Methods and compositions for treating coronavirus

Abstract

Disclosed are methods for inhibiting coronavirus infection or replication in cells, comprising exposing cells to an alternating current electric field for a period of time, the alternating current electric field having a frequency and field strength that inhibits coronavirus infection or replication. Disclosed are methods for reducing the number of coronavirus copies per cell, comprising exposing cells to an alternating current electric field for a period of time, the alternating current electric field having a frequency and field strength that reduces the number of coronavirus copies in the cell. Disclosed are methods for treating a subject infected with coronavirus, comprising applying an alternating current electric field to a target site in the subject for a period of time, the alternating current electric field having a frequency and field strength, the target site containing one or more coronavirus-infected cells.

Description

[Technical Field]

[0001] The present invention relates to a method for inhibiting coronavirus infection or replication within cells, comprising the steps of exposing cells to an alternating current electric field for a certain period of time, wherein the alternating current electric field has frequency and electric field strength, and the frequency and electric field strength of the alternating current electric field inhibit coronavirus infection or replication. The present invention also relates to a method for reducing the number of coronavirus copies per cell, comprising the steps of exposing cells to an alternating current electric field for a certain period of time, wherein the alternating current electric field has frequency and electric field strength, and the frequency and electric field strength of the alternating current electric field reduce the number of coronavirus copies in the cell. Furthermore, the present invention relates to a method for treating a coronavirus-infected object, comprising the steps of applying an alternating current electric field to a target site of the object for a certain period of time, wherein the alternating current electric field has frequency and electric field strength, and the target site includes one or more coronavirus-infected cells. [Background technology]

[0002] Viruses are small, obligate intracellular parasites. Viruses contain nucleic acids that contain the genetic information necessary for viral replication and, in the simplest viruses, for synthesizing mechanisms in the host cell for a protective protein coat.

[0003] To infect cells, a virus must attach to the cell surface, penetrate into the cell, and be sufficiently decoated to allow access to the viral or host mechanisms for transcription or translation of its genome. Viral duplication usually causes cell damage or cell death. Proliferative infection results in the formation of progeny viruses.

[0004] Coronaviruses are a group of RNA viruses that cause disease in mammals and birds. In humans and birds, they cause respiratory infections that can range from mild to fatal. Mild illnesses in humans include some cases of the common cold (which can also be caused by other viruses, mainly rhinoviruses), while more deadly variants can cause SARS, MERS, and COVID-19. In cattle and pigs, they cause diarrhea, and in mice, they cause hepatitis and encephalomyelitis.

[0005] Coronaviruses belong to the Riboviria kingdom, Nidovirales order, Coronaviridae family, and Orthocoronavirinae subfamily. They are enveloped viruses with a positive-sense single-stranded RNA genome and a helical nucleocapsid. The genome size of coronaviruses ranges from approximately 26 to 32 kilobases, making them one of the largest RNA viruses. They possess characteristic club-shaped spikes protruding from their surface, which, in electron microscopy, create an image resembling the solar corona from which their name is derived.

[0006] Over the past 20 years, emerging pathogenic coronaviruses capable of causing life-threatening diseases in humans and animals have been identified, namely Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV). In December 2019, the Wuhan Municipal Health Committee (Wuhan, China) identified an outbreak of viral pneumonia cases of unknown origin. Coronavirus RNA was identified in some of these patients. This novel coronavirus was named SARS-CoV-2, and the disease it causes was named COVID-19. Currently, there are approximately 50 million confirmed cases of COVID-19 and more than 1.2 million deaths worldwide.

[0007] Individuals of all ages are at risk of infection and severe illness. However, the probability of a severe COVID-19 illness is higher in people over 60 years of age, those living in nursing homes or long-term care facilities, and those with chronic medical conditions. The disease spectrum can range from asymptomatic infection to acute respiratory distress syndrome (ARDS) and severe pneumonia with death. COVID-19 patients may present with many different symptoms, but the main symptoms are fever, cough, or shortness of breath. Abnormalities seen on chest X-rays vary, but bilateral multi-focal opacities are the most common. Abnormalities seen on chest computed tomography (CT) scans also vary, but the most common are bilateral peripheral ground-glass opacities with compartments of sclerosis that develop later in the clinical course. Both X-ray and CT imaging may be normal in the early and asymptomatic stages of the disease. Virological testing (i.e., using molecular diagnostic tests or antigen tests to detect SARS-CoV-2) is recommended by the NIH for diagnosing SARS-CoV-2 in patients suspected of having COVID-19 symptoms.

[0008] COVID-19 patients can be grouped into the following categories based on the severity of their illness: asymptomatic or prodromal, mild, moderate, severe, and critical illness. Patients with critical illness are those with a respiratory rate greater than 30 breaths per minute, SpO2 < 94% in indoor air at sea level, arterial oxygen partial pressure ratio (PaO2 / FiO2) < 300 mmHg, or pulmonary infiltration > 50%. Management of COVID-19 patients with critical illness includes lung imaging and ECG, where applicable. Laboratory assessment includes complete blood count (CBC) with differential and metabolic profiles, including liver and kidney function tests. Measurement of inflammatory markers, such as C-reactive protein (CRP), D-dimer, and ferritin, is not part of standard care but may have prognostic value.

[0009] Nearly a year has passed since the first cases of COVID-19 pneumonia, but current treatment options are limited, consisting of symptomatic treatment, supportive care, isolation, and experimental measures. Therefore, there is an urgent unmet need to develop new therapies for the treatment of COVID-19. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] U.S. Patent No. 7,016,725 [Patent Document 2] U.S. Patent No. 7,565,205 [Patent Document 3] U.S. Patent No. 9,750,934 [Non-patent literature]

[0011] [Non-Patent Document 1] www.who.int / emergencies / diseases / novel-coronavirus-2019 [Overview of the Initiative] [Means for solving the problem]

[0012] It has been previously shown that if cells are exposed to an alternating electric field (AEF) within a specific frequency range while undergoing mitosis, the AEF can disrupt the mitotic process and induce apoptosis. As described in U.S. Patent No. 7,016,725 and U.S. Patent No. 7,565,205 (each of which is incorporated herein by reference in whole), this phenomenon has been successfully used to treat tumors (e.g., glioblastoma, mesothelioma, etc.). In the context of treating tumors, these alternating electric fields are also referred to as "TT fields" (or "tumor treating fields"). One reason why TT field therapy is well-suited for treating tumors is that TT fields appear to selectively disrupt dividing cells during mitosis and have no effect on non-dividing cells. Also, because tumor cells divide far more frequently than other cells in the human body, the application of TT fields to a target selectively attacks tumor cells while leaving other cells harmless. As described in U.S. Patent No. 9,750,934 (which is incorporated herein by reference in its entirety), the same phenomenon has also been successfully demonstrated to be useful in destroying bacteria. Again, one reason why this approach is well suited to destroying bacteria is that bacterial cells divide much more rapidly than other cells in the human body.

[0013] Disclosed herein is a method for inhibiting coronavirus infection or replication within a cell, comprising the step of exposing the cell to an alternating electric field for a period of time, wherein the alternating electric field has a frequency and an electric field intensity, and the frequency and electric field intensity of the alternating electric field inhibit the infection or replication of coronavirus.

[0014] Disclosed herein is a method for reducing the number of coronavirus copies per cell, comprising the step of exposing cells to an alternating electric field for a certain period of time, wherein the alternating electric field has a frequency and an electric field intensity, and the frequency and electric field intensity of the alternating electric field reduce the number of virus copies in the cells.

[0015] Disclosed herein is a method for treating a subject infected with a coronavirus, the method comprising the step of applying an alternating electric field to a target site of the subject over a period of time, wherein the alternating electric field has a frequency and an electric field strength, and the target site includes one or more coronavirus-infected cells.

[0016] Disclosed is a method for treating a subject at risk of infection with a coronavirus, the method comprising the step of applying an alternating electric field to a target site of the subject over a period of time, wherein the alternating electric field has a frequency and an electric field strength.

[0017] Disclosed is a method for preventing the spread of a coronavirus from an infected subject to an uninfected subject, the method comprising the step of applying an alternating electric field to a target site of the coronavirus-infected subject over a period of time, wherein the alternating electric field has a frequency and an electric field strength, and the target site includes one or more coronavirus-infected cells.

[0018] Disclosed is a method for preventing a viral infection in a subject, the method comprising the step of applying an alternating electric field to a target site of the subject over a period of time, wherein the alternating electric field has a frequency and an electric field strength.

[0019] Disclosed is a method for reducing the replication of a coronavirus in a cell, the method comprising the step of exposing the cell to an alternating electric field over a period of time, wherein the alternating electric field has a frequency and an electric field strength, and the frequency and electric field strength of the alternating electric field reduce the number of viral copies in the cell.

[0020] Additional advantages of the disclosed methods and compositions will be explained in part in the following description, in part will be understood from this specification, the drawings, and / or can be understood by the practice of the disclosed methods and compositions. The advantages of the disclosed methods and compositions are realized and achieved by the elements and combinations particularly pointed out in the appended claims. It should be understood that both the foregoing summary and the following detailed description are exemplary and explanatory only and do not limit the claimed invention.

[0021] The accompanying drawings, which are incorporated herein and constitute a part of this specification, illustrate several embodiments of the disclosed methods and compositions and, together with the specification, serve to explain the principles of the disclosed methods and compositions.

Brief Description of the Drawings

[0022] [Figure 1] Examples showing the effect of TT field application over 24 hours during the infection and replication periods on cell growth and virus replication are shown. [Figure 2] Examples showing the effect of TT field application over 48 hours during the infection and replication periods on cell growth and virus replication are shown. [Figure 3] Examples showing cell growth with and without TT field in the absence of virus are shown. [Figure 4] Examples showing the effect of TT field application on cell number and virus copy during only the infection period (TT field + infection) or during the infection and replication periods (TT field + infection + growth) are shown. [Figure 5] Examples showing 229E copies / cell and PR8 copies / cell are shown. [Figure 6] Examples showing the effect of the TT field on 229E depending on virus concentration and time are shown. In all experiments with virus, control cells are infected cells not given the TT field. [Figure 7] Examples showing the effect of 48hr TT field treatment on PR8 virus in A549 cells are shown. [Figure 8]This study demonstrates the effect of 48-hour TT field application on the concentration of replication-competent lysed virions (PFU) on MRC5 cells infected with 229E virus during viral infection and proliferation. [Figure 9] This is an exemplary experimental design for treating virus-infected cells using a TT field. [Figure 10A] This demonstrates the exemplary effect of the TT field on viral entry. This was performed as a frequency scan to evaluate the optimal frequency. MRC-5 cells were infected with 1% HCoV-229E virus while being exposed to a TT field at 100, 150, or 400 kHz, and the cellular viral load was measured at 2 hpi (hours post-infection) by RT-qPCR. RQ, relative quantification; ER = endoplasmic reticulum; DMS = bilayer vesicles; black asterisk = bilayer vesicles (DMVs). Values ​​are mean ± SD. *p<0.05, ***p<0.001, and ****p<0.0001, compared to control; one-way ANOVA for a. [Figure 10B] This demonstrates the exemplary effect of the TT field on viral entry. This was performed as a frequency scan to evaluate the optimal frequency. MRC5 cells were infected with 1% HCoV-229E virus while exposed to a 150 kHz TT field, and the cellular viral load was subsequently measured to 0.5 hpi by RT-qPCR. RQ, relative quantification; ER = endoplasmic reticulum; DMS = bimembrane vesicles; black asterisk = bimembrane vesicles (DMVs). Values ​​are mean ± SD. *p<0.05, ***p<0.001, and ****p<0.0001, compared to control. [Figure 10C]This demonstrates the exemplary effect of a TT field on viral entry. This was performed as a frequency scan to evaluate the optimal frequency. MRC-5 cells were infected with HCoV-229E virus at a multiple degree of infection (MOI) of 20 while being exposed to a 150 kHz TT field, and the number of viruses attached to the cells (indicated by yellow arrows) was determined by SEM examination at 0.5 hpi. RQ, relative quantification; ER = endoplasmic reticulum; DMS = bimembrane vesicles; black asterisk = bimembrane vesicles (DMVs). Values ​​are mean ± SD. *p<0.05, ***p<0.001, and ****p<0.0001, compared to control. Student's t-test for c. [Figure 10D] This demonstrates the exemplary effect of the TT field on viral entry. This was performed as a frequency scan to evaluate the optimal frequency. MRC-5 cells were infected with HCoV-229E virus at a multiple infection degree (MOI) of 20 while being exposed to a 150 kHz TT field, and the number of viruses attached to the cells (indicated by yellow arrows) was determined by SEM examination at 0.5 hpi. RQ, relative quantification; ER = endoplasmic reticulum; DMS = bimembrane vesicles; black asterisk = bimembrane vesicles (DMVs). Values ​​are mean ± SD. *p<0.05, ***p<0.001, and ****p<0.0001, compared to control. [Figure 10E] This demonstrates the exemplary effect of the TT field on viral entry. This was performed as a frequency scan to evaluate the optimal frequency. MRC-5 cells were infected with 3% HCoV-229E virus for 3 hours, and the TT field was applied only after washing the cells. At 48 hpi, TEM examination was performed to determine the distance of the virus from the cells (indicated by white arrows, N=150 / group). RQ, relative quantification; ER = endoplasmic reticulum; DMS = bimembrane vesicles; black asterisk = bimembrane vesicles (DMVs). Values ​​are mean ± SD. *p<0.05, ***p<0.001, and ****p<0.0001, compared to control. Mann-Whitney test for e. [Figure 10F]This section demonstrates the exemplary effect of the TT field on viral entry. This was performed as a frequency scan to evaluate the optimal frequency. MRC-5 cells were infected with 3% HCoV-229E virus for 3 hours, and the TT field was applied only after washing the cells. At 48 hpi, TEM analysis was performed to determine the distance of the virus from the cells (indicated by white arrows, N=150 / group). RQ = relative quantification; ER = endoplasmic reticulum; DMS = bilayer vesicles; black asterisk = bilayer vesicles (DMVs). Values ​​are mean ± SD. *p<0.05, ***p<0.001, and ****p<0.0001, compared to control. [Figure 11A] This study demonstrates the exemplary effect of the TT field on long-term viral exposure. MRC-5 cells were exposed to the TT field for 24, 48, or 72 hours while being infected with 0.01% HCoV-229E virus for 3 hours. Intracellular (Figure 11A) and extracellular (Figure 11B) viral loads were measured by RT-qPCR. Cell counts were also measured (Figure 11C). RQ = relative quantification, ns = not significant. Values ​​are mean ± SD. *p<0.05, **p<0.01, and ***p<0.001, compared to control; Sidac multiple comparisons. [Figure 11B] This study demonstrates the exemplary effect of the TT field on long-term viral exposure. MRC-5 cells were exposed to the TT field for 24, 48, or 72 hours while being infected with 0.01% HCoV-229E virus for 3 hours. Intracellular (Figure 11A) and extracellular (Figure 11B) viral loads were measured by RT-qPCR. Cell counts were also measured (Figure 11C). RQ = relative quantification, ns = not significant. Values ​​are mean ± SD. *p<0.05, **p<0.01, and ***p<0.001, compared to control; Sidac multiple comparisons. [Figure 11C]This study demonstrates the exemplary effect of the TT field on long-term viral exposure. MRC-5 cells were exposed to the TT field for 24, 48, or 72 hours while being infected with 0.01% HCoV-229E virus for 3 hours. Intracellular (Figure 11A) and extracellular (Figure 11B) viral loads were measured by RT-qPCR. Cell counts were also measured (Figure 11C). RQ = relative quantification, ns = not significant. Values ​​are mean ± SD. *p<0.05, **p<0.01, and ***p<0.001, compared to control; Sidac multiple comparisons. [Figure 12A] This study demonstrates the exemplary effect of the TT field on viral replication. MRC-5 cells were infected with 1% HCoV-229E virus for 3 hours, and the TT field was applied only after washing the cells. At 24 hpi, dsRNA was detected by fluorescence microscopy with green staining, and cell nuclei were imaged with blue DAPI (20x magnification). The number of foci, focus size, and focus area per infected cell were quantified. Values ​​are mean ± SD. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001, compared to control; Student's t-test. [Figure 12B] This study demonstrates the exemplary effect of the TT field on viral replication. MRC-5 cells were infected with 1% HCoV-229E virus for 3 hours, and the TT field was applied only after washing the cells. At 24 hpi, dsRNA was detected by fluorescence microscopy with green staining, and cell nuclei were imaged with blue DAPI (20x magnification). The number of foci, focus size, and focus area per infected cell were quantified. Values ​​are mean ± SD. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001, compared to control; Student's t-test. [Figure 12C]This study demonstrates the exemplary effect of the TT field on viral replication. MRC-5 cells were infected with 1% HCoV-229E virus for 3 hours, and the TT field was applied only after washing the cells. At 24 hpi, dsRNA was detected by fluorescence microscopy with green staining, and cell nuclei were imaged with blue DAPI (20x magnification). The number of foci, focus size, and focus area per infected cell were quantified. Values ​​are mean ± SD. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001, compared to control; Student's t-test. [Figure 12D] This study demonstrates the exemplary effect of the TT field on viral replication. MRC-5 cells were infected with 1% HCoV-229E virus for 3 hours, and the TT field was applied only after washing the cells. At 24 hpi, dsRNA was detected by fluorescence microscopy with green staining, and cell nuclei were imaged with blue DAPI (20x magnification). The number of foci, focus size, and focus area per infected cell were quantified. Values ​​are mean ± SD. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001, compared to control; Student's t-test. [Figure 12E] This study demonstrates the exemplary effect of the TT field on viral replication. MRC-5 cells were infected with 3% HCoV-229E virus for 3 hours, and the TT field was applied only after washing the cells. At 48 hpi, TEM analysis was performed to measure invagination (indicated by black arrows) and fusion (indicated by yellow arrows) of double-membrane vesicles (DMVs), and autophagolisosomes (indicated by white arrows) were quantified. Black asterisk = DMV; White asterisk = Intracellular viral particle in membrane-bound vacuole; AL = autophagolisosome; M = mitochondria; Lys = lysosome. Values ​​are mean ± SD. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001, compared to control; Student's T-test. [Figure 12F]This study demonstrates the exemplary effect of the TT field on viral replication. MRC-5 cells were infected with 3% HCoV-229E virus for 3 hours, and the TT field was applied only after washing the cells. At 48 hpi, TEM analysis was performed to measure invagination (indicated by black arrows) and fusion (indicated by yellow arrows) of double-membrane vesicles (DMVs), and autophagolisosomes (indicated by white arrows) were quantified. Black asterisk = DMV; White asterisk = Intracellular viral particle in membrane-bound vacuole; AL = autophagolisosome; M = mitochondria; Lys = lysosome. Values ​​are mean ± SD. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001, compared to control; Student's T-test. [Figure 12G] This study demonstrates the exemplary effect of the TT field on viral replication. MRC-5 cells were infected with 3% HCoV-229E virus for 3 hours, and the TT field was applied only after washing the cells. At 48 hpi, TEM analysis was performed to measure invagination (indicated by black arrows) and fusion (indicated by yellow arrows) of double-membrane vesicles (DMVs), and autophagolisosomes (indicated by white arrows) were quantified. Black asterisk = DMV; White asterisk = Intracellular viral particle in membrane-bound vacuole; AL = autophagolisosome; M = mitochondria; Lys = lysosome. Values ​​are mean ± SD. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001, compared to control; Student's T-test. [Figure 12H]This study demonstrates the exemplary effect of the TT field on viral replication. MRC-5 cells were infected with 3% HCoV-229E virus for 3 hours, and the TT field was applied only after washing the cells. At 48 hpi, TEM analysis was performed to measure invagination (indicated by black arrows) and fusion (indicated by yellow arrows) of double-membrane vesicles (DMVs), and autophagolisosomes (indicated by white arrows) were quantified. Black asterisk = DMV; White asterisk = Intracellular viral particle in membrane-bound vacuole; AL = autophagolisosome; M = mitochondria; Lys = lysosome. Values ​​are mean ± SD. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001, compared to control; Student's T-test. [Figure 12I] This study demonstrates the exemplary effect of the TT field on viral replication. MRC-5 cells were infected with 3% HCoV-229E virus for 3 hours, and the TT field was applied only after washing the cells. At 48 hpi, TEM analysis was performed to measure invagination (indicated by black arrows) and fusion (indicated by yellow arrows) of double-membrane vesicles (DMVs), and autophagolisosomes (indicated by white arrows) were quantified. Black asterisk = DMV; White asterisk = Intracellular viral particle in membrane-bound vacuole; AL = autophagolisosome; M = mitochondria; Lys = lysosome. Values ​​are mean ± SD. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001, compared to control; Student's T-test. [Figure 12J] This study demonstrates the exemplary effect of the TT field on viral replication. Supernatants from 48-hour long-term exposure experiments were added to MRC-5 cells (not exposed to the TT field at any stage), and plaque formation was determined for equal viral counts or calculated for equal supernatant volumes. PFU = plaque-forming units; SN = supernatant. Values ​​are mean ± SD. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001, compared to control; Student's t-test. [Figure 12K]This study demonstrates the exemplary effect of the TT field on viral replication. Supernatants from 48-hour long-term exposure experiments were added to MRC-5 cells (not exposed to the TT field at any stage), and plaque formation was determined for equal viral counts or calculated for equal supernatant volumes. PFU = plaque-forming units; SN = supernatant. Values ​​are mean ± SD. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001, compared to control; Student's t-test. [Figure 13] An example of the combined effect of remdesivir and the TT field is shown. MRC-5 cells were exposed to the TT field alone or in combination with 0.011 or 0.023 μM remdesivir for 48 hours, and then infected with 1% HCoV-229E virus for 3 hours. Intracellular (Figure 13A) and extracellular (Figure 13B) viral loads were examined by RT-qPCR. dsRNA was detected by fluorescence microscopy with green staining, and cell nuclei were imaged with blue DAPI (20x magnification) (Figure 13C). RQ = relative quantification. Values ​​are mean ± SD. **p<0.01, ***p<0.001, and ****p<0.0001, comparison with control; Sidac multiple comparison. [Figure 14] An example of a clinical research design is shown. [Figure 15] An example of viral shedding levels over time is shown. MRC-5 cells were infected with 0.01% HCoV-229E virus for 3 hours, and extracellular viral load was measured at 24 and 48 hpi by RT-qPCR. Smokeless signal = supernatant. [Figure 16] This study demonstrates the exemplary effect of a TT field on virus-free MRC-5 cells. MRC-5 cells were exposed to a TT field for 24 or 48 hours, and cell counts were measured. Values ​​are mean ± SD. Student's t-test, comparison with control; ns = not significant. Sidac multiple comparisons. [Figure 17A]This study demonstrates the exemplary effect of a TT field on A549 virus infection. A549 cells were infected with 0.01% HCoV-229E virus for 3 hours while being exposed to a TT field for 48 hours. Intracellular (Figure 17A) and extracellular (Figure 17B) viral loads were measured by RT-qPCR. Cell counts were also measured with and without viral infection (Figure 17C). RQ = relative quantification, ns = not significant, w / o = without. Values ​​are mean ± SD. ***p<0.001 and ****p<0.0001, compared to control; Student's t-test for Figure 17A. [Figure 17B] This study demonstrates the exemplary effect of a TT field on A549 virus infection. A549 cells were infected with 0.01% HCoV-229E virus for 3 hours while being exposed to a TT field for 48 hours. Intracellular (Figure 17A) and extracellular (Figure 17B) viral loads were measured by RT-qPCR. Cell counts were also measured with and without viral infection (Figure 17C). RQ = relative quantification, ns = not significant, w / o = without. Values ​​are mean ± SD. ***p<0.001 and ****p<0.0001, compared to control; Student's t-test for Figure 17B. [Figure 17C] This study demonstrates the exemplary effect of a TT field on A549 virus infection. A549 cells were infected with 0.01% HCoV-229E virus for 3 hours while being exposed to a TT field for 48 hours. Intracellular (Figure 17A) and extracellular (Figure 17B) viral loads were measured by RT-qPCR. Cell counts were also measured with and without viral infection (Figure 17C). RQ = relative quantification, ns = not significant, w / o = without. Values ​​are mean ± SD. ***p<0.001 and ****p<0.0001, compared to control; Sidac multiple comparisons for Figure 17C. [Modes for carrying out the invention]

[0023] The methods and compositions disclosed may be more readily understood by referring to the following detailed descriptions of specific embodiments and the examples contained herein, as well as the drawings and their preceding and subsequent descriptions.

[0024] The methods and compositions disclosed are not limited to specific synthesis methods, specific analytical techniques, or specific reagents unless otherwise specified, and should therefore be understood to be subject to modification. It should also be understood that the scientific terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.

[0025] Disclosed are materials, compositions, and components that can be used for, in combination with, or in preparation for the disclosed methods and compositions, or are products thereof. Where these and other materials are disclosed herein, and combinations, subsets, interactions, groups, etc., of these materials are disclosed, it is understood that each is specifically assumed and described herein, even if specific references to various individual and collective combinations and arrangements of each of these compounds are not explicitly disclosed. For example, where, in addition to the disclosure of classes A, B, and C, classes D, E, and F and examples of combined molecules, AD are disclosed, each is assumed individually and collectively, even if each is not described individually. For example, in this example, each of the combinations AE, AF, BD, BE, BF, CD, CE, and CF is specifically assumed and should be considered disclosed from the disclosure of A, B, and C; D, E, and F; and exemplary combination AD. Similarly, any subset or combination of these is also specifically assumed and disclosed. Therefore, for example, the subgroups AE, BF, and CE should be considered as particularly assumed and disclosed from the disclosures of A, B, and C; D, E, and F; and exemplary combination AD. This concept applies to all aspects of this application, encompassing but not limited to the steps in a method of producing and using the disclosed compositions. Therefore, where there are various additional steps that may be performed, each of these additional steps should be considered as being performed in any specific embodiment or combination of embodiments of the disclosed method, and each such combination should be considered as particularly assumed and disclosed.

[0026] A.Definition The methods and compositions disclosed are not limited to the specific methodologies, protocols, and reagents described, and it is understood that these may be modified. The scientific terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the invention, which is also understood to be limited only by the appended claims.

[0027] When used herein and in the appended claims, it should be noted that the singular forms “a,” “an,” and “the” refer to multiple objects unless otherwise explicitly stated in the context. For example, a reference to “a cell” includes multiple such cells, and a reference to “the cell” includes one or more cells and their equivalents known to those skilled in the art, and so on.

[0028] As used herein, “coronavirus” refers to a group of RNA viruses belonging to the kingdom Ribowilliae, order Nidovirales, family Coronaviridae, and subfamily Orthocoronimbusinae. They are enveloped viruses with a positive-sense single-stranded RNA genome and a helical nucleocapsid. Coronavirus genome sizes range from approximately 26 to 32 kilobases, making them one of the largest RNA viruses. They possess characteristic club-shaped spikes protruding from their surface, which, in electron micrographs, create an image resembling the solar corona from which their name is derived. In some embodiments, coronaviruses include Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Human Coronavirus-Erasmus University Medical Center (HCoV-EMC), SARS-CoV, or SARS-CoV-2.

[0029] As used herein, “target site” refers to a specific site or location within or on a subject or patient. For example, “target site” may refer to, but is not limited to, a cell, a group of cells, an organ, or a tissue. In some embodiments, the target site may be a cell infected with or containing the coronavirus. In some embodiments, the organ may be the lung. In some embodiments, the cell or group of cells may be one or more lung cells. In some embodiments, the “target site” may be a lung cell target site. In some embodiments, the target site may be the nasal cavity or nasopharynx. In some embodiments, the “target site” may be a specific site or location where coronavirus receptors are present. In some embodiments, the “target site” may be a specific site or location where SARS-CoV-2 receptors, particularly ACE2 receptors, are present. In some embodiments, the “target site” may be a specific site or location where dipeptidyl peptidase-4 (DPP-4) or aminopeptidase N (APN) is present.

[0030] As used herein, “alternating electric field” or “alternating electric fields” refers to a very low-intensity, directional, intermediate-frequency alternating electric field delivered to a subject, a sample obtained from a subject, or a specific location (e.g., a target site) within a subject or patient. In some embodiments, the alternating electric field may be directional or multidirectional. In some embodiments, the alternating electric field may be delivered through two pairs of transducer arrays that generate an electric field perpendicular to the lung being treated. For example, in the Optune® system (alternating electric field delivery system), one pair of electrodes is located in the left and right (LR) lungs, and the other pair of electrodes is located in the anterior and posterior (AP) lungs. Cycling the electric field between these two directions (i.e., LR and AP) ensures that the maximum range of cell orientation is targeted.

[0031] In vivo and in vitro studies have shown that the effectiveness of alternating current field therapy increases as the intensity of the electric field increases. Therefore, optimizing the array placement on the patient to increase the intensity in the desired area can be done using the Optune system. Array placement optimization may be performed by "rule of thumb" (e.g., placing the array on the chest as close as possible to the desired area of ​​the target site (e.g., lungs)), measurements describing the geometry of the patient's body, and body dimensions. Measurements used as input may be derived from imaging data. The imaging data is intended to include any kind of visual data. In certain embodiments, the image data may include 3D data obtained from or generated by a 3D scanner (e.g., point cloud data). Optimization can rely on an understanding of how the electric field is distributed in the lung as a function of the array's position, and in some embodiments, variations in the distribution of electrical properties within the target site (e.g., infected cells) of different patients can be taken into account.

[0032] The term "subject" refers to the target of administration, for example, an animal. Therefore, the subject of the disclosed method may be a vertebrate, for example, a mammal. For example, the subject may be a human. The term does not imply a specific age or sex. "Subject" may be used interchangeably with "individual" or "patient." For example, the subject of administration may mean the recipient of an alternating electric field.

[0033] "To treat" means administering or applying a treatment, such as an alternating electric field, to a subject, such as a human or other mammal (e.g., an animal model), who has, is at risk of, or has increased susceptibility to developing coronavirus infection, in order to prevent or delay the worsening of the effects of coronavirus infection, or to partially or completely reverse the effects of coronavirus infection. For example, treating a subject infected with coronavirus may include inhibiting the coronavirus from infecting or replicating cells in the subject, or reducing the number of coronavirus copies per cell in the subject.

[0034] "Preventing" means minimizing or reducing the likelihood that a person will develop coronavirus infection.

[0035] As used herein, the terms “administer” and “dosage” refer to any method of providing a treatment, such as an antiviral agent or coronavirus treatment (e.g., remdesivir or plasma therapy), to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, vaginal administration, ocular administration, intramural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injections such as intravenous, intra-arterial, intramuscular, and subcutaneous administration. Dosage may be continuous or intermittent. In various embodiments, preparations may be therapeutically administered; i.e., administered to treat an existing disease or condition. In various further embodiments, preparations may be prophylactically administered; i.e., administered to prevent a disease or condition. In one embodiment, those skilled in the art can determine an effective dose, effective schedule, or effective route of administration to treat a subject. In some embodiments, administration includes exposure. Thus, in some embodiments, exposing a subject to an alternating electric field means administering an alternating electric field to the subject.

[0036] "Optional" or "optional" means that the event, situation, or material described thereafter may or may not occur or exist, and that such description includes both cases where the event, situation, or material occurs or exists and cases where it does not occur or exist.

[0037] A range may be expressed herein as "about" one specific value and / or "about" another specific value. Where such a range is expressed, unless the context specifically indicates otherwise, the range from one specific value and / or another specific value is also considered to be specifically assumed and disclosed. Similarly, where a value is expressed as approximate by the use of the antecedent "about," unless the context specifically indicates otherwise, it will be understood that the specific value forms another, specifically assumed embodiment that should be considered to be disclosed. Unless the context specifically indicates otherwise, it will be further understood that each endpoint of a range has significance both with respect to the other endpoints and independently of the other endpoints. Finally, unless the context specifically indicates otherwise, it should be understood that all individual values ​​and partial ranges of values ​​contained within an explicitly disclosed range should also be considered to be specifically assumed and disclosed. The above applies whether, in any particular case, some or all of these embodiments are explicitly disclosed.

[0038] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as those generally understood by those skilled in the art in the field to which the disclosed methods and compositions belong. Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the methods and compositions of the present invention, but particularly useful methods, devices, and materials are described. Publications referenced herein and materials referenced in those publications are thus incorporated by particular reference. Nothing described herein should be construed as an acknowledgment that the present invention has no prior rights to such disclosures on the grounds of prior art. No acknowledgment is made that any reference constitutes prior art. Discussions of references are statements of the claims of their authors, and the applicant reserves the right to challenge the accuracy and validity of the referenced documents. Although numerous publications are referenced herein, it is clearly understood that such references do not constitute an acknowledgment that any of these documents form part of the general knowledge in the art.

[0039] Throughout the specification and claims of this application, the word “comprise” and its variations, such as “comprising” and “comprises,” mean “comprise but not limited to,” and are not intended to exclude, for example, other additives, components, integers or processes. In particular, in methods described as comprising one or more steps or operations, each step is specifically assumed to include what is listed (except where the step includes a limiting term, such as “consisting of”), i.e., each step is not intended to exclude, for example, other additives, components, integers or processes not listed in the step.

[0040] B. Methods to inhibit the infection or replication of coronavirus In some embodiments, the disclosed methods for inhibiting viral infection or replication in cells may be used for any virus that relies on electrostatic interactions with receptors. For example, the virus may be a coronavirus or a lentivirus. The coronavirus described herein is used as an example of a virus that can be inhibited from infection or replication in cells. In some embodiments, the disclosed methods for inhibiting viral infection or replication in cells may be used for any virus that relies on electrostatic interactions with receptors, and the virus is not influenza.

[0041] Disclosed is a method for inhibiting coronavirus infection or replication within a cell, comprising the step of exposing the cell to an alternating electric field for a period of time, wherein the alternating electric field has a frequency and an electric field intensity, and the frequency and electric field intensity of the alternating electric field inhibit the infection or replication of the coronavirus. In some embodiments, the coronavirus is Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Human Coronavirus-Erasmus University Medical Center (HCoV-EMC), SARS-CoV, or SARS-CoV-2. In some embodiments, the coronavirus may be alpha-coronavirus, beta-coronavirus, gamma-coronavirus, or delta-coronavirus. Examples of alphacoronaviruses may include, but are not limited to, alphacoronavirus 1, human coronavirus 229E, human coronavirus NL63, long-fingered bat (Miniopterus bat) coronavirus 1, long-fingered bat coronavirus HKU8, porcine diarrhea virus, horseshoe bat (Rhinolophus bat) coronavirus HKU2, and yellow house bat (Scotophilus bat) coronavirus 512. Examples of betacoronaviruses may include, but are not limited to, betacoronavirus 1 (bovine coronavirus, human coronavirus OC43), hedgehog coronavirus 1, human coronavirus HKU1, Middle East respiratory syndrome-related coronavirus, mouse coronavirus, Japanese house bat (Pipistrellus bat) coronavirus HKU5, Rousettus bat (Rousettus bat) coronavirus HKU9, severe acute respiratory syndrome-related coronaviruses (SARS-CoV, SARS-CoV-2), and bamboo bat (Tylonycteris bat) coronavirus HKU4. Examples of gamma coronaviruses may include, but are not limited to, avian coronaviruses and beluga whale coronavirus SW1. Examples of delta coronaviruses may include, but are not limited to, bulbul coronavirus HKU11 and swine coronavirus HKU15. In some embodiments, a coronavirus can be a variant of one or more coronaviruses.For example, a variant could be a variant of SARS-CoV-2, such as the alpha (B.1.1.7), beta (B.1.351, B.1.351.2, B.1.351.3), delta (B.1.617.2, AY.1, AY.2, AY.3), and gamma (P.1, P.1.1, P.1.2) variants.

[0042] In some embodiments, the frequency of the alternating electric field is 150 kHz.

[0043] In some embodiments, the parameters of the AC electric field are 150 kHz and 1.7 V / cm.

[0044] In some embodiments, the AC electric field is applied at a frequency of 250 kHz to 350 kHz. In some embodiments, the AC electric field is applied at a frequency of 50 to 190 kHz. In some embodiments, the AC electric field is applied at a frequency of 210 to 400 kHz. In some embodiments, the AC electric field is applied at a frequency of 50 kHz to 1 MHz. In some embodiments, the AC electric field has an electric field strength of at least 1 V / cm RMS. In some embodiments, the AC electric field has a frequency of 50 to 190 kHz. In some embodiments, the AC electric field has a frequency of 210 to 400 kHz. In some embodiments, the AC electric field has an electric field strength of at least 1 V / cm RMS. In some embodiments, the AC electric field has an electric field strength of 1 to 4 V / cm RMS.

[0045] In some embodiments, the period can be hours (hr), days, or weeks. In some embodiments, cells may be exposed to an alternating electric field for 24 or 48 hours.

[0046] In some embodiments, an alternating electric field inhibits both the transmission and replication of the coronavirus. In some embodiments, an alternating electric field inhibits the transmission of the coronavirus. In some embodiments, an alternating electric field inhibits the replication of the coronavirus.

[0047] In some embodiments, an alternating electric field promotes the fusion of autophagosomes with lysosomes, resulting in viral lysis.

[0048] In some embodiments, an alternating electric field can prevent or reduce the amount of virus shedding by cells. Thus, in some embodiments, an alternating electric field can result in infected cells producing less virus, which in turn can result in less reinfection.

[0049] In some embodiments, cells that are not infected with the coronavirus are not damaged.

[0050] In some embodiments, cell survival is maintained.

[0051] In some embodiments, the viral load in the subject is reduced, and cell proliferation is unaffected.

[0052] In some embodiments, cell survival at the target site is maintained, and viral replication or infection is reduced.

[0053] It is known that alternating electric fields induce an anti-mitotic effect by exerting bidirectional forces on highly polar intracellular elements, such as tubulin. Therefore, in some embodiments, alternating electric fields may have an effect on other highly polar elements, such as viral proteins. For example, the spike protein of coronaviruses is highly polar and electrostatic, enabling it to bind to its receptor (e.g., ACE2). In some embodiments, alternating electric fields can interfere with the ability of coronaviruses to interact with their receptors. Interference with the ability of coronaviruses to interact with their receptors can interfere with or inhibit their ability to infect cells.

[0054] In some embodiments, an alternating electric field can prevent a virus from infecting a cell. For example, in some embodiments, an alternating electric field can prevent or reduce the likelihood of a virus getting close enough to the cell membrane to infect a cell.

[0055] In some embodiments, an alternating electric field can increase the fusion of a virus with lysosomes. This fusion of a virus with lysosomes can result in the killing of the virus. In some embodiments, the virus is a coronavirus, and an alternating electric field can increase the fusion of the coronavirus with lysosomes.

[0056] C. Methods to reduce the number of virus copies In some embodiments, the disclosed method for reducing the copy number of a virus in a cell may be used for any virus that relies on electrostatic interaction with a receptor. For example, the virus may be a coronavirus or a lentivirus. As described herein, coronavirus is used as an example of a virus whose copy number can be reduced by the application of an alternating electric field.

[0057] Disclosed is a method for reducing the number of coronavirus copies per cell, comprising the step of exposing cells to an alternating electric field for a certain period of time, wherein the alternating electric field has a frequency and an electric field intensity, and the frequency and electric field intensity of the alternating electric field reduce the number of virus copies in the cells.

[0058] Also disclosed is a method for reducing the replication of coronavirus in cells, comprising the step of exposing the cells to an alternating electric field for a period of time, wherein the alternating electric field has a frequency and an electric field intensity, and the frequency and electric field intensity of the alternating electric field reduce the number of viral copies in the cells. In some embodiments, the coronavirus is Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Human Coronavirus-Erasmus University Medical Center (HCoV-EMC), SARS-CoV, or SARS-CoV-2. In some embodiments, the coronavirus may be an alpha-coronavirus, beta-coronavirus, gamma-coronavirus, or delta-coronavirus. Examples of alpha-coronaviruses may include, but are not limited to, alpha-coronavirus 1, human coronavirus 229E, human coronavirus NL63, long-fingered bat coronavirus 1, long-fingered bat coronavirus HKU8, porcine diarrhea virus, horseshoe bat coronavirus HKU2, and yellow house bat coronavirus 512. Examples of beta-coronaviruses may include, but are not limited to, beta-coronavirus 1 (bovine coronavirus, human coronavirus OC43), hedgehog coronavirus 1, human coronavirus HKU1, Middle East respiratory syndrome-associated coronavirus, mouse coronavirus, pipistrelle bat coronavirus HKU5, rosette bat coronavirus HKU9, severe acute respiratory syndrome-associated coronaviruses (SARS-CoV, SARS-CoV-2), and bamboo bat coronavirus HKU4. Examples of gamma-coronaviruses may include, but are not limited to, avian coronavirus and beluga whale coronavirus SW1. Examples of delta-coronaviruses may include, but are not limited to, bulbul coronavirus HKU11 and swine coronavirus HKU15. In some embodiments, a coronavirus can be a variant of one or more coronaviruses.For example, a variant could be a variant of SARS-CoV-2, such as the alpha (B.1.1.7), beta (B.1.351, B.1.351.2, B.1.351.3), delta (B.1.617.2, AY.1, AY.2, AY.3), and gamma (P.1, P.1.1, P.1.2) variants.

[0059] In some embodiments, the frequency of the alternating electric field is 150 kHz.

[0060] In some embodiments, the parameters of the AC electric field are 150 kHz and 1.7 V / cm.

[0061] In some embodiments, the AC electric field is applied at a frequency of 250 kHz to 350 kHz. In some embodiments, the AC electric field is applied at a frequency of 50 to 190 kHz. In some embodiments, the AC electric field is applied at a frequency of 210 to 400 kHz. In some embodiments, the AC electric field is applied at a frequency of 50 kHz to 1 MHz. In some embodiments, the AC electric field has an electric field strength of at least 1 V / cm RMS. In some embodiments, the AC electric field has a frequency of 50 to 190 kHz. In some embodiments, the AC electric field has a frequency of 210 to 400 kHz. In some embodiments, the AC electric field has an electric field strength of at least 1 V / cm RMS. In some embodiments, the AC electric field has an electric field strength of 1 to 4 V / cm RMS.

[0062] In some embodiments, the period can be hours (hr), days, or weeks. In some embodiments, cells may be exposed to an alternating electric field for 24 or 48 hours.

[0063] In some embodiments, the reduction in coronavirus copy number per cell is determined based on a comparison with the coronavirus copy number per cell in cells not treated with an alternating electric field.

[0064] In some embodiments, a reduction in the number of coronavirus copies per cell is achieved while simultaneously maintaining cell survival.

[0065] In some embodiments, cells that are not infected with the coronavirus are not damaged.

[0066] In some embodiments, cell survival is maintained.

[0067] In some embodiments, the viral load in the subject is reduced, and cell proliferation is unaffected.

[0068] In some embodiments, cell survival at the target site is maintained, and viral replication or infection is reduced.

[0069] D. Treatment Method In some embodiments, the disclosed method for treating a virus-infected object may be used for any virus that relies on electrostatic interaction with a receptor. For example, the virus may be a coronavirus or a lentivirus. As described herein, coronavirus is used as an example of a virus for which an alternating electric field may be used to treat a coronavirus-infected object.

[0070] Disclosed is a method for treating a subject infected with coronavirus, comprising the step of applying an alternating electric field to a target site of the subject for a certain period of time, wherein the alternating electric field has a frequency and an electric field intensity, and the target site comprises one or more coronavirus-infected cells.

[0071] Disclosed is a method for treating an object at risk of infection with coronavirus, comprising the step of applying an alternating current electric field to a target site of the object for a period of time, wherein the alternating current electric field has frequency and electric field intensity. In some embodiments, the object at risk of infection with coronavirus may be a first responder (e.g., a healthcare worker) or an object that has had close contact with an object known to have or be exposed to coronavirus.

[0072] Disclosed is a method for preventing a viral infection in a subject, comprising the step of applying an alternating electric field to a target site of the subject for a certain period of time, wherein the alternating electric field has frequency and electric field intensity. In some embodiments, the subject has not been previously infected with a coronavirus. In some embodiments, the subject is at risk of being infected with a coronavirus. In some embodiments, the coronavirus is Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Human Coronavirus-Erasmus University Medical Center (HCoV-EMC), SARS-CoV, or SARS-CoV-2. In some embodiments, the coronavirus may be an alpha-coronavirus, beta-coronavirus, gamma-coronavirus, or delta-coronavirus. Examples of alpha-coronaviruses may include, but are not limited to, alpha-coronavirus 1, human coronavirus 229E, human coronavirus NL63, long-fingered bat coronavirus 1, long-fingered bat coronavirus HKU8, porcine diarrhea virus, horseshoe bat coronavirus HKU2, and yellow house bat coronavirus 512. Examples of beta-coronaviruses may include, but are not limited to, beta-coronavirus 1 (bovine coronavirus, human coronavirus OC43), hedgehog coronavirus 1, human coronavirus HKU1, Middle East respiratory syndrome-associated coronavirus, mouse coronavirus, pipistrelle bat coronavirus HKU5, rosette bat coronavirus HKU9, severe acute respiratory syndrome-associated coronaviruses (SARS-CoV, SARS-CoV-2), and bamboo bat coronavirus HKU4. Examples of gamma-coronaviruses may include, but are not limited to, avian coronavirus and beluga whale coronavirus SW1. Examples of delta-coronaviruses may include, but are not limited to, bulbul coronavirus HKU11 and swine coronavirus HKU15. In some embodiments, a coronavirus can be a variant of one or more coronaviruses.For example, a variant could be a variant of SARS-CoV-2, such as the alpha (B.1.1.7), beta (B.1.351, B.1.351.2, B.1.351.3), delta (B.1.617.2, AY.1, AY.2, AY.3), and gamma (P.1, P.1.1, P.1.2) variants.

[0073] In some embodiments, the frequency of the alternating electric field is 150 kHz. In some embodiments, the alternating electric field is approximately 150 kHz to 300 kHz.

[0074] In some embodiments, the parameters of the AC electric field are 150 kHz and 1.7 V / cm.

[0075] In some embodiments, the AC electric field is applied at a frequency of 250 kHz to 350 kHz. In some embodiments, the AC electric field is applied at a frequency of 250 kHz to 350 kHz. In some embodiments, the AC electric field is applied at a frequency of 50 to 190 kHz. In some embodiments, the AC electric field is applied at a frequency of 210 to 400 kHz. In some embodiments, the AC electric field is applied at a frequency of 50 kHz to 1 MHz. In some embodiments, the AC electric field has an electric field strength of at least 1 V / cm RMS. In some embodiments, the AC electric field has a frequency of 50 to 190 kHz. In some embodiments, the AC electric field has a frequency of 210 to 400 kHz. In some embodiments, the AC electric field has an electric field strength of at least 1 V / cm RMS. In some embodiments, the AC electric field has an electric field strength of 1 to 4 V / cm RMS.

[0076] Disclosed is a method for treating COVID-19 in a subject, comprising the step of applying an alternating electric field to a target site of the subject for a period of time, wherein the alternating electric field has a frequency and an electric field intensity, and the target site comprises one or more SARS-CoV-2 infected cells.

[0077] In some embodiments, an alternating current (AC) electric field reduces the number of viral copies in one or more coronavirus-infected cells. In some embodiments, the frequency of the AC electric field is 150 kHz. In some embodiments, the parameters of the AC electric field are 150 kHz and 1.7 V / cm. In some embodiments, the AC electric field is applied at a frequency of 250 kHz to 350 kHz. In some embodiments, the AC electric field is applied at a frequency of 50 to 190 kHz. In some embodiments, the AC electric field is applied at a frequency of 210 to 400 kHz. In some embodiments, the AC electric field has an electric field strength of at least 1 V / cm RMS. In some embodiments, the AC electric field has a frequency of 50 to 190 kHz. In some embodiments, the AC electric field has a frequency of 210 to 400 kHz. In some embodiments, the AC electric field has an electric field strength of at least 1 V / cm RMS. In some embodiments, the AC electric field has an electric field strength of 1 to 4 V / cm RMS.

[0078] In some embodiments, the disclosed method further includes administering a second treatment to a target. In some embodiments, the second treatment may, but is not limited to, an antiviral treatment. In some embodiments, the second treatment is administered before the alternating electric field. In some embodiments, the second treatment is administered simultaneously with the alternating electric field. In some embodiments, the second treatment is administered after the alternating electric field. In some embodiments, an antiviral agent is delivered to a target or target region, and as a result, the antiviral agent is present in the target region while the alternating electric field is administered. In some embodiments, the antiviral treatment is a cell or gene therapy, an immunomodulator, an antibody, or a mixture of antibodies or antiviral agents.In some embodiments, antiviral therapies include remdesivir (veklury), avigan (faviravir), bamranivimab, olumiant and baricinix (baricitinib), hydroxychloroquine / chloroquine, cacilibimab and imdevimab (formerly known as REGN-COV2), PTC299, and leronlimab (PRO 140), bamranibimab (LY-CoV555), redinilumab, ivermectin, RLF-100 (aviptadil), metformin (glucophage, Glumetza, Riomet), AT-527, Actemra (tocilizumab), nicloside (niclosamide), convalescent plasma, Pepcid (famotidine), Kaletra (lopinavir-ritonavir), Remicade (infliximab), AZD7442, CT-P59, heparin (UF and LMW), VIR-7831 (GSK4182136), JS016, Kevzara (sarilumab), SACCOVID (CD24Fc), Humira (adalimumab), CO VI-GUARD (STI-1499), Dexamethasone (Dextenza, Ozurdex, etc.), PB1046, Galidesivir, Bucillamine, PF-00835321 (PF-07304814), Eliquis (Apixaban), Takhzyro (Lanadermab), Hydrocortisone, Ilaris (Canakinumab), Colchicine (Mitigare, Colcrys), BLD-2660, Avigan (Faviravir / Avifavir), Rhu-pGSN (Gelzolin), MK-4482, TXA127, LAM-002A (Apilimod dimesylate) The coronavirus is dimesylate), DNL758 (SAR443122), INOpulse, ABX464, AdMSCs, rosmapimod, maprilimumab, or calquens (acalabrutinib). In some embodiments, the coronavirus is Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Human Coronavirus-Erasmus University Medical Center (HCoV-EMC), SARS-CoV, or SARS-CoV-2.In some embodiments, coronaviruses can be alpha-coronaviruses, beta-coronaviruses, gamma-coronaviruses, or delta-coronaviruses. Examples of alpha-coronaviruses may include, but are not limited to, alpha-coronavirus 1, human coronavirus 229E, human coronavirus NL63, long-fingered bat coronavirus 1, long-fingered bat coronavirus HKU8, porcine diarrhea virus, horseshoe bat coronavirus HKU2, and yellow house bat coronavirus 512. Examples of beta-coronaviruses may include, but are not limited to, beta-coronavirus 1 (bovine coronavirus, human coronavirus OC43), hedgehog coronavirus 1, human coronavirus HKU1, Middle East respiratory syndrome-associated coronavirus, mouse coronavirus, pipistrelle bat coronavirus HKU5, rosette bat coronavirus HKU9, severe acute respiratory syndrome-associated coronaviruses (SARS-CoV, SARS-CoV-2), and bamboo bat coronavirus HKU4. Examples of gamma-coronaviruses may include, but are not limited to, avian coronaviruses and beluga whale coronavirus SW1. Examples of delta coronaviruses may include, but are not limited to, bulbul coronavirus HKU11 and swine coronavirus HKU15. In some embodiments, a coronavirus can be a variant of one or more coronaviruses. For example, a variant can be a variant of SARS-CoV-2, such as alpha (B.1.1.7), beta (B.1.351, B.1.351.2, B.1.351.3), delta (B.1.617.2, AY.1, AY.2, AY.3), and gamma (P.1, P.1.1, P.1.2) variants.

[0079] In some embodiments, the target site may be the lung. In some embodiments, the target site does not include a tumor.

[0080] Also disclosed are methods for preventing the spread of coronavirus. In some embodiments, a coronavirus-infected object may be treated by a step of applying an alternating current field to a target site of the object for a certain period of time, wherein the alternating current field has frequency and field intensity, and the target site contains one or more coronavirus-infected cells. The alternating current field may result in the production of a defective virus, which is less infectious than wild-type coronavirus, and as a result the object is less infectious than other objects. In some embodiments, the alternating current field may modify the virus produced by infected cells in the object, thereby resulting in a defective virus, or the alternating current field may modify the virus-producing cells, thereby resulting in the production of a defective virus. In some embodiments, the alternating current field may provide the replication mechanism required for coronavirus replication.

[0081] In some embodiments, cells within a subject that are not infected with the coronavirus are not damaged.

[0082] In some embodiments, cell survival at the target site is maintained.

[0083] In some embodiments, the viral load in the subject is reduced, and cell proliferation is unaffected.

[0084] In some embodiments, cell survival at the target site is maintained, and viral replication or infection is reduced.

[0085] E. Combination therapy Any disclosed method using TT fields may be performed in combination with one or more known standard treatments for coronavirus infection. Therefore, in some embodiments, TT fields may be combined with antibodies, or antibody cocktails, nanobodies, antiviral small molecules, sulfated polysaccharide polymers, and polypeptides. Frequent targets include viral spike proteins, host angiotensin-converting enzyme 2, host transmembrane protease serine 2, and clathrin-mediated endocytosis. For example, disclosed methods using TT fields include remdesivir (Veklury), nafamostat, avigan (faviravir), bamranivimab, olumiant and baricinix (baricitinib), hydroxychloroquine / chloroquine, cacilibimab and imdevimab (formerly REGN-COV2), PTC299, and leronlimab (PRO 140), bamranibimab (LY-CoV555), redinilumab, ivermectin, RLF-100 (aviptadil), metformin (glucophage, gourmetza, riomet), AT-527, Actemra (tocilizumab), nicloside (niclosamide), convalescent plasma, pepsid (famotidine), kaletra (lopinavir-ritonavir), Remicade (infliximab), AZD7442, CT-P59, heparin (UF and LMW), VIR-7831 (GSK4182136), JS016, Kevzara (sarilumab), SACCOVID (CD24Fc), Humira (adalimumab), COVI-GUARD (STI-1499), dexamethasone (dextenza, ozuldec) (S, others), PB1046, galidesivir, bucillamine, PF-00835321 (PF-07304814), Eliquis (apixaban), Taxylo (lanadermab), hydrocortisone, Ilaris (canakinumab), colchicine (mitigare, colcris), BLD-2660, Avigan (faviravir / avifavir), Rhu-pGSN (gelzolin), MK-4482, TXA127, LAM-002A (apirimod dimesylate), DNL758 (SAR443122), INOpulse, ABX464, AdMSCs, rosmapimod, maprilimumab, or Calquence (acalabrutinib), quinoline antimalarial agents ((hydroxy)-chloroquine and others),This can be done in combination with RAAS modifiers (captopril, losartan, and others), statins (atorvastatin and simvastatin), guanidino serine protease inhibitors (camostat and nafamostat), antibacterial agents (macrolides, clindamycin, and doxycycline), antiparasitic agents (ivermectin and niclosamide), cardiovascular drugs (amiodarone, verapamil, and tranexamic acid), antipsychotics (chlorpromazine), antiviral agents (umifenovir and oseltamivir), DPP-4 inhibitors (linagliptin), JAK inhibitors (baricitinib and others), sulfated glycosaminoglycans (UFH and LMWHs), and polypeptides, such as one or more of the enzymes DAS181 and rhACE2. These also include viral spike protein-targeted monoclonal antibodies REGN10933 and REGN10987. ,

[0086] F. AC electric field The methods disclosed herein involve an alternating current field. In some embodiments, the alternating current field used in the methods disclosed herein is a tumor treatment field. In some embodiments, the alternating current field may be modified depending on the type of cell or the conditions under which the alternating current field is applied. In some embodiments, the alternating current field may be applied through one or more electrodes placed on the body of the subject. In some embodiments, two or more pairs of electrodes may be present. For example, an array may be placed on the anterior / posterior and lateral sides of the patient and may be used in conjunction with the systems and methods disclosed herein. In some embodiments, when two pairs of electrodes are used, the alternating current field may alternate between the pairs of electrodes. For example, a first pair of electrodes may be placed on the anterior and posterior sides of the subject, and a second pair of electrodes may be placed on either side of the subject, and the alternating current field may then be applied, alternating between the anterior and posterior electrodes, and then between the lateral and lateral electrodes.

[0087] In some embodiments, the frequency of the alternating electric field can be 150 kHz. The frequency of the alternating electric field can also be, but is not limited to, about 150 kHz, about 200 kHz, 50-500 kHz, 100-500 kHz, 25 kHz-1 MHz, 50 kHz-1 MHz, 50-190 kHz, 25-190 kHz, or 210-400 kHz. In some embodiments, the frequency of the alternating electric field can be an electric field of 50 kHz, 100 kHz, 150 kHz, 200 kHz, 300 kHz, 400 kHz, 500 kHz, 1 MHz, or any frequency in between.

[0088] In some embodiments, the frequency of the alternating electric field is approximately 150 kHz to approximately 300 kHz, approximately 100 kHz to approximately 300 kHz, approximately 200 kHz to approximately 400 kHz, approximately 250 kHz to approximately 350 kHz, and may also be approximately 300 kHz.

[0089] In some embodiments, the electric field strength of the AC electric field can be 1 to 5 V / cm RMS. In some embodiments, different electric field strengths may be used (e.g., 0.1 to 10 V / cm). In some embodiments, the electric field strength can be 1.75 V / cm RMS. In some embodiments, the electric field strength is at least 1 V / cm. In some embodiments, combinations of electric field strengths are applied, for example, two or more frequencies are combined simultaneously, and / or two or more frequencies are applied at different time points.

[0090] In some embodiments, the AC electric field is applied at a frequency of 250 kHz to 350 kHz. In some embodiments, the AC electric field is applied at a frequency of 50 to 190 kHz. In some embodiments, the AC electric field is applied at a frequency of 210 to 400 kHz. In some embodiments, the AC electric field has an electric field strength of at least 1 V / cm RMS. In some embodiments, the AC electric field has a frequency of 50 to 190 kHz. In some embodiments, the AC electric field has a frequency of 210 to 400 kHz. In some embodiments, the AC electric field has an electric field strength of at least 1 V / cm RMS. In some embodiments, the AC electric field has an electric field strength of 1 to 4 V / cm RMS.

[0091] In some embodiments, the alternating current field may be applied over various different intervals ranging from 0.5 hours to 72 hours. In some embodiments, different durations may be used (e.g., 0.5 hours to 14 days). In some embodiments, the application of the alternating current field may be repeated periodically. For example, the alternating current field may be applied daily for a duration of 2 hours.

[0092] In some embodiments, the exposure may last for at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours or longer.

[0093] G. Device Disclosed is a method for preventing or treating a viral infection in an object, comprising the step of applying an alternating current electric field to a target site of the object for a period of time, wherein the alternating current electric field has frequency and electric field strength. In some embodiments, the disclosed method may be used for any virus that relies on electrostatic interaction with a receptor. For example, the virus may be a coronavirus or a lentivirus. Also described herein are devices that may be used to apply an alternating current electric field to a target site of an object.

[0094] Disclosed is a method for preventing or treating coronavirus infection in a subject, comprising the step of applying an alternating current electric field to a target site of the subject for a period of time, wherein the alternating current electric field has frequency and electric field intensity. Also described herein are devices that may be used to apply an alternating current electric field to a target site of a subject.

[0095] Disclosed herein are devices for use in the disclosed methods. For example, disclosed are devices for use in the disclosed methods that inhibit coronavirus from infecting or replicating within cells. Disclosed are devices for use in the disclosed methods that reduce the number of coronavirus copies per cell. Disclosed are devices for use in the disclosed methods that treat subjects infected with coronavirus. Disclosed are devices for use in the disclosed methods that prevent the spread of coronavirus from subjects infected with coronavirus to uninfected subjects.

[0096] In some embodiments, the disclosed device may be an apparatus for electrotherapy procedures. Generally, the apparatus may be a portable, battery- or power-supplied device that generates an alternating electric field in the body by means of transducer arrays or other electrodes. The apparatus may include an electric field generator and one or more electrode (e.g., transducer) arrays, each containing multiple electrodes. The apparatus may be configured to generate a tumor-treating electric field (TT field) (e.g., 150 kHz) via the electric field generator and to deliver the TT field to compartments of the body through one or more electrode arrays. The electric field generator may be a battery and / or power-supplied device.

[0097] An electric field generator may include a processor in communication with a signal generator. The electric field generator may also include control software configured to control the operation of the processor and the signal generator. While the processor and / or control software may reside within the electric field generator, they can be provided separately from the electric field generator, provided the processor is configured to be in communication with the signal generator and to run the control software.

[0098] A signal generator can produce one or more electrical signals in the shape of a waveform or a series of pulses. A signal generator may be configured to produce an AC voltage waveform (e.g., a TT field) with a frequency in the range of about 50 kHz to about 1 MHz (preferably about 100 kHz to about 300 kHz). The voltage is such that the electric field strength in the tissue being treated is typically in the range of about 0.1 V / cm to about 10 V / cm.

[0099] One or more outputs of the electric field generator may be connected at one end to one or more conductive leads attached to a signal generator. The opposite end of the conductive lead is connected to one or more electrode arrays driven by an electrical signal (e.g., a waveform). The conductive leads may include standard insulated conductors with flexible metal shielding and may be grounded to prevent diffusion of the electric field generated by the conductive leads. One or more outputs may be operated sequentially. Output parameters of the signal generator may include, for example, the electric field strength, the wave frequency (e.g., processing frequency), the maximum allowable temperature of one or more electrode arrays, and / or a combination thereof. In some embodiments, a temperature sensor may be associated with each electrode array. If the temperature sensor measures a temperature above a threshold, the current to the electrode array associated with the temperature sensor may be stopped until a second, lower threshold temperature is detected. Output parameters may be set and / or determined by control software in combination with a processor. After determining the desired (e.g., optimal) processing frequency, the control software can cause the processor to send a control signal to a signal generator, which in turn outputs the desired processing frequency to one or more electrode arrays.

[0100] One or more electrode arrays may be configured in various shapes and positions to generate an electric field of a desired configuration, direction and intensity at a target site (also referred to herein as the “target volume” or “target region”) to concentrate processing. Optionally, one or more electrode arrays may be configured to deliver two perpendicular electric field directions through the volume of interest.

[0101] In some embodiments, a first responder / healthcare professional may wear the disclosed device for use in one or more of the disclosed methods. For example, a first responder exposed to the coronavirus may be treated with an electric field within the device, thereby preventing any further possible spread of the virus.

[0102] H. Kit In addition to the materials described above, other materials may be packaged together in any suitable combination as a kit useful for performing or assisting the disclosed method. This is useful when the kit components in a given kit are designed and adapted for use together in the disclosed method. For example, disclosed are kits for imaging and / or treatment. In some embodiments, the kit may include devices and other equipment for applying alternating electric fields to a target.

[0103] Also disclosed are a system or device for administering an alternating electric field and a kit comprising, but not limited to, one or more of the second therapies, such as antiviral therapies.

[0104] I. Implementation Embodiment 1: A method for inhibiting coronavirus infection or replication within a cell, comprising the step of exposing the cell to an alternating electric field for a certain period of time, wherein the alternating electric field has a frequency and an electric field intensity, and the frequency and electric field intensity of the alternating electric field inhibit the infection or replication of coronavirus.

[0105] Embodiment 2: A method for reducing the number of coronavirus copies per cell, comprising the step of exposing cells to an alternating electric field for a certain period of time, wherein the alternating electric field has a frequency and an electric field intensity, and the frequency and electric field intensity of the alternating electric field reduce the number of virus copies in the cells.

[0106] Embodiment 3: A method for treating a subject infected with coronavirus, comprising the step of applying an alternating current electric field to a target site of the subject for a certain period of time, wherein the alternating current electric field has frequency and electric field intensity, and the target site comprises one or more coronavirus-infected cells. In some embodiments, Embodiment 3 may be the medical applications described herein. An alternating current electric field for use in the treatment of coronavirus infection, comprising the step of applying an alternating current electric field to a target site of a subject having coronavirus infection for a certain period of time, wherein the alternating current electric field has frequency and electric field intensity, and the target site comprises one or more coronavirus-infected cells.

[0107] Embodiment 4; The method according to any prior embodiment, wherein the coronavirus is SARS-CoV-2.

[0108] Embodiment 5; The method according to any prior embodiment, wherein an AC electric field is applied at a frequency of 250 kHz to 350 kHz.

[0109] Embodiment 6; The method according to any prior embodiment, wherein an alternating electric field is applied at a frequency of 50 to 190 kHz.

[0110] Embodiment 7; The method according to any prior embodiment, wherein an alternating electric field is applied at a frequency of 210 to 400 kHz.

[0111] Embodiment 8; The method according to any prior embodiment, wherein the alternating electric field has an electric field strength of at least 1 V / cm RMS.

[0112] Embodiment 9; The method according to any prior embodiment, wherein the alternating electric field has a frequency of 50 to 190 kHz.

[0113] Embodiment 10; The method according to any prior embodiment, wherein the alternating electric field has a frequency of 210 to 400 kHz.

[0114] Embodiment 11; The method according to any prior embodiment, wherein the alternating electric field has an electric field strength of at least 1 V / cm RMS.

[0115] Embodiment 12; The method according to any prior embodiment, wherein the alternating electric field has an electric field strength of 1 to 4 V / cm RMS.

[0116] Embodiment 13; The method according to any prior embodiment, wherein the frequency of the AC electric field is 150 kHz.

[0117] Embodiment 14; The method according to any prior embodiment, wherein the parameters of the AC electric field are 150 kHz and 1.7 V / cm.

[0118] Embodiment 15; The method according to any prior embodiment, wherein the parameters of the AC electric field are approximately 150 kHz and approximately 1.5 V / cm.

[0119] Embodiment 16; The method according to any prior embodiment, wherein the target site is the lung.

[0120] Embodiment 17; The method according to any prior embodiment, wherein an alternating electric field is applied in multiple directions.

[0121] Embodiment 18; the method according to Embodiment 17, wherein the multidirectionality is in at least two directions.

[0122] Embodiment 19; The method according to any prior embodiment, wherein cells not infected with coronavirus are not damaged.

[0123] Embodiment 20; The method according to any prior embodiment, wherein cell survival is maintained.

[0124] Embodiment 21; The method according to any prior embodiment, wherein the viral load in the subject is reduced and cell proliferation is unaffected.

[0125] Embodiment 22; The method according to any prior embodiment, wherein cell survival at the target site is maintained and viral replication or viral infection is reduced.

[0126] Embodiment 23; A method for preventing the spread of coronavirus from an infected subject to an uninfected subject, comprising the step of applying an alternating current electric field to a target site of a coronavirus-infected subject for a certain period of time, wherein the alternating current electric field has frequency and electric field intensity, and the target site comprises one or more coronavirus-infected cells.

[0127] Embodiment 24; The method according to Embodiment 23, wherein an alternating electric field results in a subject infected with the coronavirus producing a defective virus, the defective virus being less infectious than the wild coronavirus.

[0128] Embodiment 25; A method for reducing coronavirus replication in cells, comprising the step of exposing cells to an alternating electric field for a certain period of time, wherein the alternating electric field has a frequency and an electric field intensity, and the frequency and electric field intensity of the alternating electric field reduce the number of virus copies in the cells.

[0129] Embodiment 26; A method for preventing a viral infection in an object, comprising the step of applying an alternating current electric field to a target area of ​​the object for a certain period of time, wherein the alternating current electric field has frequency and electric field strength.

[0130] Embodiment 27; The method according to Embodiment 26, wherein the subject is not infected with the coronavirus.

[0131] Embodiment 28; the method according to Embodiment 26 or 27, wherein the subject is at risk of being infected with the coronavirus.

[0132] Embodiment 29; A method for treating an object at risk of infection with coronavirus, comprising the step of applying an alternating current electric field to a target site of the object for a certain period of time, wherein the alternating current electric field has frequency and electric field strength.

[0133] Embodiment 30; A method for preventing a virus from infecting a cell by getting close enough to it, comprising the step of applying an alternating electric field to a target site for a certain period of time, wherein the alternating electric field has frequency and electric field strength.

[0134] Embodiment 31; A method for increasing viral fusion with lysosomes, comprising the step of applying an alternating electric field to a target site for a certain period of time, wherein the alternating electric field has a frequency and an electric field intensity, and the alternating electric field results in viral fusion with lysosomes.

[0135] Embodiment 32: The method according to Embodiment 1 or 2, wherein cells are present in the target.

[0136] Embodiment 33: The method according to any prior embodiment, wherein an AC electric field is applied at a frequency of 50 kHz to 1 MHz. [Examples]

[0137] A. (Example 1) Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It was first identified in Wuhan, Hubei, China in December 2019 and has resulted in an ongoing pandemic. As of August 19, 2020, nearly 22 million cases have been reported across 188 countries and territories, resulting in more than 775,000 deaths (www.who.int / emergencies / diseases / novel-coronavirus-2019). Management involves symptomatic treatment, supportive care, isolation, and experimental measures. Supportive care may include fluid therapy, oxygen support, and support for other affected vital organs. There is an urgent unmet need to develop new therapies for the treatment of COVID-19.

[0138] The tumor therapeutic electric field (TT field) is a low-intensity (1–5 V / cm), intermediate-frequency (100–500 kHz) alternating electric field that has been shown to interfere with cancer cell division. TT field application generates an electric field inside and around cells, thus inducing forces on dipoles and polarizable objects. TT field application has been shown to interfere with the organization of cellular structural elements, whose assembly relies on electrostatic interactions between subunits with high electrical dipoles. Additionally, TT field application has been demonstrated to induce replication stress and the collapse of replication forks. The cell death induced by the TT field primed the immune system, possessing characteristics of immunogenic cell death. Clinical application of the TT field has demonstrated increased overall survival in some high-grade cancers, along with minimal adverse events, leading to FDA approval of the modality for the treatment of newly diagnosed glioblastoma, recurrent glioblastoma, and malignant pleural mesothelioma. Recent studies (unpublished) have demonstrated that TT field application can inhibit lentiviral replication and reduce the percentile of infected cells, but there is no data on the potential effects of TT fields against COVID-19.

[0139] The purpose of this study was to investigate the effect of TT fields on coronaviruses in an in vitro setting.

[0140] 1. Summary The tumor therapeutic electric field (TT field) is a low-intensity (1–5 V / cm), intermediate-frequency (100–500 kHz) alternating electric field that has been shown to interfere with cancer cell division. TT field application generates an electric field inside and around cells, thus inducing dipoles and forces on polarizable objects. Clinical TT field application has demonstrated increased overall survival in some high-grade cancers, along with minimal adverse events, leading to FDA approval of the modality for the treatment of newly diagnosed glioblastoma, recurrent glioblastoma, and malignant pleural mesothelioma (MPM). Recent studies have demonstrated that TT field application can inhibit lentiviral replication and reduce the percentile of infected cells.

[0141] The effect of a bidirectional TT field (150 kHz, 1.7 V / cm) on the number of viruses produced by previously infected MRC5 cells during or after treatment was studied using human coronavirus HCoV-229E. The number of viable cells, viruses per ml, viruses per cell, and RQ (virus particles normalized to host housekeeping genes) were determined at the end of treatment.

[0142] The results demonstrate a significant reduction in viral copy number per cell (32.4% of viral copies per cell compared to control, p<0.0001) and viral copies per housekeeping gene (RQ=0.284 in the TT field arm and 1.561 in the control arm, p<0.0001) in cells treated with the TT field for 48 hours during the 6-hour infection phase and 42-hour replication phase. A similar trend was observed when the TT field was applied for 24 hours during the 6-hour infection phase and 18-hour replication phase, but the results were not statistically significant. In cultures treated for 48 hours, the viral copy number in the supernatant (297,345 copies) was also reduced compared to the control (1,411,880 copies) (p<0.0001). Significantly more cells survived HCoV-229E infection damage in infected cultures treated with the TT field for 24 or 48 hours compared to untreated cultures.

[0143] In summary, the results of this study provide evidence that the TT field can inhibit coronavirus infection and replication. It should be noted that the TT field parameters used in this study (150 kHz, 1.7 V / cm) are similar in both frequency and intensity to the treatment delivered by the FDA-approved Optune Lua system (Novocure Ltd.) for the treatment of malignant pleural mesothelioma (MPM).

[0144] 2. Materials and Methods i. Cell lines and viruses MRC5 cells (ATCC, CCL-171®) and HCoV-229E (ATCC, VR740®) were provided by Dr. Michal Mendelbaum of Sheba Medical Center. The cells were grown in Eagle's Minimal Essential Medium (EMEM) (ATCC, 30-2003®) supplemented with 10% fetal bovine serum (FBS) (Biological Industries, 04-007-1A).

[0145] ii. Virus infection and replication MRC5 cells were placed on a glass coverslip (22 mm in diameter) in a 1.5 × 10⁶ 5 Cells were seeded at a cell / coverslip density. After 24 hours of incubation, the incubator temperature was changed from 37°C to 35°C, and the cells were transferred to inovtro dishes containing 2 ml of EMEM supplemented with 2% FBS. The cells were infected with HCoV-229E virus at a concentration of 0.01% over 6 hours. After infection, the cells were washed with PBS and maintained in EMEM supplemented with 2% FBS for an additional 18 or 42 hours. A TT field was applied during either the infection or proliferation phase. 24 or 48 hours after infection, the proliferation medium was collected, the cells were trypsinized, resuspended, and counted using a Scepter® 2.0 Cell Counter (Merck, Millipore). After resuspending, the cells were washed with PBS. The supernatant and pellet were frozen at -80°C and transferred to Sheba for viral genome extraction and qRT-PCR.

[0146] iii. Applying TT Field The TT field (150 kHz, 1.7 V / cm RMS) was applied using the Inovitro® system (Novocure Ltd.) as described, along with the following correction: "The Inovitro plate was covered with Parafilm and the culture medium was not replaced during cell culture."

[0147] iv.Statistical analysis Each experiment was repeated three times. Data from all replicates were pooled for analysis. Data are presented as mean ± SD. Statistical significance was analyzed using the Kruskal-Wallis test.

[0148] 3.Results Following preliminary testing, a virus / cell infection ratio of 0.01% was used in all experiments. A TT field (150 kHz, 1.7 V / cm) was applied for a total duration of 24 or 48 hours, during the 6 hours of infection and the 18 or 42 hours of viral replication. Three independent replicates were performed for each group. Due to variability between replicates, the following parameters were normalized to the mean control of each replicate: cell number, viral copies per cell, and viral copies in the supernatant (SN).

[0149] Infected cultures treated with a TT field for 24 hours had significantly more cells at the end of treatment compared to untreated cultures (p<0.0001). There were no significant differences in viral copies in the supernatant (SN) or in the number of viral copies (RQ) compared to the number of housekeeping gene copies, compared to the control. 24-hour application of the TT field led to some reduction in viral copy number per cell, but the results were not statistically significant (see Figure 1).

[0150] Since it is known that the effectiveness of the TT field depends on the duration of treatment, the duration of TT field application was extended to 48 hours in the following set of experiments.

[0151] Infected cultures treated with a TT field for 24 hours had significantly more cells at the end of treatment compared to untreated cultures (p<0.01). 48 hours of TT field application resulted in a significant reduction in viral copies in the supernatant (SN) compared to the control (p<0.01), as well as significant reductions in viral copy number per cell (p<0.0001) and viral copy number relative to housekeeping gene copies (RQ) (p<0.0001) (see Figure 2).

[0152] 4. Conclusion As disclosed herein, the TT field was applied to MRC5 cell cultures infected with coronavirus 229E. The frequency (150 kHz) and intensity (1.7 V / cm) used were equivalent to those applied by the Optune Lua device, which received FDA approval for the treatment of malignant pleural mesothelioma after a successful STELLAR clinical study that demonstrated the efficacy and safety of the treatment.

[0153] The viral copy number per cell was reduced after 24 hours of treatment with the TT field. Since the effectiveness of the TT field is known to depend on the duration of treatment, the TT field was applied for 48 hours in the second set of experiments. Indeed, the extended treatment duration resulted in significant reductions in viral copy numbers per cell, per housekeeping gene, and in the supernatant.

[0154] The application of a TT field at 150 kHz and 1.7 V / cm for 48 hours demonstrated effective killing of dividing cancer cells. However, in this study, the number of infected cells treated with the TT field was higher compared to infected cultures not treated with the TT field.

[0155] In summary, the results of this study provide evidence for the use of TT fields as a treatment for COVID-19 using the currently available Optune Lua system.

[0156] B. (Example 2) The objective of this study was to test the feasibility of using TT fields to treat coronaviruses. Additional experiments were conducted in cells to determine the effect of TT fields on cells that were not infected with the virus, in addition to the virus copy number per cell in infected cells.

[0157] Cells were infected with human coronavirus 229E and then exposed to a TT field, while cultures were not exposed to a TT field. Cells that were not infected with human coronavirus 229E were also exposed to a TT field.

[0158] The number of cells did not differ significantly between cultures treated with the TT field and cultures not treated with the TT field (Figure 3). Only when comparing cultures infected with 229E did MRC5 cells show a survival benefit after TT field treatment compared to infected cells not treated with the TT field.

[0159] Since uninfected cells proliferate faster with a TT field than without, it may be worthwhile to show the same results after normalizing for this effect. The results were normalized to the number of cells in the control culture.

[0160] The number of viral detector copies from the cell pellet was normalized per housekeeping gene (RNAseP). RQ - Relative Quantification Strategy for Quantitative RT-PCR Data Analysis. This method was used to calculate the relative 229E viral gene expression levels between samples from cell pellets with and without TT field treatment. The RQ method directly uses threshold cycles (CTs) generated by the qPCR system for calculation.

[0161] The report presents accumulated / normalized data from all three experiments. The results demonstrate a significant reduction in the number of viral copies per cell in cells treated with the TT field (Figure 2). Similar trends were observed in all three experiments conducted.

[0162] Cells are processed in two TT field directions. Besides human devices, all standard in vitro and in vivo studies consistently utilize two-directional TT fields.

[0163] The trend remained consistent throughout the 24-hour application period. Since TT fields are known to affect processes related to DNA replication and integration, the effects observed throughout the reported experiments are thought to relate to the intracellular activity of the virus, and the cumulative effect is expected to become statistically significant after prolonged application of the electric field, as is also seen in cancer models.

[0164] A reduction in viral load was observed after 24 hours in both the supernatant (SN) and cells, and this did not significantly differ whether the TT field was applied only during infection (6 hours) or over the entire 24 hours (Figure 4).

[0165] 48-hour application of a TT field at 150 kHz and approximately 1.5 V / cm resulted in significant reductions in viral copy number per cell, per housekeeping gene, and in the supernatant. The results of this study provide evidence for the use of TT fields as a treatment for COVID-19 using the currently available Optune Luna system.

[0166] Figure 5 shows human coronavirus 229E in MRC5 cells (human fibroblasts from the lung) and mouse-compatible influenza virus PR8 in A549 cells (human lung cancer). After 24 hours, the 229E virus showed an increase in virus copies / ml in the cell lysate, and after 48 hours, an increase in the cell culture medium. For PR8, virus copies were already detectable after 6 hours.

[0167] Figure 3 shows the effect of the TT field on MRC5 cells. Cell numbers did not change significantly between control and TT field-treated MRC5 cells.

[0168] Figure 6 shows an example of the effect of TT field on 229E in a virus concentration-dependent manner. Virus copy number / cell was measured at 24 and 48 hours after TT field treatment. Cells were infected with either 0.01% or 1% virus. After 48 hours, virus copy number / cell was significantly reduced with TT field treatment, regardless of virus concentration. It should be noted that TT field has a greater effect at lower virus-to-cell ratios, and since chronic infections can occur at lower viral loads, TT field may be used for chronic infections.

[0169] Figure 4 shows the TT field during infection or infection and replication. Cells were infected with 0.01% virus. TT field infection was performed for 6 hours. TT field infection + replication was performed for 18 hours. The amount of cells increased in TT field-treated plates was greater than in controls, but the viral copy number / cell decreased in TT field-treated cells. Healthy (uninfected) cells were not affected by the TT field. Many treatments can damage healthy cells along with "bad" cells or non-healthy / infected cells. Because the TT field shows minimal damage to healthy cells, it has the added benefit of this safety factor when used as a treatment for coronavirus.

[0170] Following preliminary testing, a virus / cell infection ratio of 0.01% was used in both experiments (Figures 1 and 2). A TT field (150 kHz, 1.5 V / cm) was applied for a total duration of 24 (Figure 1) or 48 (Figure 2) hours, during the 6 hours of infection and 18 or 42 hours of viral replication. Four independent replicates were performed for each group. Due to variability between replicates, the following parameters were normalized to the mean control for each replicate: cell number, virus copies / cell, and virus copies in supernatant (SN).

[0171] Infected cultures treated with a TT field for 24 hours had significantly more cells at the end of treatment compared to untreated cultures (p<0.0001). There were no significant differences in viral copies in the supernatant (SN) or in the number of viral copies (RQ) compared to the number of housekeeping gene copies, compared to the control. 24-hour application of the TT field led to some reduction in viral copy number per cell, but the results were not statistically significant (see Figure 12).

[0172] In the following experimental set, the duration of TT field application was extended to 48 hours. Compared to untreated cultures, infected cultures treated with TT field for 48 hours had significantly more cells at the end of treatment (p=0.0862). 48 hours of TT field application led to a significant reduction in viral copies in the supernatant (SN) compared to the control (p<0.0001), as well as significant reductions in viral copy number per cell (p<0.0001) and viral copy number relative to housekeeping gene copies (RQ) (p<0.0001) (see Figure 2).

[0173] The viral copy number per cell was slightly reduced after 24 hours of treatment with the TT field, but the result was not statistically significant. Since the effectiveness of the TT field is known to depend on the treatment duration, the TT field was applied for 48 hours in a second set of experiments. Indeed, the extended treatment duration resulted in significant reductions in viral copy numbers per cell, per housekeeping gene, and in SNs. Furthermore, TT field treatment also led to a significant reduction in the concentration of replication-competent lysed virions (PFU) (see Figure 8).

[0174] 1.5 × 10 5Nine MRC5 cells were infected with 229E virus and treated with a TT field during either the infection phase or the infection and replication phase. After 24 hours, there was approximately a 40% reduction in cell count for cells treated during both phases, approximately a 55% reduction for cells treated only during infection, and approximately a 75% reduction for control cells (Figure 4 left). The viral copy number per cell was slightly reduced after 24 hours of treatment with a TT field in both treatments (infection only or infection + replication).

[0175] Figure 7 shows an example of the effect of 48-hour TT field treatment on PR8 virus in A549 cells. In contrast to 229e virus, the PR8 virus experiment showed a decrease in cell number when TT field-treated cells were compared to controls. There was also an increase in viral copy number / cell in TT field-treated cells compared to controls. Therefore, either the PR8 virus did not behave in the same way as the 229e virus in response to the TT field, or the TT field has a different effect on the interaction between the virus and cancer or healthy cells.

[0176] C. (Example 3) 1. Plaque assay Plaque assays can be a useful quantitative method for measuring infectious coronaviruses by quantifying plaques formed in cell cultures upon infection with serially diluted viral samples. Therefore, plaque assays remain the gold standard for quantifying the concentration of replication-competent lysed virions. This example describes a plaque assay performed to quantify 229E in a supernatant portion collected after 48 hours of treatment from coronavirus-infected MRC5 cells.

[0177] i. Reagents and Solutions a. Infection medium: Eagle's Minimal Essential Medium (EMEM) (ATCC, 30-2003(trademark)) supplemented with 2% fetal bovine serum (FBS) (Biological Industries, 04-007-1A). b. Overlay diluent:

[0178] DMEM x2 - 500ml

[0179] Pen-Strep - 10ml

[0180] Glutamine - 10ml

[0181] Pyruvate - 10ml

[0182] FBS - 20ml

[0183] c. Carboxymethylcellulose (CMC), 3% (w / v) 6g carboxymethylcellulose, culture medium viscosity (solid; Sigma-Aldrich #C4888-500G),

[0184] 200 ml distilled water

[0185] Sterilization by autoclaving at 121°C for 15 minutes.

[0186] Store at room temperature for up to 2 months.

[0187] d. Crystal violet, 1% (w / v) 1g of crystal violet

[0188] 20 ml anhydrous ethanol

[0189] 80 ml distilled water

[0190] filter sterilization

[0191] Store at room temperature

[0192] 2. Method i.Cell culture (day 0) Seed plate: Cells were trypsin-treated. 1 ml of cell suspension was added to each well to create 2 x 10⁶ cells in a 12-well plate. 5Cells were seeded in wells. They were incubated overnight in a 5% CO2 incubator at 37°C until 100% confluence was achieved the following day. Cells were visualized the following day using a light microscope. Cells that had reached 95%–100% confluence were used for infection.

[0193] ii. Sample dilution, cell infection, and primary overlay (Day 1) Sample dilution: For each sample to be titrated, a 2 ml microcentrifuge tube was labeled as follows: (1.) Negative control (infection medium only); (2.) 190571 virus copies in 1 ml of infection medium; and (3.) 10 No. 2 -1 (Add 100 μl of No. 2 to 900 μl of culture medium.)

[0194] Infection: Culture medium was aspirated from the cell monolayer. Cells were washed with 1 ml of PBS. 900 μl of infection medium was added to each well. The viral sample was diluted 10-fold by transferring 100 μl of each sample to the appropriate well. Cells were incubated for 2 hours at 35°C in a 5% CO2 incubator.

[0195] Preparation of liquid overlay medium (LOM): Approximately 10 minutes before the end of incubation, the overlay diluent was incubated in a water bath at 44°C. The LOM was prepared by adding equal (1:1) volumes of overlay diluent and 3% CMC (liquid matrix).

[0196] The cells were washed twice with 1 ml of PBS. 1 ml of LOM was added to each well of the plate. The cells were incubated at 35°C in a 5% CO2 incubator for 4 days. Plate movement was minimized during this incubation.

[0197] iii. Fixation, staining, plaque counting, and titer calculation (Day 5) a. Fixed: LOM was aspirated from each well and disposed of in a waste bottle. Cells were gently washed twice with PBSx1, the wells were filled with ice-cold anhydrous ethanol and incubated at -20°C for 15 minutes.

[0198] b.Staining: The fixative was aspirated from the wells and the waste was disposed of. 300 μl of 1% CV was added to each well and incubated at room temperature for 5 minutes.

[0199] c. Cleaning: The cells were washed 3-4 times with distilled water and then blot-dried. At this point, the plates could be surface-decontaminated with an appropriate disinfectant and stored at 4°C for up to 1 month.

[0200] d. Plaque counting: Plaques appear as distinct circles on a purple monolayer of cells. Negative controls should have a homogeneous monolayer and can be used as a reference. The number of plaques observed per well in each viral dilution was recorded.

[0201] e. Potency calculation: PFU / [ml of infection] = PFU / well × dilution factor

[0202] PFU / [ml of collected SN] = PFU × 1000 / infectious volume of SN in 1 ml of infection medium.

[0203] iv. Plaque assay MRC5 cells were placed in a 12-well plate in a 2 × 10⁶ container. 5 Cells were seeded at a density of cells / well. After 24 hours of incubation, the incubator temperature was changed from 37°C to 35°C, and the medium was replaced with 1 ml of fresh EMEM supplemented with 2% FBS. 5The cells were infected with a viral copy over a period of 2 hours. After infection, the cells were washed with PBS and covered with medium supplemented with 2% FBS and 1.5% CMC. Four days after infection, the cells were fixed with ice-cold anhydrous ethanol for 15 minutes and stained with 1% Cristal Violet at room temperature for 5 minutes. Plaque-forming units (PFUs) were counted, and the PFU / ml SN was calculated (infectious volume of SN in 1ml of infection medium, PFU × 1000).

[0204] 3.Results This assay was used to quantify 229E in a portion of the supernatant collected after 48 hours of treatment from coronavirus-infected MRC5 cells. 48 hours of TT field application led to a significant reduction in PFU per equal volume of viral copies (P=0.0143) and an additional reduction in PFU per milliliter of supernatant (P=0.0002) in each treatment (see Figure 8). The stronger reduction in PFU per ml of supernatant may be explained by the reciprocal effect of reductions in the number of competent viruses and reductions in total viral copies.

[0205] D. (Example 4) Clinical trial research Disclosed is a pilot, randomized, open-label study comparing Optune-Lua® (TT field, 150 Hz) with best-in-class care in hospitalized patients with COVID-19. The objective of this study is to evaluate the efficacy and safety of best-in-class care (SOC) and the accompanying TT field compared to best-in-class care (SOC) for treating patients with COVID-19.

[0206] The data provided throughout this application demonstrate that the TT field can significantly reduce coronavirus infection and replication in vitro. The frequency (150 kHz) and intensity (1.7 V / cm) used in this model are equivalent to those applied by the Optune Lua (NovoTTF-100L) device, which received FDA approval for MPM treatment after a successful STELLAR clinical study that demonstrated the efficacy and safety of the treatment.

[0207] In summary, as a novel, safe, and topical therapy with demonstrated preclinical efficacy, TT Field (150 kHz), deliverable using the NovoTTF-100L system, has the potential to be an effective treatment for COVID-19.

[0208] This embodiment demonstrates that the addition of TT fields delivered to the thoracic cavity using the NovoTTF-100L system to SOC as a treatment for COVID-19 disease significantly improves patient clinical outcomes compared to SOC treatment alone.

[0209] In some embodiments, this study could evaluate whether a TT field to the thoracic cavity at 150 kHz with SOC in the treatment of hospitalized patients with COVID-19 shortens the time to recovery compared to SOC treatment alone.

[0210] In some embodiments, this study can evaluate whether a TT field to the thoracic cavity at 150 kHz with SOC in the treatment of hospitalized patients with COVID-19 improves the clinical status of patients on days 8, 15, 22, and 29 compared to SOC treatment alone.

[0211] In some aspects, this study could evaluate whether a TT field to the thoracic cavity at 150 kHz with SOC in the treatment of hospitalized patients with COVID-19 shortens the duration of hospitalization compared to SOC treatment alone.

[0212] In some aspects, this study could evaluate whether a TT field to the thoracic cavity at 150 kHz with SOC in the treatment of hospitalized patients with COVID-19 reduces all-cause mortality compared to SOC treatment alone.

[0213] In some embodiments, this study could evaluate whether a TT field to the thoracic cavity at 150 kHz with SOC in the treatment of hospitalized patients with COVID-19 reduces the incidence of ICU transfer compared to SOC treatment alone.

[0214] In some aspects, this study could evaluate whether a TT field to the thoracic cavity at 150 kHz with SOC in the treatment of hospitalized patients with COVID-19 reduces the duration of ICU stay compared to SOC treatment alone.

[0215] In some aspects, this study could evaluate whether a TT field to the thoracic cavity at 150 kHz with SOC in the treatment of hospitalized patients with COVID-19 reduces the occurrence of non-invasive ventilation or high-flow oxygen use compared to SOC treatment alone.

[0216] In some aspects, this study could evaluate whether a TT field to the thoracic cavity at 150 kHz with SOC in the treatment of hospitalized patients with COVID-19 shortens the duration of non-invasive ventilation or high-flow oxygen use compared to SOC treatment alone.

[0217] In some aspects, this study could evaluate whether a TT field to the thoracic cavity at 150 kHz with SOC in the treatment of hospitalized patients with COVID-19 reduces the incidence of invasive ventilation compared to SOC treatment alone.

[0218] In some aspects, this study could evaluate whether a TT field to the thoracic cavity at 150 kHz with SOC in the treatment of hospitalized patients with COVID-19 shortens the duration of invasive ventilation compared to SOC treatment alone.

[0219] In some aspects, this study could evaluate whether a TT field to the thoracic cavity at 150 kHz with SOC in the treatment of hospitalized patients with COVID-19 reduces the incidence of ECMO compared to SOC treatment alone.

[0220] In some aspects, this study could evaluate whether a TT field to the thoracic cavity at 150 kHz with SOC in the treatment of hospitalized patients with COVID-19 shortens the duration of ECMO compared to SOC treatment alone.

[0221] In some aspects, this study could evaluate whether a TT field to the thoracic cavity at 150 kHz with SOC in the treatment of hospitalized patients with COVID-19 reduces the incidence of return to hospital compared to SOC treatment alone.

[0222] In some embodiments, this study can evaluate whether a TT field to the thoracic cavity at 150 kHz with SOC in the treatment of hospitalized patients with COVID-19 increases saturation levels and stability compared to SOC treatment alone.

[0223] In some embodiments, this study could evaluate whether a TT field to the thoracic cavity at 150 kHz with SOC in the treatment of hospitalized patients with COVID-19 shortens the duration and level of body temperature compared to SOC treatment alone.

[0224] In some aspects, this study could evaluate whether a TT field to the thoracic cavity at 150 kHz with SOC in the treatment of hospitalized patients with COVID-19 shortens the duration of oxygen supplementation compared to SOC treatment alone.

[0225] In some aspects, this study could evaluate whether a TT field to the thoracic cavity at 150 kHz with SOC in the treatment of hospitalized patients with COVID-19 reduces the inflammatory state of patients compared to SOC treatment alone.

[0226] In some aspects, this study could evaluate whether a TT field to the thoracic cavity at 150 kHz with SOC in the treatment of hospitalized patients with COVID-19 improves the radiographic assessment of the patient's lungs compared to SOC treatment alone.

[0227] In some aspects, this study could evaluate whether a TT field to the thoracic cavity at 150 kHz with SOC is safer than SOC treatment alone in the treatment of hospitalized patients with COVID-19.

[0228] 1. Research Procedures All subjects in the study, regardless of their research arm, will be given the best SOC treatment for COVID-19.

[0229] i. Device This study may utilize the Optune Lua® System. The NovoTTF-100L System is an investigational medical device that delivers a 150 kHz TT field to the thoracic cavity for the treatment of patients aged 18 years or older with COVID-19. It may be used exclusively by patients in clinical studies for at least 18 hours per day on average per month.

[0230] The device is a portable, battery-powered system that delivers a 150 kHz TT field to the rib cage by means of an isolated transducer array. The NovoTTF-100L generates an electrical force intended to attenuate SARS-CoV-2 infection and replication.

[0231] ii. Application of TT fields using devices Treatment plan: The transducer array layout may be determined based on the clinical practice guidelines: Optimization of transducer arrays in TT field-treated patients (thoracic infections).

[0232] Initiation of treatment: The NovoTTF-100L treatment may be initiated by the researcher within 24 hours of admission. Subjects with hair to which the transducer array is planned to be applied may be required to shave it before transducer array placement. Transducer array placement may be performed based on a transducer array layout map selected before initiation of treatment, avoiding areas of skin damage, such as wounds.

[0233] The NovoTTF-100L System can be programmed to deliver a 150 kHz TT field to the rib cage in two sequential, vertical electric field directions at a maximum intensity of 1414 mA (RMS).

[0234] Transducer array replacement: With the assistance of a caregiver, the patient can have their transducer array replaced 2-3 times per week. During each transducer array replacement, the patient's skin may be re-shaved if necessary and treated according to the guidelines below.

[0235] iii. Duration The TT field to the thoracic cavity at 150 kHz can be continuous for at least 18 hours per day on average. Subjects may take breaks for personal needs (e.g., shower, transducer array change) as long as the average treatment remains at 18 hours per day (monthly average). The TT field may continue for 29 days, or until the subject completes hospitalization and is free from activity restrictions, or until death, an unacceptable adverse event occurs, an interventional illness preventing further administration of the treatment, the researcher decides to withdraw the subject, the subject withdraws their consent, the subject becomes pregnant, fails to comply with the research treatment or procedure requirements, or for administrative reasons.

[0236] 2. Research Procedures and Schedule i. Study-specific procedures The following data may be collected in the study.

[0237] Clinical status score: For each study day, the clinical status may be recorded on an 8-point ordinal scale as follows: Day 1 - Clinical assessment at the time of randomization; Day 2 - Assessment of the most severe condition at any point between randomization and midnight on the day of randomization; Day 3 and thereafter - Assessment of the most severe condition from midnight to midnight (00:00~23:59) on the previous day (for example, the value recorded on Day 3 may be the most severe outcome that occurred on Day 2).

[0238] The clinical status scale can be defined as follows: Not hospitalized and no activity restrictions; Not hospitalized and activity restrictions; Hospitalized and not requiring oxygen support - no longer requiring ongoing medical care; Hospitalized and not requiring oxygen support - requiring ongoing medical care (COVID-19 related or otherwise); Hospitalized and requiring oxygen support; Hospitalized and receiving non-invasive ventilation or high-flow oxygen device; Hospitalized and receiving invasive mechanical ventilation or ECMO; or dead.

[0239] The medical history may include any clinically significant history of the patient, focusing on the past five years, and any other significant history beyond five years. The history can be obtained from medical records supplemented by interviews with the patient. If medical records are unavailable, the history can be obtained by interviewing the patient. The medical history should include smoking and significant comorbidities.

[0240] Concomitant medications include both prescription and over-the-counter medications taken by patients throughout the study period, including dosage, frequency, indications, and start and stop dates.

[0241] The COVID-19 medical history includes the date of diagnosis, the date of onset of COVID-19 symptoms and signs, the dates and results of previous SARS-CoV-2 RT-PCR tests, and all past treatments.

[0242] Patient demographics may also be included.

[0243] A physical examination may also be performed. The physical examination can include heart rate, blood pressure, respiratory rate, body temperature, weight, height, blood saturation level (SpO2). The following systems may be examined and reviewed: head and neck, heart, lungs, abdomen, extremities, skin, nerves.

[0244] CT / X-ray scans can also be performed, including but not limited to chest scans, which include the collection of complete data required for the assessment of COVID-19 lung radiological status.

[0245] ii. Clinical Laboratory Evaluation Blood tests can also be performed. A complete blood count and differential can include hemoglobin, hematocrit, MCV, RBC, WBC, neutrophils, eosinophils, basophils, lymphocytes, monocytes, platelets. Chemistry can include sodium, potassium, urea / BUN, creatinine, glucose, LDH, AST, ALT, albumin, bilirubin. Coagulation can include PTT / aPTT, PT / INR. Inflammatory blood markers can include CRP, D-dimer, and ferritin. A pregnancy test can be performed using a serum beta-hCG test.

[0246] RT-PCR SARS-CoV-2 OP swab tests and RT-PCR SARS-CoV-2 blood tests can also be performed.

[0247] iii. Screening The following can be performed within 24 hours after admission: Clinical status based on an 8-point ordinal scale; COVID-19 disease history (including review of previous SARS-CoV-2 test results); RT-PCR SARS-CoV-2 oropharyngeal swab test; RT-PCR SARS-CoV-2 blood test; Chest CT / X-ray scan; Demographics and medical history; Record of concomitant medications; Physical examination (including vital signs and SpO2 level); Serum pregnancy test (if applicable); Complete blood count including differential count; Serum chemistry panel; Coagulation tests; Inflammatory blood marker tests.

[0248] iv. Randomization Patients can be centrally randomized in a 1:1 ratio to two treatment arms using the IxRS system before treatment initiation: Treatment Arm I: Patients are given a 150 kHz TT field to the chest wall using the NovoTTF-100L System together with SOC; and Treatment Arm II: Patients are given SOC alone.

[0249] 3. Schedule of Events An example of the schedule of events is shown in Table 1 below.

[0250]

Table 1A

[0251]

Table 1B

[0252]

Table 1C

[0253] 4. Analysis of Endpoints The primary outcome can use an ordinal severity scale with 8 categories (defined below). Time to recovery, defined as recovery in clinical status in state 1, 2, or 3 of the 8-point ordinal scale, truncated at day 29.

[0254] 8-point ordinal scale: Not hospitalized, no activity restrictions; Not hospitalized, activity restrictions and / or requiring home oxygen; Hospitalized, not requiring oxygen supplementation - no longer requiring ongoing medical care; Hospitalized, not requiring oxygen supplementation - requiring ongoing medical care (COVID-19 related or otherwise); Hospitalized, requiring oxygen supplementation; Hospitalized, receiving non-invasive ventilation or high-flow oxygen device; Hospitalized, receiving invasive mechanical ventilation or ECMO; Dead.

[0255] The primary endpoint may be achieved if the time to recovery is significantly shorter in the TT field + best SOC arm than in the best SOC arm alone. The statistical hypothesis can be tested by comparing the Kaplan-Meier time to recovery curves of the two groups using a one-sided stratified log-rank test.

[0256] The day of recovery may be measured from the day of randomization until the subject meets one of the following three categories on an ordinal scale: 1) not hospitalized and no activity restrictions; 2) not hospitalized, activity restrictions and / or requiring home oxygen; or 3) hospitalized, not requiring oxygen supplementation and no longer requiring ongoing medical care.

[0257] Any subject who disappears from follow-up or terminates prematurely before observation for recovery may be terminated on the date of their last observed assessment. Subjects who complete follow-up but do not experience recovery may be terminated on their 29th day visit. All deaths within 29 days (and before recovery) may be considered as having been terminated on day 28.

[0258] The median and confidence intervals from the Kaplan-Meier curve for each treatment arm can be shown. The table can show the median time to the event for each treatment arm, along with the overall 95% confidence interval, the estimated hazard ratio, and the log-rank p-value.

[0259] The clinical status at specific time points can include the evaluation of clinical status scores on days 8, 15, 22, and 29.

[0260] The number and proportion of subjects, along with the 95% confidence intervals by category of clinical status, can be shown by treatment arm on days 8, 15, 22, and 29.

[0261] The duration of hospitalization can be defined as the first day of hospitalization. The duration of hospitalization can be measured from the first day of hospitalization to the day of discharge.

[0262] The duration can be summarized in the table using the median and quartiles by treatment arm.

[0263] The occurrence of all-cause death can include the number and percentage of subjects who died by day 15 and 29 as shown by treatment arm (the denominator for the percentage can be the number of subjects in the safety population in each treatment arm). 14- and 28-day mortality rates can be calculated and shown, taking into account the amount of follow-up time for each subject. Deaths through day 29 can be measured as the time interval in days between randomization and death.

[0264] At the time of analysis, subjects who are lost to follow-up or have withdrawn consent or are still in the study can be censored at the last day known to be alive before day 29. Subjects who have completed follow-up can be censored on the day of the 29th visit. Differences in time to event endpoints can be summarized and shown with the median estimates and confidence intervals from the Kaplan-Meier curves by treatment arm. The table can show the median time to event for each treatment arm along with the overall 95% confidence interval, hazard ratio estimate, and log-rank p-value.

[0265] The 15-day and 29-day mortality rates represent the proportion of subjects who have died at 14 and 28 days, respectively. These can be derived from Kaplan-Meier estimates of survival rates at defined time points.

[0266] The occurrence of ICU transfers can be analyzed by the treatment arm. The number of patients transferred and the incidence rate (and CI) can be reported.

[0267] The duration of ICU stay can be measured from the date of ICU admission to the date of discharge from the ICU. This may include only those patients who were not admitted at the time of registration. The duration may be summarized in a table using the median and quartiles by the treatment arm.

[0268] The occurrence of non-invasive ventilation or high-flow oxygen use may be defined as a clinical status score equal to 6.

[0269] The occurrence of new non-invasive ventilation or high-flow oxygen use may be analyzed by the treatment arm. The number of subjects using non-invasive ventilation or high-flow oxygen use and the incidence rate (including CI) may be reported.

[0270] The duration of non-invasive ventilation or high-flow oxygen use can be measured from the day the clinical status score (based on an 8-point ordinal scale) is equal to 6 to the day the clinical status score is less than 6. The total duration can be the sum of all durations, regardless of whether the events occur consecutively or at separate intervals. Durations can be summarized in a table using medians and quartiles by treatment arm.

[0271] The occurrence of invasive ventilation can be analyzed by the treatment arm. The number of patients using invasive ventilation devices and the incidence (and CI) can be reported.

[0272] The duration of invasive ventilation can be measured from the day invasive ventilation began to the day invasive ventilation ceased. The total duration can be the sum of all durations, regardless of whether the events occur consecutively or at intervals.

[0273] The duration can be summarized in a table using the median and quartiles based on the treatment arm.

[0274] The occurrence of ECMO use can be analyzed by the treatment arm. The number of patients using ECMO and the incidence (and CI) can be reported.

[0275] The duration of ECMO use can be measured from the start date of ECMO use to the day of discontinuation of ECMO use. The total duration can be the sum of all durations, regardless of whether the events occur consecutively or at intervals.

[0276] The duration can be summarized in a table using the median and quartiles based on the treatment arm.

[0277] The incidence of hospital readmissions due to COVID-19 symptoms can be analyzed by the treatment arm. The number and incidence (and CI) of readmitted subjects are reported.

[0278] Saturation levels (including stability): Descriptive statistics including mean, median, standard deviation, maximum, and minimum values, as well as changes from baseline over time, can be summarized by the treatment arm. Changes from baseline values ​​can be shown over time in a line graph along with the mean and standard deviation plotted by the treatment arm.

[0279] The duration of body temperature ≥38°C can be measured from the first day after randomization when the patient had a body temperature ≥38°C until the day the body temperature decreased (<38°C). The duration can be summarized in a table using the median and quartiles by treatment arm.

[0280] Body temperature levels: Descriptive statistics including mean, median, standard deviation, maximum, and minimum values, as well as the change from baseline over time, can be summarized in the treatment arm. The change from baseline values ​​can be shown over time in a line graph along with the mean and standard deviation plotted in the treatment arm.

[0281] Oxygen supplementation may be defined as a clinical status (based on an 8-point ordinal scale) score equal to 5, 6, or 7. The duration of oxygen supplementation may be measured from the day the clinical status score equals 5, 6, or 7 to the day the clinical status score is lower than 5. The total duration can be the sum of all durations, regardless of whether the events occur consecutively or at separate intervals. Durations may be summarized in a table using medians and quartiles by the treatment arm.

[0282] Inflammatory status is measured as the difference in CRP, D-dimer, and ferritin serum levels from baseline at days 3, 8, and 15 leading up to discharge between the two study arms. Descriptive statistics, including the mean, median, standard deviation, maximum, and minimum values ​​for each inflammatory biomarker, as well as the change from baseline over time, may be summarized by the treatment arm. Changes from baseline may be shown over time in line graphs, along with the mean and standard deviation plotted by the treatment arm.

[0283] The number and percentage of patients showing improvement in radiological evaluation can be summarized by the treatment arm.

[0284] E. (Example 5) 1. Introduction Coronaviruses are enveloped RNA viruses with a single-stranded positive-sense RNA genome. Their genome is complexed with a nucleocapsid (N) protein and encapsulated by a lipid bilayer containing three embedded structural proteins: envelope (E), membrane (M), and spike (S). The S protein consists of two subunits: S1, which mediates viral binding to host cell receptors, and S2, which facilitates the fusion of the viral envelope with the host cell membrane. Since the critical step in viral infection is viral entry into host cells, therapeutic approaches to prevent infection largely focus on blocking the binding of the S protein to its receptor.

[0285] The binding affinity of a virus to a host receptor is primarily influenced by electrostatic protein-protein interactions. In the case of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the infectious agent causing the COVID-19 pandemic, entry into host cells is mediated through interaction with the human angiotensin-converting enzyme 2 (ACE2) receptor. The positively charged S trimer, in its closed conformation, engages in transient, nonspecific binding with the negatively charged ACE2 receptor, an interaction governed by electrostatic forces. The trimer then rearranges to an open state, exposing its specific receptor-binding interface, and forms a complex that is stabilized by additional interactions. The binding affinity of the SARS-CoV-2 S protein to the ACE2 receptor has been found to be higher than that of earlier SARS-CoV strains, a phenomenon attributed to the former's higher positive charge, further highlighting the importance of electrostatic interactions in coronavirus infections.

[0286] This crucial role of electrostatic interactions for viral infection suggests the use of electric fields for interference with viral host entry. The clinically approved treatment based on the application of electric fields is tumor therapeutic electric field (TT field)

[10] . TT field therapy is applied to the thoracic region of tumor patients in the United States and Europe for the treatment of two lung cancer indications: malignant pleural mesothelioma (an approved indication based on the STELLAR trial) and non-small cell lung cancer (an ongoing phase 3 study, the LUNAR trial, NCT02973789). This potent cancer treatment modality is based on the non-invasive delivery of a low-intensity (1–3 V / cm root mean square (RMS)), intermediate-frequency (100–500 kHz) alternating electric field via an array placed on the patient's skin at the tumor site. While electric fields at these frequencies are too high to stimulate nerve cells and too low to cause significant tissue heating, they are precisely sufficient to penetrate cancer cells and exert a bidirectional force therein. TT fields have been shown to induce alignment of polar tubulin and septin molecules according to the electric field, thereby impairing their proper function during cell division and causing an antimitotic effect and subsequent cell death. The high charge on coronavirus S proteins, particularly the SARS-CoV-2 S protein, suggests a possible effect of TT fields on its function.

[0287] The objective of this study was to investigate the in vitro effects of TT fields on coronavirus infection and to evaluate their safety in COVID-19 patients. Since the S protein, a major viral protein involved in viral attachment and entry into host cells, is highly conserved in all human coronaviruses, a less infectious member of this family, HCov-229E, was used in this study. Application of TT fields to human lung cells was shown to reduce viral entry and replication. Furthermore, progeny virions formed under TT fields exhibited lower infectivity. TT fields were also shown to enhance the in vitro efficacy of remdesivir, an approved treatment for COVID-19 patients with severe illness. Finally, the safety of applying TT fields to remdesivir and concomitant COVID-19 patients was demonstrated.

[0288] 2. Method i. Cell lines and viruses Human MRC-5 lung fibroblasts (ATCC, CCL-171™) and human lung cancer cell line A549 (ATCC, CCL-185™) were grown at 37°C in a 5% CO2 humidified incubator in Eagle's Minimum Essential Medium (EMEM) (ATCC, 30-2003™) and Dulbecco's Modified Eagle Medium (DMEM) (Biological Industries, 01-055-1A), respectively, supplemented with 10% fetal bovine serum (FBS) (Biological Industries, 04-007-1A). HCoV-229E (ATCC, VR740™) was handled in a Biosafety Level 2 (BSL2) facility and grown at its optimal temperature of 35°C throughout the study. For the production of a stock virus pool, commercial HCoV-229E was grown in MRC-5 cells according to the supplier's instructions, quantified by plaque assay, and stored in aliquots at -80°C.

[0289] ii. Applying TT Field The TT field was applied using the Inovitro® system (Novocure, ISR). Cell suspensions were grown in Inovitro® dishes made of high-dielectric steady-state ceramic (magnesium lead niobate-lead titanate [PMN-PT]) with two vertical pairs of transducers printed on their outer walls. The transducers were connected to a sinusoidal waveform generator that produced an AC electric field at a selected frequency and intensity, while vertically changing the electric field direction every second. The incubator temperature was set to 18°C ​​to generate a TT field at an intensity of 1.5 V / cm RMS, resulting in a temperature of 35°C inside the dish.

[0290] iii. The effect of TT fields on viral invasion Place MRC-5 cells on a glass coverslip (22 mm in diameter) in a 1.5 × 10⁶ 5 Cells were seeded at a cell / coverslip density. After 24 hours, cells were transferred to inovitol dishes containing 2 ml of EMEM supplemented with 2% FBS. Cells were then exposed to a TT field in the frequency range of 100–400 kHz and infected with HCoV-229E at a multiple infection degree (MOI) of 0.01 after 30 minutes with continued TT field application. Control cells were not treated with a TT field at any point. 0.5 or 2 hours (hpi) post-infection, cells were washed with PBS, trypsinized, resuspended, and counted using a Scepter® 2.0 Cell Counter (Merck, Millipore). Cells were then washed with PBS and frozen at -80°C until RT-qPCR analysis.

[0291] iv. Effects of TT fields on long-term viral exposure and viral replication MRC-5 cells were seeded and grown as described above, exposed to a TT field (150 kHz), and then infected with HCoV-229E at an MOI of 0.0001. Cells were washed with PBS at 3 hpi to remove unbound virus and maintained in fresh medium for a total of 24, 48, or 72 hours. The TT field was applied throughout the process, starting 30 minutes prior to infection, while control cells were not exposed to the TT field at any point. At the end of the treatment, the growth medium was collected and stored at -80°C for RT-qPCR analysis and plaque assay. Cells were treated as described above. 1.0 × 10⁶ 5 The same procedure was performed with A549 cells after seeding in cell / coverslips. Alternatively, MRC-5 cells were infected with HCoV-229E at an MOI of 0.01, and after washing the cells at 3 hpi, a TT field was applied up to 24 hpi, followed by analysis of dsRNA formation.

[0292] v. Combination effect of TT field with remdesivir As described above, MRC-5 cells were seeded, grown, exposed to a TT field (150 kHz), and then infected with HCoV-229E at an MOI of 0.01. At 3 hpi, the cells were washed with PBS and maintained in fresh medium supplemented with 0, 0.011, or 0.023 μM remdesivir (Cayman Chemicals, Cay30354) with or without TT field application. At 48 hpi, the growth medium and cells were collected and analyzed as described above.

[0293] vi. Real-time quantitative reverse transcription PCR (RT-qPCR) Total RNA was extracted using the MagnaPure 96 instrument (Roche, Germany) according to the manufacturer's instructions. The reaction was performed in a 25 μl reaction mixture prepared with AgPath-ID™ One-Step RT-PCR Reagents (Applied Biosystems, Thermo Fisher Scientific) using type-specific primers and probes for HCoV-229E selected using Primer Express software (PE Applied Biosystems) based on a highly conserved genomic region of the nucleocapsid gene: forward: 5'-CAGTCAAATGGGCTGATGCA-3'; reverse: 5'-AAAGGGCTATAAAGAGAATAAGGTATTCT-3'; probe: 5'-CCCTGACGACCACGTTGTGGTTCA-3' labeled at the 5' end with fluorescein amidite (FAM). Amplification and detection were performed on an ABI 7500 instrument using TaqMan Chemistry under the following conditions: 48 °C for 30 min (1 cycle); 95 °C for 10 min (1 cycle); and 95 °C for 10 s followed by 60 °C for 1 min (45 cycles). The amount of HCoV-229E in the supernatant (SN) was quantified per volume and expressed as a percentage compared to the control. To determine intracellular HCoV-229E, relative quantification (RQ) was used with RNaseP as the intracellular normalization gene: forward: 5'-AGATTTGGACCTGCGAGCG-3'; reverse: 5'-GAGCGGCTGTCTCCACAAGT- 3'; probe: 5'- TTCTGACCTGAAGGCTCTGCGCG-3' labeled at the 5' end with FAM.

[0294] vii. Scanning electron microscopy (SEM) MRC-5 cells were seeded at 4 × 10 on glass coverslips (13 mm diameter) 4Cells were seeded at cell / coverslip density and treated as described above. Cells were infected with HCoV-229E virus at a MOI of 20. A TT field (150 kHz) was applied throughout, starting 30 minutes prior to infection, while control cells were not treated with the TT field. Slides were transferred to clean plates at 0.5 hpi, briefly washed with PBS, and fixed at room temperature for 2 hours using 2% glutaraldehyde and 1% paraformaldehyde in 0.1 M sodium cacodylate buffer. After 15 minutes of fixation with osmium tetroxide in cacodylate buffer, the samples were dehydrated in a graded ethanol series, critical point dried (Quorum K850), and sputter-coated with 4 nm iridium (Quorum Q150T). The samples were then observed with a Zeiss Ultra Plus HR Scanning Electron Microscope.

[0295] viii. Transmission electron microscopy (TEM) MRC-5 cells are placed on a Thermox coverslip (22 mm in diameter) (Thermo, 174977) in a 3 x 10⁶ arrangement. 4 Cells were seeded at a cell / coverslip density. Cells were then grown as described above, infected with HCoV-229E at an MOI of 0.03, washed at 3 hpi, and then subjected to TT field treatment up to 48 hpi. Control cells were not treated with TT field treatment at any point. Cells were then fixed for 2 hours with 2% glutaraldehyde and 3% paraformaldehyde in 0.1 M sodium cacodylate buffer containing 5 mM CaCl2. Samples were washed, post-fixed with 2% osmium tetroxide, washed with DDW, and incubated in 2% uranyl acetate. After dehydration in a graded ethanol series, the coverslips containing cells were transferred to fresh wells filled with Epon812 for embedding. 75 nm transverse sections were cut using an ultramicrotome UC7 (Leica), transferred to a copper grid, and observed at an accelerating voltage of 120 keV using a Talos L120C Transmission Electron Microscope.

[0296] ix. Immunofluorescence imaging of dsRNA Cells were fixed for 15 minutes using ice-cold anhydrous ethanol (Millipore, 100983), followed by three washes with PBS. The cells were then blocked for 30 minutes in 1% BSA diluted in PBS (Sigma, A7906), incubated at room temperature for at least 1 hour with 1:100 diluted anti-double-stranded RNA monoclonal antibody (SCICONS, J2, 10010200) in PBS containing 1% BSA, followed by three washes with PBS. Next, the cells were incubated for 40 minutes with 1:800 diluted IgG Alexa fluor 488-conjugated donkey anti-mouse antibody in PBS containing 1% BSA and 1 μg / ml 4',6-diamidino-2-phenylindole (DAPI) (Sigma, 32670), washed three times with PBS, and mounted on slides. Images were acquired using an LSM 700 laser scanning confocal system (Zeiss, Gottingen). Image analysis and quantification were performed using FIJI software.

[0297] x. Plaque assay MRC-5 cells were placed in a 12-well plate at a rate of 2 × 10⁶ 5 Cells were seeded at a density of cells / well. After 24 hours, the incubator temperature was changed to 35°C, and the medium was replaced with 1 ml of fresh EMEM supplemented with 2% FBS. Cells were then transferred to the supernatant from a 48-hour long-term virus exposure experiment at a density of 1.9 × 10⁶ cells. 5The cells were infected with virus copies / well and subjected to five consecutive 10-fold dilutions. At 2 hpi, cells were washed with PBS to remove unbound virus and covered with EMEM supplemented with 2% FBS and 1.5% carboxymethylcellulose (CMC) (Sigma, C4888). After 4 days, the cells were fixed in ice-cold anhydrous ethanol for 15 minutes and stained with 1% Cristal Violet (Mercury, 1159400025) and 20% ethanol at room temperature for 5 minutes. Plaque-forming units (PFUs) were counted and divided by the dilution factor to obtain PFUs per equal viral load. The following formula was applied to calculate PFUs per equal supernatant (SN) volume: number of plaques formed by the same viral dilution / dilution factor × infectious volume of SN in 1 ml of infection medium.

[0298] xi.Statistical analysis Each in vitro experiment was repeated three times, and data from all replicates were pooled for analysis and presented as mean ± SD. Statistical significance was calculated using GraphPad Prism 8 software (La Jolla), along with the specific tests used, as described in the legends in the figures. Differences were considered significant at values ​​of *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0299] xii. Safety Clinical Trials The EF-37 study was a single-arm, open-label pilot trial of NovoTTF-100L (150 kHz TT field) in hospitalized patients with COVID-19.

[0300] A total of 10 patients were enrolled. Eligibility for the study was limited to hospitalized patients found to be positive for COVID-19. Inclusion criteria were as follows: age ≥ 18; hospitalization with a diagnosis of COVID-19 infection by RT-PCR within 72 hours prior to the start of treatment; SpO2 ≤ 93% at sea level; lung involvement confirmed by chest imaging; ability and willingness to comply with all study procedures; and, for female participants of childbearing age, use of highly effective contraception (failure rate of ≤ 1% per year when used consistently and accurately). Exclusion criteria included: any experimental treatment for COVID-19 prior to or during the study; assisted ventilation; dangerous illness defined as respiratory failure (SpO2 / FiO2 < 150), septic shock, and / or multiple organ dysfunction; clinically significant hematological, hepatic, and renal dysfunction (neutrophil count < 1.5 × 10⁻⁶). 9 / L and platelet count <100 × 10 9 Significant baseline comorbidities including (defined as bilirubin > 1.5 × ULN, AST and / or ALT > 2.5 × ULN, and serum creatinine > 2.5 mg / dL), a history of significant cardiovascular disease until the disease is well controlled (second / third degree heart block, significant ischemic heart disease, poorly controlled hypertension, congestive heart failure, or symptoms of heart failure at rest), a history of symptomatic or interventional arrhythmias, or a history of any psychiatric condition that may impair the patient's ability to understand or comply with the requirements of this protocol or to provide informed consent; implantable electronic medical devices in the upper torso (e.g., pacemakers, defibrillators); pregnancy or lactation; known allergies to medical adhesives or hydrogels; or unwillingness or inability to comply with the requirements of this protocol.

[0301] Written informed consent was obtained from all patients before any study-related evaluations / procedures were performed. Enrollment was performed within 48 hours of admission, and patients were treated for a duration of 29 days or until they were no longer hospitalized, using a thoracic TT field (≥18 hr / day) delivered to the chest, while receiving adjunctive care in standard COVID-19 treatment. Clinical follow-up was continued for 30 days after the end of the procedure. The safety profile was measured by the frequency and severity of procedure-related adverse events based on the Common Terminology Criteria for Adverse Events (CTCAE) V5.0. Secondary endpoints included: all-cause mortality, intensive care unit (ICU) transfer, use of non-invasive ventilation or high-flow oxygen, use of invasive ventilation, use of extracorporeal membrane oxygenation (ECMO), patient inflammatory status, duration of hospitalization, time to recovery, and clinical status on days 8, 15, 22, and 29 of the procedure.

[0302] The TT field was delivered to the patient through four insulated surface arrays placed on the patient's skin around the rib cage to generate two vertical electric fields in the patient's chest. The area where the array application was planned was shaved as needed, a layer of adhesive hydrogel was placed beneath the array, and hypoallergenic medical tape was placed on top of the array. The array was replaced 2-3 times per week to maintain optimal connectivity between the transducer array and the patient's skin. The array was mounted on an electric field generator that delivered a current of 1414 mA in two sequential, vertical directions. The time the TT field was delivered was captured by the NovoTTF-100A's internal memory unit, thereby enabling objective usage reporting. The procedure was administered continuously for at least 18 hours per day throughout the study.

[0303] 3.Results i. The effect of TT fields against viral intrusion Application of a 150 kHz TT field to MRC-5 lung fibroblasts during 2 hours of HCoV-229E infection at an MOI of 0.01 produced a significant 42% reduction in cellular viral load compared to untreated infected cells (Figure 10A). TT field frequencies of 100 and 400 kHz showed lower efficacy, exhibiting reductions of 19% and 32%, respectively. Therefore, a 150 kHz TT field frequency was selected for all consecutive experiments. Similar experiments performed with only 30 minutes of viral incubation, focusing on the cell adhesion process, showed a 43% inhibition of the TT field compared to the control (Figure 10B).

[0304] To further investigate the effect of the TT field on viral cell adhesion, SEM and TEM examinations were performed. SEM was used on MRC-5 cells exposed to HCoV-229E for 30 minutes at a high MOI of 20 to allow for easier visualization of virions. The results showed a 25% reduction in the number of virions bound to the cell surface when the TT field was applied compared to the control (Figures 10C and 10D). TEM examination at 48 hpi on control cells infected with HCoV-229E for 3 hours at an MOI of 0.03 revealed that the average distance of virions from the cells was 75 nm (range = 21-307 nm), while the average distance increased to 158 nm (range = 21-740 nm) when the TT field was applied for a certain period after infection (Figures 10E and 10F).

[0305] ii. Effects of TT fields on long-term viral exposure MRC-5 cells infected with HCoV-229E at a MOI of 0.0001 while exposed to a 150 kHz TT field during infection and up to 24, 48, or 72 hpi showed a reduction in viral cellular load of 42, 51, and 58%, respectively, compared to the control (Figure 11A). The amount of virus secreted into the culture medium was also affected by the TT field, showing a reduction of 68 and 74%, respectively, for delivery at 48 and 72 hours in the TT field compared to the control (Figure 11B). Very low levels of virus were secreted from cells within 24 hours of infection (Figure 15), and therefore no significant difference was observed between the control and the TT field in terms of extracellular viral load in this time frame. Infected MRC-5 cells showed lower cell counts than uninfected cells in the absence of the TT field, at 9% at 48 hpi and 1% at 72 hpi (FIG). When the TT field was applied to infected cells, the cell count increased by 19%, 21%, and 35% compared to control cells at 24, 48, and 72 hr treatments, respectively (Figure 11C). The cell count, however, was not affected by the delivery of the TT field to cells in the absence of the virus (Figure 17C). Together, these results indicate that the TT field protects cells from the harmful effects of the virus. Supernatants from 48-h long-term viral exposure were subjected to a plaque formation assay in MRC-5 cells without further application of the TT field. The viral titer formed under TT field application was 79% lower than that formed without the TT field when examining the same supernatant volume (Figure 11D), and the PFU per equal amount of virus was 50% lower (Figure 11E), indicating differences not only in viral load but also in pathogenicity.

[0306] The effect of the TT field on infection of A549 lung cancer cells was also investigated. These cells showed higher susceptibility to the virus compared to MRC-5 cells, with a 25% reduction in cell number at 48 hpi compared to uninfected A549 cells (MOI of 0.0001) (Figure 17B). Nevertheless, the protective effect of the 150 kHz TT field was also enhanced in these cells, with a 92% reduction in intracellular viral load (Figure 17A) and an 85% reduction in the number of extracellular viral copies (Figure 17C). It should be noted that the 150 kHz TT field is known to be cytotoxic to A549 cells as part of the anti-mitotic effect of the TT field on cancer cells. Indeed, without viral infection, A549 cells exposed to the 150 kHz TT field for 48 hours showed a 33% lower cell number compared to the control (Figure 17C). However, no differences in cell number were observed in cells exposed to a 48hr TT field in the presence of the virus compared to the control.

[0307] iii. Effects of TT fields on virus replication MRC-5 cells were infected with HCoV-229E at a MOI of 0.01 for 3 hours. After washing the cells to remove any extracellular virions, the TT field was applied up to 24 hpi, which is the time frame in which minimal virion secretion into the culture medium is expected (Figure 15), thus minimizing the level of reinfection events. This protocol allowed for the isolation of the effect of the TT field on the replication process, measured by fluorescence microscopy for the detection of dsRNA associated with viral replication (Figure 12A). Since the infection process was carried out identically without TT field delivery, the number of infected cells was equal in both groups (not shown). However, compared to the control, the application of the TT field resulted in a 24% reduction in the number of foci per infected cell (Figure 12B), a 23% reduction in focus size (Figure 12C), and a 41% reduction in the overall focus area per infected cell (Figure 12D). TEM examination was performed to allow for the investigation of intracellular events (Figures 12E and 12H). Cells infected with HCoV-229E at a MOI of 0.03 for 3 hours and examined at 48 hpi while exposed to a TT field showed 70% less double-membrane vesicle (DMV) invagination (Figure 12F) and 98% less DMV fusion (Figure 12G). On the other hand, three times higher levels of autophagolisosomes were observed in infected cells treated with a TT field compared to control cells (Figure 12I, Figure 12H).

[0308] Supernatants from 48-hour long-term viral exposure were subjected to a plaque formation assay in MRC-5 cells without further application of the TT field. When comparing the equivalent amounts of virus formed with and without the TT field, the former showed a 44% lower plaque formation capacity (Figure 12I); and a 71% reduction in PFU per equal supernatant volume (Figure 12K).

[0309] iv. Combination effect of TT field with remdesivir MRC-5 cells infected with HCoV-229E at a 0.01 MOI for 3 hours and subsequently treated with remdesivir showed a dose-dependent response, with 0.011 μM remdesivir inhibiting cellular viral load by 27% and 0.023 μM remdesivir reducing it by 65% ​​at 48 hpi (Figure 13A). Under these conditions, delivery of TT Field alone reduced the viral load by 42%, while contingent application of TT Field with remdesivir reduced the viral load by 54% at low doses and 85% at high doses. The lower viral load with the combination of TT Field and remdesivir was also evident from the reduction in intracellular dsRNA levels compared to monotherapy (Figure 13C). The number of virions secreted into the culture medium was also reduced by 31% and 75% for 0.011 and 0.023 μM remdesivir, respectively, and by 55% for TT Field alone (Figure 13B). The contingent application of the two treatments resulted in a 68% reduction for low doses and an 88% reduction for high doses.

[0310] The calculated additional effect was obtained by multiplying the remdesivir alone by the TT field alone. When the calculated effect was compared with the measured effect, a smaller additional effect was observed at higher concentrations (0.023) (see Table 2).

[0311] [Table 2]

[0312] 4. Discussion In this study, the effect of TT fields on coronavirus-induced lung infection was investigated using a less infectious strain of HCoV-229E. When TT fields were examined at several frequencies, all showed significant inhibition of viral entry into MRC-5 lung fibroblasts, as determined by RT-qPCR measurements. The effect was found to be most pronounced at 150 kHz, and this frequency was used throughout the study. The same level of inhibition by TT fields was detected at both 2 hours and 30 minutes after viral infection, suggesting that TT fields primarily interfere with the viral attachment process rather than the intracellular migration process. SEM and TEM examinations allowed for virion visualization, and when TT fields were applied, the former showed less virion attachment to cells, while the latter demonstrated a greater average distance of virions from cells. Overall, these results indicate that TT fields interfere with viral attachment to cells, leading to a reduction in viral entry.

[0313] Since the viral life cycle involves the secretion of progeny virions from cells and repeated invasion, the long-term effects of the TT field were also investigated. In these 72-hour long-term studies, the TT field effectively reduced the intracellular viral load after 24 hours and showed an increased effect over longer treatment durations. This was accompanied by a decrease in virion secretion from cells into the culture medium, as clearly demonstrated by both RT-qPCR measurements and plaque assays. Furthermore, cell numbers were higher for cells treated with the TT field than for controls, and the effect increased with longer treatment times, indicating that the TT field protects cells from the harmful consequences of viral infection. Long-term studies were also conducted on A549 lung cancer cells, and a significant decrease in cellular viral load and viral secretion into the culture medium was observed in cells treated with the TT field.

[0314] The application of an electric field has been shown to affect the secondary and tertiary structure of the SARS-CoV-2 S protein. Simulations revealed local dipole alignment and charge transfer parallel to the direction of the electric field, which resulted in irreversible disruption of the spatial organization of key residues involved in receptor binding when the electric field was delivered at a low intensity of 100 V / cm over several hundred nanoseconds. This structural alteration of the S protein after exposure to the electric field was suggested to cause virion inactivation or attenuation. The TT field used in this study was only 1.5 V / cm RMS in intensity, but it has been shown that intensity amplification occurs near the cell membrane due to its non-dielectric properties.

[0315] Thorough investigation of plaque assays from long-term experiments revealed interesting findings: the PFU for virions formed under the application of a TT field was lower than that of the control, not only for the same supernatant volume but also for the same amount of virus. This indicates that progeny virions formed under a TT field were less pathogenic than the control. One explanation for this phenomenon may be conformational changes of the S protein induced by the electric field, as described above. A second option is to link this to tubulin, which has been suggested to be involved in S protein transport, localization, and assembly to virions, and a reduction in the release of infectious viral particles has been observed when microtubule depolymerization is induced. The TT field has been shown to mediate changes in microtubule organization and dynamics in cancerous cells. This effect of the TT field may interfere with the proper assembly of virions, as it may be relevant to non-cancerous cells. The TT field may interfere with additional steps in the viral life cycle.

[0316] To investigate the effect of TT fields on viral replication, TT fields were delivered only after cells had been initially infected with HCoV-229E. The infection process was identical in both the control and treatment groups, so there was no difference in the number of infected cells. However, the amount and size of dsRNA foci formed within infected cells were lower when TT fields were applied compared to the control; DMV invagination and fusion events were less frequent. On the other hand, a greater number of autophagolysosomes (resulting in the fusion of autophagosomes with lysosomes) were observed in cells exposed to the TT field, indicating an increase in autophagy flux in these cells. It is known that cells infected with coronaviruses utilize the autophagy pathway to sense the virus, control its proliferation, and clear it. Viruses have evolved to inhibit, evade, or manipulate this host response to protect themselves. In the case of coronaviruses, upon infection, the virus hijacks autophagosomes, preventing their fusion with lysosomes and utilizing these DMVs as replication and translation niches. In later stages, DMV invagination and fusion allow for a re-purposed membrane for further virion production. Together, the results demonstrate that the TT field interferes with viral replication, and this can be attributed at least in part to the TT field's ability to enhance autophagy, as previously demonstrated in glioblastoma, Lewis lung cancer, and hepatocellular carcinoma cell lines.

[0317] On May 1, 2020, the FDA issued emergency use authorization for the use of remdesivir as a treatment for patients with severe COVID-19, as it determined that the potential benefits of remdesivir outweighed its known and potential risks. Investigations into possible combinations of remdesivir and TTfield showed superiority in both intracellular viral accumulation and secretion compared to each treatment alone, in vitro. This suggests that lower doses of remdesivir may be sufficient for contingent treatment with TTfield, thus potentially mitigating remdesivir-related risks. The safety of the TTfield and remdesivir combination was investigated in a Phase 1 clinical trial.

[0318] In conclusion, in addition to the effectiveness of TT fields in preventing coronavirus infection and replication, the safety of applying TT fields to COVID-19 patients has been demonstrated. As the COVID-19 pandemic continues to spread rapidly worldwide, the virus is mutating at a rapid pace. Changes in central residues in the S protein result in variants that exhibit enhanced viral-host cell adhesion and fusion, and therefore increased infectivity and reduced tactability of these new forms of the virus. This may be seen alongside the currently spreading, more transmissible variants, delta (B.1.617.2 series) and lambda (C.37 series), which harbor multiple mutations in the S protein receptor-binding domain and exhibit relative resistance to neutralizing antibodies induced in convalescent and vaccinated individuals. These changes may also interfere with the effectiveness of therapeutic approaches that primarily rely on the structure of the S protein and its interaction with host receptors. Since TT fields are not specifically designed for any particular S protein amino acid sequence, but rather for high protein polarity that leads to increased host receptor binding, they may be suitable for treating different viral variants, suggesting the potential of this treatment modality in the constantly changing viral landscape.

[0319] Those skilled in the art will recognize, or can confirm by common experiments, many equivalents to specific embodiments of the methods and compositions described herein. Such equivalents are intended to be encompassed by the following claims. (References) TIFF0007886853000005.tif233170TIFF0007886853000006.tif78170

Claims

1. A system comprising one or more electrodes for inhibiting coronavirus from infecting or replicating within cells, An alternating electric field is applied to the cell via the electrodes, and the alternating electric field has a frequency of 150 kHz and an electric field intensity of 1 to 4 V / cm RMS. system.

2. The system according to claim 1, wherein the coronavirus is SARS-CoV-2.

3. A system for reducing the number of coronavirus copies per cell, comprising one or more electrodes, An alternating electric field is applied to the cell via the electrodes, and the alternating electric field has a frequency of 150 kHz and an electric field intensity of 1 to 4 V / cm RMS. system.

4. The system according to claim 3, wherein the coronavirus is SARS-CoV-2.

5. A system for treating coronavirus infections, comprising one or more electrodes, An alternating current electric field is applied to a target site of a person having coronavirus infection via the electrodes, and the alternating current electric field has a frequency of 150 kHz and an electric field strength of 1 to 4 V / cm RMS. The aforementioned target site includes one or more coronavirus-infected cells, system.

6. The system according to claim 5, wherein the coronavirus is SARS-CoV-2.

7. The system according to claim 5, wherein the target site of the subject is the lung.

8. The system according to claim 6, wherein cells not infected with the coronavirus are not damaged.

9. The system according to claim 5, wherein the survival of the cells at the target site is maintained and viral replication or viral infection is reduced.

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