WO2016134422A1 - A method of treatment - Google Patents

Abstract

The present specification teaches generally a therapeutic protocol for the management of inflammation in the gut and respiratory system and to reduce intracellular bacterial persistence in blood or tissue cells. Agents, medicaments, probiotics, microbial and viral delivery vehicles and therapeutic kits for use the in the therapeutic protocol are also encompassed herein. An embodiment of the agents and medicaments of the present invention relates to agents that down-regulate the expression, level, or function of toll¬ like receptor (TLR) 7 and/or 8.

Description

A METHOD OF TREATMENT

FILING DATA

[0001] This application is associated with and claims priority from Australian Provisional Patent Application No. 2015900672, filed on 26 February 2015, entitled "A method of treatment", the entire contents of which, are incorporated herein by reference.

BACKGROUND

FIELD

[0002] The present specification teaches generally a therapeutic protocol for the management of inflammation in the gut and respiratory system and to reduce intracellular bacterial persistence in blood or tissue cells. Agents, medicaments, probiotics, microbial and viral delivery vehicles and therapeutic kits for use in the therapeutic protocol are also encompassed herein.

DESCRIPTION OF RELATED ART

[0003] Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.

[0004] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

[0005] Helicobacter pylori is a Gram-negative bacterium found in the stomach of about half the world's population. It is one of the few bacteria known to be able to colonize the stomach, since it can survive at low pH. To persist in this acidic environment, H. pylori produces urease, which neutralizes the local pH environment, and the bacterium then penetrates the fine layer of mucus lining of the stomach that protects epithelial cells from the low pH (Amieva and El-Omar (2008) Gastroenterology 134:306-23). About 20% of H. pylori bacteria present in the stomach directly adhere to the epithelial cells (Amieva and El-Omar, (2008) supra). This adhesion is responsible for conditions such as chronic inflammation, tissue damage and/or gastritis that disrupts the epithelial barrier.

[0006] Whether or not these inflammatory and tissue damaging conditions benefit the bacteria is still unclear, however, the fact that infection invariably results in chronic inflammation of the gastric mucosa strongly suggests that inflammation is an important part of the colonization process by this organism (Amieva and El-Omar (2008) supra). A direct consequence of the release of pro-inflammatory cytokines by epithelial cells is the recruitment of large numbers of macrophages and neutrophils to the site of infection (Suzuki et al. (2002) Pathol Int 52:265-71). While macrophages contribute to H. pylori- driven inflammation (Kaparakis et al. (2008) Infect Immun 7(5:2235-9), they also efficiently phagocytose H. pylori. However, macrophages are unable to eradicate the pathogen as the bacterium blocks phagosomal maturation, creating large vacuoles containing several viable organisms (Allen et al. (2000) J Exp Med 797: 115-28). The ability of the bacterium to block phagosome maturation appears essential, as it is present in all H. pylori strains (Borlace et al. (2011) Gut Pathog 3:3). This suggests that uptake by macrophages might in fact be beneficial to the life cycle of the bacterium and contribute to the chronic inflammation and immune responses leading to H. /riJorz^'-associated diseases. Intracellular survival in macrophages may also explain the resistance mechanisms to membrane-impermeable antibiotics (Engstrand et al. (1997) Am J Clin Pathol 705:504-9).

[0007] Salmonella enterica is an intracellular, Gram-negative bacterium and one of the most common causes of food poisoning worldwide. Ingestion of contaminated food is responsible for a range of diseases, from gastroenteritis with occasional bacteremia to systemic infection. Salmonella enterica serovar Typhi {Salmonella Typhi) causes typhoid fever in humans, whereas Salmonella enterica serovar Typhimurium {Salmonella Typhimurium) gives rise to gastroenteritis in humans and a systemic typhoid-like disease in susceptible mouse models. In less developed countries, Salmonella Typhimurium infections are a prominent cause of bloodstream infection (Feasey et al. (2012) The Lancet 379:2489-2499). After ingestion, Salmonella can pass the intestinal barrier and infects phagocytes, wherein they can survive and replicate within a modified endosome. During a systemic infection, Salmonella-infected phagocytes will disseminate the infection to the liver and the spleen (Vazquez-Torres et al. (1999) Nature ¥07:804-808). Importantly, Salmonella Typhi-infected individuals may become chronic carriers and represent a risk to public health.

[0008] The phagosomal persistence of H. pylori and Salmonella Typhimurium bacteria are two examples of a general phenomena of bacterial persistence in various cells such as peripheral blood mononuclear cells (PBMCs) and other cell types in the gut and respiratory system. Hence, there is a need to look at molecular mechanisms underlying bacterial persistence, especially in situations leading to chronic and even acute conditions.

[0009] Human toll-like receptor 7 and 8 (TLR7/8) and its murine homog, TLR7 (referred to herein collectively as "TLR7/8") are intracellular sensors localized in the endosomal compartment of phagocytes such as macrophages and plasmacytoid dendritic cells (pDCs). Both receptors are highly homologous, each encoded on chromosome X and are specialized in the sensing of sequence-specific motifs in foreign RNAs (Sarvestani et al. (2014) J Virol 55:799-810). TLR7/8 is selectively induced amongst other TLRs by interferon (∑FN)y priming of macrophages (Gantier et al. (2008) J Immunol, 180.2117-24). TLR7/8 recognizes distinct motifs/structures in RNA sequences, suggesting a cooperative sensing of a broad range of foreign RNAs by human phagocytes (Gantier et al. (2008) supra; Gantier et al. (2010b) Mol Ther, 75:785-95; Sarvestani et al. (2014) supra). Critically, TLR7/8 distinguish between self and foreign RNAs by sensing RNA modifications, such as 2'O-methyl groups (present in mRNA 5'caps), which strongly inhibit TLR7/8 sensing, or inosine residues (present in viral RNAs), which potentiate TLR7/8 sensing (Sarvestani et al. (2014) supra). TLR7/8 recruits MyD88 to transduce signal and instigate the immune response of macrophages to pathogenic RNA through the rapid production of pro-inflammatory cytokines, such as TNFa, IL6 and IL12. Although TLR8 activity predominates in human macrophages (Gantier et al. (2008) supra), mouse TLR8 does not directly contribute to RNA sensing. Rather, mouse TLR7 recapitulates human TLR8 activity in mouse macrophages (Sarvestani et al. (2012) J Interferon Cytokine Res 32:350-61). Importantly, TLR7/8 needs to shuttle from the endoplasmic reticulum to the endosomal compartment for sensing activity. This is achieved by interaction with UNC93B1 (Kim et al. (2008) Nature ¥52:234-8, Itoh et al. (2011) PLoS One 6:e28500).

[0010] Although originally described as a sensor of viral RNA (Sarvestani et al. (2012) supra, Sarvestani et al. (2014) supra), it is now well established that TLR8 is an important sensor of bacterial RNA located within phagosomes (Kariko et al. (2005) Immunity 23: 165-75, Cervantes et al. (2013) J Leukoc Biol 94: 1231-41, Sarvestani et al. (2012) supra). Critically, genetic variations in the promoter/initiation codon of TLR8 were found to correlate with protection against active tuberculosis (TB) triggered by M. tuberculosis, suggesting an important contribution of TLR8 sensing to M. tuberculosis infection (Davila et al. (2008) PLoS Genet 4: el 000218).

[0011] There is a need to develop a therapeutic protocol for the management of bacterial- induced inflammatory responses of the gut and respiratory system and to reduce intracellular bacterial persistence in blood and tissue cells.

SUMMARY

[0012] The present invention is predicated in part on the determination that certain bacteria such as but not limited to Helicobacter sp, Mycobacterium sp and Salmonella sp can survive in macrophage phagosomes and within or on epithelial tissue for extended periods of time. In addition, H pylori infections are associated with elevated gastric levels of IFNy. It is proposed herein that toll-like receptor 7/8 (TLR7/8) is involved in persistent bacterial infection of blood cells such as PBMCs and is associated with bacterial -induced inflammatory responses of the gut and respiratory system.

[0013] It has been determined that TLR8 levels are induced by H pylori in human macrophages, resulting in potentiated TLR8 signaling. In addition, genetic variations (e.g. rs3764880:A>G; p.MetlVal) associated with protection against active tuberculosis (TB), result in a change of Kozak context and decreased protein translation of the predominant isoform of TLR8 [Gantier et al. (2010a) Hum Mutat 37: 1069-79]. Counter intuitively, these findings show that less TLR8 is in fact protective against endogenous microbial persistence. This is in line with the observation that the protective G allele of rs3764880 is highly prevalent in Asian populations (e.g. 85% in Japanese versus 18% in Italian populations), where M. tuberculosis and H. pylori infections prevail.

[0014] Hence enabled herein is a therapeutic protocol for the treatment or prophylaxis of a bacterial-induced inflammatory response in the gut or respiratory system of a subject, or to reduce intracellular bacterial persistence in a gut or respiratory cell or a peripheral blood mononuclear cell (PBMC), the therapeutic protocol comprising administering to the subject an agent which down-regulates expression, level or function of toll-like receptor (TLR) 7/8 in the cell for a time and under conditions sufficient to reduce, inhibit or prevent development of an inflammatory immune response or to promote clearance of the bacterium. Reference to "TLR7/8" means human TLR7 and 8 and the murine homolog, TLR7. For brevity, the term "TLR7/8" is used in a singular form but means TLR7 and/or TLR8. In an embodiment, the gut or respiratory cell or PBMC is a macrophage or antigen- presenting cell. [0015] In an embodiment, the agent is a nucleic acid molecule which targets expression of a TLR7/8 gene or a gene encoding a component of the TLR7/8 signaling pathway or which inhibits sensing by a TLR7/8 ligand. In an embodiment, the nucleic acid is an RNA species or a chemically modified form thereof such as but not limited to a 2'-0-methyl ribose modification, a micro RNA, sense RNA, anti-sense RNA, double stranded RNA, short interfering RNA, hairpin RNA, a modified tRNA, a methylated RNA, synthetic RNA construct or an RNA hybrid molecule. Alternatively, the nucleic acid molecule is a nucleic acid in a DNA species such as a double stranded DNA, single stranded DNA, a synthetic DNA construct or a DNA hybrid molecule. In addition, the nucleic acid molecule may comprise a clustered regularly interspaced short palindromic repeat (CRISPR) DNA.

[0016] Yet in another alternative, the agent is an antibody specific for TLR7/8 or a component of the TLR7/8 signaling pathway or which otherwise inhibits sensing by a TLR7/8 ligand or is a protein or small chemical molecule which inhibits the function of TLR7/8-mediated signaling or which otherwise inhibits sensing by a TLR7/8 ligand. Examples of chemical molecules include agents which block TLR7/8 via endosomal acidification and/or autophagy and various anti-malarial drugs and anti -protozoan drugs. These include thiostrepton, chloroquine and its analogs such as quinacrine, amodiaquine, pamaquine, mefloquine, piperaquine, trioxaquine (SARI 16242), 7-chloro-N-(3-(4-(7- (trifluoromethyl)quinolin-4-yl)piperazin-l-yl)propyl)quinolin-4-amine and {3-[4-(7-chloro- quinolin-4-yl)-piperazin-l-yl]-propyl}-(7-trifluoromethyl-quinolin-4-yl)-amine. Also useful is bafilomycin Al (BafAl) and other functionally related plecomacrolide subclass of macrolide antibiotics. Two or more chemical molecules may also be employed.

[0017] The agent may also be in the form of a microorganism genetically modified or selected to produce a modified RNA species such as a modified methyltransferase. In an embodiment, the methyltransferase is selected from the list consisting of a Gm-2'-0- methyltransf erase (spoU/trmH), uracil -5 -methyltransferase (trmA), and a guanine-7- methyltransferase (uggh/trmB). Such as a microorganism is encompassed by the term "probiotic" although may also be a vehicle to deliver the modified mRNA species and not intended to function as a probiotic. In relation to the latter embodiment, encompassed herein are microbial and viral delivery vehicles to provide nucleic and protein agents useful in modulating TLR7/8 expression and/or activity.

[0018] The bacterial-induced inflammatory response contemplated herein includes responses mediated by but not limited to a Helicobacter species, a Mycobacterium species, a Salmonella species, a Campylobacter species, a Francisella species and/or a Chlamydia species. In an embodiment, the Helicobacter species is Helicobacter pylori; the Mycobacterium species is Mycobacterium tuberculosis; the Salmonella species is Salmonella enterica such as but not limited to serovar Typhimurium {Salmonella Typhimurium) or serovar Typhi {Salmonella Typhi); the Campylobacter species is Campylobacter jejuni; the Francisella species is Francisella tularensis. In an embodiment, the intracellular bacterium is selected from the list consisting of Mycobacterium leprae, Mycobacterium bovis, Yersinia pestis, Tropheryma whipplei, Brucella melitensis, Legionella pneumophila and Coxiella brunetii. The present invention extends, however, to a therapeutic management protocol against an inflammatory condition associated with any form of microbial persistence.

[0019] Whilst the present invention extends predominately to therapeutic protocols for human subjects, that are also veterinary applications.

[0020] Reference herein to the "gut" includes any of the stomach, small and large intestine and bowel including PBMC within the gut. Reference to the respiratory system comprises the lung and alveolus including various cell types such as alveolar macrophages.

[0021] Inflammatory responses contemplated herein include those leading to inter alia gastritis, gastroenteritis, Crohn's disease, inflammatory bowel syndrome, cancer and/or pre-cancerous lesions. The present invention extends to the amelioration of symptoms of persistent infection or where the bacterial infection is secondary to another condition such as a viral infection (e.g. flu) or an immune-compromising event. The treatment of sepsis and septic shock is also contemplated herein. [0022] Bacterial persistence can also lead to pneumonia or Q Fever.

[0023] Reference to a "PBMC" includes a macrophage, monocyte, antigen -presenting cell (such as a dendritic cell) and lymphocyte.

[0024] Further contemplated herein is the use of an agent which down regulates expression, level or function of TLR7/8 in a PBMC in the manufacture of a medicament to treat or prevent a bacterial-induced inflammatory response in the gut or respiratory system of a subject or to reduce intracellular bacterial persistence in a gut or respiratory cell or a PBMC.

[0025] In a related embodiment, taught herein is an agent which down regulates expression, level or function of TLR7/8 in a PBMC for use in the treatment or prevent of a bacterial-induced inflammatory response in the gut or respiratory system of a subject or to reduce intracellular bacterial persistence in a gut or respiratory cell or PBMC.

[0026] The present specification is instructional for a therapeutic kit for treating or preventing a bacterial -induced inflammatory response in a subject or to reduce intracellular bacterial persistence in a PBMC, the kit comprising an agent which down regulates expression, level or function of TLR7/8 and instructions for treating the subject according to the therapeutic protocol as herein described. Hence, the agent includes a nucleic acid, antibody and a chemical molecule as well as a microorganism genetically modified or selected to produce a modified RNA species such as a modified methyltransferase.

[0027] As indicated above, the agent may be delivered as part of a probiotic which comprises a genetically modified or selected gut or respiratory system flora microorganism which produces a modified RNA species which reduces TLR7/8 -mediated signaling. The probiotic may be in the form of a medicament, dosage or other formulation. Non-probiotic microbial and viral delivery vehicles may also be employed. [0028] A list of abbreviations used in this specification is provided in Table 1.

Table 1 - List of Abbreviations

Abbreviation Definition

BMM Bone marrow macrophage

cfu Colony forming units

CRISPR Clustered regularly interspaced short palindromic repeat DNA

CXCL1 chemokine (C-X-C motif) ligand 1

DC Dendritic cell

IFNy Gamma interferon

Ig Immunoglobulin

IL6 Interleukin 6

IL10 Interleukin 10

MOI Multiplicity of infection

PBMC Peripheral blood mononuclear cell

pDC Plasmacytoid dendritic cell

PMA Phorbol 12-myri state- 13 -acetate

Q Fever Illness caused by Coxiella brunetii

Salmonella Typhi Salmonella enterica serovar Typhi

Salmonella Typhimurium Salmonella enterica serovar Typhimurium

spoU/trm H Gm -2 '-O-methyl transferase

TB Tuberculosis

trmA Uracil-5-methyltransferase

TLR Toll-like receptor

TLR7 Toll-like receptor 7

TLR8 Toll-like receptor 8

TLR7/8 Human Toll-like receptor 7, human Toll-like receptor 8 and murine Toll-like receptor 7

TNFa Tumor necrosis factor alpha

Uggh/trmB Guanine-7-methyltransferase

WT Wild type BRIEF DESCRIPTION OF THE FIGURES

[0029] Some figures contain color representations or entities. Color photographs are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.

[0030] Figure 1 is a representation showing TLR7/8-mediated bacterial phagosomal survival and TLR7-dependent macrophage responses to bacterial infection, a) PMA- activated human THP-1 macrophages, pre-treated or not with IFNy, are infected with H. pylori bacteria (MOI 10). Cells are rinsed after 1 h and incubated for a further 5 h with gentamicin containing medium and intracellular viable bacteria are counted by gentamicin protection assay, b) Primary BMMs for wild-type (WT) and TLR7 -deficient (TLR7^"/_) mice are infected with H. pylori (MOI 10) or Salmonella Typhimurium (MOI 1) and intracellular viable bacteria are counted by gentamicin protection assay. BMMs are isolated from 3 different mice, b) WT or TLR7-/- BMMs are infected with H. pylori (MOI 10) or Salmonella Typhimurium (MOI 1) for 6 or 4 hours, respectively, and supernatants are analyzed by ELISA for IL10 levels. BMMs are isolated from 3 different mice, c) WT or TLR7^_/"-/- BMMs are infected with H. pylori (MOI 10) or Salmonella Typhimurium (MOI 1) for 24 hours and arginase 1 gene expression is measured by qRT-PCR. BMMs are isolated from 3 or 6 mice, respectively.

[0031] Figure 2 is a representation showing the impact of TLR7 -deficiency in a mouse model of H. pylori infection. WT and TLR7-deficient mice are infected with lxlO⁷ H. pylori bacteria for 30 days, a) Serum anti-H. pylori IgG titres are measured by ELISA (n=16 mice). The dotted line indicates the detection limit of the assay, b) Cumulative histology scores for chronic and acute inflammation in upper gastric sections from infected mice (n=5 mice), c) IL6 level in gastric homogenates is measured by ELISA and normalized to tissue weight (n=13 non-infected mice, 16 infected mice).

[0032] Figure 3 is a representation showing the impact of TLR7 -deficiency in a mouse model of Salmonella Typhimurium infection. WT (n=6) and TLR7 (n=6)-deficient mice are infected with lxlO⁶ Salmonella Typhimurium bacteria for 6 days, a) Mouse weight is monitored daily, b) Bacterial count in spleen and liver is measured by colony-forming unit (CFU) assay. The dotted line indicates the detection limit of the assay, c) Spleen weight and gross anatomy of infected liver. Scale bar is 1 cm. d) ΠΤΝΓγ, TNFa and CXCLl levels in serum from mice are measured by ELISA. e) Serum anti-^. Typhimurium IgG and IgM titres are measured by ELISA. The dotted line indicates the detection limit of the assay.

[0033] Figure 4 is a graphical representation showing the minimal direct effect of thiostrepton on Salmonella enterica serovar Typhimurium.

[0034] Figure 5 is a graphical representation on survival of macrophages infected with Salmonella enterica serovar Typhimurium following exposure to thiostrepton. Salmonella Typhimurium survival is lower in thiostrepton-treated macrophages than their control counterparts.

[0035] Figure 6 is a graphical representation showing the thiostrepton inhibited TLR7/8 signaling in macrophages.

DETAILED DESCRIPTION

[0036] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or method step or group of elements or integers or method steps but not the exclusion of any other element or integer or method steps or group of elements or integers or method steps.

[0037] As used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a bacterium" includes a single bacterium, as well as two or more bacteria; reference to "an agent" includes a single agent, as well as two or more agents; reference to "the disclosure" includes a single and multiple aspects taught by the disclosure; and so forth. Aspects taught and enabled herein are encompassed by the term "invention". All such aspects are enabled within the width of the present invention.

[0038] The present invention is predicated on the counter intuitive proposal that certain bacteria potentiate toll-like receptors (TLR) in particular cell types and this leads to a protective effect from innate mechanisms designed to protect against infection. The result is bacterial persistence in macrophages and PBMCs and other cells and tissues leading to chronic infection and various disease conditions.

[0039] In an embodiment, the TLR is human TLR7 and/or 8 or the murine homolog, TLR7. For convenience, these TLR receptors are referred to herein collectively as "TLR7/8" or individually as "TLR7" or "TLR8". The term "TLR7/8" is used in a singular context but means TLR7 and/or TLR8. Enabled herein is a therapeutic protocol for the treatment or prophylaxis of a bacterial-induced inflammatory response in the gut or respiratory system of a subject, or to reduce intracellular bacterial persistence in a PBMC, the therapeutic protocol comprising administering to the subject an agent which down- regulates expression, level or function of toll-like receptor (TLR) 7/8 in a gut or respiratory cell or in the PBMC for a time and under conditions sufficient to reduce, inhibit or prevent development of an inflammatory immune response or to promote clearance of the bacterium. As indicated above, reference to "TLR7/8" means human TLR7 and or 8 and its murine homolog, TLR7.

[0040] In an embodiment, the present specification teaches down-regulation of TLR7/8 to treat or prevent a bacterial-induced gut inflammatory response.

[0041] Hence, taught herein is a therapeutic protocol for the treatment or prophylaxis of a bacterial-induced inflammatory response in the gut of a subject, the therapeutic protocol comprising administering to the subject an agent which down-regulates expression, level or function of TLR7/8 in a gut cell or in a PBMC for a time and under conditions sufficient to reduce, inhibit or prevent development of an inflammatory immune response or to promote clearance of the bacterium.

[0042] The present specification also teaches managing respiratory inflammation caused or exacerbated by intracellular or intra-tissue bacterial persistence including sepsis and septic shock.

[0043] According to this embodiment, the subject specification is instructional for a therapeutic protocol for the treatment or prophylaxis of a bacterial -induced inflammatory response in the gut or respiratory system of a subject, the therapeutic protocol comprising administering to the subject an agent which down -regulates expression, level or function of TLR7/8 in a respiratory cell or in a PBMC for a time and under conditions sufficient to reduce, inhibit or prevent development of an inflammatory immune response or to promote clearance of the bacterium.

[0044] The bacterial-induced inflammatory response contemplated herein includes responses mediated by but not limited to a Helicobacter species, Mycobacterium species, a Salmonella species, a Campylobacter species, a Francisella species or a Chlamydia species. In an embodiment, the Helicobacter species is Helicobacter pylori; the Mycobacterium species is Mycobacterium tuberculosis; the Salmonella species is Salmonella enterica such as but not limited to serovar Typhimurium {Salmonella Typhimurium) or serovar Typhi {Salmonella Typhi); the Campylobacter species is Campylobacter jejuni; the Francisella species is Francisella tularensis. In an embodiment, the intracellular bacterium is selected from the list consisting of Mycobacterium leprae, Mycobacterium bovis, Yersinia pestis, Tropheryma whipplei, Brucella melitensis, Legionella pneumophila and Coxiella brunetii. The present invention extends, however, to a chronic or acute inflammatory condition in the gut or respiratory system which is exacerbated by any bacterial persistence. The bacterial infection may be a primary or secondary infection such as following a viral infection (e.g. flu) or an immune system-compromising event. The bacterial infection may also lead to sepsis or sceptic shock.

[0045] In a particular embodiment, the bacterium is a species of Helicobacter.

[0046] Hence, further taught herein is a therapeutic protocol for the treatment or prophylaxis of a Helicobacter species-induced inflammatory response in the gut of a subject, or to reduce intracellular Helicobacter species persistence in PBMC, the therapeutic protocol comprising administering to the subject an agent which down- regulates expression, level or function of TLR7/8 in the PBMC for a time and under conditions sufficient to reduce, inhibit or prevent development of an inflammatory immune response or to promote clearance of the Helicobacter species.

[0047] Without limiting the present invention to any one species, in an embodiment the species of Helicobacter is Helicobacter pylori.

[0048] Accordingly, another aspect taught herein is a therapeutic protocol for the treatment or prophylaxis of a Helicobacter pylori-induced inflammatory response in the gut or respiratory system of a subject, or to reduce intracellular Helicobacter pylori persistence in PBMC, the therapeutic protocol comprising administering to the subject an agent which down-regulates expression, level or function of TLR7/8 in a gut cell or in the PBMC for a time and under conditions sufficient to reduce, inhibit or prevent development of an inflammatory immune response or to promote clearance of the Helicobacter pylori.

[0049] In a particular embodiment, the bacterium is a species of Salmonella.

[0050] Hence, further taught herein is a therapeutic protocol for the treatment or prophylaxis of a Salmonella species-induced inflammatory response in the gut of a subject, or to reduce intracellular Salmonella species persistence in PBMC, the therapeutic protocol comprising administering to the subject an agent which down-regulates expression, level or function of TLR7/8 in a gut or respiratory cell or in the PBMC for a time and under conditions sufficient to reduce, inhibit or prevent development of an inflammatory immune response or to promote clearance of the Salmonella species.

[0051] In an embodiment, the species of Salmonella is Salmonella enterica serovar Typhimurium {Salmonella Typhimurium) or serovar Typhi {Salmonella Typhi).

[0052] Accordingly, another aspect taught herein is a therapeutic protocol for the treatment or prophylaxis of a Salmonella enterica serovar Typhimurium- or serovar Typhi-induced inflammatory response in the gut of a subject, or to reduce intracellular Salmonella enterica serovar Typhimurium or serovar Typhi persistence in PBMC, the therapeutic protocol comprising administering to the subject an agent which down -regulates expression, level or function of TLR7/8 in a gut or in the PBMC for a time and under conditions sufficient to reduce, inhibit or prevent development of an inflammatory immune response or to promote clearance of the Salmonella.

[0053] As indicated above, any bacteria which can result in chronic or even short term persistence via a TLR7/8 protective mechanism is encompassed by the therapeutic protocol enabled by the present invention. Reference to a "gut or respiratory cell" includes a gut or respiratory PBMC such as a macrophage or antigen-presenting cell.

[0054] In an embodiment, the agent is a nucleic acid molecule which targets expression of a TLR7/8 gene or a gene encoding a component of the TLR7/8 signaling pathway or which inhibits sensing by a TLR7/8 ligand. The TLR7/8 gene may be present in a PBMC or epithelial cell to which the bacteria may adhere. In an embodiment, the nucleic acid is an RNA species or a chemically modified form thereof such as but not limited to a 2'-0- methyl ribose modification, a micro RNA, sense RNA, anti-sense RNA, double stranded RNA, short interfering RNA, hairpin RNA, a modified tRNA, a methylated RNA, synthetic RNA construct or an RNA hybrid molecule. Alternatively, the nucleic acid molecule is a nucleic acid in a DNA species such as a double stranded DNA, single stranded DNA, a synthetic DNA construct or a DNA hybrid molecule.

[0055] Yet in another alternative, the agent is an antibody specific for TLR7/8 or a component of the TLR7/8 signaling pathway or which otherwise inhibits sensing by a TLR7/8 ligand or is a protein or small chemical molecule which inhibits the function of TLR7/8-mediated signaling or which otherwise inhibits sensing by a TLR7/8 ligand. In an embodiment, the chemical molecule is an anti-malarial or anti -protozoan drug or otherwise inhibits TLR7/8 via endosomal acidification (Shacka et al. (2006) Mol. Pharmacol. 69: 1125-1136; Chao-Yang Lai et al. (2015) J Immunol 795^:3912-3921; Kunzik et al. (2011) J Immunol 7S<5(¾⁾:4794-4804). In an embodiment, the chemical molecule is thiostrepton. In another embodiment, the chemical molecule is chloroquine or an analog thereof including quinacrine (Kunzik et al. (2011) J Immunol 7S<5(¾⁾:4794-4804), amodiaquine, pamaquine, mefloquine, piperaquine, trioxaquine (SARI 16242), 7-chloro-N- (3-(4-(7-(trifluoromethyl)quinolin-4-yl)piperazin-l-yl)propyl)quinolin-4-amine and {3-[4- (7-chloro-quinolin-4-yl)-piperazin-l-yl]-propyl}-(7-trifluoromethyl-quinolin-4-yl)-amine. Other agents include bafilomycin Al (BafAl) and other macrolide antibiotics of the plecomacrolide subclass. Such agents inhibit vacuolar ATPase (V-ATPase), cause a decrease in endosomal H, promote the accumulation of autophagic vacuoles and trigger Bax-dependent apoptosis. In this regard, their action is similar to chloroquine. The present invention further contemplates the use of two or more of the above chemical molecules or at least one of these molecules and at least one other agent. Reference to "functionally related" in relation to bafilomycin Al means a macrolide antibiotic of the plecomacrolide subclass which acts via autophagic vacuoles and modulation of endosomal pH as described above.

[0056] The agent may also be in the form of a microorganism genetically modified or selected to produce a modified RNA species such as a modified methyltransferase. In an embodiment, the methyltransferase is selected from the list consisting of a Gm-2'-0- methyltransf erase (spoU/trmH), uracil -5 -methyltransferase (trmA), and a guanine-7- methyltransferase (uggh/trmB). Such as a microorganism is encompassed by the term "probiotic" although may also be a vehicle to deliver the modified MRNA species and not intended to function as a probiotic. Furthermore, the agent may be a non-probiotic microorganism or viral delivery vehicle.

[0057] The term "oligonucleotide" includes an oligodeoxynucleotide (ODN) or a ribonucleotide (ORN) which may be antisense to the TLR7/8 coding sequence or sense to it. The term generally refers to a plurality of linked nucleoside units.

[0058] Such oligonucleotides can be obtained from existing nucleic acid sources, including genomic or cDNA or mRNA or produced by synthetic methods. In exemplarily embodiments, each nucleoside unit can encompass various chemical modifications and substitutions as compared to wild-type oligonucleotides, including but not limited to modified nucleoside base and/or modified sugar unit. Examples of chemical modifications are known to the person skilled in the art and are described, for example, in Uhlmann et al. (1990) Chem. Rev. 90:543; Hunziker. et al. (1995) Mod. Syn. Methods 7:331-417; and Crooke et al. (1996) Ann. Rev. Pharm. Tox. 3(5: 107-129. The nucleoside residues can be coupled to each other by any of the numerous known internucleoside linkages. Such internucleoside linkages include, without limitation, phosphodiester, phosphorothioate, phosphorodithioate, alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate, siloxane, carbonate, carboalkoxy, acetamidate, carbamate, morpholino, borano, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate, and sulfone internucleoside linkages. The term "oligonucleotide" also encompasses polynucleosides having one or more stereospecific internucleoside linkage (e.g. phosphorothioate, alkylphosphonate, or phosphotriester linkages). As used herein, the term "oligonucleotide" includes polynucleosides, having any such internucleoside linkage, whether or not the linkage comprises a phosphate group. In an embodiment, these internucleoside linkages may be phosphodiester, phosphorothioate or phosphorodithioate linkages, or combinations thereof.

[0059] The nucleosides may be 2'-substituted. The term "2'-substituted" generally includes nucleosides in which the hydroxyl group at the 2' position of the pentose moiety is substituted to produce a 2'-substituted or 2'-0-substituted nucleoside. In an embodiment, such a substitution is with a lower hydrocarbyl group containing 1 -6 saturated or unsaturated carbon atoms, with a halogen atom, or with an aryl group having 6-10 carbon atoms, wherein such hydrocarbyl, or aryl group may be unsubstituted or may be substituted, for example, but not limited to substitution with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carboalkoxy or amino groups. Examples of 2'-0-substituted nucleosides include, without limitation 2'-amino, 2'- fluoro, 2'-allyl, 2'-0-alkyl (e.g. 2'-OMethyl [2'-OMe]) and 2'-propargyl ribonucleosides or arabinosides, 2'-0-methylribonucleosides or 2'-0-methylarabinosides and 2'-0- methoxyethoxyribonucleosides or 2'-0-methoxyethoxyarabinosides.

[0060] Reference to an "antisense" or "sense" oligonucleotide includes from about 6 to the entire nucleotide or complementary nucleotide or antisense nucleotide sequence of TLR mRNA transcript. The term "about" generally means that the exact number is not critical. Thus, the number of from about 6 to about the total nucleotide sequence in an antisense or sense TLR7/8 sequence in an oligonucleotide according to this aspect of the present invention is not necessarily critical, and oligonucleotides having fewer or more nucleoside residues, or from one to several, fewer or additional nucleoside residues are contemplated as equivalents of each of the embodiments described above. Oligonucleotides which target non-coding 5' and 3' ends of the TLR7/8 mRNA transcript or a corresponding portion in the gene are also contemplated herein. Furthermore, the oligonucleotide may be coupled to a DNA or RNA promoter and/or 3' terminal sequence heterologous to the naturally occurring TLR7/8 genomic sequence. [0061] The term "antisense oligonucleotide" generally refers to strands of DNA or RNA or combinations thereof that are complementary to a chosen nucleic acid sequence such as mRNA transcribed from the TLR7/8 gene. In an embodiment the target nucleic acid is to TLR7/8 mRNA transcript. When introduced into macrophage or PBMC or other cell carrying an endogenous bacterium, an antisense oligonucleotide can bind to and cause the reduction in the translation of TLR7/8 RNA to which it is complementary. If binding takes places, this nucleic acid complex can be degraded by endogenous enzymes. Antisense oligonucleotides include, but are not limited to, traditional antisense oligonucleotides but also include, as indicated above, short interfering RNA (siRNA), micro RNA (miRNA), single stranded RNAs, hairpin RNAs and ribozymes, and deoxyribonucleotide equivalents of any of these.

[0062] Useful oligonucleotides also include clustered regularly interspaced short palindromic repeat (CRISPR) DNAs. These are DNA loci comprising short repetitions of nucleotide sequences interspersed with spacer DNA. CRISPRs in association with Cas genes, are used for gene editing by the insertion, deletion or substitution of target nucleotide sequences in coding, non-coding and regulatory regions. For example, CRISPRs can deliver the Cas9 endonuclease into a cell using guide RNAs (Wang et al. (2013) Cell 753(^:910-918).

[0063] Hence, enabled herein, is a method for the treatment or prophylaxis of a bacterial- induced inflammatory response in the gut or respiratory system of a subject or to reduce intracellular bacterial persistence in PBMC, the method comprising administering to the subject, a CRISPR/Cas agent which enters the gut or respiratory system and targets the TLR7/8 gene in a gut or respiratory cell or PBMC thereby reducing its ability to express and/or produce a functional protein. The amount of CRISPR/Cas agent is effective to ameliorate symptoms or prevent development of symptoms or minimize further progression of symptoms of the gut or respiratory inflammatory response.

[0064] Expression vectors comprising nucleic acid molecules may encode a sense or antisense oligonucleotide or a protein antagonist of TLR7/8. These may be present in a bacterium or virus or viroid for use to introduce to a target cell. The nucleic acid is operably linked to regulatory elements needed for gene expression. Accordingly, incorporation of the DNA or RNA molecule into a delivery virus or other vehicle results in the expression of the DNA or RNA encoding the oligonucleotide or protein when the virus introduces the expression vector to the target cell. A "target cell" means a cell in a subject carrying the bacterium which persists in that cell. A bacteria delivery vehicle may export a nucleic acid which is then taken up by the cell carrying the persistent bacterium.

[0065] Hence, the nucleic acid molecule that includes the nucleotide sequence encoding the oligonucleotide or protein operably linked to the regulatory elements may be introduced to a target cell via a bacterial or viral vector or agent. Alternatively, linear DNA or RNA which can integrate into the chromosome may be introduced into the target cell. When introducing DNA or RNA into a cell, reagents which promote DNA or RNA integration into chromosomes or transcriptome may be added.

[0066] The necessary elements of an expression vector include a nucleotide sequence that encodes the oligonucleotide or a protein antagonist of TLR7/8 and the regulatory elements necessary for expression of that sequence in the target cells. The regulatory elements are operably linked to the nucleotide sequence that encodes the oligonucleotide or protein antagonist to enable expression inside the target cell. The nucleotide sequence may be cDNA, genomic DNA, synthesized DNA or a hybrid thereof or an RNA molecule such as mRNA.

[0067] The regulatory elements necessary for gene expression include: a promoter, an initiation codon, a stop codon, and a polyadenylation signal. It is necessary that these elements be operable in the target cell. Moreover, it is necessary that these elements be operably linked to the nucleotide sequence that encodes the oligonucleotide or protein antagonist such that the nucleotide sequence can be expressed in the target cells and thus the oligonucleotide or protein antagonist can be produced. Reference to a "target cell" includes a gut cell, macrophage such as a gut or alveolar macrophage, respiratory cell and a PBMC. [0068] Other agents contemplated herein include small chemical molecules, antibodies optionally modified to enhance their half life in body fluid, small peptides, cyclohexene derivatives, lipid derivatives, and vehicles used to transport these agents, such as liposomes and genetically modified viral agents which infect target cells and facilitate delivery and expression or production of an agent such as an oligonucleotide.

[0069] The term "small molecule" generally refers to small organic compounds that are biologically active. Small molecules may exist naturally or may be created synthetically. Small molecules include compounds that down-regulate the expression, function or activity of TLR7/8. They may be administered directly into the gut or respiratory system or via the blood system or via the bacterial or viral vector.

[0070] Generally, the agents are delivered with a physiologically or pharmaceutically acceptable carrier, diluent or excipient. Hence, formulations, medicaments, therapeutic agents and pharmaceutical compositions comprising the agent and a physiologically or pharmaceutically acceptable carrier, diluent or excipient are contemplated herein.

[0071] The term "physiologically acceptable" generally refers to a material that does not interfere with the effectiveness of the agent and that is compatible with a biological system such as a cell, cell culture, tissue, or organism. Generally, the organism is a mammal such as a human. Both human and veterinary applications are encompassed by the present invention.

[0072] The term "pharmaceutically acceptable" generally refers to compositions that are suitable for use in humans and animals without undue toxicity.

[0073] The term "carrier" generally encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid containing vesicle, microspheres, liposomal encapsulation, or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient, or diluent will depend on the route of administration for a particular application. The preparation of pharmaceutically acceptable formulations containing these materials is described in, e.g. Remington's Pharmaceutical Sciences, 18th Edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990. The "carrier" may facilitate delivery via oral administration or inhalation. Coated pills for delivery to the gut or enteric system are also contemplated herein.

[0074] The terms an "effective amount," "pharmaceutically effective amount" or "therapeutically effective amount" generally refer to an amount sufficient to affect a desired biological effect, such as beneficial results including reducing TLR7/8 producing, reducing TLR7/8 function, activity or gene expression or ameliorating or mitigating the effects of bacterial-induced inflammation. Thus, an "effective amount" or "sufficient amount" or "pharmaceutically effective amount" or "therapeutically effective-amount" will depend upon the context in which it is being administered. In the context of administering a composition that reduces TLR7/8 gene expression or TLR7/8 protein function or activity is an amount sufficient to achieve the desired amelioration of inflammatory symptoms. Hence, the amount of agent administered is effective to inhibit or reduce the function or level or activity of TLR7/8. Generally, the amount is effective to mitigate the symptoms or underlying cause of the inflammation. Hence, in an embodiment, the amount is effective to reduce bacterial persistence in the gut or respiratory system.

[0075] The agent may be administered in any way which enables it to reach its target in the gut or respiratory system by any mechanism.

[0076] Enabled herein is a therapeutic protocol for treating or preventing bacterial-induced gut or respiratory inflammation or bacterial persistence in PBMC subject, the protocol comprising:

(i) identifying and selecting a subject based on symptoms of inflammation or genetic testing; (ii) administering to the subject an agent or vehicle carrying the agent which enters the gut or respiratory system and inhibits expression of the gene in a cell carrying an endogenous bacterium in a persistent manner encoding TLR7/8 or inhibits TLR7/8 function or activity in an amount effective to ameliorate symptoms, prevent development of the symptoms or minimize further progression of the symptoms of the inflammation;

(iii) monitoring the symptoms and behavior of the subject;

(iv) provide further agent or other medicaments as required to maintain the health of the subject.

[0077] In an embodiment, the cell is a macrophage or antigen-presenting cell or PBMC.

[0078] Further enabled herein is the use of an agent which antagonizes TLR7/8 activity or function or TLR7/8 gene expression in the manufacture of a medicament to treat or ameliorate the symptoms of bacterial-induced inflammation of the gut or respiratory system or bacterial persistence in a gut or respiratory cell or in a PBMC.

[0079] The agent may be given alone or in combination with other medicaments to assist the patient in mitigating the severity of symptoms or ameliorate the symptoms.

[0080] Hence, combination therapy is further contemplated herein comprising a first therapeutic protocol comprising administration of a TLR7/8 antagonistic agent and one or more other medicaments.

[0081] In a related embodiment, taught herein is an agent which down regulates expression, level or function of TLR7/8 in a PBMC for use in the treatment or prevent of a bacterial-induced inflammatory response in the gut or respiratory system of a subject or to reduce intracellular bacterial persistence in a gut or respiratory cell or in a PBMC.

[0082] The present specification is instructional for a therapeutic kit for treating or preventing a bacterial -induced inflammatory response in a subject or to reduce intracellular bacterial persistence in a PBMC, the kit comprising an agent which down regulates expression, level or function of TLR7/8 and instructions for treating the subject according to the therapeutic protocol as herein described. The kit may deliver multiple doses for from 2 days to 100 days. The kit may also comprise the TRL7/8 antagonist alone also designed to dispense multiple doses from 2 days to 100 days. Alternatively, a package is provided with TLR7/8 antagonists in pill form which might last for months.

[0083] Dosing will be dependent on the person's disorder, severity of symptoms and the like. Hence, dosing may be from a single or multiple daily doses to weekly or monthly doses may be required, generally for the life of the subject.

EXAMPLES

[0084] Aspects disclosed herein are further described by the following non-limiting Examples.

[0085] In work leading to the present invention, a genotyping study was carried out on a small cohort of patients with or without H. pylori infection (112 infected patients and 58 controls) to determine if TLR8 is an important factor in H. pylori infection. Two TLR8 genetic variations were studied, the first one impacting the TLR8 promoter region (rs3764879 :C>G), the second changing the initiating methionine and resulting in a change of Kozak context and decreased TLR8 levels (rs3764880:A>G) [Gantier et al. (2010a) Hum Mutat 37: 1069-79]. The two single nucleotide polymorphisms (SNPs) are in linkage disequilibrium, rs3764879:C found with rs3764880:A and rs3764879:G found with rs3764880:G. There was no association between these TLR8 SNPs and the development of premalignant gastric atrophophy/lesions, unlike previously reported for TLR9 SNPs in this cohort (Ng et al. (2010) Infect Immun 75: 1345-52). Critically, however, there was an association of the G alleles of the two SNPs with protection against H pylori infection, indicating that, similar to M. tuberculosis, less TLR8 is protective against H. pylori infection.

EXAMPLE 1

TLR8 contributes to H. pylori phagosomal survival

[0086] In order to characterise the putative mechanism of action of TLR8 on H. pylori infection suggested above, the intracellular levels and localization of TLR8 are investigated in Phorbol 12-myri state- 13 -acetate (PMA)-activated human THP-1 macrophages following infection with a variant of H. pylori (Gantier et al. (2010a) supra). In accordance with induction of TLR8 at the mRNA level, TLR8 protein is induced upon infection. Confocal data indicate that TLR8 co-localizes with the bacterium within the cell. Given the ability of H. pylori to block phagosomal maturation and the suggestion that low TLR8 levels associate with protection against infection, it is proposed herein that TLR8 is beneficial to the phagosomal survival of bacterium. In this study, PMA-activated human THP-1 macrophages are pre-treated or not with IFNy to strongly up-regulate TLR8 expression and are infected (Gantier et al. (2008) supra). Cells are rinsed after 1 h to remove extracellular bacteria and incubated for a further 5 h with gentamicin containing medium to kill the bacteria that are not phagocytosed. Intracellular bacteria are then recovered by gentle saponin lysis of the cell walls, and viability determined by colony forming units (cfu) on blood-agar plates. ΠΤΝΓγ priming resulted in a 2-fold increased phagosomal survival of H. pylori, suggesting involvement of TLR8 in H. pylori intracellular survival (Figure la).

EXAMPLE 2

Mice macrophages lacking endosomal TLR7 clear more efficiently phagosomal H.

pylori and Salmonella Typhimurium

[0087] In light of the results in Example 1 in human macrophages which indicated a role for human TLR8 in H. pylori phagosomal survival, the direct requirement for mouse TLR7 in macrophages during H. pylori infection is investigated since mouse TLR7 recapitulates human TLR8 function. Infection with another phagosomal bacterium, Salmonella enterica serovar Typhimurium {Salmonella Typhimurium) that leads to acute infections in vivo, is also investigated in parallel. Infection of primary TLR7-deficient macrophages with H. pylori or Salmonella Typhimurium results in significantly more rapid intracellular clearance of the bacteria compared to wild type mice (Figure lb). This activity is accompanied with increased IL10 production with both bacteria in TLR7 -deficient cells (Figure lc). In addition, TLR7-deficient cells increase Arginasel expression with both bacteria suggesting that TLR7 inhibition favours polarisation of macrophages towards an M2 (anti -inflammatory) phenotype (Figure Id).

EXAMPLE 3

TLR7 -deficient mice have impaired anti-H. pylori antibody response and less

inflammation

[0088] To define further the impact of TLR7 during chronic H. pylori infection in vivo, a short-term infection (30 days) in TLR7^" /^" mice is performed with high levels (107 bacteria) of mouse-adapted H. pylori SSI . Every infected mouse is colonized, but there is no significant difference in the bacterial counts recovered from stomach homogenates of WT and TLR7^{_ "} mice after 30 days, presumably relating to the saturating dose of bacteria used. Nonetheless, lack of TLR7 resulted in a strong decrease of anti-H. pylori antibodies (Figure 2a). In addition, lack of TLR7 decreased inflammation in stomach sections - with fewer infiltrating immune cells and lower pro-inflammatory IL6 in gastric homogenates (Figures 2b and 2c). This suggests that lack of TLR7 in mice infected by H. pylori is protective against the inflammation/immune responses driven by the bacterium, in line with the observation that low TLR8 levels are predominant in human populations with high exposure to H. pylori. Collectively, these findings indicate that decreased TLR7/8 levels can protect from colonization in populations exposed to low doses of H. pylori, and are beneficial to colonized hosts by reducing chronic inflammation.

EXAMPLE 4

TLR7 -deficient mice are protected against Salmonella Typhimurium infection.

[0089] To determine the role of TLR7 during acute Salmonella Typhimurium infection, TLR7^_/" mice are infected by oral gavage with high levels (10⁶ bacteria) of Salmonella Typhimurium. The infection is kept for 6 days, time at which the wild-type mice have lost >10% body weight - which is absent in TLR7-deficient mice (Figure 3a). TLR7-deficiency protects the mice from infection, with more than 1000 times fewer bacteria present in the spleen and liver of infected wild-type mice (Figure 3b). This is readily visible with increased spleen size (and weight) and necrotic pale liver in wild-type mice, absent in TLR7-deficient mice (Figure 3c). In addition, circulating pro-inflammatory serum cytokines (IFNy, TNFa, CXCLl) are strongly decreased in TLR7-deficient mice (Figure 3d). In addition, lack of TLR7 resulted in a strong decrease of anti-Salmonella Typhimurium antibodies (Figure 3e). These results demonstrate that TLR7 is essential for systemic spreading of Salmonella Typhimurium in vivo, thus highlighting how inhibition of TLR7 could help protect from infection.

EXAMPLE 5

Characterization of TLR7/8 inhibitors with therapeutic potential against bacterial infection

[0090] RNA sensing by TLR7/8 can be inhibited in trans by the transfection of 2'0-methyl RNA (2'Ome), which has a strong affinity to these receptors and antagonises the sensing of immunostimulatory RNA (Hamm et al. (2010) Immunobiology 275:559-69). This property of 2'Ome is believed to be used by the innate immune sensor to distinguish self and non-self RNA, given that 2'Ome ribonucleotides are incorporated in the 5'cap of self mRNA, resulting in the inhibition of several innate immune sensors (TLR7/8, MDA5 and IFITl/5). A comparison made on the TLR7/8 inhibitory activity of different sequences of 2'Ome RNA. Relying on a panel of synthetic 2'Ome RNA sequences designed as microRNA inhibitors, sequences are sought with high inhibitory activity on TLR7/8 in PBMCs. Surprisingly, strong differences were found between select 2'Ome sequences in their ability to inhibit TNFa and/or IFNa production (Sarvestani et al. (2015) Nucleic Acids Res. 43 : 1177-88) [TNFa is indicative of TLR8 recruitment, while IFNa reflects TLR7 activation (Sarvestani et al. (2012) supra)]. Such RNA molecules are already used in humans as microRNA inhibitors, and have strong therapeutic potential (Janssen et al. (2013) N Engl J Med 3(55: 1685-94). The use of highly potent 2'Ome RNAs targeting TLR7/8 against H. pylori survival in phagosomal cells is therefore proposed herein.

EXAMPLE 6

Effect of thiostrepton

[0091] Thiostrepton has only minimal direct antibiotic effect against Salmonella enterica serovar Typhimurium. This is shown in Figure 4. An aliquot of ΙΟΟμΙ of a 4 hour liquid culture of Salmonella enterica serovar Typhimurium was used to infect 5ml of LB broth containing increasing concentrations of thiostrepton (0.001 μΜ to 10μΜ). Absorbance at 600nm was assessed 4 hours post injection. From Figure 4, it is observed that thiostrepton at concentrations from 0.001 μΜ to 10μΜ did not directly inhibit Salmonella activity.

[0092] Notwithstanding that thiostrepton is not a strong direct acting antibiotic, it nevertheless protected macrophages infected with Salmonella enterica serovar Typhimurium. Mouse bone marrow-derived macrophages were pre-treated with 5μΜ thiostrepton or DMSO 30 minutes before infection with Salmonella enterica serovar Typhimurium (MOL5). At 30 minutes post infection, cells were washed with complete medium supplemented with 100μg/ml gentamycin to kill extracellular bacteria. At a time of 30 minutes later, medium was changed with medium supplemented with 10μg/ml gentamycin and DMSO or thiostrepton (5μΜ). 2 hours post infection, cells were lysed with 180μ1 0.2% v/v lgepal CA-630 in PBS and plated on LB agar plates after dilution in LB broth. Colonies were counted 20 hours post plating. From Figure 5, it is observed that thiostrepton at 5μΜ inhibited Salmonella inside the macrophages. This is indicative of thiostrepton having a host cell-induced effect on the survivability of Salmonella.

[0093] Given that thiostrepton is not a direct acting antibiotic (Figure 4) and it appears to be mediating an affect against Salmonella via a host cell effect (Figure 5), the effect of thiostrepton on TLR7/8 signaling was assayed. Mouse bone marrow-derived macrophages were pre-treated with increasing concentration of thiostrepton (0.5, 2 or 5μΜ) or DMSO 30 minutes before stimulation with TLR agonists. R848 is a TLR7/8 agonist (used at Ι μΜ). Gardiquimod is a TLR7 agonist (used at 2^g/ml). Cells were incubated 20 hours in presence of thiostrepton and TLR agonists. Supernatants were collected and activation of macrophages was assessed by measuring the production of TNF-a. The results are shown in Figure 6 and indicate that thiostrepton is mediating an effect on TLR7/8. Thiostrepton is a TLR7/8 antagonist (Figure 6). Hence, this supports the proposition that a TLR7/8 antagonist is useful in inhibiting microbial infection of a cell. In particular, thiostrepton is an inhibitor of TLR7/8 signaling in macrophages. Taken together, these results indicate that thiostrepton activity against Salmonella is mediated by an effect on the host wherein this effect comprises inhibition of TLR7/8 signaling.

[0094] A similar protocol is proposed for other potential TLR7/8 antagonists such as nucleic acid agents or a chemical agent such as chloroquine, quinacrine, amodiaquine, pamaquine, mefloquine, piperaquine, trioxaquine (SARI 16242), 7-chloro-N-(3-(4-(7- (trifluoromethyl)quinolin-4-yl)piperazin-l-yl)propyl)quinolin-4-amine and {3-[4-(7-chloro- quinolin-4-yl)-piperazin-l-yl]-propyl}-(7-trifluoromethyl-quinolin-4-yl)-amine. In addition, bafilomycin Al (BafAl) may be used or another plecomacrolide subclass of a macrolide antibiotic. Various combinations of any of the above agents or use of at least one of these agents with another compound can also be readily tested.

EXAMPLE 7

Therapeutic protocol

[0095] The therapeutic use of synthetic nucleic acids such as 2'Ome and LNA/DNA is illustrated by several current clinical trials using such molecules to inhibit select microRNAs such as miR-122 in humans (Janssen et al. (2013) supra). Some of these molecules have strong TLR7/8 inhibitory activity (Sarvestani et al. (2015) supra).

[0096] In addition, pre-clinical modeling defines the ability of TLR7/8 inhibitors to antagonize H. pylori survival within macrophages, and how this impacts on ability of the bacterium to colonize the host and regulates the chronic inflammation associated with infection. This innovative approach to control H. pylori enables bypassing antibiotic resistance, by reducing intracellular reservoirs of the bacterium.

[0097] Example 6 also shows that thiostrepton is a TLR7/8 antagonist but is not a direct antibiotic. However, by antagonizing TLR7/8, it is able to cause the inhibition of intracellular Salmonella infection.

[0098] Importantly, this work also identifies the existence of a novel branch of TLR7/8 signaling, with immune-suppressive capacities, which benefits intracellular parasites such as Salmonella species and Mycobacterium species. This defines a new paradigm where TLRs can also repress inflammation, with widespread consequence in the research world of host-pathogens interactions.

[0099] The present invention has the potential to impact research on several other phagosomal parasites of national and international relevance such as C. burnetii and M. tuberculosis. Given the growing burden of drug-resistant M. tuberculosis across the world, using TLR7/8 inhibitors present a new way to treat infected patients, for which other treatments have failed. The present invention further has potential to treat Salmonella infection such as resulting in sepsis or septic shock. The therapeutic protocol comprises administering a TLR7/8 signaling antagonist such as but not limited to a nucleic acid, an antibody or a chemical molecule such as thiostrepton or chloroquine or its analogs including quinacrine, amodiaquine, pamaquine, mefloquine, piperaquine, trioxaquine (SARI 16242), 7-chloro-N-(3-(4-(7-(trifluoromethyl)quinolin-4-yl)piperazin-l- yl)propyl)quinolin-4-amine and {3-[4-(7-chloro-quinolin-4-yl)-piperazin-l-yl]-propyl}-(7- trifluoromethyl-quinolin-4-yl)-amine or a macrolide antibiotic such as but not limited to bafilomycin Al or a combination of any two or more of the above or at least one together with another chemical/agent.

[0100] Those skilled in the art will appreciate that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure contemplates all such variations and modifications. The disclosure also enables all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features or compositions or compounds.

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Claims

CLAIMS:

1. A therapeutic protocol for the treatment or prophylaxis of a bacterial -induced inflammatory response in the gut or respiratory system of a subject, or to reduce intracellular bacterial persistence in a peripheral blood mononuclear cell (PBMC), said therapeutic protocol comprising administering to said subject an agent which down- regulates expression, level or function of toll-like receptor (TLR) 7/8 in a gut or respiratory cell or in said PBMC for a time and under conditions sufficient to reduce, inhibit or prevent development of an inflammatory immune response or to promote clearance of the bacterium.

2. The therapeutic protocol of Claim 1 wherein the agent is a nucleic acid molecule which targets expression of a TLR7/8 gene or a gene encoding a component of the TLR7/8 signaling pathway or which inhibits sensing by a TLR7/8 ligand.

3. The therapeutic protocol of Claim 2 wherein the nucleic acid is an RNA species or a chemically modified form thereof.

4. The therapeutic protocol of Claim 3 wherein the nucleic acid comprises a 2'-0- methyl ribose modification.

5. The therapeutic protocol of Claim 3 or 4 wherein the nucleic acid is a micro RNA, sense RNA, anti-sense RNA, double stranded RNA, short interfering RNA, hairpin RNA, a modified tRNA, a methylated RNA, synthetic RNA construct or an RNA hybrid molecule.

6. The therapeutic protocol of Claim 2 wherein the nucleic acid is a double stranded DNA, single stranded DNA, a synthetic DNA construct or a DNA hybrid molecule.

7. The therapeutic protocol of Claim 1 wherein the agent is an antibody specific for TLR7/8 or a component of the TLR7/8 signaling pathway or which otherwise inhibits sensing by a TLR7/8 ligand.

8. The therapeutic protocol of Claim 1 wherein the agent is a protein or small chemical molecule which inhibits the function of TLR7/8-mediated signaling or which otherwise inhibits sensing by a TLR7/8 ligand.

9. The therapeutic protocol of Claim 8 wherein the chemical molecule is thiostrepton.

10. The therapeutic protocol of Claim 8 wherein the chemical molecule is chloroquine or an analog thereof.

11. The therapeutic protocol of Claim 10 wherein the chloroquine analog is selected from quinacrine, amodiaquine, pamaquine, mefloquine, piperaquine, trioxaquine (SARI 16242), 7-chloro-N-(3-(4-(7-(trifluoromethyl)quinolin-4-yl)piperazin-l- yl)propyl)quinolin-4-amine and {3-[4-(7-chloro-quinolin-4-yl)-piperazin-l-yl]-propyl}-(7- trifluoromethyl-quinolin-4-yl)-amine or a combination of two or more.

12. The therapeutic protocol of Claim 8 wherein the chemical molecule is bafilomycin Al or other functionally related macrolide antibiotic of the plecomacrolide subclass.

13. The therapeutic protocol of Claim 1 wherein the agent is a microorganism genetically modified or selected to produce a modified RNA species.

14. The therapeutic protocol of Claim 1 wherein the agent is a microorganism genetically modified or selected to produce a modified methyltransferase.

15. The therapeutic protocol of Claim 14 wherein the methyltransferase is selected from the list consisting of a Gm-2'-0-methyltransferase (spoU/trmH), uracil-5- methyltransferase (trmA), and a guanine-7-methyltransferase (uggh/trmB).

16. The therapeutic protocol of Claim 15 wherein the methyltransferase is spoU/trmH.

17. The therapeutic protocol of Claim 1 wherein the bacterial -induced inflammatory response is mediated by a Helicobacter species, Mycobacterium species, a Salmonella species, a Campylobacter species, a Francisella species or a Chlamydia species.

18. The therapeutic protocol of Claim 17 wherein the Helicobacter species is Helicobacter pylori.

19. The therapeutic protocol of Claim 17 wherein the Mycobacterium species is Mycobacterium tuberculosis.

20. The therapeutic protocol of Claim 17 wherein the Salmonella species is Salmonella enter ica serovar Typhimurium or serovar Typhi.

21. The therapeutic protocol of Claim 17 wherein the Campylobacter species is Campylobacter jejuni.

22. The therapeutic protocol of Claim 17 wherein the Francisella species is Francisella tularensis.

23. The therapeutic protocol of Claim 1 wherein the intracellular bacterium is selected from the list consisting of Mycobacterium leprae, Mycobacterium bovis, Yersinia pestis, Tropheryma whipplei and Brucella melitensis.

24. The therapeutic protocol of Claim 1 wherein the intracellular bacterium is selected from the list consisting of Legionella pneumophila and Coxiella brunetii.

25. The therapeutic protocol of any one of Claims 1 to 24 wherein the subject is a human.

26. The therapeutic protocol of Claim 1 wherein the gut comprises any of the stomach, small and large intestine and bowel.

27. The therapeutic protocol of Claim 1 wherein the respiratory system comprises the lung or alveolar macrophage.

28. The therapeutic protocol of Claim 25 or 26 wherein the inflammatory response leads to gastritis, gastroenteritis, Crohn's disease or inflammatory bowel syndrome.

29. The therapeutic protocol of Claim 25 wherein the inflammatory response leads to cancer or pre-cancerous lesion.

30. The therapeutic protocol of Claim 27 wherein the intracellular bacterium leads to pneumonia, Q Fever, Typhoid fever, sepsis or septic shock.

31. The therapeutic protocol of Claim 1 wherein the gut or respiratory cell or PBMC is a macrophage, monocyte, antigen-presenting cell or lymphocyte.

32. The therapeutic protocol of Claim 31 wherein the antigen-presenting cell is a dendritic cell.

33. Use of an agent which down regulates expression, level or function of toll-like receptor (TLR) 7/8 in a gut or respiratory cell or a PBMC in the manufacture of a medicament to treat or prevent a bacterial-induced inflammatory response in the gut or respiratory system of a subject or to reduce intracellular bacterial persistence in the cell.

34. Use of Claim 33 wherein the agent is a nucleic acid molecule which targets expression of a TLR7/8 gene or a gene encoding a component of the TLR7/8 signaling pathway or which inhibits sensing by a TLR7/8 ligand.

35. Use of Claim 34 wherein the nucleic acid is an RNA species or a chemically modified form thereof.

36. Use of Claim 35 wherein the nucleic acid comprises a 2'-0-methyl ribose modification.

37. Use of Claim 35 or 36 wherein the nucleic acid is a micro RNA, sense RNA, anti- sense RNA, double stranded RNA, short interfering RNA, hairpin RNA, a modified tRNA, a methylated RNA, synthetic RNA construct or an RNA hybrid molecule.

38. Use of Claim 34 wherein the nucleic acid is a double stranded DNA, single stranded DNA, a synthetic DNA construct or a DNA hybrid molecule.

39. Use of Claim 33 wherein the agent is an antibody specific for TLR7/8 or a component of the TLR7/8 signaling pathway or which otherwise inhibits sensing by a TLR7/8 ligand.

40. Use of Claim 33 wherein the agent is a protein or small chemical molecule which inhibits the function of TLR7/8-mediated signaling or which otherwise inhibits sensing by a TLR7/8 ligand.

41. Use of Claim 40 wherein the chemical molecule is thiostrepton.

42. Use of Claim 40 wherein the chemical molecule is chloroquine or an analog thereof.

43. Use of Claim 42 wherein the chloroquine analog is selected from quinacrine, amodiaquine, pamaquine, mefloquine, piperaquine, trioxaquine (SARI 16242), 7-chloro-N- (3-(4-(7-(trifluoromethyl)quinolin-4-yl)piperazin-l-yl)propyl)quinolin-4-amine and {3-[4- (7-chloro-quinolin-4-yl)-piperazin-l-yl]-propyl}-(7-trifluoromethyl-quinolin-4-yl)-amine or a combination of two or more.

44. Use of Claim 40 wherein the chemical molecule is bafilomycin Al or other functionally related macrolide antibiotic of the plecomacrolide subclass.

45. Use of Claim 33 wherein the agent is a microorganism genetically modified or selected to produce a modified RNA species.

46. Use of Claim 33 wherein the agent is a microorganism genetically modified or selected to produce a modified methyltransferase.

47. Use of Claim 46 wherein the methyltransferase is selected from the list consisting of a Gm-2'-0-methyltransferase (spoU/trmH), uracil-5-methyltransferase (trmA), and a guanine-7-methyltransferase (uggh/trmB).

48. Use of Claim 47 wherein the methyltransferase is spoU/trmH.

49. Use of Claim 33 wherein the bacterial-induced inflammatory response is mediated by a Helicobacter species, a Mycobacterium species, a Salmonella species, a Campylobacter species, a Francisella species or a Chlamydia species.

50. Use of Claim 49 wherein the Helicobacter species is Helicobacter pylori.

51. Use of Claim 50 wherein the Mycobacterium species is Mycobacterium tuberculosis.

52. Use of Claim 50 wherein the Salmonella species is Salmonella enterica serovar Typhimurium or serovar Typhi.

53. Use of Claim 50 wherein the Campylobacter species is Campylobacter jejuni.

54. Use of Claim 50 wherein the Francisella species is Francisella tularensis.

55. Use of Claim 33 wherein the intracellular bacterium is selected from the list consisting of Mycobacterium leprae, Mycobacterium bovis, Yersinia pestis, Tropheryma whipplei and Brucella melitensis .

56. Use of Claim 33 wherein the intracellular bacterium is selected from the list consisting of Legionella pneumophila and Coxiella brunetii.

57. Use of any one of Claims 33 to 56 wherein the subject is a human.

58. Use of Claim 33 wherein the gut comprises any of the stomach, small and large intestine and bowel.

59. Use of Claim 58 wherein the inflammatory response leads to gastritis, gastroenteritis, Crohn's disease or inflammatory bowel syndrome.

60. Use of Claim 58 or 59 wherein the inflammatory response leads to cancer or precancerous lesion.

61. Use of Claim 33 wherein the respiratory system comprises the lung or alveolar macrophage.

62. Use of Claim 61 wherein the intracellular bacterium leads to pneumonia, Q Fever, sepsis or septic shock.

63. Use of Claim 33 wherein the gut or respiratory cell or PBMC is a macrophage, monocyte, antigen-presenting cell or lymphocyte.

64. Use of Claim 63 wherein the antigen-presenting cell is a dendritic cell.

65. A therapeutic kit for treating or preventing a bacterial -induced inflammatory response in a subject or to reduce intracellular bacterial persistence in a gut or respiratory cell or PBMC, said kit comprising an agent which down regulates expression, level or function of toll-like receptor 7/8 and instructions for treating the subject according to the therapeutic protocol of any one of Claims 1 to 32.

66. A probiotic comprising a genetically modified or selected gut or respiratory system flora microorganism which produces a modified RNA species which reduces toll-like receptor (TLR) 7/8-mediated signaling.

67. The probiotic of Claim 66 wherein the microorganism is genetically modified to produce a methyltransferase.

68. The probiotic of Claim 67 wherein the methyltransferase is selected from the list consisting of Gm-2'-0-methyltransferase (spoU/trmH), uracil -5 -methyltransferase (trmA), and a guanine-7-methyl transferase (uggh/trmB).

69. The probiotic of Claim 68 wherein the methyltransferase is spoU/trmH.

70. A probiotic formulation comprising the probiotic of any one of Claims 66 to 69.