitate the generation of high-fidelity peptide sequences. Peptides and lipopeptides were purified by reverse-phase high-performance liquid chromatography, and product authenticity was determined by mass spectrometry. The procedures used for peptide assembly, purification, and characterization have been described in detail elsewhere (References 1, 2, 3). A soluble form of Pam2Cys was synthesized using O-(N-Fmoc-2-aminoethyl)-O'-(2-carboxyethyl)-undecaethyleneglycol (Fmoc-PEG). 11 -OH, Merck Ltd) to Pam2Cys, resulting in a PEGylated form of the lipid, Pam2Cys-PEG. 11 The lipopeptide constructs and epitopes contained in the individual lipopeptide compositions are shown in Table 1.

[0061] Animals. Male and female C57BL / 6, BALB / c, B6.IFN-γ, B6.IFNAR, and HHD mice were used, aged 6–8 weeks. HHD “knockout” mice express the α1–α2 domains of HLA-A2.1 and H-2D. b These mice express a chimeric single chain of the α3 and cytoplasmic and transmembrane domains of H-2D. b Double knockout H-2D, unable to express the molecule bThe HHD mice were constructed into B6.IFN-γ-β2m- / - mice (references 4, 5). HHD mice were developed at the Institut Pasteur, Paris, and kindly provided by the Queensland Institute for Medical Research. Mice were bred and maintained at the Animal House Facility, Department of Microbiology. - / - The mice are interferon-γ deficient and express B6.IFNAR - / - The mice lack type 1 interferon receptors. TLR2-deficient mice were kindly provided by Dr. Shizuo Akira, Osaka University. All procedures involving mice were approved by The University of Melbourne Animal Experimentation Ethics Committee.

[0062] Inoculation Procedure. Mice were anesthetized with Penthrane™ or isoflurane inhalation and inoculated intranasally with 25 nmol of lipopeptide, 25 nmol of non-lipidated peptide, or 2-20 nmol of Pam2Cys-containing constructs. Pam2Cys-containing constructs, lipopeptides, and peptides were dissolved in saline and administered in a volume of 50 μl, while the saline control group received 50 μl of saline alone.

[0063] Influenza A virus challenge. Mice were challenged intranasally with live IAV on days 1, 3, or 7 after lipopeptide inoculation. For mild IAV infection, mice were challenged intranasally with live IAV for 10 days. 4.5 PFU of H3N1 virus, Mem71, a genetic reassortant of A / Memphis / 1 / 71[H3N2]xA / Bellamy / 42[H1N1], was administered. On day 5 of challenge, lungs were harvested for viral titer determination, and spleens were analyzed by CD8 +Mice were harvested for characterization of T cell responses. Highly pathogenic IAV challenge was performed via the intranasal route using 50 PFU (HHD mice), 200 PFU (C57BL / 6 mice), or 500 PFU (BALB / c mice) of the H1N1 virus A / Puerto Rico / 8 / 34 (PR8). This highly pathogenic virus induces symptomatic infection characterized by weight loss and dehydration. Mice were monitored daily for signs of morbidity and were terminated when necessary at a euthanasia endpoint determined using a combination of clinical symptoms and the degree of weight loss as approved by The University of Melbourne Animal Ethics Committee.

[0064] Legionella pneumophila challenge. C57BL / 6 mice were pre-treated intranasally with 20 nmol of PEG-Pam2Cys, and 3 days later, 1x10 6 Mice were challenged intranasally with CFU of L. pneumophila (JR32Δfla strain). Bacterial burden in the lungs of mice was assessed 1, 2, and 3 days after infection.

[0065] Contact transmission studies. To assess virus transmission in BALB / c mice, "donor" mice (n=2) were given 10 4.5 pfu of H3N2 Udorn virus (A / Udorn / 72) was administered intranasally. One day after challenge, donor mice were co-housed with naive "recipient" mice (n=3) for 24 hours, after which the donor mice were removed, and their nasal turbinates, trachea, and lungs were harvested for viral titer assessment. Three and a half days after exposure to the donor mice, the nasal turbinates, trachea, and lungs of the recipient mice were harvested for viral titer assessment. This protocol is based on the contagious infection model developed by Edenborough et al. (in preparation).

[0066] Assessment of viral titers in nasal turbinates, trachea, and lungs. Mouse nasal turbinates, trachea, and lungs were homogenized in 3 ml of RPMI, and IAV viral titers in the lung supernatant were determined using the Madine Darby Canine Kidney (MDCK) Plaque Assay described previously (Reference 6).

[0067] Preparation of single-cell suspensions from organs. After CO2 asphyxiation, mouse lungs were perfused with 10 ml of PBS through the right ventricle of the heart to remove circulating cells. Lungs were minced and subjected to enzymatic digestion with collagenase A (2 mg / ml, Roche, Mannheim, Germany) in RPMI for 30 minutes. Digested lung fragments were strained through a sieve and treated with 3 ml of prewarmed Tris-buffered ammonium chloride solution (ATC) for 2 minutes at room temperature to lyse red blood cells. The lung cells were then washed twice in RP10 (RPMI 1640 medium [Gibco, USA] supplemented with 10% FCS [CSL, Parkville, Australia], 7.5 mM HEPES, 2 mM L-glutamine, 76 μM 2-mercaptoethanol, 150 U / ml penicillin, 150 μg / ml streptomycin, and 150 μM non-essential amino acids [Gibco]). The spleens were collected in 10 ml of RP10, and a single-cell suspension was prepared by crushing through a sieve and then treated with ATC for 5 minutes at 37°C. The cells were washed twice with RP10 before use. To obtain bronchoalveolar lavage (BAL) fluid, the trachea of ​​the mice was cannulated with a syringe, and the airspace was flushed with three separate 1-ml washes of RPMI, with the final 1 ml rinsing the syringe. Supernatants from BAL lavage fluids were stored at −70° C. for later cytokine analysis. Supernatants from BAL lavage fluids were stored at −70° C. for later cytokine analysis. Viable cells were counted using a hemocytometer and trypan blue exclusion.

[0068] Characterization of the pulmonary cytokine environment. Cytokine levels in BAL supernatants were determined using the BD™ Cytometric Bead Array (CBA) (Biosciences) Mouse Inflammation Kit according to the manufacturer's instructions, except that 2 μl of each capture bead was used for each 50 μl BAL sample. Standard curves (20–5000 pg / ml) were prepared for the following cytokines: interleukin-6 (IL-6), interleukin-10 (IL-10), monocyte chemoattractant protein-1 (MCP-1), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-12p70 (IL-12p70). Cytokine concentrations were determined from undiluted or 1 / 10 dilutions of BAL supernatants. Samples were analyzed using a Becton Dickinson FACSCalibur flow cytometer and FlowJo software.

[0069] Characterization of lung cells: 5 x 10 5 Lung cells were stained with a combination of the following anti-mouse antibodies: FITC-labeled anti-CD11b, PerCP-Cy5.5 anti-GR-1 (Ly-6G and Ly-6GC), PE-labeled anti-CD11c, APC anti-F4 / 80, FITC anti-IA / IE class 2, PerCP-Cy5.5 anti-CD8, and PE anti-CD4 (BD Pharminigen). Lung cell subsets were classified as follows: neutrophils: CD11b. hi ,GR1 hi , CD11c - , F4 / 80 - ;Alveolar macrophages: CD11c hi , F4 / 80 + , CD11b int / lo , GR1 int / lo and CD11c hi , Autofluorescence hi ;Dendritic cells: CD11c hi and MHC class 2 hi , GR1 int Monocytes and interstitial macrophages: CD11b hi , GR1 int , CD11cint / lo , F4 / 80 + ;CD8 + T cells: CD8 + ;CD4 + T cells: CD4 + (7, 8, 9).

[0070] Intracellular cytokine staining (ICS) assay. Single-cell suspensions of lung or spleen cells were stimulated with 1 μg / ml (C57BL / 6 and BALB / c) or 10 μg / ml (HHD) peptide for 6 h at 37°C in a total volume of 200 μl of RP10 in the presence of 5 μg / ml GolgiPulg (BD Biosciences Pharmingen) 25 U / ml and recombinant human IL-2 (Roche, Indianapolis, USA). Cells were stained with PerCP (Cy5,5) labeled with rat anti-mouse CD8α antibody (BD Biosciences Pharmingen) for 30 min on ice. Cells were fixed and permeabilized using the BD Cytofix / Cytoperm kit™ (BD) according to the manufacturer's instructions, and stained with FITC-labeled anti-IFN-γ and APC-labeled anti-TNF-α (BD Biosciences Pharmingen) for 30 min at 4°C. Samples were analyzed using a Becton Dickinson FACSCalibur flow cytometer and analyzed using FlowJo software.

[0071] Statistical analysis. For time point comparisons, one-way ANOVA (post-hoc Dunnett's multiple comparison test) was used to determine differences between the pre-vaccination (day 0) and post-vaccination (days 1, 3, 6, and 8) groups. For other studies, two-tailed unpaired Student's t-test or one-way ANOVA (post-hoc Tukey's multiple comparison test) was used to determine statistical differences between two or more groups, respectively. A P value <0.05 was considered significant.

[0072] result Pam2Cys-based lipopeptide vaccination expands lung cell populations. The effect of Pam2Cys-based lipopeptides on the lung cell environment was examined by administering the T helper epitope (Th)OT2 and herpes simplex virus 1-derived CD8 + T cell epitope-containing lipopeptide OT2-P2C-gB 498-505 (gB 498-505 (see Table 1) was examined in intranasally inoculated C57BL / 6 mice. Lung-resident cell populations in PBS-perfused lungs were characterized using cell flow cytometry.

[0073] OT2-P2C-gB 498-505 Intranasal inoculation of OT2-gB induced a dramatic increase in the total number of lung cells, reaching a maximum on day 3 and continuing to increase until day 8 (Figure 2A). In contrast, the peptide OT2-gB 498-505 Mice receiving lipopeptide-inoculated mice (lacking Pam2Cys) showed no significant changes in the total number of cells or the proportion of cell types present in the lungs, indicating Pam2Cys as a mediator of cellular influx (Figure 2A). In lipopeptide-inoculated mice, the cellular infiltrate at 3 days post-inoculation consisted mostly of neutrophils and interstitial macrophages (Figure 2B). Microscopic examination with Giemsa staining revealed that neutrophils displayed a highly vacuolated phenotype, indicative of activation, whereas F480 + The monocyte / interstitial macrophage population was found to be composed predominantly of interstitial macrophages with large nuclear morphology, with few cells identified possessing the doughnut- or kidney-shaped nuclei characteristic of monocytes (data not shown). Although alveolar macrophages (AMs) are present at very low levels in the lung at steady state (day 0), a significant increase in this population was evident after lipopeptide inoculation. Finally, we demonstrated that CD4 + and CD8 + Lymphocytes and CD11c hi We also observed an increase in dendritic-like cells between days 3 and 8 post-inoculation. Examination of pulmonary cellular influx in BALB / c and HHD mice revealed an early infiltration of neutrophils, followed by interstitial macrophages and CD11b hiA similar pattern of expansion of the alveolar macrophage population was evident (data not shown).

[0074] Administration of PEGylated Pam2Cys expands lung cell populations. 11 Intranasal administration of IFN-γ-producing NK cells and γδ T cells also resulted in significant increases in the overall lung population (including those contained in the lung interstitium and BAL fluid) of neutrophils, interstitial and alveolar macrophages, and lymphocytes in C57BL / 6 (Fig. 7) and BALB / c mice (data not shown). Increases in the levels of activated IFN-γ-producing NK cells and γδ T cells were also observed (Fig. 7).

[0075] Prophylaxis with Pam2Cys is effective against highly pathogenic IAV challenge. To determine whether the antiviral activity of Pam2Cys is effective against virulent strains of IAV, PEGylated Pam2Cys (P2C-PEG) was administered. 11 Mice pretreated with saline (pre-treated with PEG-Pam2Cys) were challenged with a lethal dose of H1N1 virus PR8 72 hours or 7 days later. Saline-treated (post-challenge) mice experienced substantial weight loss, the onset of clinical symptoms of infection, and by day 8, all mice had succumbed to the infection (Figure 8). In contrast, mice pretreated with PEG-Pam2Cys experienced substantially less weight loss, and all mice survived the infection (Figure 8).

[0076] Prophylaxis with Pam2Cys can reduce the rate of transmission. To determine whether influenza-infected mice pretreated with Pam2Cys had a reduced ability to transmit the virus, a mouse model of contact transmission was utilized (Edenborough et al., in press). PEG-Pam2Cys-pretreated "donor" mice were subsequently challenged with 10 4.5Mice were challenged with pfu of H3N2 Udorn virus. The results show that prophylaxis with Pam2Cys reduced viral titers in the nose, trachea, and lungs (Figure 9). All recipient mice co-housed with saline-treated donor mice were infected, confirming the ability of the donor mice to transmit virus. Mice given Pam2Cys 5 or 7 days before challenge transmitted virus to recipient mice, whereas mice given Pam2Cys 24 or 72 hours before virus challenge did not transmit infection to co-housed recipient mice.

[0077] Intranasal delivery of lipopeptides provides immediate protection against IAV challenge. To determine whether the lung changes induced by intranasal delivery of lipopeptides can reduce the effects of IAV challenge, we examined the protective effects of lipopeptide vaccination against mild (H3N1) and highly virulent PR8 (H1N1) IAV viruses. Three strains of mice were transfected with lipopeptide-containing IAV-specific CD8 markers restricted to specific mouse strains. + T cell epitopes (IAV-LP) or irrelevant non-IAV-derived CD8 + T cell epitopes (non-IAV-LP) were administered (Table 1). All lipopeptides stimulated CD4 + The Th cell epitopes were unrelated to influenza virus (Table 1). IAV-LPs stimulated CD8 T cells against the delivered IAV-derived epitopes. + It was possible to induce T cell responses, whereas IAV-specific epitopes (CD8 + T or CD4 + ) indicates that no IAV-specific response is induced upon inoculation.

[0078] C57BL / 6, BALB / c, and HHD mice were inoculated with either IAV-LP or non-IAV-LP at either 3 or 7 days after inoculation. 4.5PFU of H3N1 virus, Mem71, was challenged intranasally, and lung viral titers were assessed on day 5 postinfection. The results in Figure 3A show that in all mouse strains, inoculation with IAV-LP and non-IAV-LP resulted in a significant reduction in lung viral titers compared with animals that did not receive lipopeptide. In the non-IAVLP group, viral clearance was most pronounced when challenge occurred 3 days postinoculation (Figure 3A).

[0079] IAV-specific epitopes in non-IAV-LP (CD8 + T or CD4 + The absence of Pam2Cys suggests that the antiviral activity of non-IAV-LPs is mediated by the Pam2Cys moiety. To confirm this theory, we investigated the antiviral activity of non-IAV-LPs in C57BL / 6 mice by using the immunodominant IAV-specific target, PA 224-233 IAV-specific CD8 + The presence of T cell responses was examined, and the same epitope was contained in IAV-LP but not in non-IAV-LP. In lipopeptide-vaccinated C57BL / 6 mice, only mice given IAV-LP elicited significant levels of IFN-γ. + or IFN-γ + TNF-alpha + PA 224-233 -specific CD8 + T cells, whereas neither the saline nor the non-IAV-LP group elicited detectable responses to these epitopes (Figure 3B). + This same pattern of T cell responses was observed in BALB / c and HHD mouse strains. The absence of IAV-specific cells in mice inoculated with non-IAV-LP demonstrates that the observed early antiviral effect is due to the action of Pam2Cys and not its promoting ability to mount an IAV-specific adaptive immune response.

[0080] In contrast to IAV-LP, non-IAV LP express IAV-specific CD8 +It should be noted that IAV-LP does not provide long-term protection related to the induction of T cell responses. When challenged with H3N1 6-8 weeks after inoculation, only BALB / c and C57BL / 6 mice inoculated with IAV-LP showed significant levels of viral clearance (98±1% and 65±14%, respectively) (data not shown). Thus, in the absence of an antigen-specific response, the antiviral activity of non-IAV-LP diminishes over time, and the lipopeptide CD8 + This demonstrates that the T cell epitope component is essential for long-term protection by IAV-LP.

[0081] Lipopeptide prophylaxis is effective against highly pathogenic IAV challenge. To determine whether the antiviral activity of Pam2Cys was effective against highly pathogenic infection, lipopeptide-inoculated mice were challenged with the highly pathogenic H1N1 virus, PR8, 7 days later. Inoculation with both IAV-LP and non-IAV-LP lipopeptides dramatically increased the survival rate of PR8-challenged mice (Figure 4). While the majority of saline-inoculated mice succumbed to infection, 100% of IAV-LP-inoculated animals and 80% of non-IAV-LP-inoculated animals survived the infection. In addition to improved survival, the severity of weight loss and clinical symptoms commonly associated with infection was also reduced in the non-IAV-LP group (Figure 4).

[0082] Cytokine spectrum induced by soluble Pam2Cys. To eliminate the influence of peptide- or epitope-specific responses from this system, we constructed a soluble form of Pam2Cys by conjugating the normally insoluble Pam2Cys to polyethylene glycol (PEG). Pam2Cys-PEG 11 To determine the effect of Pam2Cys-PEG on the lung environment, we administered 20 nmol of Pam2Cys-PEG to 11 The concentrations of inflammation-related cytokines in the bronchoalveolar lavage (BAL) fluid of C57BL / 6 mice administered (in) were measured by cytometric bead array analysis (Figure 5). On day 3 after administration, the inventors measured the concentrations of Pam2Cys-PEG 11In inoculated mice, we detected significant increases in the concentrations of IL-6, IL-10, MCP-1, IFN-γ, TNF-α, and IL-12p70 compared to naive or saline-inoculated mice. By day 7, Pam2Cys-PEG 11 Cytokine concentrations in the group normalized to pre-treatment levels and were not significantly different from the naive group (Figure 5).

[0083] The antiviral activity of Pam2Cys is antigen-independent. To confirm that the Pam2Cys moiety is primarily responsible for the early antiviral activity of the lipopeptide, mice were treated with 20 nmol of Pam2Cys-PEG. 11 The vaccine was administered intranasally, and 10 days later, 1, 3, or 7 days later, 4.5 The results shown in Figure 6 show that Pam2Cys-PEG 11 We demonstrate that inoculation with Pam2Cys-PEG reduces pulmonary viral load to the same extent as non-IAV-LP, confirming that the observed early antiviral activity of lipopeptides is mediated by Pam2Cys. 11 Prophylaxis with Pam2Cys-PEG reduced post-challenge viral load almost immediately, on days 1 and 7 after administration, and at least 7 days of protection was observed. 11 It has become clear that prevention by

[0084] Pam2Cys protects against IAV when delivered intranasally in a single dose. PEGylated Pam2Cys (PEG-Pam2Cys) (Pam2Cys-PEG) in mice 11 or P2C-PEG 11 After prophylactic administration of PEG-Pam2Cys (also referred to as PEG-Pam2Cys) via the intranasal (in), subcutaneous (sc), or intravenous (iv) routes, followed 3 days later by a lethal challenge with PR8 virus, only mice receiving PEG-Pam2Cys intranasally were protected against death and weight loss associated with PR8 infection (Figure 10).

[0085] [Table 1]

[0086] Multiple Pam2Cys mutants confer protection against IAV challenge. Mice administered prophylactically via the intranasal route with 20 nmol of various Pam2Cys-containing constructs were protected against weight loss (Fig. 11A) and death (Fig. 11B) after challenge with a lethal dose of PR8 virus. Mice were also protected against other clinical symptoms associated with PR8 infection (data not shown).

[0087] Pam2Cys is effective when given in repeated doses. Balb / c mice administered a single dose of PEG-Pam2Cys (or two doses of PEG-Pam2Cys separated by 3 weeks) were protected against weight loss (Fig. 12B), death, and other clinical symptoms after challenge with a lethal dose of PR8. Viral loads in PEG-Pam2Cys-treated mice were significantly lower at the time of sacrifice (Fig. 12A).

[0088] PEG-Pam2Cys is effective at lower doses. When mice were prophylactically treated with a lower dose of PEG-Pam2Cys and challenged 3 days later with a lethal dose of PR8 virus, further protection against weight loss and death was achieved in all mice compared to the saline group (Figure 13).

[0089] Protection against IAV challenge is not dependent on IFN-γ or type 1 interferon (i.e., IFN-α). Mice deficient in IFN-γ (B6.IFN-γ- / -) or lacking the ability to respond to type 1 interferons (e.g., interferon-α; IFNAR- / -) treated with PEG-Pam2Cys were protected against weight loss and lethality associated with PR8 infection (Figure 14).

[0090] PEG-Pam2Cys is effective as a therapeutic agent. 4.5When mice were challenged with PFU of Udorn virus (influenza A) (in) and 4 hours later were administered 20 nmol of PEG-Pam2Cys (in), a reduction in viral load was observed in the nose, oropharynx, trachea, and lungs. In particular, the viral load in the lungs was Log 1.0 compared to the saline group. 10 A 4 or 10,000 fold reduction was observed (Figure 15).

[0091] Pam2Cys is effective as an antibacterial agent. When mice were pretreated with PEG-Pam2Cys (in), they showed a significant reduction in bacterial burden in the lungs and trachea after in challenge with L. pneumophila (Figure 16A). Bacterial burden in the lungs peaked on days 2 and 3. Reduction in bacterial burden was also achieved by day 7 after PEG-Pam2Cys administration, and bacterial burdens on day 3 post-infection are shown in mice given PEG-Pam2Cys prophylaxis 3 (Figure 16B) or 7 days (Figure 16C) before challenge.

[0092] Consideration The crucial role that the innate immune response plays in controlling infectious diseases suggests that early activation of the innate immune system before infection can provide enhanced protection against challenge with infectious agents, such as viruses or bacteria. The results of this study demonstrated that administration of a soluble TLR2 moiety, including a TLR2 agonist, enhanced the innate immune response in treated subjects, demonstrating that the immune response was non-antigen specific. Furthermore, lung changes induced by intranasal prophylactic administration of a composition according to the present invention were associated with increased resistance to subsequent exposure to viruses and bacteria. These findings suggest that such compositions are suitable as prophylactics against viral and bacterial infections, particularly in situations where there is a high risk of epidemic or pandemic outbreaks. The prophylactic and therapeutic methods according to the present invention also have the advantage of not requiring prior knowledge of the infectious agent (or its antigenic components or specific strains) and thus may be particularly useful, for example, during influenza pandemics. The stability of the compositions according to the present invention, which can be lyophilized and are stable at room temperature, also means that they are highly suitable for stockpiling in pandemic situations.

[0093] [References]

Table 2

Claims

1. 1. A method of treating or preventing a disease by enhancing an innate immune response in a subject, comprising: administering to a subject an effective amount of a composition comprising a TLR2 moiety in solution; the TLR2 portion comprises a TLR2 agonist; A method wherein the disease is not treated or prevented by a humoral or cellular immune response against the TLR2 moiety.

2. 1. A method of treating or preventing cancer by enhancing an innate immune response in a subject, comprising: administering to a subject a therapeutically effective amount of a composition comprising a TLR2 moiety in solution; the TLR2 portion comprises a TLR2 agonist; The method, wherein the TLR2 portion does not induce a specific cellular or humoral immune response against the cancer.

3. 1. A method for treating or preventing a disease caused by an infectious agent, comprising: administering to a subject in need thereof an effective amount of a composition comprising a TLR2 moiety in solution; the TLR2 portion comprises a TLR2 agonist; The method, wherein the TLR2 moiety does not induce a specific cellular or humoral immune response against an infectious agent.

4. The method of any one of claims 1 to 3, wherein the TLR2 moiety comprises a TLR2 agonist conjugated to a solubilizing agent.

5. The method of any one of claims 1 to 4, wherein the TLR2 agonist is selected from the group consisting of Pam2Cys, Pam3Cys, Ste2Cys, Lau2Cys and OctCys.

6. 6. The method of claim 5, wherein the TLR2 agonist is Pam2Cys.

7. The method according to any one of claims 4 to 6, wherein the solubilizing agent is PEG (polyethylene glycol) or a polar polypeptide.

8. 8. The method of claim 7, wherein the polar polypeptide is selected from the group consisting of R4, H4, E8 and H8.

9. 8. The method of claim 7, wherein the solubilizing agent is PEG.

10. 8. The method of claim 7, wherein the solubilizing agent comprises PEG and any one of R4, H4, H8, EB8 and E8.

11. The method of any one of claims 1 to 10, wherein the composition is administered to the subject intranasally.

12. The method of any one of claims 3 to 11, wherein the infectious agent is a virus.

13. 13. The method of claim 12, wherein the virus is influenza A virus (IAV).

14. The method according to any one of claims 3 to 11, wherein the infectious agent is Mycobacterium tuberculosis or Legionella pneumophila.

15. 3. The method of claim 2, wherein the cancer is caused by an infectious agent.

16. 16. The method of claim 15, wherein the cancer is caused by human papillomavirus (HPV), hepatitis C virus (HCV), or Epstein-Barr virus (EBV).

17. 1. A pharmaceutical composition for treating or preventing disease by enhancing the innate immune response in a subject, comprising an effective amount of a TLR2 moiety in solution, together with a pharmaceutically acceptable carrier or excipient, the TLR2 portion comprises a TLR2 agonist; A pharmaceutical composition wherein the disease is not treated or prevented by a humoral or cellular immune response against the TLR2 moiety.

18. 1. Use of an effective amount of a TLR2 moiety in solution for the manufacture of a medicament for treating or preventing a disease in a subject, comprising: the TLR2 portion comprises a TLR2 agonist; a TLR2 agonist that enhances an innate immune response in a subject; The use wherein the disease is not treated or prevented by a humoral or cellular immune response against the TLR2 moiety.

19. The method of any one of claims 1 to 16 or the composition of claim 17, wherein the composition does not comprise a TLR9 agonist.

20. 19. The use of claim 18, wherein the medicament does not comprise a TLR9 agonist.

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