Treatment method

Abstract

The invention provides a treatment method. Teaching herein are agents for inhibiting immunostimulation mediated by Toll-like receptors that are useful for the treatment of viral and microbial diseases, diseases involving autoimmune and inflammatory elements, and cancer. The agent antagonizes the formation of a disulfide bond between C98 and C475 of Toll-like receptor 7 (TLR7), thereby preventing activation of TLR7. Pharmaceutical compositions are also implemented herein.

Description

[0001] This application is a divisional application of patent application 2018800569343 entitled "A method of treatment" filed on June 29, 2018. This application claims priority to Australian provisional patent application No. 2017902545 entitled "A method of treatment" filed on June 30, 2017. Technical Field

[0002] The present invention relates generally to the field of immunomodulation. Agents for inhibiting immune stimulation mediated by Toll-like receptors are taught herein, which can be used to treat viral and microbial diseases, diseases involving autoimmune and inflammatory elements, and cancer. Pharmaceutical compositions are also realized herein. Background Art

[0003] Bibliographic details of publications cited by the authors in this description are collected alphabetically at the end of this description.

[0004] Reference in this specification to any prior publication (or information derived therefrom) or any known matter is not, and should not be regarded as, an admission or recognition or any form of implication that the prior publication (or information derived therefrom) or known matter constitutes part of the common general knowledge in the field of work involved in this specification.

[0005] Immune stimulation is a major component of protection against viral and microbial diseases. However, regulation of the immune system is complex, sensitive, and multifaceted. Unwanted stimulation can lead to autoimmune diseases. A more comprehensive understanding of the regulatory processes that control immune responses is needed if medical professionals are to meet the threat of impending viral epidemics and pandemics and manage autoimmunity. New therapeutic approaches that target pathologies are urgently needed, regardless of the infectious strain or autoimmune condition.

[0006] The generation of reactive oxygen species (ROS) is a highly coordinated process accomplished by enzymes of the NADPH oxidase (NOX) family. NOX enzymes evolved in unicellular eukaryotes 1.5 billion years ago and are present in most eukaryotic groups, including amoebas, fungi, algae and plants, nematodes, echinoderms, tunicates, 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 in eukaryotes are diverse, but a common function is the generation of ROS by innate immune cells in response to pathogens. Indeed, orthologs of NADPH oxidases in plants (ArtbohD and ArtbohF), fungi (NOXA / B), and invertebrate nematodes (Celegans) (Duox orthologs), fruit flies (Drosophila melongaster) (NOX5 orthologs, d-NOX and DUOX), and mosquitoes (Aedes aegypti) (NOXM and DUOX) produce ROS with bactericidal activity that protects the host (Kawahara et al. (2007) supra; Aguirre (2010) supra). Vertebrates (including bony fish, amphibians, birds, and mammals) possess the NOX2 NADPH oxidase, which generates a cascade of ROS within the phagosome to kill invading pathogens, especially bacteria. However, the impact of ROS on viral infection is largely unknown.

[0007] ROS, such as superoxide anions and hydrogen peroxide (H2O2), are produced by mouse and human inflammatory cells in response to viral infection and enhance pathology caused by viruses of low to high pathogenicity, including influenza A virus (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 major source of ROS in inflammatory cells is NOX2 oxidase (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 killing bacteria and fungi through the generation of phagosomal ROS, NOX2 oxidase does not appear to eliminate viruses in a manner similar to that of bacteria. Indeed, in the absence of NOX2, influenza A virus causes substantially less lung damage and dysfunction and results in lower viral loads, suggesting that NOX2 oxidase-derived ROS promote rather than inhibit 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 induce ROS production is allusive, as how this highly reactive oxygen molecule causes disease appears to be largely localized to the site of its generation.

[0008] After binding to the plasma membrane (Cossart and Helenius (2014) Cold Spring Harbor Perspectives in Biology 6(8)), the virus enters the cell through a variety of mechanisms and ultimately enters the endosome, leading to the expression of endosomal pattern recognition receptors, including Toll-like receptor 3 (TLR3), TLR7, and TLR9 (Iwasaki and Pillai (2014) Nature Reviews Immunology 14(5):315-328) for the detection of viral RNA. Specific receptor interactions depend on the viral group (I to V) or genomic orientation (i.e., ssRNA, dsRNA, or DNA) and trigger an immune response characterized by the production of type I IFN and IL-1 as well as B cell-dependent antibody production (Iwasaki and Pillai (2014) supra). Endosomal TLRs also detect host nucleic acids and self-antigens, mediating similar type I IFN responses and stimulating antibody production against self-RNA and antigens in autoimmune diseases. Notably, mice chronically deficient in NOX2 oxidase have an increased tendency to develop autoantibodies (Campbell et al. (2012) Science Translational Medicine 4(157):157ra141), and patients with chronic granulomatous disease who have a defect in the ability to generate ROS via NOX2 oxidase have elevated circulating type I IFN and autoantibodies (Kelkka et al. (2014) Antioxidants & Redox Signaling 21(16):2231-2245). These observations support the view that low levels of ROS lead to an enhanced immune response. However, until the present invention, it was unclear how ROS regulate inflammation and pathology caused by pathogens, nor whether targeted (and acute) clearance of ROS could actually be beneficial in treating infections and other conditions associated with immune responses, such as autoimmune diseases. Summary of the invention

[0009] Reactive oxygen species (ROS) promote the pathogenicity of viruses and microorganisms and other parasites. In the work leading to the present invention, the sites and enzyme sources of subcellular ROS production, as well as the effects of ROS on immunostimulation were determined. According to the present invention, it was determined that the TLR7 protein was activated by the formation of a disulfide bond between the 98th (C98) and 475th (C475) cysteine ​​residues of TLR7, which resulted in the production of toxic ROS by NADPH oxidase (NOX2). ROS in turn inhibit antiviral and antimicrobial activity. Therefore, the present invention confirms that TLR7 is a therapeutic target. The reduction of TLR7 activity also enables the treatment of autoimmune diseases, inflammation and cancer and other diseases aggravated by TLR7 activity.

[0010] Provided herein is a method for inhibiting TLR7-mediated immunostimulatory activity in a subject, the method comprising contacting a cell from a subject expressing TLR7 with an effective amount of an agent, the agent antagonizing the formation of a disulfide bond between C98 and C475 of human (or mouse) TLR7 (or its corresponding position in other TLR7). Further taught herein is a method for treating an autoimmune disease, a viral or microbial disease, an inflammation or a cancer in a subject, the method comprising contacting a cell from a subject expressing TLR7 with an effective amount of an agent, the agent antagonizing the formation of a disulfide bond between C98 and C475 of human (or mouse) TLR7 (or its corresponding position in other TLR7). The present invention can also reduce hypoimmunostimulation caused by ROS, and improve conditions associated with or aggravated by TLR7 activity. In one embodiment, the agent inhibits TLR7 activity and can be used to treat autoimmune diseases.

[0011] In one embodiment, the agent comprises a peptide, referred to herein as a "bait peptide", which is about 4 to about 190 amino acids in length and has an amino acid sequence having at least 70% amino acid sequence similarity to up to 190 consecutive amino acids between amino acids 4 and 194 of human (or mouse) TLR7, including peptides having an amino acid sequence having at least 70% amino acid sequence similarity to up to 100 consecutive amino acids between amino acids 48 and 148 of TLR7, and also including peptides having an amino acid sequence having at least 70% amino acid sequence similarity to up to 40 consecutive amino acids between amino acids 78 and 118 of TLR7, provided that the peptide contains a cysteine ​​residue (C98) at a position equivalent to position 98 of TLR7.

[0012] In one embodiment, the decoy peptide comprises an amino acid sequence RCNC corresponding to amino acids R97 to C100 of TLR7 (using a single amino acid code). In one embodiment, the decoy peptide comprises 10 amino acids in length from positions 95 to 104 of human TLR and has an amino acid sequence having at least 70% similarity to DX1RCNCX2PX3X4 (SEQ ID NO: 27), wherein

[0013] X1 is L, F or M;

[0014] X2 is V or I;

[0015] X3 is V or I or A or P; and

[0016] X4 is P or L or K or R.

[0017] In one embodiment, the decoy peptide is DFRCNCVPIP (SEQ ID NO: 26) corresponding to between D95 and P104 of human TLR7.

[0018] In another embodiment, the peptide comprises the amino acid sequence DLRCNCVPVL (SEQ ID NO: 1) corresponding to D95 to L104 of murine TLR7.

[0019] To facilitate the uptake of the peptide into cells, the peptide may further comprise a portion connected to the N-terminus or C-terminus of the peptide, the portion comprising a hydrophilic or cationic peptide, an amphiphilic or amphipathic peptide or a peptide having a periodic amino acid sequence. Alternatively, the peptide is conjugated to cholesterol.

[0020] Examples of hydrophilic peptides include peptides selected from 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 protein (SEQ ID NO: 7), BMVGag-(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).

[0021] Examples of amphipathic peptides include peptides selected from transporter 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 (NLS) (SEQ ID NO: 17), Pep-1 (SEQ ID NO: 18) and Pep-2 (SEQ ID NO: 19).

[0022] Examples of peptides with periodic amino acid sequences include peptides with polyarginine or polylysine sequences. 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. NOX2-cholesterol-linker (PEG)-gp91ds-TAT constructs can also promote cellular uptake. Connecting cholesterol and PEG to gp91ds-TAT helps deliver gp91ds-TAT to endosomes.

[0023] In one embodiment, the subject is a human or non-human mammal.

[0024] Also implemented herein is the use of an agent that antagonizes the formation of a disulfide bond between C98 and C475 of TLR7 or between its corresponding positions in the preparation of a medicament for inhibiting an autoimmune disease, a viral or microbial disease, inflammation or cancer in a subject. In a related embodiment, the present invention teaches an agent that antagonizes the formation of a disulfide bond between C98 and C475 of TLR7 or between its corresponding positions, which is used to inhibit an autoimmune disease, a viral or microbial disease, inflammation or cancer in a subject.

[0025] This article teaches a pharmaceutical composition, which includes an agent and one or more drug carriers, excipients and / or diluents that antagonize the disulfide bond formation between C98 and C475 of TLR7 or between its corresponding positions. In one embodiment, this agent includes the following agent, which includes a length of about 4 to 190 amino acids and a peptide having an amino acid sequence with at least 70% amino acid sequence similarity between up to 190 consecutive amino acids and amino acids 4 to 194 of TLR7. In another embodiment, the agent includes a length of about 4 to 100 amino acids and a peptide having an amino acid sequence with at least 70% amino acid sequence similarity between up to 100 consecutive amino acids and amino acids 48 to 148 of TLR7. In another embodiment, the agent includes a length of about 4 to 40 amino acids and a peptide having an amino acid sequence with at least 70% amino acid sequence similarity between up to 40 consecutive amino acids and amino acids 78 to 118 of TLR7. Each of these embodiments has the following conditions: the peptide contains a cysteine ​​residue (C98) at a position equivalent to position 98 of TLR7. Alternatively or additionally, the peptide comprises the amino acid sequence RCNC corresponding to R97 to C100 of TLR7. The N- and / or C-terminus of H2N-RCNC-COOH may comprise a total of 5 to 500 foreign amino acids together with RCNC. Chemical mimetics of TLR7 inhibitory peptides also form part of the present invention.

[0026] Abbreviations used herein are defined in Table 1.

[0027] Table 1 Abbreviations

[0028] abbreviation definition AA Amino Acids BMDM Bone marrow-derived macrophages C98i Decoy peptide corresponding to amino acids 95 to 104 of TLR7 EEA1 Early endosomal antigen 1 <![CDATA[H2O2]]> Hydrogen Peroxide HIV-TAT Human immunodeficiency virus TAT IAV Influenza A virus IFN Interferon MOI Multiple infections NOX NADPH oxidase NP Nucleoprotein PFU Plaque forming unit ROS Active oxygen TAT transactivating transcription factor TLR Toll-like receptors TLR1 Toll-like receptor 1 TLR2 Toll-like receptor 2 TLR3 Toll-like receptor 3 TLR4 Toll-like receptor 4 TLR5 Toll-like receptor 5 TLR6 Toll-like receptor 6 TLR7 Toll-like receptor 7 TLR8 Toll-like receptor 8 TLR9 Toll-like receptor 9 TLR10 Toll-like receptor 10 WT wild type X31 Mouse-adapted H3N2 influenza A virus

[0029] Amino acid sequences are referred to by a sequence identifier number (SEQ ID NO). SEQ ID NO corresponds numerically to the sequence identifier <400> 1 (SEQ ID NO: 1), <400> 2 (SEQ ID NO: 3), etc. Unless otherwise indicated, the 3 or single letter code or the fully written amino acid sequence is provided in N-terminal to C-terminal direction (left to right, respectively).

[0030] Table 2 provides a summary of sequence identifiers used throughout this specification.

[0031] Table 2 Summary of sequence identifiers

[0032]

[0033]

[0034] Table 3 defines the one-letter and three-letter codes for amino acids used herein.

[0035] Table 3 Amino Acid Three-letter and Single-letter

[0036]

[0037]

[0038] The list of primers and their sources and reference sequences is shown in Table 4. The influenza polymerase primer was custom synthesized and the sequence is shown below.

[0039] Table 4 Primers

[0040] BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Some of the drawings contain color representations or entities. Color photographs may be obtained from the patent owner or the appropriate patent office upon request. If obtained from the patent office, a fee may be charged.

[0042] Figures 1A to 1J are photographs and graphic representations showing that seasonal and pandemic influenza A viruses induce endosomal ROS production by activating the NOX2 oxidase. (AB) Confocal microscopy of primary alveolar macrophages from wild-type (WT) mice infected with influenza A virus strain HKx31 (MOI of 10) for 1 h and labeled with antibodies against early endosomal antigen 1 (EEA1) and A) antibodies against influenza A virus nucleoprotein (NP) or B) antibodies against NOX2, followed by the addition of 4',6'-diamidino-2-phenylindole (DAPI; blue). Quantification of the results is also shown (n=5). (CD) Time-dependent increase in endosomal ROS control levels in primary alveolar macrophages from mice, assessed by OxyBURST (100M) confocal fluorescence microscopy and labeled with DAPI (n=5). (EF) WT, NOX2 - / y(A) and superoxide dismutase (SOD; 300 U / mL) treated WT mouse primary alveolar macrophages, assessed by OxyBURST confocal fluorescence microscopy in the absence or presence of HKx31 virus, and labeled with DAPI (n=5). (G) Uninfected and HKx31 virus infected mouse primary alveolar macrophages were labeled with OxyBURST and the acidified endosomal marker Lysotracker (50 nM). Some cells were treated with bafilomycin A (Baf-A; 100 nM) to inhibit endosomal acidification (n=4). (H) Human alveolar macrophages infected with seasonal H3N2 (A / New York / 55 / 2004, A / Brisbane / 9 / 2007), seasonal H1N1 (A / New Caledonia / 20 / 1999, A / Solomon Islands / 3 / 2006), and pandemic A(H1N1)pdm09 strains (A / California / 7 / 2009, A / Auckland / 1 / 2009) and labeled with OxyBURST for endosomal ROS (n=4). (IJ) Endosomal ROS production in primary alveolar macrophages from WT mice, exposed to heat (56OC)-inactivated HKx-31 virus (to prevent viral fusion) or UV-inactivated HKx-31 virus (to block replication) and labeled with DAPI (n=4) assessed by OxyBURST fluorescence microscopy. (A, B, C, E, G, H, and I) Images represent >150 cells analyzed in each experiment. Original magnification X100. (A, B, D, F and J) Data are expressed as mean ± SEM. (A and B) Student's unpaired t-test *P < 0.05. (D, F and J) One-way ANOVA followed by Dunnett's post hoc test for multiple comparisons. *P < 0.05 and **P < 0.01.

[0043] FIG. 2A to FIG. 2I are photographs and graphic representations showing that co-localization of TLR7 with influenza A virus, NOX2, and EEA1 is a signaling platform for the generation of endosomal ROS against influenza A virus through a TLR7- and PKC-dependent mechanism. (AC) Confocal microscopy of mouse primary alveolar macrophages that were untreated or infected with influenza A virus HKx31 (MOI of 10) and labeled with TLR7 antibodies and A) influenza A virus NP, B) NOX2, or C) EEA1, followed by the addition of 4',6'-diamidino-2-phenylindole (DAPI). Quantitative data from multiple experiments are also shown (n=5). (D) WT and TLR7 - / -Endosomal ROS production in mouse primary alveolar macrophages was assessed by Oxyburst (100M) fluorescence microscopy in the absence or presence of HKx31 virus and labeled with DAPI (n=6). (E) Immunofluorescence microscopy for assessment of NOX2 and p47phox binding. WT and TLR7 primary alveolar macrophages were left untreated or infected with HKx31 virus (MOI of 10) in the absence or presence of bafloxacin A (Baf-A; 100 nM) or dynasore (Dyna; 100 M). - / - Immortalized bone marrow-derived macrophages (BMDM) were then labeled with antibodies against NOX2 and p47phox. Quantification of the results is also shown (n=5). (FI) WT and NOX2 - / y Endosomal ROS production in mouse primary alveolar macrophages was assessed by Oxyburst fluorescence microscopy in the absence or presence of F) imiquimod (Imiq; 10 μg / ml) and G) ssRNA (100 μg / ml) and co-labeled with DAPI (n=5). (H, I) Endosomal ROS production in WT and TLR7 - / - Cytoplasmic PKC activity was assessed by FRET analysis in BMDM. Cells were treated with empty vector control or bafloxacin A (100nM) or dynasore (100M) and then exposed to influenza A virus (HKx31, MOI of 10) or imiquimod (10g / ml) for 25 minutes (n=3). (AF and H) Images represent >150 cells analyzed in each experiment. Original magnification X100. All data are expressed as mean ± SEM. (A, B, C, F, and G) Student's unpaired t-test *P<0.05. (D, E, H, and I) One-way ANOVA followed by Dunnett's post hoc test for multiple comparisons. *P<0.05.

[0044] FIG. 3A to FIG. 3B are photographs and graphical representations showing endosomal ROS generation in response to ssRNA and DNA viruses via TLR7- and TLR9-dependent mechanisms, respectively. (A) In WT and TLR7 - / -Endosomal ROS production in bone marrow-derived macrophages was assessed by OxyBURST (100M) fluorescence microscopy in the absence or presence of influenza A virus (HKx31 virus), rhinovirus (rhino), respiratory syncytial virus (RSV), human parainfluenza virus (PIV), human metapneumovirus (HMPV), Sendai virus, dengue virus, human immunodeficiency virus (HIV), mumps virus (MuV), Newcastle disease virus (NDV), rotavirus (British Wagyu strain), herpes simplex virus 2 (HSV-2), and vaccinia virus, and labeled with 4',6'-diamidino-2-phenylindole (DAPI). Quantification of the results is also shown (n=5). (B) WT and TLR9 - / - Endosomal ROS production in mouse primary alveolar macrophages was assessed by OxyBURST fluorescence microscopy in the absence or presence of HKx31 virus, rhinovirus, Sendai virus, dengue virus, and herpes simplex virus 2 (HSV-2) and labeled with DAPI (n=5). (A and B) Images represent >150 cells analyzed in each experiment. Original magnification X100. All data are expressed as mean ± SEM. One-way ANOVA followed by Dunnett's post hoc test for multiple comparisons. #P<0.05 compared to WT control. *P<0.05 comparisons are indicated by horizontal bars.

[0045] FIG. 4A to FIG. 4B are photographs and graphic representations showing that bacterial-induced ROS production is distinct from virus-dependent ROS mechanisms (A) WT and TLR7 - / - Phagosomal superoxide production against Haemophilus influenzae and Streptococcus pneumoniae in immortalized bone marrow-derived macrophages, as assessed by OxyBURST (100M) fluorescence microscopy. Images represent >150 cells analyzed in each experiment. Original magnification x100. (B) is quantification of results (n=5). All data are expressed as mean ± SEM. One-way ANOVA followed by Dunnett post hoc test for multiple comparisons. *P < 0.05 compared to WT control.

[0046] FIG. 5A to FIG. 5C Figure 2 is a graphical representation showing that endosomal NOX2 oxidase inhibits cytokine expression in response to TLR7 activation in vitro and in vivo. (AB) WT and TLR7 in the absence or presence of A) apocynin (Apo; 300 M) or B) bafloxacin A (Baf-A; 100 nM). - / -Immortalized bone marrow-derived macrophages (BMDM) were untreated or treated with imiquimod (Imiq; 10 μg / mL), and IFN-, IL-1, TNF-, and IL-6 mRNA expression was measured by QPCR 24 hours later (n=6). - / y Mice were administered imiquimod (50 μg / mouse, intranasal), and C) total airway inflammation was quantified by bronchoalveolar lavage fluid analysis, and D) cytokine expression was assessed 24 hours later (n=5). (A, B, D) Responses are relative to GAPDH and then expressed as fold change relative to WT control. (AD) Data are expressed as mean ± SEM. (A, B and D) Kruskal-Wallis test with Dunn post hoc analysis for multiple comparisons. (C) One-way ANOVA followed by Dunnett post hoc test for multiple comparisons. Statistically significant when P < 0.05. *P < 0.05; **P < 0.01.

[0047] FIG. 6A to FIG. 6L are photographs and graphical representations showing that endosomal NOX2 oxidase-derived hydrogen peroxide (H2O2) inhibits cytokine expression in response to TLR7 activation in vitro and in vivo. (A) Primary alveolar macrophages from WT mice were either untreated or treated with FITC-labeled catalase for 5 minutes prior to infection with HKx31 virus (MOI of 10). Cells were labeled with Lysotracker (50 nM) and co-localization of Lysotracker and FITC catalase was assessed by confocal microscopy. Images represent >100 cells analyzed in each experiment. Original magnification X100 (n=3). (B) WT and TLR7 - / - Immortalized bone marrow-derived macrophages (BMDM) were untreated or treated with catalase (1000 U / mL) for 1 hour, and IFN- and IL-1 mRNA expression was determined by QPCR 24 hours later (n=7). (C) WT BMDM were untreated or treated with imiquimod (Imiq) for 1 hour in the absence or presence of catalase (1000 U / mL), and IFN- and IL-1 mRNA expression was assessed by QPCR 24 hours later (n=6). (D) WT BMDM were treated with DMSO (0.1%) or dynasore (Dyna; 100 M) for 30 minutes and then treated with catalase (1000 U / mL) for 1 hour. Cytokine mRNA expression was determined by QPCR 24 hours later (n=6). (E) WT and TLR2 were treated with catalase (1000 U / mL) - / -Immortalized BMDM were treated with catalase (1000 U / mL) for 1 hour and cytokine mRNA expression was determined by QPCR 24 hours later (n=6). - / - Immortalized BMDM for 1 hour, and cytokine mRNA expression was determined by QPCR after 24 hours (n=6). (GI) WT BMDM were treated with catalase or imiquimod (10g / ml) for 1 hour, and G) TLR7, H) NLRP3 or I) TREML4 mRNA expression was determined by QPCR after 24 hours (n=6). (J) Mice were treated intranasally with catalase (1000U / mouse), and then lung expression of TREML4 was determined by QPCR (n=5). (K and L) Catalase (1000U / mouse, intranasal) was administered to WT mice, and K) total BALF airway inflammation and L) lung cytokine expression were assessed after 24 hours (n=5). (BH and L) Responses were relative to GAPDH, and then expressed as fold changes relative to WT controls. (BH and L) Kruskal-Wallis test with Dunn post hoc analysis was used for multiple comparisons. (I and J) Mann-Whitney Wilcoxon test. All data are expressed as mean ± SEM. Statistically significant when P < 0.05. *P < 0.05.

[0048] FIG. 7A to FIG. 7B Graphical representation showing that C98 on TLR7 regulates receptor activity and is a target for endosomal H2O2 (A) Transfection of TLR7 with empty vector, WT TLR7, or TLR7 with cysteine ​​98, 260, 263, 270, 273, and 445 mutated to alanine (TLR76mut), TLR7 with cysteine ​​98 and 445 mutated to alanine (TLR7C98A / 445A), or TLR7 with cysteine ​​445 mutated to alanine (TLR7C445A), or TLR7 with cysteine ​​98 mutated to alanine (TLR7C98A). - / - BMDM. After 48 hours, cells were left untreated or treated with catalase (1000 U / mL) or imiquimod (Imiq, 10 g / ml) for 1 hour, and cytokine expression was assessed 24 hours later (n=6). Responses were relative to GAPDH and then expressed relative to TLR7 - / - Fold change of control. Data are expressed as mean ± SEM. One-way ANOVA followed by Dunnett's post hoc test for multiple comparisons. Statistically significant when P < 0.05. *P < 0.05. (ns) indicates not significant. (B) Multiple sequence alignment performed using CLUSTAL OMEGA shows interspecies conservation of Cys 98 on TLR7.

[0049] FIG. 8A to FIG. 8I is a graphical representation showing that inhibition of NOX2 oxidase increases the expression of type I IFN and IL-1, as well as antibody production against influenza A virus infection (A) from WT and NOX2 - / y Alveolar macrophages from mice were either untreated (unimmunized) or infected with HKx31 influenza A virus (MOI 10) to analyze IFN-, IL-1, TNF-, and IL-6 mRNA expression by QPCR after 24 hours (n=8). (BC) WT and NOX2 - / y Mice were infected with live HKx31 influenza A virus (1×105 PFU per mouse), and B) cytokine mRNA expression and IFN-protein expression in C) BALF or D) serum were assessed 3 days later (n=5). (EI) WT and NOX2 - / y Mice were infected with inactivated HKx31 influenza A virus (equivalent to 1×104 PFU per mouse) to measure on day 7: E) body weight; F) airway inflammation and differential cell counts (i.e., macrophages, neutrophils, and lymphocytes); G) cytokine expression in whole lung (responses shown as fold change relative to GAPDH) and H) serum, and I) BALF antibody levels (n=6). Data are shown as mean ± SE. (A) Kruskal-Wallis test with Dunn post hoc analysis for multiple comparisons. (BI) Unpaired t-test; statistically significant when P<0.05. *P<0.05. **P<0.01.

[0050] FIG. 9A to FIG. 9MIt is a photograph and a graphical representation showing that endosomally targeted delivery of NOX2 oxidase inhibitors protects mice after influenza A virus infection in vivo (AE). Alveolar macrophages from WT mice were treated with Cy5 cholesterol-PEG linker fluorophore (Cy5-chol; 100nM) for 30 minutes and infected with HKx31 influenza A virus (MOI of 10). The cells were then relatively visualized with antibodies against: A) and B) EEA1, C) NOX2 or D) influenza A virus nucleoprotein (NP). All cells were then stained with 4', 6'-diamidino-2-phenylindole (DAPI) and imaged with a confocal microscope. B) Cells were pretreated with dynasore (100M) for 30 minutes before exposure to Cy5-cholesterol. E) Quantification of data from (AD, n = 5). (F) RAW 264.7 macrophages were untreated or treated with various concentrations of cholesterol-conjugated gp91ds-TAT (Cgp91), ethyl-conjugated gp91ds-TAT (Egp91), or unconjugated gp91ds-TAT (Ugp91) for 30 minutes, and ROS production was quantified by L-O12 (100M)-enhanced chemiluminescence (n=7). (G) Superoxide production was measured by xanthine / xanthine oxidase cell-free assay in the absence or presence of Ugp91ds-TAT (1M) or Cgp91ds-TAT (1M) (n=6). (HI) Ugp91ds-TAT (0.02 mg / kg / day) or Cgp91ds-TAT (0.02 mg / kg / day) was delivered intranasally to WT mice once daily for 4 days. 24 hours after the first dose of inhibitor, mice were treated with saline or infected with HKx31 influenza A virus (1×105 PFU per mouse). Mice were selected on day 3 post-infection, and H) airway inflammation was assessed by BALF cell counts, I) lung IFN-mRNA was determined by QPCR (n=7). (ns) indicates not significant. (JM) Mice were subjected to NOX2 inhibitor treatment regimen and viral infection regimen as H), except that NOX2 inhibitor was delivered at a dose of 0.2 mg / kg / day (n=7). J) Airway inflammation analysis by BALF counts; K) Body weight analysis (% weight change from the value measured on day -1); L) ROS produced by BALF inflammatory cells was determined by L-O12 enhanced chemiluminescence; M) Viral mRNA was determined by QPCR. Data are expressed as mean ± SEM. E) Unpaired t-test; statistically significant when P<0.05 (F, G, H, J, K, L). One-way ANOVA followed by Dunnett's post hoc test for multiple comparisons. (I and M) Kruskal-Wallis test with Dunn's post hoc analysis for multiple comparisons. Statistical significance was considered when P < 0.05.*P<0.05; **P<0.01.

[0051] FIG. 10A to FIG. 10D Multiple sequence alignment analysis is shown, which demonstrates the position of all cysteine ​​residues on human TLR7. The individual sequences of human TLRs were obtained from the NCBI GenBank protein database with accession numbers as follows: TLR1 (CAG38593.1), TLR2 (AAH33756.1), TLR3 (ABC86910.1), TLR4 (AAF07823.1), TLR5 (AAI09119.1), TLR6 (BAA78631.1), TLR7 (AAZ99026.1), TLR8 (AAZ95441.1), TLR9 (AAZ95520.1) and TLR10 (AAY78491.1), and then sequence alignment was performed using CLUSTAL OMEGA (EMBL-EBI). The cysteine ​​residues on human TLR7 are shown in the red dotted rectangular box, and their respective positions are indicated.

[0052] FIG. 11A to FIG. 11C Multiple sequence alignment analysis of vertebrate TLR7 is shown. The individual sequences of TLRs were obtained from the NCBI GenBank protein database with accession numbers Ontario salmon (CCX35457.1), tropical African clawed frog (AAI66280.1), jungle fowl (ACR26243.1), mouse (AAI32386.1), brown rat (NP_001091051.1), human (AAZ99026.1), wild boar (ABQ52583.1) and cattle (NP_001028933.1), and then sequence alignment was performed using CLUSTAL OMEGA (EMBL-EBI). The cysteines on human TLR7 are shown in red dashed rectangles, and their respective positions are indicated.

[0053] Fig.12 is a graphical representation showing data on IL-1β cytokine expression produced by bone marrow-derived macrophages following exposure to the TLR7 agonist imiquimod in the absence or presence of C98i. The duration of C98i treatment was 1 hour, followed by exposure to imiquimod, and cytokines were measured by real-time quantitative PCR after a 24 hour period.

[0054] Fig.13 is a graphical representation showing data on IL-1β cytokine expression produced by bone marrow-derived macrophages following exposure to the TLR7 agonist gardiquimod in the absence or presence of C98i. The duration of C98i treatment was 1 hour, followed by exposure to imiquimod, and cytokines were measured by real-time quantitative PCR after a 24 hour period.

[0055] Fig.14 is a graphical representation showing data on IL-1β cytokine expression produced by bone marrow-derived macrophages following exposure to C98i. The duration of C98i treatment was 1 hour, and cytokines were measured by real-time quantitative PCR after a 24 hour period.

[0056] Fig.15 is a graphical representation showing data showing protein expression of the IL-1β cytokine produced by bone marrow-derived macrophages following exposure to the TLR7 agonist imiquimod in the presence or absence of C98i in vitro. The duration of C98i treatment was 1 hour, followed by exposure to imiquimod, and the cytokine was measured by ELISA after a 24 hour period.

[0057] Fig.16A and Fig. 16B Schematic representation of IL-1β production (a) and cell viability (b) in response to the TLR7 agonist imiquimod and C98i without TAT (no TAT).

[0058] Fig.17 is a schematic diagram showing that C98i inhibits influenza A (X31) virus response (IL-6 GAPDH) in vitro (C98i 30 μM + X31-IL-6 mRNA expression).

[0059] Fig.18 is a schematic diagram showing that the scrambled amino acid sequence of C98i has no effect on TLR7 agonist (imiquimod). The scrambled amino acid sequence is YGRKKRRQRRRCLVPNDCRLV-NH2 (SEQ ID NO: 44).

[0060] Fig.19 Schematic diagram showing that the short motif in C98i RCNC (SEQ ID NO: 45) or any of its modified forms (RANC, SEQ ID NO: 46; RANA, SEQ ID NO: 47; or RCNA, SEQ ID NO: 48) cannot inhibit the response of TLR7 agonist (imiquimod) in vitro. Peptides were used at 100 μM + 10 μg / ml imiquimod; IL-6 mRNA expression was measured. DETAILED DESCRIPTION

[0061] Throughout the specification, unless the context requires otherwise, the word "comprise" or variations such as "comprising" or "containing", will be understood to imply the inclusion of stated one 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.

[0062] As used in this 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 of the disclosure teachings; and so forth. Aspects taught and practiced herein are encompassed by the term "invention." Any variants and derivatives contemplated herein are included in "forms" of the invention. All aspects of the invention can be practiced within the scope of the claims.

[0063] The present invention is based in part on determining that the virus enters the endosomal compartment of the cell and triggers the generation of NADPH oxidase (NOX)-dependent reactive oxygen species (ROS) in the endosome. It is determined that endosomal ROS is a negative regulator of the molecular mechanism that confers antiviral immunity, which depends on Toll-like receptor 7 (TLR7) activation. The ability to limit ROS production by antagonizing TLR7 activation enables the production of universal antiviral therapies and antipathogen therapies more widely. Antagonizing TLR7 activation limits the generation of ROS, otherwise ROS will antagonize immune stimulation. Considering that TLR7 itself is related to inflammatory forms, the present invention has a wider range of significance in the treatment of microbial infections (such as bacteria and fungal microorganisms), autoimmune diseases, inflammation and cancer. Therefore, antagonizing TLR7 activation helps to improve diseases and disorders associated with TLR7 activation or aggravated by TLR7 activation.

[0064] Therefore, the present invention is able to reduce the level of activated TLR7, which leads to:

[0065] (i) Reduce endosomal ROS production via NOX2;

[0066] (ii) reduction of active TLR7 results in reduced inflammatory symptoms;

[0067] (iii) reduced immune stimulation;

[0068] (iv) decreased negative regulation of humoral immune response networks;

[0069] (v) reduced pathogen infectivity;

[0070] (vi) reduced inflammation;

[0071] (vii) reduced cancer growth ability; and / or

[0072] (viii) thereby ameliorating an autoimmune disorder or disease.

[0073] Thus, provided herein is a method of 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 that antagonizes disulfide bond formation between C98 and C475 of TLR7 or positions corresponding thereto.

[0074] Also provided herein is a method for treating an autoimmune disease, a viral or microbial disease, inflammation or cancer in a subject, the method comprising contacting a cell from the subject expressing TLR7 with an effective amount of an agent that antagonizes disulfide bond formation between C98 and C475 or corresponding positions thereof of TLR7.

[0075] Also provided herein is a method for treating excessive production of reactive oxygen species in a subject, the method comprising contacting a cell from the subject expressing TLR7 with an effective amount of an agent that antagonizes disulfide bond formation between C98 and C475 of TLR7 or positions corresponding thereto.

[0076] Provided herein is a method for treating TLR7-mediated inflammation in a subject, the method comprising contacting a cell from the subject that expresses TLR7 with an effective amount of an agent that antagonizes disulfide bond formation between C98 and C475 of TLR7 or corresponding positions thereof.

[0077] Also provided herein is a method for treating an autoimmune disorder in a subject, the method comprising contacting a cell from the subject that expresses TLR7 with an effective amount of an agent that antagonizes disulfide bond formation between C98 and C475 of TLR7 or corresponding positions thereof.

[0078] Still further provided herein is a method for treating cancer in a subject, the method comprising contacting a cell from the subject that expresses TLR7 with an effective amount of an agent that antagonizes disulfide bond formation between C98 and C475 of TLR7 or positions corresponding thereto.

[0079] Reference to TLR7 includes any TLR7 from any species. Typically, the TLR7 referred to is the TLR7 from the subject being treated. Thus, where the subject is a human, the antagonist will be directed against human TLR7 expressed by cells. The amino acid sequence of human TLR7 is set forth in SEQ ID NO: 20. FIG. 11A to FIG. 11CA comparison of TLR7 amino acid sequences from different species is listed in. Important is the cysteine ​​residue (Cys; C) at amino acid position 98 (C98), which forms a disulfide bond with the cysteine ​​at amino acid position 475 (C475) or the corresponding position in homologues from different species. These amino acid positions are conserved between TLR7 molecules between species. In one embodiment, the present invention antagonizes the formation of this disulfide bond, resulting in a decrease in activated TLR7 levels. It then reduces TLR7-mediated immune stimulation and reduces NADPH oxidase-mediated ROS formation.

[0080] It is proposed herein to use an antagonist of the disulfide bond forming pair. The agent may be proteinaceous or non-proteinaceous. In one embodiment, it is proposed that the peptide decoy forms a disulfide bond with C475 of TLR7, which prevents the formation of the C98-C475 disulfide bond. In one embodiment, the agent is a chemical mimetic of the peptide decoy.

[0081] Examples of microbial pathogens include: Helicobacter pyloris, Borrelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g., 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 (Streptococcus pyogenes), Streptococcus pyogenes (Streptococcus pyogenes), Streptococcus galactiae (Streptococcus pyogenes), Streptococcus pyogenes (Streptococcus pyogenes), Streptococcus galactiae (Streptococcus pyogenes), and Streptococcus pyogenes. agalactiae (group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic), 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, Pasturella multocida 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.

[0082] Examples of viral pathogens include: Retroviridae (including but not limited to human immunodeficiency virus (HIV)); Picornaviridae (e.g., poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, echovirus); Caliciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviviridae (e.g., dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (e.g., coronavirus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza virus); Bunyaviridae (e.g., hantaviruses, bunyaviruses, phleboviruses, and nairoviruses); =viruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviruses, and rotaviruses); BiRNA viruses; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvoviruses); Papillomaviridae (papillomaviruses, polyomaviruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and HSV-2, varicella-zoster virus, cytomegalovirus (CMV)); Poxviridae (smallpox virus, vaccinia virus, poxviruses); and Iridoviridae (e.g., African swine fever virus); as well as unclassified viruses (e.g., the causative agent of spongiform encephalopathies, the causative agent of hepatitis delta (thought to be a defective satellite of hepatitis B virus), the causative agent of non-A, non-B hepatitis (category 1 = internal transmission; category 2 = parenteral transmission (i.e., hepatitis C); Noroviruses and related viruses, and Astroviruses).

[0083] Cancers or tumors include acute lymphoblastic leukemia, B-cell lymphoma, glioma, bladder cancer, biliary tract cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, choriocarcinoma, epithelial cell cancer, gastric cancer, hepatocellular carcinoma, Hodgkin lymphoma, lung cancer, lymphoid cell leukemia, melanoma, myeloma, non-small cell lung cancer, nasopharyngeal cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, squamous cell carcinoma of the cervix and esophagus, testicular cancer, T-cell leukemia and melanoma.

[0084] Autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, Sjögren's syndrome, polymyositis, vasculitis, Wegener's granulomatosis, sarcoidosis, ankylosing spondylitis, Reiter's syndrome, psoriatic arthritis and Behcet's disease. In one embodiment, the autoimmune disease is an immune complex-associated disease. Immune complex-associated diseases specifically include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, polyarteritis nodosa, post-streptococcal glomerulonephritis, cryoglobulinemia, and acute and chronic serum sickness.

[0085] Examples of inflammatory disease conditions contemplated by the present invention include, but are not limited to, those diseases and conditions that result in redness, swelling, pain, and a sensation of heat in certain areas in response to protect tissue from injury or pathology. Inflammatory diseases that may be treated using the methods of the present invention include, but are not limited to, acne, angina, arthritis, asthma, aspiration lung disease, chronic obstructive pulmonary disease (COPD), colitis, consumptive disease, gastroenteritis, intestinal influenza, necrotizing enterocolitis, pelvic inflammatory disease, pharyngitis, pleurisy, sore throat, rash, sore throat, urinary tract infection, chronic inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyneuritis.

[0086] The peptide decoy comprises an amino acid sequence RCNC of 4 to 190 in length and corresponding to R97 to C100 of TLR7 in one embodiment. This sequence is conserved between species. It is important that C98 in TLR7 or its equivalent or corresponding position in a TLR7 homologue or variant. In one embodiment, the peptide decoy comprises:

[0087] (a) 4 to 190 amino acids in length;

[0088] (b) the amino acid sequence RCNC, which corresponds to R97 to C100 of TLR7; and

[0089] (c) Cysteine ​​at position corresponding to C98 of TLR7.

[0090] With the exception of the cysteine ​​corresponding to C98, amino acids may be substituted for conformationally or functionally equivalent or similar amino acids.

[0091] In one embodiment, the agent comprises a peptide, referred to herein as a "decoy peptide", having a length of 4 to 190 amino acids and having an amino acid sequence having at least 70% amino acid sequence similarity to up to 190 consecutive amino acids between amino acids 4 and 194 of TLR7, including a peptide having an amino acid sequence having at least 70% amino acid sequence similarity to up to 100 consecutive amino acids between amino acids 48 and 148 of TLR7, and also including a peptide having an amino acid sequence having at least 70% amino acid sequence similarity to up to 40 consecutive amino acids between amino acids 78 and 118 of TLR7, provided that the peptide comprises a cysteine ​​residue (C98) at a position equivalent to position 98 of TLR7. The peptide may be 4 to 190 amino acids in length.

[0092] With respect to the length of the bait peptide, the expression "4 to 190" 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 5, 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 2, 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 area between 78 and 118 includes 78, 79, 80, 81, 82. 2, 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. With respect to percentage similarity, the expression "at least 70%" 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%.

[0093] As used herein, the term "similarity" includes exact identity between the sequences being compared at the amino acid level. Where there is no identity at the amino acid level, "similarity" includes amino acids that are still related to each other at the structural, functional, biochemical and / or conformational levels. In a particularly preferred embodiment, nucleotide and sequence comparisons are performed at the level of identity rather than similarity.

[0094] Terms used to describe the sequence relationship between two or more polypeptides include "reference sequence", "comparison window", "sequence similarity", "sequence identity", "sequence similarity percentage", "sequence identity percentage", "substantially similar" and "substantially identical". The length of a "reference sequence" is at least 4 or more, including amino acid residues. Because two polypeptides can each contain: (1) a sequence that is similar between the two polypeptides (i.e., only a portion of the complete amino acid sequence of TLR7); and (2) an amino acid sequence that differs between the two polypeptides, sequence comparisons between two (or more) polypeptides are typically performed by comparing the 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 consecutive amino acid residues that is compared to a reference sequence. The comparison window may contain about 20% or less additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) to optimally align the two sequences. Optimal sequence alignment for the comparison window can be performed by computer-implemented algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Version 7.0, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA), or by inspection and optimal alignment (i.e., producing the highest homology percentage in the comparison window) generated by any of the various methods selected. Reference can also be made to the BLAST family of programs disclosed, for example, by Altschul et al. (1997) Nucl. Acids. Res. 25: 3389. A detailed discussion of sequence analysis is found in Ausubel et al., Unit 19.3 (in: Current Protocols in Molecular Biology, John Wiley & Sons Inc. 1994-1998. A comparison of the TLR7 amino acid sequence is shown in FIG. 10A to FIG. 10D middle.

[0095] As used herein, the terms "sequence similarity" and "sequence identity" refer to the degree of identical or functionally or structurally similar sequences on an amino acid to amino acid basis over a comparison window. Thus, for example, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions of identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) present in the two sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and then multiplying the result by 100 to obtain the percentage of sequence identity. For purposes of the present invention, "sequence identity" is understood to refer to a "percentage of matches" calculated by the DNASIS computer program (Version 2.5 for Windows; provided by Hitachi Software Engineering, Inc., South San Francisco, California, USA), using the standard default values ​​used in the reference manual accompanying the software. With respect to sequence similarity, a similar description applies.

[0096] In one 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.

[0097] Therefore, the reagent includes a peptide or peptide decoy and a non-protein chemical agent that antagonizes the formation of a disulfide bond between C98 and C475. The non-protein chemical agent includes a chemical mimetic of C98i. The peptide decoy contains a cysteine ​​equivalent to C98. The peptide decoy can also contain an amino acid sequence RCNC corresponding to R97 to C100 of human TLR7.

[0098] In one embodiment, the peptide decoy comprises 10 amino acids having the amino acid sequence DX1RCNCX2PX3X4 (SEQ ID NO: 27), wherein:

[0099] X1 is L, F or M;

[0100] X2 is V or I;

[0101] X3 is V or I or A or P; and

[0102] X4 is P or L or K or R,

[0103] Or an amino acid sequence having at least about 70% similarity to SEQ ID NO: 27 after optimal alignment, including at least about 80%, 85%, 90%, 95% or 99% similarity to SEQ ID NO: 27. The above sequences correspond to amino acid positions 95 to 104 of human TLR or their equivalents. Human TLR7 contains a P at position 104. In mouse TLR7, the amino acid L is at position 104. Both have a D at position 95.

[0104] In one embodiment, the peptide decoy comprises 4 to 40 amino acids selected from amino acids 78 to 118 of human TLR7, provided that the peptide comprises a cysteine ​​residue at a position corresponding to C98 of human TLR7, or the peptide comprises the amino acid sequence RCNC at a position corresponding to R97 to C100 of human TLR7.

[0105] 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, provided that the peptide comprises a cysteine ​​at the position corresponding to C98 of TLR7. In a specific embodiment, the peptide decoy comprises the amino acid sequence set forth in SEQ ID NO: 20. The corresponding mouse TLR7 decoy peptide sequence is DLRCNCVPVL (SEQ ID NO: 1) or has 70% similarity to SEQ ID NO: 1 after optimal alignment, provided that the peptide comprises a cysteine ​​at the position corresponding to C98 of TLR7.

[0106] One or more amino acids can be substituted with one or more amino acid analogs, or one or more side chains can be modified. Such modifications can improve serum half-life and improve stability.

[0107] Examples of side chain modifications contemplated by the present invention include modifications of the amino group, such as reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidation with acetimidate; acylation with acetic anhydride; carbamidomethyl esterification of the amino group with cyanate; trinitrobenzylation of the amino group with 2,4,6-trinitrobenzenesulfonic acid (TNBS); acylation of the amino group with succinic anhydride and tetrahydrophthalic anhydride; pyridine oxidation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH4.

[0108] The guanidinium group of arginine residues can be modified by forming heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

[0109] The carboxyl group can be modified by activation of the carbodiimide via formation of an O-acylisourea followed by derivatization to, for example, the corresponding amide.

[0110] Thiol groups can be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; oxidation to cysteine ​​with performic acid; formation of mixed disulfides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimides; formation of mercury derivatives using 4-chloromercaptobenzoate, 4-chloromercaptobenzenesulfonic acid, phenylmercuric chloride, 2-chloromercapto-4-nitrophenol and other mercury; and carbamidomethylation with cyanate esters at alkaline pH.

[0111] Tryptophan residues can be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulfinyl halides. On the other hand, tyrosine residues can be modified by nitration with tetranitromethane to form 3-nitrotyrosine derivatives.

[0112] Modification of the imidazole ring of the histidine residue can be achieved by alkylation with iodoacetic acid derivatives or by N-ethoxylation with diethyl pyrocarbonate.

[0113] Examples of incorporation of unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, the use of norleucine, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, tert-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine, and / or the D-isomers of amino acids.

[0114] For example, cross-linkers can be used to stabilize the 3D conformation, using homobifunctional cross-linkers, such as bifunctional imide esters with (CH2)n spacer groups (where n=1 to n=6), glutaraldehyde, N-hydroxysuccinimide esters, and heterobifunctional reagents, which generally contain an amino-reactive portion, such as N-hydroxysuccinimide and another group-specific reactive portion, such as maleimide or a disulfide portion (SH) or carbodiimide (COOH). In addition, the peptide can be conformationally constrained by, for example, incorporating C and N-methyl amino acids, introducing double bonds between the C and C atoms of amino acids, and by introducing covalent bonds (e.g., between the N and C termini, between two side chains, or between a side chain and the N or C termini to form an amide bond), forming a cyclic peptide or the like.

[0115] Mimics are another group of useful reagents for testing the ability of antagonizing TLR7 activation by C98-C475 disulfide bonds. The term is intended to refer to a substance that has a certain chemical similarity with the bait peptide it simulates but antagonizes it and the target (i.e., C475 of human (or mouse) TLR7) interaction. Peptide mimics can be peptide-containing molecules (Johnson et al. (1993) Peptide Turn Mimetics in Biotechnology and Pharmacy, Pezzuto et al. Eds., Chapman and Hall, New York) that simulate protein secondary structure elements. The basic principle behind using peptide mimics is that the presence of the peptide backbone of the protein is mainly used for the orientation of the amino acid side chain, to promote the molecular interaction with the C475 of TLR7. Therefore, peptide mimics are designed to be similar to bait peptides, to allow molecular interaction. Chemical mimics are also considered herein, which are non-proteinaceous, but have the same effect and / or some conformations as C98i.

[0116] The peptide decoy may further comprise a moiety that promotes cellular uptake of the peptide. Any number or type of cellular uptake moieties 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 protein (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 amphipathic (or amphipathic) peptides, such as, but not limited to, transporter 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 (NLS) (SEQ ID NO: 17), Pep-1 (SEQ ID NO: 18) and Pep-2 (SEQ ID NO: 19). In another alternative, the uptake moiety comprises a periodic amino acid sequence, such as polyarginine or polylysine. Other uptake peptides are those listed in Table 1 of Guitoti et al. (2017) supra, the contents of which are incorporated herein by reference.

[0117] Although subjects to be treated include humans by targeting human TLR7, the present invention has veterinary applications, such as treating livestock animals (e.g., cattle, sheep, pigs, goats, horses), domestic pets (e.g., cats, dogs), and captive wild animals and laboratory test animals (e.g., mice, rats, guinea pigs, hamsters, rabbits). In one embodiment, the subject is a human.

[0118] Thus, provided herein is a method of 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 that antagonizes disulfide bond formation between C98 and C475 of TLR7 or positions corresponding thereto.

[0119] Further provided herein is a method of treating an autoimmune disease, a viral or microbial disease, inflammation or cancer in a human subject, the method comprising contacting a cell from the subject that expresses TLR7 with an effective amount of an agent that antagonizes disulfide bond formation between C98 and C475 of TLR7 or positions corresponding thereto.

[0120] Also provided herein is a method of treating excessive production of reactive oxygen species in a human subject, the method comprising contacting a cell from the subject that expresses TLR7 with an effective amount of an agent that antagonizes disulfide bond formation between C98 and C475 of TLR7 or positions corresponding thereto.

[0121] Provided herein is a method for treating TLR7-mediated inflammation in a human subject, the method comprising contacting a cell from the subject that expresses TLR7 with an effective amount of an agent that antagonizes disulfide bond formation between C98 and C475 of TLR7 or positions corresponding thereto.

[0122] Also provided herein is a method of treating an autoimmune disorder in a human subject, the method comprising contacting a cell from the subject that expresses TLR7 with an effective amount of an agent that antagonizes disulfide bond formation between C98 and C475 of TLR7 or positions corresponding thereto.

[0123] Still further provided herein is a method for treating cancer in a human subject, the method comprising contacting a cell from the subject that expresses TLR7 with an effective amount of an agent that antagonizes disulfide bond formation between C98 and C475 of TLR7 or positions corresponding thereto.

[0124] When the agent is a peptide decoy, the amino acid sequence is generally derived from the TLR7 of the same species being treated (e.g., human TLR7 for treating a human subject). This is referred to as autologous therapy. However, in the case of use in another species, there is substantial amino acid sequence similarity between TLRs of certain species, and therefore, the present invention contemplates and encompasses heterologous therapy.

[0125] Further taught herein is the use of an agent that antagonizes disulfide bond formation between C98 and C475 of TLR7 or positions corresponding thereto for the preparation of a medicament for inhibiting an autoimmune disease, a viral or microbial disease, inflammation or cancer in a subject.

[0126] Provided herein are agents that antagonize disulfide bond formation between C98 and C475 of TLR7 or positions corresponding thereto for use in inhibiting autoimmune diseases, viral or microbial diseases, inflammation, or cancer in a subject.

[0127] Still further provided herein is a pharmaceutical composition comprising an agent that antagonizes disulfide bond formation between C98 and C475 of TLR7 or between corresponding positions thereof and one or more pharmaceutical carriers, excipients and / or diluents.

[0128] Also provided herein are uses and agents for treating excessive production of reactive oxygen species or TLR7-mediated inflammation in a subject.

[0129] The agents include pharmaceutically acceptable salts of the agents. The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the agents of the invention: that is, salts that retain the desired biological activity of the parent agent and do not impart undesired toxicological effects thereto.

[0130] The pharmaceutical composition of the present invention can be applied in a variety of ways, depending on whether local or systemic treatment is needed and the area to be treated. Application can be local (including eye and mucosal administration, including vaginal and rectal delivery), pulmonary administration, such as by inhalation or blowing into powder or aerosol, including by sprayer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral administration. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial administration, such as intrathecal or intraventricular administration. Pharmaceutical compositions and preparations for topical application may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powdered or oily bases, thickeners, etc. may be necessary or desired.

[0131] The pharmaceutical preparation of the present invention can be prepared according to conventional techniques well known in the pharmaceutical industry, which can be conveniently presented in unit dosage form. Such techniques include the step of combining the active ingredient with one or more pharmaceutical carriers or excipients. Typically, the preparation is prepared by uniformly and closely combining the active ingredient with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, the product is molded.

[0132] The composition of the present invention can 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 composition of the present invention can also be formulated into suspensions in aqueous, non-aqueous or mixed media. The aqueous suspension can further include substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension can also include a stabilizer.

[0133] The formulations of the present invention include liposome formulations. As used in the present invention, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in one or more spherical bilayers. Liposomes are single-layer or multi-layer vesicles having a membrane formed by a lipophilic material and an aqueous interior containing the composition to be delivered.

[0134] The preparation of therapeutic compositions and their subsequent administration (dosing) are considered to be within the capabilities of those skilled in the art. Administration depends on the severity and responsiveness of the disease state to be treated, and the course of treatment, which lasts from several days to several months, or until a cure or a reduction in the disease state is achieved. The optimal dosing regimen can be calculated based on the measured results of drug accumulation in the patient. The optimal dose, method of administration, and repetition rate can be easily determined by a person of ordinary skill. The optimal dose can vary depending on the relative potency of the individual oligonucleotides, and can generally be determined based on the effective EC found in in vitro and in vivo animal models. 50S The dosage is estimated. Typically, the dosage is 0.01 μg to 100 g of peptide decoy per kg body weight, and can be administered once or more daily, weekly, monthly or annually, or even once every 2 to 20 years. One of ordinary skill in the art can easily estimate the repetition rate of administration based on the measured residence time and concentration of the drug in body fluids or tissues. After successful treatment, it may be desirable to have the patient receive maintenance therapy to prevent recurrence of the disease state, wherein the agent is administered at a maintenance dose of 0.01 μg to 100 g per kg body weight for several days, weeks or months.

[0135] The targeted cells are typically innate and adaptive immune cells or any cell expressing TLR7. Examples include phagocytes (e.g., macrophages, neutrophils, and dendritic cells), NK cells, mast cells, eosinophils, basophils, lymphocytes (including B- and T-lymphocytes), and epithelial cells.

[0136] Example

[0137] Aspects disclosed herein are further described by the following non-limiting examples.

[0138] Materials and methods

[0139] Virus

[0140] Influenza A virus (IAV) vaccine strains HKx31(H3N2) and BJxl09(H3N2) were provided by the Faculty of Medicine, Deakin University, the Department of Immunology and Microbiology, and the Peter Doherty Institute for Infection and Immunity, University of Melbourne. The viruses were expressed at 6.7 x 10 8 Plaque forming units / ml (PFU / ml) were provided and stored at -80° C. Aliquots were thawed on the day of use and diluted with phosphate buffered saline (PBS; Sigma Aldrich, St. Louis, USA, No. D837) when needed for in vitro infection. Human IAV viruses, 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 syncytial virus (A2 strain), human parainfluenza virus type 3 (C243), human metapneumovirus (CAN97-83 strain), mumps virus (Enders strain) and Newcastle disease virus (V4 strain) were provided by the Department of Immunology and Microbiology, Peter Doherty Institute for Infection and Immunity, University of Melbourne. Other 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, Peter Doherty Institute for Infection and Immunity); Sendai virus (Cantell strain, Hudson Institute for Medical Research, Monash University), herpes simplex virus type 2 (strain 186, Hudson Institute for Medical Research, Monash University), vaccinia virus (Western Reserve strain, WR NIH-TC; Australian National University) and HIV (NL4-3 (AD8)-EGFP strain, Peter Doherty Institute for Infection and Immunity, University of Melbourne). Viruses were provided in phosphate-buffered saline (PBS, catalog #D8537, Sigma, USA) and stored at -80°C until use. On the day of use, the virus was quickly thawed and incubated at 37°C before infection. Where indicated, the HKx31 virus was inactivated by heating (56°C) for 30 minutes or ultraviolet light (30 minutes).

[0141] bacteria

[0142] In all experiments, Streptococcus pneumoniae EF3030 (capsule type 19F) was used as the parental S. pneumoniae strain (provided by the University of Melbourne, Australia). The EF3030 strain is a clinical isolate that is often used as a model for human transport because it usually colonizes the nasopharynx without bacteremia. For infection experiments, pneumococci were grown statically in Todd-Hewitt broth at 37°C and supplemented with 0.5% w / v yeast extract to an optical density (600nm) of 0.4-0.45. The culture was placed on wet ice for 5 minutes and frozen in 8% v / v glycerol at -70°C. Viable bacterial counts were confirmed before each experiment. As we have previously shown (King et al. (2013) The Journal of Allergy and Clinical Immunology 131(5): 1314-1321e1314), a clear non-typeable Haemophilus influenzae (NTHi; MU / MMC-1) strain was previously typed and sequenced to prove that it was NTHi.

[0143] Custom C98i Peptide

[0144] 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-no TAT; 10 amino acid TLR7 inhibitor, except HIV-TAT). DFRCNCVPIP-NH2 (SEQ ID NO: 26) (human C98i-no TAT; 10 amino acid TLR7 inhibitor, except HIV-TAT). RCNC-NH2 (SEQ ID NO: 45) (4AA C98i-no TAT; 4 amino acid TLR7 inhibitor, except HIV-TAT). RANC-NH2 (SEQ ID NO: 46) (4AA 98M C98i-no TAT; cysteine ​​98 mutation, 4 amino acid TLR7 inhibitor, except HIV-TAT). RANA-NH2 (SEQ ID NO: 47) (4AA 98100M C98i-no TAT; cysteine ​​98 and 100 mutations, 4 amino acid TLR7 inhibitor, except HIV-TAT). RCNA-NH2 (SEQ ID NO: 48) (4AA 100M C98i-no TAT; cysteine ​​100 mutation, 4 amino acid TLR7 inhibitor, except HIV-TAT). All peptides were dissolved in endotoxin-free water and prepared as 10mM stock solutions in 20μL, 50μL and 100μL aliquots. Store at -20°C.

[0145] Conjugation of NOX2 oxidase inhibitors

[0146] Preparation of gp91 ds-tat (YGRKK-RRQRR-RCSTR-IRRQL-NH2-SEQ ID NO: 23) was performed by standard Fmoc solid phase peptide synthesis (SPPS) on Fmoc-PAL-PEG-PS resin (LifeTechnologies, USA, loading at 0.17 mmol / g). Fmoc deprotection reactions were performed using 20% ​​v / v piperidine in N,N dimethylformamide (DMF). Coupling reactions were performed using Fmoc protected amino acids with O13 (6-chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HCTU) as coupling agent and N,N-diisopropylethylamine (DIPEA) as activator. The reaction was monitored using 2,4,6-trinitrobenzenesulfonic acid (TNBS) test to indicate the presence or absence of free amino groups. All 20 amino acid residues were manually subjected to alternating deprotection and coupling reactions using the appropriate Fmoc and side-chain protected amino acids. After the final deprotection step, a small portion of the peptide was cleaved from the resin using trifluoroacetic acid (TFA) / triisopropylsilane (TIPS) / 1,2-ethanedithiol (EDT) / water (92.5:2.5:2.5:2.5) for 4 hours, removing the side-chain protecting groups in the process. The peptide was then analyzed by reverse phase high pressure liquid chromatography (HPLC) using a Phenomenex Luna 5C8 (2) AXIA Column( The crude peptide was purified using a 4% 4-nitropropene precipitate (250 x 21.2 mm) and 0.1% TFA / water and 0.1% v / v TFA / ACN as buffer solutions. ESI-MS analysis confirmed that the purified gp91 ds-tat peptide had the correct molecular weight: calculated for C109H207N52O25S [M+5H+]: m / z 535.3, observed: m / z 535.7; C 109 H 208 N 52 O 25 Calcd. for S[M+6H+]: m / z 446.3, observed: m / z 446.6; C 109 H 209 N 52 O 25 Calcd. for S[M+7H+]: m / z 382.7, observed: m / z 382.9.

[0147] Preparation of cholesteryl-conjugated gp91 ds-tat (cgp91 ds-tat; Ac-Asp(OChol)-PEG4-PEG3-PEG4-gp91-NH2) was performed from resin-bound gp91 ds-tat by manual SPPS using Fmoc-PEG4-OH, Fmoc-PEG3-OH, Fmoc-PEG4-OH, and Fmoc-Asp(OChol)-OH as amino acids (described above). 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 the resin using TFA / TIPS / EDT / water (92.5:2.5:2.5:2.5). The crude peptide was purified as described above to yield cgp91 ds-tat: C 173 H 319 N 56 O 43 Calcd. for S[M+5H+]: m / z 780.3, observed: m / z 780.6; C 173 H 320 N 56 O 43 Calcd. for S[M+6H+]: m / z 650.4, observed: m / z 650.7; C 173 H 321 N 56 O 43 Calcd. for S[M+7H+]: m / z 557.6, observed: m / z 558.0.

[0148] Preparation of ethyl ester-conjugated gp91 ds-tat (egp91 ds-tat; Ac-Asp(OEt)-PEG4-PEG3-PEG4-gp91-NH2) was performed in the same manner as cgp91 ds-tat, except that Fmoc-Asp(OEt)-OH was substituted for Fmoc-Asp(OChol)-OH in the final coupling step: C 148 H 277 N 56 O 43 Calcd. for S[M+5H+]: m / z 711.8, observed: m / z 712.1; C 148 H 278 N 56 O 43 Calcd. for S[M+6H+]: m / z 593.3, observed: m / z 593.7; C 148 H 279 N 56 O 43Calcd. for S[M+7H+]: m / z 508.7, observed: m / z 509.0.

[0149] 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 performed by manual SPPS as described above for the non-scrambled gp91 ds-tat. The resin-bound sgp91 ds-tat was then conjugated to cholesterol via a PEG linker using the same method as described above for the non-scrambled cgp91 ds-tat. The crude peptide was purified in the same manner to yield cgp91 ds-tat: C 162 H 307 N 54 O 40 S[M+5H+] calculated value: m / z 736.3, observed value: m / z 736.5; C 162 H 308 N 54 O 40 Calcd. for S[M+6H+]: m / z 613.7, observed: m / z 614.0; C 162 H 309 N 54 O 40 Calcd. for S[M+7H+]: m / z 526.2, observed: m / z 526.3.

[0150] Influenza A virus infection and drug treatment in vivo

[0151] Age-matched (6-12 weeks) littermate males were non-immunized WT controls and NOX2 - / y mouse (also known as gp91phox - / -[Pollock et al. (1995) Nature Genetics 9(2): 202-209]) Mice were anesthetized by pentane inhalation and infected intranasally (in) with 1×104 or 1×105 plaque forming units (PFU) of Hkx31 in a volume of 35 L diluted with PBS. Mice were euthanized on days 1, 3, or 7 after influenza infection. In some experiments, anesthetized mice were treated with dimethyl sulfoxide (DMSO, control; Sigma), unconjugated gp91dstat (0.02 mg / kg, 0.2 mg / kg), cholesterol-conjugated gp91dstat (0.02 mg / kg, 0.2 mg / kg), or cholesterol-conjugated scrambled gp91ds-TAT (0.02 mg / kg) delivered intranasally one day before Hk-x31 infection and then treated daily for 3 days. In other experiments, anesthetized mice were treated with imiquimod (50 μg / mouse, in) or catalase (1000 U / mouse, in) and then euthanized on day 1 for analysis.

[0152] Airway inflammation and counts of different cells

[0153] Mice were killed by intraperitoneal (ip) injection of a ketamine / xylene (100 mg / kg) mixture. An incision was made from the mandible to the top of the thorax, where the salivary glands were separated to expose the tracheal surface. The smooth muscle layer on the trachea was removed and a small incision was made near the top of the trachea. A sheathed 21-gauge needle was inserted into the lumen and lavage was repeated with 300-400 μl PBS (4 times). The total number of cells in the BALF was stained with 0.4% w / v trypan blue solution (Thermofisher Scientific, USA), and live cells were assessed using Countess (registered trademark) automatic cell counter (Invitrogen, catalog number # C10227). Different cell analyses were prepared from BALF (5x104 cells), which was centrifuged at 3xg for 5 minutes on Cytospin 3 (Shandon, UK). Thereafter, the slides were fixed in 100% v / v propanol for 1 minute and then dried overnight. Finally, the sample was dyed with RapidI Aqueous Red StainTM (AMBER Scientific, Australia) and Rapid II Blue StainTM (AMBER Scientific, Australia) for 10 minutes, then immersed in 70% v / v ethanol and absolute ethanol twice, and then placed in xylene for 5 minutes (2 times). The sample was then encapsulated in DPX encapsulation medium (Labchem, NSW, Australia), and a cover glass was firmly placed on top. By standard morphological criteria, 500 cells of each sample from a random field of view were divided into macrophages, neutrophils, eosinophils and lymphocytes. Data are expressed as the total cell number calculated by each cell type multiplied by the total number of living cells, and the percentage of cell population.

[0154] Cell culture and primary cell isolation

[0155] Human alveolar macrophages were obtained from subjects undergoing bronchoscopy at Monash Medical Center, Monash University, Clayton, Australia, to investigate underlying lung disease. The bronchoscope was wedged into the right middle lobe and the airways were flushed with 25-50 mL of saline followed by aspiration. The cells were washed twice with PBS before use and then suspended in culture medium (Roswell Park Memorial Institute (RPMI, Life Technologies, catalog #21870-076) containing 10% v / v FCS, 100 U / mL penicillin, and 100 g / mL streptomycin) for approximately 24 hours.

[0156] The pulmonary lavage was performed on age-matched (6-12 weeks) male C57Bl / 6J (WT), NOX2 - / y 、NOX4- / - (Provided by the Eye Research Centre, University of Melbourne, Australia), TLR7 - / - (Courtesy of School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, The University of Newcastle, Hunter Research Institute of Medical Research, New South Wales, Australia), TLR9 - / - (Provided by Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia) or SOD3 - / - Alveolar macrophages were isolated from mice (provided by the School of Health and Biomedical Sciences, RMIT University). A thin, shallow midline incision was made from the mandible to the top of the thorax, and the larynx was separated to expose the top of the trachea. The smooth muscle layer covering the trachea was removed, a small incision was made and a sheathed 21-gauge needle was inserted into the lumen. The lungs were repeatedly lavaged (3 times) with 300-400L PBS. The cell suspension was centrifuged (200xg at 4°C for 5 minutes). The supernatant was removed, and the cells were resuspended in 1mL of sterile PBS and counted using a Countess (registered trademark) automated cell counter (Invitrogen, catalog number #C10227). The cells were then seeded into 24-well plates (1×105 cells / well) for immunocytochemistry and fluorescence microscopy as described below.

[0157] Immortalized cell line RAW 264.7 cells (RAW 264.7 (ATCC (registered trademark) TIB-71 (trademark)) and immortalized bone marrow-derived macrophages (BMDM; provided by the Hudson Institute for 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). TLR2 - / - , TLR3 - / - , TLR4 - / - 、TLR7 - / - TRIF - / - 、RIG-I - / - 、MyD88 - / - NLRP3 - / - and UNC93B1 - / -Immortalized BMDM were maintained in RPMI culture medium supplemented with glucose (4500 mg / L), non-essential amino acids, sodium pyruvate, streptomycin and FBS (10% v / v) and DMEM (20% v / v) (including all supplements as described above). All cells were maintained at 37° C. in a humidified mixture of 5% CO2 and 95% v / v air. The culture medium was changed 2 to 3 times per week, and when the confluence was about 80-90%, the cells were subcultured by scraping and counted using a Countess (registered trademark) automated cell counter.

[0158] Immortalized cell line RAW 264.7 cells (derived from mouse peritoneum) and immortalized bone marrow-derived macrophages (BMDM) were maintained in Dulbecco's modified Eagle's medium (DMEM: Sigma), which was supplemented with L-glutamine, glucose (4500 mg / L), sodium pyruvate (110 mg / L) and fetal bovine serum (FBS; 10%). All cells were maintained in a humidified mixture of 5% CO2 and 95% air at 37°C. The culture medium was changed 2 to 3 times a week, and when the confluence was about 80-90%, the cells were subcultured by scraping and counted using a Countess (registered trademark) automated cell counter (Invitrogen).

[0159] Confocal fluorescence microscopy

[0160] Cells were seeded onto glass coverslips of 24-well plates and allowed to adhere in DMEM for 24 hours. Cells were then incubated at different time points (5 minutes, 15 minutes, 30 minutes and 1 hour) in serum-free 16 culture medium in the absence or presence of HKx31 influenza A virus (MOI 0.1, 1 or 10). In some cases, cells were pretreated for 30 minutes before infection with Dynasore (100M) or Dynasore's empty vector DMSO (0.1% w / v). Next, cells were washed with PBS (0.01M) and fixed with 4% v / v paraformaldehyde (PFA) for 15 minutes. Cells were treated with PBS containing Triton X-100 (0.25% v / v) for 10 minutes, then washed 3 times with PBS for 15 minutes. Samples were then incubated for 2 hours with PBS containing 10% v / v goat serum, and / or in mouse IgG blocking agent (catalog number #MKB-2213, Vector Laboratories). Subsequently, a primary antibody to nucleoprotein (1:1000) was added for 24 hours at 4°C to localize influenza A virus, purified mouse anti-NOX2 (1:500) was added to localize NOX2, rabbit anti-TLR7 (1:1000) was added to localize TLR7, or mouse anti-early endosomal antigen 1 (EEA1; 1000) was added to localize early endosomes. In some experiments, a combination of antibodies was used at the indicated concentrations to determine the co-localization of proteins. The cells were washed three times with PBS (0.01 M) within 30 minutes. After washing, secondary antibodies goat anti-rabbit alexa 594 (1:1000), goat anti-rabbit red 647 (1:500, 1:1000) and / or biotinylated anti-mouse IgG were added to appropriate wells for 2 hours in the dark. Finally, the cells were washed three times with PBS (0.01 M) within 30 minutes; and where appropriate (mouse primary and secondary antibodies Fluroscein Avidin DCS were applied for 5 minutes). Coverslips were mounted onto microscope slides using 10-20 μL of diamidino-2-phenylindole (DAPI) for 3 minutes. Slides were observed and photographed on a Nikon upright inverted confocal fluorescence microscope (Nikon D-eclipse C1). All immunohistochemistry was evaluated by two observers blinded to treatment groups throughout the analysis, and all appropriate controls were performed for all combinations of primary and secondary antibodies used to ensure that no cross-reactivity occurred.

[0161] By examining the - / - and NOX2 - / - The staining degree in mouse alveolar macrophages verifies the specificity of TLR7 and NOX2 antibodies. - / - There was no TLR7 staining in macrophages ( Figure 2C). Similarly, the expression of NOX2 in NOX2 cells was significantly higher than that in WT cells using NOX2 antibody. - / y No staining was observed in mouse alveolar macrophages. Further evidence for the specificity of this NOX2 antibody can be found in Judkins et al. (2010) American Journal of Physiology Heart and Circulatory Physiology 298(1):H24-32.

[0162] Endosomal ROS production

[0163] Human alveolar macrophages; WT, TLR7 - / - , TLR2 - / - , TLR3 - / - , TLR4 - / - TRIF - / - 、MyD88 - / - 、RIG-I - / - and NLRP3 - / - BMDM; primary mouse WT, NOX2 - / y 、NOX4 - / - 、TLR7 - / - , TLR9 - / - or SOD3 - / - Alveolar macrophages and RAW264.7 cells were inoculated (1×10 5Cells were plated at 400 μg / ml and plated on glass coverslips in 24-well plates (100 cells / well), allowed to adhere for 24 h in DMEM or RPMI medium, and then pretreated with OxyBURST Green H2HFF (100 M) and / or LysoTracker Deep Red (50 nM) for 5 min. The cells were then incubated with PBS (control group; 0.01 M), imiquimod (10 g / mL), single-stranded RNA (ssRNA; 100 M), or infected with H3N2 influenza virus (A / New York / 55 / 2004, A / Brisbane / 9 / 2007), seasonal H1N1 influenza A virus (A / Brazil / 11 / 1978, A / New Caledonia / 20 / 1999, A / Solomon Islands / 3 / 2006), A(H1N1)pdm influenza A virus (A / California / 7 / 2009, A / Auckland / 1 / 2009) in serum-free medium at different time points (5 min, 15 min, 30 min and 1 h), or with the reassortant vaccine strains HKx31 (MOI 0.1-10) or BJxl09 (MOI 10). 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 rotavirus (MOI 10 each), 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 M), gp91dstat (50 M), or bafloxacin A (100 nM) for 30 minutes before infection. Next, cells were washed with PBS (0.01 M) and fixed with 4% PFA for 15 minutes. After fixation, cells were then washed 3 times with PBS within 30 minutes. Coverslips were then mounted on microscope slides with 10-20 μL DAPI for 3 min and analyzed and photographed on a Nikon upright confocal fluorescence microscope (Nikon D-eclipse C1).

[0164] NOX2 oxidase assembly

[0165] To measure NOX2 oxidase activity, we assessed p47phox and NOX2 assembly using confocal fluorescence microscopy. - / -Alveolar macrophages were treated as described above under "confocal fluorescence microscopy". In additional experiments, WT cells were treated with Dynasore (100M) or bafloxacin A (100nM) for 30 minutes before viral infection. After exposing samples for 2 hours with 10% v / v PBS containing goat serum, rabbit anti-p47phox antibody (1:1000) and mouse anti-NOX2 antibody (1:500) were added, followed by the addition of appropriate secondary antibodies as described above.

[0166] L-O12 enhanced chemiluminescence

[0167] L-O12 enhanced chemiluminescence was used to quantify ROS production. RAW264.7 cells and primary mouse alveolar macrophages were seeded into 96-well OptiView plates (5 × 10 4 Cells / well). RAW264.7 cells were treated with DMSO (control, appropriate concentration), unconjugated gp91dstat (100 nM-30 M), cholesterol-conjugated gp91dstat (100 nM-30 M) or ethyl-conjugated gp91dstat (100 nM-30 M) for 1 h. BALF was collected from mice treated with DMSO (control), unconjugated gp91dstat (0.02 mg / kg, 0.2 mg / kg), cholesterol-conjugated gp91dstat (0.02 mg / kg, 0.2 mg / kg) and / or infected with Hkx31 influenza A virus (1×105 PFU). The cells were then washed with a medium of 37°C Krebs-HEPES buffer and then exposed to Krebs-HEPES buffer containing L-O12 (10-4mol / L) in the absence (i.e., basal ROS production) or presence (stimulated ROS production) of PKC and NADPH oxidase activator phorbol dibutyrate (PDB; 10-6mol / L). The same treatment was performed in blank wells (i.e., without cells), which served as a control for background luminescence. All treatment groups were performed in triplicate. Photon emission [relative light units (RLU) / s] was detected using a Chameleon (trademark) luminescence detector (Hidex, model 425105, Finland), and 1s per well was recorded for 60 cycles. Each data point for each group was derived from the mean of three replicates minus the corresponding blank control. Data are expressed as control % or original value (in vitro experiments) in dose-response curves.

[0168] In order to test whether unconjugated or cholesterol-conjugated gp91dstat exhibits ROS scavenging properties, a xanthine oxidase cell-free assay was used. In brief, Krebs-HEPES buffer containing L-012 (100M) was added to a 96-well Optiview plate. Subsequently, 0.1% w / v DMSO, unconjugated gp91dstat (Ugp91ds-TAT, 1M) or cholesterol-conjugated gp91ds-TAT (1M) was added in combination with xanthine (100M). After adding xanthine oxidase (0.03U / mL), Chameleon (trademark) luminescence detector (Hidex, model 425105, Finland) was used to detect photon emission [relative light unit (RLU) / s] immediately, and 1s was recorded from each well for 60 cycles. Each data point of each group was obtained by subtracting the corresponding blank control from the mean of three replicates. The data are expressed as raw values.

[0169] Site-directed mutagenesis, sequencing, and transfection

[0170] HA-TLR7 cDNA was purchased from Sino Biological (mouse TLR7; catalog #MG50962-NY, gene bank reference sequence number NM_133211.3). Key cysteine ​​residues in TLR7 (Cys260, Cys263, Cys270, Cys273, Cys98, and Cys445) were mutated to alanine using the QuikChange multisite directed mutagenesis kit (catalog #200514, Agilent Technologies). The sequences of WT and mutant HA-TLR7 have been confirmed by the Australian Genome Research Institute. Cells were transfected using linear polyethyleneimine (PEI) [Halls et al. (2015) Methods in Molecular Biology 1335: 131-161].

[0171] High-content ratiometric FRET imaging

[0172] Cells were plated in black, optically clear 96-well plates and transfected with PEI in a suspension of 200 ng / well FRET biosensor DNA for 48 hours. Prior to the experiment, cells were partially serum starved overnight in 0.5% v / v FBS medium. Fluorescence imaging was performed using a high-content GE Healthcare INCell 2000 analyzer and a Nikon Plan Fluor ELWD 40x (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), where emission was measured using YFP (535 / 30) and CFP (470 / 24) filters, and polychromaticity was 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), where emission was measured using dsRed (605 / 52) and FITC (525 / 36) filters, and polychromaticity was optimized for the FITC / dsRed filter pair (Quad4). Cells were imaged every 100 seconds for 20 minutes (2 fields of view were captured in 12 wells every 100 seconds). Data were analyzed using an in-house script written for the FIJI distribution of Image J 34, as described (Halls et al. (2015) supra.

[0173] Quantification of mRNA by QPCR

[0174] Cells were treated with imiquimod (10 g / ml), poly I:C (100 ng / ml), CpG (10 g / mL), ssRNA (500 g / mL) or catalase (1000 U / ml) for 24 hours. Where indicated, cells were pretreated with apocynin (300 M), SOD (300 U / mL) or bafloxin A (100 nM) for 30 minutes. RNA was extracted from lung tissues of mice treated with DMSO (control), unconjugated gp91dstat (0.02 mg / kg, 0.2 mg / kg), cholesterol-conjugated gp91dstat (0.02 mg / kg, 0.2 mg / kg), scrambled cholesterol-conjugated gp91dstat (0.02 mg / kg) and / or infected with Hk-x31 influenza A virus (1×105 PFU) 3 days after infection to evaluate the expression of viral mRNA and cytokines. The right lung lobe was placed in an Eppendorf tube containing a mixture of BufferRLT (Qiagen, USA) and -mercaptoethanol (Sigma; 1%) and cut into small pieces with curved scissors. Thereafter, the lung sample was homogenized using an ultrasonic homogenizer (Hielscher Ultrasonics GmBH, Teltow, Germany) and centrifuged at 14000rpm for 5 minutes. The lysate in a 1:1 ratio was mixed with 70% v / v RNase-free ethanol and transferred to an RNeasy centrifuge column (RNeasy Minikit; catalog number #74104, Qiagen). The sample was rotated at 10000rpm for 15 seconds and then washed with RW1 buffer. After discarding the effluent, 5 1DNase I (catalog number #79254, Qiagen) was mixed with 35l RDD buffer, directly pipetted onto the membrane of the centrifuge column, and incubated at room temperature for 15 minutes. RPE buffer was added and centrifuged at 10000rpm for 15 seconds. After discarding the effluent, RPE buffer was added again and rotated at 10000rpm for 2 minutes. Rotated at 14000rpm for another 1 minute to remove the residue from the spin column. Finally, RNase-free water was added and centrifuged to elute RNA into Eppendorf tubes. RNA samples were measured using Nanodrop 1000 spectrophotometer (Thermo Scientific, the U.S.). High-capacity cDNA reverse transcription kit (catalog number #4368814, Applied Biosystems, FosterCity, California, USA) was used to synthesize cDNA using 1.0-2.0g total RNA. RNA was added to the reagent mixture in the high-capacity cDNA RT kit to make the final volume 20 l.BioRad Mycycler (trademark) thermal cycler (BioRad, USA) was used to set up transcription according to the following settings: 25°C for 10 minutes, 37°C for 120 minutes, 85°C for 5 minutes and static 4°C. Samples were stored at -20°C before use. Quantitative polymerase chain reaction was performed using TaqMan Universal PCR Master Mix (Catalog #4304437, Applied Biosystems, Foster City, California, USA) or SYBR Green PCR Master Mix (Catalog #4367659, Applied Biosystems, Foster City, California, USA) and analyzed on ABI Step One TM and StepOnePlus TM real-time PCR systems (Perkin-Elmer Applied Biosystems, Foster City, California, USA). PCR primers for TNF-, IL-1, IFN- and IL-6 were included in Assayon-Demand Gene Expression Assay Mix. In addition, the forward and reverse primers of the influenza virus fragment 3 polymerase (PA) designed by custom design were used to measure viral titers (Table 4). PCR program run settings: 50 ° C 2 minutes, then 95 ° C 1 hour, then 95 ° C 15 s + 60 ° C 60 s + plate reading (40 cycles). Quantitative value 129 is obtained from the threshold cycle (Ct) number. The target gene expression level of each sample is normalized for 18s or GAPDH mRNA expression, and the data are expressed as relative to the control.

[0175] RAW264.7 or BMDM cells were seeded into 6-well plates (5 × 10 5 cells / well). For all iterations of C98i peptide, cells were pretreated 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 treatment, cells were washed with PBS, medium was supplemented with fresh DMEM (10% FBS), and left to incubate for 24 hours.

[0176] Total RNA was prepared using RNeasy Mini Kit (Qiagen, Hilden, Germany), and then purified by extraction with double distilled water. The synthesis of cDNA was performed using a high-capacity cDNA RT kit (P / N4322171, Applied Biosystems, Foster City, California, USA), using 1.0-3.0 μg of total RNA. Quantitative polymerase chain reaction was performed using TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, California, USA), and analyzed on ABI StepOneTM and StepOnePlusTM real-time PCR systems (Perkin-Elmer Applied Biosystems, Foster City, California, USA). PCR primers for IL-1 and IL-6 were included in Assayon-Demand Gene Expression Assay Mix (Applied Biosystems, Foster City, California, USA). The running settings of the PCR program were: 50°C for 2 minutes, then 95°C for 1 hour, then 95°C for 15s+60°C for 60s+ plate readings (40 cycles). Quantitative values ​​were obtained from the threshold cycle (Ct) number. The target gene expression level of each sample was normalized to 18s or GAPDH mRNA expression, and the data were expressed relative to WT without immunization.

[0177] ELISA and multiplex immunoassays

[0178] Secretion of wild-type and NOX2 into HKx31 cells infected with 1 × 104 PFU was measured using ELISA - / y Protein levels of IFN-(VeriKine HM mouse IFN serum ELISA kit; catalog #42410-1, PBL Assay Science), IL-1 (Quantikine ELISA mouse IL-1 / IL-IF2; catalog #MLB00C, R&D Systems), TNF-α (Quantikine ELISA mouse TNFα, catalog #MTA00B, R&D Systems) and IL-6 (Quantikine ELISA mouse IL-6, catalog #M6000B, R&D Systems) in mouse BALF were determined using commercially available kits according to the manufacturer's instructions. The cytokine titers in the samples were determined by plotting the optical density using a 4-parameter fit of the standard curve.

[0179] Antibody assay

[0180] HKx31-infected (1×10 4 PFU)WT and NOX2 - / y Serum and BALF levels of different antibody isotypes (IgA, IgE, IgG1, Ig2a, IgG2b, IgG3, IgM and total IgG) in mice. In brief, magnetic beads conjugated to antibodies were added to 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 1:20000 in universal assay buffer) and / or standards were added to appropriate wells of a 96-well plate containing magnetic beads conjugated to antibodies. Then, the detection antibody mixture was added to each well, and the plate was incubated at room temperature for 30 minutes in the dark on a microplate shaker (500 rpm). After washing, read buffer was added to all wells. The plate was read with a Magpix (registered trademark) multiplex reader (Luminex, USA) and xPONENT (registered trademark) software (Luminex, USA). The serum and BALF antibody concentrations in each sample were interpolated from the standard curve using Procartaplex (trademark) Analyst 1.0 software (eBioscience, USA).

[0181] Data are presented as mean + standard error of the mean (SEM). Cytokine mRNA expression and antibody titers were analyzed using one-way analysis of variance (ANOVA) followed by Tukey post hoc test for multiple comparisons. All tests were performed by Graphpad Prism 7.0b (San Diego, CA, USA), and statistical significance was set at P < 0.05.

[0182] Statistical analysis and image analysis

[0183] To quantify the fluorescence microscopy data, images obtained from the confocal system were analyzed in Image J. Unless otherwise indicated in the figure numbers, approximately 100-150 cells per treatment group were analyzed by at least three independent experiments to calculate the fluorescence in each cell, then averaged and expressed as a percentage of the fluorescent area. All statistical tests were performed using GraphPad Prism (GraphPad Software Version 6.0, San Diego, CA, USA). P < 0.05 indicates significance. For separate cell culture work, n represents a single experiment in which cells from different passages were used.

[0184] Chemicals

[0185] Imiquimod (Cat. #tlrl-imq, Invivogen), HMW poly I:C (Cat. #tlrl-pic, Invivogen), and CpG ODN (Cat. #tlrl-1668, Invivogen) were dissolved in endotoxin-free water and prepared as 5-10 mg / mL stock solutions in 30 L and 100 L aliquots and stored at -20°C. ssRNA (Cat. #trl1-lna40, Invivogen) was dissolved in endotoxin-free water and prepared as 5 mM stock solutions in 50 L aliquots and stored at -20°C. Dynasore (Cat. #D7693, Sigma) (prepared fresh that day) was dissolved in DMSO (100%) to make a 10 mM stock solution. FBS (Cat. #12003C, Sigma) was stored in 50 ml aliquots at -20°C. Penicillin-streptomycin solution (Catalog # P4333, Sigma) was stored at -20°C. SOD (Catalog # S2515, Sigma) was dissolved in distilled water to make a stock solution of 30,000 units / ml (10 1) and stored at -20°C. Stock solutions (1 mg / mL) of OxyBURSTGreen H2HFF bovine serum albumin (BSA) (Catalog # 1329, Molecular probes, Life Technologies) and LysoTracker Deep Red (Catalog # L12492, Molecular probes, Life Technologies) were generated immediately before use by dissolving in PBS. Baflomycin A (from Streptomyces, Catalog # B 1793, Sigma) was prepared as a 100 M stock solution in 10 L aliquots and stored at -20°C. Apocynin (4'-hydroxy-3'-methoxyacetophenone, catalog #A10809; Sigma) and gp91dstat (catalog #AS-63818; Anaspec) were prepared fresh on the day as 100 mM and 50 mM stock solutions in 100% v / v DMSO, respectively. Phorbol dibutyrate (catalog #P1239; Sigma) was dissolved in 100% v / v DMSO as a 10 mM stock solution and prepared fresh on the day of use. Catalase (catalog #C1345, Sigma) was prepared as a 10 6U / ml stock solution in distilled water and stored at -20°C. 5 mM MitoSOX (Catalog #M3600850; Molecular Probes, Life Technologies) was prepared by dissolving the contents of one vial of MitoSOX (trademark) mitochondrial superoxide indicator (Component A) (50 g) in 13 L DMSO. Xanthine oxidase (Catalog #X1875; Sigma) was prepared fresh on the same day by dissolving in distilled water to 30 U / ml, and xanthine (Catalog #X0626; Sigma) was prepared as a 100 mM stock solution in 0.1 M NaOH.ML171 (Cat #492002; Calbiochem).

[0186] Antibodies to influenza virus nucleoprotein (mAb to influenza A virus nucleoprotein [AA5H]; catalog #120-20343, AbCAM), antibody to early endosomal antigen 1 (catalog #120-02900, AbCAM), mouse anti-gp91phox antibody (catalog #611415, BD Transduction Laboratorie, purified mouse anti-gp91[phox] clone 53 / gp91[phox] (RUO), rabbit anti-TLR7 (catalog #NBP2-24906, Novus Biologicals), rabbit anti-p47phox antibody (catalog #scl4015, Santa Cruz), FITC goat anti-mouse IgG (catalog #A-11029; Invitrogen), goat anti-rabbit alexa 594 (catalog #A-11037; Invitrogen), goat anti-rabbit far red 647 (Catalog #A-21244, Invitrogen) and DAPI (Catalog #H-1200, Vector Laboratories) were stored at -20°C.

[0187] Example 1

[0188] Influenza virus drives endosomal ROS

[0189] To address the potential role of endosomal ROS generation in viral pathology, we first considered influenza A virus, which belongs to the group IV reverse-stranded ssRNA virus of the family Orthomyxoviridae and is internalized via endocytosis. Exposure of mouse alveolar macrophages (AMs), mouse peritoneal RAW264.7 cells, or bone marrow-derived macrophages (BMDMs) to influenza A strain HKx31(H3N2) resulted in a dose- and time-dependent increase in influenza nucleoprotein (NP) fluorescence, which was nearly abolished by the dynamin inhibitor, Dynasore (100 M), suggesting a clathrin-coated pit- or caveolin-dependent internalization mechanism. Internalized virus showed strong co-localization with the early endosomal marker EEA1 ( Figure 1A However, not all NPs co-localized with EEA1, suggesting that influenza A virus is not exclusively present in early endosomes ( Figure 1A ), which may have entered late endosomes and / or lysosomes. NOX2 co-localized with EEA1 in control and influenza-infected cells ( Figure 1B ). Thus, an enzymatic machinery for ROS generation exists in early endosomes and is significantly enhanced in influenza A virus infection, promoting co-localization with internalized virus. Endosomal ROS production in response to viral uptake was assessed using OxyBURST16. Exposure to a range of seasonal and pandemic influenza A viruses of low to high pathogenicity resulted in a decrease in primary AM in mice ( Figure 1C and Figure 1D ) and human alveolar macrophages ( Figure 1H ) increased rapidly and dose-dependently. The OxyBURST-derived signal was eliminated by the addition of superoxide dismutase (SOD; 300 U / mL), which is internalized into endosomes along with the virus 17 and converts superoxide to H2O2 ( Figure 1E , Figure 1F In contrast, in mice from endosomal SOD-deficient mice (SOD3 - / - In the AM of mice, the ROS signal was significantly increased, thus establishing the detection of superoxide derivatives. To confirm that ROS generation occurred in acidic endosomes, co-localization of OxyBURST fluorescence was confirmed with LysoTracker (50 nM) in the presence of influenza virus ( Figure 1G Inhibition of the alveolar V-ATPase pump with bafloxacin A (100 nM), thereby inhibiting endosomal acidification, abolished LysoTracker fluorescence and endosomal ROS generated in response to influenza A virus infection ( Figure 1G ). Endosomal ROS in NOX2 - / y Minimum in alveolar macrophages, but in NOX4 - / -Macrophages and macrophages treated with the NOX1 inhibitor ML171 (100 nM) were not affected ( Figure 1E and Figure 1F ). - / y Internalization of influenza A virus into AM was not impaired in cells, indicating that reduced endosomal ROS production was not due to reduced viral entry. In addition, heat-inactivated and UV-inactivated forms of influenza virus (replication-deficient) resulted in an increase in endosomal ROS production that was similar to that of live virus controls ( Fig. 1I and Figure 1J ). Thus, regardless of subtype, strain, and pathogenicity, influenza A virus stimulates NOX2, but not NOX4 or NOX1 oxidase-dependent ROS production in endosomes, which is involved in endosomal acidification but is not required for viral replication.

[0190] Example 2

[0191] Endosomal TLR7-NOX2 signaling axis

[0192] RNA viruses are regulated 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 (for dsRNA viruses), as well as the cytoplasmic sensors retinoic acid-inducible gene 1 (RIG-1) (which can detect viral RNA with a 5' triphosphate (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 is hypothesized that influenza A virus enters acidified endosomes, leading to the release of viral RNA, activation of TLR7, and stimulation of NOX2 oxidase-dependent ROS production. Consistent with this proposal, TLR7 is associated with influenza A virus ( Figure 2A )、NOX2( Figure 2B ) and EEA1( Figure 2C ) co-localizes with TLR7 - / - Primary AMs from mice as well as TLR7- and MyD88-deficient BMDMs showed minimal endosomal ROS production in response to influenza A virus ( Figure 2D ). TLR7- / - and MyD88 - / - The lack of endosomal ROS production in cells in response to the virus was not due to reduced capacity of the NOX2 oxidase itself, as activation of NOX2 by the PKC activator phorbol dibutyrate (PDB; 10-6 M) in these cells was similar to that in WT control cells. As a second measure of NOX2 oxidase activity, we examined the binding of the NOX2 catalytic subunit to p47 phox The extent of subunit association was regulated to assess enzyme assembly. In unstimulated cells, NOX2 and p47 phox There is little co-localization ( Figure 2E ). However, influenza virus induces NOX2 and p47 phox Strong co-localization was reduced by pretreatment with Dynasore or bafloxacin A and was also observed in TLR7 - / - Co-localization in cells almost disappeared ( Figure 2E To provide further evidence that TLR7 activation leads to the generation of endosomal ROS, the specific TLR7 agonist imiquimod (10 μg / mL) was used. Imiquimod significantly increased endosomal ROS in AMs of humans and wild-type mice, but did not increase NOX2 - / y Mouse ( Figure 2F ) or endosomal ROS in macrophages lacking TLR7 or MyD88. Finally, AM or RAW264.7 cells were pulsed with a guanidine- and uridine-rich ssRNA sequence (ssRNA40; 100M). At concentrations that increase IL-1, IL-6, and TNF-mRNA via a TLR7-dependent mechanism, ssRNA40 led to elevated endosomal ROS production ( Figure 2G In contrast, endosomal ROS production in response to influenza A virus is expressed in RIG-I - / - NLRP3 - / - , TLR2 - / - and TLR4 - / - The expression of β-actin was retained in macrophages and macrophages treated with TLR3 inhibitor (50 M).

[0193] We then examined how TLR7 triggers the assembly and activation of endosomal NOX2 oxidase, which can be activated by protein kinase C, triggering p47 phox To define the spatiotemporal regulation of PKC signaling and assess its regulation by TLR7, we expressed the FRET biosensor cytoCKAR to detect the expression of WT and TLR7 proteins. - / -Cytoplasmic PKC in macrophages (Jensen et al. (2014) supra; Violin et al. (2003) The Journal of Cell Biology 161(5):899-909; Halls et al. (2015) supra). Treatment of WT macrophages with influenza A virus or imiquimod increased cytoplasmic PKC activity within 5 minutes, but did not activate PKC in the presence of TLR7. - / - This response was absent in WT macrophages and in Dynasore or bafloxacin A-treated WT macrophages ( Figure 2H and Fig.2I ). FRET biosensor methods for cytoplasmic pERK1 / 2 activity (Allen et al. (2009) supra; Halls et al. (2015) supra) showed that both influenza virus and imiquimod increased cytoplasmic pERK1 / 2 in a TLR7-dependent manner. In contrast, blocking pERK1 / 2 with PD98059 (30M) did not affect the production of endosomal ROS in response to influenza or the binding of NOX2 to p47phox. These data suggest that influenza A virus increases endosomal NOX2 oxidase activity and downstream PKC activation via TLR7 (but not via pERK1 / 2). It is concluded that viral infection triggers the production of NOX2 oxidase-dependent ROS in endosomes through a process that depends on low pH. Indeed, the following experimental evidence supports this conclusion. First, reduced endosomal acidification is known to impair TLR7 activation by viral RNA18,19. NOX2-dependent ROS production in response to viral infection and the TLR7 agonist imiquimod is regulated in TLR7 - / - Second, bafloxacin A inhibits PKC activation due to influenza virus and imiquimod treatment, which is upstream of acute NOX2 activation (Drummond et al. (2011) supra; Bedard and Krause (2007) supra). Third, bafloxacin A inhibits p47 phox -NOX2 binding, which is a key step in NOX2 assembly and activation.

[0194] Example 3

[0195] Virus strain independence of endosomal ROS

[0196] 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 viruses) resulted in a significant increase in endosomal ROS, which was activated by TLR7. - / - was significantly inhibited in macrophages, but in TLR9 - / - The cells were not affected ( Figure 3A and Figure 3B Neither mumps virus (paramyxoviridae, group V) nor Newcastle disease virus (NDV, paramyxoviridae, group V) produced significant endosomal ROS ( Figure 3A and Figure 3B ), it is noteworthy that these viruses enter cells mainly through the process of cell membrane fusion rather than endocytosis. Macrophages exposed to rotavirus (rhesus monkey strain or bovine UK strain, (Reoviridae, group III)) also did not produce endosomal ROS ( Figure 3A and Figure 3B The DNA viruses herpes simplex virus-2 (herpesviridae, group I) and vaccinia virus (poxyviridae, group I) both elicited expression of WT macrophages and TLR7 - / - Elevation of endosomal ROS in macrophages without induction of TLR9 - / - Increased endosomal ROS in macrophages ( Figure 3A and Figure 3B ). In conclusion, specific recognition of ssRNA viruses by TLR7 or DNA viruses by TLR9 leads to a NOX2 oxidase-dependent burst of endosomal ROS.

[0197] Example 4

[0198] Bacteria and viruses activate different ROS pathways

[0199] Plasma membrane TLRs, particularly TLR1, TLR2, and TLR4, but not those present in endosomes (e.g., TLR7), sense bacteria leading to recruitment of mitochondria to macrophage phagosomes and mitochondria-dependent production of ROS (West et al. (2011) Nature 472(7344):476-480). However, stimulation of endosomal TLRs failed to increase mitochondrial ROS (West et al. (2011), supra). TLR7 activation by imiquimod resulted in a significant increase in endosomal ROS ( Figure 2F ), failed to increase macrophage mitochondrial superoxide production. Phagosomal ROS production in response to the Gram-positive bacteria Streptococcus pneumoniae (SP) or the Gram-negative nontypeable Haemophilus influenzae (NTHI) was examined. Both SP and NTHI induced ROS production in WT mouse macrophages ( FIG. 4A to FIG. 4B ), which is in SOD3 - / - cells, but not in TLR7 - / - Macrophages were not affected ( FIG. 4A to FIG. 4B ). Thus, endosomal ROS production is not a feature of endocytosis per se, but rather a “pathogen (cargo) specific” response. ROS produced for antimicrobial purposes engage the necessary role of mitochondria as a central hub promoting innate immune signaling. In contrast, ssRNA viruses do not use these antimicrobial ROS production pathways.

[0200] Example 5

[0201] Endosomal H2O2 inhibits TLR7 immunity

[0202] To determine the functional importance of endosomal ROS, we evaluated the effect of NOX2 inhibition on cytokine production, which is endosomal TLR7-dependent and thus associated with viral pathogenicity (Diebold et al. (2004), supra). - / - Macrophages ( Figure 5A ) or macrophages treated with bafloxacin A (100 nM) ( Figure 5B ), confirming endosomal and TLR7-dependent signaling. Second, pretreatment with a NOX2 oxidase inhibitor and H2O2 scavenger apocynin (300 M) significantly upregulated the expression of IFN-, IL-1, TNF-, and IL-6 in WT macrophages, but not TLR7-dependent macrophages. - / - The expression of IFN-, IL-1, TNF-, and IL-6 in macrophages in response to imiquimod was significantly enhanced, indicating that the inhibitory effect of NOX2 oxidase-derived ROS on cytokine expression is dependent on TLR7 ( Figure 5AIn contrast, apocynin pretreatment inhibited the expression of IFN-, IL-1, TNF-, and IL-6 in response to the TLR3 agonist, poly I:C (25 g / mL), whereas the increase in these same cytokines triggered by the TLR9 agonist CpG (10 g / mL) was unaffected by apocynin. We further tested whether NOX2 oxidase affects TLR7 immunity in vivo. WT and NOX2 mice were treated with a single dose of imiquimod (50 g / mouse, intranasal) - / y Mice were treated with imiquimod to measure lung IFN-, IL-1, IL-6, and TNF-α after 24 hours. This time point was chosen to reflect the early stages of RNA infection. There were no significant changes in airway inflammation in response to imiquimod ( Figure 5C ), however, imiquimod treatment resulted in NOX2 - / y The levels of IFN-, IL-1, IL-6, and TNF-α were increased in mice.

[0203] We sought to determine how endosomal NOX2 oxidase activity leads to inhibition of TLR7-dependent responses and hypothesized that the parent superoxide species and its immediate downstream product H2O2 are the primary mediators. Inactivation of superoxide by addition of exogenous SOD (300 U / mL) did not affect basal or imiquimod-stimulated expression of IFN-, IL-1, TNF-, and IL-6, suggesting that superoxide itself plays a minor role in regulating TLR7 responses. To examine H2O2, catalase was used to inactivate H2O2 generated in endosomes. Within 30 minutes, exposure to FITC-labeled catalase was found to result in colocalization with LysoTracker, confirming internalization into acidified endosomal compartments ( Fig. 6A ). A 1-hour “pulse” exposure to catalase (1000 U / mL) resulted in a significant increase in IFN- and IL-1 expression in WT macrophages after 24 hours, whereas in TLR7 - / - In macrophages, there is no Figure 6B ). In addition, imiquimod-dependent responses were significantly increased in the presence of catalase ( Figure 6C In WT macrophages treated with Dynasore, the increase in catalase-dependent cytokines was significantly suppressed ( Fig.6D ), but in TLR2 - / - Macrophages were not affected ( Fig. 6E ). The translocation of TLR7 to endosomes is governed by the action of the chaperone UNCB93. Indeed, in the absence of UNCB93, there is a substantial signaling defect as nucleotide-sensitive TLRs fail to reach endolysosomes, where they initiate the MyD88 / TRIF-dependent signaling pathway. - / - In cells, catalase treatment resulted in a significantly smaller increase in cytokines than that observed in WT cells ( Fig. 6F ). Thus, the inhibitory effects of H2O2 are most likely to occur when TLR7 is localized within endosomal compartments. Catalase had no effect on the expression of TLR7, TREML4, or NLRP3, suggesting that H2O2 does not modulate the expression of TLR7, which is a positive regulator of TLR7 activity (i.e., TREML4 26), or NLRP3, which similarly to TLR7 drives antiviral cytokine production ( Figure 6G-6J ). Therefore, the effect of H2O2 may be post-translational. We examined whether endosomal NOX2 oxidase-derived H2O2 affects TLR7 responses in vivo. Intranasal administration of catalase (1000 U / mouse) to WT mice showed a 3- to 4-fold increase in lung IFN-, IL-1, TNF-, and IL-6 after 24 h, and this occurred before overt airway inflammation ( Figure 6K and Figure 6L ).

[0204] A question arises as to whether H2O2 released by the endosomal NOX2 oxidase targets cysteine ​​residues on TLR7 protein domains that modulate receptor activity and become exposed upon activation in the endosomal compartment (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 unique to TLR7, Cys98 and Cys445 ( FIG. 10A to FIG. 10D and FIG. 11A to FIG. 11C Site-directed mutagenesis was performed to generate a series of TLR7 mutants, including: 1) a mutant with all six cysteine ​​residues replaced by alanine; 2) a mutant with double mutations at Cys98 and Cys445 (TLR 7C98A / C445A ); and 3) Cys98 (TLR 7C98A ) and Cys445 (TLR7 C445A ) single mutation. C445A Transfection to TLR7 - / - Macrophages restored the ability of imiquimod to stimulate cytokine expression in these cells; however, expression of imiquimod in TLR7, TLR7 C98A / C445A TLR7 C98A Transfection cannot Fig. 7A ). Catalase (1000 U / mL) treatment had an effect on the expression of mutant TLR7 and TLR7 C98A / C445A TLR7 C98A There was little or no effect on cytokine expression in cells with either WT TLR7 or TLR7C445A In cells, it significantly increased the expression of cytokines ( Fig. 7A ). Sequence analysis was performed using multiple sequence analysis algorithms (i.e., CLUSTALOMEGA) and pairwise sequence analysis (NCBI, Blast) with human TLR7 as the reference point. Using multiple sequence analysis, it was found that Cys98 is unique to TLR7 and is completely conserved in vertebrate TLR7, including from bony fish to humans ( Figure 7B , FIG. 10A to FIG. 10D and FIG. 11A to FIG. 11C ). Pairwise sequence alignment revealed that, among the 27 cysteines on TLR7, Cys98 is the only cysteine ​​residue that is unique to TLR7 and completely conserved in vertebrates. It is proposed that H2O2 generated by the endosomal NOX2 oxidase may modify a single and evolutionarily conserved unique cysteine ​​residue, namely, Cys98 located on the endosomal face of TLR7, leading to a weakened antiviral cytokine response. This may indicate that Cys98 of TLR7 is a novel redox sensor that controls immune function during viral infection.

[0205] Example 6

[0206] NOX2 oxidase attenuates antibody production

[0207] We examined the inhibitory effect of endosomal NOX2 oxidase activity on type I IFN and IL-1 expression, which also occurs after influenza A virus infection. First, the virus triggered the translocation of the transcription factor IRF-7 to the nucleus of WT BMDMs, but in the presence of TLR7 - / - In BMDM, there was no expression of NOX2, indicating that influenza A virus activated TLR7-dependent antiviral signaling in macrophages. - / y In AM, the virus increased the expression of IFN-, IL-1, IL-6, and TNF-( Fig. 8A ). Third, infection of mice with influenza A virus (Hkx31; 105 PFU / mouse) for 24 h resulted in NOX2 - / y Lung IFN-, IL-1, TNF- and IL-6 mRNA in mice ( Figure 8B ), and serum ( Figure 8C ) and lung IFN-protein ( Fig.8D) was increased more. Therefore, fully functional NOX2 oxidase inhibits the production of antiviral cytokines triggered by influenza A virus. TLR7 is essential for the activation of B cells and antibody production. To test whether NOX2 oxidase inhibits TLR7-dependent immunity to influenza A virus in vivo, heat-inactivated, replication-deficient influenza A virus was used as a stimulus, which is therefore expected to be a viral form that mainly triggers TLR7 PRR engagement without much contribution from RIG-I and NLRP3 20. Intranasal inoculation of inactivated virus has an effect on 7-day weight loss ( Fig. 8E ) or airway BALF inflammation ( Fig.8F ) had no effect. NOX2 deficiency led to a significant increase in the levels of lung IFN-, IL-1, and TNF-mRNA ( Figure 8G ), the levels of IgA, total IgG, IgG1, IgG2b, and IgG3 in serum and BALF were significantly increased ( Figure 8H and Figure 8I ). Thus, activation of endosomal NOX2 oxidase following influenza A virus infection could inhibit antiviral cytokine and humoral immunity by suppressing antibody production—processes required for optimal clearance of the virus and protection against reinfection.

[0208] Example 7

[0209] Endosomal-targeted NOX2 inhibitors

[0210] A new molecular targeting system was synthesized to deliver a specific NOX2 oxidase inhibitor (i.e., gp91ds-TAT) directly to endosomes, thereby disrupting the viral signaling platform by eliminating ROS production. To this end, a triple construct was generated that contained gp9l ds-tat conjugated to membrane-anchored cholesterol via a PEG-linker at the N-terminal region of the peptide. Similar constructs have previously been shown to enhance endosomal localization of inhibitors of the enzyme β-secretase (Rajendran et al. (2008) Science 320(5875):520-523). Cy5 fluorophores conjugated to cholesterol using the same PEG linker resulted in cytoplasmic fluorescence in the perinuclear region and co-localized with EEA1, NOX2, and influenza virus NPs in a dynasore (100M)-sensitive manner after viral infection, providing evidence that endocytosis is the primary mode of cell entry ( Figure 9A-9E ). With unconjugated drug (Ugp91ds-TAT; Fig.9F ), cholesterol-conjugated gp91ds-TAT (Cgp91ds-TAT) inhibited superoxide production in macrophages in vitro with at least 10-fold potency compared to Cgp91ds-TAT, which was not attributed to the enhanced ROS scavenging properties of the compound ( Figure 9G ).

[0211] We examined whether Cgp91ds-TAT suppresses disease severity after influenza A virus infection in vivo. Daily intranasal administration of Cgp91 ds-TAT (0.02 mg / kg / d) from 1 day before to 3 days after influenza A virus infection reduced airway inflammation by approximately 40% ( Figure 9H ), while Ugp91ds-TAT had no effect ( Figure 9H Compared with the control virus group, Cgp91ds-TAT significantly increased lung type I IFN-mRNA levels, while Ugp91ds-TAT did not ( Fig.9I ). To eliminate the possibility that this improvement in NOX2 inhibition by the gp91ds-TAT cholesterol conjugate was due to the cholesterol-PEG linker itself, the cholesterol PEG-linker was conjugated to a scrambled gp91ds-TAT (Sgp91ds-TAT) and examined for its effect on influenza infection in vivo. Sgp91ds-TAT had no effect on airway inflammation, lung IFN-mRNA levels, and superoxide production. Increasing the dose of Ugp91ds-TAT 10-fold to 0.2 mg / kg / day significantly reduced weight loss caused by influenza A virus on day 3 and nearly eliminated airway inflammation, as well as superoxide production in BALF inflammatory cells, similar to the same dose of Cgp91ds-TAT ( Figure 9J-Figure 9L Strikingly, Cgp91ds-TAT (0.2 mg / kg / day) and Ugp91ds-TAT (0.2 mg / kg / day) caused a nearly 10,000-fold reduction in influenza A virus load in the lungs ( Figure 9M ). Thus, inhibition of endosomal NOX2 oxidase by intranasal administration of gp91ds-TAT resulted in a substantial reduction in the pathogenicity of influenza A virus. This is an innovative approach to inhibit NOX2 oxidase activity occurring within the endosomal compartment. Due to the conjugation of cholesterol, the tailored inhibitor was specifically and preferentially delivered through the endocytic compartment. In support of this, Fig. 9A and Fig. 9B The results showed that cholesterol conjugation produces a drug delivery system that promotes endosomal delivery, i.e., the drug showed a strong degree of co-localization with EEA1+ endosomes, which was abolished by dynasore pretreatment. This delivery system brings the NOX2 inhibitor to the major site of action associated with viral infection ( Fig.9D (showing strong co-localization of the viral nucleoprotein and our NOX2 inhibitor). This paper proposes that after internalization into the endosome, the drug is most likely on the luminal side of the endosomal membrane and since the TAT moiety can penetrate this membrane and inhibit NOX2 activity. The drug may still be able to diffuse to other sites or locations of NOX2, however, given that the drug appears to be selectively delivered via the endocytic pathway, it is believed that the direct and primary site of action is NOX2 activity on the endosome.

[0212] Example 8

[0213] The role of NOX2

[0214] The evidence presented here suggests that viral entry into the endosomal compartment triggers NOX2 oxidase-dependent ROS production in endosomes. It is proposed here that the major contributor to endosomal superoxide concentrations is superoxide generated directly in this compartment. Superoxide is the major product of NOX2 and can only be generated within the endosomal compartment due to the topology of NOX2 and the unidirectional transfer of electrons through this catalytic subunit. In line with this, it is well accepted that superoxide does not travel far from the site of its generation due to its negative charge. Unlike superoxide, hydrogen peroxide has some membrane permeability and diffusion capacity, and therefore, it is conceivable that NOX2 expressed in other parts of the cell (such as the plasma membrane) may generate some endosomal H2O2 elsewhere. There is some evidence that it is very likely that very little remotely generated H2O2 enters the endosomal compartment. PKC activation (critical for NOX2 activation) after viral infection is significantly impaired if 1) viral entry into the cell is prevented ( Figure 2H and Fig.2I ); 2) blocking endosomal acidification by bafloxacin A ( Figure 2H and Fig.2I ); or 3) if TLR7 is absent (i.e., using TLR7 - / - macrophages). Therefore, ROS generation caused by endosomal NOX2 occurs only after the virus enters the endosome and activates the endosomal-specific pathway, further demonstrating that endosomal NOX2 serves as the major site of H2O2 generation.

[0215] Here, we demonstrate that endosomal ROS are key negative regulators of the fundamental molecular mechanisms of viral pathogenicity, affecting antiviral immunity and the host's ability to resist and clear viral infection. Importantly, this effect is conserved for all viruses that enter cells via the endocytic pathway, regardless of viral class, and is TLR7-dependent. This provides a target for antiviral therapy against a wide range of viruses that cause significant morbidity and mortality worldwide.

[0216] Example 9

[0217] Generation of decoy peptides containing TLR7 C98

[0218] A decoy peptide was generated that included D95 ​​to L104 of murine TLR7 (DLRCNCVPVL—SEQ ID NO: 1) operably linked to a HIV-TAT uptake peptide portion (YGRKKRRQRRR—SEQ ID NO: 2). The decoy peptide (referred to herein as C98i) prevents disulfide bond formation between C98 and C475, thereby preventing TLR7 activation.

[0219] Example 10

[0220] C98i blocks response to TLR7 agonist (imiquimod)

[0221] Fig.12 (raw data) and Fig.12 (Normalized data) show that the production of the cytokine IL-1β by bone marrow-derived macrophages after exposure to imiquimod (TLR7 agonist) was elevated in the presence of C98i. Fig.15 Similar results were shown. The data indicate that C98i blocks TLR7 activity.

[0222] Embodiment 11

[0223] C98i blocks response to TLR7 agonist (gadquimod)

[0224] Fig.13 C98i blocks TLR7 in response to the TLR7 agonist desquimod. No effect on basal, unstimulated IL-1β levels was shown. Fig.13 There was also no effect on TLR9 (a closely related family member with a tryptophan at position 98) or TLR4 agonist responses or TLR2 agonist responses. There is little sequence homology between TLR7 and TLR4 and between TLR7 and TLR2.

[0225] Similar results were observed for TLR5 agonist responses. C98i had no adverse effects on TLR7 expression or cell viability.

[0226] The data showed that C98i blocked the activity of TLR7 and did not affect the activity of TLR2, TLR4, TLR5, and TLR9. Because of the uniqueness of the sequence, it is unlikely to affect other members of the TLR family (i.e., TLR1, TLR3, TLR6, and TLR8). It was found that C98i did not affect the activity of TLR2, TLR4, TLR5, and TLR9, which also indicates that the drug has no nonspecific properties on cell functions that affect the production of the cytokine IL-1β.

[0227] TLR7 is a target involved in viruses, autoimmune diseases and cancer. New drugs targeting TLR7 have great potential.

[0228] Example 12

[0229] The role of the C98i version without TAT

[0230] In the absence of TAT, the C98i peptide retained its ability to inhibit the response to the TLR7 agonist imiquimod in vitro. The absence of TAT is referred to as “TAT-free”. The results are shown in Fig.16ANo adverse effect on cell viability ( Fig. 16B ).

[0231] Embodiment 13

[0232] C98i inhibits influenza A virus response

[0233] Fig.17 C98i was shown to inhibit influenza A virus (X31) response (IL-6-mRNA expression) in vitro. In the absence of C98i, IL-6 mRNA expression was significantly higher compared to C98i+influenza A virus (X31).

[0234] Embodiment 14

[0235] Effects of the scrambled C98i amino acid sequence

[0236] The amino acid sequence of C98i was scrambled to generate YGRKKRRQRRRCLVPNDCRLV-NH2 (SEQ ID NO: 44). The scrambled C98i peptide sequence was unable to inhibit the TLR7 agonist (imiquimod). Fig.18 shown.

[0237] Embodiment 15

[0238] Effect of short peptides on the response to TLR7 agonist (imiquimod)

[0239] 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 without TAT to determine their effects on the TLR7 agonist imiquimod. Fig.19 The results showed that none of RANC, RCNC, RANC, RANA, or RCNA inhibited the in vitro response to the TLR7 agonist imiquimod.

[0240] Example 16

[0241] Examining the antioxidant and immunomodulatory effects of C98i in vitro and in vivo after viral infection

[0242] In vitro experiments using isolated macrophages: Assays examining oxidative stress and viral replication in the absence or presence of C98i. The following viruses were examined: low to high pathogenicity influenza A virus, respiratory syncytial virus, rhinovirus, dengue virus.

[0243] It is expected that endosomal oxidative stress and viral replication can be inhibited by C98i.

[0244] In vivo experiments: C98i was delivered intranasally to mice following infection with influenza A virus (via intranasal delivery) [To et al. (2017) Nature Communications 8(69): 1-17]. Potential modulation of pulmonary oxidative stress, inflammation, injury, viral load, and immune cell responses in the lungs and systemic circulation was assessed.

[0245] Embodiment 17

[0246] Examining the immunomodulatory effects of C98i in vivo in an autoimmune lupus-like model test

[0247] Wild-type C57BL / 6 mice were treated with C98i and then treated with epidermal topical TLR7 agonist imiquimod (Aldara Cream) to the ears three times a week. After treatment, the mice were examined for serum autoantibody and creatinine levels, as well as histopathology of the kidney, spleen, liver, heart, and skin. Immunological abnormalities were analyzed by immunohistochemistry, quantitative reverse transcription-polymerase chain reaction, and fluorescence-activated cell sorting.

[0248] Those skilled in the art will appreciate that the disclosure described herein may be varied and modified in addition to the specifically described content. It should be understood that the disclosure contemplates all such variations and modifications. The disclosure also provides all steps, features, compositions and compounds mentioned or indicated in this specification individually or collectively, and any and all combinations of any two or more steps or features or compositions or compounds.

[0249] All patents, applications, publications, test methods, literature, and other materials cited herein are incorporated by reference in their entirety, as if physically present in this specification.

[0250] References

[0251] Aguirre(2010)Free Radical Biology and Medicine 49(9):1342-1353

[0252] Allen et al. (2009) Immunity 30(4): 556-565

[0253] Altschul et al. (1997) Nucl. Acids. Res. 25: 3389

[0254] Ausubel et al. (In: Current Protocols in Molecular Biology, John Wiley & Sons Inc. 1994-1998

[0255] Bedard and Krause (2007) Physiological Reviews 87(1): 245-313

[0256] Campbell et al. (2012) Science Translational Medicine 4(157):157ra141

[0257] Cossart and Helenius (2014) Cold Spring Harbor Perspectives in Biology 6(8)

[0258] Diebold et al. Science 303(5663):1529-1531

[0259] Drummond et al (2011) Nature Reviews Drug Discovery 10(6): 453-471

[0260] Guidotti et al (2017) Trends in Pharmacological Science 38(4): 406-424

[0261] Halls et al. (2015) Methods in Molecular Biology 1335: 131-161

[0262] Ichinohe et al. (2009) The Journal of Experimental Medicine 206(1): 79-87

[0263] Imai et al. (2008) Cell 133(2): 235-249

[0264] Iwasaki and Pillai (2014) Nature Reviews Immunology 14(5): 315-328

[0265] Jensen et al. (2014) The Journal of Biological Chemistry 289(20): 20283-20294

[0266] Johnson et al (1993) Peptide Turn Mimetics in Biotechnology and Pharmacy

[0267] Judkins et al (2010) American Journal of Physiology Heart and CirculatoryPhysiology 298(1):H24-32

[0268] Kanno et al. (2013) International Immunology 25(7): 413-422

[0269] Kawahara et al. (2007) BMC Evolutionary Biology 7:109

[0270] Kelkka et al. (2014) Antioxidants & Redox Signaling 21(16): 2231-2245

[0271] King et al (2013) The Journal of Allergy and Clinical Immunology 131(5): 1314-1321e1314

[0272] Lund et al. (2004) Proceedings of the National Academy of Sciences of the United States of America 101(15):5598-5603

[0273] Pezzuto et al. Eds., Chapman and Hall, New York

[0274] Pollock et al. (1995) Nature Genetics 9(2): 202-209

[0275] Rajendran et al. (2008) Science 320(5875):520-523

[0276] Selemidis et al (2008) Pharmacology & Therapeutics 120(3):254-291

[0277] Snelgrove et al. (2006) Eur J Immunol 36(6): 1364-1373

[0278] To et al. (2014) Free Radical Research 48(8): 940-947

[0279] To et al. (2017) Nature Communications 8(69): 1-17

[0280] Violin et al. (2003) The Journal of Cell Biology 161(5): 899-909

[0281] Vlahos et al. (2011) PLoS pathogens 7(2): e1001271

[0282] Vlahos et al. (2012) Trends in Pharmacological Sciences 33(1): 308

[0283] Vlahos and Selemidis (2014) Molecular Pharmacology 86(6):747-759

[0284] West et al. (2011) Nature 472(7344): 476-480.

Claims

1. 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 that antagonizes disulfide bond formation between C98 and C475 of TLR7 or between corresponding positions thereof.

2. The method of claim 1, wherein the agent comprises a peptide having a length of about 4 to about 190 amino acids and having an amino acid sequence having at least 70% amino acid sequence similarity to up to 190 consecutive amino acids between amino acids 4 and 194 of TLR7, provided that the peptide comprises a cysteine ​​residue at a position equivalent to position 98 of TLR7.

3. The method of claim 2, wherein the peptide comprises an amino acid sequence having at least 70% amino acid sequence similarity to up to 100 consecutive amino acids between amino acids 48 and 148 of TLR7, provided that the peptide comprises a cysteine ​​residue at a position equivalent to position 98 of TLR7.

4. The method of claim 3, wherein the peptide comprises an amino acid sequence having at least 70% amino acid sequence similarity to up to 40 consecutive amino acids between amino acids 78 to 118 of TLR7, provided that the peptide comprises a cysteine ​​residue at a position equivalent to position 98 of TLR7.

5. The method of claim 1, wherein the agent comprises a peptide having an amino acid sequence having 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, which corresponds to amino acids 95 to X104 of human TLR7, provided that the peptide contains a cysteine ​​residue at a position equivalent to position 98 of TLR7.

6. The method of claim 5, wherein the peptide comprises the amino acid sequence DFRCNCVPIP (SEQ ID NO: 26).

7. The method of claim 5, wherein the peptide comprises the amino acid sequence DLRCNCVPVL (SEQ ID NO: 1).

8. The method according to any one of claims 2 to 7, wherein the peptide further comprises a moiety attached to the N-terminus or C-terminus of the peptide which enables the peptide to be taken up into cells.

9. The method of claim 8, wherein the moiety is a hydrophilic peptide, an amphiphilic peptide, a peptide having a periodic amino acid sequence, or a conjugate with cholesterol.

10. The method of claim 9, wherein the hydrophilic 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 protein (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).

11. The method of claim 9, wherein the amphipathic peptide is selected from the group consisting of transporter 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 (NLS) (SEQ ID NO: 17), Pep-1 (SEQ ID NO: 18) and Pep-2 (SEQ ID NO: 19).

12. The method of claim 9, wherein the peptide having a periodic amino acid sequence comprises a polyarginine or polylysine sequence.

13. The method according to any one of claims 1 to 12, wherein the subject is a human or a non-human mammal.

14. The method of claim 13, for inhibiting autoimmune diseases, viral or microbial diseases, inflammation or cancer.

15. Use of an agent that antagonizes disulfide bond formation between C98 and C475 or corresponding positions of TLR7 in the preparation of a medicament for inhibiting autoimmune diseases, viral or microbial diseases, inflammation or cancer in a subject.

16. A method for treating an autoimmune disease, a viral or microbial disease, inflammation or cancer in a subject, the method comprising contacting a cell from the subject expressing TLR7 with an effective amount of an agent that antagonizes disulfide bond formation between C98 to C475 or corresponding positions of TLR7.

17. The method of claim 16, wherein the agent comprises a peptide having a length of about 4 to about 190 amino acids and having an amino acid sequence having at least 70% amino acid sequence similarity to up to 190 consecutive amino acids between amino acids 4 and 194 of TLR7, provided that the peptide comprises a cysteine ​​residue at a position equivalent to position 98 of TLR7.

18. The method of claim 17, wherein the peptide has an amino acid sequence having at least 70% amino acid sequence similarity to up to 100 consecutive amino acids between amino acids 48 and 148 of TLR7, provided that the peptide comprises a cysteine ​​residue at a position equivalent to position 98 of TLR7.

19. The method of claim 18, wherein the peptide has an amino acid sequence having at least 70% amino acid sequence similarity to up to 40 consecutive amino acids between amino acids 78 and 118 of TLR7, provided that the peptide comprises a cysteine ​​residue at a position equivalent to position 98 of TLR7.

20. The method of claim 16, wherein the agent is a peptide having an amino acid sequence having at least 70% similarity to DFRCNCVPIP (SEQ ID NO: 26) corresponding to D95 to X104, wherein X is P (human) or L (mouse), provided that the peptide comprises a cysteine ​​residue at a position equivalent to position 98 of TLR7.

21. The method of claim 20, wherein the peptide comprises the amino acid sequence DFRCNCVPIP (SEQ ID NO: 26).

22. The method of any one of claims 17 to 21, wherein the peptide further comprises a moiety attached to the N-terminus or C-terminus of the peptide which enables the peptide to be taken up into cells.

23. The method of claim 20, wherein the moiety is a hydrophilic peptide, an amphiphilic peptide, or a peptide having a periodic amino acid sequence.

24. The method of claim 23, wherein the hydrophilic 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 protein (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).

25. The method of claim 23, wherein the amphipathic peptide is selected from the group consisting of transporter chimera (SEQ ID NO: 12), MAP (SEQ ID NO: 13), SBP (SEQ ID NO: 14), FBP (SEQ ID NO: 15), MPG (SEQ ID NO: 16), Pep-1 (SEQ ID NO: 17) and Pep-2 (SEQ ID NO: 18).

26. The method of claim 23, wherein the peptide having a periodic amino acid sequence comprises a polyarginine or polylysine sequence or a conjugate with cholesterol.

27. The method of any one of claims 16 to 26, wherein the subject is a human or a non-human mammal.

28. An agent that antagonizes disulfide bond formation between C98 and C475 of TLR7 or between corresponding positions thereof, for use in inhibiting autoimmune diseases, viral or microbial diseases, inflammation or cancer in a subject.

29. The use according to claim 15 or the agent according to claim 28, wherein the agent comprises a peptide of about 4 to about 190 amino acids in length and having an amino acid sequence having at least 70% amino acid sequence similarity to up to 190 consecutive amino acids between amino acids 4 and 194 of TLR7, provided that the peptide comprises a cysteine ​​residue at a position equivalent to position 98 of TLR7.

30. The use or agent according to claim 29, wherein the agent comprises a peptide having an amino acid sequence having 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, which correspond to amino acids 95 to 104 of human TLR7, provided that the peptide contains a cysteine ​​residue at a position equivalent to position 98 of TLR7.

31. The use or agent of claim 30, wherein the peptide comprises the amino acid sequence DFRCNCVPIP (SEQ ID NO: 26).

32. The use or agent according to any one of claims 29 to 31, wherein the peptide further comprises a portion linked to the N-terminus or C-terminus of the peptide which enables the peptide to be taken up into cells.

33. The use or agent of claim 32, wherein the moiety is a hydrophilic peptide, an amphiphilic peptide or a peptide having a periodic amino acid sequence or a conjugate with cholesterol.

34. The use or agent of claim 33, wherein the hydrophilic peptide is selected from 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 protein (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).

35. The use or agent of claim 33, wherein the amphipathic peptide is selected from the group consisting of transporter chimera (SEQ ID NO: 12), MAP (SEQ ID NO: 13), SBP (SEQ ID NO: 14), FBP (SEQ ID NO: 15), MPG [MAGac] (SEQ ID NO: 16), MPG (NLS) (SEQ ID NO: 17), Pep-1 (SEQ ID NO: 18) and Pep-2 (SEQ ID NO: 19).

36. The use or agent according to claim 33, wherein the peptide having a periodic amino acid sequence comprises a polyarginine or polylysine sequence.

37. The use or agent of any one of claims 29 to 36, wherein the subject is a human or a non-human mammal.

38. A pharmaceutical composition comprising an agent that antagonizes disulfide bond formation between C98 and C475 of TLR7 or between corresponding positions thereof and one or more pharmaceutical carriers, excipients and / or diluents.

39. A pharmaceutical composition according to claim 38, wherein the agent comprises a peptide having a length of about 4 to about 190 amino acids and having an amino acid sequence having at least 70% amino acid sequence similarity to up to 190 consecutive amino acids between amino acids 4 and 194 of TLR7, provided that the peptide comprises a cysteine ​​residue at a position equivalent to position 98 of TLR7.

40. A pharmaceutical composition according to claim 39, wherein the agent comprises a peptide having an amino acid sequence having 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, which corresponds to amino acids 95 to 104 of human TLR7, provided that the peptide comprises a cysteine ​​residue at a position equivalent to position 98 of TLR7.

41. The pharmaceutical composition of claim 40, wherein the peptide comprises the amino acid sequence DFRCNCVPIP (SEQ ID NO: 26).

42. The pharmaceutical composition according to any one of claims 39 to 41, wherein the peptide further comprises a portion linked to the N-terminus or C-terminus of the peptide that enables the peptide to be taken up into cells.

43. The pharmaceutical composition of claim 42, wherein the moiety is a hydrophilic peptide, an amphiphilic peptide, or a peptide having a periodic amino acid sequence or a conjugate with cholesterol.

44. A pharmaceutical composition according to claim 43, wherein the hydrophilic peptide is selected from 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 coating protein (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).

45. The pharmaceutical composition of claim 39, wherein the amphipathic peptide is selected from the group consisting of transporter 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 (NLS) (SEQ ID NO: 17), Pep-1 (SEQ ID NO: 18) and Pep-2 (SEQ ID NO: 19).

46. ​​The pharmaceutical composition of claim 43, wherein the peptide having a periodic amino acid sequence comprises a polyarginine or polylysine sequence.

47. A pharmaceutical composition according to any one of claims 38 to 46 for use in a human or non-human mammal.

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