Patent Application
20220213143
The instant application contains a Sequence Listing which has been submitted electronically in ANSI format and is hereby incorporated by reference in its entirety. The ANSI copy is named 103712.000033_SL.txt and is 152 kB in size.
The present invention relates generally to the field of immunomodulation. Taught herein is an agent for inhibiting immunostimulation mediated by a Toll-like receptor useful in the treatment of viral and microbial pathogenesis, diseases involving elements of autoimmunity and inflammation as well as cancer. Pharmaceutical compositions are also enabled herein.
Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.
The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgement or admission or any form of suggestion that the prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavor to which this specification relates.
Immune stimulation is a major component in the prevention of viral and microbial pathogenesis. However, the regulation of the immune system is complex, sensitive and multifaceted. Unwarranted stimulation can lead to autoimmune diseases. A fuller understanding of the regulatory processes controlling the immune response is required if medical professionals are to deal with the imminent threat of viral epidemics and pandemics and to control autoimmunity. There is an urgent need for novel therapeutic approaches to target pathology irrespective of the infecting strain or autoimmune condition.
The production of reactive oxygen species (ROS) is a highly coordinated process achieved by enzymes of the NADPH oxidase (NOX) family. NOX enzymes evolved in single cell eukaryotes over 1.5 billion years ago and are present in most eukaryotic groups including amoeba, fungi, algae and plants, nematodes, echinoderms, urochordates, insects, fish, reptiles and mammals (Kawahara et al. (2007) BMC Evolutionary Biology 7:109; Aguirre (2010) Free Radical Biology and Medicine 49(9):1342-1353). The functions of NADPH oxidases within eukaryotes are diverse, however, a common function is the generation of ROS by innate immune cells in response to pathogens. Indeed, orthologs of NADPH oxidase in plants (ArtbohD and ArtbohF), fungi (NOXA/B), and invertebrates Celegans (Duox orthologs), Drosophila melongaster (NOX5 homolog, d-NOX and DUOX) and mosquito Aedes aegypti (NOXM and DUOX) generate ROS with bactericidal activity that protects the host (Kawahara et al. (2007) supra; Aguirre (2010) supra). Vertebrates including teleosts, amphibians, birds and mammals possess a NOX2 NADPH oxidase that generates a burst of ROS within phagosomes to kill invading pathogens especially bacteria. However, the impact of ROS on virus infection is largely unknown.
ROS, such as superoxide anion and hydrogen peroxide (H2O2), are produced by mouse and human inflammatory cells in response to viral infection and enhance the pathology caused by viruses of low to high pathogenicity, including influenza A viruses (Imai et al. (2008) Cell 133(2):235-249; Snelgrove et al. (2006) Eur J Immunol 36(6):1364-1373; To et al. (2014) Free Radical Research 48(8):940-947; Vlahos et al. (2011) PLoS Pathogens 7(2):e1001271; Vlahos et al. (2012) Trends in Pharmacological Sciences 33(1):308; Vlahos and Selemidis (2014) Molecular Pharmacology 86(6):747-759). The primary source of inflammatory cell ROS is the NOX2 oxidase enzyme (Vlahos and Selemidis (2014) supra; Selemidis et al. (2008) Pharmacology & Therapeutics 120(3):254-291; Drummond et al. (2011) Nature Reviews Drug Discovery 10(6):453-471; Bedard and Krause (2007) Physiological Reviews 87(1):245-313). Although NOX2 oxidase plays a role in the killing of bacteria and fungi via phagosomal ROS production, NOX2 oxidase does not appear to eliminate viruses in a manner analogous to that for bacteria. In fact, in the absence of NOX2, influenza A virus causes substantially less lung injury and dysfunction, and leads to lower viral burden suggesting that NOX2 oxidase-derived ROS promotes rather than inhibits viral infection Imai et al. (2008) supra; Snelgrove et al. (2006) supra; To et al. (2014) supra; Vlahos et al. (2011) supra; Vlahos et al. (2012) supra; Vlahos and Selemidis (2014) supra). However, identifying how viruses cause ROS production has been allusive, as is how these highly reactive oxygen molecules, which appear to be largely confined to their site of generation, contribute to disease.
After binding to the plasma membrane (Cossart and Helenius (2014) Cold Spring Harbor Perspectives in Biology 6(8)), viruses enter cells and ultimately endosomes by a variety of mechanisms resulting in viral RNA detection by endosomal pattern recognition receptors, including Toll-like receptor 3 (TLR3), TLR7 and TLR9 (Iwasaki and Pillai (2014) Nature Reviews Immunology 14(5):315-328). The specific receptor interaction depends upon either the Group (I to V) or genomic orientation (i.e. ssRNA, dsRNA or DNA) of the virus and triggers an immune response characterized by Type I IFN and IL-1β production, and B-cell-dependent antibody production (Iwasaki and Pillai (2014) supra). Host nucleic acids and self-antigens are also detected by endosomal TLRs, and in autoimmune disease, mediate similar Type I IFN responses and stimulate antibody production against self-RNA and antigen. Notably, mice that are chronically deficient in NOX2 oxidase have an increased tendency to develop self-antibodies (Campbell et al. (2012) Science Translational Medicine 4(157):157ra141) and patients with chronic granulomatous disease, who have a defective capacity to generate ROS via the NOX2 oxidase, have elevated circulating Type I IFNs and autoantibodies (Kelkka et al. (2014) Antioxidants & Redox Signaling 21(16):2231-2245). These observations are supportive of the notion that low levels of ROS result in an enhanced immune response. However, until the advent of the present invention, it was unknown how ROS modulate inflammation and the pathology caused by pathogens and whether targeted (and acute) abrogation of ROS may actually be beneficial in treating infection as well as other immune response-related conditions such as autoimmune disease conditions.
Reactive oxygen species (ROS) promote the pathogenicity of viruses and microorganisms and other parasites. In work leading up to the present invention, the site and enzymatic source of subcellular ROS generation were determined together with the impact on ROS on immunostimulation. In accordance with the present invention, it is determined that TLR7 protein is activated by a disulfide bond forming between cysteine residues at positions 98 (C98) and 475 (C475) of TLR7 and this leads to toxic ROS production via NADPH oxidase (NOX2). The ROS in turn suppress antiviral and antimicrobial activity. The present invention therefore validates TLR7 as a therapeutic target. Reduction in TLR7 activity also enables treatment of autoimmune disease, inflammation and cancer and other conditions exacerbated by TLR7 activity.
Enabled herein is a method for inhibiting TLR7-mediated immunostimulatory activity in a subject, the method comprising contacting a cell from the subject expressing TLR7 with an effective amount of an agent which antagonizes disulfide bond formation between C98 and C475 of human (or murine) TLR7 or their corresponding positions in other TLR7s. Further taught herein is a method for treating a subject for autoimmune disease, viral or microbial pathogenesis, inflammation or cancer, the method comprising contacting a cell from the subject expressing TLR7 with an effective amount of an agent which antagonizes disulfide bond formation between C98 and C475 of TLR7 human (or murine) or their corresponding positions in other TLR7s. The present invention also enables a reduction in hypoimmunostimulation induced by ROS and ameliorates conditions associated with or exacerbated by TLR7 activity. In an embodiment, the agent suppresses TLR7 activity and is useful in the treatment of autoimmune disease condition.
In an embodiment, the agent comprises a peptide, referred to herein as a “decoy peptide” of from about 4 to about 190 amino acids in length and having an amino acid sequence with at least 70% amino acid sequence similarity to up to 190 contiguous amino acids between amino acids 4 to 194 of TLR7 human (or murine) which includes a peptide having an amino acid sequence with at least 70% amino acid sequence similarity to up to 100 contiguous amino acids between amino acids 48 to 148 of TLR7 and which also includes a peptide having an amino acid sequence with at least 70% amino acid sequence similarity to up to 40 contiguous amino acids between amino acid 78 to 118 of TLR7 with the proviso that the peptide comprises a cysteine residue at the equivalent of position 98 of TLR7 (C98).
In an embodiment, the decoy peptide comprises the amino acid sequence RCNC (SEQ ID NO:45) (using single amino acid code) corresponding to amino acids R97 to C100 of TLR7. In an embodiment, the decoy peptide comprises 10 amino acids in length from positions 95 to 104 of human TLR and has an amino acid sequence with at least 70% similarity to DX1RCNCX2PX3X4 (SEQ ID NO:27) wherein:
X1 is L, F or M;
X2 is V or I;
X3 is V or I or A or P; and
X4 is P or L or K or R.
In an embodiment, the decoy peptide is DFRCNCVPIP (SEQ ID NO:26) corresponding to human TLR7 between D95 and P104.
In another embodiment, the peptide comprises the amino acid sequence DLRCNCVPVL (SEQ ID NO:1) corresponding to D95 to L104 of murine TLR7.
To facilitate uptake of the peptide into the cell, the peptide may further comprise a moiety attached to the N-terminal or C-terminal end of the peptide which moiety includes a hydrophilic or cationic peptide, an amphiphilic or amphipathic peptide or a peptide with a periodic amino acid sequence. Alternatively, the peptide is conjugated to cholestanol.
Examples of a hydrophilic peptide include a peptide is selected from the group consisting of TAT (SEQ ID NO:2), SynB1 (SEQ ID NO:3), SynB3 (SEQ ID NO:4), PTD-4 (SEQ ID NO:5), PTD-5 (SEQ ID NO:6), FHV coat (SEQ ID NO:7), BMV Gag-(7-25) (SEQ ID NO:8), HTLV-II Rex-(4-16) (SEQ ID NO:9), D-Tat (SEQ ID NO:10) and R9-Tat (SEQ ID NO:11).
Examples of an amphiphilic peptide include a peptide is selected from the group consisting of Transportan chimera (SEQ ID NO:12), MAP (SEQ ID NO:13), SBP (SEQ ID NO:14), FBP (SEQ ID NO:15), MPG [MPGac] (SEQ ID NO:16), MPG (ΔNLS) (SEQ ID NO:17), Pep-1 (SEQ ID NO:18) and Pep-2 (SEQ ID NO:19).
Examples of a peptide with a periodic amino acid sequence include a peptide with a polyarginine or a polylysine sequence. Other examples are provided in Table 1 of Guidotti et al. (2017) Trends in Pharmacological Sciences 38(4):406-424, the contents of which are incorporated by reference. Cell uptake can also be facilitated by a NOX2-cholestanol-linker (PEG)-gp91ds-TAT construct. Linking cholestanol and PEG to gp91ds-TAT facilities delivery of gp91ds-TAT to the endosome.
In an embodiment, the subject is a human or non-human mammal.
Further enabled herein is use of an agent which antagonizes disulfide bond formation between C98 and C475 of TLR7 or their corresponding positions in the manufacture of a medicament to inhibit autoimmune disease, viral or microbial pathogenesis, inflammation or cancer in a subject. In a related embodiment, the present invention teaches an agent which antagonizes disulfide bond formation between C98 and C475 of TLR7 or their corresponding positions for use in inhibiting autoimmune disease, viral or microbial pathogenesis, inflammation or cancer in a subject.
Taught herein is a pharmaceutical composition comprising an agent which antagonizes disulfide bond formation between C98 and C475 of TLR7 or their corresponding positions and one or more pharmaceutical carriers, excipients and/or diluents. Such an agent, in an embodiment, includes an agent which comprises a peptide of from about 4 to 190 amino acids in length and having an amino acid sequence with at least 70% amino acid sequence similarity to up to 190 contiguous amino acids between amino acids 4 and 194 of TLR7. In another embodiment the agent comprises a peptide of from about 4 to 100 amino acids in length and having an amino acid sequence with at least 70% amino acid sequence similarity to up to 100 contiguous amino acids between amino acids 48 and 148 of TLR7. In another embodiment, the agent comprises a peptide of from about 4 to 40 amino acids in length and having an amino acid sequence with at least 70% amino acid sequence similarity to up to 40 contiguous amino acids between amino acids 78 and 118 of TLR7. Each of these embodiments is with the proviso that the peptide comprises a cysteine residue at the equivalent of position 98 of TLR7 (C98). Alternatively, or in addition, the peptide comprises the amino acid sequence RCNC (SEQ ID NO:45) corresponding to R97 to C100 of TLR7. Extraneous amino acids totaling, with RCNC (SEQ ID NO:45), from 5 to 500 may be included on the N- and/or C-terminal ends of H2N-RCNC-C00H. Chemical mimetics of the TLR7-inhibitory peptides also form part of the present invention.
Abbreviations used herein are defined in Table 1.
Amino acid sequences are referred to by a sequence identifier number (SEQ ID NO). The SEQ ID NOs correspond numerically to the sequence identifiers <400>1 (SEQ ID NO:1), <400>2 (SEQ ID NO:3), etc. An amino acid sequence in 3 or single letter code or written in full is provided in the N-terminal to C-terminal direction (left to right, respectively), unless otherwise specified.
A summary of sequence identifiers used throughout the subject specification is provided in Table 2.
Single and three letter codes for amino acids used herein are defined in Table 3.
List of primers and their sources and reference sequences are shown in Table 4. The influenza polymerase primers were custom synthesized and the sequences are shown.
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.
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 step or group of elements or integers or method steps.
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 pathogen” includes a single pathogen, as well as two or more pathogens; reference to “an agent” includes a single agent, as well as two or more agents; reference to “the disclosure” includes single and multiple aspects taught by the disclosure; and so forth. Aspects taught and enabled herein are encompassed by the term “invention”. Any variants and derivatives contemplated herein are encompassed by “forms” of the invention. All aspects of the invention are enabled across the width of the claims.
The present invention is predicated in part on the determination that virus entry into the endosomal compartment of a cell triggers NADPH oxidase (NOX)-dependent production of reactive oxygen species (ROS) in the endosome. It is determined that endosomal ROS are negative regulators of molecular mechanisms conferring antiviral immunity and is dependent on Toll-like receptor 7 (TLR7) activation. The ability to limit ROS production by antagonizing TLR7 activation enables production of a universal antiviral therapy as well as anti-pathogen therapy more generally. Antagonizing TLR7 activation limits production of ROS which would otherwise antagonize immunostimulation. Given that TLR7 is itself associated with forms of inflammation, the present invention has wider implications in the treatment of microbial infection (e.g. bacteria and fungal microorganisms), autoimmune disease conditions, inflammation and cancer. Hence, antagonizing TLR7 activation assists in ameliorating diseases and conditions associated with or exacerbated by TLR7 activation.
Hence, the present invention enables a reduction in levels of activated TLR7 which leads to:
(i) reduced endosomal ROS production via NOX2;
(ii) reduced active TLR7 leading to reduced inflammatory conditions;
(iii) reduced hypoimmunostimulation;
(iv) reduced negative regulation of humoral immune response networks;
(v) reduced capacity for pathogen infection;
(vi) reduced inflammation;
(vii) reduced capacity for cancer growth; and/or
(viii) amelioration of autoimmune conditions or diseases arising therefrom.
Accordingly, enabled herein is a method for inhibiting TLR7-mediated immunostimulatory activity in a subject, the method comprising contacting a cell from the subject expressing TLR7 with an effective amount of an agent which antagonizes disulfide bond formation between C98 and C475 of TLR7 or their corresponding positions.
Further enabled herein is a method for treating a subject for autoimmune disease, viral or microbial pathogenesis, inflammation or cancer, the method comprising contacting a cell from the subject expressing TLR7 with an effective amount of an agent which antagonizes disulfide bond formation between C98 and C475 of TLR7 or their corresponding positions.
Also enabled herein is a method for treating a subject for excessive production of reactive oxygen species, the method comprising contacting a cell from the subject expressing TLR7 with an effective amount of an agent which antagonizes disulfide bond formation between C98 and C475 of TLR7 or their corresponding positions.
Enabled herein is a method for treating a subject for TLR7-mediated inflammation, the method comprising contacting a cell from the subject expressing TLR7 with an effective amount of an agent which antagonizes disulfide bond formation between C98 and C475 of TLR7 or their corresponding positions.
Further enabled herein is a method for treating a subject for an autoimmune condition, the method comprising contacting a cell from the subject expressing TLR7 with an effective amount of an agent which antagonizes disulfide bond formation between C98 and C475 of TLR7 or their corresponding positions.
Still further enabled herein is a method for treating a subject for cancer, the method comprising contacting a cell from the subject expressing TLR7 with an effective amount of an agent which antagonizes disulfide bond formation between C98 and C475 of TLR7 or their corresponding positions.
Reference to TLR7 includes any TLR7 from any species. Generally, the TLR7 referred to is the TLR7 from the subject being treated. Hence, where the subject is a human, the antagonist would be for human TLR7 expressed by the cell. The amino acid sequence of human TLR7 is set forth in SEQ ID NO:20. Comparisons of amino acid sequence of TLR7 form different species is set forth in
It is proposed herein to use an antagonist of this disulfide bond forming pair. The agent may be proteinaceous or non-proteinaceous. In an embodiment, a peptide decoy is proposed to form a disulfide bond with C475 of TLR7 which prevents a C98-C475 disulfide bond forming. In an embodiment, the agent is a chemical mimetic of the peptide decoy.
Examples of microbial pathogens include: Helicobacter pyloris, Borrelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (such as. M. tuberculosis, M. avium, M. intracellulare, M. kansasii and M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidum, Treponema pertenue, Leptospira and Actinomyces israelii. Examples of pathogenic fungi include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis and Candida albicans.
Examples of viral pathogens include: Retroviridae (including but not limited to human immunodeficiency virus (HIV)); Picornaviridae (for example, polio viruses, hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (such as strains that cause gastroenteritis); Togaviridae (for example, equine encephalitis viruses, rubella viruses); Flaviviridae (for example, dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (for example, coronaviruses); Rhabdoviridae (for example, vesicular stomatitis viruses, rabies viruses); Filoviridae (for example, ebola viruses); Paramyxoviridae (for example, parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (for example, influenza viruses); Bunyaviridae (for example, Hantaan viruses, bunya viruses, phleboviruses, and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g. reoviruses, orbiviurses, and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adeno viruses); Herpesviridae (herpes simplex virus (HSV) 1 and HSV-2, varicella zoster virus, cytomegalovirus (CMV)); Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (such as African swine fever virus); and unclassified viruses (for example, the etiological agents of spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class 1=internally transmitted; class 2=parenterally transmitted (i.e. Hepatitis C); Norwalk and related viruses, and astro viruses).
Cancers or tumors include acute lymphoblastic leukemia, B cell lymphoma, glioma, bladder cancer, biliary cancer, breast cancer, cervical carcinoma, colon carcinoma, colorectal cancer, choriocarcinoma, epithelial cell cancer, gastric cancer, hepatocellular cancer, Hodgkin's lymphoma, lung cancer, lymphoid cell-derived leukemia, melanoma, myeloma, non-small cell lung carcinoma, nasopharyngeal cancer, ovarian cancer, prostate cancer, pancreatic cancer, renal cancer, squamous cell cancers of cervix and esophagus, testicular cancer, T-cell leukemia and melanoma.
Autoimmune disorders include systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, Sjogren's syndrome, polymyositis, vasculitis, Wegener's granulomatosis, sarcoidosis, ankylosing spondylitis, Reiter's syndrome, psoriatic arthritis and Behcet's syndrome. In an embodiment, the autoimmune disorder is an immune complex associated disease. Immune complex associated diseases specifically include, without limitation, systemic lupus erythematosus, rheumatoid arthritis, polyarteritis nodosa, poststreptococcal glomerulonephritis, cryoglobulinemia, and acute and chronic serum sickness.
Examples of inflammatory disease conditions contemplated by the present invention include but are not limited to those diseases and disorders which result in a response of redness, swelling, pain, and a feeling of heat in certain areas that is meant to protect tissues affected by injury or disease. Inflammatory diseases which can be treated using the methods of the present invention, include, without being limited to, acne, angina, arthritis, asthma, aspiration pneumonia disease, chronic obstructive pulmonary disease (COPD), colitis, empyema, gastroenteritis, intestinal flu, necrotizing enterocolitis, pelvic inflammatory disease, pharyngitis, pleurisy, raw throat, rubor, sore throat, urinary tract infections, chronic inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyradiculoneuropathy.
The peptide decoy comprises from 4 to 190 amino acids in length and comprises, in an embodiment, the amino acid sequence RCNC (SEQ ID NO:45) corresponding to R97 to C100 of TLR7. This sequence is conserved across species. Critical is C98 in TLR7 or its equivalent or corresponding position in a TLR7 homolog or variant. In an embodiment, the peptide decoy comprises:
(a) from 4 to 190 amino acids in length;
(b) the amino acid sequence RCNC (SEQ ID NO:45) corresponds to R97 to C100 of a TLR7; and
(c) a cysteine at a position corresponding to C98 of TLR7.
Amino acids may be substituted for conformationally or functionally equivalent or similar amino acids except for the cysteine corresponding to C98.
In an embodiment, the agent comprises a peptide, referred to herein as a “decoy peptide” of from 4 to 190 amino acids in length and having an amino acid sequence with at least 70% amino acid sequence similarity to up to 190 contiguous amino acids between amino acids 4 and 194 of TLR7 which includes a peptide having an amino acid sequence with at least 70% amino acid sequence similarity to up to 100 contiguous amino acids between amino acids 48 and 148 of TLR7 and which also includes a peptide having an amino acid sequence with at least 70% amino acid sequence similarity to up to 40 contiguous amino acids between amino acids 78 and 118 of TLR7 with the proviso that the peptide comprises a cysteine residue at the equivalent of position 98 of TLR7 (C98). The peptide may be from 4 to 190 amino acids in length.
The expression “4 to 190” in terms of length of the decoy peptide includes 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189 and 190 amino acids in length. The region between amino acids 48 and 148 includes positions 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147 and 148. The region between 78 to 118 includes 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117 and 118. The expression “at least 70%” in relation to percentage similarity includes 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%.
The term “similarity” as used herein includes exact identity between compared sequences at the amino acid level. Where there is non-identity at the amino acid level, “similarity” includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, nucleotide and sequence comparisons are made at the level of identity rather than similarity.
Terms used to describe sequence relationships between two or more polypeptides include “reference sequence”, “comparison window”, “sequence similarity”, “sequence identity”, “percentage of sequence similarity”, “percentage of sequence identity”, “substantially similar” and “substantial identity”. A “reference sequence” is at least 4 or above, inclusive of amino acid residues, in length. Because two polypeptides may each comprise: (1) a sequence (i.e. only a portion of the complete TLR7 amino acid sequence) that is similar between the two polypeptides; and (2) an amino acid sequence that is divergent between the two polypeptides, sequence comparisons between two (or more) polypeptides are typically performed by comparing sequences over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of typically 4 contiguous amino acid residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al. (1997) Nucl. Acids. Res. 25:3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. (In: Current Protocols in Molecular Biology, John Wiley & Sons Inc. 1994-1998. Comparisons of TLR7 amino acid sequence are presented in
The terms “sequence similarity” and “sequence identity” as used herein refers to the extent that sequences are identical or functionally or structurally similar on an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity”, for example, is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e. the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, “sequence identity” will be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, Calif., USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity.
In an embodiment, the percentage similarity to the amino acid sequence between amino acids 4 to 194 of human TLR7 is at least about 80% or 85% or 90% or 95% or 99%. This also applies to the percentage similarity between amino acids 48 and 148 and between 78 and 118 of human TLR7.
The agent includes, therefore, a peptide or peptide decoy as well as a non-proteinaceous chemical agent which antagonizes disulfide bond formation between C98 and C475. A non-proteinaceous chemical agent includes a chemical mimetic of C98i. A peptide decoy comprises a cysteine at the equivalent of C98. A peptide decoy may also comprise the amino acid sequence RCNC (SEQ ID NO:45) corresponding to R97 to C100 of human TLR7.
In an embodiment, the peptide decoy comprises 10 amino acids having the amino acid sequence DX1RCNCX2PX3X4 (SEQ ID NO:27) wherein:
X1 is L, F or M;
X2 is V or I;
X3 is V or I or A or P; and
X4 is P or L or K or R,
or an amino acid sequence having at least about 70% similarity to SEQ ID NO:27 after optimal alignment which includes at least about 80%, 85%, 90%, 95% or 99% similarity to SEQ ID NO:27. The above sequence corresponds to amino acid positions 95 to 104 of human TLR or its equivalent. Human TLR7 comprises a P at position 104. In murine TLR7, the amino acid L is at position 104. Both have a D at position 95.
In an embodiment, the peptide decoy comprises from 4 to 40 amino acids selected from between amino acids 78 to 118 of human TLR7 with the proviso that either the peptide comprises a cysteine residue at a position corresponding to C98 of human TLR7 or the peptide comprises the amino acid sequence RCNC (SEQ ID NO:45) at positions corresponding to R97 to C100 of human TLR7.
In yet another embodiment, the peptide decoy comprises the amino acid sequence: DFRCNCVPIP (SEQ ID NO:26) which corresponds to D95 to P104 of human TLR7 or an amino acid sequence having at least 70% similarity to SEQ ID NO:20 after optimal alignment with the proviso that the peptide comprises a cysteine at a position corresponding to C98 of TLR7. In a particular embodiment, the peptide decoy comprises the amino acid sequence set forth in SEQ ID NO:20. The corresponding murine TLR7 decoy peptide sequence is DLRCNCVPVL (SEQ ID NO:1) or having 70% similarity to SEQ ID NO:1 after optimal alignment with the proviso that the peptide comprises a cysteine at a position corresponding to C98 of TLR7.
One or more amino acids may be substituted by one or more amino acid analogs or one or more side chains may be modified. Such modifications can improve serum half life and improve stability.
Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH4.
The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.
Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulfides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
Crosslinkers can be used, for example, to stabilize 3D conformations, using homo-bifunctional crosslinkers such as the bifunctional imido esters having (CH2)n spacer groups with n=1 to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformationally constrained by, for example, incorporation of Ca and N a-methylamino acids, introduction of double bonds between Cα and Cβ atoms of amino acids and the formation of cyclic peptides or analogs by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
Mimetics are another useful group of agents to test for an ability to antagonise TLR7 activation via the C98-C475 disulfide bond. The term is intended to refer to a substance which has some chemical similarity to the decoy peptide it mimics but which antagonizes its interaction with a target (i.e. C475 of human (or murine) TLR7). A peptide mimetic may be a peptide-containing molecule that mimics elements of protein secondary structure (Johnson et al. (1993) Peptide Turn Mimetics in Biotechnology and Pharmacy, Pezzuto et al, Eds., Chapman and Hall, New York). The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions with C475 of TLR7. A peptide mimetic, therefore, is designed to permit molecular interactions similar to the decoy peptide. A chemical mimetic is also contemplated herein which is non-proteinaceous yet has the same effect and/or some conformation as C98i.
The peptide decoy may further comprise a moiety to facilitate peptide uptake by a cell. Any number or type of cell uptake moiety may be employed such as but not limited to TAT (SEQ ID NO:2); SynB1 (SEQ ID NO:3), SynB3 (SEQ ID NO:4), PTD-4 (SEQ ID NO:5), AD-5 (SEQ ID NO:6), FHV Coat (SEQ ID NO:7), BMV Gag (7-25) (SEQ ID NO:8), HTLV-III Rex-(4-16) (SEQ ID NO:9), D-Tat (SEQ ID NO:10) and R9-Tat (SEQ ID NO:11). Such moieties are referred to as hydrophilic or cationic peptides. Other uptake peptides include amphiphilic (or amphipathic) peptides such as but not limited to Transportan chimera (SEQ ID NO:12), MAP (SEQ ID NO:13), SBP (SEQ ID NO:14), FBPC (SEQ ID NO:15), MPG [MPGac] (SEQ ID NO:16), MPG (ΔNLS) (SEQ ID NO:17), Pep-1 (SEQ ID NO:18) and Pep-2 (SEQ ID NO:19). In yet another alternative, the uptake moiety comprises a periodic amino acid sequence such as comprising a polyarginine or a polylysine. Other uptake peptides are those listed in Table 1 of Guidotti et al. (2017) supra, the contents of which are incorporated herein by reference.
Whilst the subject to be treated includes a human by targeting hum TLR7, the present invention has a veterinary application such as the treatment of livestock animals (e.g. cows, sheep, pigs, goats, horses), domestic pets (e.g. cats, dogs) as well as captive wild animals and laboratory test animals (e.g. mice, rats, guinea pigs, hamsters, rabbits). In an embodiment, the subject is a human.
Accordingly, enabled herein is method for inhibiting TLR7-mediated immunostimulatory activity in a human subject, the method comprising contacting a cell from the subject expressing TLR7 with an effective amount of an agent which antagonizes disulfide bond formation between C98 and C475 of TLR7 or their corresponding positions.
Further enabled herein is a method for treating a human subject for autoimmune disease, viral or microbial pathogenesis, inflammation or cancer, the method comprising contacting a cell from the subject expressing TLR7 with an effective amount of an agent which antagonizes disulfide bond formation between C98 and C475 of TLR7 or their corresponding positions.
Also enabled herein is a method for treating a human subject for excessive production of reactive oxygen species, the method comprising contacting a cell from the subject expressing TLR7 with an effective amount of an agent which antagonizes disulfide bond formation between C98 and C475 of TLR7 or their corresponding positions.
Enabled herein is a method for treating a human subject for TLR7-mediated inflammation, the method comprising contacting a cell from the subject expressing TLR7 with an effective amount of an agent which antagonizes disulfide bond formation between C98 and C475 of TLR7 or their corresponding positions.
Further enabled herein is a method for treating a human subject for an autoimmune condition, the method comprising contacting a cell from the subject expressing TLR7 with an effective amount of an agent which antagonizes disulfide bond formation between C98 and C475 of TLR7 or their corresponding positions.
Still further enabled herein is a method for treating a human subject for cancer, the method comprising contacting a cell from the subject expressing TLR7 with an effective amount of an agent which antagonizes disulfide bond formation between C98 and C475 of TLR7 or their corresponding positions.
When the agent is a peptide decoy, the amino acid sequence is generally derived from a TLR7 of the same species being treated (e.g. human TLR7 to treat a human subject). This is referred to an autologous treatment. However, wherein there is substantial amino acid sequence similarity between a TLR of some species for use in another species, heterologous treatment is contemplated and encompassed by the present invention.
Further taught herein is use of an agent which antagonizes disulfide bond formation between C98 and C475 of TLR7 or their corresponding positions in the manufacture of a medicament to inhibit autoimmune disease, viral or microbial pathogenesis, inflammation or cancer in a subject.
Enabled herein is an agent which antagonizes disulfide bond formation between C98 and C475 of TLR7 or their corresponding positions for use in inhibiting autoimmune disease, viral or microbial pathogenesis, inflammation or cancer in a subject.
Still further enabled herein is a pharmaceutical composition pharmaceutical composition comprising an agent which antagonizes disulfide bond formation between C98 and C475 of TLR7 or their corresponding positions and one or more pharmaceutical carriers, excipients and/or diluents.
Also enabled herein is the use and agent for treating a subject for excessive production of reactive oxygen species or TLR7-mediated inflammation.
The agent includes a pharmaceutically acceptable salt of the agent. The term “pharmaceutically acceptable salt” refers to physiologically and pharmaceutically acceptable salts of the agents of the present invention: i.e. salts that retain the desired biological activity of the parent agent and do not impart undesired toxicological effects thereto.
The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g. by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g. intrathecal or intraventricular, administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
Formulations of the present invention include liposomal formulations. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered.
The formulation of therapeutic compositions and their subsequent administration (dosing) is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 μg to 100 g of peptide decoy per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the agent is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight, over days, weeks or months.
The cells targeted are generally innate and adapted immune cells or any cell which expresses a TLR7. Examples include phagocytic cells (e.g. macrophages, neutrophils and dendritic cells), NK cells, mast cells, eosinophils, basophils, lymphocytes include B- and T-lymphocytes and epithelial cells.
Aspects disclosed herein are further described by the following non-limiting Examples.
The influenza A virus (IAV) vaccine strains HKx31 (H3N2) and BJx109 (H3N2) were provided by the School of Medicine, Deakin University and the Department of Immunology and Microbiology, University of Melbourne, The Peter Doherty Institute for Infection and Immunity. Virus provided at 6.7×108 plaque-forming units/ml (PFU/ml) and stored at −80° C. Aliquots were thawed and diluted on the day of use with phosphate buffered aline (PBS; Sigma Aldrich, St Louis, USA (at No. D837) when required for in vitro infections). Human IAV virus, including seasonal H3N2 (A/New York/55/2004, A/Brisbane/9/2007), seasonal H1N1 (A/Brazil/11/1978, A/New Caledonia/20/1999, A/Solomon Islands/3/2006), A(H1N1)pdm09 strains (A/California/7/2009, A/Auckland/1/2009), rhinovirus (RV16 strain), respiratory synctitial virus (strain A2), human parainfluenza virus type-3 (C243), human metapneumovirus (strain CAN97-83), mumps virus (strain Enders) and Newcastle disease virus (strain V4) were provided by the Department of Immunology and Microbiology, University of Melbourne, The Peter Doherty Institute for Infection and Immunity. Additional viruses were provided by the following Institutions: dengue virus serotype 2 (Vietnam 2005 isolate, Monash University, Clayton, Victoria); rotavirus (Rhesus and UK strains, Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity); sendai virus (Cantell strain, Hudson Institute of Medical Research, Monash University), herpes simplex virus type-2 (strain 186; Hudson Institute of Medical Research, Monash University), vaccinia virus (Western Reserve strain, WR NIH-TC; Australia National University) and HIV (NL4-3(AD8)-EGFP strain, The Peter Doherty Institute for Infection and Immunity, The University of Melbourne). The viruses were provided in phosphate buffered saline (PBS, Cat #D8537, Sigma, USA) and stored at −80° C. until used. On the day of use, virus was thawed quickly and incubated at 37° C. prior to infection. Where indicated, HKx31 virus was inactivated by heat (56° C.) for 30 min or UV light (30 min).
Streptococcus pneumoniae EF3030 (capsular type 19F) was used as the parent S. pneumoniae strain in all experiments (provided by University of Melbourne, Australia). Strain EF3030 is a clinical isolate that is frequently used as a model of human carriage as it typically colonizes the nasopharynx in the absence of bacteremia. For infection experiments, pneumococci were grown statically at 37° C. in Todd-Hewitt broth, supplemented with 0.5% w/v yeast extract, to an optical density (600 nm) of 0.4-0.45. Cultures were placed on wet ice for 5 min and frozen in 8% v/v glycerol at −70° C. Live bacterial counts were confirmed prior to each experiment. A defined strain of non-typeable Haemophilus influenzae (NTHi; MU/MMC-1) was previously typed and sequenced and demonstrated to be NTHi, as we have previously shown (King et al. (2013) The Journal of Allergy and Clinical Immunology 131(5):1314-1321 e1314).
The following custom peptides were purchased from GenicBio Limited: YGRKKRRQRRRDLRCNCVPVL-NH2 (SEQ ID NO:57) (C98i-TAT; 10 amino acid TLR7 inhibitor). YGRKKRRQRRRCLVPNDCRLV-NH2 (SEQ ID NO:44) (Scrambled C98i-TAT; 10 amino acid TLR7 inhibitor). DLRCNCVPVL-NH2 (SEQ ID NO:1) (C98i-noTAT; 10 amino acid TLR7 inhibitor excluding HIV-TAT). DFRCNCVPIP-NH2 (SEQ ID NO:26). (Human C98i-noTAT; 10 amino acid TLR7 inhibitor excluding HIV-TAT). RCNC-NH2 (SEQ ID NO:45) (4AA C98i-noTAT; 4 amino acid TLR7 inhibitor excluding HIV-TAT). RANC-NH2 (SEQ ID NO:46) (4AA 98M C98i-noTAT; Cysteine 98 mutation, 4 amino acid TLR7 inhibitor excluding HIV-TAT). RANA-NH2 (SEq ID NO:47) (4AA 98100M C98i-noTAT; Cysteine 98 and 100 mutation, 4 amino acid TLR7 inhibitor excluding HIV-TAT). RCNA-NH2 (SEQ ID NO:48) (4AA 100M C98i-noTAT; Cysteine 100 mutation, 4 amino acid TLR7 inhibitor excluding HIV-TAT). All peptides were dissolved in endotoxin free water and prepared as stock solutions of 10 mM in aliquots of 20 μL, 504 and 1004 and stored at −20° C.
Preparation of gp91 ds-tat (YGRKK-RRQRR-RCSTR-IRRQL-NH2—SEQ ID NO:23) was carried out by standard Fmoc solid-phase peptide synthesis (SPPS) on Fmoc-PAL-PEG-PS resin (Life Technologies, USA, loading 0.17 mmol/g). Fmoc deprotection reactions were carried out using 20% v/v piperidine in N,Ndimethylformamide (DMF). Coupling reactions were carried out using Fmoc-protected amino acids with 013 (6-chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HCTU) as coupling agent and N,N-diisopropylethylamine (DIPEA) as activating agent. Reactions were monitored using the 2,4,6-trinitrobenzenesulfonic acid (TNBS) test to indicate the absence or presence of free amino groups. The alternating sequence of deprotection and coupling reactions was carried out manually for all 20 amino acid residues using the appropriate Fmoc- and side-chain protected amino acids. After a final deprotection step, a small portion of the peptide was cleaved from resin using trifluoroacaetic acid (TFA)/triisopropylsilane (TIPS)/1,2-ethanedithiol (EDT)/water (92.5:2.5:2.5:2.5) for 4 h, during which time the side-chain protecting groups were simultaneously removed. The crude peptide was then purified by reverse-phase high-pressure liquid chromatography (HPLC) using a Phenomenex Luna 5 C8 (2) 100 Å AXIA column (10 Å, 250×21.2 mm) with 0.1% TFA/water and 0.1% v/v TFA/ACN as the buffer solutions. The purified gp91 ds-tat peptide was confirmed as having the correct molecular weight by ESI-MS analysis: calcd. for C109H207N52O25S [M+5H+] m/z 535.3, obs. m/z 535.7; calcd. for C109H208N52O25S [M+6H+] m/z 446.3, obs. m/z 446.6; calcd. for C109H209N52O25S [M+7H+] m/z 382.7, obs. m/z 382.9.
Preparation of cholestanol-conjugated gp91 ds-tat (cgp91 ds-tat; Ac-Asp(OChol)-PEG4-PEG3-PEG4-gp91-NH2) was carried out by manual SPPS from resin-bound gp91 ds-tat (as described above), using Fmoc-PEG4-OH, Fmoc-PEG3-OH, Fmoc-PEG4-OH and Fmoc-Asp(OChol)-OH as the amino acids. After the final deprotection step, the N-terminus was capped using a mixture of acetic anhydride and DIPEA in DMF and the peptide construct was cleaved from resin using TFA/TIPS/EDT/water (92.5:2.5:2.5:2.5). The crude peptide was purified as described previously to give cgp91 ds-tat: calcd. for C173H319N56O43S [M+5H+] m/z 780.3, obs. m/z 780.6; calcd. for C173H320N56O43S [M+6H+] m/z 650.4, obs. m/z 650.7; calcd. for C173H321H56O43S [M+7H+] m/z 557.6, obs. m/z 558.0.
Preparation of ethyl ester-conjugated gp91 ds-tat (egp91 ds-tat; Ac-Asp(OEt)-PEG4-PEG3-PEG4-gp91-NH2) was carried out in the same way as for cgp91 ds-tat, except for replacement of Fmoc-Asp(OChol)-OH with Fmoc-Asp(OEt)-OH in the final coupling step: calcd. for C148H277N56O43S [M 5H+] m/z 711.8, obs. m/z 712.1; calcd. for C148H278N56O43S [M 6H+] m/z 593.3, obs. m/z 593.7; calcd. for C148H279N56O43S [M+7H+] m/z 508.7, obs. m/z 509.0.
Preparation of the 18 amino acid scrambled gp91 ds-tat (Sgp91 ds-tat; Ac-Asp(OChol)-PEG4-PEG3-PEG4-RKK-RRQRR-RCLRI-TRQSR-NH2— SEQ ID NO:24) peptide was carried out by manual SPPS as described above for unscrambled gp91 ds-tat. The resin-bound sgp91 ds-tat was then conjugated to cholestanol via a PEG linker using the same method described above for unscrambled cgp91 ds-tat. The crude peptide was purified in the same way to give cgp91 ds-tat: calcd. for C162H307N54O40S [M+5H+] m/z 736.3, obs. m/z 736.5; calcd. for C162H308N54O40S [M+6H+] m/z 613.7, obs. m/z 614.0; calcd. for C162H309N54O40S [M+7H+] m/z 526.2, obs. m/z 526.3.
In Vivo Infection with Influenza a Virus and Drug Treatments
Aged matched (6-12 weeks) littermate male naïve WT control and NOX2−/y mice (also known as gp91phox−/− [Pollock et al. (1995) Nature Genetics 9(2):202-209]) were anaesthetized by penthrane inhalation and infected intranasally (i.n.) 1×104 or 1×105 plaque forming units (PFU) of Hkx31 in a 35 μL volume, diluted in PBS. Mice were euthanized at Day 1, 3 or 7 following influenza infections. In some experiments, anaesthetized mice were treated via intranasal delivery with either dimethyl sulfoxide (DMSO, control; Sigma), unconjugated gp91dstat (0.02 mg/kg, 0.2 mg/kg), cholestenol conjugated-gp91dstat (0.02 mg/kg, 0.2 mg/kg) or cholestanol conjugated-scrambled gp91ds-TAT (0.02 mg/kg) one day prior to infection with Hk-X31 and everyday thereafter for 3 days. In additional experiments, anaesthetized mice were treated with imiquimod (50 μg/mouse, i.n.) or catalase (1000 U/mouse, i.n.) and then euthanized for analysis at Day 1.
Mice were killed by an intraperitoneal (i.p) injection of ketamine/xylazene (100 mg/kg) mixture. An incision was made from the lower jaw to the top of the rib cage, where the salivary glands were separated to expose the surface of the trachea. The layer of smooth muscle on the trachea was removed, allowing a small incision to be made near the top of the trachea. A sheathed 21-gauge needle was inserted to the lumen and 300-400 μl of PBS was lavaged repeatedly (4 times). The total number cells in the BALF were stained with 0.4% w/v trypan blue solution (Thermofisher Scientific, USA) and viable cells were evaluated using the Countess (Registered Trademark) automated cell counter (Invitrogen, Cat #C10227). Differential cell analysis was prepared from BALF (5×104 cells) that were centrifuged at 3×g for 5 min on the Cytospin 3 (Shandon, UK). Following this, slides were fixed in 100% v/v propanol for one minute and allowed to dry overnight. Finally, samples were stained with Rapid I Aqueous Red Stain™ (AMBER Scientific, Australia) and Rapid II Blue Stain™ (AMBER Scientific, Australia) for 10 mins, then submerged in 70% v/v ethanol and absolute ethanol twice before being placed into xylene for 5 min (2 times). Samples were then mounted in DPX mounting medium (Labchem, NSW, Australia) and coverslips were firmly placed on top. 500 cells per sample from random fields were differentiated into macrophages, neutrophils, eosinophils and lymphocytes by standard morphological criteria. Data are represented as total cell numbers that was calculated by the respective cell type multiplied by the total live cell numbers and as a percentage of the cell population.
Human alveolar macrophages were obtained from subjects undergoing a bronchoscopy at Monash Medical Centre, Monash University, Clayton, Australia, to investigate underlying lung disease. The bronchoscope was wedged in the right middle lobe and 25-50 mL of saline was washed into the airway then aspirated. Cells were washed twice with PBS before being suspended in culture medium (Roswell Park Memorial Institute (RPMI, Life Technologies, Cat #21870-076) with 10% v/v FCS with 100 U/mL penicillin and 100 μg/mL streptomycin) for ˜24 h before use.
Alveolar macrophages were isolated by lung lavage from age-matched (6-12 weeks) male C57Bl/6J (WT), NOX2−/y, NOX4−/− (provided by Centre for Eye Research, The University of Melbourne, Australia, TLR7−/− (provided by the School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, The University of Newcastle, and Hunter Medical research Institute, New South Wales, Australia) TLR9−/− (provided by the Baker IDI Heart & Diabetes Institute, Melbourne, Victoria, Australia) or SOD3−/− mice (provided by the School of Health and Biomedical Sciences, RMIT University). A thin shallow midline incision from the lower jaw to the top of the rib cage was made and the larynx was separated to expose the top of the trachea. The layer of smooth muscle covering the trachea was removed, a small incision made and a sheathed 21-gauge needle was inserted into the lumen. The lungs were repeatedly (3 times) lavaged with 300-400 μL of PBS. Cell suspensions were spun down by centrifugation (200×g at 4° C. for 5 min). Supernatant was removed, then cells were re-suspended in 1 mL of sterile PBS and counted using the Countess (Registered Trademark) automated cell counter (Invitrogen, Cat #C10227). Cells were then seeded into 24-well plates (1×105 cells/well) for immunocytochemistry and fluorescence microscopy, as stated below.
The immortalized cell line RAW 264.7 cells (RAW 264.7 (ATCC (Registered Trademark) TIB-71 (Trademark)) and immortalized bone marrow-derived macrophages (BMDMs; courtesy of the Hudson Institute of Medical Research Monash University and the Institute of Innate Immunity, University of Bonn, Germany) were maintained in Dulbecco's Modified Eagle's Medium (DMEM: Sigma) supplemented with L-glutamine, glucose (4500 mg/L), sodium pyruvate (110 mg/L) and fetal bovine serum (FBS; 10% v/v. The TLR2−/−, TLR3−/−, TLR4−/−, TLR7−/−, MyD88−/−, NLRP3−/− and UNC93B1−/− immortalized BMDMs were maintained in RPMI medium supplemented with glucose (4500 mg/L), non essential amino acids, sodium pyruvate, streptomycin and FBS (10% v/v) and DMEM (20% v/v) (containing all supplements, as stated above). All cells were kept at 37° C. with a humidified mixture of 5% CO2 and 95% v/v air. The medium was changed two to three times a week, cells were sub-cultured by scraping when ˜80-90% confluent, and counted using the Countess (Registered Trademark) automated cell counter.
The immortalized cell line RAW 264.7 cells (derived from mouse peritoneum) and immortalized bone marrow-derived macrophages (BMDMs) were maintained in Dulbecco's Modified Eagle's Medium (DMEM: Sigma) supplemented with L-glutamine, glucose (4500 mg/L), sodium pyruvate (110 mg/L) and fetal bovine serum (FBS; 10%). All cells were kept at 37° C. with a humidified mixture of 5% CO2 and 95% air. The medium was changed two to three times a week, cells were sub-cultured by scraping when ˜80-90% confluent, and counted using the Countess (Registered trademark) automated cell counter (Invitrogen).
Cells were seeded onto glass cover slips in 24-well plates, and allowed to adhere for 24 h in DMEM. Cells were then incubated in the absence or presence of HKx31 influenza A virus (MOI 0.1, 1 or 10) in serum 16 free medium at varying time points (5 min, 15 min, 30 min and 1 h). In some cases, cells were pretreated for 30 min prior to infection with Dynasore (100 μM) or the vehicle for Dynasore, DMSO (0.1% w/v). Next, the cells were washed with PBS (0.01 M) and fixed with 4% v/v paraformaldehyde (PFA) for 15 min. Cells were treated for 10 min with PBS-containing Triton X-100 (0.25% v/v) and then washed three times over 15 min with PBS. The samples were then incubated with 10% v/v goat serum-containing PBS for 2 h and/or mouse on mouse IgG blocking reagent (Cat #MKB-2213, Vector Laboratories). This was followed by the addition of a primary antibody for nucleoprotein (1:1000) to localize influenza A virus, purified mouse anti-NOX2 (1:500) to localize NOX2, rabbit anti-TLR7 (1:1000) to localize TLR7, or mouse anti-early endosome antigen 1 (EEA1; 1000) to localize early endosomes for 24 h at 4° C. In some experiments, combinations of antibodies were used at the indicated concentrations to determine protein co-localization. Cells were washed three times over 30 min with PBS (0.01 M). Following the washes, a secondary antibody goat anti-rabbit alexa 594 (1:1000), goat anti rabbit red 647 (1:500, 1:1000) and/or biotinylated anti-mouse IgG was added to appropriate wells in the dark for 2 h. Finally, the cells were washed three times over 30 min with PBS (0.01 M); and where appropriate (mouse primary and secondary anti Fluroscein Avidin DCS was applied for 5 minutes). Cover slips were mounted onto microscope slides with 10-20 μL of diamidino-2-phenylindole (DAPI) for 3 min. Slides were viewed and photographed on a Nikon upright inverted confocal fluorescence microscope (Nikon D-eclipse C1). All immunohistochemistry was assessed by two observers blinded as to the treatment groups throughout the analysis process and all of the appropriate controls were performed, in that all combinations of primary and secondary antibodies were used to ensure no cross reactivity occurred.
The specificity of both the TLR7 and NOX2 antibodies were verified by examining the degree of staining in alveolar macrophages taken from WT, TLR7−/− and NOX2−/− mice respectively. There was no staining for TLR7 in the TLR7−/− macrophages (
Human alveolar macrophages; WT, TLR7−/−, TLR2−/−, TLR3−/−, TLR4−/−, MyD88−/−, and NLRP3−/− BMDMs; mouse primary WT, NOX24Y, NOX4−/−, TLR7−/−, TLR9−/− or SOD3−/− alveolar macrophages and RAW264.7 cells were seeded (1×105 cells/well) onto glass coverslips in 24-well plates allowing the cells to adhere for 24 h in DMEM or RPMI medium before being pretreated with OxyBURST Green H2HFF (100 μM) and/or LysoTracker Deep Red (50 nM) for 5 min. This was followed by incubation with PBS (control group; 0.01 M), imiquimod (10 μg/mL), single stranded RNA (ssRNA; 100 μM), or infected with either H3N2 influenza viruses (A/New York/55/2004, A/Brisbane/9/2007), seasonal H1N1 influenza A viruses (A/Brazil/11/1978, A/New Caledonia/20/1999, A/Solomon Islands/3/2006), A(H1N1)pdm influenza A viruses (A/California/7/2009, A/Auckland/1/2009), or with re-assortant vaccine strains HKx31 (MOI 0.1-10) or BJx109 (MOI 10) in serum-free medium at varying time points (5 min, 15 min, 30 min and 1 hr). Other wells were infected with dengue virus (MOI 10), Sendai virus (40 HAU/mL), human parainfluenza virus (MOI 10), human metapneumovirus (MOI 10), rhinovirus (MOI 10), respiratory syncytial virus (MOI 10), HIV (MOI 10), Newcastle disease virus (MOI 10), mumps virus (MOI 10), rhesus or UK rotaviruses (each at MOI 10) or herpes simplex virus-2 (MOI 10) under similar conditions. In some cases, cells were pretreated with superoxide dismutase (SOD; 300 U/mL), apocynin (300 μM), gp91dstat (50 μM) or bafilomycin A (100 nM), for 30 min prior to infection. Next, the cells were washed with PBS (0.01 M) and fixed with 4% PFA for 15 min. After fixation, cells were then washed three times with PBS over 30 min. Cover slips were then mounted onto microscope slides with 10-20 μL of DAPI for 3 min, then analyzed and photographed on an Nikon upright confocal fluorescence microscope (Nikon D-eclipse C1).
To measure NOX2 oxidase activity we assessed p47phox and NOX2 assembly using confocal fluorescence microscopy. Control and HKx31 virus-infected WT and TLR7−/− alveolar macrophages were processed as indicated above under “confocal fluorescence microscopy”. In additional experiments, WT cells were treated with Dynasore (10004) or bafilomycin A (100 nM) for 30 min prior to virus infection. After exposing samples with 10% v/v goat serum-containing PBS for 2 hr, the rabbit anti-p47phox antibody (1:1000) and the mouse anti-NOX2 antibody (1:500) were added followed by addition of appropriate secondary antibodies, as specified above.
ROS production was quantified using L-012-enhanced chemiluminescence. RAW264.7 cells and primary mouse alveolar macrophages were seeded into a 96-well OptiView plate (5×104 cells/well). RAW264.7 cells were either treated with DMSO (control, appropriate concentration), unconjugated gp91dstat (100 nM-30 μM), cholestanol-conjugated gp91dstat (100 nM-30 μM) or ethyl-conjugated gp91dstat (100 nM-30 μM) for 1 h. BALF was collected from mice treated with DMSO (control), unconjugated gp91dstat (0.02 mg/kg, 0.2 mg/kg), cholestanol conjugated-gp91dstat (0.02 mg/kg, 0.2 mg/kg) and/or infected with Hkx31 influenza A virus (1×105 PFUs). Cells were then washed of media with 37° C. Krebs-HEPES buffer, then exposed to a Krebs-HEPES buffer containing L-012 (10-4 mol/L) in the absence (i.e. basal ROS production) or presence (stimulated ROS production) of the PKC and NADPH oxidase activator phorbol dibutyrate (PDB; 10-6 mol/L). The same treatments were performed in blank wells (i.e. with no cells), which served as controls for background luminescence. All treatment groups were performed in triplicates. Photon emission [relative light units (RLU)/s] was detected using the Chameleon (Trademark) luminescence detector (Hidex, model 425105, Finland) and recorded from each well for 1 s over 60 cycles. Individual data points for each group were derived from the average values of the three replicates minus the respective blank controls. Data are represented as a % of the control in the dose-response curves or as raw values (ex vivo experiments).
To test whether the unconjugated or cholestanol conjugated gp91dstat exhibited ROS scavenging properties, the xanthine oxidase cell free assay was used. Briefly, Krebs-HEPES buffer containing L-012 (100 μM) was added into a 96-well Optiview plate. Following this, 0.1% w/v DMSO, unconjugated gp91dstat (Ugp91ds-TAT, 1 μM) or cholestanol-conjugated gp91ds-TAT (1 μM) were added in combination with Xanthine (100 μM). Immediately after xanthine oxidase (0.03 U/mL) was added, photon emission [relative light units (RLU)/s] was detected using the Chameleon (Trademark) luminescence detector (Hidex, model 425105, Finland) and recorded from each well for 1 s over 60 cycles. Individual data points for each group were derived from the average values of the three replicates minus the respective blank controls. Data are represented as raw values.
HA-TLR7 cDNA was purchased from Sino Biological (mouse TLR7; Cat #MG50962-NY with Gene Bank Ref Seq number NM 133211.3). Mutation of the key cysteine residues in TLR7 (Cys260, Cys263, Cys270, Cys273, Cys98 and Cys445) to alanine was performed using the QuikChange Multi Site-Directed Mutagenesis kit (Cat #200514, Agilent Technologies). Sequences of WT and mutant HA-TLR7 were confirmed by the Australian Genome Research Facility. Cells were transfected using linear polyethyleneimine (PEI) [Halls et al. (2015) Methods in Molecular Biology 1335:131-161].
Cells were plated and transfected in suspension with 200 ng/well FRET biosensor DNA using PEI, in black, optically clear 96-well plates for 48 hr. Prior to the experiment, cells were partially serum-starved overnight in 0.5% v/v FBS media. Fluorescence imaging was performed using a high-content GE Healthcare INCell 2000 Analyzer with a Nikon Plan Fluor ELWD 40× (NA 0.6) objective and FRET module as described (Jensen et al. (2014) The Journal of Biological Chemistry 289(29):20283-20294). For CFP/YFP (CKAR) emission ratio analysis, cells were sequentially excited using a CFP filter (430/24) with emission measured using YFP (535/30) and CFP (470/24) filters, and a polychroic optimized for the CFP/YFP filter pair (Quad3). For GFP/RFP (EKAR) emission ratio analysis, cells were sequentially excited using a FITC filter (490/20) with emission measured using dsRed (605/52) and FITC (525/36) filters, and a polychroic optimized for the FITC/dsRed filter pair (Quad4). Cells were imaged every 100 sec for 20 min (image capture of 2 fields of view in 12 wells per 100 sec). Data were analyzed using in-house scripts written for the FIJI distribution of Image J 34, as described (Halls et al. (2015) supra.
Quantification of mRNA by QPCR
Cells were treated with imiquimod (10 μg/ml), poly I:C (100 ng/ml), CpG (10 μg/mL), ssRNA (500 μg/mL) or catalase (1000 U/ml) for 24 hours. Where indicated, cells were pre-treated with apocynin (300 μM), SOD (300 U/mL) or bafilomycin A (100 nM) for 30 mins. RNA was extracted from the lung tissue of mice that were treated with either DMSO (control), unconjugated gp91dstat (0.02 mg/kg, 0.2 mg/kg), cholestanol conjugated-gp91dstat (0.02 mg/kg, 0.2 mg/kg), scrambled cholestanol conjugated gp91dstat (0.02 mg/kg) and/or infected with Hk-X31 influenza A virus (1×105 PFUs) 3 days post infection for the assessment of viral mRNA and cytokine expression. The right lung lobe was placed in Eppendorf tubes containing a mixture of Buffer RLT (Qiagen, USA) and β-mercaptoethanol (Sigma; 1%), which was minced into small pieces using curved scissors. Following this, lung samples were homogenized using the ultrasound homogenizer (Hielscher Ultrasonics GmBH, Teltow, Germany) and centrifuged at 14,000 rpm for 5 mins. A 1:1 ratio of lysate was mixed with 70% v/v RNase free ethanol transferred to RNeasy spin columns (RNeasy Minikit; Cat #74104, Qiagen). Samples were spun at 10,000 rpm for 15 seconds and then washed with Buffer RW1. After discarding the flow-through, 5 μl of DNase I (Cat #79254, Qiagen) was mixed with 35 μl of Buffer RDD was pipetted directly onto the membrane of the spin column and incubated at room temperature for 15 mins. Buffer RPE was added and centrifuged for 10,000 rpm for 15 seconds. After discarding the flow-through, Buffer RPE was re-added and spun for 10,000 rpm for 2 mins. An additional spin at 14,000 rpm for 1 min was done to remove residual flow-through from the spin column. Finally, RNase free water was added and centrifuged to elute the RNA into an Eppendorf tube. RNA samples were measured using the Nanodrop 1000 Spectrophotometer (Thermo Scientific, USA). cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit (Cat #4368814, Applied Biosystems, Foster City, Calif., USA) using 1.0-2.0 μg total RNA. RNA was added to a mixture of reagents in the High-Capacity cDNA RT kit to make a final volume of 20 μl. This was transcribed using the BioRad Mycycler (Trademark) thermal cycler (BioRad, USA) at the following settings: 25° C. for 10 mins, 37° C. for 120 mins, 85° C. for 5 mins and 4° C. at rest. Samples were stored at −20° C. prior to use. Quantitative polymerase chain reaction was carried out using the TaqMan Universal PCR Master Mix (Cat #4304437, Applied Biosystems, Foster City, Calif., USA) or SYBR Green PCR Master Mix (Cat #4367659, Applied Biosystems, Foster City, Calif., USA) and analyzed on ABI Step One™ and StepOnePlus™ Real-time PCR Systems (Perkin-Elmer Applied Biosystems, Foster City, Calif., USA). The PCR primers for TNF-α, IL-1β, IFN-β and IL-6 were included in the Assayon-Demand Gene Expression Assay Mix. Additionally, a custom designed forward and reverse primer of the segment 3 polymerase (PA) of influenza virus was used to measure viral titres (Table 4). The PCR program run settings: 50° C. for 2 min, followed by 95° C. for 1 hr, then 95° C. for 15 s+60° C. for 60 s+plate read (40 cycles). Quantitative values 129 were obtained from the threshold cycle (Ct) number. Target gene expression level was normalized against 18s or GAPDH mRNA expression for each sample and data was expressed relative to the control.
RAW264.7 or BMDM cells were seeded into a 6-well plate (5×105 cells/well). For all iterations of the C98i peptides, cells were either pre-treated with Phosphate buffered saline (PBS; control, appropriate concentration) or the appropriate peptide for 1 hour. Cells were then treated with imiquimod (10 μg/ml) for an additional hour. After treatments, cells were washed with PBS, media was replenished with fresh DMEM (10% FBS) and left to incubate over 24 hours.
Total RNA was prepared using RNeasy Mini Kit (Qiagen, Hilden, Germany), and then total RNA was purified via extraction with double distilled H2O. Synthesis of cDNA was performed using the High-Capacity cDNA RT kit (P/N4322171, Applied Biosystems, Foster City, Calif., USA) using 1.0-3.0 μg total RNA. Quantitative polymerase chain reaction was carried out using the TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, Calif., USA) and analyzed on ABI StepOne™ and StepOnePlus™ Real-time PCR Systems (Perkin-Elmer Applied Biosystems, Foster City, Calif., USA). The PCR primers for IL-1 and IL-6 were included in the Assayon-Demand Gene Expression Assay Mix (Applied Biosystems, Foster City, Calif., USA). The PCR program run settings: 50.0 for 2 min, followed by 95.0 for 1 hr, then 95.0 for 15 s+60.0 for 60 s+plate read (40 cycles). Quantitative values were obtained from the threshold cycle (Ct) number. Target gene expression level was normalized against 18s or GAPDH mRNA expression for each sample and data was expressed relative to the WT naïve.
Protein levels of IFN-β (VeriKine HM mouse IFN β Serum ELISA kit; Cat #42410-1, PBL Assay Science), IL-1β (Quantikine ELISA Mouse IL-1β/IL-IF2; Cat #MLB00C, R & D Systems,), TNF-α (Quantikine ELISA Mouse TNF α, Cat #MTA00B, R & D Systems,) and IL-6 (Quantikine ELISA Mouse IL-6, Cat #M6000B, R & D Systems) secreted into the BALF of HKx31-infected (1×104 PFUs) wild-type and NOX2−/y mice were measured using ELISAs and performed using commercially available kits according to the manufacturer's instructions. The cytokine titres in samples were determined by plotting the optical densities using a 4-parameter fit for the standard curve.
Serum and BALF levels of various antibody isotypes (IgA, IgE, IgG1, Ig2a, IgG2b, IgG3, IgM and total IgG) were quantified in HKx31-infected (1×104 PFUs) WT and NOX2−/y mice using the ProcartaPlex Multiplex Immunoassay (Mouse Isotyping 7plex, Cat #EPX070-2815-901, eBioscience) according to the manufacturer's instructions. Briefly, antibody-conjugated magnetic beads were added into each well of a 96-well plate. Antibody standards were serially diluted (1:4) in universal assay buffer to construct a 7-point standard curve. Serum and BALF samples (diluted 1:20,000 in universal assay buffer) and/or standards were added to appropriate wells of the 96-well plate containing the antibody-conjugated magnetic beads. Following this, a detection antibody mix was added to each well and the plate was incubated for 30 min at room temperature on a microplate shaker (500 rpm) in the dark. After washing, a reading buffer was added to all wells. The plate was read by a Magpix (Registered Trademark) multiplex reader (Luminex, USA) with xPONENT (Registered Trademark) software (Luminex, USA). Procartaplex (Trademark) Analyst 1.0 software (eBioscience, USA) was used to interpolate serum and BALF antibody concentrations in each sample from the standard curve.
Data were represented as the mean±standard error of the mean (SEM). Cytokine mRNA expression and antibody titres were analyzed using one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. All tests were performed by Graphpad Prism 7.0b (San Diego, Calif., USA) and statistical significance was taken at P<0.05.
In order to quantify the fluorescence microscopy data, images acquired from confocal systems were analyzed in Image J. Approximately 100-150 cells per treatment group from at least three independent experiments were analyzed unless otherwise stated in the figure legend to calculate the fluorescence in each cell, which was then averaged and expressed as a percentage of the area fluorescence. All statistical tests were performed using GraphPad Prism (GraphPad Software Version 6.0, San Diego Calif., USA). P<0.05 was taken to indicate significance. For isolated cell culture work, n is representative of a separate experiment where cells were used from a different passage.
Imiquimod (Cat #tlrl-imq, Invivogen), H.M.W poly I:C (Cat #tlrl-pic, Invivogen) and CpG ODN (Cat #tlrl-1668, Invivogen) were dissolved in endotoxin-free water and prepared as stock solutions of 5-10 mg/mL in aliquots of 30 μL and 100 μL and stored at −20° C. ssRNA (Cat #tlrl-lma40, Invivogen) was dissolved in endotoxin-free water and prepared as a stock solution of 5 mM in aliquots of 50 μL and stored at −20° C. Dynasore (Cat #D7693, Sigma) (freshly prepared on the day) was dissolved in DMSO (100%) and prepared as 10 mM stock solutions. FBS (Cat #12003C, Sigma) was stored in 50 ml aliquots at −20° C. Penicillin-streptomycin solution (Cat #P4333, Sigma) was stored at −20° C. SOD (Cat #S2515, Sigma) was dissolved in distilled water and prepared as stock solutions (10 μl) of 30,000 units/ml and stored at −20° C. OxyBURST Green H2HFF bovine serum albumin (BSA)(Cat #1329, Molecular probes, Life Technologies) and LysoTracker Deep Red (Cat #L12492, Molecular probes, Life Technologies) in stock solutions (1 mg/mL) were generated immediately before use by dissolving in PBS. Bafilomycin A (from Streptomyces, Cat #B1793, Sigma) was prepared as a stock solution of 100 μM in aliquots of 10 μL and stored at −20° C. Apocynin (4′-Hydroxy-3′-methoxy acetophenone, Cat #A10809; Sigma) made freshly on the day of use and gp91dstat (Cat #AS-63818; Anaspec) were prepared as stock solutions of 100 mM and 50 mM respectively, in 100% v/v DMSO. Phorbol dibutyrate (Cat #P1239; Sigma) was dissolved in 100% v/v DMSO as 10 mM stocks and made fresh on the day of use. Catalase (Cat #C1345, Sigma) was prepared as stock solutions of 106 U/ml in distilled water and stored at −20° C. MitoSOX (Cat #M3600850; Molecular Probes, Life Technologies) was prepared at 5 mM by dissolving the contents (50 μg) of one vial of MitoSOX (Trademark) mitochondrial superoxide indicator (Component A) in 13 μL of DMSO. Xanthine oxidase (Cat #×1875; Sigma) was prepared fresh on the day by dissolving in distilled water to 30 U/ml and xanthine (Cat #×0626; Sigma) was prepared as a stock of 100 mM in 0.1M NaOH. ML171 (Cat #492002; Calbiochem).
Antibodies for influenza nucleoprotein (mAb to Influenza A Virus Nucleoprotein [AASH]; Cat #120-20343, AbCAM), early endosome antigen 1 (Cat #120-02900, AbCAM), mouse anti-gp91phox (Cat #611415, BD Transduction Laboratories, Purified Mouse Anti gp9l [phox] Clone 53/gp91[phox](RUO), rabbit anti-TLR7 (Cat #NBP2-24906, Novus Biologicals), rabbit anti-p47phox antibody (Cat #sc14015, Santa Cruz), FITC goat anti-mouse IgG (Cat #A-11029; Invitrogen), goat anti-rabbit alexa 594 (Cat #A-11037; Invitrogen), goat anti-rabbit far red 647 (Cat #A-21244, Invitrogen) and DAPI (Cat #H-1200, Vector Laboratories) were stored at −20° C.
To address the potential role of endosomal ROS production in virus pathology, influenza A viruses, which belong to the Group IV negative sense, ssRNA viruses of the Orthomyxoviridae family and are internalized by endocytosis were first considered. Exposure of mouse alveolar macrophages (AMs), mouse peritoneal RAW264.7 cells or bone marrow-derived macrophages (BMDMs) to influenza A virus strain HKx31 (H3N2) resulted in a dose and time-dependent increase in influenza nucleoprotein (NP) fluorescence, which was almost abolished by the dynamin inhibitor, Dynasore (100 μM) indicating a clathrin-coated pit or caveolin-dependent mechanism of internalization. Internalized virus displayed a strong co-location with the early endosomal marker EEA1 (
RNA viruses are recognized by endosomal TLR7 (for ssRNA viruses) [Lund et al. (2004) Proceedings of the National Academy of Sciences of the United States of America 101 (15):5598-5603, Diebold et al. Science 303(5663):1529-1531] and TLR3 (dsRNA viruses), as well as the cytosolic sensors retinoic acid inducible gene I (RIG-I) (which can detect viral RNA bearing 5′ triphosphates (Lund et al. (2004) supra) and NOD-like receptors (NLRs) [Iwasaki and Pillai (2014) supra; Ichinohe et al. (2009) The Journal of Experimental Medicine 206(1):79-87; Allen et al. (2009) Immunity 30(4):556-565]. It was hypothesized that influenza A virus entry into acidified endosomes results in the liberation of viral RNA, activation of TLR7 and stimulation of NOX2 oxidase-dependent ROS production. Consistent with this suggestion, TLR7 co-locates with influenza A virus (
How TLR7 elicits the assembly and activation of endosomal NOX2 oxidase was then examined. NOX2 oxidase can be activated by protein kinase C, which triggers robust phosphorylation of key serine residues on p47phox, resulting in a NOX2 oxidase-dependent oxidative burst (Drummond et al. (2011) supra). To define the spatiotemporal regulation of PKC signaling and to assess its regulation by TLR7, the FRET biosensor cytoCKAR was expressed to detect cytosolic PKC (Jensen et al. (2014) supra; Violin et al. (2003) The Journal of Cell Biology 161(5):899-909; Halls et al. (2015) supra) in WT and TLR7−/− macrophages. The treatment of WT macrophages with influenza A virus or imiquimod elevated cytosolic PKC activity within 5 min, but this response was absent in TLR7−/− macrophages and in WT macrophages treated with Dynasore or bafilomycin A (
Exposure of macrophages to rhinovirus (picornaviridae, Group IV), respiratory syncytial virus (paramyxoviridae, Group V), human parainfluenza virus (paramyxoviridae, Group V), human metapneumovirus (paramyxoviridae, Group V), Sendai virus (paramyxoviridae, Group V), Dengue virus (flavoviridae Group IV), or HIV (retroviridae, Group VI, ssRNA-RT virus) resulted in a significant elevation of endosomal ROS that was markedly suppressed in TLR7−/− macrophages, but unaffected in TLR9−/− cells (
Plasma membrane TLRs, especially TLR1, TLR2 and TLR4, and not those present within endosomes (such as TLR7), sense bacteria resulting in the recruitment of mitochondria to macrophage phagosomes and mitochondrial dependent ROS production (West et al. (2011) Nature 472(7344):476-480). However, the stimulation of endosomal TLRs failed to augment mitochondrial ROS (West et al. (2011) supra). TLR7 activation with imiquimod, which caused a significant elevation in endosomal ROS (
To establish the functional importance of endosomal ROS, the impact of NOX2 inhibition was assessed on the production of cytokines that are endosome TLR7-dependent and thus relevant to virus pathogenicity (Diebold et al. (2004) supra). An endosome- and TLR7-dependent signal was confirmed by showing that imiquimod caused a significant elevation in IFN-β, IL-1β, TNF-α and IL-6 expression in WT macrophages, but not in TLR7−/− macrophages (
It was sought to establish how endosomal NOX2 oxidase activity results in the suppression of TLR7-dependent responses and hypothesized that the parent species superoxide and its immediate downstream product, H2O2 are culprit mediators. Inactivation of superoxide by adding exogenous SOD (300 U/mL) failed to influence either basal or imiquimod-stimulated expression of IFN-β, IL-1β, TNF-α and IL-6, suggesting little role for superoxide itself in modulating TLR7 responses. To examine H2O2, catalase was utilized to inactivate the H2O2 generated within endosomes. Within 30 min, it was found that exposure to a FITC-labeled catalase resulted in co-localization with LysoTracker, confirming internalization into acidified endosomal compartments (
The question arose whether H2O2 released by endosomal NOX2 oxidase targets cysteine residues on protein domains of TLR7 that regulate receptor activity and are exposed upon activation within endosomal compartments (Kanno et al. (2013) International Immunology 25(7):413-422). These include Cys260, Cys263, Cys270 and Cys273 within the leucine repeat region as well as two additional cysteines, Cys98 and Cys445 that are unique to TLR7 (
The suppressive effect of endosomal NOX2 oxidase activity was examined on Type I IFN and IL-1β expression also occurs following influenza A virus infection. First, virus triggered translocation of the transcription factor, IRF-7, to the nucleus of WT BMDMs, but not TLR7−/− BMDMs, indicating that influenza A virus activates TLR7-dependent antiviral signaling in macrophages. Second, virus elevated IFN-β, IL-1β, IL-6, and TNF-α expression to a greater extent in NOX2−/y AMs (
An innovative molecular targeting system was synthesized, to deliver a specific NOX2 oxidase inhibitor (i.e. gp91ds-TAT) directly to endosomes, so as to disrupt the viral signaling platform by abrogating ROS production. To do this, a tripartite structure was generated comprising gp91ds-tat conjugated to the membrane anchor cholestanol via a PEG-linker at the N-terminal region of the peptide. Similar constructs have been shown previously to enhance endosome localization for inhibitors of the enzyme beta secretase (Rajendran et al. (2008) Science 320(5875):520-523). A Cy5 fluorophore conjugated to cholestanol using the same PEG linker resulted in cytosolic fluorescence in the peri-nuclear region and co-localization with EEA1, NOX2 and influenza virus NP following viral infection in a dynasore (100 μM)-sensitive manner providing evidence for endocytosis as its primary mode of cell entry (
It was examined whether Cgp91ds-TAT suppresses disease severity following influenza A virus infection in vivo. Daily intranasal administration of Cgp91ds-TAT (0.02 mg/kg/d) from 1 day prior, until day 3 post-influenza A virus infection resulted in a ˜40% reduction in airways inflammation (
Evidence is provided here that virus entry into endosomal compartments triggers a NOX2 oxidase-dependent production of ROS in endosomes. It is proposed here that the major contributor to endosomal concentrations of superoxide is superoxide generated directly in this compartment. Superoxide is the primary product of NOX2 and it will only be generated within the endosome compartment owing to the topology of the NOX2 and the unidirectional transfer of electrons through this catalytic subunit. In keeping with this, it is well regarded that superoxide does not travel far from its site of generation due to its negative charge. By contrast to superoxide, hydrogen peroxide has some capacity to permeate membranes and diffuse, and as such, it can be envisaged that some endosome H2O2 might have been generated elsewhere by NOX2 expressed in other sites of the cell such as the plasma membrane. There are several lines of evidence that indicate that it is very likely that little remotely generated H2O2 is finding its way into the endosome compartment. PKC activation following virus infection, which is critical for NOX2 activation, is significantly impaired if: 1) the virus is prevented from entering cells (
Here, it is demonstrated that endosomal ROS are essential negative regulators of a fundamental molecular mechanism of viral pathogenicity that impacts on antiviral immunity and the capacity of the host to fight and clear viral infections. Importantly, this effect is conserved, regardless of viral classification, for all viruses that enter cells via the endocytic pathway, and is TLR7 dependent. This provides a target for antiviral therapy for a range of viruses that cause significant morbidity and mortality worldwide.
A decoy peptide is generated comprising the D95 to L104 of murine TLR7 (DLRCNCVPVL—SEQ ID NO:1) operably linked to the HIV-TAT uptake peptide moiety (YGRKKRRQRRR—SEQ ID NO:2). The decoy peptide (referred to herein as C98i) prevents the disulfide bond forming between C98 and C475, thus preventing TLR7 activation.
Similar results were noted with TLR5 agonist response. C98i had no adverse effect on TLR7 expression or on cell viability.
The data show that C98i blocks TLR7 activity and does not influence TLR2, TLR4, TLR5, TLR9 activity. It is unlikely to influence the other members of the TLR family (i.e. TLR1, TLR3, TLR6 and TLR5) due to the uniqueness of the sequence. The findings that C98i does not influence the activity of TLR2, TLR4, TLR5 and TLR9 also suggests that the drug does not have non-specific properties on cell function that impact the production of the cytokine IL-1β.
TLR7 is a target that is involved in viral, autoimmune diseases and cancer. A novel drug targeting TLR7 has huge potential.
In the absence of TAT, the C98i peptide retains its ability to inhibit TLR7 agonist, imiquimod, responses in vitro. The absence of TAT is referred to as “no TAT”. The results are shown in
The amino acid sequence of C98i was scrambled to produce YGRKKRRQRRRCLVPNDCRLV-NH2 (SEQ ID NO:44). The scrambled C98i peptide sequence was not able to inhibit TLR7 agonist (imiquimod). The results are shown in
The Arg-Cys-Asn-Cys (RCNC) (SEQ ID NO:45) motif of the decoy peptide in C98i, was modified to form RANC (SEQ ID NO:46), RANA (SEQ ID NO:47) and RCNA (SEQ ID NO:48). These short peptides were tested with no TAT to ascertain their effects on TLR7 agonist, imiquimod.
Experimentation in vitro using isolated macrophages: Assays for examining oxidative stress and viral replication in the absence or presence of C98i. The following viruses are examined: low to high pathogenic Influenza A virus, respiratory synctitial virus, rhinovirus, Dengue virus.
It is expected that there is suppression of endosome oxidative stress and viral replication by C98i.
Experimentation in vivo: Delivery of C98i intranasally in mice, followed by Influenza A virus infection (via intranasal delivery) [To et al. (2017) Nature Communications 8(69):1-17]. The potential regulation of lung oxidative stress, inflammation, injury, viral burden and immune cell responses in lung and systemic circulation are assessed.
Wild-type C57BL/6 mice are treated with C98i and then treated with epicutaneous topical TLR7 agonist imiquimod (Aldara Cream) to the ear 3 times weekly. Following treatment, the mice are examined for serum autoantibody and creatinine levels as well as histopathology of the kidneys, spleens, livers, hearts and skin. Immunologic abnormalities are analyzed by immunohistochemistry, quantitative reverse transcription-polymerase chain reaction, and fluorescence-activated cell sorting.
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.
All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2017902545 | Jun 2017 | AU | national |
This application is a continuation of U.S. application Ser. No. 16/624,367, filed on Dec. 19, 2019, which is the National Stage Application of International Patent Application No. PCT/AU2018/050667, filed 29 Jun. 2018, which claims priority from Australian Provisional Patent Application No. 2017902545, filed on 30 Jun. 2017, entitled “A method of treatment”, the entire contents of each of which are incorporated herein by reference, in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16624367 | Dec 2019 | US |
| Child | 17703111 | US |
