How to Treat the Effects of a Cytokine Storm

Abstract

The present invention provides a novel approach to the prevention and treatment of respiratory or non-respiratory infections and the effects of mild to severe cytokine storm, including multi-organ effects, and reducing mortality. More specifically, the present invention describes specific combinations or cytokines or soluble receptors that must be depleted to eliminate or reduce mortality as a result of severe viral cytokine storm.

Description

[Technical field]

[0001] Government Subsidy Statement This invention was made with government support under several grants (1R01DK109713, 1R01DK111102, 1R01DK129522, 1R01DK128203) awarded by the National Institutes of Health. The Government has certain rights.

[0002] The general field of the present disclosure is a novel approach to the prevention and treatment of the effects of cytokine storm. The present invention describes specific combinations or cytokines or soluble receptors that must be depleted to eliminate or reduce mortality as a result of severe viral cytokine storm. [Background technology]

[0003] A striking feature of the COVID-19 pandemic is multisystem involvement involving the respiratory tract, kidneys, brain, liver, heart, gastrointestinal tract, eyes, and many other organs. See, for example, Huang et al., "Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China," (2020) Lancet 395:497-506; Wang et al., "Clinical features of 69 cases with coronavirus disease 2019 in Wuhan, China," (2020) Clin Infect Dis. 71:769-777. The virus was not always detected in affected organs, and in a cardiac autopsy study, the presence or absence of virus did not appear to affect the degree of inflammatory cell infiltration. See Spudich et al., "Nervous system consequences of COVID-19," (2022) Science 375:267-269; Topol, "COVID-19 can affect the heart," (2020) Science 370:408-409; Lindner et al., "Association of cardiac infection with SARS-CoV-2 in confirmed COVID-19 autopsy cases.", (2020) JAMA Cardiol. 5:1281-1285; Gupta et al., "Extrapulmonary manifestations of COVID-19," (2020) Nat Med. 26:1017-1032.

[0004] Viral infection triggers the production of cytokines as part of the innate and adaptive immune responses. We previously suspected that the widespread cytokine storm documented early in the pandemic may be involved in organ damage and developed a novel evidence-based model of cytokine-mediated end-organ damage. See Huang et al., 2020. Of the three organs studied, the literature on cardiac involvement shows elevated cardiac troponin I levels (similar to acute myocardial infarction), myocarditis, myocardial necrosis, pericarditis, arrhythmias and heart failure4,7. See Topo 2020; Inciardi et al., "Cardiac involvement in a patient with coronavirus disease 2019 (COVID-19)", (2020) JAMA Cardiol. 5:819-824. Evidence of liver damage includes elevated aminotransferase levels, hepatocellular injury, inflammation and hepatic steatosis8. See Herta et al., "COVID-19 and the liver - Lessons learned," (2021) Liver Int. 41 Suppl 1: pp. 1-8. Renal manifestations are highly prevalent in hospitalized COVID-19 patients, with nearly 40% developing proteinuria and about one-third developing acute kidney injury (AKI). See Cheng et al., "Kidney disease is associated with in-hospital death of patients with COVID-19," (2020) Kidney Int. 97:829-838; Hirsch et al., "Acute kidney injury in patients hospitalized with COVID-19," (2020) Kidney Int. 98:209-218. Renal biopsy studies of COVID-19 patients with severe proteinuria and / or impaired renal function have most commonly documented the collapsing variant of focal and segmental glomerulosclerosis (FSGS) and acute kidney injury.See Kudose et al., "Kidney Biopsy Findings in Patients with COVID-19," (2020) J Am Soc Nephrol. 31:1959-1968; Nasr et al., "Kidney Biopsy Findings in Patients With COVID-19, Kidney Injury and Proteinuria," (2021) Am J Kidney Dis. 77:465-468 (2021). Early autopsy studies suspected viral particles, but kidney biopsies of surviving patients did not confirm any viral particles. See also Bradley et al., "Histopathology and ultrastructural findings of fatal COVID-19 infections in Washington State: a case series," (2020) Lancet 396:320-332.

[0005] The advantage of building a COVID-19 cytokine storm model centered around kidney disease is that it can be mechanistically compared to the rare manifestation of cytokine storm in the common cold, which shares some elements. Basnet et al., “Rhinoviruses and Their Receptors”, (2019) Chest 155:1018–1025; Wine et al., “Cytokine responses in the common cold and otitis media”, (2012) Curr Allergy Asthma Rep.12:574–581; Nieters et al., “Cross-sectional study on cytokine polymorphisms, cytokine production after T-cell stimulation and clinical parameters in a random sample of a German population", (2001) Hum Genet. 108:241-248; Noah et al., "Nasal cytokine production in viral acute upper respiratory infection of childhood", (1995) J Infect Dis. 171:584-592; van Kempen et al., "An update on the pathophysiology of rhinovirus upper respiratory tract infections", (1999) Rhinology 37:97~103; Whiteman et al., “IFN-gamma regulation of ICAM-1 receptors in bronchial epithelial cells: soluble ICAM-1 release inhibits human rhinovirus infection”, (2008) J Inflamm (Lond).5:8; Jartti et al., “Systemic T-helper and T-regulatory cell type cytokine responses in rhinovirus vs.See, for example, "respiratory syncytial virus induced early wheezing: an observational study," (2009) Respir Res. 10:85; Hershey et al., "The association of atopy with a gain-of-function mutation in the alpha subunit of the interleukin-4 receptor," (1997) N Engl J Med. 337:1720-1725; Abdel-Hafez et al., "Idiopathic nephrotic syndrome and atopy: is there a common link?", (2009) Am J Kidney Dis. 54:945-953.

[0006] The common cold, often caused by rhinovirus, causes approximately 70% of episodes of recurrent glomerular diseases MCD and FSGS. See Passioti et al., "The common cold: potential for future prevention or cure," (2014) Curr Allergy Asthma Rep. 14:413; Takahashi et al., "Triggers of relapse in steroid-dependent and frequently relapsing nephrotic syndrome," (2007) Pediatr Nephrol. 22:232-236.

[0007] This relapse pathway is unclear, but we have long suspected that cytokine storm plays a major role. Because the COVID-19 cytokine storm is more extensive than that of the common cold, subtractive analysis may identify key contributors in specific aspects of each disease. Human and experimental MCD and most forms of FSGS are associated with reduced podocyte expression of the transcription factor ZHX2. See Mace et al., "ZHX2 and its interacting proteins regulate upstream pathways in podocyte diseases," (2020) Kidney Int. 97:753-764. In contrast, experimental evidence suggests that basal podocyte ZHX2 expression is high in the collapsed form of FSGS. Mace et al., 2020. In contrast to many other cells, podocytes express the majority of all ZHX proteins in a plasma membrane (non-nuclear) distribution. In the setting of appropriate combinations and altered ZHX2 expression status, systemic cytokine release may induce translocation of ZHX proteins from normal (aminopeptidase A / APA, ephrin Bl) or putative alternative plasma membrane anchors to the podocyte nucleus.

[0008] However, despite efforts to understand the resulting cytokine storm seen after COVID-19 infection, there is still a need to understand the mechanisms underlying this phenomenon and the resulting clinical manifestations, which would facilitate the design of therapeutic approaches to reduce organ damage associated with the cytokine storm.

[0009] The present invention addresses these needs. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Huang et al., "Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China", (2020) Lancet 395:497 - 506 [Non - Patent Document 2] Wang et al., "Clinical features of 69 cases with coronavirus disease 2019 in Wuhan, China", (2020) Clin Infect Dis. 71:769 - 777 [Non - Patent Document 3] Spudich et al., "Nervous system consequences of COVID - 19", (2022) Science 375:267 - 269 [Non - Patent Document 4] Topol, "COVID - 19 can affect the heart", (2020) Science 370:408 - 409 [Non - Patent Document 5] Lindner et al., "Association of cardiac infection with SARS - CoV - 2 in confirmed COVID - 19 autopsy cases.", (2020) JAMA Cardiol. 5:1281 - 1285 [Non - Patent Document 6] Gupta et al., "Extrapulmonary manifestations of COVID - 19", (2020) Nat Med. 26:1017 - 1032 [Non - Patent Document 7] Inciardi et al., "Cardiac involvement in a patient with coronavirus disease 2019 (COVID - 19)", (2020) JAMA Cardiol. 5:819 - 824 [Non - Patent Document 8] Herta et al., "COVID-19 and the liver - Lessons learned", (2021) Liver Int. 41 Suppl 1: pp. 1-8 [Non-Patent Document 9] Cheng et al., “Kidney disease is associated with in-hospital death of patients with COVID-19”, (2020) Kidney Int. 97:829–838 [Non-Patent Document 10] Hirsch et al., "Acute kidney injury in patients hospitalized with COVID-19," (2020) Kidney Int. 98:209-218 [Non-Patent Document 11] Kudose et al., “Kidney Biopsy Findings in Patients with COVID-19”, (2020) J Am Soc Nephrol. 31:1959~1968 [Non-Patent Document 12] Nasr et al., "Kidney Biopsy Findings in Patients With COVID-19, Kidney Injury and Proteinuria", (2021) Am J Kidney Dis. 77:465-468 (2021) [Non-Patent Document 13] Bradley et al., “Histopathology and ultrastructural findings of fatal COVID-19 infections in Washington State: a case series”, (2020) Lancet 396:320–332 [Non-Patent Document 14] Basnet et al., "Rhinoviruses and Their Receptors," (2019) Chest 155:1018-1025 [Non-Patent Document 15] Wine et al., “Cytokine responses in the common cold and otitis media”, (2012) Curr Allergy Asthma Rep.12:574–581 [Non-Patent Document 16] Nieters et al., "Cross-sectional study on cytokine polymorphisms, cytokine production after T-cell stimulation and clinical parameters in a random sample of a German population", (2001) Hum Genet. 108:241-248 [Non-Patent Document 17] Noah et al., "Nasal cytokine production in viral acute upper respiratory infection of childhood", (1995) J Infect Dis. 171:584-592 [Non-Patent Document 18] van Kempen et al., "An update on the pathophysiology of rhinovirus upper respiratory tract infections", (1999) Rhinology 37:97-103. [Non-Patent Document 19] Whiteman et al., “IFN-gamma regulation of ICAM-1 receptors in bronchial epithelial cells: soluble ICAM-1 release inhibits human rhinovirus infection”, (2008) J Inflamm (Lond).5:8. [Non-Patent Document 20] Jartti et al., “Systemic T-helper and T-regulatory cell type cytokine responses in rhinovirus vs. respiratory syncytial virus induced early wheezing: an observational study”, (2009) Respir Res.10:85 [Non-Patent Document 21] Hershey et al., “The association of atopy with a gain-of-function mutation in the alpha subunit of the interleukin-4 receptor”, (1997) N Engl J Med.337:1720–1725 [Non-Patent Document 22] Abdel-Hafez et al., “Idiopathic nephrotic syndrome and atopy: is there a common link?” (2009) Am J Kidney Dis.54:945-953 [Non-Patent Document 23] Passioti et al., “The common cold: potential for future prevention or cure”, (2014) Curr Allergy Asthma Rep.14:413 p. [Non-Patent Document 24] Takahashi et al., “Triggers of relapse in steroid-dependent and frequently relapsing nephrotic syndrome”, (2007) Pediatr Nephrol.22:232–236 [Non-Patent Document 25] Mace et al., “ZHX2 and interacting its proteins regulate upstream pathways in podocyte diseases”, (2020) Kidney Int.97:753-764 Summary of the Invention [Problem to be solved by the invention]

[0011] Summary of the Invention The present invention provides a mechanism for targeting various cytokines. The present invention describes specific combinations of cytokines or soluble factors that must be depleted to eliminate or reduce the effects of cytokine storm, including mortality and end-organ damage.

[0012] Viral diseases, including respiratory viruses such as SARS-CoV-1 and SARS-CoV-2, have pathological effects on non-respiratory organs even in the absence of obvious direct viral infection. In addition, diseases caused by other respiratory and non-respiratory viruses, such as influenza, parainfluenza, respiratory syncytial virus, adenovirus, enterovirus, other coronaviruses, cytomegalovirus (CMV), Epstein-Barr virus (EBV), Middle East respiratory syndrome (MERS), and Ebola hemorrhagic fever, are also known to trigger cytokine storms, leading to death and multi-organ damage.

[0013] To study and compare the role of viral cytokine storm in extrapulmonary manifestations of SARS-CoV-2, novel COVID-19 and cytokine combination "cocktails" were developed from clinical data and injected into mice. Previous studies by the inventors have demonstrated efficacy in a rhinovirus cold infection model using a combination "cocktail" of cold cytokines.

[0014] In earlier studies, we found that Zhx2 flox / floxand NPHS2 promoter-driven Cre mice were utilized. However, we then used BALB / cJ and BALB / c mice (Zhx2+ / +), which are established models of the Zhx2 hypomorphic condition.See Mace et al. 2020; Perincheri et al., "Hereditary persistence of alpha - fetoprotein and H19 expression in liver of BALB / cJ mice is due to a retrovirus insertion in the Zhx2 gene." (2005) Proc Natl Acad Sci USA 102: 396 - 401; Perincheri et al., "Characterization of the ETnII - alpha endogenous retroviral element in the BALB / cJ Zhx2 (Afrl) allele", (2008) Mamm Genome 19: 26 - 31; Gargalovic et al., "Quantitative trait locus mapping and identification of Zhx2 as a novel regulator of plasma lipid metabolism", (2010) Circ Cardiovasc Genet. 3: 60 - 67; Creasy et al., "Zinc Fingers and Homeoboxes 2 (Zhx2) Regulates Sexually Dimorphic Cyp Gene Expression in the Adult Mouse Liver", (2016) Gene Expr. 17: 7 - 17; Jiang et al., "Zhx2 (zinc fingers and homeoboxes 2) regulates major urinary protein gene expression in the mouse liver", (2017) J Biol Chem 292: 6765 - 6774 (2017); Erbilgin et al., "Transcription Factor Zhx2 Deficiency Reduces Atherosclerosis and Promotes Macrophage Apoptosis in Mice", (2018) Arterioscler Thromb Vase Biol. 38: 2016 - 2027.

[0015] At low doses, the COVID-19 cocktail, but not individual cytokines, induced glomerular injury in mice and albuminuria similar to COVID-19-associated proteinuria. The cytokine cocktail activated STAT6 signaling in cultured glomerular epithelial cells, but reduced it in CRISPR B Zhx2 hypomorphic glomerular epithelial cells. Depletion of selected single cytokines ameliorated glomerular injury and albuminuria. At high doses, the COVID-19 cocktail, but not individual cytokines, induced common clinical manifestations of SARS-CoV-2 disease in mice, including acute cardiac injury, myocarditis, pericarditis, liver and kidney injury, as well as high mortality. STAT5, STAT6 and NFκB pathways were activated in these organs. Dual depletion after model induction of TNF-α and selected combinations of IL-2 or IL-13 or IL-4 in BALB / c. In summary, the systemic manifestations of viral cytokine storm, mechanisms of disease, and therapeutic principles to reduce morbidity and mortality have been identified. [Means for solving the problem]

[0016] In an embodiment of the invention, there is provided a method of inhibiting, treating or preventing the effects of a cytokine storm as a result of a viral infection in a patient comprising inhibiting, neutralizing or depleting one or more cytokines from the patient.

[0017] In any embodiment, the cytokine storm can be induced by viral infection caused by any respiratory or non-respiratory virus. Thus, the viral infection can be caused by viral illnesses, including respiratory viruses such as SARS-CoV-1 and SARS-CoV-2, which have pathological effects on non-respiratory organs even in the absence of obvious direct viral infection. In addition, the viral infection can be caused by other respiratory and non-respiratory viruses, such as influenza, parainfluenza, respiratory syncytial virus, adenovirus, enterovirus, other coronaviruses, cytomegalovirus (CMV), Epstein-Barr virus (EBV), Middle East respiratory syndrome (MERS), and Ebola hemorrhagic fever, which are also known to cause cytokine storms and cause death and multi-organ damage.

[0018] In another embodiment, the cytokine storm is caused by a non-viral infection, for example an infection caused by a bacteria, a fungus, or a protozoa.

[0019] In yet another embodiment, the cytokine storm is of non-infectious etiology, such as that associated with cancer or its treatment, organ transplantation, or changes in the stable cytokine milieu of a systemic disorder such as diabetes.

[0020] Furthermore, since the inventors have discovered that a major difference between the common cold (which causes a mild illness) and the more severe cytokine storm profile caused by viral infections, e.g. caused by SARS-CoV-2, is the presence of concomitant acute activation of the "allergic pathways" related to IL-4, IL-13, the present invention also includes viral infections that involve significant concomitant activation of allergic pathways.

[0021] In some embodiments, the present invention provides methods of depleting two or more cytokines to reduce mortality caused by severe cytokine storm.

[0022] In another embodiment of the present invention, a method for treating the effects of acute cardiac injury, acute liver injury, and acute kidney injury caused by a cytokine storm is provided. In some embodiments, the cytokine storm is caused by a viral infection.

[0023] In yet another embodiment of the present invention, a method of reducing mortality caused by a cytokine storm is provided. In some embodiments, the cytokine storm is caused by a viral infection.

[0024] In an embodiment of the disclosure, a method is provided for preventing multiple organ damage induced by cytokine storm, comprising inhibiting, neutralizing or depleting two or more cytokines.

[0025] In yet another embodiment of the present invention, there is provided a method for treating or preventing the effects of post-acute sequelae following SARS-Cov-2 infection comprising inhibiting, neutralizing or depleting one or more cytokines.

[0026] In an embodiment of the disclosure, a method of preventing recurrence of a viral infection is provided. In certain embodiments, the method comprises providing a treatment that inhibits, neutralizes, or depletes one or more cytokines.

[0027] In yet another embodiment of the invention, a method is provided for treating or preventing the effects of a SARS-CoV-2 viral mRNA vaccine, comprising inhibition, neutralization or depletion of one or more cytokines.

[0028] In other embodiments, animal models of cytokine storm caused by viral infections and other disease conditions are provided for testing methods of treating or preventing the effects of said cytokine storm.

[0029] Thus, in any of the methods provided herein, one or more cytokines can be inhibited, neutralized or depleted by administration to the patient of an agent comprising an adeno-associated virus (AAV) or lentivirus containing a short hairpin RNA (shRNA) against one or more cytokines.

[0030] In some embodiments, the shRNA is commercially available and may be associated with or be part of any vector known in the art, including plasmids, viral vectors, bacteriophages, cosmids, and artificial chromosomes.

[0031] In other embodiments, the agent comprises a monoclonal or polyclonal antibody against one or more cytokines. In yet other embodiments, the agent comprises a monoclonal or polyclonal antibody against one or more cytokines. In yet other embodiments, the agent is a siRNA or antisense oligonucleotide targeting one or more cytokines.

[0032] In yet other embodiments, the agent is an antagonist that binds to a cytokine-mediated receptor and prevents binding of one or more cytokines.

[0033] In an embodiment of the disclosure, a method for treating a viral infection is provided, hi certain embodiments, the method comprises providing a therapy that inhibits, neutralizes or depletes one or more cytokines.

[0034] In any of the disclosed embodiments, the one or more cytokines inhibited, neutralized or depleted include TNFα, IL-2, IL-4, IL-13, IFN-γ, or IL-6.

[0035] It will be appreciated from the disclosure herein that depending on the severity of the viral infection or other condition being treated, inhibition, neutralization or depletion of two or more cytokines may be more effective than depletion of a single cytokine. [Brief description of the drawings]

[0036] [Figure 1a] Figure 1a-g show the development and characterization of the COVID-19 cytokine storm model. (Figure 1a) Schematic representation of COVID-19 induced cytokine storm in the context of human disease. [Figure 1b] Composition of dose X of COVID cocktail A-D. [Figure 1c] Albuminuria after injection of different doses of Cocktail D in BALB / cJ mice (n=4 mice per group). Half-times (X / 2) is the threshold nephritogenic dose in BALB / cJ mice. [Figure 1d] Albuminuria after injection of dose X of individual COVID cocktail components in BALB / cJ mice (n=4 mice per group). [Figure 1e] Albuminuria after injection of dose X / 2 of COVID cocktail A-D in BALB / c mice (n = 6 mice per group). [Figure 1f] Albuminuria after injection of dose X / 2 of COVID cocktail A-D in BALB / cJ mice (n = 6 mice per group). [Figure 1g] Albuminuria after injection of BALB / c mice with dose X / 2 of intact cocktail C or with dose X / 2 of cocktail C lacking individual podocyte-targeting components (n=6 mice per group). P<0.05; **P<0.01; ***P<0.001. All significance values ​​are two-sided. [Figure 2a]Figures 2a-i: Evaluation of systemic injury induced by a high dose of Cocktail D (3X) compared with low or 3X doses of individual components in BALB / c mice. (Figure 2a) Acute myocardial injury assessed by cardiac troponin I (cTPI3) levels (n=8 mice per group). [Figure 2b] Acute liver injury assessed by alanine aminotransferase (ALT) activity levels (n = 8 mice per group). [Figure 2c] Acute kidney injury assessed by serum creatinine measured using mass spectrometry (n=8 mice per group). [Figure 2d] Histological characterization of acute cardiac injury using H&E stained sections from mice injected with 3x dose of cocktail D (n=3 mice per group), showing myocardial lysis (red arrows), inflammation (black arrows), fibrosis (blue arrows), eosinophilia (green arrows), and pericarditis (orange arrows). [Figure 2e] Histological characterization of acute liver injury using H&E stained sections from mice injected with 3x dose of cocktail D (n=3 mice per group), showing hepatocellular injury (red arrows), inflammation (black arrows), prominent Kupffer cells (green arrows), regenerative changes (yellow arrows), and pericentral venous injury (blue arrows). [Figure 2f] Histological evaluation of acute kidney injury using PAS-stained sections (rows 1, 2, 4) and kidney electron microscopy (row 3) from mice injected with 3x dose of cocktail D (n=3 mice per group). The first three rows show the proximal tubules and the last row shows the distal tubules. In the proximal tubules, there was vacuolization (red arrows), brush border destruction (green arrows), and tubular degeneration (black arrows). In the distal tubules, there was evidence of desquamation (blue arrows). Foam cells were also seen (white arrows). Light microscopy scale bar 20 μm; electron microscopy scale bar 2.66 μm. [Figure 2g] Table showing morphometric analysis of histological changes in the hearts of BALB / c mice. [Figure 2h] Table showing morphometric analysis of histological changes in the liver of BALB / c mice. [Figure 2i] Table showing morphometric analysis of histological changes in the kidneys of BALB / c mice. *P<0.05; **P<0.01; ***P<0.001, all values ​​are based on two-tailed tests. [Figure 3a] Figure 3a-h shows the treatment strategies for the impact of mild and moderate cytokine storm on glomerular and systemic diseases. All depleting antibodies or control IgG were injected intravenously 1 hour after model induction. (Figure 3a) Albuminuria after Cocktail C model induction in BALB / c mice (n=6 mice per group, dose X / 2), followed by control IgG or depleting antibodies. Groups are ordered by potency from left to right. [Figure 3b] Urinary albumin to creatinine ratios at baseline and day 1 in the 1.8x Cocktail D dose model (n=8 BALB / c mice per group) after depletion of one or more components with antibodies. [Figure 3c] Serum cardiac troponin I (cTPI3) levels on day 1 in the cocktail D 1.8x dose model (n=8 BALB / c mice per group) after depletion of one or more components with antibodies. Control and cocktail D 1.8x + IgG injected BALB / cJ mice are shown for comparison. [Figure 3d] Serum ALT activity on day 1 in the cocktail D 1.8x dose model (n=8 BALB / c mice per group) after depletion of one or more components with antibodies. Control and cocktail D 1.8x + IgG injected BALB / cJ mice are shown for comparison. [Figure 3e] Serum creatinine on day 1 in the cocktail D 1.8x dose model (n=8 BALB / c mice per group) after depletion of one or more components with antibodies. Control and cocktail D 1.8x + IgG injected BALB / cJ mice are shown for comparison. [Figure 3f] Table showing a comparison of morphometric analysis and histological changes in the hearts between control IgG and antibody-treated BALB / c mice. [Figure 3g]Table showing a comparison of morphometric analysis and histological changes in the liver between control IgG and antibody-treated BALB / c mice. [Figure 3h] Table showing comparison of morphometric analysis and histological changes in kidneys between control IgG and antibody-treated BALB / c mice. n=3 mice per morphometric analysis group. *P<0.05; **P<0.01; ***P<0.001, all values ​​are based on two-tailed tests. [Figure 4a] Figure 4a-g show possible therapeutic strategies for the impact of severe cytokine storm on systemic disease in BALB / c mice. The number of mice injected per group is shown in panel a. All depleting antibodies or control IgG were injected intravenously 1 hour after model induction. A large (red) asterisk indicates universal mortality. (Figure 4a) Mortality table of BALB / c mice injected with cocktail D 3X together with control IgG or depleting antibodies. Timed urine collection for albuminuria was not performed in these studies because mortality was higher with metabolic cages (5 / 6) than without them (2 / 6) in the control IgG group. [Figure 4b] Serum cardiac troponin I (cTPI3) levels on day 1 in surviving mice injected with 3x doses of cocktail D followed by injection of control IgG antibody or depleting antibody. [Figure 4c] Serum ALT activity levels on day 1 in surviving mice injected with 3x doses of cocktail D followed by injection of control IgG antibody or depleting antibody. [Figure 4d] Serum creatinine levels on day 1 in surviving mice injected with 3x doses of cocktail D followed by injection of control IgG antibody or depleting antibody. [Figure 4e] Morphometric comparison of cardiac histology between control and cytokine-depleted groups. [Figure 4f] Morphometric comparison of liver histology between control and cytokine-depleted groups. [Figure 4g]Morphometric comparison of kidney histology between control and cytokine-depleted groups. Morphometric analysis. n=3 mice per group. *P<0.05; **P<0.01; ***P<0.001, all values ​​are based on two-tailed tests. [Figure 5a] Figure 5a-e shows signaling pathway activation and disease mechanisms by COVID cocktail. Examples of qualitative studies of NFκB / p-p65 (liver, 30 min), pSTAT6 (kidney, 60 min) and pSTAT5 (heart, 15 min) activation by Western blot of whole organ protein extracts from mice (n=3 per group) injected with cocktail D3x or control saline. [Figure 5b] Western blot of a quantitative study assessing activation of pSTAT6 signaling in wild-type and ZHX2 hypomorph (CRISPR B) cultured human podocytes incubated with the human counterpart of cocktail C (final concentration X / 100,000, n=3 plates per condition). [Figure 5c] Densitometry of western blots of wild-type and CRISPR B podocytes from panel b incubated with cocktail C. [Figure 5d] Albuminuria in Il4a- / - and control BALB / cJ mice after injection with cocktail C dose X / 2 (left panel) and percentage increase from baseline in albuminuria on day 1 (right panel) (n = 5–8 mice per group). [Figure 5e] Schematic diagram of the binding potential of COVID cocktail components to specific receptors already described in glomerular endothelial cells, mesangial cells, and podocytes, and the feedback loops between these cells (red). *P<0.05; **P<0.01; ***P<0.001, all values ​​are based on two-tailed tests, except for the right panel of Fig. 5d, which is one-tailed. [Figure 6a]Figures 6a-c show supplemental human data and the additive effect of the cytokine cocktail. (Figure 6a) Plasma IL-4Rα levels assessed by ELISA in general COVID-19 patients, age-, sex-, and race-matched healthy controls, and COVID-19 patients with proteinuria. The number of patient samples assayed is shown below. [Figure 6b] Electron microscopy image of a BALB / cJ mouse glomerulus 1 day after injection of cocktail D dose X / 2, showing focal areas of foot process effacement (black arrow), endothelial vacuolization (green circle), and endothelial hypertrophy (blue circle). [Figure 6c] Serum creatinine measured by mass spectrometry is not increased in the COVID cytokine cocktail dose X / 2 model (BALB / c and BALB / cJ mice; n=6 mice per group). Scale bar 0.5 μm. *P<0.05; ***P<0.001. [Figure 7a] Plasma creatine kinase, a marker of skeletal muscle damage, in BALB / cJ mice (n=4 mice per group) 24 hours after injection of different doses of Cocktail D. [Figure 7b] Serum cardiac troponin I level data from Figure 2a replotted to show with higher resolution the smaller increase in levels in some of the single cytokine injection groups. [Figure 7c] Serum ALT level data taken from Figure 2b replotted to show with higher resolution the smaller increase in levels in some of the single cytokine injection groups. [Figure 7d] 18 h albuminuria of BALB / c mice injected with a single 3x dose of cytokines, corresponding to Figure 2a-c. Considering their high mortality after cocktail D 3x, metabolic cage housing for timed urine collection is not feasible for BALB / c mice at this dose. [Figure 7e] Electron micrograph of a kidney glomerulus from a BALB / c mouse 24 hours after injection of a triple dose of cocktail D. Extensive foot process effacement (red arrow), endothelial hypertrophy (green arrow), and remodeling of the glomerular basement membrane (GBM) (blue arrow) were observed. [Figure 7f] Hematoxylin and eosin stained skeletal muscle from a BALB / cJ mouse 24 hours after injection of a triple dose of cocktail D. Focal inflammation (black arrows) was noted in some sections. [Figure 7g] Albuminuria after cocktail C induction in BALB / c mice (n=6 mice per group, dose X / 2) and receptor blockade using antibodies against IL-4Rα, TNFR1, IL-10Rβ, or control IgG. Scale bars (e) 0.5 μm, (f) 20 μm. *P<0.05; **P<0.01; ***P<0.001. [Figure 8a] Figure 8a-e show the histology of the intermediate dose injury. Histological sections from a study of BALB / c mice (n=3 mice per group) euthanized 24 hours after injection of Cocktail D 1.8x dose and additional antibody or control IgG 1 hour after model induction (see Figure 3). Number code for each group is as follows: "1"=control IgG; "2"=anti-TNFα antibody; "3"=anti-IL-6 antibody; "4"=anti-IL-10 antibody; "5"=anti-TNFα+anti-IFNγ+anti-IL-4 antibody; "6"=anti-IL4 antibody; "7"=anti-TNFα+anti-IL-4+anti-IL-10 antibody; "8"=anti-IFNγ antibody; "9"=anti-TNFα+anti-IL-4 antibody. (Figure 8a) Two rows of H&E stained sections of heart and pericardium. Myocardial lysis (red arrow), inflammation (black arrow), eosinophilia (green arrow), pericarditis (orange arrow), and pericardial microcalcifications (blue arrow) were observed. [Figure 8b] H&E stained section of the liver showing hepatocellular injury (red arrow), inflammation (black arrow), degenerative changes (green arrow), and regenerative changes (yellow arrow). [Figure 8c] Toluidine blue stained Epon section of kidney showing gross tubular morphology. Tubular vacuolization (red arrow) and tubular degeneration (black arrow) were noted in the proximal tubules. [Figure 8d] Electron micrograph of a kidney tubule. Tubular vacuolization (red arrow) and tubular degeneration (black arrow) were observed in the proximal tubule. [Figure 8e]Electron micrographs of glomeruli. Areas of podocyte foot process effacement (black arrows) were observed. Scale bars: (a) 20 μm, (b) 20 μm, (c) 20 μm, (d) 0.5 μm, (e) 0.5 μm. [Figure 9a] Figure 9a-d show histology of the severe injury model. Histological sections from a study of BALB / c mice (n=3 mice per group) injected with 3x Cocktail D doses and euthanized 24 hours after injection of additional antibody or control IgG 1 hour after model induction (see Figure 4). The number codes for each group are as follows: "1" = control IgG; "2" = anti-IL-2 antibody; "3" = anti-TNFα + anti-IL-2 antibody; "4" = anti-TNFα + anti-IL-13 antibody; "5" = anti-TNFα + anti-IL-4 antibody; "6" = anti-TNFα antibody; "7" = anti-IL-13; "8" = anti-IL-4 antibody; "9" = anti-TNFα + anti-IFNγ antibody; "10" = anti-TNFα + anti-IL-6 antibody; "11" = anti-IFNγ antibody; "12" = anti-TNFα + anti-ACE2 antibody; "13" = anti-TNFα + anti-IL-10 antibody; "14" = anti-IL-6 antibody. (Fig. 9a) Two rows of H&E stained sections of heart and pericardium. Myocardial lysis (red arrow), inflammation (black arrow), eosinophilia (green arrow), and pericarditis (orange arrow) were observed. [Figure 9b] Two rows of H&E stained sections of liver showing hepatocellular injury (red arrows), inflammation (black arrows), degenerative changes (green arrows), and regenerative changes (yellow arrows). [Figure 9c] Two rows of toluidine blue stained kidney sections showing gross tubular morphology. Tubular vacuolization (red arrows) and tubular degeneration (black arrows) were observed in the proximal tubules. [Figure 9d] Two rows of electron micrographs of kidneys showing images of glomeruli. Areas of podocyte foot process effacement (black arrows) were observed. Scale bars: (a) 20 μm, (b) 20 μm, (c) 20 μm, (d) 0.5 μm. [Figure 10a]Confocal expression of cytokine receptors in BALB / c mouse glomeruli. White arrows indicate receptor expression in podocytes (P), endothelial cells (E), and mesangial cells (M). TNFR1 is expressed in podocytes and endothelial cells, so there is only partial colocalization with the podocyte protein nephrin (blue). Green is the nuclear stain. [Figure 10b] Confocal expression of ACE-2 and cytokine receptors (red) in renal tubules of BALB / c mice. Most images show proximal tubules, whereas IL-10Rβ images show collecting ducts. [Figure 10c] Western blot characterization of antibodies used in depletion studies with recombinant proteins constituting the cytokine cocktail. Scale bars (a) 20 μm (b) 20 μm. [Figure 11a] FIG. 11a is a schematic diagram of data compiled from the Human Protein Atlas Project (https: / / www.proteinatlas.org / ) showing the approximate distribution and semi-quantitative expression of cytokine receptors and ACE2 renal tubular segments. [Figure 11b] Approximate distribution and semi-quantitative expression of cytokine receptors and myocardium are shown. [Figure 11c] Approximate distribution and semi-quantitative expression of cytokine receptors and liver are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The present invention provides a mechanism for targeting various cytokines. The present invention describes specific combinations of cytokines or soluble factors that must be depleted to eliminate or reduce the effects of cytokine storm, including mortality and end-organ damage. Overview of the methods provided The present invention provides a method of inhibiting, treating or preventing the effects of a cytokine storm as a result of viral infection in a patient comprising inhibiting, neutralizing or depleting one or more cytokines from the patient.

[0038] The inventors believe that in any embodiment, the cytokine storm can be induced by a viral infection caused by any respiratory or non-respiratory virus. Thus, the viral infection can be caused by viral diseases, including respiratory viruses such as SARS-CoV-1 and SARS-CoV-2, which have pathological effects on non-respiratory organs even in the absence of obvious direct viral infection. In addition, the viral infection can be caused by other respiratory and non-respiratory viruses, such as influenza, parainfluenza, respiratory syncytial virus, adenovirus, enterovirus, other coronaviruses, cytomegalovirus (CMV), Epstein-Barr virus (EBV), Middle East respiratory syndrome (MERS), and Ebola hemorrhagic fever, which are also known to cause a cytokine storm and cause mortality and multi-organ damage. Furthermore, since the inventors have discovered that a major difference between the common cold (which causes a mild illness) and the more severe cytokine storm profile caused by viral infections, e.g. caused by SARS-CoV-2, is the presence of concomitant acute activation of the "allergic pathways" related to IL-4, IL-13, the present invention also includes viral infections that involve significant concomitant activation of allergic pathways.

[0039] Embodiments of the present invention provide the following:

[0040] A method for depleting two or more cytokines to reduce mortality caused by severe cytokine storm; A method of treating the effects of acute cardiac injury, acute liver injury and acute kidney injury caused by a cytokine storm. The inventors believe that in some embodiments, a cytokine storm may be caused by a viral infection; A method of reducing mortality caused by a cytokine storm. The inventors believe that in some embodiments, a cytokine storm may be caused by a viral infection; A method for preventing cytokine storm-induced multi-organ damage, comprising inhibiting, neutralizing or depleting two or more cytokines; A method for treating or preventing the effects of post-acute sequelae following SARS-Cov-2 infection, comprising inhibiting, neutralizing or depleting one or more cytokines; A method for preventing recurrence of a viral infection comprising providing a treatment that inhibits, neutralizes or depletes one or more cytokines; · A method for treating or preventing the effects of a SARS-CoV-2 viral mRNA vaccine, comprising inhibiting, neutralizing or depleting one or more cytokines; Animal models of cytokine storm induced by viral infections and other disease states to test methods of treating or preventing the effects of cytokine storm; and The method is for treating a viral infection.

[0041] The inventors also believe that the methods of the invention can be used in any disease state in which a cytokine storm occurs, including diseases of non-viral origin, e.g. diseases resulting from alterations in the systemic cytokine milieu of bacterial, fungal or parasitic infections, cancer, organ transplants, or multi-organ diseases such as diabetes or metabolic syndrome.

[0042] The inventors believe that in any of the disclosed methods, one or more cytokines can be inhibited, neutralized or depleted.One possible method is to administer to a patient a drug comprising an adeno-associated virus (AAV) or lentivirus containing short hairpin RNA (shRNA) against one or more cytokines.The shRNA can be made or purchased, and it can be attached to or be part of any vector known in the art, including plasmids, viral vectors, bacteriophages, cosmids, and artificial chromosomes.

[0043] Another possible method of depleting one or more cytokines is by administration of monoclonal or polyclonal antibodies against one or more cytokines. In yet another embodiment, the agent comprises a monoclonal or polyclonal antibody against one or more cytokines.

[0044] The agent may also be an siRNA or an antisense oligonucleotide targeted to one or more cytokines.

[0045] In yet other embodiments, the agent is an antagonist that binds to a cytokine-mediated receptor and prevents binding of one or more cytokines.

[0046] In any of the disclosed embodiments, the one or more cytokines inhibited, neutralized or depleted include TNFα, IL-2, IL-4, IL-13, IFN-γ or IL-6. It will be appreciated from the disclosure herein that depending on the severity of the viral infection or other condition being treated, inhibition, neutralization or depletion of more than one cytokine may be more effective than depletion of a single cytokine.

[0047] Throughout this disclosure, various quantities, e.g., amounts, sizes, dimensions, ratios, etc., are presented in range format. It should be understood that the description of quantities in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiment. Thus, the description of a range shall be considered to have specifically disclosed all possible subranges and all individual numerical values ​​within that range, unless the context clearly indicates otherwise. For example, description of a range such as 1-6 shall be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., and individual values ​​within that range, e.g., 1.1, 2, 2.3, 4.62, 5, 5.9, etc. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges and are encompassed within the scope of the disclosure, subject to any specifically excluded limitations within the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, unless the context clearly indicates otherwise.

[0048] The terminology used herein is merely for the purpose of describing certain embodiments and is not intended to limit any embodiment. As used herein, the singular forms "a", "an" and "the" are intended to include the plural unless the context clearly indicates otherwise. It will be further understood that the terms "includes", "comprises", "including" and / or "comprising" as used herein specify the presence of the stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. It will further be noted that items included in a list in the format "at least one of A, B, and C" can mean (A); (B); (C); (A and B); (B and C); (A and C); or (A, B, and C). Similarly, an item listed in the format "at least one of A, B, or C" can mean (A); (B); (C); (A and B); (B and C); (A and C); or (A, B and C).

[0049] Unless otherwise specified or clear from the context, as used herein, the term "about" in reference to a numerical value or numerical range is understood to mean, for the stated numerical value and values ​​+ / - 10% thereof, or for values ​​recited as ranges, 10% below the recited lower limit and 10% above the recited upper limit.

[0050] In any embodiment disclosed herein, the term "treating" or "to treat" includes inhibiting, slowing, halting, or reversing the progression or severity of an existing condition or disorder.

[0051] In any embodiment disclosed herein, the term "patient" refers to a human.

[0052] Cytokine inhibitors The present invention contemplates that various cytokines can be neutralized or inhibited by several different non-limiting methods. For example, as described herein, target cytokines can be neutralized or inhibited by administration of a therapeutically effective amount of an agent comprising an adeno-associated virus (AAV) or lentivirus containing short hairpin RNA (shRNA) against one or more cytokines (sh-"cytokines"). In some embodiments, sh-"cytokines" are commercially available and can be attached to or part of any vector known in the art, including plasmids, viral vectors, bacteriophages, cosmids, and artificial chromosomes.

[0053] Alternatively, as described herein, one or more target cytokines may be neutralized or inhibited by administration of a therapeutically effective amount of an agent comprising an antibody, bivalent antibody, or monoclonal antibody against one or more specific target cytokines.

[0054] Furthermore, as described herein, one or more target cytokines may be neutralized or inhibited by administration of a therapeutically effective amount of an agent comprising an siRNA or antisense oligonucleotide targeted to one or more target cytokines.

[0055] Also, as contemplated herein, one or more target cytokines may be neutralized or inhibited by administration of a therapeutically effective amount of an agent, including an antagonist, that binds to a target cytokine-mediated receptor and prevents binding of one or more target cytokines.

[0056] The one or more targeted cytokine inhibitors or compositions thereof can be administered once a day, more than once a day, or once a week. The one or more targeted cytokine inhibitors or compositions containing same can be administered to a subject by any conventional means, including oral, intramuscular, intraperitoneal, or intravenous administration. When injected, they can be injected at a single site per administration or at multiple sites per administration.

[0057] Cytokine antibodies and related inhibitors More specifically, the cytokine inhibitor is an antibody against any cytokine as disclosed herein. Examples of suitable antibodies against one or more target cytokines are disclosed herein and known to those skilled in the art. The cytokine antibody may also include an antibody fragment or a bivalent antibody or a fragment thereof that inhibits one or more target cytokines. As described herein, the cytokine inhibitor may be part of a pharmaceutical composition, which may include either an antibody against one or more target cytokines or a fragment thereof.

[0058] The anti-cytokine antibodies described herein can be produced or obtained by any means known in the art, including commercially available. It is also contemplated that antibodies can react specifically with a particular cytokine protein or polypeptides can also be used as antagonists. The anti-cytokine antibodies herein can be antibodies or fragments thereof that bind to a cytokine, or bivalent antibodies that bind to two different cytokines.

[0059] As used herein, the term "antibody" refers to an immunoglobulin (Ig), whether natural or partially or wholly synthetically produced. The term also covers any polypeptide or protein having a binding domain that is, or is homologous to, an antigen-binding domain. The term further includes "antigen-binding fragments" and other interchangeable terms that refer to similar binding fragments, such as those described below.

[0060] Native antibodies and native immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is typically linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced disulfide bridges within the chain. Each heavy chain contains a variable domain ("V") at one end. H " or "VH") followed by several constant domains ("C H Each light chain has at one end a variable domain ("V" or "CH"). L " or "VL") at one end, and a constant domain ("C L " or "CL"). The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light-chain variable domain and the heavy-chain variable domain.

[0061] The cytokine inhibitors described herein may be "synthetic polypeptides" derived from "synthetic polynucleotides" derived from "synthetic genes", meaning that the corresponding polynucleotide sequence or a portion thereof, or the amino acid sequence or a portion thereof, is derived from a designed or newly synthesized or modified sequence compared to the equivalent natural sequence. Synthetic polynucleotides (antibodies or antigen-binding fragments) or synthetic genes can be prepared by methods known in the art, including but not limited to chemical synthesis of nucleic acid or amino acid sequences. Synthetic genes typically differ from natural genes at the amino acid level or polynucleotide level (or both) and are typically placed in the context of synthetic expression control sequences. Synthetic gene polynucleotide sequences do not necessarily code for proteins with different amino acids compared to natural genes. For example, these sequences may also include synthetic polynucleotide sequences that incorporate different codons but code for the same amino acids (i.e., the nucleotide changes represent silent mutations at the amino acid level).

[0062] With respect to anti-cytokine antibodies, the term "antigen" refers to any of the cytokine proteins disclosed herein or any fragment of that protein molecule, respectively.

[0063] The terms "antigen-binding portion of an antibody," "antigen-binding fragment," "antigen-binding domain," "antibody fragment," or "functional fragment of an antibody" are used interchangeably herein to refer to one or more fragments of an antibody that retain the ability to specifically bind to one or more cytokines.

[0064] Cytokine antibodies are also contemplated to include "diabodies," which refer to small antibody fragments with two antigen-binding sites, which comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. See, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444 6448 (1993).

[0065] It is contemplated that cytokine antibodies may also include "chimeric" forms of non-human (e.g., murine) antibodies, including chimeric antibodies that contain minimal sequence derived from non-human Ig. In most cases, chimeric antibodies are murine antibodies into which at least a portion of the immunoglobulin constant region (Fc), usually of a human immunoglobulin, has been inserted in place of the mouse Fc. See, e.g., Jones et al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

[0066] It is contemplated that cytokine antibodies may also include "monoclonal antibodies," which refer to antibodies obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that may contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies may be produced by hybridoma methods, recombinant DNA methods, or isolated from phage antibodies.

[0067] As used herein, "immunoreactive" refers to a binding agent, antibody or fragment thereof that is specific for a sequence of amino acid residues ("binding site" or "epitope") on a cytokine protein, but is non-toxic at levels formulated for administration to human use, even if cross-reactive with other peptides / proteins. The term "binding" refers to a direct association between two molecules, e.g., by covalent, electrostatic, hydrophobic, ionic and / or hydrogen bonding interactions under physiological conditions, including interactions such as salt bridges and water bridges, as well as other conventional binding means. The term "preferentially binds" means that the binding agent binds to the binding site with higher affinity than it binds to an unrelated amino acid sequence.

[0068] As used herein, the term "affinity" refers to the equilibrium constant of reversible binding of two agents, expressed as Kd. The affinity of a binding protein to a ligand, e.g., the affinity of an antibody to an epitope, can be, for example, about 100 nanomolar (nM) to about 0.1 nM, about 100 nM to about 1 picomolar (pM), or about 100 nM to about 1 femtomolar (fM). As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation after dilution. Apparent affinity can be determined by methods such as enzyme-linked immunosorbent assay (ELISA), or any other technique familiar to those of skill in the art. Avidity can be determined by methods such as Scatchard analysis, or any other technique familiar to those of skill in the art.

[0069] "Epitope" refers to that portion of an antigen or other macromolecule capable of forming a binding interaction with the variable region binding pocket of an antibody.

[0070] The term "specific" refers to the situation where the antibody does not show any significant binding to molecules other than the antigen containing the epitope recognized by the antibody. The term is also applicable, for example, when the antigen-binding domain is specific for a particular epitope carried by some antigens, in which case the antibody can bind to various antigens carrying the epitope. The term "preferentially binds" or "specifically binds" means that the antibody binds to the epitope with higher affinity than it binds to an unrelated amino acid sequence, and is non-toxic at the levels formulated for administration to human use, even if there is cross-reactivity with other polypeptides containing the epitope.

[0071] The term "binding" refers to a direct association between two molecules, e.g., by covalent, electrostatic, hydrophobic, ionic and / or hydrogen bonding interactions under physiological conditions, and includes interactions such as salt bridges and water bridges as well as any other conventional binding means.

[0072] As discussed herein, targeted cytokine inhibitors can be produced by gene expression technology. The term "RNA interference" or "RNAi" refers to the silencing or reduction of gene expression by siRNA. It is a process of sequence-specific post-transcriptional gene silencing in animals and plants, initiated by siRNA that is homologous in the duplex region to the sequence of the silenced gene. The gene may be endogenous or exogenous to the organism, and may be present integrated in a chromosome or present in a transfection vector that is not integrated in the genome. The expression of the gene is completely or partially inhibited. RNAi may also be considered to inhibit the function of the target RNA. The function of the target RNA may be complete or partial.

[0073] The term "siRNA" refers to small interfering RNA. In some embodiments, siRNA comprises a duplex or double-stranded region of about 18-25 nucleotides in length. Often, siRNA contains about 2-4 unpaired nucleotides at the 3' end of each strand. At least one strand of the duplex or double-stranded region of the siRNA is substantially homologous or substantially complementary to a target RNA molecule. The strand complementary to the target RNA molecule is the "antisense strand" and the strand homologous to the target RNA molecule is the "sense strand" and is also complementary to the siRNA antisense strand. siRNA may also contain additional sequences. Non-limiting examples of such sequences include linking sequences, or loops, and stems, as well as other folded structures. siRNA appears to function as an important intermediary in triggering RNA interference in invertebrates and vertebrates, and in triggering sequence-specific RNA degradation during post-transcriptional gene silencing in plants.

[0074] It is also contemplated that any cytokine gene can be silenced or "turned off" using CRISPR technology, as disclosed in the Examples herein.

[0075] "Post-acute sequelae" "Post-acute sequelae" of SARS-CoV-2 or COVID-19, also known as "Long COVID", is used herein to describe any of the long-term symptoms or effects described as part of this invention that may be experienced weeks to months after primary infection with SARS-CoV-2, the virus that causes COVID-19.

[0076] Common methods COVID and cold cytokine cocktails and related animal studies All animal studies performed were approved by the IACUC of Rush University or the University of Alabama at Birmingham. All animals were treated humanely according to protocols. Methods for isolation of mouse glomeruli by Dynabeads, isolation of rat glomeruli by sieving, tissue preservation for tissue sections, 18-hour urine collection in unfed metabolic cages, evaluation of albuminuria and proteinuria, real-time PCR, confocal imaging, electron microscopy and sample processing, histology for light microscopy, western blotting and co-immunoprecipitation have been previously described and are known. The following were assayed using commercially available kits using serum samples; mouse ALT (BioVision K752-100), mouse cardiac troponin I type 3 (Novus Biologicals NBP3-00456), mouse creatine kinase (Abeam ab155901) and human IL-4Rα ELISA (Abeam ab46022). The following antibodies were purchased for Western blot: anti-pSTAT6 (Cell Signaling Technology, Inc. Danvers MA, USA; cat # 56554, 1:500 dilution); anti-STAT6 (Cell Signaling Technology, Inc. Cat # 5397, 1:500 dilution). Antibodies against ZHX1, ZHX2, and ZHX3 have been described previously. Mace et al. 2020; Liu et al., "ZHX proteins regulate podocyte gene expression during the development of nephrotic syndrome," (2006) J. Biol. Chem. 281:39681-39692; Clement et al., "Early changes in gene expression that influence the course of primary glomerular disease," (2007) Kidney Int. 72:337-347.

[0077] All cytokines, soluble receptors and antibodies were injected intravenously into rodents and are listed below:

[0078] [Table 1]

[0079] Antibodies used in the depletion studies were characterized by Western blot using the corresponding recombinant proteins. Each dose of cytokine cocktail was dissolved in a final volume of 100 μL of sterile 0.9% saline. BALB / cJ (Jackson Labs) and BALB / c (Envigo) mice were purchased at 8 weeks of age and allowed to acclimate for 2 weeks before baseline 18-h urine and tail blood sample collection. Additional baseline urine collections were performed on BALB / cJ mice. Most in vivo studies were performed between 10 and 15 weeks of age. Enpep - / -25 ;Zhx2 def / def The mixed background of Enpep - / -25 The dose spectrum of the cytokine cocktail was determined by crossing between BALB / cJ, BALB / c, IL4r and Zhx2-deficient BALB / cJ mice. - / - The threshold nephritogenic dose was established for mice (Jackson Labs). - / - During mouse cytokine studies using mice with BALB / cJ background (X / 2), 100 μL of 0.9% saline was administered intraperitoneally immediately after intravenous administration of the cytokine cocktail to maintain intravascular hydration. In the intermediate and high cocktail models, two additional intraperitoneal injections of 100 μL of 0.9% saline were administered at 6 and 23 hours. In cytokine depletion studies, different groups of mice were administered 50 μg of control IgG or the respective antibody or antibody combination intravenously 1 hour after administration of the mouse cytokine cocktail.

[0080] Mass spectrometric assay of plasma creatinine Serum creatinine was measured by LC / MS / MS using an Agilent 1290 Infinity II LC system coupled with a 2x50mm, 2μm Tosoh Bioscience TSK-GEL amide-80 LC column connected to an Agilent 6495 triple quadrupole mass spectrometer. The oven temperature was fixed at 40°C. The mobile phase consisted of 10mM ammonium acetate in LCMS grade water (35%) and LCMS grade acetonitrile (ACN; 65%). Synthetic creatinine (20μg / ml-0.16μg / ml range; Sigma) and isotopically labeled creatinine (D3-creatinine, 10μg / ml; Sigma) were used as standards and internal standards, respectively. 10ul of sample or standard was then combined with 5ul of internal standard and 235ul of 100% ACN, vortexed, and centrifuged at 15000rpm for 15min at 4°C. The supernatant was transferred to a new tube containing 200 μL of 10 mM ammonium acetate and 65% acetonitrile in LCMS grade water, vortexed, and centrifuged at 15,000 rpm for 15 min at 4° C. before measurement. All samples were measured in duplicate.

[0081] Sources of human genomic DNA and human kidney biopsies Genomic DNA samples from 36 patients with nephrotic syndrome, 33 control subjects, and 16 patients with diabetic nephropathy were obtained from the following sources: (a) Immortalized monocytes from plasma of patients with nephrotic syndrome at the University of Alabama at Birmingham, obtained via IRB-approved protocol X080813001 for collecting DNA, blood, and urine samples; (b) IRB-approved studies at the Instituto Nacional De Cardiologia, Mexico City, including archived kidney biopsies from patients with glomerular disease or pre-transplant kidney biopsies from healthy living related kidney donors (CONACYT 34751M, CONACYT 11-05, DPAGA-UNAM IN-201902); (c) IRB-exempt archived kidney biopsies from Hospital Nacional Alberto Sabogal Essalud, Lima, Peru; (d) Duke Molecular Physiology Institute, Duke University ... (e) the Coriell Cell Repositories, which store DNA from the 1000 Genomes Project and the HAPMAP Project. For analytical comparison between cases and controls, the 1000 Genomes Project Phase 3 Ensambl v84 was included as a single additional control.

[0082] Agilent Custom Capture and High-Throughput Illumina Sequencing A custom capture sequencing panel was created to isolate the genomic interval between HAS2 and ZHX2 on chromosome 8. Target intervals were uploaded to the SureDesign website for Agilent SureSelect capture probe design and synthesis (Agilent Technologies, Santa Clara CA). Genomic DNA library preparation and interval capture were performed using the QXT SureSelect kit according to the manufacturer's instructions (Agilent Technologies). The resulting DNA libraries were quantified by QPCR (Kapa Biosystems, Wilmington MA) and sequenced by paired-end 100 bp sequencing on an Illumina HiSeq 2500 or NextSeq 500 according to standard protocols. Approximately 15 million sequences were obtained per reaction. Generation of FASTQ files was performed using the bcl2fastq converter from Illumina (Illumina, Inc., San Diego CA). Paired Illumina sequences were compared to the hg38 database (GRCh38.pl3 Primary Assembly) using CLC Genomics software (version 12, Qiagen, Venlo, The Netherlands). Insertions and deletions ≥ 3 bp in size and a minimum of 20 sequence reads were selected for analysis. Fisher test comparisons of insertions and deletions in study and control subjects were exported in Excel format, after which all insertions and deletions present in the controls were excluded, both software-assisted and manually. Only insertions and deletions that were subsequently confirmed using IGV browser software (Broad Institute, Boston MA) were included. Establishment of homozygosity required the presence of InDels in > 85% of sequences, followed by confirmation by IGV. Minor discrepancies (1–2 base pair position differences) at the sites of insertions or deletions were occasionally observed between the two software and were resolved by Sanger sequencing during the design of the CRISPR Cas9 study.All genome numbering is based on hg38 and CLC genomics software.

[0083] Genome editing of cultured human podocytes using CRISPR / Cas9 The basic methodology for CRISPR Cas9 has been previously published, see Cong et al., "Multiplex Genome Engineering using CRISPR / Cas Systems," (2013) Science 339:819-823. Single-cell derived cell clones were generated from an established early passage immortalized human podocyte cell line51 and used for genome editing studies. The oligonucleotides and primers used are listed in Table 4.

[0084] [Table 2] TIFF2024541980000004.tif111169

[0085] CRISPR B Generation of sgRNA plasmids: To introduce a 10 bp insertion (CACACACACA), sgRNAs recognizing specific sites (Chr8-122, 533, 694-122, 533, 695) 45 bp downstream of the insertion site were designed using the Benchling website (https: / / benchling.com). Oligos G0016 and G0017 were phosphorylated and annealed using T4 polynucleotide kinase (NEB), digested with BbsI, and ligated into the pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid (kindly provided by Feng Zhang, Addgene plasmid #42230) using T7 DNA ligase (New England Biolabs). The ligation products were treated with PlasmidSafe exonuclease (Epicentre) to prevent unwanted recombination products and then transformed into One Shot TOP10 cells (Invitrogen). Ten colonies were picked and plasmids were isolated using the QIAprep Spin Miniprep kit (QIAgen). Plasmid DNA was sequenced using primer KI145 (see Table 4). Donor Plasmid Creation: The human genomic sequence of patient E58-13 containing the insert of interest was amplified using the KAPA HiFi HotStart PCR Kit (Kapa Biosystems), specific patient genomic DNA, and primers KI195 and KI196, and cloned into the pBlueScript II KS+ vector between the BamHI and HindIII restriction sites. Plasmid DNA was sequenced using KI207 to confirm the presence of the insert. A single mutation was created in the PAM sequence using the Quikchange Mutagenesis Kit (Agilent Technologies) and primers K1215 and K1216 to prevent cleavage of the donor template plasmid, and the change was confirmed by sequencing. The plasmid was then linearly amplified using primers K1219 and K1220, and the PCR product was digested with DpnI to remove any remaining circular template plasmid. An antibiotic selection cassette (puromycin resistance and truncated thymidine kinase) flanked by ITR sequences was amplified by PCR from the PB-MV1 Puro-TK plasmid (Transposagen) using primers K1217 and K1218 and ligated into the linearized plasmid (see above) at the TTAA region 78 bp upstream of the insertion using Gibson assembly Master Mix (NEB). NEB® 5-alpha competent E. coli cells were transformed with 2 μl of the assembly reaction product. Plasmid DNA was isolated from 10 colonies and sequenced using primer K1217 to confirm correct assembly. Genome editing using sgRNA and donor plasmid: To in vitro replicate the InDels identified in kidney disease patients, cultured human podocytes derived from single cells were transfected by electroporation (Biorad Gene Pulser Xcell™ electroporation system, 0.2 cm cuvette, square wave mode, 150V, 10 ms pulse) with CRISPR / Cas9 vectors containing specific sgRNAs and donor plasmids containing donor sequences and antibiotic selection cassettes. After 15 days of incubation with 1 μg / ml puromycin dihydrochloride (Gibco) to remove non-transfected cells, 10 μg of an excision-only piggyBac transposase expression vector (Transposagene) was transfected to remove the antibiotic selection cassette without scarring. Four days after transfection, cells were incubated with 2.5 μM ganciclovir (Sigma) to remove cells with residual truncated thymidine kinase activity. Single cells were picked, clones were established, genomic DNA was extracted using the QIAamp DNA Mini Kit (QIAgen), and the target region was PCR amplified using Platinum HiFi DNA polymerase (Invitrogen) and primers KI189 and KI188. PCR products were gel purified using the QIAquick Gel Extraction Kit (QIAgen), cloned into pCR2.1 vector using the TA Cloning™ kit (Invitrogen), and inserts were sequenced using the M13 forward sequencing primer. Sequences were aligned by BLAST with the native podocyte genome sequence and the donor template sequence.

[0086] CRISPR A The overall method was the same as for CRISPR B, except for the primers and oligonucleotides used, as well as the following site-specific details. An 8 bp insert (TGGATGGA) was introduced into Chr8-122, 304, 094-122, 304, 095) and sgRNA was designed to recognize a specific site 73 bp upstream of the insertion site. To generate the donor plasmid, patient-specific genomic DNA (patient SF3) was cloned between the SpeI and BamHI sites of the pBlueScript II KS+ vector. During Gibson assembly, an antibiotic resistance cassette was ligated with the linearized plasmid at the TTAA region 51 bp upstream of the insertion.

[0087] In vitro STAT6 signaling assay Wild-type (precursors of CRISPR-modified podocytes) and CRISPR-B podocytes were cultured at 33°C in RPMI 1640 medium containing heat-inactivated 10% fetal bovine serum, 1% insulin-transferrin-selenium (ITS-G, Thermo Fisher Scientific-cat. no. 41400045), and 1% penicillin-streptomycin (Thermo Fisher Scientific, cat. no. 15140122). Cells were subcultured and seeded at 50,000 cells / dish in 10 cm culture dishes and cultured at 37°C for 3 days. The culture medium was then replaced with RPMI 1640 containing heat-inactivated 0.2% FBS and 1% penicillin-streptomycin. After 24 h, cells were treated with cocktail C or cold cocktail (X / 100,000) for 10, 20 and 30 min. Proteins were isolated using RIPA buffer (Thermo Fisher Scientific, Catalog No.: 89900) containing protease inhibitors (Thermo Fisher Scientific, Catalog No.: A32953) and phosphatase inhibitors (Thermo Fisher Scientific, Catalog No.: A32957). (10 ml of RIPA buffer contained one tablet each of protease inhibitor and phosphatase inhibitor.) Protein concentration was assessed using the Bradford protein assay.

[0088] Human plasma from COVID-19 and control patients for IL-4Rα assay 100 μL aliquots of human plasma were obtained from the following sources: (a) de-identified IRB-approved hospitalized COVID patient samples from the Rush University COVID-19 Registry and Biorepository, (b) de-identified IRB-approved hospitalized COVID patient samples from the Rush University COVID-19 Registry and Biorepository selected for the presence of proteinuria, and (c) de-identified plasma samples purchased from Zenbio (Durham NC, USA) matched to group a for age, sex, and race.

[0089] statistical analysis All graph values ​​are mean + standard error. For differences in proteinuria, albuminuria, or gene expression involving two groups, unpaired Student's t-tests were used in Microsoft Excel 2013. All significance is two-sided unless otherwise stated.

[0090] See further experimental examples below. EXAMPLES

[0091] The following examples are provided for the purpose of illustrating various embodiments of the present invention and are not intended to limit the disclosure in any manner. The examples, together with the methods described herein, are representative of currently preferred embodiments and are provided by way of illustration only and are not intended to limit the scope of the present invention. Modifications therein and other uses encompassed within the spirit of the disclosure as defined by the claims will be apparent to those skilled in the art.

[0092] [Example 1] Development of a novel COVID-19 cytokine storm cocktail Figure 1a-g shows the development and characterization of the COVID-19 cytokine storm model. (Figure 1a) Schematic of COVID-19 induced cytokine storm in the context of human disease. (Figure 1b) Composition of dose X of COVID cocktail A-D. (Figure 1c) Albuminuria after injection of different doses of cocktail D in BALB / cJ mice (n=4 mice per group). X / 2 is the threshold nephritogenic dose in BALB / cJ mice. (Figure 1d) Albuminuria after injection of dose X of individual COVID cocktail components in BALB / cJ mice (n=4 mice per group). (Figure 1e) Albuminuria after injection of dose X / 2 of COVID cocktail A-D in BALB / c mice (n=6 mice per group). (Figure 1f) Albuminuria after injection of dose X / 2 of COVID cocktail A-D in BALB / cJ mice (n=6 mice per group). (FIG. 1g) Albuminuria after injection of BALB / c mice with dose X / 2 of intact cocktail C or with dose X / 2 of cocktail C lacking individual podocyte-targeting components (n=6 mice per group). P<0.05; ** P < 0.01; *** P<0.001. All significance values ​​are two-sided.

[0093] Figure 6a-c show supporting human data and the additive effect of the cytokine cocktail. (Figure 6a) Plasma IL-4Rα levels assessed by ELISA in general COVID-19 patients, age-, sex-, and race-matched healthy controls, and COVID-19 patients with proteinuria. Number of patient samples assayed are indicated below. (Figure 6b) Electron microscopy image of a BALB / cJ mouse glomerulus 1 day after injection of cocktail D dose X / 2. Areas of focal foot process effacement (black arrow), endothelial vacuolization (green circle) and endothelial hypertrophy (blue circle) were observed. (Figure 6c) Serum creatinine measured by mass spectrometry is not increased in the COVID cytokine cocktail dose X / 2 model (BALB / c and BALB / cJ mice; n=6 mice per group). Scale bar 0.5 μm. * P < 0.05; *** P<0.001.

[0094] COVID cocktails A-D were developed stepwise to model COVID-19 patients admitted to the intensive care unit (Fig. 1b, c). The first five cytokines (Fig. 1b) are common to all cocktails. Circulating IL-4Rα levels are also elevated in COVID patients with proteinuria (Fig. 6a). The COVID-19 receptor, ACE2, was included in the COVID-19 cocktail because plasma sACE2 levels were significantly higher in COVID-19 patients in the intensive care unit and in elderly and metabolic syndrome patients who are more susceptible to severe COVID-19 disease. High plasma IL-13 and IL-4 levels in COVID-19 patients suggest acute activation of allergic pathways in this disease. Cocktail B was created by removing sIL-4Rα from cocktail A and adding IL-4 and IL-13, while cocktail C was obtained by adding IL-4 to cocktail A. Cocktail D was obtained by adding IL-13 to cocktail C.

[0095] [Example 2] Systemic manifestations of synergistic multi-cytokine injury induced by COVID-19 cocktail Injection of a high dose (3-fold) of cocktail D induced albuminuria and elevated serum cardiac troponin type I3 ​​(cTPI3; myocardial injury, Fig. 2a), serum alanine aminotransferase (ALT; acute liver injury, Fig. 2b), serum creatinine (acute kidney injury, AKI; Fig. 2c), and plasma creatine kinase (CK, skeletal muscle injury; Fig. 7a).

[0096] Figure 2a-i: Evaluation of systemic injury induced by a high dose of Cocktail D (3x) compared to low or 3x doses of individual components in BALB / c mice. (Figure 2a) Acute myocardial injury assessed by cardiac troponin I (cTPI3) levels (n=8 mice per group). (Figure 2b) Acute liver injury assessed by alanine aminotransferase (ALT) activity levels (n=8 mice per group). (Figure 2c) Acute kidney injury assessed by serum creatinine measured using mass spectrometry (n=8 mice per group). (Figure 2d) Histological characterization of acute cardiac injury using H&E stained sections from mice injected with Cocktail D 3x dose (n=3 mice per group). Cardiolysis (red arrow), inflammation (black arrow), fibrosis (blue arrow), eosinophilia (green arrow), and pericarditis (orange arrow) were observed. (Fig. 2e) Histological characterization of acute liver injury using H&E stained sections from mice injected with 3x Cocktail D dose (n=3 mice per group). Hepatocellular injury (red arrow), inflammation (black arrow), prominent Kupffer cells (green arrow), regenerative changes (yellow arrow), and pericentral vein injury (blue arrow) were observed. (Fig. 2f) Histological evaluation of acute kidney injury using PAS stained sections (columns 1, 2, 4) and kidney electron microscopy (column 3) from mice injected with 3x Cocktail D dose (n=3 mice per group). The first three columns show the proximal tubules, and the last column shows the distal tubules. In the proximal tubules, vacuolization (red arrow), brush border destruction (green arrow), and tubular degeneration (black arrow) were observed. In the distal tubules, evidence of desquamation (blue arrow) was present. Foam cells were also observed (white arrow). Electron microscopy scale bar BALB / c, 2.66 μm. (FIG. 2g) Table showing morphometric analysis of histological changes in the heart of BALB / c mice. (FIG. 2h) Table showing morphometric analysis of histological changes in the liver of BALB / c mice. (FIG. 2i) Table showing morphometric analysis of histological changes in the kidney of BALB / c mice. Light microscope scale bar 20 μm. * P < 0.05; ** P < 0.01; *** P<0.001, all values ​​are based on two-tailed tests.

[0097] Figure 7a-g: (Figure 7a) Plasma creatine kinase, a marker of skeletal muscle damage, in BALB / cJ mice (n=4 mice per group) 24 hours after injection with different doses of Cocktail D. (Figure 7b) Serum cardiac troponin I level data taken from Figure 2a, replotted to show with higher resolution the smaller increase in levels in some single cytokine injection groups. (Figure 7c) Serum ALT level data taken from Figure 2b, replotted to show with higher resolution the smaller increase in levels in some single cytokine injection groups. (Figure 7d) 18-hour albuminuria of BALB / c mice injected with a single triple dose of cytokines, corresponding to Figures 2a-c. Given their high mortality after triple dose of Cocktail D, metabolic cage housing for timed urine collection is not feasible for BALB / c mice at this dose. (Figure 7e) Electron micrograph of kidney glomeruli of BALB / c mice 24 hours after injection with triple dose of Cocktail D. There was extensive foot process effacement (red arrows), endothelial hypertrophy (green arrows), and remodeling of the glomerular basement membrane (GBM) (blue arrows). (Fig. 7f) Hematoxylin and eosin stained skeletal muscle from a BALB / cJ mouse 24 hours after injection of a triple dose of cocktail D. Focal inflammation (black arrows) was noted in some sections. (Fig. 7g) Albuminuria after receptor blockade using antibodies against IL-4Rα, TNFR1, and IL-10Rβ or control IgG after cocktail C induction in BALB / c mice (n=6 mice per group, dose X / 2). Scale bars (e) 0.5 μm, (f) 20 μm. * P < 0.05; ** P < 0.01; *** P<0.001.

[0098] cTPI3, ALT and albuminuria were also increased at the 3x dose for several individual cytokines, albeit at significantly lower levels than the cocktail (Fig. 2a, b; Fig. 7b-e). Timed urine collection in metabolic cages for albuminuria assessment was not performed given the high mortality in BALB / c mice injected with the 3x dose of cocktail D (see below). The 3x dose of cocktail D induced severe cardiac, liver and acute kidney injury in BALB / c mice. Cardiac histology (Fig. 2d) revealed myocardial lysis, focal fibrosis and eosinophilia, inflammation (myocarditis) and pericarditis. Liver histology (Fig. 2e) showed parenchymal hepatocellular injury, prominent Kupffer cells, frequent degenerative and regenerative changes and mild inflammation. Histological evaluation of the kidney tubulo-interstitial compartment (Fig. 2f) revealed evidence of proximal tubule injury in the form of frequent vacuolization, luminal enlargement, brush border destruction and detachment of tubular epithelial cells. Epithelial cell desquamation, foam cells, and vacuolation were also observed in the distal tubules. Morphometric changes in these organs 24 hours after triple injection of cocktail D in BALB / cJ mice are also depicted in Figure 2g, h, i. No evidence of severe or widespread inflammation was seen.

[0099] [Example 3] Therapeutic cytokine depletion to disrupt synergy in mild and moderate cytokine storms Figure 3a-h shows the treatment strategies for the impact of mild and moderate cytokine storm on glomerular and systemic diseases. All depleting antibodies or control IgG were injected intravenously 1 hour after model induction. (Figure 3a) Albuminuria after Cocktail C model induction in BALB / c mice (n=6 mice per group, dose X / 2), followed by control IgG or depleting antibodies. Groups are ordered by potency from left to right. (Figure 3b) Ratio of urinary albumin to creatinine at baseline and day 1 of Cocktail D 1.8x dose model (n=8 BALB / c mice per group) after depletion of one or more components with antibodies. (Figure 3c) Serum cardiac troponin I (cTPI3) levels at day 1 of Cocktail D 1.8x dose model (n=8 BALB / c mice per group) after depletion of one or more components with antibodies. Control and Cocktail D 1.8x+IgG injected BALB / cJ mice are shown for comparison. (Fig. 3d) Serum ALT activity on day 1 in the cocktail D 1.8x dose model (n=8 BALB / c mice per group) after depletion of one or more components with antibodies. Control and cocktail D 1.8x + IgG injected BALB / cJ mice are shown for comparison. (Fig. 3e) Serum creatinine on day 1 in the cocktail D 1.8x dose model (n=8 BALB / c mice per group) after depletion of one or more components with antibodies. Control and cocktail D 1.8x + IgG injected BALB / cJ mice are shown for comparison. (Fig. 3f) Table showing morphometric analysis and histological changes in the heart between control IgG and antibody treated BALB / c mice. (Fig. 3g) Table showing morphometric analysis and histological changes in the liver between control IgG and antibody treated BALB / c mice. (Fig. 3h) Table showing morphometric analysis and histological changes in the kidney between control IgG and antibody treated BALB / c mice. Morphometric analysis n=3 mice per group. * P < 0.05; ** P < 0.01; *** P<0.001, all values ​​are based on two-tailed tests.

[0100] Figure 8a-e shows the histology of the intermediate dose injury. Histological sections from a study of BALB / c mice (n=3 mice per group) injected with 1.8x dose of cocktail D and euthanized 24 hours after model induction with additional antibody or control IgG injected 1 hour after model induction (see Figure 3). Number codes for each group are as follows: "1" = control IgG; "2" = anti-TNFα antibody; "3" = anti-IL-6 antibody; "4" = anti-IL-10 antibody; "5" = anti-TNFα + anti-IFNγ + anti-IL-4 antibody; "6" = anti-IL4 antibody; "7" = anti-TNFα + anti-IL-4 + anti-IL-10 antibody; "8" = anti-IFNγ antibody; "9" = anti-TNFα + anti-IL-4 antibody. (Figure 8a) Two rows of H&E stained sections of heart and pericardium. Cardiolysis (red arrow), inflammation (black arrow), eosinophilia (green arrow), pericarditis (orange arrow), and pericardial microcalcifications (blue arrow) were observed. (Fig. 8b) H&E stained section of liver. Hepatocellular injury (red arrow), inflammation (black arrow), degenerative changes (green arrow), and regenerative changes (yellow arrow) were observed. (Fig. 8c) Epon section of kidney stained with toluidine blue showing gross tubular morphology. Tubular vacuolization (red arrow) and tubular degeneration (black arrow) were observed in the proximal tubule. (Fig. 8d) Electron micrograph of kidney tubule. Tubular vacuolization (red arrow) and tubular degeneration (black arrow) were observed in the proximal tubule. (Fig. 8e) Electron micrograph of glomerulus. Areas of podocyte foot process effacement (black arrow) were observed. Scale bar (a) 20 μm (b) 20 μm (c) 20 μm (d) 0.5 μm (e) 0.5 μm.

[0101] Injection of low doses (X / 2) of cocktail C in BALB / c mice (Fig. 3a) followed by cytokine depletion alone or in combination showed that anti-TNF-α, anti-IL-10, anti-IFN-γ and selected anti-TNFα antibody-based combinations significantly reduced albuminuria. In many cases, depleting more cytokines is not necessarily better, especially in mild cytokine storm models, suggesting that excessive manipulation of the cytokine milieu can be counterproductive. In an intermediate dose (1.8-fold) model of cocktail D in BALB / c mice, anti-IL-4, anti-IL-6, TNF-α and several anti-TNFα antibody-based combinations were effective in reducing albuminuria (Fig. 3b), cTPI3 (except anti-IL-4, Fig. 3c), serum ALT levels (Fig. 3d) and normalizing serum creatinine (Fig. 3e). Morphometric analysis of these studies showed significant improvements in histological changes in the most effective regimens mentioned above. (Fig. 3f–h; Fig. 8a–e).

[0102] [Example 4] Therapeutic cytokine depletion blocks synergy, prevents mortality, and reduces multiorgan toxicity in severe cytokine storms Injection of BALB / c mice with cocktail D 3x resulted in high mortality at 24 h (Figure 4a) and was used as a model for COVID-19-affected patients requiring intensive care. Figure 4a-g shows possible treatment strategies for the impact of severe cytokine storm on systemic disease in BALB / c mice. The number of mice injected per group is shown in panel a. All depleting antibodies or control IgG were injected intravenously 1 h after model induction. A large red asterisk indicates universal mortality. (Figure 4a) Mortality table of BALB / c mice injected with cocktail D 3x together with control IgG or depleting antibodies. Timed urine collection for albuminuria was not performed in these studies because mortality was higher with metabolic cages (5 / 6) than without them (2 / 6) in the control IgG group. (Figure 4b) Serum cardiac troponin I (cTPI3) levels on day 1 in surviving mice injected with cocktail D 3x dose followed by mice injected with control IgG or depleting antibodies. (Fig. 4c) Serum ALT activity levels on day 1 in surviving mice injected with 3x Cocktail D dose followed by control IgG or depleting antibodies. (Fig. 4d) Serum creatinine levels on day 1 in surviving mice injected with 3x Cocktail D dose followed by control IgG or depleting antibodies. (Fig. 4e) Morphometric comparison of cardiac histology between control and cytokine depleted groups. (Fig. 4f) Morphometric comparison of liver histology between control and cytokine depleted groups. (Fig. 4g) Morphometric comparison of kidney histology between control and cytokine depleted groups. Morphometric analysis n=3 mice per group. * P < 0.05; ** P < 0.01; *** P<0.001, all values ​​are based on two-tailed tests.

[0103] Figure 9a-d shows histology of the severe injury model. Histological sections from a study of BALB / c mice (n=3 mice per group) injected with a triple dose of Cocktail D and euthanized 24 hours after injection of additional antibody or control IgG 1 hour after model induction (see Figure 4). The number codes for each group are as follows: "1" = control IgG; "2" = anti-IL-2 antibody; "3" = anti-TNFα + anti-IL-2 antibody; "4" = anti-TNFα + anti-IL-13 antibody; "5" = anti-TNFα + anti-IL-4 antibody; "6" = anti-TNFα antibody; "7" = anti-IL-13; "8" = anti-IL-4 antibody; "9" = anti-TNFα + anti-IFNγ antibody; "10" = anti-TNFα + anti-IL-6 antibody; "11" = anti-IFNγ antibody; "12" = anti-TNFα + anti-ACE2 antibody; "13" = anti-TNFα + anti-IL-10 antibody; "14" = anti-IL-6 antibody. (Fig. 9a) Two rows of H&E stained sections of heart and pericardium. Myocardial lysis (red arrow), inflammation (black arrow), eosinophilia (green arrow), and pericarditis (orange arrow) were observed. (Fig. 9b) Two rows of H&E stained sections of liver showing hepatocellular injury (red arrow), inflammation (black arrow), degenerative changes (green arrow) and regenerative changes (yellow arrow). (Fig. 9c) Two rows of toluidine blue stained kidney sections showing gross tubular morphology. Tubular vacuolization (red arrow) and tubular degeneration (black arrow) were observed in the proximal tubules. (Fig. 9d) Two rows of electron micrographs of kidney showing images of glomeruli. Areas of podocyte foot process effacement (black arrow) were observed. Scale bars: (a) 20 μm, (b) 20 μm, (c) 20 μm, (d) 0.5 μm.

[0104] To avoid near total mortality in the control IgG group (5 / 6 deaths), metabolic cages for urine collection were not used in this study. The combination of TNF-α depletion with IL-2, IL-13 or IL-4, or TNF-α, IL-13 or IL-4 depletion alone were the most effective in blocking the synergistic action of the cocktail components, eliminating mouse deaths at 24 h and normalizing overall activity (Fig. 4a). These interventions, especially the specific anti-TNF-α antibody-based combination, were the most effective in reducing serum levels of cTPI3, ALT and creatinine (Fig. 4b, c, d). IL-2 depletion alone effectively reduced organ damage, but mice were still partially piloerector and showed mild distress even at 24 h. IL-6, IL-10 and IFN-γ depletion alone were all counterproductive. Morphometric analysis of the heart, liver, and kidneys showed significant improvements in the most effective regimen (Figures 4e, f, g; Figures 9a–d).

[0105] Figure 5a-e shows activation of signaling pathways and disease mechanisms by COVID cocktail. (Figure 5a) Example of NFκB / p-p65 (liver, 30 min, qualitative test), pSTAT6 (kidney, 60 min) and pSTAT5 (heart, 15 min) activation by Western blot of whole organ protein extracts of mice (n=3 mice per group) injected with cocktail D 3x or control saline. (Figure 5b) Western blot of quantitative test evaluating activation of pSTAT6 signaling in wild type and ZHX2 hypomorph (CRISPR B) cultured human podocytes incubated with the human counterpart of cocktail C (final concentration X / 100,000, n=3 plates per condition). (Figure 5c) Densitometry of Western blot of wild type and CRISPR B podocytes from panel b incubated with cocktail C. (Figure 5d) Il4a activation after injection of cocktail C dose X / 2. - / -Albuminuria (left panel) and percentage increase from baseline in albuminuria on day 1 (right panel) in IgE mice and control BALB / cJ mice (n=5–8 mice per group). (Fig. 5e) Schematic representation of possible binding of COVID cocktail components to specific receptors already described in glomerular endothelial cells, mesangial cells, and podocytes, as well as the feedback loops (red) between these cells. * P < 0.05; ** P < 0.01; *** All values ​​are based on two-tailed tests, except for P < 0.001, right panel of Fig. 5d, which is a one-tailed test.

[0106] Figure 10a-c. (Figure 10a) Confocal expression of cytokine receptors in BALB / c mouse glomeruli. White arrows indicate receptor expression in podocytes (P), endothelial cells (E), and mesangial cells (M). TNFR1 is expressed in podocytes and endothelial cells, so only partial colocalization with the podocyte protein nephrin (blue) is observed. Green is nuclear staining. (Figure 10b) Confocal expression of ACE-2 and cytokine receptors (red) in renal tubules of BALB / c mice. Most images show proximal tubules, except for the image of IL-10Rβ, which is a collecting duct. (Figure 10c) Western blot characterization of antibodies used in depletion studies with recombinant proteins constituting the cytokine cocktail. Scale bars (a) 20 μm (b) 20 μm.

[0107] Consideration SARS-CoV-2 infection begins in the respiratory tract and elicits a prominent immune response, occasionally involving other organs through direct infection. The extent of extrapulmonary lesions is often out of proportion to the direct infection, suggesting that innate and adaptive immune responses to the primary infection may play an important role in pathogenesis. This study highlights the multisystem pathogenic effects of the widespread cytokine storm documented early in the pandemic.

[0108] The de novo construction of a two-viral cytokine storm model (SARS-CoV-2 / COVID-19) focuses on the synergistic rather than individual action of the components. Glomerular disease was used as the model of choice for mild cytokine storm because it allowed us to compare the impact of two common viral infections on rare (e.g., relapse of MCD due to cold) and common clinical scenarios (COVID-19-induced proteinuria) in the absence of other end-organ damage.

[0109] When a 1.8-fold or 3-fold dose of Cocktail D was used to recreate the severe COVID-19 cytokine storm in BALB / c mice, effects other than glomerular damage were observed in mice injected with the 3-fold dose, including acute myocarditis, pericarditis, liver and kidney damage, and significant acute overall mortality. Because the origin of the cytokine storm was extrinsic to these organs, there was only mild to moderate inflammation, which is often seen in patients infected with SARS-CoV-2. Compared to BALB / c mice, Zhx2 hypo / hypo BALB / cJ mice showed less severe cardiac, hepatic and renal injury and lower mortality, while the extent of glomerular injury was similar. As suggested by the literature, the differences between glomerular injury and other forms of injury are likely related to the predominant plasma membrane localization of ZHX proteins in podocytes and their extensive nuclear expression in cells of the heart, liver and kidney.

[0110] The depleting antibody was administered 1 hour after injection of the cytokine cocktail, a sufficient time to initiate multi-pathway injury, as all mice injected with the high dose of cocktail D were similarly ill at 6 hours. Improvement or disappearance at 24 hours reflected the therapeutic effect of the depletion regimen.

[0111] The most effective regimen against severe cytokine storm in BALB / c mice, which corresponds to most ICU-admitted patients, was a combination of TNFα and IL-2, IL-4 or IL-13 depletion. Using these combinations, there were no deaths, the mice's overall activity normalized, and serum cTPI3, ALT and creatinine levels were closest to normal. IL-2, TNFα, IL-4 and IL-13 depletion alone also eliminated deaths and improved overall activity, but biomarker levels tended to be higher than in the combination group or mild distress, such as hair standing on end, persisted. IL-6 depletion or IL-10 depletion alone performed worse than the other groups. A 1.8-fold intermediate dose of cocktail D was also tested in BALB / c mice to mimic the non-intensive care environment of hospitalized patients, with no deaths and less multiorgan damage. These mice responded well to the selected single cytokine depletion. In the mild cocktail C model (dose X / 2), anti-TNF-α, anti-IL-10, anti-IFN-γ and selected anti-TNFα antibody-based combinations were effective in reducing albuminuria.

[0112] The concept of synergy between different cytokines within a cytokine cocktail, rather than through equal or higher doses of individual cytokines, is clearly demonstrated. The cytokine depletion regimen described in this study may work by reducing this synergy.

[0113] Of the many pathways that may be activated in podocytes during the cytokine storm, at least two were defined: translocation of ZHX proteins from the plasma membrane and pSTAT6 signaling were activated downstream of IL-4Rα by the COVID-19 cocktail. Furthermore, the pathogenic action of circulating sACE-2 in the COVID-19 cocktail was mediated by the integrin 41 This may be mediated by interactions with sIL-4Rα. Elevated plasma levels of sIL-4Rα in COVID-19 patients with proteinuria suggest that this pathway is likely activated in this subset of patients.

[0114] As will be appreciated from the description herein, a wide variety of aspects and embodiments are contemplated by the present disclosure, examples of which include, but are not limited to, the aspects and embodiments listed below: A method for inhibiting, treating or preventing the effects of a cytokine storm as a result of a viral infection in a patient comprising inhibiting, neutralizing or depleting one or more cytokines from the patient; A method for depleting two or more cytokines to reduce mortality caused by severe cytokine storm; Methods of treating the effects of acute cardiac injury, acute liver injury and acute kidney injury caused by cytokine storm; the inventors believe that in some embodiments, cytokine storm may be caused by viral infection; A method of reducing mortality caused by a cytokine storm; the inventors believe that in some embodiments, a cytokine storm may be caused by a viral infection; A method for preventing cytokine storm-induced multi-organ damage, comprising inhibiting, neutralizing or depleting one or more cytokines; A method for treating or preventing the effects of post-acute sequelae following SARS-Cov-2 infection, comprising inhibiting, neutralizing or depleting one or more cytokines; A method for preventing recurrence of a viral infection comprising providing a treatment that inhibits, neutralizes or depletes one or more cytokines; A method for treating or preventing the effects of a SARS-CoV-2 viral mRNA vaccine, comprising inhibiting, neutralizing or depleting one or more cytokines; Animal models of cytokine storm induced by viral infections and other disease states to test methods of treating or preventing the effects of cytokine storm; and The method is for treating a viral infection.

[0115] A method of treating a cytokine storm that is not of viral origin, including bacterial, fungal or parasitic infections, cancer, organ transplants, or that results from an altered systemic cytokine environment in multi-organ diseases such as diabetes or metabolic syndrome.

[0116] A method of inhibiting, neutralizing or depleting one or more cytokines by administering to a patient an agent comprising an adeno-associated virus (AAV) or lentivirus containing a short hairpin RNA (shRNA) against one or more cytokines. The shRNA can be made or purchased and can be attached to or be part of any vector known in the art, including plasmids, viral vectors, bacteriophages, cosmids, and artificial chromosomes.

[0117] The method of depleting one or more cytokines is by administration of a monoclonal or polyclonal antibody against one or more cytokines. In yet another embodiment, the agent comprises a monoclonal or polyclonal antibody against one or more cytokines.

[0118] A method of depleting one or more cytokines is by administration of siRNA or antisense oligonucleotides targeted to one or more cytokines.

[0119] A method of depleting one or more cytokines is by administration of an antagonist that binds to the cytokine-mediated receptor and prevents binding of one or more cytokines.

[0120] In any of the disclosed methods, the one or more cytokines inhibited, neutralized or depleted include TNFα, IL-2, IL-4, IL-13, IFN-γ or IL-6. It will be understood in the disclosure herein that depending on the severity or other symptoms of the viral infection being treated, inhibition, neutralization or depletion of more than one cytokine may be more effective than depletion of a single cytokine.

[0121] Although embodiments of the present disclosure are described herein, it should be understood by those skilled in the art that such embodiments are provided by way of example only. Numerous variations, modifications, and alternatives will occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in implementing the present invention. The following claims define the scope of the present invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. A composition for use in inhibiting, treating, or preventing the effects of a mild to moderate cytokine storm in a patient, the composition comprising a first inhibitor of TNFα and a second inhibitor selected from an inhibitor of IL-2, an inhibitor of IL-13, and an inhibitor of IL-4, wherein the first and / or second inhibitor neutralize or deplete the cytokine or its effects from the patient, thereby alleviating the symptoms of a mild cytokine storm.

2. 1. A composition for use in inhibiting, treating, or preventing the effects of a severe cytokine storm in a patient, the composition comprising a first inhibitor of TNFα and a second inhibitor selected from an inhibitor of IL-2, an inhibitor of IL-13, and an inhibitor of IL-4, wherein the first and / or second inhibitor neutralize or deplete these cytokines or the effects thereof from the patient, thereby alleviating the symptoms of the severe cytokine storm.

3. 1. A composition for use in reducing mortality following a severe cytokine storm in a patient, said composition comprising a first inhibitor of TNFα and a second inhibitor selected from an inhibitor of IL-2, an inhibitor of IL-13, and an inhibitor of IL-4, wherein said first and / or second inhibitor neutralize or deplete these cytokines or their effects from the patient, thereby reducing mortality.

4. The composition of claim 1, wherein the composition comprises a third inhibitor that neutralizes or depletes IFN-γ.

5. The composition described in claim 2 or 3, wherein the composition comprises a third inhibitor that neutralizes or depletes IFN-γ or IL-6.

6. The composition described in claim 1, wherein the cytokine storm is the result of a disease state selected from the group consisting of bacterial infection, fungal infection, parasitic infection, cancer, organ transplant, diabetes and metabolic syndrome.

7. The composition described in claim 2 or 3, wherein the cytokine storm is the result of a disease state selected from the group consisting of bacterial infection, fungal infection, parasitic infection, cancer, organ transplant, diabetes and metabolic syndrome.

8. A composition for use in treating or preventing the effects of post-acute sequelae of SARS-CoV-2 infection caused by a cytokine storm, said composition comprising a first inhibitor of TNFα and a second inhibitor selected from an inhibitor of IL-2, an inhibitor of IL-13, and an inhibitor of IL-4, wherein said first and / or second inhibitor neutralize or deplete one or more cytokines from a patient.

9. A composition for use in treating or preventing the effects of an mRNA vaccine for SARS-CoV-2 virus in a patient, said composition comprising a first inhibitor of TNFα and a second inhibitor selected from an inhibitor of IL-2, an inhibitor of IL-13, and an inhibitor of IL-4, wherein said first and / or second inhibitor neutralize or deplete one or more cytokines from the patient.

10. A composition described in any one of claims 1 to 3, wherein the cytokine storm is the result of a viral infection.

11. A composition described in any one of claims 1 to 4, 6, 8 and 9, wherein each inhibitor is an antibody or antibody fragment against one or more cytokines selected from a polyclonal antibody, a monoclonal antibody, a bivalent antibody, and an antibody fragment of any of them.

12. The composition of claim 11, wherein the antibody or antibody fragment is bivalent and targets two different cytokines.

13. The composition of claim 10, wherein the viral infection is caused by an infection selected from the group consisting of SARS-CoV-1, SARS-CoV-2, influenza, parainfluenza, respiratory syncytial virus (RSV), adenovirus, enterovirus, other coronaviruses, cytomegalovirus (CMV), Epstein-Barr virus (EBV), Middle East respiratory syndrome (MERS), and Ebola virus.

14. A composition described in any one of claims 1 to 3, wherein the effects of cytokine storm are non-respiratory effects.

15. The composition described in claim 14, wherein the non-respiratory effects are selected from acute cardiac injury, acute liver injury, and acute kidney injury.

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