UV A light exposure and its use to increase mitochondrial antiviral protein expression in tracheal cells via cell-cell communication

Abstract

Mitochondrial antiviral signaling (MAVS) protein mediates the innate antiviral response and is a key component of the response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Provided herein are methods for increasing expression of mitochondrial antiviral signaling (MAVS) protein in epithelial cells by use of UVA therapy to expose epithelial cells to UVA therapy, or to contact a first set of epithelial cells with a second set of epithelial cells exposed to UVA therapy, or to contact the first set of epithelial cells with a cell lysate of the second set of epithelial cells exposed to UVA therapy. TIFF2024521637000002.tif84128

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 USC §119(e) to U.S. Provisional Patent Application No. 63 / 184,749, filed May 5, 2021, which is incorporated by reference in its entirety.

[0002] FIELD OF THEINVENTION The present invention relates to a system and method for ultraviolet light therapy for treating respiratory infections. [Background technology]

[0003] background All publications in this specification are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description contains information that may be useful in understanding the present invention. No admission is made that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] The human body has many defenses against infection, the best known of which involves the innate immune response, in which immune cells are recruited to the site of infection via cytokine signaling. However, intracellular responses to infection are also important, especially in defense against viruses. Over the past decade, it has become clear that mitochondria can mediate the establishment and maintenance of innate and adaptive immune responses, including the production of mitochondrial antiviral (MAVS, or mitochondrial antiviral signaling) proteins.

[0005] The MAVS protein is primarily localized in the outer membrane of mitochondria and transduces signals from RIG-I-like receptors (RLRs), cytoplasmic receptors that recognize viral RNA. Specifically, after recognition and binding of viral components, the RLRs, retinoic acid-inducible gene I (RIG-I), and melanoma differentiation-associated gene 5 (MDA5), interact with MAVS and activate transcription factors that induce the expression of proinflammatory and antiviral genes. However, some viruses have developed mechanisms to antagonize MAVS activation and evade this innate immune response. For example, the SARS-CoV-2 transmembrane glycoprotein M is thought to antagonize MAVS and thus impair the MAVS-mediated innate antiviral response. Summary of the Invention

[0006] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods that are intended to be exemplary and illustrative, not limiting in scope.

[0007] A method of increasing expression of mitochondrial antiviral signaling (MAVS) protein in epithelial cells is provided, comprising exposing epithelial cells to an effective amount of ultraviolet A (UVA) light to increase expression of MAVS protein in the epithelial cells or in distal epithelial cells not exposed to an effective amount of UVA. Preferably, the method is performed in a subject in need thereof; and the method uses UV exposure within at least 335-350 nm, does not cause UV-induced DNA damage in the exposed cells, and does not require administration of anesthesia to the subject before, during, or after exposure to UVA light. In various aspects, the increase in MAVS protein expression is compared to one that has not been exposed to an effective amount of UVA or compared to a control. The control can be a baseline value of epithelial cells before exposure to UVA, a baseline value of epithelial cells before contact with a pathogen, or a baseline value of a population of epithelial cells that have not been exposed to the amount of UVA and are not infected with a pathogen.

[0008] In some embodiments, the epithelial cell comprises a tracheal epithelial cell, a nasopharyngeal epithelial cell, a ciliated epithelial cell. In some embodiments, the epithelial cell is one or more of a mammalian nasal epithelial cell, a mammalian oral epithelial cell, a mammalian olfactory epithelial cell, a mammalian tracheal epithelial cell, a mammalian pharyngeal epithelial cell, a mammalian lung epithelial cell. In additional embodiments, the epithelial cell is a urethral epithelial cell, a bladder epithelial cell, a vaginal epithelial cell, a urogenital epithelial cell, a gastrointestinal epithelial cell (e.g., a rectal epithelial cell or a gastrointestinal epithelial cell other than a rectal epithelial cell), an outer ear epithelial cell, and / or a middle ear epithelial cell.

[0009] In some embodiments of the method, exposing the subject's tracheal epithelial cells to UVA light or irradiating the tracheal epithelium with UVA light increases MAVS protein expression in the subject's trachea.

[0010] In some embodiments of the method, exposing a subject's nasal epithelial cells, olfactory epithelial cells, oral epithelial cells, or a combination thereof to UVA light, or irradiating the subject's nasal epithelium, olfactory epithelium, and / or oral mucosal epithelium with UVA light, increases MAVS protein expression in the subject's nasal epithelium, olfactory epithelium, and / or oral mucosal epithelium, as well as in the subject's lungs.

[0011] As an additional example of increasing expression of MAVS protein in distal epithelial cells (including those not exposed to UVA light), the methods may, in some embodiments, include: exposing urethral epithelial cells to UVA light, increasing MAVS protein expression in epithelial cells in the subject's bladder; exposing vaginal epithelial cells to UVA light, increasing MAVS protein expression in epithelial cells in the subject's uterus; exposing penile epithelial cells to UVA light, increasing MAVS protein expression in epithelial cells in the subject's urethra or bladder; exposing rectal epithelial cells to UVA light, increasing MAVS protein expression in epithelial cells in the subject's rectum or colon; exposing outer ear epithelial cells to UVA light, increasing MAVS protein expression in epithelial cells in the subject's middle ear or inner ear; and / or exposing middle ear epithelial cells to UVA light, increasing MAVS protein expression in epithelial cells in the subject's inner ear.

[0012] In some embodiments, the subject who needs UVA light exposure or has been exposed to UVA light does not have symptoms or signs of microbial infection or has never been exposed to microbial infection.In some embodiments, the subject who needs UVA light exposure or has been exposed to UVA light exhibits symptoms or signs of microbial infection for 3 days, 5 days, 7 days, or 10 days or less.In additional embodiments, the method further comprises selecting a subject who exhibits symptoms or signs of microbial infection as a subject who needs UVA light exposure before exposing the epithelial cells of the subject to an effective amount of UVA light.

[0013] Some embodiments provide that an effective amount of UVA light increases MAVS protein levels to reduce the proliferation of microorganisms that infect epithelial cells, or to pretreat epithelial cells prior to microbial infection so that the microbial infection has a lower proliferation rate or even a reduced amount when it infects epithelial cells.An effective amount of UVA light administration can include one or more continuous exposures, or one or more pulsed exposures, in some embodiments.

[0014] Also provided is a method of evaluating UVA treatment in a subject in need thereof, comprising assaying a biological sample obtained from the subject exposed to UVA treatment for MAVS protein expression levels, where a MAVS protein expression level higher than the subject's baseline level or higher than a control level indicates that the treatment is effective. The biological sample, in various implementations, comprises epithelial cells.

[0015] Further provided is a method of administering UVA treatment to a subject in need thereof, comprising assaying MAVS protein expression in a biological sample obtained from the subject exposed to UVA treatment, and continuing to administer UVA treatment to the subject if MAVS protein expression is lower than the subject's baseline level, compared to a control, or compared to a target level.

[0016] In some embodiments, the method of administering ultraviolet A (UVA) treatment in a subject in need thereof comprises exposing epithelial cells to an effective amount of ultraviolet A (UVA) radiation in a subject having low MAVS protein expression compared to a control, indicating that the subject is in need of UVA treatment, where the exposure increases expression of MAVS protein in the epithelial cells or in distal epithelial cells not exposed to the effective amount of UVA. In other embodiments, the method of administering ultraviolet A (UVA) treatment in a subject in need thereof comprises exposing epithelial cells to an effective amount of ultraviolet A (UVA) radiation in a subject having MAVS protein expression higher than the subject's baseline level or higher compared to a control, indicating that UVA treatment will be effective.

[0017] Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of aspects of the invention. [Brief description of the drawings]

[0018] Exemplary embodiments are illustrated in the reference figures, in which: The embodiments and figures disclosed herein are to be considered illustrative rather than restrictive. [Figure 1A] FIG. 1 depicts a schematic diagram showing the experimental design in which 100% confluent monolayer plates of primary tracheal epithelial cells (HTEpCs) were partially exposed to 2 mW / cm2 NB-UVA for 20 min. NB-UVA was applied only to region 1. After UVA treatment, cells were collected from regions 4, 3, 2, and 1, in that order. [Figure 1B] Depicts normalized MAVS levels in 30-40% confluent HTEpCs exposed to 2mW / cm2 NB-UVA for 20 min and in unexposed controls. Y-axis units are AU (arbitrary units) normalized by Ponceau. [Figure 1C] Normalized MAVS levels are depicted in 100% confluent HTEpC region 1 exposed to 2mW2 mW / cm2 NB-UVA for 20 min, and in unexposed monolayer controls. Y-axis units are AU (arbitrary units) normalized by Ponceau. [Figure 1D] Depicts normalized MAVS levels in 30–40% confluent naive HTEpCs treated with supernatant from 30–40% confluent NB-UVA-exposed HTEpCs, and in controls incubated with supernatant from unexposed 30–40% confluent HTEpCs. [Figure 1E] Depicts western blots of proteins extracted from 30–40% confluent naive HTEpCs treated with supernatant from 30–40% confluent NB-UVA-exposed HTEpCs (lanes 1, 2, and 3) and from controls treated with supernatant from 30–40% confluent unexposed HTEpCs (lanes 4, 5, and 6). [Figure 1F] Depicts normalized MAVS levels in 30–40% confluent naive HTEpCs treated with lysate from 30–40% confluent NB-UVA-exposed HTEpCs, and in controls incubated with lysate from 30–40% confluent unexposed HTEpCs. [Figure 1G]Depicts western blots prepared directly from lysates of 30–40% confluent naive HTEpCs incubated with lysates from 30–40% confluent NB-UVA-exposed cells (lanes 1–4) and from control lysates incubated with lysates from 30–40% confluent unexposed HTEpCs (lanes 5–8). [Figure 1H] Normalized MAVS levels are depicted in 100% confluent HTEpCs partially exposed to 2mW / cm2 NB-UVA for 20 minutes. Region 1 was directly exposed to NB-UVA, whereas regions 2, 3, and 4 were not exposed to NB-UVA. [Figure 1I] Depicts Western blots prepared from cell lysates of 100% confluent HTEpC from three experiments that were exposed to NB-UVA (region 1 - lanes 1, 5, and 9) and from lysates of confluent HTEpC from the same culture plate that were not exposed to NB-UVA (region 2 - lanes 2, 6, and 10; region 3 - lanes 3, 7, and 11; region 4 - lanes 4, 8, and 12). [Figure 1J] Depicts a Western blot of proteins extracted from 100% confluent HTEpCs exposed to NB-UVA (lanes 1, 2, and 4) and 100% confluent HTEpCs not exposed to NB-UVA (lanes 5, 6, and 7). Lane 3 (exposed to NB-UVA) was discarded due to insufficient total protein magnification. [Diagram 2] Depicts normalized MAVS levels in 30-40% confluent HTEpC cells exposed to 2mW / cm2 NB-UVA for 20 min (1, 2, and 3 times), in HTEpC cells exposed to 5mW / cm2 NB-UVA for 20 min (1 time), and in unexposed controls. [Diagram 3]Figure 3A depicts normalized MAVS levels in 100% confluent HTEpC cells exposed to 2 mW / cm2 NB-UVA for 20 min. Figure 3B shows a Western blot of proteins extracted from 100% confluent HTEpC cells exposed to NB-UVA (lanes 1-4) and 100% confluent HTEpC cells not exposed to NB-UVA (lanes 5-7). [Figure 4] Figure 4A depicts normalized MAVS levels in HTEpC cells incubated with lysates from NB-UVA-exposed HTEpC cells and in controls incubated with lysates from unexposed HTEpC cells. Figure 4B depicts western blots prepared directly from lysates of HTEpC cells incubated with lysates from NB-UVA-exposed cells (lanes 1-5) and from lysates of control HTEpC cells incubated with lysates from unexposed cells. [Diagram 5] Figure 5A depicts normalized MAVS levels in 100% confluent HTEpC cells partially exposed to 2 mW / cm2 NB-UVA for 20 min. Region 1 was directly exposed to NB-UVA, whereas regions 2, 3, and 4 were not exposed to NB-UVA. Figure 5B shows Western blots prepared from cell lysates of 100% confluent HTEpC cells exposed to NB-UVA (region 1 - lanes 1, 5, and 9) and from lysates of confluent HTEpC cells not exposed to NB-UVA from the same culture plate (regions 2, 3, and 4 - lanes 2, 3, 4, 6, 7, 8, and 10, 11, and 12, respectively). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Description of the Invention All references cited herein are incorporated by reference in their entirety as if fully set forth. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3 rd ed., Revised, J. Wiley & Sons (New York, NY 2006); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 7 th ed., J. Wiley & Sons (New York, NY 2013); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4 th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provides those of skill in the art with a general guide to many of the terms used in this application.

[0020] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

[0021] As used herein, the term "about" when used in conjunction with a reference numerical indication means, unless otherwise specified herein, the reference numerical indication plus or minus up to 5% of the reference numerical indication. For example, the language "about 50%" covers a range of 45% to 55%. In various embodiments, the term "about" when used in conjunction with a reference numerical indication can mean, unless otherwise specified in the claims, the reference numerical indication plus or minus up to 4%, 3%, 2%, 1%, 0.5%, or 0.25% of the reference numerical indication.

[0022] As used herein, the term "distal" with respect to an epithelial cell refers to an epithelial cell that is either directly connected (e.g., by a gap junction, tight junction, or desmosome) or indirectly connected to a UVA-exposed epithelial cell. Indirect connection in this context refers to a cell that is intercellularly connected to a cell that is ultimately directly connected to the UVA-exposed epithelial cell.

[0023] In various embodiments, the distal epithelial cells are up to 30 cm away from the periphery of the UVA light exposed / irradiated area or volume of epithelial cells. In various embodiments, the distal epithelial cells are up to 20 cm away from the periphery of the UVA light exposed / irradiated area or volume of epithelial cells. In various embodiments, the distal epithelial cells are up to 10 cm away from the periphery of the UVA light exposed / irradiated area or volume of epithelial cells. In various embodiments, the distal epithelial cells are up to 5 cm away from the periphery of the UVA light exposed / irradiated area or volume of epithelial cells.

[0024] "Mitochondrial antiviral signaling protein (MAVS)" (protein accession number Q7Z434), also known as IPS1, KIAA1271, VISA, or CARDIF, is a 540 amino acid protein that contains one caspase recruitment domain (CARD) and several transmembrane domains and is localized to the outer mitochondrial membrane. Without wishing to be bound by a particular theory, MAVS is thought to function downstream of proteins such as retinoic acid-inducible gene I (RIG-I) that detect double-stranded (ds) virus replication and is required for an appropriate immune response to ds virus infection.

[0025] As part of our studies exploring the potential of ultraviolet A (UVA) therapy in the treatment of infectious diseases, we recently showed that application of UVA light under certain conditions to human ciliated tracheal epithelial cells infected with coronavirus-229E significantly improved cell viability and prevented virus-induced cell death, which was accompanied by reduced levels of the CoV-229E spike (S) protein. Furthermore, cells treated with UVA light showed significantly increased levels of MAVS protein, indicating that UVA can activate MAVS. Furthermore, in a first-in-human clinical trial on the safety of using intratracheally delivered UVA light to treat mechanically ventilated subjects with coronavirus disease 2019 (COVID-19), subjects showed a significant reduction in SARS-CoV-2 viral load in endotracheal aspirates by day 6 of therapy, indicating that UVA light can reduce SARS-CoV-2 infection. Furthermore, viral load in nasal swabs was also reduced, despite the fact that only a small portion of the trachea was exposed to UVA light.

[0026] In this study, we explored the effect of UVA light (e.g., narrow band, NB, UVA light) on MAVS expression in human ciliated tracheal epithelial cells in vitro. We also explored whether the effect of UVA light was limited to cells directly exposed to UVA or was also seen in cells that were not directly exposed to UVA.

[0027] In general, narrow band (NB) UV-A (or UVA, or UV A) light can have wavelengths centered around 345 nm and can include a range of ±1 nm, ±2 nm, ±3 nm, ±4 nm, or ±5 nm. In some embodiments, NB UV-A has a peak wavelength in the range of 343 nm to 345 nm. In some embodiments, NB-UVA LEDs are used, which emit peak wavelengths in the range of 343 nm to 345 nm. In some embodiments, the UV-A light is 315 nm to 400 nm, or 320 nm to 410 nm, or 335 nm to 350 nm. In some embodiments, the UV-A light peaks at 335 nm to 345 nm. In some examples, light sources are used that are LEDs with peak wavelengths of 341 nm, 342 nm, 343 nm, 344 nm, 345 nm, 346 nm, 347 nm, 348 nm, and / or 349 nm. In some instances, the peak wavelengths of the LEDs may have a + / - 3 nm, 2 nm, or 1 nm error therearound. In some embodiments, only UV-A light is exposed to the subject's tracheal cells.

[0028] Various embodiments provide a method of increasing expression of mitochondrial antiviral signaling (MAVS) protein in epithelial cells in a subject in need thereof, comprising exposing epithelial cells to an effective amount of ultraviolet A (UVA) radiation to increase expression of MAVS protein in the epithelial cells or in distal epithelial cells not exposed to an effective amount of UVA, wherein the increased expression of MAVS protein is compared to those not exposed to an effective amount of UVA or compared to a control.

[0029] In some implementations, a method is provided for increasing MAVS protein expression in epithelial cells in a subject in need thereof by exposing the epithelial cells to an effective amount of UVA to increase expression of MAVS protein in the epithelial cells.

[0030] In some implementations, a method is provided for increasing MAVS protein expression in epithelial cells in a subject in need thereof by exposing the epithelial cells to an effective amount of UVA to increase expression of MAVS protein in distal epithelial cells that have not been exposed to an effective amount of UVA.

[0031] In another embodiment, a method is provided for increasing MAVS protein expression in epithelial cells in a subject in need thereof by exposing epithelial cells to an effective amount of UVA to increase expression of MAVS protein in the epithelial cells and in distal epithelial cells that have not been exposed to the effective amount of UVA.

[0032] In some embodiments of the method, the epithelial cells include or are tracheal epithelial cells and / or nasopharyngeal epithelial cells. The tracheal epithelial cells and / or nasopharyngeal epithelial cells may be exposed to or irradiated with UVA in some implementations of the method.

[0033] In some embodiments of the method, the epithelial cells include or are ciliated epithelial cells. The ciliated epithelial cells may be exposed to or irradiated with UVA in some implementations of the method.

[0034] In some embodiments of the method, the epithelial cells include or are ciliated tracheal epithelial cells and / or ciliated nasopharyngeal epithelial cells. The ciliated tracheal epithelial cells and / or ciliated nasopharyngeal epithelial cells may be exposed to or irradiated with UVA in some implementations of the method.

[0035] In some embodiments of the method, the epithelial cells include or are human nasal epithelial cells. In some embodiments of the method, the epithelial cells include or are human tracheal epithelial cells. Furthermore, in some embodiments of the method, the epithelial cells include or are human nasal epithelial cells and human tracheal epithelial cells. In some implementations of the method, the human nasal epithelial cells, the human tracheal epithelial cells, or both can be exposed to UVA or irradiated with UVA.

[0036] In some embodiments of the method, the epithelial cells include or are human lung epithelial cells. The human lung epithelial cells may be exposed to or irradiated with UVA in some implementations of the method.

[0037] In one aspect, a method for increasing expression of mitochondrial antiviral signaling (MAVS) protein in epithelial cells in a subject includes exposing nasal epithelial cells, olfactory epithelial cells, oral epithelial cells, or a combination thereof to an effective amount of UVA to increase MAVS protein levels in at least the exposed epithelial cells.

[0038] In another embodiment, a method for increasing expression of mitochondrial antiviral signaling (MAVS) protein in epithelial cells in a subject comprises exposing nasal epithelial cells, olfactory epithelial cells, oral epithelial cells, or a combination thereof to an effective amount of UVA to increase MAVS protein levels in the trachea, bronchi, or both of the subject.

[0039] In yet another aspect, a method for increasing expression of mitochondrial antiviral signaling (MAVS) protein in epithelial cells in a subject comprises exposing nasal epithelial cells, olfactory epithelial cells, oral epithelial cells, or a combination thereof to an effective amount of UVA to increase MAVS protein levels in epithelial cells in the lungs of the subject.

[0040] In yet further embodiments, epithelial cells can also include or be one or more of urethral epithelial cells, bladder epithelial cells, vaginal epithelial cells, urogenital epithelial cells, rectal epithelial cells, gastrointestinal epithelial cells other than rectal epithelial cells, external ear epithelial cells, and middle ear epithelial cells.The gastrointestinal system includes the organs of the mouth, pharynx (throat), esophagus, stomach, small intestine, large intestine, rectum, and anus.Thus, gastrointestinal epithelial cells other than rectal epithelial cells can include one or more of oral mucosa epithelial cells, pharyngeal epithelial cells, esophageal epithelial cells, secretory epithelial cells that cover the surface of the stomach, and intestinal epithelial cells.Thus, in some embodiments, the method of increasing the expression of mitochondrial antiviral signaling (MAVS) protein in epithelial cells in a subject, comprising exposing epithelial cells to an effective amount of UVA, comprises exposing urethral epithelial cells, bladder epithelial cells, vaginal epithelial cells, urogenital epithelial cells, gastrointestinal epithelial cells other than rectal epithelial cells, external ear epithelial cells, middle ear epithelial cells, or a combination thereof, to an effective amount of UVA.

[0041] Preferably, exposing a region of epithelial cells to a dose of UVA, or narrowband UVA, is effective to increase MAVS protein levels not only in this UVA-exposed region of the epithelial cells, but also in distal regions of the epithelial cells, including immediately adjacent but unexposed and more distal regions of the epithelial cells, and more preferably, the continuum of epithelial cells from the exposed region to the distal region (where cell-cell contact is involved) all exhibits increased MAVS protein levels.

[0042] One implementation provides that exposing (or irradiating) urethral epithelial cells in the methods disclosed herein increases MAVS protein expression in epithelial cells in the bladder of a subject.

[0043] Another implementation provides that exposing (or irradiating) vaginal epithelial cells increases MAVS protein expression in epithelial cells in the uterus of the subject.

[0044] Another implementation provides that exposing (or irradiating) urogenital epithelial cells increases MAVS protein expression in epithelial cells in the urethra or bladder of a subject.

[0045] Another implementation provides that exposing (or irradiating) rectal epithelial cells increases MAVS protein expression in epithelial cells in the rectum or colon of the subject.

[0046] Another implementation provides that exposing (or irradiating) gastrointestinal epithelial cells other than rectal epithelial cells increases MAVS protein expression in epithelial cells in the gastrointestinal tract of a subject.

[0047] Another implementation provides that exposing (or irradiating) outer ear epithelial cells increases MAVS protein expression in epithelial cells in the subject's middle or inner ear.

[0048] Yet another implementation provides that exposing (or irradiating) middle ear epithelial cells increases MAVS protein expression in epithelial cells in the inner ear of the subject.

[0049] The subject in need of the method is, in some embodiments, a mammal, including a human, experiencing a microbial infection. In some embodiments, the subject in need of the method is a mammal, including a human, at risk of developing a microbial infection, or has been exposed to or in contact with another person infected with or suspected of having a microbial infection, or has been in contact with an object in which the presence of a microorganism has been detected or is suspected of being present. In yet some other embodiments, the subject in the method exhibits one or more symptoms (or signs) of a microbial infection for 10 days or less, or for about 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or for less than 24 hours. For example, the subject may exhibit one or more symptoms (or signs) of a microbial infection for 7 days or less and is selected to be subjected to the method disclosed herein. In another embodiment, the subject may exhibit one or more symptoms (or signs) of a microbial infection for 5 days or less and is selected to be subjected to the method disclosed herein. In another embodiment, a subject may exhibit one or more symptoms (signs) of a microbial infection for three days or less and is selected to be subjected to the methods disclosed herein.

[0050] The microbial infection may be one or more of a viral infection, a bacterial infection, and a fungal infection, or may be caused by a parasite (e.g., Trichomonas vaginalis). Symptoms and signs of microbial infections are known or accessible to those skilled in the medical field. In many cases, symptoms of microbial infections may be associated with an inflammatory response.

[0051] Exemplary viral infections may be caused by or may be caused by the presence of Coxsackievirus B, coronavirus (e.g., coronavirus-229E), HIV, respiratory syncytial virus, parainfluenza virus, respiratory adenovirus, human herpes virus (HHV), herpes simplex virus (HSV), human papilloma virus (HPV). Exemplary viral infections include, but are not limited to, cold, influenza, herpes, chickenpox, mumps, HPV infection, genital herpes, genital warts, measles, and rubella.

[0052] Exemplary bacterial infections include those caused by Klebsiella pneumoniae, Escherichia coli, Clostridioides difficile, M. catarrhalis, Streptococcus pneumoniae, Haemophilus species, Streptococcus pyogenes, Staphylococcus aureus, Mycobacterium tuberculosis, Haemophilus influenza, group B Streptococcus, Staphylococcus, S saprophyticus, Proteus species, Enterococcus faecalis, and the like. faecalis, Streptococcus pneumoniae, or Salmonella. Exemplary bacterial infections include, but are not limited to, whooping cough, streptococcal pharyngitis, sinusitis, bacterial rhinosinusitis, nasal vestibulitis, folliculitis, furuncles, pneumonia, tuberculosis, ear infections, otitis media, bacterial vaginosis, chlamydia, gonorrhea, urinary tract infections (UTIs), and cystitis.

[0053] Exemplary fungal infections may be infected by or caused by the presence of Candida (e.g., Candida albicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis), Blastomyces, Cryptococcus gattii, Paracoccidioides, Coccidioides, Histoplasma, Aspergillus, or Cryptococcus neoformans. Exemplary yeast infections include, but are not limited to, athlete's foot, tinea, ringworm, yeast infections (in one or more body parts such as the vagina, mouth, throat, esophagus, ears, eyes), candidiasis, thrush, onychomycosis, pneumocystic pneumonia, mucormycosis, and talaromycosis.

[0054] In some cases, the subject of the methods disclosed herein is not infected with Klebsiella pneumoniae, Escherichia coli, Clostridioides difficile, Candida albicans, Coxsackievirus B, or coronavirus. In some cases, the subject of the methods disclosed herein is infected with a microorganism other than Klebsiella pneumoniae, Escherichia coli, Clostridioides difficile, Candida albicans, Coxsackievirus B, and coronavirus.

[0055] The methods disclosed herein for a subject in need thereof may involve the administration of another drug, such as an antibiotic, antiviral, antifungal, or painkiller to relieve pain associated with the symptoms of an infection, although in some embodiments, the step of irradiating epithelial cells with UVA or exposing a body part of a subject to UVA preferably does not require the administration of general anesthesia, regional anesthesia, local anesthesia, twilight anesthesia, or sedative. In some embodiments, the methods disclosed herein do not include the administration of anesthesia or sedative to the subject before, during, and / or after UVA exposure.

[0056] Different doses or durations of UV radiation can be administered depending on the type, severity and location of infection.For example, in some embodiments, higher intensity UVA radiation can be administered for a shorter duration, or lower intensity UVA radiation can be administered for a longer duration to achieve a dose in one type of epithelial cell, to produce a desired increase in MAVS protein level in epithelial cells.In another case, the light source can be manipulated to be placed at different distances from the target epithelial cell, based on UVA intensity and / or limited by the space in the tissue, organ or body part to be irradiated.

[0057] The UVA light source intensity should be at least 1,000 microwatts / cm, depending on the application and other factors related to treatment efficacy. 2 (1,000μW / cm 2 , i.e., 1 milliwatt / cm 2 ), 1,100 microwatts / cm 2 (i.e., 1.1 milliwatts / cm 2 ), 2,000 microwatts / cm 2 , 2,100 microwatts / cm 2 , 2,200 microwatts / cm 2 , 2,300 microwatts / cm 2 , 2,400 microwatts / cm 2 , 2,500 microwatts / cm 2 , 2,600 microwatts / cm 2, 2,700 microwatts / cm 2 , 2,800 microwatts / cm 2 , 2,900 microwatts / cm 2 , 3,100 microwatts / cm 2 , 3100 microwatts / cm 2 , 3,200 microwatts / cm 2 , 1,000 to 5,000 microwatts / cm 2 or other suitable intensity. We have found that the application of UV-A light is at least about 5,000 microwatts / cm 2 It was confirmed that the strength was safe.

[0058] Various implementations of the methods disclosed herein do not cause UV-induced damage to UV-exposed epithelial cells at least at 335-350 nm. For example, the absence of UV-induced damage can be assessed by quantifying the viable cell number or proliferation of cells after UV light exposure compared to that before exposure or compared to control epithelial cells not exposed to UV light exposure; and wherein a similar level of viable cell number or proliferation to that of control epithelial cells not exposed to UV light indicates the absence of UV-induced damage. As another example, the absence of UV-induced damage can be assessed by measuring 8-oxo-2'-deoxyguanosine (8-OHdG) in cells, and wherein a similar level of 8-OHdG in UV-exposed cells to that in cells not exposed to UV light indicates the absence of UV-induced damage. In yet another example, the absence of UV-induced damage can be assessed in vivo (or in a mammalian subject whose internal tissues have been exposed to UV light) by endoscopic evaluation for the absence of macroscopic evidence of mucosal erythema, fragility, ulceration, or bleeding, and / or by histological analysis of specimens for the absence of chronic / acute inflammation, cystitis, crypt abscesses, granulomas, ulceration, or dysplasia.Thus, in some embodiments, the methods disclosed herein further comprise a step for assessing or detecting the absence of UV-induced damage to epithelial cells exposed to UVA light and / or to distal epithelial cells not exposed to UVA light.

[0059] In some instances, the light will be delivered continuously. In other instances, the light will be incorporated into a pulsed regimen. In additional instances, the light will be delivered repeatedly, for example, for two or more consecutive exposures with a pause (no UVA exposure) between exposures.

[0060] In some embodiments, the UVA light is administered for 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, or 160 minutes, any range of minutes between 1 and 160 minutes, or other suitable time periods during each successive exposure. In some embodiments, the threshold duration of UVA exposure is at least 20 minutes. In some embodiments, the threshold duration of UVA exposure is at least 15 minutes. In some embodiments, the threshold duration of UVA exposure is at least 10 minutes. In some embodiments, the threshold duration of UVA exposure is at least 5 minutes. In some embodiments, the threshold duration of UVA exposure is at least 3 minutes.

[0061] Additionally, methods of the invention may include administering UVA light for a threshold duration of at least 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23, minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, or 60 minutes, or for a total threshold duration if two or more exposures are combined.

[0062] Some embodiments provide that the method includes exposing epithelial cells for a first period of time, and thereafter exposing epithelial cells for one or more additional periods of time, where each period of time is independently about 1-5 seconds, 5-10 seconds, 10-30 seconds, 30-60 seconds, 1-5 minutes, 5-10 minutes, 10-15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, 25-30 minutes, 30-40 minutes, 40-50 minutes, 50-60 minutes, 60-90 minutes, 90-120 minutes, or 120-240 minutes; and where the period of time and the period immediately preceding or following it can have an lapse period (wherein UVA is not administered) independently selected from seconds, minutes, hours, days, or other suitable lapse periods.

[0063] In some embodiments, UVA exposure, therapy or treatment is administered via a UVA light-emitting diode (LED)-based catheter device that can be inserted into body parts (e.g., endotracheal tube; nasopharyngeal airway; auditory tract; reproductive tract, etc.) to deliver UVA light.For example, the UVA LED-based catheter device can be controlled by the system to adjust and monitor the intensity and duration of the wavelength emitted from the UVA light source.

[0064] Additional embodiments provide that an effective amount of UVA therapy for epithelial cells is one to which the epithelial cells, upon exposure, have at least a 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% increase in MAVS protein levels compared to a baseline of the epithelial cells prior to exposure to UVA ("baseline"), or compared to a baseline of an otherwise identical epithelial cell that is not exposed to UVA. In other words, an effective amount of UVA therapy for epithelial cells is one to which the MAVS protein expression level in the epithelial cells, upon exposure, reaches a target level (e.g., is measured to be at least a target level). In some aspects, the target level is at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% higher than the baseline value of epithelial cells before exposure to UVA ("baseline") or compared to a control. In some aspects, the target level of MAVS protein expression is at least 20%, 21%, or 22% higher than the baseline value of epithelial cells before exposure to UVA ("baseline") or compared to a control.

[0065] The level of MAVS protein in epithelial cells, for example the relative level of MAVS protein after UVA exposure compared to that before UVA exposure, as well as the relative level of MAVS protein after microbial infection but before UVA exposure / therapy compared to that after UVA exposure / therapy after microbial infection, can in some embodiments be used as an indicator for evaluating the effectiveness of UVA exposure and / or determining the need for additional UVA exposure.

[0066] In some embodiments, the threshold increase in MAVS protein levels in epithelial cells (e.g., human ciliated tracheal epithelial cells) is at least 20%, 21%, or 22% after UVA exposure compared to baseline levels (i.e., before UVA exposure) or compared to otherwise identical epithelial cells that were not exposed to UVA.

[0067] In some aspects, epithelial cells subject to UVA-induced threshold increase are not infected with a microorganism or are derived from a subject that is not infected, at least in the organ in which the epithelial cells are present, where a threshold increase in MAVS protein levels can provide a prophylactic effect.

[0068] In an additional aspect, UVA-induced threshold increase is the increase in MAVS protein level when epithelial cells are infected with microorganisms.For example, the MAVS protein level in epithelial cells after UVA exposure / therapy after microorganism infection has a threshold increase compared to that in epithelial cells after microorganism infection but before UVA therapy, or compared to that in epithelial cells before microorganism infection and before UVA therapy.In this way, threshold increase can reduce apoptosis in epithelial cells infected with microorganisms and / or reduce proliferation or microbial load (e.g., viral load) in epithelial cells.

[0069] For example, 2 mW / cm 2 Human tracheal epithelial cells exposed to or irradiated with a 20 minute narrowband UVA exposure at an intensity of 2 mW / cm had, in some embodiments, an increase in MAVS protein levels of about 20%, 21%, 22%, or 23% when the cells were at 100% confluency (as shown in FIG. 1C), which mimics healthy tracheal epithelium in mammals. 2 Human tracheal epithelial cells exposed to or irradiated with a narrowband UVA exposure for 20 minutes at an intensity of 100 nm had, in some embodiments, an increase in MAVS protein levels of about 8%, 9%, or 10% when the cells were at 30-40% confluency (as shown in FIG. 1B), which mimics damaged tracheal epithelium in mammals (possibly due to a pre-existing infection or other prior damage).

[0070] Thus, in some cases, the method of prevention may include irradiating mammalian tracheal epithelial cells, or the trachea or mammal, with an amount of UVA therapy effective to increase MAVS protein levels in tracheal epithelial cells by at least 20%, 21%, 22%, or 23% before the trachea is exposed to an infectious microorganism. In some other cases, the method of prevention may include irradiating mammalian tracheal epithelial cells, or the trachea or mammal, with an amount of UVA therapy effective to increase MAVS protein levels in tracheal epithelial cells by at least 5%, 6%, 7%, 8%, 9%, or 10% before the trachea is exposed to an infectious microorganism. Optionally, the UVA intensity, duration, and peak wavelength may be adjusted to induce a target level of MAVS protein expression or a threshold increase in MAVS protein expression.

[0071] In another example, the intervention method can include irradiating mammalian tracheal epithelial cells, or trachea or mammals, with an amount of UVA therapy effective to increase MAVS protein levels in tracheal epithelial cells by at least 20%, 21%, 22%, or 23%, where the trachea has been exposed to an infectious microorganism, such that the irradiation reduces apoptosis of the infected epithelium. In yet another example, the intervention method can include irradiating mammalian tracheal epithelial cells, or trachea or mammals, with an amount of UVA therapy effective to increase MAVS protein levels in tracheal epithelial cells by at least 5%, 6%, 7%, 8%, 9%, or 10%, where the trachea has been exposed to an infectious microorganism, such that the irradiation reduces apoptosis of the infected epithelium. Optionally, the UVA intensity, duration, and peak wavelength can be adjusted to induce a target amount of increased MAVS protein expression.

[0072] In a further aspect, if the MAVS protein level falls below the target level or does not reach a threshold increase, the method may include further exposing the epithelium or epithelial cells to UVA, and optionally assaying the MAVS protein expression level from a sample of the epithelium or epithelial cells.In some implementations, if the assayed MAVS protein level continues to fall below the target level or continues to fall below a threshold increase, or if epithelial cell apoptosis continues or microbial growth continues, the method may include further exposing the epithelium or epithelial cells to UVA or to UVA of higher intensity and / or longer duration than before; and optionally treating the subject with one or more antimicrobial agents.In other implementations, if the assayed MAVS protein level reaches the target level or reaches a threshold increase, and / or epithelial cell apoptosis decreases or stops, or microbial growth decreases or stops, the method may discontinue UVA exposure, or provide the same UVA exposure as before for routine or preventive benefits.

[0073] In additional embodiments, a method for administering UVA treatment to a subject in need thereof comprises assaying MAVS protein expression levels in a biological sample obtained from a subject exposed to UVA treatment, and continuing to administer UVA treatment to the subject if MAVS protein expression falls below the subject's baseline level or compared to a control. In another embodiment, a method for administering UVA treatment to a subject in need thereof comprises assaying MAVS protein expression levels in a biological sample obtained from a subject exposed to UVA treatment, and continuing to administer UVA treatment to the subject if MAVS protein expression falls below a target level. In some implementations, the subject is exposed to a first dose of UVA treatment before being assayed from the MAVS protein expression level in the subject's biological sample, and a second dose of UVA treatment is administered (as a "continuation" administration) if the assayed MAVS protein expression falls below baseline, compared to a control, or compared to a target level; wherein the first and second doses of UVA treatment may be the same or different. For example, the second dose may be higher in intensity, duration, or both intensity and duration than the first dose.

[0074] Thus, some embodiments provide a method of evaluating UVA treatment in a subject in need thereof, comprising assaying MAVS protein expression in a biological sample obtained from a subject exposed to UVA treatment, wherein a MAVS protein expression level higher than the subject's baseline level or higher than a control level indicates that the treatment is effective. In various embodiments, the biological sample includes an epithelial cell or part of an epithelium in a respiratory cavity or tract (e.g., trachea, nasopharynx, oral cavity, bronchus), in an auditory tract or sensory epithelium, in a reproductive tract or uterine lumen epithelium or bladder, in an eye, in a rectal / colonic cavity, or another epithelium from the subject.

[0075] Techniques for assaying MAVS protein expression levels are available in the art and include, but are not limited to, based on one or more of the following: anti-MAVS antibodies or conjugated anti-MAVS antibodies with a detectable label for protein level quantification, such as Western blotting, ELISA, immunoprecipitation, isotopically labeled MAVS proteins or peptides for mass spectrometry in protein quantification, or gene transcription levels, e.g., mRNA quantification. In some implementations, protein level quantification is performed using a biopsy sample obtained from the mammal.

[0076] The control in the methods disclosed herein can be a baseline value of epithelial cells before exposure to UVA, a baseline value of epithelial cells before contact with a pathogen, or a baseline value of a population of epithelial cells that has not been exposed to the amount of UVA and is not infected with a pathogen.

[0077] Various embodiments provide a method for increasing expression of mitochondrial antiviral signaling (MAVS) protein in epithelial cells, comprising exposing the epithelial cells to an effective amount of ultraviolet A (UVA) radiation to increase expression of MAVS protein in the epithelial cells. In various aspects, the increase in MAVS protein expression is compared to those not exposed to the effective amount of UVA or compared to a control.

[0078] In some embodiments, the epithelial cells comprise or consist of tracheal epithelial cells. In some embodiments, the epithelial cells comprise or consist of ciliated epithelial cells. In some embodiments, the epithelial cells comprise or consist of ciliated epithelial cells. In some embodiments, the epithelial cells comprise or consist of one or more combinations of tracheal epithelial cells, ciliated epithelial cells, and ciliated epithelial cells. In some embodiments, the epithelial cells are human tracheal epithelial cells. In some embodiments, the epithelial cells are human lung epithelial cells. In some embodiments, the epithelial cells are human nasal epithelial cells. In some embodiments, the epithelial cells are one or more combinations of human tracheal epithelial cells, human lung epithelial cells, and human nasal epithelial cells.

[0079] Various embodiments provide a method for increasing expression of MAVS protein in a population of epithelial cells, the method comprising: (1) exposing a first portion of a population of epithelial cells to an effective amount of UVA to increase expression in the first portion of the population of epithelial cells, and (2) contacting the first portion exposed to UVA with a second portion of the population of epithelial cells that has not been exposed to UVA to increase expression in the second population.

[0080] Some embodiments provide a method for increasing expression of MAVS protein in a population of epithelial cells, the method comprising: (1) exposing a first portion of the population of epithelial cells to an effective amount of UVA; and (2) providing a second portion of the population of epithelial cells, where the second portion has not been exposed to UVA, or not exposing the second portion of the population of epithelial cells to UVA, where the first portion and the second portion are in cell-to-cell contact in the population, such that both the first portion and the second portion of the population of epithelial cells have increased expression of MAVS protein compared to those that have not been exposed to the effective amount of UVA or compared to a control.

[0081] Another embodiment provides a method for increasing expression of MAVS protein in a population of epithelial cells, the method comprising exposing a first portion of the population of epithelial cells to an effective amount of UVA, obtaining a cell lysate from the first portion of the population of epithelial cells after exposure to UVA, and exposing a second portion of the population of epithelial cells to the cell lysate obtained from the first portion of the population, where the second portion of the population has not been exposed to UVA, such that both the first and second portions of the population of epithelial cells have increased expression of MAVS protein compared to those that have not been exposed to the effective amount of UVA or compared to a control.

[0082] A further embodiment provides exposing a portion of a subject's trachea, lungs (bronchi of the lungs), or nasal cavity to an effective amount of UVA therapy by irradiating a portion of the trachea, lungs (bronchi of the lungs), or nasal cavity with UVA therapy, which results in increased expression of MAVS protein in the ciliated epithelium of the exposed portion as well as adjacent portions not exposed to UVA therapy, thereby treating, reducing the severity of, and / or reducing the risk of a respiratory microbial infection.

[0083] In some aspects, the second portion not previously exposed to UVA has a surface area that is 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-100%, 100%-200%, or 200%-300% of the size of the first portion that was exposed to UVA. In some aspects, the second portion not previously exposed to UVA has a volume of cells that is 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-100%, 100%-200%, or 200%-300% of the volume in the first portion that was exposed to UVA.

[0084] In some embodiments, an effective amount of UVA therapy is between 2 and 5 milliwatts / cm 2 (or mW / cm 2 ), 5~10mW / cm 2 , 0.5~2mW / cm 2 , or 10-20mW / cm 2In various embodiments, an effective amount of UVA therapy includes two or more doses / exposures, each administered for a period ranging from, for example, 30 seconds to 3 minutes, 3 minutes to 10 minutes, 10 minutes to 20 minutes, or 20 minutes to 30 minutes. In some embodiments, UVA therapy is exposed to the target tissue at a distance of 0-1 cm, 0-1.5 cm, 0-2 cm, 0-2.5 cm, 0-3.0 cm, 0-3.5 cm, 0-4.0 cm, 0-5.0 cm, or 0-10 cm, or other similar and suitable ranges based on the light intensity and the target pathogen. Thus, in some embodiments, exposing epithelial cells to UVA light (e.g., as a therapy, treatment, or administration) to increase expression of MAVS protein in distal epithelial cells includes increasing expression of MAVS protein in epithelial cells that are between 0-1 cm, 0-1.5 cm, 0-2 cm, 0-2.5 cm, 0-3.0 cm, 0-3.5 cm, 0-4.0 cm, 0-5.0 cm, 0-10 cm, or 0-30 cm from the UVA light exposed area / volume, or, in some cases, from the periphery of the UVA light exposed area / volume of the epithelial cells.

[0085] In some aspects, the control is a baseline value of epithelial cells prior to exposure to UVA. In some aspects, the control is a baseline value from epithelial cells prior to contact with a pathogen. In some aspects, the control is a baseline value from a population of epithelial cells that have not been exposed to the amount of UVA and that are not infected with a pathogen. EXAMPLES

[0086] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. To the extent that specific materials are mentioned, they are merely for illustration and are not intended to limit the invention. Those skilled in the art can produce equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

[0087] Example 1: Ultraviolet A light increases mitochondrial antiviral signaling proteins in confluent human tracheal cells even at a distance from the light source The mitochondrial antiviral signaling (MAVS) protein mediates innate antiviral responses, including responses to certain coronaviruses such as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We previously showed that ultraviolet A (UVA) therapy can prevent virus-induced cell death in human ciliated tracheal epithelial cells (HTEpCs) infected with coronavirus-229E, and that UVA treatment increases intracellular levels of MAVS. In this study, we sought to determine the mechanism by which UVA light can activate MAVS, and whether localized UVA light application can activate MAVS at locations distant from the light source (e.g., via cell-cell communication). MAVS levels were measured at 30-40% and 100% confluency at 2 mW / cm 2 MAVS levels were compared in HTEpCs exposed to narrow band (NB)-UVA for 20 min and in unexposed controls. MAVS levels were also compared in unexposed HTEpCs treated with supernatants or lysates from UVA-exposed cells or unexposed controls. MAVS was also assessed in different sections of confluent monolayer plates where only one section was exposed to NB-UVA. The results show that UVA increases the expression of MAVS protein. Cells in confluent monolayers exposed to UVA were able to give rise to MAVS in cells adjacent to the exposed section and even in cells in the most distant section that was not exposed to UVA. In this study, human ciliated tracheal epithelial cells exposed to UVA showed an increase in MAVS protein and appear to transmit this effect to distal confluent cells that were not exposed to light.

[0088] introduction The human body has various defense mechanisms against infections, the best known of which involves the innate immune response, in which immune cells are recruited to the site of infection via cytokine signaling. Host intracellular responses to infection are also important, especially in defense against viruses. In the past decade, it has been discovered that mitochondria can mediate innate and adaptive immune responses through several mechanisms, including the production of mitochondrial antiviral signaling (MAVS) proteins.

[0089] The MAVS protein is primarily localized to the outer membrane of mitochondria and transduces signals from cytoplasmic retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) that recognize viral RNA. Specifically, after recognition and binding of viral components, RLRs, RIG-I, and melanoma differentiation-associated gene 5 (MDA5) interact with MAVS and activate transcription factors that induce the expression of proinflammatory and antiviral genes. However, some viruses have developed mechanisms to antagonize MAVS activation and evade this innate immune response. For example, the SARS-CoV-2 transmembrane glycoprotein M is thought to antagonize MAVS and thus impair the MAVS-mediated innate antiviral response.

[0090] We have recently shown that application of UVA light under certain conditions to human ciliated tracheal epithelial cells infected with CoV-229E significantly improved cell viability and prevented virus-induced cell death, which was accompanied by a reduction in the levels of CoV-229E spike (S) protein. Furthermore, cells treated with UVA light showed significantly increased levels of MAVS protein, indicating that UVA can activate MAVS. Furthermore, in a first-in-human clinical trial in ventilated subjects with coronavirus disease 2019 (COVID-19), daily 20-min intratracheal UVA treatment for 5 days significantly reduced respiratory SARS-CoV-2 viral load. Interestingly, despite time-limited topical UVA therapy in this study, the mean log intratracheal viral load from baseline to day 6 was significantly reduced by 1.5%. 10The change was -3.2, indicating a potential antiviral phenomenon beyond the immediate local effect.

[0091] In this study, we explore the effect of narrowband (NB)-UVA light on MAVS expression in uninfected human ciliated tracheal epithelial cells in vitro. We also explore whether the effect of UVA light is restricted to cells directly exposed to UVA or is also seen in cells that were not directly exposed to UVA.

[0092] Materials and Techniques NB-UVA effect on MAVS Primary human tracheal epithelial cells (HTEpC, lot no. 454Z019.11, PromoCell GmbH, Heidelberg, Germany) isolated from the surface epithelium of the human trachea were cultured at 37°C (5% CO2) in 60x15mm standard tissue culture dishes (cat. 351007, Corning, NY, USA) containing Airway Epithelial Cell Growth Medium (cat. C-21060, PromoCell) prepared with SupplementMix (cat. C-39165, PromoCell) and Gibco antibiotic-antimycotic solution (cat. 15240096, ThermoFisher Scientific, MA, USA).

[0093] 10 cells per plate 5 Once the cells reached confluency (30–40%), the HTEpCs were washed three times with sterile 1x PBS pH 7.4 (cat. 10010072, ThermoFisher) and fresh medium was added to each plate. The cells were incubated at 2 mW / cm 2 UVA light, based on previously validated ideal UVA irradiation levels. 2The cells were exposed to 100 µL of NB-UVA for 20 min. Unexposed cells were used as control. After 24 h, the supernatant was collected and the cells were washed three times with sterile 1x PBS, pH 7.4. After removing the residual PBS, the cells were lysed in the plate using 1 mL of RTL buffer from the AllPrep DNA / RNA / Protein Isolation Kit (Qiagen, Hilden, Germany). Experiments were performed in triplicate.

[0094] NB-UVA effects on MAVS signaling to unexposed UVA-naive cells To determine whether MAVS activation caused by exposure to NB-UVA light could be transmitted to naive unexposed HTEpCs and begin to elucidate the mechanisms involved, three experiments were performed: - To determine whether extracellular mediators were involved, supernatants from 30-40% confluent HTEpCs exposed to NB-UVA were transferred to 30-40% confluent naive HTEpCs. - To determine whether intracellular mediators were involved, cell lysates (after removal of supernatant) from 30-40% confluent HTEpCs exposed to NB-UVA were transferred to 30-40% confluent naive HTEpCs. - To determine whether cell-cell signaling was involved, we analyzed areas of 100% confluent HTEpCs that were or were not exposed to NB-UVA.

[0095] NB-UVA effects on MAVS signaling via extracellular mediators Supernatants collected from UVA-exposed and control HTEpCs from the previous experiment were cultured at 10 5100 mL of naïve HTEpCs (i.e., cells that were not exposed to UVA) were transferred to a new 60x15mm tissue culture dish containing 100 mL of naïve HTEpCs (i.e., cells that were not exposed to UVA). Naïve HTEpCs were washed three times with sterile 1x PBS, pH 7.4, before receiving supernatant from UVA-exposed or control cells. The PBS was completely removed, and 4 mL of supernatant collected from UVA-exposed or control HTEpCs was added to the naïve cells. After 24 hours of incubation, the cells were washed three times and then lysed in the plate using 1 mL of RTL buffer from the AllPrep DNA / RNA / Protein Isolation Kit (Qiagen). Experiments were performed in triplicate.

[0096] NB-UVA effects on MAVS signaling via intracellular mediators HTEpCs were cultured at 37°C (5% CO2) in 60x15mm standard tissue culture dishes (cat. 351007, Corning, NY, USA) in airway epithelial cell growth medium (cat. C-21060, PromoCell) containing SupplementMix (cat. C-39165, PromoCell) and Gibco antibiotic-antimycotic solution (cat. 15240096, ThermoFisher Scientific, MA, USA).

[0097] 10 cells per plate 5 Once the cells reached confluency (30–40%), the HTEpCs were washed three times with sterile 1x PBS pH 7.4 (cat. 10010072, ThermoFisher) and fresh medium was added to each plate. The cells were incubated at 2 mW / cm 2 The cells were exposed to 100 µL of NB-UVA for 20 min. Unexposed cells were used as a control. After 24 h, cells were washed three times with sterile 1x PBS, pH 7.4, scraped off the culture dish, and transferred to a 15 mL sterile tube. Cells were pelleted and new fresh airway epithelial cell growth medium was added. Cells were lysed by adding a single sterile 5 mm stainless steel bead (Qiagen) to each tube and vortexing the tubes for 5 min. Lysates from UVA-exposed and control HTEpCs were diluted with 10 µL of NB-UVA for 20 min.5 100 mL of naïve HTEpCs (i.e., HTEpCs that had never been exposed to UVA) were transferred to a new 60x15mm tissue culture dish containing 100 mL of naïve HTEpCs (i.e., HTEpCs that had never been exposed to UVA). The naïve HTEpCs were washed three times with sterile 1x PBS, pH 7.4, before receiving lysates from UVA-exposed or control cells. The PBS was completely removed, and 4 mL of lysate from either UVA-exposed or control HTEpCs was added to the naïve cells. After 24 hours of incubation, the cells were washed three times with sterile 1x PBS and then lysed in the plate using 1 mL of RTL buffer from the AllPrep DNA / RNA / Protein Isolation Kit (Qiagen). Experiments were performed in quadruplicate.

[0098] NB-UVA effect on MAVS signaling via cell-cell signaling HTEpCs were cultured at 37°C (5% CO2) in 150 mm dishes (cat. 430599, Corning) containing airway epithelial cell growth medium (cat. C-21060, PromoCell) prepared with SupplementMix (cat. C-39165, PromoCell) and Gibco antibiotic-antimycotic solution (cat. 15240096, ThermoFisher) until they reached 100% confluence.

[0099] On the day of NB-UVA therapy, cells were washed twice with sterile 1x PBS, pH 7.4, and fresh medium was added. Each 150 mm dish containing a 100% confluent monolayer of HTEpCs was divided longitudinally into four sections, designated as regions 1, 2, 3, and 4, respectively (Figure 1A). The NB-UVA light-emitting device was placed 2.3 cm from the bottom of the dish and emitted approximately 2 mW / cm. 2 of NB-UVA was applied for 20 min to area 1. The experiment was performed four times.

[0100] To prevent UVA leakage to other parts of the plate during therapy, regions 2, 3, and 4 were covered with a sterile barrier that blocked the passage of light through the top and sides of the plate. During the course of therapy, NB-UVA intensity was constantly checked in unexposed regions (top, bottom, and sides) of the culture plate using a UV meter (SDL470, Extech, NH) to ensure that these regions were free of UVA light. UVA-treated plates were then re-incubated at 37° C. (5% CO2) for 24 hours.

[0101] The UVA-treated HTEpC plate was washed three times with sterile 1x PBS, pH 7.4, before harvesting the cells. 10mL of sterile 1x PBS, pH 7.4 was added to the plate and the cells from region 4 were carefully scraped with a sterile Corning Cell Lifter (cat. 3008, Corning) and immediately transferred to a 15mL sterile tube. The cells were pelleted at low speed (approximately 1000 RPM) and lysed in 1mL RTL buffer from the AllPrep DNA / RNA / Protein Isolation Kit (Qiagen).

[0102] The remaining UVA-exposed HTEpCs (still attached to the plate) from regions 1, 2, and 3 were washed three times with sterile 1x PBS, pH 7.4. 10 mL of sterile 1x PBS, pH 7.4 was added to the plate and cells from region 3 were carefully scraped off and lysed as above. The same process was used to harvest cells from regions 2 and 1 (in that order).

[0103] Protein extraction and western blotting Total protein was extracted from UVA-exposed and non-exposed HTEpCs from all experiments using the AllPrep DNA / RNA / Protein Mini Kit (Qiagen) according to the manufacturer's protocol. Total protein was quantified using Qubit Protein Assays (ThermoFisher) and equal loading of total protein was separated on NuPAGE 4-12% Bis-Tris minigels (NP0336BOX, ThermoFisher) and then transferred onto Biotrace NT nitrocellulose membranes (27376-991, VWR). Total protein was stained with Ponceau S solution (P7170, Sigma-Aldrich). The membrane was blocked with Tris-buffered saline (TBS-T) containing 3% bovine serum albumin (cat. A7030, Sigma-Aldrich) and 0.1% Tween 20 (P1379, Sigma-Aldrich) and incubated overnight at 4°C with mouse anti-MAVS antibody (1:200; SC-166583, Santa Cruz Biotechnology) diluted in blocking solution. After washing in TBS-T, the membrane was then covered with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (1:300; 5220-0286, SeraCare), washed in TBS-T, and exposed to enhanced chemiluminescence solution (RPN2235, GE Healthcare). Immunoreactive protein bands were imaged using an iBright FL1500 instrument (ThermoFisher) and analyzed using iBright analysis software (ThermoFisher). Samples were normalized to total protein determined from Ponceau S staining (MilliporeSigma, St. Louis, MO, US).

[0104] statistical analysis Graph construction and statistical analysis were performed with GraphPad Prism V. 9 (GraphPad Software, CA, USA). For all experiments, immunoreactive MAVS bands from nitrocellulose membranes were normalized to total protein (Ponceau S) before statistical analysis using iBright analysis software (ThermoFisher). MAVS relative densities (obtained after normalization) were compared between groups applying unpaired t-tests. Comparisons between each area from experiments with 100% confluent cell cultures were performed using paired t-tests and ANOVA tests. The significance level was set at p<0.05.

[0105] result Narrow-band UVA (NB-UVA) increases MAVS protein levels in human non-confluent and confluent ciliated tracheal epithelial cells 2mW / cm 2 We analyzed levels of MAVS in primary tracheal epithelial cells (HTEpCs) at 30-40% confluency exposed to NB-UVA for 20 min, and in unexposed controls. Normalized MAVS levels, detected by Western blot, were increased in NB-UVA-exposed cells compared to unexposed controls (P=0.0193, Figure 1B).

[0106] In addition, when primary tracheal epithelial cells were grown to 100% confluent monolayers (as opposed to 30-40% confluency), the levels of 2 mW / cm were observed when compared to levels in unexposed monolayers. 2 After 20 min of exposure to NB-UVA, normalized MAVS levels in area 1 were also significantly increased (P=0.0006, Figures 1C, 1J).

[0107] MAVS is activated by cell-cell signaling after NB-UVA exposure When naive 30-40% confluent HTEpCs were treated with supernatant from NB-UVA-exposed 30-40% confluent HTEpCs, no change in MAVS levels was observed (P = 0.4022, Figure 1D, 1E). However, when naive 30-40% confluent HTEpCs were incubated with cell lysates from NB-UVA-exposed 30-40% confluent HTEpCs, there was a trend toward increased normalized levels of MAVS (Figure 1F, 1G, P = 0.1256).

[0108] Next, only a part of the plate (area 1) is powered at 2 mW / cm 2 After 20 min exposure to NB-UVA, the levels of MAVS in different regions of a culture plate containing a 100% confluent monolayer of HTEpCs were analyzed (Figure 1A). Normalized MAVS levels increased gradually from region 4 (the furthest unexposed region) to region 1 (exposed to NB-UVA) (ANOVA P=0.08, Figures 1H, 1I), with a statistically significant increase in MAVS levels in region 1 (exposed to NB-UVA) when compared to unexposed region 4 (P=0.0382, Figures 1H, 1I). Importantly, the levels of MAVS were also significantly increased in unexposed regions 2 and 3 when compared to the control from an unexposed plate (P=0.0289 and P=0.0402, respectively, Figure 1H). Normalized MAVS levels in region 4 (the furthest unexposed region) also appeared to be higher than in the control, but did not reach statistical significance (P=0.1262, Figure 1H).

[0109] In this study, we show that narrowband UVA light increases the expression of MAVS protein in uninfected human ciliated tracheal epithelial cells in vitro. In addition, in confluent monolayer cultures of these cells, induction of MAVS protein is transmitted to cells that were not directly exposed to NB-UVA light. This transmission does not appear to be due to secreted extracellular mediators, but likely results from direct cell-cell signaling and possibly cytoplasmic mediators.

[0110] External UVA therapy has long been used in the treatment of skin conditions such as psoriasis, eczema, and cutaneous lymphoma, for which it has received FDA approval. To explore the potential of internal UVA light therapy to treat microbial infections, we recently tested the efficacy of UVA against various pathogens in vitro and found that under controlled and monitored conditions, UVA light effectively reduced various bacterial species (including Klebsiella pneumoniae, Escherichia coli, Clostridioides difficile, etc.), yeast Candida albicans, Coxsackievirus group B, and coronavirus-229E. Importantly, we found that human ciliated tracheal epithelial cells infected with coronavirus-229E and then treated by NB-UVA light in vitro showed increased MAVS protein and survived infection. These results indicated that the increased cell viability of coronavirus-229E-infected and UVA-treated cells compared to infected but untreated controls could be attributed to the activation of MAVS-mediated antiviral signaling pathways. In this study, human ciliated tracheal epithelial cells were exposed to UVA light without viral infection, and the results confirmed that exposure to UVA light alone increased the levels of MAVS protein in these cells, demonstrating that this was a response to UVA light.

[0111] It is well recognized that the common cold, influenza, and other viruses are seasonal, occurring more frequently in the winter and less frequently in the summer. Although the mechanism for this is unclear, data indicate that sunlight and vitamin D production may be important. Sunlight has historical importance in medicine - for example, during the 1918-1919 H1N1 influenza pandemic, it was shown that the combination of sunlight and access to fresh air, with strict hygiene and the use of face masks, may have reduced mortality among patients and staff at a "field" hospital in Boston. A systematic review of data on vitamin D levels and the current COVID-19 pandemic indicates that sunlight and increased vitamin D levels may improve outcomes. Although trials selected for inclusion in later studies had heterogeneous results, these and other past data indicate that exposure to sunlight, and therefore UVA, may be beneficial in fighting viral infections.

[0112] Under normal physiological conditions, MAVS protein levels are low, due in part to binding of human antigen R and microRNAs to elements in the 3'UTR of MAVS mRNA. Following recognition and binding of viral components, the N-terminal caspase recruitment domain (CARD) of RIG-I-like receptors (RLRs) is ubiquitinated and binds to the CARD of MAVS, leading to MAVS aggregation and activation of inflammatory cytokines and antiviral interferon genes. However, viruses can also circumvent these pathways - for example, the membrane glycoprotein M of SARS-CoV-2, the virus that causes COVID-19, can interact with MAVS and impair MAVS aggregation and activation of antiviral responses. In our preclinical studies, tracheal cells infected with CoV-229E and treated with UVA light also showed a reduction in CoV-229E spike protein, indicating to us that UVA light may also be an effective treatment for SARS-CoV-2.

[0113] The primary site of SARS-CoV-2 infection is the ciliated epithelial cell, with characteristic bilateral ground-glass opacities downstream. Acute respiratory viral infection and subsequent inflammatory response can result in decline of lung function and death. Secondary bacterial and fungal infections are also common, and ventilator-associated pneumonia (VAP) occurs in 31% of mechanically ventilated patients. To test the safety and efficacy of UVA light as a potential treatment for SARS-CoV-2, we developed a novel UVA light-emitting diode (LED)-based catheter device that can be inserted into an endotracheal tube to deliver UVA light to severely ill COVID-19 subjects. In a first-in-human study of mechanically ventilated COVID-19 subjects, all of whom had a World Health Organization (WHO) symptom severity score of 9 at baseline (10 being death), subjects treated with endotracheally delivered UVA light (treated daily for 20 min for 5 days) had a mean log 2.0 SARS-CoV-2 viral load of 3.2 in endotracheal aspirates by day 6 of therapy. 10 (p<0.001), and these accelerated reductions in viral load correlated with 30-day improvements in WHO symptom severity scores. Furthermore, despite the fact that only a small portion of the trachea was exposed to UVA light, the magnitude of improvement suggested the possibility that the antiviral effects of UVA light are not limited to cells directly exposed to UVA, but may be transmitted to neighboring cells.

[0114] To explore the potential mechanisms underlying this transfer, we first harvested supernatants from UVA-exposed cells and added them to fresh plates of cells that had not been exposed to UVA light. No increase in MAVS protein levels was observed in these cells, indicating that secreted extracellular mediators were not involved. Next, to explore whether cytoplasmic mediators were involved, we lysed UVA-exposed cells and unexposed controls and added the lysates to fresh plates of cells that had not been exposed to UVA light. There was a trend toward increased MAVS protein levels in naive HTEpCs incubated with lysates from UVA-exposed cells, but this did not reach significance. In contrast, when comparing MAVS levels in confluent monolayers of HTEpCs directly exposed to UVA light and in adjacent regions from the same plates that were shielded from UVA light, we found that MAVS was not only increased in cells in region 1 (directly exposed to UVA light), but also in cells in adjacent regions 2, 3, and 4 that were shielded from direct UVA light, with a gradient that decreased with increasing distance from the UVA-exposed cells. These findings confirm that the increase in MAVS in response to UVA light can be transmitted from directly exposed cells to adjacent unexposed cells, indicating that intercellular signaling is involved, although further studies are needed to determine the mechanisms involved.

[0115] SARS-CoV-2 inhibits MAVS, but UVA light exposure is shown here to override this inhibition, and the mechanisms by which UVA light overrides this inhibition and potentially further damage to single-stranded viral RNA may be further explored. This effect of MAVS activation may be important to study in in vivo models. Limited data indicate that MAVS and the resulting intracellular production of interferon-α may attract circulating immune cell responses to attack infected cells. Interestingly, in our previous in vitro studies, CoV-229E caused rapid cell death, which was mitigated by UVA. This increase in cell survival perhaps indicates that MAVS is a cell salvage pathway. This is also supported by the first human study of UVA in intubated critically ill subjects with COVID-19. Two patients underwent bronchoscopy after 5 days of UVA application. There was no macroscopic evidence of inflammation. Further studies are needed to explore these concepts, which were not addressed in this study.

[0116] In conclusion, this study begins to elucidate a possible mechanism by which UVA light can affect innate intracellular immunity. The data herein show that NB-UVA increases MAVS protein levels in human ciliated tracheal epithelial cells. This increase in MAVS protein appears to be transmissible to neighboring cells that were not directly exposed to UVA light. Furthermore, our results indicate that MAVS signaling involves cell-cell communication and possibly cytoplasmic (but not secreted extracellular) mediators. This finding may be the basis for the benefits of UVA seen in vitro and in human studies of critically ill patients with COVID-19. This finding may have broad implications for the treatment of SARS-CoV-2, other coronaviruses, and other RNA respiratory viruses such as influenza. Further studies are needed to determine whether this mechanism is an important factor in the seasonality of certain respiratory viral diseases.

[0117] Example 2. Study of NB-UVA effects on mitochondrial antiviral signaling (MAVS) protein Primary human tracheal epithelial cells (HTEpC, lot no. 454Z019.11, PromoCell GmbH, Heidelberg, Germany) isolated from the surface epithelium of the human trachea were cultured at 37°C (5% CO2) in 60x15mm standard tissue culture dishes (cat. 351007, Corning, NY, USA) containing Airway Epithelial Cell Growth Medium (cat. C-21060, PromoCell) prepared with SupplementMix (cat. C-39165, PromoCell) and Gibco antibiotic-antimycotic solution (cat. 15240096, ThermoFisher Scientific, MA, USA).

[0118] 10 cells per plate 6 Upon reaching the cells, HTEpC cultures were washed three times with sterile 1x PBS, pH 7.4 (cat. 10010072, ThermoFisher) and fresh medium was added to each plate. Cells were incubated with 0 (control) or 2000 μW / cm for 20 min every 24 h for 1, 2, and 3 days. 2 NB-UVA. 24 h after the last day of UVA treatment, the supernatant was collected and the cell cultures were washed three times with sterile 1x PBS, pH 7.4. After removing the residual PBS, the cells were lysed in the plate using 1 mL of RTL buffer from the AllPrep DNA / RNA / Protein Isolation Kit (Qiagen, Hilden, Germany).

[0119] Protein extraction and western blotting Total protein was extracted from UVA-exposed and unexposed tracheal cells using the AllPrep DNA / RNA / Protein Mini Kit (Qiagen) according to the manufacturer's protocol. Proteins were loaded onto NuPAGE 4-12% Bis-Tris minigels (NP0336BOX, ThermoFisher) and transferred onto Biotrace NT nitrocellulose membranes (27376-991, VWR). Total protein was stained with Ponceau S solution (P7170, Sigma-Aldrich). Membranes were blocked with Tris-buffered saline (TBS-T) containing 3% bovine serum albumin (cat. A7030, Sigma-Aldrich) and 0.1% Tween 20 (P1379, Sigma-Aldrich) and incubated overnight at 4°C with mouse anti-MAVS antibody (1:200; SC-166583, Santa Cruz Biotechnology) diluted in blocking solution. After washing in TBS-T, the membrane was then covered with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (1:300; 5220-0286, SeraCare), washed in TBS-T, and exposed to enhanced chemiluminescence solution (RPN2235, GE Healthcare). Immunoreactive protein bands were imaged using an iBright FL1500 instrument (ThermoFisher).

[0120] NB-UVA effect on MAVS via secreted molecules Supernatants collected from UVA-exposed and control HTEpC cells were cultured at 10 6 The naïve HTEpCs were then transferred to a new 60x15mm tissue culture dish containing 1000 pieces of UVA-unexposed HTEpCs (naïve). The naïve HTEpCs were washed three times with sterile 1x PBS, pH 7.4, before receiving the supernatant from UVA-exposed or control cells. The PBS was completely removed, and 4mL of the supernatant collected from the UVA-exposed or control HTEpC cells was added to the naïve cells. After 24h of incubation, the cells were washed three times and proteins were extracted and analyzed as described above.

[0121] NB-UVA effect on MAVS via intercellular signaling HTEpCs were cultured at 37°C (5% CO2) in 150 mm dishes (cat. 430599, Corning) containing airway epithelial cell growth medium (cat. C-21060, PromoCell) prepared with SupplementMix (cat. C-39165, PromoCell) and Gibco antibiotic-antimycotic solution (cat. 15240096, ThermoFisher) until they reached 100% confluence.

[0122] On the day of NB-UVA therapy, cells were washed twice with sterile 1x PBS, pH 7.4, and fresh medium was added. Each 150 mm dish containing a monolayer of HTEpC cells was divided longitudinally into four sections, designated as areas 1, 2, 3, and 4, respectively (Figure 1A). The NB-UVA light-emitting device was placed 2.3 cm into the bottom of the dish, delivering approximately 2,000 µW / cm. 2 of NB-UVA was applied to area 1 for 20 minutes.

[0123] To avoid UVA leakage to other parts of the plate during therapy, regions 2, 3, and 4 were covered with a sterile device that blocked the passage of light through the top and sides of the plate (Figure 1B). During the course of therapy, NB-UVA intensity was constantly checked in unexposed regions (top, bottom, and sides) of the culture plate using a UV meter (SDL470, Extech, NH) to ensure that these regions were free of UVA light. The UVA-treated plates were then re-incubated at 37°C (5% CO2) for 24 hours.

[0124] The UVA-treated HTEpC plate was washed three times with sterile 1x PBS, pH 7.4, before harvesting the cells. 10mL of sterile 1x PBS, pH 7.4 was added to the plate and the cells from region 4 were carefully scraped with a sterile Corning Cell Lifter (cat. 3008, Corning) and immediately transferred to a 15mL sterile tube. The cells were pelleted at low speed (approximately 1000 RPM) and lysed in 1mL RTL buffer from the AllPrep DNA / RNA / Protein Isolation Kit (Qiagen).

[0125] The remaining UVA-exposed HTEpC cells (still attached to the plate) from regions 1, 2, and 3 were washed three times with sterile 1x PBS, pH 7.4. 10 mL of sterile 1x PBS, pH 7.4 was added to the plate and cells from region 3 were carefully scraped off and lysed as above. The same process was used to harvest cells from regions 2 and 1 (in that order). Total protein was extracted and analyzed using an AllPrep DNA / RNA / Protein Mini Kit (Qiagen).

[0126] Example 3. Narrow-band UVA light (NB-UVA) increases MAVS levels in human ciliated tracheal epithelial cells 20 minutes per treatment, 2mW / cm 2 NB-UVA (1-3 times) or 5mW / cm 2 MAVS levels were analyzed in primary tracheal epithelial (HTEpC) cells at 30–40% confluency exposed to 5 mW / cm (once). Normalized MAVS levels, as detected by Western blot, were calculated as 100% for cells at 30–40% confluency exposed to 5 mW / cm (once). 2 When these cells were treated with NB-UVA light at 2 mW / cm (P = 0.0026), 2 No effect was observed when exposed to NB-UVA (P>0.05, Figure 2).

[0127] In contrast, when primary tracheal epithelial cells were grown to a 100% confluent monolayer (as opposed to 30-40% confluency), normalized MAVS levels were 2 mW / cm when compared to levels in unexposed monolayers. 2 There was a significant increase after just a single 20 min treatment with NB-UVA (P=0.0079, Figures 3A and 3B).

[0128] Example 4. MAVS is activated by intercellular signaling after NB-UVA exposure To determine whether MAVS activation caused by exposure to NB-UVA light could be transmitted to naive unexposed HTEpC cells and to begin to elucidate the mechanisms involved, three experiments were performed: (1) To determine whether secreted factors were involved, supernatants from confluent NB-UVA-exposed HTEpC cells were transferred to naive HTEpC cells; (2) to determine whether cytoplasmic factors were involved, cell lysates (after removal of the supernatant) from exposed NB-UVA HTEpC cells were transferred to naive HTEpC cells; and (3) To determine whether cell-cell signaling was involved, regions of cells that were or were not exposed to NB-UVA were analyzed from culture plates, where a portion of the plate was exposed to NB-UVA (Figure 1A).

[0129] No change in MAVS levels was observed when supernatants from NB-UVA-exposed HTEpC cells were transferred to naive HTEpC cells, however, normalized levels of MAVS tended to increase in naive HTEpC cells incubated with cell lysates prepared from NB-UVA-exposed HTEpC cells (Figures 4A and 4B, P = 0.2787).

[0130] Next, only a part of the plate (area 1) is powered at 2 mW / cm 2After 20 min exposure to NB-UVA, the levels of MAVS in different regions of a culture plate containing a 100% confluent monolayer of HTEpC cells were analyzed (Figure 1A). Normalized MAVS levels appeared to increase gradually from region 4 (the furthest unexposed region) to region 1 (exposed to NB-UVA) (ANOVA P=0.07, Figures 5A and 5B). MAVS levels were increased in region 1 (exposed to NB-UVA) when compared to unexposed region 3 (P=0.04) and to a lesser extent when compared to unexposed regions 2 and 4 (P=0.1329 and P=0.1068, respectively) (Figure 4A). Importantly, the levels of MAVS also appeared to be increased in unexposed region 2 when compared to the control from an unexposed plate (Figure 5A), although the P value only reached statistical significance when one outlier was omitted from the analysis (P=0.02).

[0131] In this study, the data show that UVA light increases the expression of MAVS protein in human ciliated tracheal epithelial cells in vitro. In addition, in confluent monolayers of these cells, induction of MAVS protein is transmitted to cells that were not exposed to UVA light. This transmission does not appear to be due to factors secreted into the medium, but likely results from direct cell-cell signaling, and possibly also from cytoplasmic factors.

[0132] External UVA therapy has long been used in the treatment of skin conditions such as psoriasis, eczema, and cutaneous lymphoma, for which it has received FDA approval. To explore the potential of internal UVA light therapy to treat microbial infections, we recently tested the efficacy of UVA against various pathogens in vitro and found that under controlled and monitored conditions, UVA light effectively reduced various bacterial species (including Klebsiella pneumoniae, Escherichia coli, Clostridioides difficile, etc.), yeast Candida albicans, Coxsackievirus group B, and coronavirus-229E. Importantly, we found that human ciliated tracheal epithelial cells infected with coronavirus-229E and then treated by UVA light in vitro showed an increase in MAVS protein. These results indicated that the increased cell viability of coronavirus-229E-infected and UVA-treated cells compared to infected but untreated controls could be attributed to the activation of MAVS-mediated antiviral signaling pathways. In this study, human ciliated tracheal epithelial cells were exposed to UVA light without viral infection, and the results confirmed that exposure to UVA light alone increased the levels of MAVS protein in these cells, confirming that this was a response to UVA light.

[0133] The common cold, influenza, and other viruses appear to be seasonal; in particular, they occur more frequently in the winter and less frequently in the summer. Although the mechanism is unclear, data indicate that sunlight and vitamin D production may be important. Sunlight has historical importance in medicine - for example, during the 1918-1919 H1N1 influenza pandemic, it was shown that the combination of sunlight and access to fresh air, with strict hygiene and the use of face masks, may have reduced mortality among patients and staff at a "field" hospital in Boston. A systematic review of data on vitamin D levels and the current COVID-19 pandemic indicates that sunlight and increased vitamin D levels may improve outcomes. Although trials selected for inclusion in later studies have had mixed results, these and other past data indicate that exposure to sunlight, and therefore UV exposure, may be beneficial in fighting viral infections.

[0134] Under normal physiological conditions, MAVS protein levels are low, due in part to the binding of human antigen R and microRNAs to elements in the 3'UTR of MAVS mRNA. Following recognition and binding of viral components, the N-terminal caspase recruitment domain (CARD) of RLR is ubiquitinated and binds to the CARD of MAVS, leading to MAVS aggregation and activation of inflammatory cytokines and antiviral interferon genes. However, viruses can also circumvent these pathways - for example, the membrane glycoprotein M of SARS-CoV-2, the virus that causes COVID-19, can interact with MAVS and impair MAVS aggregation and activation of antiviral responses. In our preclinical studies, tracheal cells infected with coronavirus-229E and treated with UVA light also showed a reduction in coronavirus-229E spike protein, indicating to us that UVA light can also be an effective treatment for SARS-CoV-2.

[0135] The primary site of SARS-CoV-2 infection is the lungs, which show characteristic bilateral ground-glass opacities, and acute respiratory viral infection and subsequent inflammatory responses can lead to decline in lung function and death. Secondary bacterial and fungal infections are also common, and ventilator-associated pneumonia (VAP) occurs in 31% of mechanically ventilated patients. To test the safety and efficacy of UVA light as a potential treatment for SARS-CoV-2, we developed a novel UVA LED-based catheter device that can be inserted into an endotracheal tube to deliver UVA light to severe COVID-19 subjects. In a small pilot study of mechanically ventilated COVID-19 subjects, all of whom had a World Health Organization (WHO) symptom severity score of 9 at baseline (10 being death), subjects treated with endotracheally delivered UVA light (treated for 20 min daily for 5 days) showed a mean log10 reduction in SARS-CoV-2 viral load of 3.2 in endotracheal aspirates by day 6 of therapy (p<0.001), and these reductions in viral load correlated with improvements in WHO symptom severity scores. Furthermore, despite the fact that only a small portion of the trachea was exposed to UVA light, the magnitude of improvement suggested the possibility that the effects of UVA light were not limited to cells directly exposed to UVA, but could be transmitted to neighboring cells. In this study, we found that exposure of cells at 30-40% confluency to NB UVA light at lower UVA intensities (2 mW / cm 2 ), no increase in MAVS was observed, and a higher UVA intensity (5 mW / cm) was observed before an increase in MAVS was detected. 2 In contrast, when cells were grown in confluent monolayers, MAVS levels were suppressed by lower intensities of UVA light (2 mW / cm 2 ), which supported the hypothesis that the increase in MAVS in response to UVA light could be transmitted to other cells.

[0136] To explore the potential mechanisms underlying this transfer, we first harvested supernatants from UVA-exposed cells and added them to fresh plates of cells that were not exposed to UVA light. No increase in MAVS expression levels was observed in these cells, indicating that secreted factors were not involved. Next, to explore whether cytoplasmic factors were involved, we lysed UVA-exposed cells and unexposed controls and added the lysates to fresh plates of cells that were not exposed to UVA light. There was a trend toward increased MAVS in naive HTEpC cells incubated with lysates from UVA-exposed cells, but this did not reach significance in this study. When comparing MAVS levels in HTEpC cells directly exposed to UVA light and in adjacent regions from the same plate that were shielded from UVA light, we found that MAVS was not only increased in cells in region 1 (directly exposed to UVA light), but also in cells in adjacent regions 2, 3, and 4 that were shielded from direct UVA light, with a decreasing gradient with increasing distance from the UVA-exposed cells. These findings confirm that the increase in MAVS in response to UVA light can be transmitted from directly exposed cells to adjacent unexposed cells, indicating that intercellular signaling is involved, although further studies are needed to determine the mechanisms involved.

[0137] UVA may have the potential to enhance innate cellular immunity against viruses. For example, SARS-CoV-2 suppresses MAVS, and therefore it will be important to understand the mechanism by which UVA light overrides this suppression. This may include damage to single-stranded viral RNA. In addition, the effect of this MAVS activation may be important to study in in vivo models. Limited data indicate that MAVS and the resulting intracellular production of interferon-gamma may attract circulating immune cell responses to attack infected cells. Interestingly, in our previous in vitro studies, coronavirus 229E caused rapid cell death, which was mitigated by UVA. This cell salvage indicates that perhaps MAVS is a cell salvage pathway (not cell lysis). This is also supported by the first human study of UVA in intubated critically ill subjects with COVID-19. Two patients underwent bronchoscopy after 5 days of UVA application. There was no macroscopic evidence of inflammation of cell detachment.

[0138] In conclusion, this study begins to elucidate a possible mechanism by which UVA light can affect innate cellular immunity. In this study, UVA appears to increase the expression of MAVS in human ciliated tracheal epithelial cells. This expression appears to be transmissible to neighboring cells that were not exposed to light. Furthermore, our results indicate that this transmission of increased MAVS involves cell-to-cell communication and possibly cytoplasmic (but not secreted) factors. This finding may support the benefits of UVA seen in vitro and in human studies of critically ill patients with COVID-19. This finding may have broad implications for the treatment of SARS-CoV-2, other coronaviruses, and other RNA respiratory viruses such as influenza. Further studies are needed to determine whether this mechanism is an important factor in the seasonality of certain respiratory viral diseases.

[0139] Various aspects of the present invention have been described in the above detailed description. Although these descriptions directly describe the above aspects, it is understood that those skilled in the art may conceive of modifications and / or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the scope of this description are intended to be included therein as well. Unless otherwise specified, it is the intention of the inventors that the words and phrases in this specification and claims are given the ordinary and familiar meaning to those skilled in the applicable technical field.

[0140] The foregoing description of the various aspects of the present invention known to the applicant at the time of filing this application has been presented and is intended for purposes of illustration and description. This description is not intended to be exhaustive or to limit the invention to the precise form disclosed, as many modifications and variations are possible in light of the above teachings. The described aspects serve to explain the principles of the invention and its practical application, and to enable those skilled in the art to utilize the invention in various aspects and with various modifications suited to the particular use contemplated. Therefore, it is not intended that the invention be limited to the specific aspects disclosed for carrying out the invention.

[0141] While particular embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that, based on the teachings herein, changes and modifications may be made without departing from the present invention and its broader aspects, and therefore, the appended claims will encompass within their scope all such changes and modifications that are within the true spirit and scope of the present invention. In general, it will be understood by those skilled in the art that the terms used herein are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "comprising" should be interpreted as "including but not limited to," etc.).

[0142] As used herein, the term "comprising" or "comprises" is used in reference to compositions, methods, and each component(s) thereof that are useful in an embodiment, but is open to the inclusion of unspecified elements, whether useful or not. In general, it will be understood by those skilled in the art that the terms used herein are generally intended as "open" terms (e.g., the term "comprise" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "comprising" should be interpreted as "including but not limited to," etc.). The open-ended term "comprising" is used herein to describe and claim the present invention as synonymous with terms such as including, containing, or having, although the present invention or aspects thereof may instead be described using alternative terms such as "consisting of" or "consisting essentially of."

Claims

1. 1. A method for increasing expression of a mitochondrial antiviral signaling (MAVS) protein in an epithelial cell in a subject in need thereof, comprising: exposing epithelial cells to an effective amount of ultraviolet A (UVA) radiation to increase expression of MAVS protein in the epithelial cells or in distal epithelial cells not exposed to the effective amount of UVA radiation, wherein the increased expression of MAVS protein is compared to those not exposed to the effective amount of UVA radiation or compared to a control. A method comprising:

2. The method of claim 1, wherein the epithelial cells comprise tracheal epithelial cells.

3. The method of claim 1, wherein the epithelial cells comprise ciliated epithelial cells.

4. The method of claim 1, wherein the epithelial cells comprise ciliated tracheal epithelial cells.

5. The method of claim 1, wherein the epithelial cells comprise human nasal epithelial cells, human tracheal epithelial cells, or both.

6. The method of claim 1, wherein the epithelial cells comprise human lung epithelial cells.

7. 2. The method of claim 1, wherein the step of exposing epithelial cells to an effective amount of UVA comprises exposing nasal epithelial cells, oral epithelial cells, olfactory epithelial cells, or a combination thereof, to an effective amount of UVA.

8. The method of claim 7, wherein exposing nasal epithelial cells, oral epithelial cells, olfactory epithelial cells, or a combination thereof increases MAVS protein expression in epithelial cells in the trachea, bronchi, or both of the subject.

9. The method of claim 7, wherein exposing nasal epithelial cells, olfactory epithelial cells, oral epithelial cells, or a combination thereof increases MAVS protein expression in epithelial cells in the lungs of the subject.

10. 2. The method of claim 1, wherein the step of exposing an epithelial cell to an effective amount of UVA comprises exposing a urethral epithelial cell, a bladder epithelial cell, a vaginal epithelial cell, a urogenital epithelial cell, a rectal epithelial cell, a gastrointestinal epithelial cell other than a rectal epithelial cell, an outer ear epithelial cell, a middle ear epithelial cell, or a combination thereof to an effective amount of UVA.

11. exposing urethral epithelial cells to the subject increases MAVS protein expression in epithelial cells in the bladder of the subject. exposing vaginal epithelial cells to the subject increases MAVS protein expression in epithelial cells in the uterus; exposing urogenital epithelial cells increases MAVS protein expression in epithelial cells in the urethra or bladder of the subject; exposing rectal epithelial cells increases MAVS protein expression in epithelial cells in the rectum or colon of the subject; exposing gastrointestinal epithelial cells other than rectal epithelial cells increases MAVS protein expression in epithelial cells in the gastrointestinal tract of the subject; exposing the outer ear epithelial cells increases MAVS protein expression in epithelial cells in the middle or inner ear of the subject; exposing middle ear epithelial cells increases MAVS protein expression in epithelial cells in the inner ear of the subject; or The method of claim 10, or a combination thereof.

12. 10. The method of claim 1, wherein the subject exhibits one or more symptoms of a microbial infection for 10 days or less.

13. 10. The method of claim 1, wherein the subject exhibits one or more symptoms of a microbial infection for 7 days or less.

14. 10. The method of claim 1, wherein the subject exhibits one or more symptoms of a microbial infection for 5 days or less.

15. 10. The method of claim 1, wherein the subject exhibits one or more symptoms of a microbial infection for three days or less.

16. 10. The method of claim 1, wherein the subject is not administered general anesthesia, regional anesthesia, local anesthesia, twilight anesthesia, or a sedative.

17. The method of claim 1, further comprising the step of selecting a subject exhibiting one or more symptoms of a microbial infection as a subject in need thereof prior to the step of exposing the epithelial cells to an effective amount of UVA.

18. 18. The method of claim 17, wherein the microbial infection is a viral infection, a bacterial infection, or a fungal infection.

19. An effective dose of UVA of 5 milliwatts / cm for a duration of at least 1 minute 2 (mW / cm 2 ) or more, or the effective dose of UVA is 5mW / cm 2 2. The method of claim 1, comprising a UVA intensity of at least 100 nm.

20. An effective dose of UVA is 2-5mW / cm for a duration of at least 20 minutes. 2 or an effective dose of UVA of 2-5mW / cm 2 2. The method of claim 1, comprising a UVA intensity of

21. 10. The method of claim 1, wherein exposing to an effective amount of UVA comprises exposing for a first period of time, followed by exposing for a subsequent period of time.

22. The method of claim 1, wherein the control is a baseline value of epithelial cells before exposure to UVA, a baseline value of epithelial cells before contact with a pathogen, or a baseline value of a population of epithelial cells that has not been exposed to the amount of UVA and is not infected with a pathogen.

23. 1. A method for evaluating ultraviolet A (UVA) treatment in a subject in need thereof, comprising: Assaying the biological sample obtained from the subject exposed to UVA treatment for mitochondrial antiviral signaling (MAVS) protein expression levels. Including, wherein a MAVS protein expression level higher than the subject's baseline level or higher than the control level indicates that the treatment is efficacious.

24. 1. A method of administering ultraviolet A (UVA) treatment to a subject in need thereof, comprising: Assaying mitochondrial antiviral signaling (MAVS) protein expression in a biological sample obtained from the subject exposed to UVA treatment; and continuing to administer UVA treatment to the subject if MAVS protein expression is lower compared to the subject's baseline level, or compared to a control, or compared to a target level. A method comprising:

25. 1. A method of administering ultraviolet A (UVA) treatment to a subject in need thereof, comprising: Carrying out the method of any one of claims 1 to 22 in a subject having a low mitochondrial antiviral signaling (MAVS) protein expression compared to a control, indicating that the subject is in need of UVA treatment; or Carrying out the method of any one of claims 1 to 22 in a subject with MAVS protein expression higher than the subject's baseline level or higher compared to a control, indicating that UVA treatment is effective. A method comprising:

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