Treating inflammatory responses

Abstract

The present invention relates to a method of treating inflammation in a mammal in need thereof that involves providing a mammal with elevated host endogenous reverse transcriptase produced by host endogenous retroviruses (ERVs), and thereafter contacting in vivo the reverse transcriptase with a reverse transcriptase inhibitor in an amount sufficient to ameliorate ERV- induced inflammatory response.

Description

[0001] TREATING INFLAMMATORY RESPONSES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims benefit of U.S. Provisional Application No. 63 / 619,435, filed on January 10, 2024, which is incorporated herein by reference in its entirety for any purpose.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under 5R01 AI042347-28 awarded by National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND OF THE INVENTION

[0007] This invention relates to inflammatory responses and methods and compositions for treating the same.

[0008] Inflammatory responses may exacerbate damage to host tissues due to an overactive immune system that is concomitant with autoimmune disease or infection. These diseases may drive inflammation to the point of severe organ damage, sepsis, the formation of abscesses, or even death. Understanding pathways involved in inflammatory responses is useful for developing therapeutics and treatments for autoimmune diseases and pathogenic infections.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention provides methods and compositions for treating inflammation. For example, the invention provides methods for treating tissue damage in a subject experiencing endogenous retrovirus (ERV)-induced reverse transcription or, alternatively, long interspersed nuclear element (LINE)-induced reverse transcription.

[0011] Accordingly, in one aspect, there is presented a method of treating inflammation in a mammal in need thereof, said method including:

[0012] (i) providing a mammal with elevated host endogenous reverse transcriptase produced by host endogenous retroviruses (ERVs); and

[0013] (ii) following step (i), contacting in vivo the reverse transcriptase with a reverse transcriptase inhibitor in an amount sufficient to ameliorate ERV-induced inflammatory response.

[0014] In some embodiments, the ERV-induced inflammatory response is concomitant with sepsis in the mammal.

[0015] In some embodiments, the ERV-induced inflammatory response is concomitant with tissue damage in the mammal.

[0016] In some embodiments, the ERV-induced inflammatory response is concomitant with autoimmune disease in the mammal.

[0017] In some embodiments, the ERV-induced inflammatory response is concomitant with bacterial or fungal or non-retrovirus infection in the mammal. In some embodiments, the ERV-induced inflammatory response is concomitant with cytokine storm in the mammal.

[0018] In another aspect, there is presented a method of treating pathogen-induced inflammation in a mammal in need thereof, said method including:

[0019] (i) providing a mammal with elevated host endogenous reverse transcriptase; and

[0020] (ii) following step (i), contacting in vivo the reverse transcriptase with a reverse transcriptase inhibitor in an amount sufficient to ameliorate the pathogen-induced inflammation.

[0021] In some embodiments, the host endogenous reverse transcriptase is produced by a retroelement.

[0022] In some embodiments, the retroelement is an endogenous retrovirus (ERV) or a long interspersed nuclear element (LINE).

[0023] In some embodiments, the pathogen-induced inflammation is concomitant with sepsis in the mammal.

[0024] In some embodiments, the pathogen-induced inflammation is concomitant with tissue damage in the mammal.

[0025] In some embodiments, the pathogen-induced inflammation is concomitant with bacterial or fungal or non-retrovirus infection in the mammal.

[0026] In some embodiments, the reverse transcriptase inhibitor is a nucleoside / nucleotide reverse transcriptase inhibitor.

[0027] In some embodiments, the nucleoside / nucleotide reverse transcriptase inhibitor is tenofovir, abacavir, adefovir, alovudine, azvudine, censavudine, didanosine, emtricitabine, entecavir, islatravir, lamivudine, stavudine, zalcitabine, or zidovudine.

[0028] In some embodiments, the nucleoside / nucleotide reverse transcriptase inhibitor is tenofovir.

[0029] In some embodiments, the nucleoside / nucleotide reverse transcriptase inhibitor is abacavir.

[0030] In some embodiments, the nucleoside / nucleotide reverse transcriptase inhibitor is censavudine.

[0031] In some embodiments, the reverse transcriptase inhibitor includes two or more nucleoside / nucleotide reverse transcriptase inhibitors.

[0032] In some embodiments, the two or more nucleoside / nucleotide reverse transcriptase inhibitors are tenofovir and emtricitabine.

[0033] In some embodiments, the two or more nucleoside / nucleotide reverse transcriptase inhibitors are tenofovir and abacavir.

[0034] In some embodiments, the two or more nucleoside / nucleotide reverse transcriptase inhibitors are tenofovir, emtricitabine, abacavir, and zidovudine.

[0035] In some embodiments, the two or more nucleoside / nucleotide reverse transcriptase inhibitors are selected from the group consisting of abacavir, adefovir, alovudine, azvudine, censavudine, didanosine, emtricitabine, entecavir, islatravir, lamivudine, stavudine, tenofovir, zalcitabine, and zidovudine.

[0036] In some embodiments, the contacting includes administering to the mammal a pharmaceutical composition including a nucleoside / nucleotide reverse transcriptase inhibitor selected from the group consisting of abacavir, adefovir, alovudine, azvudine, censavudine, didanosine, emtricitabine, entecavir, islatravir, lamivudine, stavudine, tenofovir, zalcitabine, zidovudine, and prodrugs thereof. In some embodiments, the contacting includes administering to the mammal a pharmaceutical composition including a prodrug selected from adefovir dipivoxil, tenofovir disoproxil, tenofovir disoproxil hemifumarate, tenofovir disoproxil fumarate, tenofovir alafenamide, tenofovir alafenamide hemifumarate, tenofovir alafenamide fumarate, tenofovir amibufenamide, and tenofovir exalidex.

[0037] In some embodiments, the reverse transcriptase inhibitor is a non-nucleoside / non-nucleotide reverse transcriptase inhibitor.

[0038] In some embodiments, the non-nucleoside / nucleotide reverse transcriptase inhibitor is delavirdine, doravirine, efavirenz, elsulfavirine, etravirine, fosdevirine, lersivirine, nevirapine, or rilpivirine.

[0039] In some embodiments, the contacting includes administering to the mammal a pharmaceutical composition including a non-nucleoside / non-nucleotide reverse transcriptase inhibitor selected from the group consisting of delavirdine, doravirine, efavirenz, elsulfavirine, etravirine, fosdevirine, lersivirine, nevirapine, and rilpivirine.

[0040] In some embodiments, the mammal is a human.

[0041] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0042] BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0044] FIG. 1A - FIG. 1 E show that ERV expression correlates with abscess susceptibility. FIG. 1 A is a schematic of variation in abscess susceptibility in BALB / cJ, B6J, and B6N mice. FIG1 B is a schematic of RNA- Sequencing experiment. FIG. 1 C shows Z-score normalized gene expression of transcripts that are induced by infection in B6N mice and whose expression correlates with abscess susceptibility. FIG. 1 D shows a maximum likelihood tree of abscess-associated and representative ERV loci in B6J. Loci identified in this study are in bold and those identical to previously known loci are in parentheses (e.g Xmv13 (Chr13: 67.9Mb)). 4 loci lack envand do not match previously known ERV loci (10). For clarity, one xenotropic and several polytropic MLVs are represented as single, collapsed branches (triangles). FIG. 1 E shows a sequence alignment of the 11 abscess-associated MLV loci compared to their consensus.

[0045] FIG. 2A - FIG. 2C show NRTI treatment prevents abscess formation. FIG. 2A shows B6N females were inoculated IV with E. co / / ' and NRTIs or vehicle control was injected IP at indicated times (bottom table) and doses (top table). CFUs are shown per liver, along with whether the animal possessed (blue) or lacked (black) abscesses, as well as the fraction of animals with abscesses (red). P values are derived from one-tailed Mann Whitney U tests (mw, to compare CFUs) or Fisher Exact tests (fe, to compare the fraction of animals that developed abscesses), ‘historical data from (1 ) for reference. FIG. 2B shows growth curves of E. co / / exposed to NRTIs in LB (n =4, mean ± SD). FIG. 2C shows an affect of NRTI treatment (all four drugs IP, one dose immediately following inoculation) on the formation of necrotic lesions caused by formalin-killed E. coli (middle and bottom rows, blue arrows). Abscesses formed from inoculation with live E. coli are shown for reference (top row, blue arrows)..

[0046] FIG. 3A - FIG. 3F show that NRTIs diminish proinflammatory gene expression in the liver. FIG. 3A shows a schematic of RNA-sequencing experiment. FIG. 3B shows a PCA plot of hepatic transcriptomes of E. coli infected mice at 4 hpi administered NRTIs (red) or PBS (blue). FIG. 3C shows a volcano plot of genes differentially expressed in NRTI-treated E. coli infected mice compared to PBS- treated E. coli infected mice. Red points denote genes with adjusted Rvalues less than 0.001 and absolute values of fold changes greater than 0.9. FIG. 3D shows a Gene Set Enrichment Analysis using fold-changes from panel B with Hallmark categories. The top 10 pathways (by adjusted Rvalue) are shown. FIG. 3E shows fold-changes of genes differentially regulated by E. coli infection (x-axis) plotted against genes differentially regulated by NRTIs in E. coli infected mice. FIG. 3F shows flow cytometry analysis for relative abundance of indicated cell types at 4 hpi in E. coli infected mice administered PBS or NRTIs. No statistical significance is observed via one-tailed students t-test. For all panels, NRTIs were administered i.p. immediately following i.v. E. coli inoculation at 1 .6 mg / drug / mouse.

[0047] FIG. 4A - FIG. 4C show that NRTIs diminish systemic proinflammatory responses. FIG. 4A shows that cytokine abundances in serum from mice administered various stimuli, across indicated mouse strains and time points, with or without NRTIs. NRTIs were administered i.p. (1 .6 mg / drug / mouse) at a single dose immediately following inoculation of indicated stimuli. R-values were obtained from Browne-Forsythe and Welch ANOVA tests and corrected for multiple comparisons using the false discovery rate (FDR). Rvalue calculations were omitted for conditions where values exceeded limits of detection, and minimum or maximum values are plotted. Axes are modified to visualize all conditions. FIG. 4B shows mice were administered 200 pg of LPS i.v. and either PBS or NRTIs i.p.. R values were obtained by multiple t-tests followed by FDR correction. FIG. 4C shows survival curves from mice in (FIG. 4B). Rvalue was obtained by log-rank test. For B and C, NRTIs were administered at 1 .6 mg / mouse / drug immediately following inoculation; in addition, NRTIs and vehicle were also administered 3 h prior to LPS inoculation, 3 h post LPS inoculation, and 18 h post LPS inoculation, for a total of 4 doses.

[0048] FIG. 5 shows the relative mRNA expression of nitric oxide synthase 2 (Nos2) in bone marrow- derived macrophages induced in response to treatment with 0.1 pg / ml lipopolysaccharide (LPS) alone or with LPS and either 400 pg / ml tenofovir or 400 pg / ml abacavir, as compared to the expression of p-actin (ActB). mRNA expression was measured using qPCR at 4 hours post stimulation.

[0049] DETAILED DESCRIPTION OF THE INVENTION

[0050] The present disclosure provides methods for reducing inflammatory responses in a subject (e.g., a mammal such as a human) in need thereof by administering nucleoside / nucleotide reverse transcriptase inhibitors. Exemplary diseases which may be treated include those caused by pathogenic infection, autoimmune diseases, cancer, or other chronic conditions. The invention further provided methods for treating tissue damage. The sections that follow describe exemplary methods for reducing inflammatory responses, compounds and compositions for reducing inflammation in a subject, and methods of treatment of inflammation that utilize the compounds and compositions of the disclosure either alone or in combination.

[0051] Definitions

[0052] As used herein the singular forms “a,” “an,” and, “the” include plural reference unless the context clearly dictates otherwise.

[0053] The term “about” when modifying a numerical value or range herein includes normal variation encountered in the field, and includes plus or minus 1 -10% (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%) of the numerical value or end points of the numerical range. Thus, a value of 10 includes all numerical values from 9 to 11 . All numerical ranges described herein include the endpoints of the range unless otherwise noted, and all numerical values in-between the end points, to the first significant digit.

[0054] As used herein, the term “tissue damage” refers to a disruption of a body’s tissues which typically results from injury, infection, or disease. Such tissue damage, regardless of its anatomical site, results from an inflammatory response. In some instances, white blood cells accumulate and destroy local tissue. An abscess is one example of such tissue damage. Abscesses may form almost anywhere in the body. When an area of the body, for example, becomes infected, the body's immune system fights the infection. White blood cells infiltrate the infected area, collect within the damaged tissue, and cause inflammation. During this process, pus forms. Pus is a mixture of living and dead white blood cells, germs, and dead tissue. Abscesses may form on the skin, in the mouth, and internally, and are diagnosed according to standard methods. Tissue damage and the subsequent inflammatory response are a common outcome of a wide variety of insults, including microbial invasion, trauma (e.g., physical injury), chemical injury, thermal injury (e.g., burns), ischemic injury (lack of blood flow), and immunologic injury.

[0055] As used herein, the term “cytokine” refers to the collection of proteins or signaling molecules that coincide with the immune response and are involved in the regulation of inflammation. In response to infection, such as by a bacterium, virus, or fungi, the expression of cytokines may become dysregulated and ultimately result in pathologically worsened inflammation or sepsis. Hypercytokinemia, a “cytokine storm,” may result from immune system hyperactivation in response to an administered therapy, pathogens, cancers, or autoimmune conditions, and indicates the host is producing an excess (e.g. elevated level) of inflammatory signaling molecules.

[0056] As used herein, the term "effective amount," refers to a quantity of a pharmaceutical composition sufficient to, when administered to the subject, for example a human subject, affect beneficial or desired results, such as clinical results. For example, in the context of inflammation, described herein, this term refers to an amount of the composition sufficient to achieve a reduction in the inflammation as compared to the response obtained without administration of the composition. The quantity of a given composition described herein that will correspond to such an amount depending upon various factors, such as the given agent, the pharmaceutical formulation, the route of administration, the identity of the subject (e.g., age, sex, weight) being treated, and the like.

[0057] As used herein, the terms “endogenous retrovirus” and “ERVs” refer to integrated proviruses from ancient retroviral infections that have coevolved with eukaryotic genomes. Functional ERVs can be transcribed, packaged, and secreted as mature viral particles, as well as reverse-transcribed to DNA and reintegrated into the chromosome. ERV reverse transcription generates cytosolic DNA, which can induce immune signaling through cytoplasmic nucleic acid sensors. ERVs may be contrasted with “exogenous retroviruses,” such as human immunodeficiency virus (HIV) and human T-cell lymphotropic virus (HTLV), which are transmitted to a host by infection and from an external source.

[0058] As used herein, the term “gene” refers to the segment of deoxyribonucleic acids (DNA) involved in producing or encoding a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). Alternatively, the term “gene” can refer to the segment of DNA involved in producing or encoding a non-translated RNA, such as an rRNA, tRNA, gRNA, or micro-RNA.

[0059] As used herein, the term “level” refers to the measure of a compound, metabolite, transcript, or enzyme activity, or property thereof, as might be measured to a reference. The reference can be any useful reference, as defined herein. By a “decreased level” or an “increased level” of a compound, protein, or enzyme activity is meant a decrease or increase in level, as compared to a reference (e.g., a decrease or an increase by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, or more; a decrease or an increase of more than about 10%, about 15%, about 20%, about 50%, about 75%, about 100%, or about 200%, as compared to a reference; a decrease or an increase by about 0.01 -fold, about 0.01 -fold, about 0.02-fold, about 0.1 -fold, about 0.3-fold, about 0.5- fold, about 0.8-fold, about 0.9-fold, or less; or an increase by more than about 1 .1 -fold, about 1 .2-fold, about 1 .3-fold, about 1 .4-fold, about 1 .5-fold, about 1 .8-fold, about 2.0-fold, about 3.0-fold, about 3.5-fold, about 4.5- fold, about 5.0-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50- fold, about 100-fold, or more). A level of a compound, protein, or transcript may be expressed in mass / vol (e.g., g / dl, mg / ml, pg / ml, ng / ml), concentration or molarity (e.g., M, mM, pM, nM, pM), or percentage relative to total compound in a sample, or by any other suitable units of measure as described by the disclosure. For example, “elevated host endogenous reverse transcriptase” may refer to increased levels of the reverse transcriptase itself, or increased levels of the genes / retroelements prior to translation. The detection of increase or decreased levels may be performed by any appropriate assay; in the case of detecting a retroelement, polymerase chain reaction (PCR) may be utilized in order to measure transcript levels thereof.

[0060] As used herein, the terms “LINEs” and “long interspersed nuclear elements” refer to mobile genetic elements (retrotransposons) within a host genome that are sometimes expressed and later copied elsewhere into the host genome by retrotransposition. LINEs are regulated in expression by intracellular mechanisms, but upon translation of LINE mRNA, proteins having reverse transcriptase and endonuclease activity serve to integrate LINE DNA elsewhere within the host genome. LINEs compose about 20% of genomic sequences in humans, although these genes may become inadvertently silenced by random mutations. The active lineage of LINEs expressed in mammals, for example humans, belong to the LINE-1 class, and LINE-1 reverse transcriptase activity is thought to be susceptible to inhibition in accordance with the methods herein. As used herein, the term “pathogen-induced inflammation” refers to the inflammation induced within a host organism following bloodstream infection by a pathogen. Rapidly following bloodstream infection, the host immune system may react in such a manner that a subject’s tissues are damaged in response to the infection. For example, in the context of the present disclosure, bacterial endotoxin may activate immune cells, leading to potential necrosis of the infection site. The damaged tissue serves as a vehicle for furthering bacterial growth, immune cell recruitment, and may ultimately result in abscess formation or severe tissue damage. Within the context of the disclosure, the inflammatory response may be endogenous retrovirus (ERV)-induced due to a bacterial toxin stimulating the immune system and resulting in elevated levels cytokines, thereby driving the proinflammatory response. As such, the methods of the present invention may be for ameliorating ERV-induced inflammatory responses. The inflammation induced by a pathogen is not limited to induction by ERVs, and may be any pathogen (e.g. as described herein).

[0061] As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms, which are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human) without excessive toxicity, irritation, allergic response, and other problem complications commensurate with a reasonable benefit / risk ratio.

[0062] As used herein, the term “sepsis” refers to a condition characterized by an inflammatory immune response, and which may arise in response to infection, trauma, or disease. Sepsis may be associated with a bacterial infection, a viral infection, a fungal infection, or a parasitic infection (e.g., as described herein). Common locations for the primary infection include the lungs, brain, urinary tract, skin, and abdominal organs. Risk factors include very young age, older age, a weakened immune system from conditions such as cancer or diabetes, major trauma, or burns. Sepsis may also arise independent from an infection and is then referred to as sterile sepsis. Sepsis therefore may also be associated with trauma, burns, pancreatitis, or ischaemic reperfusion. Common signs and symptoms include fever, increased heart rate, increased breathing rate, and confusion. There may also be symptoms related to a specific infection, such as a cough with pneumonia, or painful urination with a kidney infection. In the very young, old, and people with a weakened immune system, there may be no symptoms of a specific infection and the body temperature may be low or normal, rather than high. Severe sepsis may be characterized by poor organ function or insufficient blood flow. Insufficient blood flow may be evident by low blood pressure, high blood lactate, or low urine output. Septic shock may be characterized by low blood pressure due to sepsis that does not improve after fluid replacement. Sepsis may be characterized by an increase in pro-inflammatory cytokines in a subject, e.g., an increase in include one or more interleukin (e.g., IL-1 , IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, or IL-18), tumor necrosis factor (TNF, e.g., TNFa), interferon gamma (IFNy) , or granulocyte macrophage colony stimulating factor (GMCSF). Other symptoms associated with sepsis include, e.g., increased white blood cell count, immature white blood cells in the circulation, elevated plasma C-reactive protein, elevated procalcitonin (PCT), low blood pressure, low central venous or mixed venous oxygen saturation, high cardiac index, low oxygen level, low urine output, high creatinine in the blood, coagulation (clotting) abnormalities, absent bowel sounds, low platelets in the blood, high bilirubin levels, high lactate in the blood, or decreased capillary filling or mottling. As used herein, the term “retroelement” refers to the sequences integrated within a host genome that encode genetic elements; these genetic elements are reverse transcribed for the purpose of reintegration within the host genome. Examples of retroelements within the context of the disclosure include, e.g., endogenous retroviruses (ERVs) and long interspersed nuclear elements (LINEs). Each retroelement encodes a functional domain to assist with reverse transcription of a retroelement RNA transcript into DNA, which may then be inserted elsewhere within the host genome by endonucleases.

[0063] As used herein, the term “reverse transcriptase inhibitor” refers to antiretroviral drugs that inhibit the activity of reverse transcriptase, a polymerase enzyme which transcribes pathogenic genes from RNA to DNA and facilitates the integration of the pathogenic DNA into the host genome. Reverse transcriptase inhibitors in the context of the disclosure may be nucleoside / nucleotide reverse transcriptase inhibitors (NRTIs) or non-nucleoside / non-nucleotide reverse transcriptase inhibitors (NNRTIs), which are separated by whether they resemble naturally occurring nucleobases. NRTIs suitable for use in the methods of treatment disclosed herein may be, e.g., abacavir, adefovir, alovudine, azvudine, censavudine, didanosine, emtricitabine, entecavir, islatravir, lamivudine, stavudine, tenofovir, tenofovir exalidex (CMX- 157), zalcitabine, or zidovudine (ZDV, also known as azidothymine or AZT). NNRTIs suitable for use in the methods of treatment disclosed herein may be, e.g., delavirdine, doravirine, efavirenz, elsulfavirine, etravirine, fosdevirine, lersivirine nevirapine, RDEA806, rilpivirine, RPT-A, or RPT-2950. Analogs, isomers, tautomers, salts, prodrugs or other derivatives of any of the above reverse transcriptase inhibitors are contemplated to be within the scope of the methods of treatment herein.

[0064] As used herein, the term “subject” refers to a subject receiving any of the methods of treatment disclosed herein or being administered any of the pharmaceutical compositions described herein. The terms “patient” and “subject” may be interchangeable with respect to the methods of treatment herein described. As used in the context of the present invention, a subject to be treated is preferably a human subject who is diagnosed as suffering from one or more of the diseases / conditions / disorders, as disclosed herein, by a licensed professional in accordance with accepted medical practice. In some instances, the subject may have previously been determined to be at risk of said disease or disorder.

[0065] As used herein, “treatment” and “treating” in reference to a disease or condition, refer to an approach for obtaining beneficial or desired results, e.g., clinical results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions; diminishment of extent of disease or condition; stabilized (i.e., not worsening) state of disease, disorder, or condition; preventing spread of disease or condition; delay or slowing the progress of the disease or condition; amelioration or palliation of the disease or condition; and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0066] Host Endogenous Retroelements

[0067] Endogenous Retroelements (ERVs) The compositions of the disclosure have effects in reducing the inflammatory responses in a subject that are induced by host endogenous retroviruses (ERVs). ERVs are coevolved proviruses integrated with eukaryotic genomes that may be functionally transcribed, packaged, or secreted as particles, or alternatively integrated into the host genome by reverse-transcription to DNA.

[0068] Host inflammatory response pathways may be triggered by bacterial infection, thereby leading to downstream effects such as abscess formation resulting from the inflammatory response to bacterial molecules. In subjects infected with Escherichia coli (E. coli), a positive correlation is observed between abscess formation in the liver and the levels of ERV expression in response to infection. It is thought that bloodstream bacterial exposure results in an immune response that activates ERV reverse transcription and results in generation of cytosolic DNA. The cytosolic DNA may then stimulate host immune signaling due to cytoplasmic DNA sensors; thus, the creation of cytosolic DNA by ERV transcription in response to infection drives the immune response and may further bacterial growth at the site of infection if said immune response results in damage to host tissues. It is contemplated that administration of nucleoside / nucleotide reverse transcriptase inhibitors (NRTIs) reduces the immunostimulatory activity of ERVs by inhibiting ERV reverse transcriptase activity that is induced by infection, thereby attenuating the host immune response. Use of reverse transcriptase inhibitors results in suppression of ERV reverse transcription and is thought to reduce the bacterial burden in a subject after infection. It is further contemplated that alternative reverse transcriptase inhibitors, such as non-nucleoside / non-nucleotide reverse transcriptase inhibitors (NNRTIs), are suitable for the methods of treatment described herein.

[0069] Reduction of inflammatory markers in response to reverse transcriptase inhibitor treatment has been previously demonstrated to be independent of bacterial strain, as mice inoculated with either grampositive or gram-negative bacteria experienced a reduction in inflammatory cytokine production following NRTI administration at 4 hours post-inoculation. These effects are not solely limited to liver infection, as serum levels of inflammatory cytokines are observed to be lower in mice after treatment with the compounds and compositions of the disclosure. In mice which do not develop abscesses in response to infection (e.g. due to the strain of mouse), similar cytokine level reductions are observed, which indicates the effect of reverse transcriptase inhibition to not be limited by either the site of liver necrosis or to a particular strain of mouse.

[0070] Long Interspersed Nuclear Elements (LINEs)

[0071] The methods of the disclosure have effects in reducing inflammatory responses in a subject and may coincide with increased levels of long interspersed nuclear element (LINE) transcript expression or increased LINE-associated reverse transcriptase activity. LINEs are retrotransposable (i.e. transposed by reverse transcription) genetic elements within a host genome. Within the functional domains encoded by many LINEs, proteins with reverse transcriptase and endonuclease activity are produced to facilitate reintegration into the host genome. The methods of the invention are further applicable to inhibiting the reverse transcriptase activity associated with LINE transcription, particularly if they become elevated in response to a pathogen or disorder.

[0072] Pharmaceutical Compositions Reverse Transcriptase Inhibitors

[0073] The compositions of the disclosure may include one or more nucleoside / nucleotide reverse transcriptase inhibitors (NRTIs) or non-nucleoside / non-nucleotide reverse transcriptase inhibitors (NNRTIs).

[0074] NRTIs are compounds which mimic endogenous nucleobases, and upon binding to enzymes capable of reverse transcription, they inhibit the transcriptase activity in order to treat a disease or disorder, typically a viral infection. By binding reverse transcriptase and reducing its activity, RNA transcripts are prevented from being reverse-transcribed to DNA; the bound NRTI prevents elongation and incorporation of alternative nucleosides / nucleotides into a developing DNA transcript. In the context of the present disclosure, it is contemplated that retroelement cytosolic DNA expression is reduced by administering NRTIs, thereby serving to mitigate host inflammatory responses which might otherwise promote bacterial reproduction.

[0075] The compounds of the present disclosure include NRTIs and analogs or derivatives thereof. Such compounds may be formulated into compositions together or administered separately in accordance with the needs of a subject and as determined by the physician. NRTIs suitable for use in the methods of the present disclosure include, but are not limited to, e.g., abacavir, adefovir, alovudine, azvudine, censavudine, didanosine, emtricitabine, entecavir, islatravir, lamivudine, stavudine, tenofovir, tenofovir exalidex (CMX-157), zalcitabine, or zidovudine (ZDV, also known as azidothymidine or AZT).

[0076] Compounds of the present disclosure are not limited to NRTIs. It is further contemplated that non- nucleoside / non-nucleotide reverse transcriptase inhibitors (NNRTIs) may also serve to reduce the reverse transcriptase activity of ERVs in response to infection. NNRTIs similarly inhibit the activity of reverse transcriptase, but the molecular structure of the active therapeutic agent is not meant to resemble that of endogenous nucleosides or nucleotides. NNRTIs suitable for use in the methods of the present disclosure include, but are not limited to, e.g., delavirdine, doravirine, efavirenz, elsulfavirine, etravirine, fosdevirine, lersivirine nevirapine, RDEA806, or rilpivirine.

[0077] Additionally, LINE-1 reverse transcriptase inhibitors are useful in the methods disclosed herein. Exemplary inhibitors of LINE-1 reverse transcriptase are disclosed in US application no. 18 / 359,297, US application no. 18 / 561 ,644, US application no. 18 / 572,316, WO / 2022 / 271880, WO / 2024 / 107859, WO / 2023 / 178133, WO / 2023 / 178128, WO / 2023 / 164472, WO / 2023 / 205786, WO / 2023 / 178127, WO / 2024 / 107850, WO / 2024 / 103016, WO / 2022 / 245814, WO / 2022 / 271878, W02022 / 066880, WO2022 / 197689, WO / 2022 / 256625 and WO2023 / 192491 (each of which is incorporated herein with respect to the disclosure of LINE-1 reverse transcriptase inhibitors).

[0078] In some cases, analogs of any of the above compounds may be utilized in the methods of the present disclosure. Such analogs include, but are not limited to, stereoisomers, geometric isomers, tautomers, or alternative isotopic forms (e.g. where one or more atoms has been substituted with a different isotope of the same atom, such as hydrogen substituted for deuterium).

[0079] In some instances, one or more compounds for use in the methods of the present disclosure may exist in different tautomeric forms. As will be clear from context, unless explicitly excluded, references to such compounds encompass all such tautomeric forms. In some embodiments, tautomeric forms result from the swapping of a single bond with an adjacent double bond and the concomitant migration of a proton. In certain embodiments, a tautomeric form may be a prototropic tautomer, which is an isomeric protonation states having the same empirical formula and total charge as a reference form. Examples of moieties with prototropic tautomeric forms are ketone — enol pairs, amide — imidic acid pairs, lactam — lactim pairs, amide — imidic acid pairs, enamine — imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, such as, 1 H- and 3H-imidazole, 1 H-, 2H- and 4H- 1 ,2,4-triazole, 1 H- and 2H-isoindole, and 1 H- and 2H-pyrazole. In some embodiments, tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution. In the context of the present disclosure, synthetic nucleotide analogs may be present in multiple tautomeric forms, and it is contemplated that any tautomer from such compound is within the scope of the present methods.

[0080] Methods of the disclosure may utilize compounds administered in the form of prodrugs, wherein a chemically modified version of any of the aforementioned compounds is administered to the subject. Following administration, intracellular enzymes or native physiological conditions may cause cleavage of the moiety introduced as a chemical modification, thereby releasing the unmodified (parent) compound and result in therapeutic activity. Derivatives which act as prodrugs of compounds are transformable in vivo or in vitro into one of the parent compounds. Typically, at least one of the biological activities of a compound will be reduced in the prodrug form of the compound and can be increased following conversion of the prodrug to release the compound or a metabolite thereof. Examples of prodrugs include the use of protecting groups which may be removed in situ releasing active compounds or serve to inhibit clearance of the drug in vivo. Prodrugs can take on many different functional forms, and may include, but are not limited to, acetals, esters, or phosphates.

[0081] In the context of the present disclosure, an exemplary derivative of a compound herein might include tenofovir disoproxil fumarate, wherein the hydroxyl groups of the tenofovir phosphate are further modified as isopropoxycarbonyloxymethyl esters; the tenofovir is furthermore modified to be the fumaric acid salt of the prodrug. Compounds of the present disclosure are thus not limited to a singular modification in order to prepare derivatives and may utilize combinations of steps to prepare the form suitable for administration (e.g. a salt of a prodrug of an isomer). Under physiological conditions, the ester groups of tenofovir disoproxil fumarate become hydrolyzed, resulting in release of tenofovir and allowing binding to reverse transcriptase. It is contemplated that an active form of any compounds described by the present disclosure might be released in a similar manner, regardless of the moiety that is cleaved under physiological conditions.

[0082] Salts compatible with the compounds of the present disclosure as used in the methods described herein include, but are not limited to, hydrochlorides, acetates, citrates, salicylates, nitrates, and phosphates. Examples of salts of compounds suitable for use in the present methods may be formed with acetic, citric, oxalic, tartaric or mandelic acid, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo- or phosphor-acids or N-substituted sulfamic acids, for example, acetic acid, phenylacetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid, isonicotinic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane 1 ,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, 2-naphthalenesulfonic acid, naphthalene-1 ,5-disu Ifonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate, or N- cyclohexylsulfamic acid (with the formation of cyclamates); with other acid organic compounds, such as ascorbic acid; or with amino acids, such as the 20 alpha amino acids involved in the synthesis of proteins in nature, e.g., glutamic acid or aspartic acid. Thus, it is contemplated that compounds including, but not limited to, adefovir, tenofovir, adefovir dipivoxil, tenofovir disoproxil, tenofovir disoproxil hemifumarate, tenofovir disoproxil fumarate, tenofovir alafenamide, tenofovir alafenamide hemifumarate, tenofovir alafenamide fumarate, and tenofovir amibufenamide are suitable for use in the methods of treatment described herein, regardless of the compound being in free base or salt form.

[0083] Combination Therapy

[0084] The compositions and methods of treatment of the disclosure are not intended to be limited to a single active agent. Multiple reverse transcriptase inhibitors are thought to be appropriate for inhibiting transcription of retroelements and reducing or treating inflammation as described herein, and the effects of administering more than one single compound may be greater than administering an individual compound. Thus, a compound of the disclosure may be administered with one or more further active agents, wherein the one or more further active agents is another compound of the disclosure. Such combinations may indicate two or more total active agents, e.g., two compounds, three compounds, four compounds, five compounds, six compounds, seven compounds, eight compounds, nine compounds, or ten compounds suitable for use in the methods of treatment described herein. For example, tenofovir may be administered alone or in combination with emtricitabine (or any other compounds described herein) in accordance with the present methods. In some cases, one may administer at least a second, if not more preferrable two or more additional active agents, for the purposes of inhibiting a broad spectrum of isoforms and improving inhibitory activity and the therapeutic result achieved.

[0085] Administering two or more compounds of the disclosure as part of a combination therapy is not limited to a single pharmaceutical composition and may include the active agents within the same or in separate compositions. The first reverse transcriptase inhibitor may be administered substantially simultaneously (e.g., in the same pharmaceutical composition or in separate pharmaceutical compositions) as any additional reverse transcriptase inhibitor or may be administered prior to or following the administration of the first reverse transcriptase inhibitor (e.g., within a period of 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, or more).

[0086] Formulations

[0087] Methods of the present disclosure may include administering compounds that can be formulated into pharmaceutical compositions using techniques well known to those in the art. Suitable pharmaceutically acceptable carriers, outside those mentioned herein, are known in the art; for example, see Remington, The Science and Practice of Pharmacy, 20th Edition, 2000, Lippincott Williams & Wilkins, (Editors: Gennaro et al.). While it is possible that, for use in the prophylaxis or treatment, a compound of the disclosure may, in an alternative use, be administered as a raw or pure chemical, it is preferable however to present the compound or active ingredient as a pharmaceutical formulation or composition further comprising at least one pharmaceutically acceptable excipient.

[0088] Formulations comprising the active compounds described herein alongside one or more inert reagents (excipients) that improve the bioavailability, pharmacokinetic properties, ease of administration, or processability during manufacture are envisioned to be used for the methods of the present disclosure. Pharmaceutically acceptable excipients that may be present in the compositions of the present invention include, but are not limited to, diluents, binders, disintegrants, fillers, lubricants, colorants, flavors, pH adjusters, buffers, stabilizers, viscolizers, antiadherents, preservatives, glidants, acidulants, artificial and natural sweeteners, and the like. Pharmaceutical compositions can be formulated, for example, for oral administration in unit dosage form (e.g., a tablet, pill, capsule, caplet, gelcap, suspension, solution, or syrup); for intravenous administration (e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use); or in any other pharmaceutically acceptable formulation. Some embodiments include methods for treatment of diseases or disorders in a subject which include administering compounds of the present disclosure in combination with at least one pharmaceutically acceptable excipient. Formulations may be prepared by any suitable method, typically by uniformly mixing the active compound(s) with liquids or finely divided solid carriers, or both, in the required proportions.

[0089] Fillers (also referred to as diluents or binders) employed in the compositions of the present invention include, but are not limited to, microcrystalline cellulose, maltodextrin, calcium hydrogen phosphate, polyethylene glycol, polyvinylpyrrolidone, maize starch, xanthan gum, corn starch, Cutina HR, pregelatinized starch, partially pregelatinized starch, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, lactose monohydrate, or combinations thereof.

[0090] Disintegrants employed in the compositions of the present invention include, but are not limited to, sodium starch glycolate, sodium carboxymethyl cellulose, carmellose, carmellose calcium, croscarmellose sodium, cross-linked alginic acid, crospovidone, polyvinylpyrrolidone, methyl cellulose, starch, crosslinked starch, pregelatinized starch, partially pregelatinized starch, glycerin fatty acid ester, or combinations thereof.

[0091] Lubricants that may be employed in the compositions of the present invention include, but are not limited to, magnesium stearate, sodium stearate, hydrogenated vegetable oils, silicon dioxide, Aerosil 200, talc, boric acid, sodium benzoate, sodium acetate, sodium chloride, DL-leucine, polyethylene glycol, waxes, calcium stearate, zinc stearate, sodium stearyl fumarate, sodium oleate, sodium lauryl sulfate, magnesium lauryl sulfate, or combinations thereof.

[0092] A glidant is an excipient that improves the flow properties powders or granulates by decreasing interparticle friction and cohesion. Potentially suitable glidants include, without limitation, silicon dioxide, in particular colloidal silicon dioxide, anhydrous colloidal silica, peptized silica, talc, magnesium trisilicate, powdered cellulose, or combinations thereof. In some embodiments, the glidant is colloidal silicon dioxide. If two or more glidants are present in the tablet, these amounts refer to the total amount of the glidants.

[0093] Pharmaceutical compositions are sometimes in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules and powders in vials, sachets or ampoules. Also, the unit dosage form can be a capsule, tablet, a pill, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

[0094] The compounds of the disclosure may be prepared as liquid formulations to be suitable for the methods described herein, such as in the form of a solution, suspension, or emulsion. Liquid preparations for oral administration may be in the form of solutions, emulsions, aqueous or oily suspensions, and syrups. Additional additives such as suspending or emulsifying agents, non-aqueous vehicles (including edible oils), preservatives and flavorings and colorants may be added to the liquid preparations. Formulations suitable for oral administration can consist of (a) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (b) powders; (c) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice; (d) suspensions in an appropriate liquid; and (e) suitable emulsions. Preferred are solid oral dosage forms such as capsule forms, tablet forms, and powder forms. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and cornstarch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers. Lozenge forms can comprise the active ingredient and a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, other carriers. In some instances, the composition is administered as a dry powder that is reconstituted as a liquid prior to administration to the subject, thereby forming a solution or suspension. Parenteral (e.g. injectable) dosage forms may be prepared by dissolving a compound of the disclosure in a suitable liquid vehicle and filter sterilizing the solution before filling and sealing an appropriate vial or ampule. The solutions may then be administered to a subject to treat a disease or condition by injection into a blood vessel or to the target site, as appropriate.

[0095] Dosage

[0096] Dosage Amounts

[0097] In one aspect, provided herein are dosage amounts and regimens for administration of reverse transcriptase inhibitors in appropriate amounts to treat a disorder in a subject. The dose when using the inhibitors in the present methods may be selected from a range as is customary and known to the physician and is to be tailored to an individual subject in each case. It depends, for example, on the nature and severity of the illness to be treated, on the condition of the subject, on the compound (or compounds or compositions) employed, on whether an acute or chronic disease state is treated, if prophylactic administration is conducted, or on whether further active compounds are administered in addition to the compounds and compositions described by the present disclosure. Representative doses of the present disclosure include, but are not limited to, from 0.1 to 1000 mg. Depending on the individual and as deemed appropriate from the subject's physician or caregiver, it may be necessary to deviate upward or downward from the doses described herein. For example, the dose administered to the subject may be about 0.1 mg (100 pg), about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 .0 mg, about 1 .5 mg, about 2.0 mg, about 2.5 mg, about 3.0 mg, about 4.0 mg, about 5 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1000 mg. Accordingly, the acceptable amount of reverse transcriptase inhibitor to be administered may be present in an amount from 0.1 mg to 1000 mg in total across all reverse transcriptase inhibitors administered, not accounting for the weight of pharmaceutically acceptable excipients. In some instances, smaller doses may be administered or prepared from a single unit dose form by dividing the larger dose.

[0098] In some cases, the dose amount to be administered includes a combination of reverse transcriptase inhibitors, such that multiple active compounds may total the amount of reverse transcriptase inhibitor to be administered. For example, a subject receiving 600 mg of reverse transcriptase inhibitor in accordance with the present methods may be receiving one or more reverse transcriptase inhibitors in combination, such as 400 mg of a first compound and 200 mg of a second compound from the disclosure. In some cases, more than one reverse transcriptase inhibitor is administered (e.g. one, two, three, four, five, six, seven, eight, nine, or ten different inhibitors). The doses to be administered may be determined by the physician and adjusted to be suitable for the needs of the subject.

[0099] The amount of active ingredient required for use in treatment will vary not only with the compounds or composition selected but also with the route of administration, the nature of the condition being treated, and the age and condition of the subject; the amount will ultimately be at the discretion of the attendant physician or clinician. In general, one skilled in the art understands how to extrapolate in v / 'vo data obtained in a model system, typically an animal model, to another, such as a human. In some circumstances, these extrapolations may merely be based on the weight of the animal model in comparison to another, such as a mammal, preferably a human. More often, these extrapolations are not simply based on weights but instead incorporate representative factors including the type, age, weight, sex, diet and medical condition of the subject, the severity of the disease, the route of administration, pharmacological considerations such as the activity, efficacy, pharmacokinetic and toxicology profiles of the particular compounds or composition employed, whether a drug delivery system is utilized, on whether an acute or chronic disease state is being treated or if prophylaxis conducted, or on whether further active compounds are administered that are incompatible with reverse transcriptase inhibitor administration, such that the dose amounts administered to a subject may be reduced in order to mitigate the effects of simultaneous treatment of disorders unrelated to the present disclosure.

[0100] Routes of Administration

[0101] Nonlimiting examples of the routes of administration considered within the scope of the invention include, but are not limited to, oral, intravenous, subcutaneous, transdermal, topical, intranasal, or transmucosal. In some embodiments, the pharmaceutical compositions are self-administered (e.g. by consuming a tablet or other oral dosage form). In other embodiments, the pharmaceutical compositions may be administered by a medical practitioner (e.g. by intravenous administration or other injection). Automated devices capable of delivering controlled doses of the pharmaceutical compositions over time to the subject are also contemplated to be within the scope of the present invention. In some cases, administration of the pharmaceutical composition may be facilitated by a kit the provides the composition and instructions for use. In some embodiments, the kit further comprises a device that enables intravenous infusion of the pharmaceutical composition. Administration of the pharmaceutical compositions to a subject may be co-administered or administered at substantially different times than another drug substance or medicament which the subject is concurrently receiving, such as for combination therapy, and the route of administration need not be identical for each active compound. For instance, a subject of the methods of treatment described herein may be orally administered 200 mg of tenofovir as a tablet, while concurrently receiving 400 mg zidovudine intravenously (totaling 600 mg of reverse transcriptase inhibitor), the timing of administration for each individual reverse transcriptase inhibitor may be determined by the physician and to total a desired dose amount in accordance with the present methods.

[0102] Dosing Regimen

[0103] The dosing regimen used for the treatment methods described herein can vary depending on many factors, e.g., the age, health, and weight of the recipient; the nature and extent of the symptoms of edema; and the frequency and type of concurrent treatment, if any. One of skill in the art can determine the appropriate dosage based on the above factors. The compounds used in the methods described herein may be administered initially in a suitable dosage that may be adjusted as required, depending on the individual response.

[0104] In some embodiments, the compound is administered according to a dosing regimen having a dosing periodicity ranging from about six doses daily to about one dose weekly. However, the pharmaceutical compositions may also be formulated for administration at any frequency of administration, including once every 5 days, once every 3 days, once every 2 days, once a day, twice a day, three times a day, four times a day, five times a day, eight times a day, every hour, or any greater frequency. In some embodiments, the compound is administered according to a dosing regimen having a dosing periodicity of one dose once daily.

[0105] In some embodiments, the desired dose is administered as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations. If appropriate, depending on individual behavior, it may be necessary to deviate upward or downward from the daily dose indicated.

[0106] The dosage regimen for treating a disease with the compounds and / or compositions of this disclosure is selected in accordance with a variety of factors as cited above. Thus, the actual dosage regimen employed may vary widely and may deviate from a preferred dosage regimen, and one skilled in the art will recognize that a dosage and dosage regimen outside these typical ranges can be tested and, where appropriate, be used in the methods of this disclosure.

[0107] Methods of Treatment

[0108] The invention features, generally, a method of treating tissue damage in a mammal in need thereof, including:

[0109] (i) providing a mammal with elevated host endogenous reverse transcriptase; and

[0110] (ii) following step (i), contacting in vivo the reverse transcriptase with a reverse transcriptase inhibitor in an amount sufficient to ameliorate the pathogen-induced inflammation.

[0111] In some embodiments, the host endogenous reverse transcriptase is produced by a retroelement. In other embodiments, the retroelement is an endogenous retrovirus (ERV) or a long interspersed nuclear element (LINE).

[0112] Details on treating various types of tissue damage are described below.

[0113] Treatment of Pathogenic Infection and Pathogen-induced Inflammation

[0114] Pathogens associated with generating an inflammatory response or inflammation in a subject may be bacterial, viral, or fungal. It is envisioned that administering compounds or compositions described herein in accordance with the disclosed methods are suitable for alleviating the inflammatory response in a subject. Inhibiting transcription of retroelements with the methods of the invention may have effect at ameliorating inflammation. Following infection, inflammatory immune cells infiltrate the liver, leading to necrosis of liver tissue. Bacteria within the necrotic regions replicate, resulting in further immune cell recruitment and abscess formation. Within this framework, an attenuated inflammatory response is expected to reduce development of tissue damage or abscesses, or even prevent their formation if the present methods are utilized early enough in the timeline of infection; mice lacking TLR4, the membrane-bound receptor for lipopolysaccharide, do not develop abscesses. Pathogenic bacteria causative of infections may furthermore be concomitant with sepsis in a subject.

[0115] Bacteria are typically categorized into two major types: gram-positive bacteria and gram-negative bacteria. Gram-positive bacteria possess a thick cell wall containing multiple layers of peptidoglycan and teichoic acids, while Gram-negative bacteria have a relatively thin cell wall containing fewer layers of peptidoglycan that are surrounded by a second lipid membrane containing lipopolysaccharides (LPS) and lipoproteins. LPS, also called endotoxins, are composed of polysaccharides and lipid A. Molecules such as LPS are the dominant immunostimulatory molecule from E. coli, and administration of the compounds in accordance with the methods of treatment herein serve to reduce to inflammatory response by exposure to LPS or any other bacterial immunostimulatory molecules. Examples of Gram-positive bacteria include, but are not limited to, bacteria in the genus Streptococcus (e.g., Streptococcus pyogenes), bacteria in the genus Staphylococcus (e.g., Staphylococcus aureus), bacteria in the genus Corynebacterium (e.g., Corynebacterium auris), bacteria in the genus Listeria (e.g., Listeria monocytogenes), bacteria in the genus Bacillus (e.g., Bacillus anthracis), and bacteria in the genus Clostridium (e.g., Clostridium dificille). Examples of Gram-negative bacteria include, but are not limited to, bacteria in the genus Escherichia (e.g., Escherichia coli), bacteria in the genus Enterobacter (e.g. Enterobacter cloacae, Enterobacter aerogenes, Enterobacter gergoviae, Enterobacter asburiae, and Enterobacter hormaechei), bacteria in the genus Serratia (e.g. Serratia marcescens, Serratia plymuthica, Serratia liquefaciens, Serratia rubidaea, Serratia odorifera, and Serratia fonticola), bacteria in the genus Proteus (e.g. Proteus hauseri, Proteus mirabilis, Proteus myxofaciens, Proteus penned, and Proteus vulgaris), bacteria in the genus Providencia (e.g. Providencia stuartii, Providencia sneebia, Providencia rettgeri, Providencia rustigianii, and Providencia alcalifaciens), bacteria in the genus Shigella (e.g. Shigella boydii, Shigella dysenteriae, Shigella flexneri, and Shigella sonnei), bacteria in the genus Klebsiella (e.g., Klebsiella granulomatis, Klebsiella oxytoca, Klebsiella pneumoniae, Klebsiella terrigena, and Klebsiella variicola), bacteria in the genus Acinetobacter (e.g., Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter kookii, and Acinetobacter junii), bacteria in the genus Pseudomonas (e.g., Pseudomonas aeruginosa), bacteria in the genus Neisseria (e.g., Neisseria gonorrhoeae, Neisseria meningitits), bacteria in the genus Yersinia (e.g., Yersinia pestis), bacteria in the genus Vibrio (e.g., Vibrio cholerae), bacteria in the genus Campylobacter (e.g., Campylobacter jejuni), and bacteria in the genus Salmonella (e.g., Salmonella enterica).

[0116] Exemplary viruses include, but are not limited to, coronaviruses, pneumoviridae viruses, picornaviridae viruses, enteroviruses, flaviviridae viruses, filoviridae viruses, orthomyxoviruses, or paramyxoviridae viruses.

[0117] Exemplary fungi include, but are not limited to, Epidermophyton floccusum, Microsporum audouini, Microsporum canis, Microsporum distortum, Microsporum equinum, Microsporum gypsum, Microsporum nanum, Trichophyton concentricum, Trichophyton equinum, Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini, Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophyton rubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophyton verrucosum, T. verrucosum var. album, var. discoides, var. ochraceum, Trichophyton violaceum, and / or Trichophyton faviforme, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowi, Aspergillus flavatus, Aspergillus glaucus, Blastoschizomyces capitatus, Candida auris, Candida albicans, Candida enolase, Candida tropicalis, Candida glabrata, Candida krusei, Candida parapsilosis, Candida stellatoidea, Candida kusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis, Candida guilliermondi, Cladosporium carrionii, Coccidioides immitis, Blastomyces dermatidis, Cryptococcus neoformans, Geotrichum clavatum, Histoplasma capsulatum, Microsporidia, Encephalitozoon spp., Septata intestinalis and Enterocytozoon bieneusi, Brachiola spp, Microsporidium spp., Nosema spp., Pleistophora spp., Trachipleistophora spp., Vittaforma spp Paracoccidioides brasiliensis, Pneumocystis carinii, Pythiumn insidiosum, Pityrosporum ovale, Sacharomyces cerevisae, Saccharomyces boulardii, Saccharomyces pombe, Scedosporium apiosperum, Sporothrix schenckii, Trichosporon beigelii, Toxoplasma gondii, Penicillium marneffei, Malassezia spp., Fonsecaea spp., Wangiella spp., Sporothrix spp., Basidiobolus spp., Conidiobolus spp., Rhizopus spp, Mucor spp, Absidia spp, Mortierella spp, Cunninghamella spp, Saksenaea spp., Alternaria spp, Curvularia spp, Helminthosporium spp, Fusarium spp, Aspergillus spp, Penicillium spp, Monolinia spp, Rhizoctonia spp, Paecilomyces spp, Pithomyces spp, or Cladosporium spp.

[0118] Treatment of Autoimmune Diseases It is contemplated that the methods of the disclosure may be suitable for the treatment of autoimmune diseases. Examples of autoimmune diseases to be treated include autoimmune disease is selected from the list consisting of inflammatory bowel disease, type I diabetes, aplastic anemia, Graves’ disease, systemic lupus erythematosus, celiac disease, multiple sclerosis, psoriasis, scleroderma, chronic inflammatory demyelinating polyneuropathy, pernicious anemia, polyarteritis nodosa, Sjogren’s syndrome, premature ovarian failure, alopecia (baldness), polyglandular failure, hypothyroidism, polymyosititis, Crohn’s disease, ulcerative colitis, autoimmune hepatitis, hypopituitarism, myocardititis, Addison’s disease, autoimmune skin diseases, uveititis, polymyalgia rheumatica, Goodpasture’s syndrome, hypoparathyroidism, Hashimoto’s thyoriditis, Raynaud’s phenomenon, and Guillain-Barre syndrome.

[0119] Liver Abscess Formation

[0120] The methods of the disclosure may be for use in treatment of subjects at risk of developing a liver abscess in response to an infection (e.g., a pathogenic infection). Abscesses are regions of severe necrosis and consist of millions of bacteria surrounded by inflammatory immune cells. Administration of NRTIs prevents formation of liver abscesses in response to bloodstream infection with E. coli. It is thought that the inhibition of retroelement reverse transcription restricts the buildup of cytosolic DNA, thereby diminishing pro-inflammatory responses. Administration of a cocktail of tenofovir, emtricitabine, zidovudine, and abacavir as NRTIs prevented the formation of necrotic lesions in B6N mice following inoculation with formalin-killed E. coli, whereas live E. coli resulted in measurable abscess formation without treatment.

[0121] Methods of Modulating Inflammatory Cytokine Expression

[0122] Levels of inflammatory cytokines may become altered in response to the methods of treatment disclosed herein. Host inflammatory pathways become activated in response to infection, causing an increase in the expression of cytokines. Administration of reverse transcriptase inhibitors, such as NRTIs, causes a reduction in the expression in many inflammatory cytokines, with the notable exception being IL- 10. In E. coli, only IL-10 increased in expression in response to NRTI treatment. By contrast, cytokines such as IFN-y, CXCL1 , TNF-a, MIP-1 a, MIP-1 p, MCP-1 , KC, IP-10, IL-6, or IL-1 p had statistically significant reductions in expression. This trend held following infection with either the gram-positive bacteria E. coli or the gram-negative bacteria S. aureus, indicating that the signaling pathways effected by reverse transcriptase administration broadly diminish inflammatory responses regardless of the pathogenic species.

[0123] The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided above. The Examples are therefore intended to illustrate, not limit, the scope of the invention.

[0124] EXAMPLES We profiled hepatic transcriptomes in mice with varying susceptibility to liver abscess formation. We found that transcripts from endogenous retroviruses (ERVs) are robustly induced in the liver by E. coli infection and ERV expression positively correlates with the frequency of abscess formation. We tested if nucleoside / nucleotide reverse transcriptase inhibitors (NRTIs) influence abscess formation. Strikingly, as described in the below examples, we found that a single NRTI dose administered immediately following E. coli inoculation prevented abscess formation, led to a concomitant 100,000-fold reduction in bacterial burden. We further provide evidence indicating that NRTIs inhibit abscess formation by preventing the tissue necrosis that facilitates bacterial replication. Together, our findings indicate that endogenous reverse transcriptases drive inflammatory responses during bacterial bloodstream infection to promote abscess formation.

[0125] Example 1. Expression of endogenous retroviruses correlates with abscess susceptibility.

[0126] Following IV E. coli inoculation, -90% of B6N female mice develop liver abscesses while BALB / cJ mice are entirely resistant. B6J females display an intermediate susceptibility phenotype, where abscesses are less numerous and less likely to develop, leading to fewer colony forming units (CFU) in B6J relative to B6N (Figure 1 A, (Hullahalli, et al. Genetic and immune determinants of E.coli liver abscess formation, bioRxiv, 2023, 543319). To identify host factors that control abscess formation, we performed RNA-Sequencing on livers of BALB / cJ (resistant), B6J (intermediate), and B6N (sensitive) females at 4 hours post inoculation (hpi), a time point prior to bacterial replication but when inflammatory responses are triggered (Figure 1 B). We reasoned that genes that drive abscess formation would be 1 ) upregulated following infection, 2) more highly expressed in B6N relative to B6J, and 3) more highly expressed in B6J relative to BALB / cJ. These criteria (p-adj < 0.05, Iog2 FC > 1 ) identified 13 genes, but none appeared to function together in a common biological pathway. However, two of these genes (Gm42031 and Gm43305) mapped to endogenous retroviruses (ERVs). By reexamining all murine retroelements in the gEVE database (Nakagawa, et al. gEVE: a genome-based endogenous viral element database provides comprehensive viral protein-coding sequences in mammalian genomes, Database (Oxford) 2016), which include loci not present in the initial analysis, we identified 21 transcripts derived from 11 distinct ERV loci whose abundance increased following infection in a manner correlated with abscess susceptibility (Figure 1 C).

[0127] ERVs are integrated proviruses from past retroviral infections and have coevolved with eukaryotic genomes for millennia (Johnson, WE Nat Rev Microbiol 2018, 17, 355-370). Functional ERVs can be transcribed, packaged, and secreted as mature viral particles, as well as reverse- transcribed to DNA and reintegrated into the chromosome. Mice possess several classes of ERVs that reflect varying histories of viral infections (Stocking, et al. Cell Mai Life Sci 2008, 65, 3383-3398; Young, et al. Nature 2012, 491 , 774-778). Comparative analysis of the 11 abscess- associated ERV loci against known murine ERVs revealed that 10 loci cluster with xenotropic murine leukemia viruses (MLV) and related ERV loci (Xmvs), while one clusters with polytropic MLVs and related ERV loci (Pmvs) (Figure 1 D). 4 MLV loci encode full-length proviruses with intact reading frames for all viral proteins (Figure 1 E), including the major structural polyprotein (Gag), reverse transcriptase / integrase (Pol), and envelope protein (Env). These data together raise the possibility that E. coli bloodstream infection leads to the production of MLV particles and / or the production of cytosolic DNA.

[0128] Example 2. Nucleoside / nucleotide reverse transcriptase inhibitors prevent abscess formation.

[0129] ERV reverse transcription can generate cytosolic cDNA or DNA:RNA hybrids, which can induce immune signaling through cytoplasmic nucleic acid sensors (Lima-Junior, et al. Cell 2021 , 184, 3794- 3811 ). We hypothesized that reverse transcriptases stimulate inflammatory responses through creation of cytosolic DNA, ultimately driving abscess formation. To assess the role of reverse transcription in abscess formation, we treated B6N mice with a cocktail of nucleoside / nucleotide reverse transcriptase inhibitors (NRTIs), which are also used in the treatment and prevention of HIV infection (Lima-Junior, et al. Cell 2021 , 184, 3794-3811 , Holec, et al. Curr HIV Res 2017, 15, 411-421 )

[0130] Abscess formation was entirely prevented when a cocktail consisting of 1 .6 mg each of tenofovir, emtricitabine, zidovudine, and abacavir was delivered intraperitoneally 1 day prior to inoculation, 4 hours prior to inoculation, immediately following inoculation, and 1 day post inoculation (dpi) (Figure 2A, column 3). The high efficacy of the NRTI cocktail was also observed with a single dose administered immediately following E. coli inoculation (Figure 2A, column 5). To determine whether any individual constituent of the cocktail was responsible for preventing abscess formation, we treated mice with tenofovir / emtricitabine (which are used together clinically), zidovudine, or abacavir alone. Surprisingly, no individual constituent phenocopied the high efficacy of the NRTI cocktail (Figure 2A, columns 7-9). However, at higher doses equal to the total dose of the NRTI cocktail, both tenofovir / emtricitabine and abacavir alone significantly reduced abscess formation (Figure 2A, columns 10-11 ). Timing was critical, as administration of both drugs 1 dpi did not prevent abscess formation (Figure 2A, columns 12-13). These observations together reveal that preventing abscess formation with NRTIs requires a specific dose administered during a critical window early after infection, the time frame where inflammatory host pathways are triggered (Hullahalli, et al. Genetic and immune determinants of E.coli liver abscess formation, bioRxiv, 2023, 543319). Since both tenofovir / emtricitabine and abacavir reduced abscess formation when administered alone, their efficacies are likely due to shared downstream consequences, which may include similar off-target effects or, as we hypothesize, direct inhibition of endogenous reverse transcriptases.

[0131] Tenofovir, emtricitabine, and abacavir do not inhibit E. coli growth in vitro (Figure 2B). Thus, the efficacy of tenofovir / emtricitabine and abacavir alone (Figure 2A, columns 10- 11 ) is likely not due to direct bacterial killing. In contrast, zidovudine strongly inhibits E. co / / growth (Figure 2B). However, despite the presence of zidovudine in the NRTI cocktail, zidovudine alone did not reduce abscess susceptibility (Figure 2A, column 8), and therefore the efficacy of the NRTI cocktail is likely not due to bacterial killing, However, it is possible that NRTIs are uniquely bactericidal in vivo. To provide further evidence that NRTI efficacy does not require direct bacterial killing, we examined whether NRTI treatment prevents formation of the necrotic liver lesions that form following inoculation with formalin-killed bacteria; these lesions are smaller, less abundant, and morphologically distinct from abscesses containing live E. coli, and presumably result from inflammation-induced necrosis without sustained immune cell infiltration following bacterial replication (Figure 2C, middle row). NRTI treatment prevented the formation of the necrotic lesions (Figure 2C, bottom row), revealing that NRTIs are modulating the host response to bacterial molecules. Although it is formally possible that NRTIs heighten bacterial killing in vivo in a manner specific to the use of live bacteria, these data together indicate that NRTIs prevent liver abscesses by inhibiting the formation of the necrotic region that serves as the site for bacterial replication.

[0132] In sum, here we demonstrate that NRTIs prevent liver abscess formation during E. coli bloodstream infection. We propose that NRTI efficacy is due to inhibition of ERV reverse transcription, leading to a reduction in the levels of cytosolic DNA and diminished proinflammatory responses. Since liver abscesses are highly tissue-specific and sex- dependent, we speculate that ERV expression and downstream signaling may be regulated in a sex- and tissue-dependent manner. Future work will be targeted towards dissecting the role of ERVs in liver abscess formation, including identifying the host cell types that mediate NRTI-dependent responses and characterizing upstream pathways and downstream consequences of ERV expression.

[0133] In homeostatic conditions, silencing ERVs is critical for the host, since the presence of viral epitopes or ERV-mediated insertional mutagenesis can promote autoimmune diseases or cancer (Kassiotis, G. Annu Rev Immunol 2023, 41 , 99-125, Geis, et al. Viruses 2020, 12). In mice, phenotypes resembling lupus are associated with polymorphisms in SNERV1 / 2, which encode suppressors of ERV expression (Treger, et al. Immunity 2019, 50, 334-347). However, during infection, ERVs and potentially other retroelements (e.g., (long interspersed nuclear elements [LINEs]) may amplify inflammatory responses by stimulating innate immune pathways that recognize DNA. In our model for liver abscess formation, inflammation leads to hepatic necrosis which facilitates bacterial replication. However, in other infection contexts, it is possible that inflammatory responses mediated by reverse transcription may promote pathogen control. Thus, whether ERVs - and NRTI treatment - are beneficial or detrimental for the host is likely context-dependent. Together, our data suggest that NRTIs and potentially other antiretrovirals should be reexamined for the treatment of other diseases where reverse transcription may drive detrimental inflammatory responses.

[0134] The above results described in Examples 1 -2 were obtained using the following materials and methods.

[0135] MATERIALS AND METHODS

[0136] Animals

[0137] All animal experiments were conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the Animal Welfare Act of the United States Department of Agriculture using protocols reviewed and approved by Brigham and Women's Hospital Committee on Animals (Institutional Animal Care and Use Committee (IACUC) protocol number 2016N000416 and Animal Welfare Assurance of Compliance number A4752-01 ).8-12- week-old female mice were used for all experiments. Mice were obtained from Jackson Laboratories (C57BL / 6J (B6J, #000664), C57BL / 6NJ (B6N, #005304), BALB / cJ (#000651 )). Animals were maintained at 68-75QC with 50% humidity in 12 hour day-night cycles. For infections, defined volumes of frozen CHS7-STAMP library (derived from strain CFT073, isolated from the blood of a patient with pyelonephritis (Welch, et al. Proc Natl Acad Sci 2002, 99, 17020-17024 , Hullahalli et al. Elite, 10, e70910)) from overnight cultures were thawed, diluted in PBS, and immediately used to inoculate mice at a dose of 5x106. For injection of killed bacteria, frozen bacteria were resuspended in 4% paraformaldehyde and incubated at room temperature for 20 minutes, washed twice in PBS, and resuspended in PBS. NRTIs were dissolved in PBS and administered at doses indicated by intraperitoneal injection in a total volume of 200p1 (Tenofovir disoproxil fumarate: Fisher Scientific AC461250010, Emtricitabine: Sigma PHR2120-500MG, Zidovudine: Sigma PHR1292-1 G, Abacavir: Fisher Scientific AC458860010). For intravenous injections, animals were restrained using a Broome-style restrainer (Plas- Labs) and inoculated via the lateral tail vein with 100p1 using a 27G needle. A heating pad was used to facilitate dilation of the tail vein. For intraperitoneal injections, animals were injected with 200p1 into the abdominal cavity with a 25G needle. Animals were euthanized by isoflurane inhalation and cervical dislocation. To quantify CFU, organs were excised and homogenized with 2 x 3.2 mm stainless steel beads for 2 minutes with a bead beater (BioSpec). Organs were plated and diluted on LB + 50pg / ml Kanamycin. Abscess were defined as the visible appearance of at least one white lesion and CFU > 104, which are the criteria established and used in our previous study (Hullahalli, et al. Genetic and immune determinants of E.coli liver abscess formation, bioRxiv, 2023, 543319).

[0138] RNA Sequencing

[0139] BALB / cJ, B6J, and B6N females were infected IV and livers were immediately flash frozen in liquid nitrogen 4 hours post inoculation. Total RNA was extracted with the Direct-zol RNA miniprep kit (Zymo) and library preparation was performed with the NEB Ultra II Directional RNA Library Prep Kit (New England Biolabs). Libraries were sequenced on a NovaSeq 6000 instrument at Harvard Medical School Biopolymers Core Facility as 1 x101 nt reads. Reads were trimmed with T rimGalore using default settings and mapped using HISAT2 to the GRCm38 (mm10) mouse genome. FeatureCounts (Liao, et al. Bioinformatics 2014, 30, 923-930) was used to quantify transcript abundance. Only uniquely mapped reads were counted, and therefore highly repetitive loci with no unique sequences were excluded. ERV loci were counted using .gtf files from the Genome-based Endogenous Viral Element (gEVE) database (2). Counts were analyzed using DESeq2 for differential expression analysis (Love, et al. Genome Biol 20 4, 15, 550). To identify infection-induced transcripts that correlated with abscess frequency, we quantified differentially regulated genes with adjusted P- value less than 0.05 and Iog2 fold change greater than 1 , between infected B6J vs infected BALB / cJ, infected B6N vs infected B6J, and infected B6N vs uninfected B6N.

[0140] Growth Curves

[0141] Approximately ~10^ CFU were collected from E. coli colonies on LB plates and resuspended in 10ml of LB. Bacterial suspensions were diluted 1 / 3 in NRTIs with indicated concentrations in LB and cultured overnight with shaking in a microplate reader (BioTek). CD600 was monitored every 10 minutes. Sequence analysis

[0142] Nucleotide sequences correspond to MLV genomes and MLV-related ERV loci were aligned using CLUSTAL-omega. A segment corresponding to the start of the gag gene through the end of the envgene (relative to Moloney MLV) was extracted and used to identify the best-fit tree by maximum likelihood using PHYML, as implemented in Geneious Prime (2023.1 .2). The tree image was created in FigTree (v.1 .4.4). HEMV, an ERV from Mus spiceligus, was designated as an outgroup based on previously reported relationship to MLVs (Tipper, et al. J Viral 2005, 79, 8316-8329).

[0143] EXAMPLE 3 - Reverse transcriptase inhibitors diminish systemic proinflammatory responses to bacterial pathogens

[0144] In this example, we characterize the effects of nucleotide / nucleoside reverse transcriptase inhibitors (NRTIs) on the innate immune response to bacteria. In the liver, NRTI administration following E. coli inoculation reduced the expression of a large repertoire of proinflammatory transcripts. NRTIs also had systemic anti-inflammatory effects, including reducing proinflammatory cytokine levels in serum in response to E. coli 'n different mouse strains. The anti-inflammatory effects of NRTIs were also apparent in response to lipopolysaccharide (LPS) and Staphylococcus aureus, indicating that the molecular mechanisms underlying the immunomodulatory functions of NRTIs are conserved across distinct immune signaling pathways. Moreover, in a model of lethal LPS shock, NRTI administration prevented hypothermia and death. Together, these results revealed that NRTIs impede systemic inflammatory responses during Gram-positive and Gramnegative bacterial infections.

[0145] Our results demonstrate that treatment of mice with reverse transcriptase inhibitors leads to broad reductions in systemic proinflammatory responses during bacterial infections and protects mice from acute death in a lethal model of sepsis.

[0146] RESULTS

[0147] NRTIs diminish proinflammatory gene expression in the liver

[0148] We tested whether NRTI treatment modulates early hepatic inflammatory responses to E. coli infection by characterizing the influence of NRTIs on the liver transcriptome. Female C57BL / 6NJ mice, which are highly susceptible to E. coli mediated liver abscess formation, were inoculated i.v. with E. coli and then administered a single dose of NRTIs or vehicle control i.p. Four hours post inoculation (hpi), the livers were harvested and subject to RNA-sequencing (Fig. 3A).

[0149] Principal component analysis demonstrated that mice infected with E. coli and administered vehicle control were clearly separated along the first principal component (79.4% of variance) from mice infected with E. coli and subsequently administered the NRTI cocktail (Fig. 3B). Differential expression analysis revealed that 221 genes had higher expression (adjusted R value < 0.001 , Iog2 fold change >0.9) and 469 genes had lower expression following NRTI treatment in infected mice (Fig. 3C). Several major components of the innate immune response to bacteria had reduced expression following NRTI treatment, including components of the NFkB signaling apparatus (Relb, Rel, Nfkbie, Nfkbiz), numerous proinflammatorycytokines (Tnf, Ccl2, Ccl4, Cxcl2), and other hallmarks of inflammation (Nos2, Myd88, Gsdmd, Saa3). Gene Set Enrichment Analysis (Subramanian, et al. Proc Natl Acad Sci 2005, 102, 15545-15550) corroborated these observations, as inflammatory pathways engaged during bacterial infection were significantly and negatively enriched in NRTI-treated infected mice (Fig. 3D). Transcripts that were more abundant following NRTI administration were primarily associated with growth factor signaling (Vegfd, Fgf21 , Bmp10, Kdr).

[0150] RNA sequencing described above identified hepatic genes induced by E. coli infection (Hullahalli, et al. Proc Natl Acad Sci 2024, 121 , e2319162121 ). By intersecting the genes that are differentially regulated by E. coli infection and those that are sensitive to NRTIs in infected mice, we assessed how NRTI treatment modulates E. co / / -induced gene expression in the liver (Fig. 3E). Among the 3,078 genes that are upregulated by E. coli infection, 275 (9%) had reduced expression following NRTI treatment, while only 5 (0.16%) had increased expression following NRTI treatment. Among the 2,984 genes that are downregulated by E. coli infection, 174 (5.8%) had increased expression following NRTI treatment, while 29 (0.9%) had lower expression following NRTI treatment. Notably, comparing genome-wide fold changes revealed that the effects of NRTI treatment and E. coli infection are significantly anti-correlated (slope = -0.13, P < 2.2e-16 by Spearman correlation, Fig. 3E). These observations indicate that NRTI treatment partially reverses the hepatic differential gene expression profile resulting from E. coli infection, where among the 483 genes differentially regulated by both NRTIs and E. coli, 93% are inversely correlated in their expression. In particular, the expression of numerous components of the inflammatory response is diminished in the presence of NRTIs. A notable exception is 1110, a prototypical anti-inflammatory cytokine whose expression increases following E. coli infection and increases further following NRTI administration (Couper, et al. J Immunol 2008, 180, 5771-5777; Agag, et al. Brain Behav Immun 2018, 74, 176-185).

[0151] Rapidly following bloodstream infection, the liver captures bacterial pathogens and recruits inflammatory immune cells, particularly macrophages, monocytes, and neutrophils (Llorente, et al Cell Host Microbe 2016, 20, 1-2). Therefore, the reduction in inflammatory gene expression in the liver could potentially be attributable to reduced numbers of infiltrating cells. Flow cytometry on mice infected with E. coli and administered NRTIs or vehicle control was used to determine whether the number of liver-infiltrating cells is altered following NRTI administration (Fig. 3F). The numbers of monocytes (CD11 b+, Ly6C+, Ly6G-), monocyte-derived macrophages (CD11 bhi, F4 / 80+, Ly6C-, Ly6G-), and neutrophils (CD11 b+, Ly6C+, Ly6G+) relative to total CD45+ immune cells were identical in vehicle- and NRTI-treated infected mice. Thus, early after infection, NRTIs appear to diminish inflammatory gene expression in the liver without altering the number of infiltrating cells, potentially accounting for the fact that NRTI treatment does not impair E. coli clearance from the liver (Hullahalli, et al. Proc Natl Acad Sci 2024, 121 , e2319162121 ). NRTIs diminish systemic proinflammatory responses

[0152] We next examined whether NRTIs modulate systemic inflammatory responses by profiling the abundance of proinflammatory cytokines in serum (Fig. 4A). In C57BL / 6NJ mice infected with E. coli, NRTI administration led to significant reductions in several proinflammatory cytokines in serum at 4 hpi (black / gray points, Fig. 4A). This trend continued at 8 hpi (green points), although the amounts of these inflammatory markers were lower at 8 vs 4 hpi, regardless of NRTI treatment. Thus, the ability of NRTIs to blunt inflammatory cytokine levels does not appear to result from a delay their production. Similar reductions were observed at 4 hpi in BALB / cJ mice (blue points), a strain that does not develop liver abscess from E. coli infection, indicating that the anti-inflammatory effects of NRTIs are not mouse strain-specific or limited to animals that develop liver necrosis associated with E. coli bacteremia. Interestingly, the abundance of IL10 was consistently increased in NRTI-treated mice, corroborating its RNA expression pattern in the liver (Fig. 4C). These results together indicate that NRTIs broadly diminish systemic proinflammatory cytokine production in mice during E. coli systemic infections. Notably, since BALB / cJ and C57BL / 6NJ are distantly related among inbred mice (Beck, et al. Nat Genet 2000, 24, 23-25), the observation that NRTIs dampen proinflammatory responses in both strains may suggest that the underlying molecular mechanisms are conserved across mice.

[0153] The dominant immunostimulatory molecule on E. coins lipopolysaccharide (LPS) (Miller, et al. Nat Rev Microbiol 2005, 3, 36-46), and we tested whether NRTIs modulate the inflammatory response to purified E. coli LPS alone. Mice were inoculated with 250 pg of LPS i.v. and administered either NRTIs or PBS control i.p. NRTI treatment reduced the abundance of most proinflammatory markers that were reduced with E. coli infection and increased IL10 production (Fig. 4A, pink points). However, the levels of some cytokines, such as KC and IL-6, that were altered by NRTIs during E. coli infection were unchanged during LPS stimulation. We also tested whether NRTIs diminish inflammatory responses induced by the Gram-positive pathogen Staphylococcus aureus. Although the stimulation of inflammatory cytokines by S. aureus was not as broad or pronounced as observed with E. coli, NRTIs significantly reduced the abundance of MCP-1 , IP-10, and MIP-i p (Fig. 4A, brown points). Finally, we examined whether NRTI treatment could improve clinical outcomes during lethal LPS shock, a commonly used model for sepsis (Stortz, et al. ILAR J, 2017, 58, 90-105). Mice were administered 200 pg of LPS i.v. (50% lethal dose) and either NRTIs or vehicle control. NRTIs prevented LPS-induced hypothermia (Fig. 4B) and death (Fig. 4C). Together, these data indicate that NRTIs broadly diminish inflammatory responses to bacteria and can protect mice from lethal endotoxin shock. Since NRTIs diminish inflammatory cytokine production in the context of both Gram-negative and Gram-positive infection, the molecular mechanisms underlying the anti-inflammatory effects of NRTIs may be shared between diverse immune signaling pathways.

[0154] In Figure 3C, we demonstrate that the NRTI cocktail consisting of tenofovir disoproxil fumarate, emtricitabine, zidovudine, and abacavir reduces the expression of Nos2 in infected mice. Nos2 encodes inducible nitric oxide synthase (iNOS) and is notable since it is also a classical marker for the activation of macrophages by proinflammatory signals such as bacteria, raising the possibility that NRTIs elicit their anti-inflammatory functions in mice in part by directly modulating the activation of macrophages during bacterial infections. To directly test whether NRTI treatment modulates macrophage activity, we cultured bone-marrow derived macrophages (BMDMs) and quantified the impact of NRTI treatment during lipopolysaccharide (LPS) stimulation by measuring mRNA expression of Nos2 using RT-qPCR. At 400 pg / ml, tenofovir (disoproxil fumarate) and abacavir both individually abolished Nos2 induction. These observations indicate that two distinct NRTIs both elicit similar anti-inflammatory effects in macrophages.

[0155] In addition, in a Nos2 expression in vitro assay, tenofovir as well as abacavir each attenuated the effects of LPS (Fig. 5).

[0156] The above results in this Example were obtained using the following materials and methods.

[0157] MATERIALS AND METHODS

[0158] Animal experiments

[0159] All mice used in this study were 8- to 10-week-old females obtained from The Jackson Laboratories (C57BL / 6NJ — #5304 and BALB / cJ — #651 ). For intravenous inoculation, mice were restrained in a Broome-style restrainer and 100 pL of inoculum was injected into the tail vein with a 27G needle. A heating pad was used to facilitate the dilation of the tail vein. PBS was used as a vehicle for all inocula in this study. NRTIs were prepared from 100 mg / mL stocks in PBS and freshly diluted to 8 mg / mL for each drug (Tenofovir disoproxil fumarate, emtricitabine, zidovudine, and abacavir). Two hundred microliters was administered intraperitoneally. E. coli (strain CHS7- STAMP) and S. aureus (strain HG003) were prepared by diluting frozen stocks in PBS and were immediately used for inoculation at a dose of 5 x 106 CFU per mouse (Hullahalli et al. Elife, 10, e70910; Sit, et al. Nature New Biol 2023, 613, 721-728). LPS (Sigma L2630) was stored frozen as 8 mg / mL solutions in PBS and diluted freshly before use. For euthanasia, mice were overdosed with isoflurane and cervical dislocation or exsanguination (for collecting serum) was performed. Body temperature was monitored with a rectal probe.

[0160] Serum collection and cytokine measurements

[0161] Blood was collected by cardiac puncture and was left at room temperature for 20 min. Serum was obtained by centrifugation of clotted blood at 1 ,000 x g for 10 min in a refrigerated centrifuge and filtered using a 0.22 pm centrifugal filter. Cytokine abundance was measured using Luminex technology (EveTechnologies) in duplicate.

[0162] RNA-sequencing Livers were harvested and the right lobe was flash frozen in liquid nitrogen. RNA was extracted with Trizol and the Direct Zol RNA Miniprep Kit (Zymo). Illumina-compatible libraries were prepared with the NEBNext Poly(A) mRNA Magnetic Isolation Module and the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). Libraries were sequenced on a NextSeq 1000 as 1 x 101 nt reads. Reads were processed with TrimGalore and mapped to the reference C57BL / 6J chromosome (mm10, GRCm38) using HISAT2 (Kim, et al. Nat. Biotech. 2019, 37, 907-915). Reads counts were obtained with FeatureCounts and differential expression was performed with DESeq2 (Love, et al. Genome Biol, 2014, 15, 550). Gene Set Enrichment Analysis was performed using the fgsea package in R (Korotkevich, et al. Fast gene set enrichment analysis, bioRxiv, 2021 .).

[0163] Flow cytometry

[0164] Livers were dissected from mice and coarsely chopped with scissors and stored in HBSS + 10 mM EDTA until all mice were dissected. Tissue chunks were washed 3x with 50 mL PBS to remove EDTA and incubated in 10 mL DMEM containing 0.2 mg / mL DNase (Roche 10104159001 ) and 1 mg / mL Collagenase (Sigma-Aldrich C5138) for 15 min at 37°C. Dissociated cells were passed through a 70 pm filter, which was further flushed with ~20 mL of DMEM. Cells were spun at 50 x g for 2 min to spin down hepatocytes. The supernatant containing nonparenchymal cells was harvested in a 15 mL conical and centrifuged at 500g for 5 min. Cells were resuspended in 1 mL of red blood cell lysis buffer (Sigma 11814389001 ) and incubated for 2 min. Ten milliliters of PBS was added, and cells were centrifuged at 500 x g for 5 min. To remove debris, cells were washed three times with PBS + 10 mM EDTA + 2% FBS (FACS Buffer) and finally resuspended in 2 mL of FACS buffer. Two hundred microliters of cell suspensions were used for flowcytometry. Cell surface Fc receptors were blocked with anti-CD16 / 32 (Biolegend 101302) for 5 min and stained with the following antibody cocktail in FACS buffer for 30 min at room temperature; Live / Dead Aqua, anti- Cd45 + Alexa Fluor 700 (BD Bioscience 560510), anti-Ly6c PerCP-Cy5.5 (Biolegend 128011 ), anti- Ly6g APC-Cy7 (Biolegend 127623), anti-Cd11 b APC (Biolegend 553312), anti-Cd11c PE-Cy7 (Biolegend 558079), anti-Cd3e Super Bright 600 (ThermoFisher 63-0031 -82), anti-F4 / 80 PE-Fire 640 (Biolegend 157320), anti-B220 PE-Fire 810 (Biolegend 103287). Cells were washed in FACS buffer twice, fixed in 4% PFA-PBS, and analyzed with the Cytek Northern Lights instrument (Cytek, MA).

[0165] Statistical analyses

[0166] Statistical analyses were performed in R or Graphpad Prism. Descriptions of specific methods are provided in respective figure legends.

[0167] Nos2 in vitro assay Bone marrow macrophages were cultured using M-CSF as previously described (Mendoza et al 2022, Methods Mol Biol). After differentiation, LPS (0.1 pg / ml), tenofovir disoproxil fumarate (400pg / ml) and abacavir (400pg / ml) were added to 200,000 macrophages in 24-well plates. After 4 hours post stimulation, RNA was isolated with Trizol and RT-qPCR was performed to quantify Nos2 expression using the Luna OneStep RT-qPCR Kit (New England Biolabs) and with the Applied Biosystems StepOnePlus Real-Time PCR System.

[0168] Enumerated Embodiments

[0169] The subject matter of the present disclosure is also exemplified by the following enumerated embodiments:

[0170] Embodiment 1 . A method of treating or preventing a liver abscess in a patient by administering a reverse transcriptase inhibitory composition to the patient in an amount effective to prevent the development of the abscess.

[0171] Embodiment 2. A method of treating sepsis in a mammal by administering a reverse transcriptase inhibitory composition to the patient in an amount effective to treat the sepsis.

[0172] Embodiment 3. A method of treating an inflammatory condition in a patient comprising administering a reverse transcriptase inhibitory composition to the patient with the condition.

[0173] Embodiment 4. The method of embodiment 3 wherein the inflammatory conditions is caused by an infectious agent.

[0174] Embodiment 5. The method of embodiment 3 wherein the administering of the reverse transcriptase inhibitory composition prevents the formation of liver abscesses.

[0175] Embodiment 6. A method of reducing the levels of proinflammatory cytokines in a patient comprising administering a reverse transcriptase inhibitory composition to the patient in an amount effective to cause reduction in the levels.

[0176] Embodiment 7. The method of any one of embodiments 1 -5 wherein the reverse transcriptase inhibitory composition is a nucleotide / nucleoside reverse transcriptase inhibitory composition.

[0177] Embodiment 8. The method of any of embodiment 6 wherein the nucleotide / nucleoside reverse transcriptase inhibitory composition is selected from tenofovir, emtricitabine, abacavir, zidovudine, zalcitabine, stavudine, lamivudine, entecavir truada, azvudine, tenofovir, adefovir, and islatravir. Embodiment 9. The method of any of embodiments 1-5 wherein the reverse transcriptase inhibitory composition is a non-nucleotide / nucleoside reverse transcriptase inhibitory composition. Embodiment 10. The method of embodiment 8 wherein the non-nucleotide / nucleoside reverse transcriptase inhibitory composition is selected from efavirenz, nevirapine, delavirdine, etravirine, rilpivirine, doravirine, and elsulfavirine.

[0178] Embodiment 11 . Any and all compositions, articles of manufacture, methods, and uses disclosed and / or described in the above specification.

[0179] The subject matter of the present disclosure is further exemplified by the following enumerated embodiments:

[0180] Embodiment 12. A method of reducing an inflammatory response or reducing the risk of developing inflammation resulting from a pathogen or the presence of a toxin in a mammal, the method comprising administering to the mammal a composition comprising at least one reverse transcriptase inhibitor, in an amount sufficient to reduce the inflammatory response or reduce the risk of developing the inflammation.

[0181] Embodiment 13. The method of embodiment 12, wherein the composition comprises two or more reverse transcriptase inhibitors.

[0182] Embodiment 14. The method of embodiment 12, wherein the inflammation or inflammatory response causes tissue damage.

[0183] Embodiment 15. The method of embodiment 12, wherein the inflammatory response or inflammation causes sepsis.

[0184] Embodiment 16. The method of embodiment 12, wherein the inflammatory response or the inflammation results from the presence of a bacterial toxin.

[0185] Embodiment 17. The method of embodiment 16, wherein the bacterial toxin is a component of a gram-negative bacterium.

[0186] Embodiment 18. The method of embodiment 16, wherein the bacterial toxin is a component of a gram-positive bacterium.

[0187] Embodiment 19. The method of embodiment 17, wherein the bacterial toxin is lipopolysaccharide. Embodiment 20. The method of embodiment 12, wherein the administering reduces a systemic proinflammatory response.

[0188] Embodiment 21 . The method of embodiment 12, wherein the administering decreases a cytokine response.

[0189] Embodiment 22. The method of embodiment 21 , wherein the cytokine response comprises a decrease in at least one of IFN-y, CXCL1 , TNF-a, MIP-1a, MIP-10, MCP-1 , KC, IP-10, IL-6, or IL- 1 .

[0190] Embodiment 23. The method of embodiment 12, wherein the administering increases a cytokine response.

[0191] Embodiment 24. The embodiment of claim 23, wherein the cytokine response comprises an increase in IL-10.

[0192] Embodiment 25. The method of embodiment 12, wherein the pathogen is a bacterial, fungal, or viral pathogen.

[0193] Embodiment 26. The method of embodiment 12, wherein the reverse transcriptase inhibitor is a nucleoside / nucleotide reverse transcriptase inhibitor.

[0194] Embodiment 27. The method of embodiment 26, wherein the nucleoside / nucleotide reverse transcriptase inhibitor is abacavir, adefovir, alovudine, azvudine, censavudine, didanosine, emtricitabine, entecavir, islatravir, lamivudine, stavudine, tenofovir, zalcitabine, or zidovudine.

[0195] Embodiment 28. The embodiment of claim 12, wherein the composition comprises tenofovir, tenofovir alafenamide fumarate, tenofovir disoproxil fumarate, or tenofovir exalidex.

[0196] Embodiment 29. The method of embodiment 12, wherein the composition comprises censavudine.

[0197] Embodiment 30. The method of embodiment 12, wherein the composition comprises tenofovir.

[0198] Embodiment 31. The method of embodiment 12, wherein the composition comprises abacavir.

[0199] Embodiment 32. The method of embodiment 13, wherein the composition comprises emtricitabine and tenofovir. Embodiment 33. The method of embodiment 13, wherein the composition comprises abacavir and tenofovir.

[0200] Embodiment 34. The method of embodiment 13, wherein the composition comprises abacavir, emtricitabine, tenofovir, and zidovudine.

[0201] Embodiment 35. The method of embodiment 12, wherein the reverse transcriptase inhibitor is a non-nucleoside / nucleotide reverse transcriptase inhibitor.

[0202] Embodiment 36. The method of embodiment 35, wherein the non-nucleoside / nucleotide reverse transcriptase inhibitor is delavirdine, doravirine, efavirenz, elsulfavirine, etravirine, fosdevirine, lersivirine, nevirapine, or rilpivirine.

[0203] Embodiment 37. The method of embodiment 12, wherein the reverse transcriptase inhibitor reduces expression of an endogenous retrovirus (ERV).

[0204] Embodiment 38. The method of embodiment 12, wherein the mammal is a human.

[0205] Other Embodiments

[0206] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.

[0207] What is claimed is:

Claims

CLAIMS1 . A method of treating inflammation in a mammal in need thereof, said method comprising:(i) providing a mammal with elevated host endogenous reverse transcriptase produced by host endogenous retroviruses (ERVs); and(ii) following step (i), contacting in vivo the reverse transcriptase with a reverse transcriptase inhibitor in an amount sufficient to ameliorate ERV-induced inflammatory response.

2. The method of claim 1 , wherein the ERV-induced inflammatory response is concomitant with sepsis in the mammal.

3. The method of claim 1 , wherein the ERV-induced inflammatory response is concomitant with tissue damage in the mammal.

4. The method of claim 1 , wherein the ERV-induced inflammatory response is concomitant with autoimmune disease in the mammal.

5. The method of claim 1 , wherein the ERV-induced inflammatory response is concomitant with bacterial or fungal or non-retrovirus infection in the mammal.

6. The method of claim 1 , wherein the ERV-induced inflammatory response is concomitant with cytokine storm in the mammal.

7. A method of treating pathogen-induced inflammation in a mammal in need thereof, said method comprising:(i) providing a mammal with elevated host endogenous reverse transcriptase; and(ii) following step (i), contacting in vivo the reverse transcriptase with a reverse transcriptase inhibitor in an amount sufficient to ameliorate the pathogen-induced inflammation.

8. The method of claim 7, wherein the host endogenous reverse transcriptase is produced by a retroelement.

9. The method of claim 8, wherein the retroelement is an endogenous retrovirus (ERV) or a long interspersed nuclear element (LINE).

10. The method of claim 7, wherein the pathogen-induced inflammation is concomitant with sepsis in the mammal.11 . The method of claim 7, wherein the pathogen-induced inflammation is concomitant with tissue damage in the mammal.

12. The method of claim 7, wherein the pathogen-induced inflammation is concomitant with bacterial or fungal or non-retrovirus infection in the mammal.

13. The method of any one of claims 1 to 12, wherein the reverse transcriptase inhibitor is a nucleoside / nucleotide reverse transcriptase inhibitor.

14. The method of claim 13, wherein the nucleoside / nucleotide reverse transcriptase inhibitor is tenofovir, abacavir, adefovir, alovudine, azvudine, censavudine, didanosine, emtricitabine, entecavir, islatravir, lamivudine, stavudine, zalcitabine, or zidovudine.

15. The method of claim 14, wherein the nucleoside / nucleotide reverse transcriptase inhibitor is tenofovir.

16. The method of claim 14, wherein the nucleoside / nucleotide reverse transcriptase inhibitor is abacavir.

17. The method of claim 14, wherein the nucleoside / nucleotide reverse transcriptase inhibitor is censavudine.

18. The method of claim 13, wherein the reverse transcriptase inhibitor comprises two or more nucleoside / nucleotide reverse transcriptase inhibitors.

19. The method of claim 18, wherein the two or more nucleoside / nucleotide reverse transcriptase inhibitors are tenofovir and emtricitabine.

20. The method of claim 18, wherein the two or more nucleoside / nucleotide reverse transcriptase inhibitors are tenofovir and abacavir.21 . The method of claim 18, wherein the two or more nucleoside / nucleotide reverse transcriptase inhibitors are tenofovir, emtricitabine, abacavir, and zidovudine.

22. The method of claim 18, wherein the two or more nucleoside / nucleotide reverse transcriptase inhibitors are selected from the group consisting of abacavir, adefovir, alovudine, azvudine, censavudine, didanosine, emtricitabine, entecavir, islatravir, lamivudine, stavudine, tenofovir, zalcitabine, and zidovudine.

23. The method of claim 13, wherein the contacting comprises administering to the mammal a pharmaceutical composition comprising a nucleoside / nucleotide reverse transcriptase inhibitor selected from the group consisting of abacavir, adefovir, alovudine, azvudine, censavudine, didanosine,emtricitabine, entecavir, islatravir, lamivudine, stavudine, tenofovir, zalcitabine, zidovudine, and prodrugs thereof.

24. The method of claim 23, wherein the contacting comprises administering to the mammal a pharmaceutical composition comprising a prodrug selected from adefovir dipivoxil, tenofovir disoproxil, tenofovir disoproxil hemifumarate, tenofovir disoproxil fumarate, tenofovir alafenamide, tenofovir alafenamide hemifumarate, tenofovir alafenamide fumarate, tenofovir amibufenamide, and tenofovir exalidex.

25. The method of any one of claims 1 to 12, wherein the reverse transcriptase inhibitor is a non- nucleoside / non-nucleotide reverse transcriptase inhibitor.

26. The method of claim 25, wherein the non-nucleoside / nucleotide reverse transcriptase inhibitor is delavirdine, doravirine, efavirenz, elsulfavirine, etravirine, fosdevirine, lersivirine, nevirapine, or rilpivirine.

27. The method of claim 25, wherein the contacting comprises administering to the mammal a pharmaceutical composition comprising a non-nucleoside / non-nucleotide reverse transcriptase inhibitor selected from the group consisting of delavirdine, doravirine, efavirenz, elsulfavirine, etravirine, fosdevirine, lersivirine, nevirapine, and rilpivirine.

28. The method of any of the aforementioned claims, wherein the mammal is a human.

Images