DESC:TECHNICAL FIELD
The present disclosure relates to the field of managing viral infections/diseases. Particularly, the present disclosure relates to a composition and methods for managing viral infections/diseases caused by viruses such as HIV, Coronavirus, influenza, etc. The composition comprises H2S donor either alone or in combination with antiviral agents, and methods include a method of modulating viral replication/reactivation, method of modulating virus latency, method of treating viral infections/diseases, etc.

BACKGROUND AND PRIOR ART
Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immuno-Deficiency Syndrome (AIDS), is responsible for 0.6 million deaths and 1.7 million new infections in 2019 (https://www.who.int/gho/hiv/epidemic_status/deaths_text/en/). Despite advances in antiretroviral therapy (ART), the persistence of latent but replication-competent HIV in cellular reservoirs is a major barrier to cure. Understanding host factors and signaling pathways underlying HIV latency and rebound upon cessation of ART is of the highest importance in the search for an HIV cure. Effective ARVs treatment suppresses plasma HIV levels and improves the quality and life expectancy of infected patients. Contrastingly, ARVs remain unsuccessful in curing HIV infection. Highly active antiretroviral therapy (HAART) comprises a combination of three ARVs, targeting distinct stages of HIV-infection, but fails to eradicate HIV completely. Under HAART, using ultrasensitive assays, it has been revealed that HIV persists in latently infected reservoirs of HIV in long-lived memory CD4+ T cells. Later, upon HAART interruption HIV rebounds and leads to the development of active disease with immunodeficiency and infection with opportunistic pathogens, e.g., Mycobacterium tuberculosis. The latent HIV reservoirs containing integrated viral genome serves as a stable source of virus that can be reactivated to produce viral particles and transmit new infection. Thus, there is an unmet need to treat millions of patients who are on anti-HIV therapy across the globe.

The Covid-19 pandemic caused by SARS-CoV-2 infection has resulted in the most severe health emergency of the decade. With the few clinically approved vaccines, only limited therapeutic options are currently available. There is thus a need for new and effective therapeutics for the management of SARS-CoV-2.
The present disclosure thus, aims to achieve the above objectives by providing compositions and methods to treat HIV, SARS-CoV-2 and other viral infections such as Influenza.

STATEMENT OF THE DISCLOSURE
The present disclosure relates to a composition comprising at least one H2S donor and at least one antiviral agent, optionally along with pharmaceutically acceptable excipient.

The present disclosure also relates to a method of preparing a composition comprising at least one H2S donor and at least one antiviral agent, optionally along with pharmaceutically acceptable excipient, said method comprising: (a) obtaining at least one H2S donor, obtaining at least one antiviral agent and combining the at least one H2S donor and the at least one antiviral agent optionally along with pharmaceutically acceptable excipient to obtain the composition; or (b) assembling at least one H2S donor, at least one antiviral agent, optionally along with pharmaceutically acceptable excipient and an instruction manual.

The present disclosure also relates to an invitro method of modulating viral reactivation, viral replication or viral latency in a cell, said method comprising contacting the cell with an H2S donor optionally along with a pharmaceutically acceptable excipient or the composition of the present disclosure.

The present disclosure also relates to a method of managing a viral infection or a viral disease, said method comprising administering an H2S donor optionally along with a pharmaceutically acceptable excipient or the composition of the present disclosure to a subject in need thereof.

The present disclosure further relates to use of an H2S donor optionally along with a pharmaceutically acceptable excipient or the composition of the present disclosure for managing a viral infection or viral disease in a subject in need thereof

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure where:

Figure 1: HIV-1 reactivation diminishes expression of H2S metabolic enzymes. (A) Schematic showing H2S producing enzymes in mammalian cells. (B) Experimental strategy for measuring HIV-1 reactivation and H2S production in U1. (C) U1 cells were stimulated with 5 ng/ml PMA and the expression of gag transcript was measured at indicated time points. (D and E) Time-dependent changes in the expression of CTH, CBS and MPST at mRNA and protein level during HIV-1 latency (-PMA) and reactivation (+PMA [5 ng/ml]) in U1 cells. (F and G) Time-dependent changes in the expression of CTH, CBS and MPST at mRNA and protein level in U937 cells with or without PMA treatment. Results were quantified by densitometric analysis for CTH, CBS and MPST band intensities and normalized to GAPDH, using ImageJ software. Results are expressed as mean ± standard deviation and are representative of data from three independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001, by two-way ANOVA with Tukey’s multiple comparison test.

Figure 2: HIV-1 reactivation decreases levels of H2S metabolizing enzymes in a T cell line model of latency. (A-B) J1.1 and Jurkat cells were stimulated with 5 ng/ml PMA for 6 h, 12 h, 24 h, and 36 h and total cell lysates were prepared to analyze CTH, CBS, and MPST protein levels. HIV-1 reactivation was assessed by immunoblotting for intracellular viral protein, p24. The results are representative of data from three independent experiments. Results were quantified by densitometric analysis for CTH, CBS, and MPST band intensities and normalized to GAPDH.

Figure 3: HIV reactivation reduces endogenous H2S levels. (A) U1 and U937 cells treated with 5 ng/ml PMA for 24 h or left untreated, stained with AzMC for 30 min at 37?C, and images were acquired using Leica TCS SP5 confocal microscope. Scale bar represents 40 ?m. (B) Average fluorescence intensity was quantified by ImageJ software. Results are expressed as mean ± standard deviation and are representative of data from three independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001, by two-way ANOVA with Tukey’s multiple comparison test.

Figure 4: Genetic silencing of CTH reactivates HIV-1 in U1 cells. (A) Total RNA was isolated from shCTH E8, shCTH E9, and non-targeting shRNA (shNT) lentiviral vectors transduced U1 cells and change in CTH mRNA was examined by RT-qPCR. (B) Cell lysates of U1, shNT, and shCTH E8 were assessed for CTH abundance using immuno-blotting. The CTH band intensities were quantified by densitometric analysis and normalized to GAPDH. (C) U1 cells stably transduced with non-targeting (shNT) and shCTH lentiviral vectors were stained with AzMC for 30 min at 37?C, and images were acquired using ZEISS LSM 880 confocal microscope (B). Scale bar represents 20 ?m. (D) Average fluorescence intensity was quantified by ImageJ software. (E) shCTH and shNT were treated with 5 ng/ml PMA or left untreated for 24 h and HIV-1 reactivation was determined by gag RT-qPCR. (F and G) shCTH and shNT were treated with 5 ng/ml PMA or left untreated. At the indicated time points, HIV-1 reactivation was measured by flow-cytometry using fluorescently tagged (PE-labeled) antibody specific to intracellular p24 (Gag) antigen and p24 ELISA in the supernatant. Results are expressed as mean ± standard deviation and are representative of data from two independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001, by two-way ANOVA with Tukey’s multiple comparison test.

Figure 5: Genetic silencing of CTH reactivates HIV-1 in J1.1 cells. (A) Total cell lysates were prepared from un-transduced (J1.1), non-targeting (shNT), shCBS, and shCTH knockdown J1.1 cells. Expression of CBS, CTH, MPST and HIV-1 p24 levels was assessed by immuno-blotting. Results are representative of data from two independent experiments.

Figure 6: Genetic silencing of CTH alters gene expression associated with redox stress, apoptosis, and mitochondrial function. (A) Total RNA isolated from untreated or PMA (5 ng/ml; 24 h)-treated U1-shNT and U1-shCTH were examined by NanoString Technology to assess the expression of genes associated with HIV infection and oxidative stress response. Heatmap showing functional categories of significantly differential expressed genes (DEGs). Gene expression data obtained were normalized to internal control ß2 microglobulin (B2M), and fold changes were calculated using the nSolver 4.0 software. Genes with fold changes values of >1.5 and P <0.05 were considered as significantly altered.

Figure 7: CTH maintains redox homeostasis and mitochondrial bioenergetics to promote HIV-1 latency. (A) Total and oxidized cellular glutathione (GSSG) content was assessed in U1-shCTH and U1-shNT cell lysates using glutathione assay kit. (B) U1-shNT and U1- shCTH cells were left untreated or treated with 5 ng/ml PMA for 6 h, stained with CM-H2DCFDA dye (5 µM) analyzed using flow cytometry. (C) U1-shNT and U1-shCTH were stained with MitoSOX? Red dye for 30 mins at 37?C and analyzed by flow cytometry (Ex-510 nm, Em-580 nm). (D) Schematic representation of Agilent Seahorse XF Cell Mito Stress test profile to assess key parameters related to mitochondrial respiration. (E) U1-shNT and U1-shCTH (5×104) were seeded in triplicate wells of XF microplate and incubated for 1 h at 37?C in a non-CO2 incubator. Oxygen consumption was measured without adding any drug (basal respiration), followed by measurement of OCR change upon sequential addition of 1 µM oligomycin (ATP synthase inhibitor) and 0.25 µM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), which uncouples mitochondrial respiration and maximizes OCR. Lastly, rotenone (0.5 µM) and antimycin A (0.5 µM) were injected to completely inhibit respiration by blocking complex I and complex III, respectively. (F) Various respiratory parameters derived from OCR measurement were determined by Wave desktop software. nmOCR; non-mitochondrial oxygen consumption rate and SRC; spare respiratory capacity. Error bar represent standard deviations from mean. Results are representative of data from three independent experiments. *, P<0.05; **, P<0.01; ***,P<0.001; ns, nonsignificant, by two-way ANOVA with Bonferroni’s multiple comparison test.

Figure 8: H2S donor (GYY4137) suppresses PMA induced HIV-1 reactivation in U1. (A) Chemical structure of GYY4137. (B) U1 cells were treated with NaHS or GYY4137 and media supernatant was harvested to assess H2S production by methylene blue assay over time. (C) U1 cells were pretreated with 5 mM GYY4137 or 5 mM NAC for 24 and then stimulated with 5 ng/ml PMA for 24 h or 48 h. Cells were stained with 3 ?M propidium iodide (PI) for 15 min in dark, washed, and analyzed using flow cytometry. (D and E) U1 cells were pre-treated with 5 mM GYY4137 or 5 mM NAC for 24 h and then stimulated with 5 ng/ml PMA for 24 h and 48 h. Total RNA was isolated and HIV-1 reactivation assessed by gag RT-qPCR (D). Culture supernatant was harvested to monitor HIV-1 release by p24 ELISA (E). (F) Decomposed GYY4137 (spent GYY4137) which was aerated for at least 180 days and left at room temperature was used to pretreat U1 cells. Cells pretreated with 5 mM spent GYY4137 for 24 h or left untreated were stimulated with 5 ng/ml PMA for 24 h and HIV-1 reactivation was assessed by gag RT-qPCR. Results are expressed as mean ± standard deviation and data are representative of three independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001, by two-way ANOVA with Tukey’s multiple comparison test.

Figure 9: H2S donor (GYY4137) suppresses TNF-? and CTH knockdown mediated HIV-1 reactivation in U1. (A) U1 cells were pretreated with 5 mM GYY417, 5 mM NAC for 24 h or left untreated and then stimulated with 100 ng/ml TNF-? for 24 h and 48 h. HIV-1 reactivation was assessed by gag RT-qPCR. (B) U1-shCTH and U1-shNT were pretreated with 5 mM GYY4137 for 24 h and stimulated with 5 ng/ml PMA for 24 h. Culture supernatant was harvested to determine HIV-1 reactivation by HIV-1 p24 ELISA. Results are expressed as mean ± standard deviation and data are representative of three independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001, by two-way ANOVA with Tukey’s multiple comparison test.

Figure 10: H2S donor (GYY4137) suppresses HIV-1 reactivation in J1.1 cells. (A) J1.1 cells were pretreated with indicated concentrations of GYY4137 or 5 mM NAC for 24 h and then stimulated with 5 ng/ml PMA for 12 h. Cells were harvested to isolate total RNA and HIV-1 reactivation was assessed by gag RT-qPCR. (B) J1.1 cells were pretreated with GYY4137 for 24 h and then stimulated with PMA for 12 h. Cells were then harvested, stained with PI, and subjected to flow cytometry to assess viability. Error bar represent standard deviations from mean. Results are representative of data from two independent experiments. *, P<0.05; **,P<0.01; ***,P<0.001; ****, P<0.0001; ns, nonsignificant, by two-way ANOVA with Tukey’s multiple comparison test.

Figure 11: H2S donor (GYY4137) suppresses HIV-1 reactivation in J-Lat cells. (D) J-Lat 6.3 cells were pretreated with 5 mM NAC or indicated concentrations of GYY4137 for 24 h and then stimulated with 2.5 ?M prostratin for 24 h. HIV-1 reactivation was determined by estimating GFP expressing cells using flow cytometry. Error bar represent standard deviations from mean. Results are representative of data from two independent experiments. *, P<0.05; **,P<0.01; ***,P<0.001; ****, P<0.0001; ns, nonsignificant, by two-way ANOVA with Tukey’s multiple comparison test.

Figure 12: H2S donor (GYY4137) reduces HIV replication in T cells. (A) Jurkat cells were treated with 5 mM, 2.5 mM, 1.2 mM, 0.6 mM and 0.3 mM of GYY4137 for 24 h, 48 h, 72 h, and 96 h. Samples were harvested and stained with PI to determine cells viability by flow cytometry. (B and C) Jurkat cells were infected with HIV-NL4.3 at 0.2 MOI for 4 h, cells were washed, seeded in fresh media, and cultured in presence or absence of 300 µM GYY4137. HIV-1 replication was monitored by measuring intracellular gag transcript levels (B) and p24 protein levels in culture supernatant by ELISA (C). Results are expressed as mean ± standard deviation and data are representative of three independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001, by two-way ANOVA with Tukey’s multiple comparison test.

Figure 13: GYY4137 modulates Nrf2, NF-?B, and YY1 pathways. (A) U1 cells pre-treated with 5 mM GYY4137 for 24 h or left untreated and then stimulated with 5 ng/ml PMA for 24 h or left unstimulated. Total RNA was isolated and expression of genes associated with HIV infection and oxidative stress response was assessed by nCounter NanoString technology. Heatmap showing functional categories of significant DEGs in all four conditions: untreated (U1), GYY4137 alone (U1-GYY) or PMA- alone (U1-PMA) and GYY4137 +PMA (U1-GYY+PMA). Gene expression data obtained were normalized to internal control ß2 microglobulin (B2M), and fold changes were calculated using the nSolver 4.0 software. Genes with fold changes values of >1.5 and P <0.05 were considered as significantly altered. (B) Total cell lysates were used to analyze the expression levels of Nrf2 and HMOX1 by immuno-blotting. Results were quantified by densitometric analysis of Nrf2 and HMOX1 band intensities and normalized to GAPDH. (C) U1 cells were pretreated with 5 mM GYY4137 for 6 h and then stimulated with 30 ng/ml PMA for 4 h or left unstimulated. Cells were harvested to prepare total cell lysate. Levels of phosphorylated NF-?B p65 (Ser536), NF-?B p65, and YY1 were determined by immuno-blotting. Results were quantified by densitometric analysis for each blot and were normalized to GAPDH.

Figure 14: GYY4137 suppresses NF-?B and induces YY1 activity. (A) Schematic depiction of the binding sites for NF-?B (p65-p50 heterodimer) and YY1 on HIV-1 5´-LTR. Highlighted arrow in red indicates the regions targeted for genomic qPCR; ER site for NF-?B, and RBEI and RBEIII sites for YY1 enrichments, respectively. (B and C) U1 cells were pretreated with 5 mM GYY4137 for 6 h, stimulated with PMA (30 ng/ml) for 4 h, fixed with formaldehyde, and lysed. Lysates were subjected to immunoprecipitation for p65 and YY1 and protein-DNA complexes were purified using protein-G magnetic beads. The enrichment of NF-?B p65 and YY1 on HIV-1 LTR was assessed by qPCR for designated regions using purified DNA as a template. Results are expressed as mean ± standard deviation and data are representative of three independent experiments *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, nonsignificant, by two-way ANOVA with Tukey’s multiple comparison test.

Figure 15: GYY4137 subverts HIV-1 reactivation in latent CD4+ T cells derived from HIV-1 infected patients. (A) Schematic representation of PBMCs extraction from blood samples of ART-treated HIV-1 infected subjects. CD4+ T cells were sorted and activated with PHA (1 µg/ml), IL-2 (100 U/ml), feeder PBMCs (gamma-irradiated) from healthy donor. CD4+ T cells were activated in presence of ART or ART in combination with 100 ?M GYY4137. Post-activation cells were culture with ART alone or ART plus GYY4137 treatment in IL-2 containing medium. (B) Total RNA was isolated from five patients CD4+ T cells expanded in ART or ART plus GYY4137. HIV-1 RNA levels were measured every 7 days by ultrasensitive semi-nested RT-qPCR with detection limit of three viral RNA copies per million cells. (C) On day 28, cells were washed of any treatment and both ART or ART+GYY4137 treatment groups were stimulated with 1 µM prostratin for 24 h. HIV-1 RNA copies were assessed by RT-qPCR. Reduction in viral stimulation in GYY4137 treated samples are represented as percentage values. ND - non-determined. (D) Aggregate plot for 5 patients from data shown in C. (E-F) Primary CD4+ T cells expanded and cultured in presence of ART or ART+GYY4137 were analyzed overtime for the expression of activation (CD38) and quiescence markers (CD127) by flow cytometry. (G) Total HIV-1 DNA content was determined up to 28 days in ART or ART+GYY4137 treated groups. Results are expressed as mean ± standard deviation. **, P<0.01; ns, nonsignificant, by two-way ANOVA with Tukey’s multiple comparison test.

Figure 16: Phenotypic features of CD4+ T cells were preserved upon prolong treatment with GYY4137. (A) Flow cytometry-gating strategy used to analyze the expression of activation (CD38), quiescence (CD127), and frequency of different memory subsets of CD4+ T cells. The figure represents expression of different markers at Day 7 post activation of CD4+ T cells from a single patient sample. (B-E) Primary human CD4+ T cells from HIV infected patients cultured with ART or ART with GYY4137 were analyzed by flow cytometry overtime to determine frequency of different subsets of CD4+ T cells- TN (naive [B]) and TCM (central memory [C]), TTM (transition memory [D]), TEM (effector memory [E]). (F) Cell viability was assessed by Live-Dead staining overtime. Results obtained suggest no difference in viability between ART alone and ART with GYY4137 treated group overtime. Error bar represent standard deviations from mean. Results are representative of data from four patients samples.

Figure 17: Effect of H2S on mitochondrial respiration and ROS generation in latent CD4+ T cells derived from HIV-1 patients. (A) Primary human CD4+ T cells from HIV infected subjects were activated and cultured ex vivo with ART or ART+GYY4137. On day 28, cells from ART or ART+GYY4137 treatment groups were harvested to assess mitochondrial respiration by using Seahorse XF mito-stress test as described in materials and methods. (B) Cells from ART and ART+GYY4137 treated groups were stimulated with 1 ?M prostratin 6 h. Post-stimulation mitochondrial respiration profile was determined by Seahorse XF mito-stress test. (C) Various mitochondrial respiratory parameters derived from OCR measurement were determined by Wave desktop software. nmOCR; non-mitochondrial oxygen consumption rate and SRC; spare respiratory capacity. (D) On day 28, Cells from both ART and ART+GYY4137 treatment groups were stimulated with 1 µM Prostratin for 6 h. Cells were harvested and stained with 5 µM MitoSOX-Red dye for 30 min followed by washing. Samples were analyzed by flow cytometry. Unstimulated- cells cultured under ART alone. (E) Both ART and ART+GYY4137 treated cells at day 14 post-activation were stimulated with 1 µg/ml PMA and 100 µg/ml ionomycin (Iono) for 6 h. Cells were harvested post-stimulation and stained with 5 µM MitoSOX-Red dye. Samples were analyzed by flow cytometry to assess mitoROS generation. Unstimulated- cells cultured under ART alone. (F) CD4+ T cells from ART and ART+GYY4137 treated groups were stimulated with 1 ?M prostratin on 28th day for 24 h or left unstimulated. Cells were harvested to isolate total RNA and expression of hmox1, txnrd1, gclc and prdx1 were determined by RT-qPCR. Data obtained were normalized to internal control ß2 microglobulin (B2M). Error bar represent standard deviations from mean. Results are representative of data from three patients samples. *, P<0.05; **, P<0.01, by two-way ANOVA with Tukey’s multiple comparison test.

Figure 18: H2S and Coronavirus. (A, B): VeroE6 cells stably expressing Grx1-roGFP2 were infected with SARS-CoV-2 at an MOI of 0.01 in presence or absence of 5 mM GYY4137. Post infection at 24 h and 48 h, cells were harvested and fixed with 1% NEM and 4% PFA. Cells were analyzed in FACS and oxidative stress was measured in terms of roGFP2 response. Increase in 405/488 ratio indicates increase in oxidative stress (A). Culture supernatant containing virus particles was processed for determining infectious viral titre by plaque assay and for viral RNA isolation. Levels of SARS-CoV-2 N gene was assessed by qRT-PCR (B). (C): Gold Syrian hamsters were infected intranasally with 105 PFU of SARS-CoV-2 in presence or absence of Na-GYY4137. Treatment strategy involved administration of Na-GYY4137 (50 mg/kg) 1 h before and 6 h after infection. Total body weight was recorded each day during the entire course of the experiment until the animals were sacrificed at 4 dpi. Viral RNA load in lung tissue specimens was detected by Plaque Assay.

DETAILED DESCRIPTION OF THE DISCLOSURE
The foregoing has broadly outlined the features and technical advantages of the present disclosure in order that the detailed description of the disclosure that follows may be better understood. Additional features and advantages of the disclosure will be described hereinafter which form the subject of the description of the disclosure. It should also be realized by those skilled in the art that such equivalent product/methods do not depart from the scope of the disclosure.

Definitions:
Unless otherwise defined, all terms used in the disclosure, including technical and scientific terms, have meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the singular forms ‘a’, ‘an’ and ‘the’ include both singular and plural referents unless the context clearly dictates otherwise.

With respect to the use of any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. Various singular/plural permutations may be expressly set forth herein for the sake of clarity. The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results.

The term ‘comprising’, ‘comprises’ or ‘comprised of’ as used herein are synonymous with ‘including’, ‘includes’, ‘containing’ or ‘contains’ and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. Likewise, certain terms may be interchangeably used throughout the specification and thus have the same meaning even when they are referred interchangeably. For example, H2S donor is interchangeably used as Hydrogen sulfide donor, ARVs as antiretroviral drugs, Coronavirus as SARS-CoV-2 etc. throughout the specification.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

Reference throughout this specification to “some embodiments”, “one embodiment”, “an embodiment” or “a preferred embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment may be included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases “in some embodiments”, “in one embodiment”, “in an embodiment” or “a preferred embodiment” in various places throughout this specification may not necessarily all refer to the same embodiment. It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

The term ‘about’ as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of ±10% or less, preferably ±5% or less, more preferably ±1% or less and still more preferably ±0.1% or less of and from the specified value, insofar such variations are appropriate to perform the present disclosure. It is to be understood that the value to which the modifier ‘about’ refers is itself also specifically, and preferably disclosed.

The present disclosure relates to a composition and methods for the management of viral infections/diseases. The composition comprises H2S donor either alone or in combination with antiviral agents. Methods include but not limiting to a method of modulating viral reactivation, method of modulating viral latency, method of treating viral infections/diseases, etc.

The present disclosure relates to a composition comprising at least one H2S donor and at least one antiviral agent, optionally along with pharmaceutically acceptable excipient.

In an embodiment of the present disclosure, the at least one H2S donor and the at least one antiviral agent are provided in a single composition.

In some embodiments of the present disclosure, the composition is in the form of a combination or a kit.

In some embodiments of the present disclosure, the at least one H2S donor is provided as a separate component from the at least one antiviral agent.

In an embodiment of the present disclosure, the H2S donor is present at a concentration ranging from about 100 ?M to about 5 mM and the antiviral agent is present at a concentration ranging from about 100 nM to about 500 nM.

In an embodiment, the present disclosure provides a composition having the H2S donor and the antiviral agent at any concentration within the ranges as defined above.

In an embodiment of the present disclosure, the H2S donor is present at a concentration of about 100 ?M, about 200 ?M, about 300 ?M, about 400 ?M, about 500 ?M, about 600 ?M, about 700 ?M, about 800 ?M, about 900 ?M, about 1mM, about 1.5mM, about 2mM, about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM or about 5mM.n an embodiment of the present disclosure, the antiviral agent is present at a concentration of about 100nM, about 150nM, about 200nM, about 250nM, about 300nM, about 350nM, about 400nM, about 450nM or about 500nM.

In a preferred non-limiting embodiment of the present disclosure, the H2S donor is present at a concentration ranging from about 100?M to about 5mM and the antiviral agent is present at a concentration ranging from about 100 nM to about 200 nM.

In an embodiment of the present disclosure, the H2S donor is a chemically synthesized H2S donor, a naturally occurring H2S donor, or a combination thereof.

In an embodiment of the present disclosure, the chemically synthesized H2S donor is selected from a group comprising morpholin-4-ium 4-methoxyphenyl(morpholino)phosphinodithioate (GYY4137), 5-amino-2-hydroxy-benzoic acid 4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenyl ester hydrochloride (ATB-429), Diallyl disulfide, Na-morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate (Na-GYY4137), Otenaproxesul (ATB-346), ACS-15, Na2S, and combinations thereof.

In an embodiment of the present disclosure, the naturally occurring H2S donor is selected from a group comprising S-allyl-L-cysteine, Diallyl trisulfide, S-propyl-L-cysteine, Sulphoraphane and combinations thereof.

In a preferred non-limiting embodiment of the present disclosure, the H2S donor is morpholin-4-ium 4-methoxyphenyl(morpholino)phosphinodithioate (GYY4137) or Na-morpholin-4-ium 4-methoxyphenyl(morpholino)phosphinodithioate (Na-GYY4137).

In an embodiment of the present disclosure, the antiviral agent is an antiviral agent for HIV, SARS-CoV-2 and other viral infections such as Influenza.

In an embodiment of the present disclosure, the antiviral agent is an antiviral drug.
In an embodiment of the present disclosure, the antiviral drug is an antiretroviral drug for HIV and is selected from a group comprising Efavirenz, Zidovudine, Raltegravir, Lamivudine, Tenofovir disoproxil and combinations thereof.

In an embodiment of the present disclosure, the pharmaceutically acceptable excipient is selected from a group comprising additives, gums, sweeteners, coatings, binders, disintegrants, lubricants, disintegration agents, suspending agents, solvents, colorants, glidants, anti-adherents, anti-static agents, surfactants, plasticizers, emulsifying agents, flavors, viscocity enhancers, antioxidants, and combination thereof.

In an embodiment of the present disclosure, the composition is in a form selected from a group comprising liquid, powder, capsule, tablet, injectable, patch, ointment, gel, emulsion, cream, lotion, dentifrice, spray, drop and combinations thereof.

In a non-limiting embodiment of the present disclosure, the composition comprises at least one H2S donor and at least one antiretroviral drug, optionally along with a pharmaceutically acceptable excipient.

In a preferred non-limiting embodiment of the present disclosure, the composition comprises GYY4137 and at least one antiretroviral drug.

In another preferred non-limiting embodiment of the present disclosure, the composition comprises Na-GYY4137 and at least one antiretroviral drug.

In an embodiment of the present disclosure, the GYY4137 or Na-GYY4137 and the antiretroviral drug may be present in a single composition or as separate components.

In an embodiment of the present disclosure, the composition comprising GYY4137 or Na-GYY4137 and the antiretroviral drug is in the form of a combination or a kit.

The present disclosure also relates to a kit comprising at least one H2S donor, at least one antiviral agent, optionally along with pharmaceutically acceptable excipient and an instruction manual.

In an embodiment of the present disclosure, the at least one H2S donor and the at least one antiviral agent are provided in a single composition.

In an embodiment of the present disclosure, the at least one H2S donor is provided as a separate component from the at least one antiviral agent.

In an embodiment of the present disclosure, the H2S donor and the antiviral agent are as defined earlier.

The present disclosure also relates to a method of preparing the composition, said method comprising:
(a) obtaining at least one H2S donor, obtaining at least one antiviral agent and
combining the at least one H2S donor and the at least one antiviral agent optionally along with pharmaceutically acceptable excipient to obtain the composition; or
(b) assembling at least one H2S donor, at least one antiviral agent, optionally along with pharmaceutically acceptable excipient and an instruction manual.

In an embodiment of the present disclosure, the method involves step (a) when the at least one H2S donor and the at least one antiviral agent are present in a single composition.

In an embodiment of the present disclosure, the method involves step (b) when the at least one H2S donor is present as a separate component from the at least one antiviral agent.

The present disclosure also relates to a method for preparing the kit, said method comprising assembling at least one H2S donor, at least one antiviral agent, optionally along with pharmaceutically acceptable excipient and an instruction manual to obtain the kit. Alternatively, the method comprises assembling a composition comprising at least one H2S donor, at least one antiviral agent and optionally along with pharmaceutically acceptable excipient and an instruction manual to obtain the kit.

The present disclosure also relates to an invitro method of modulating viral reactivation, viral replication and/or viral latency in a cell, said method comprising contacting the cell with an H2S donor optionally along with a pharmaceutically acceptable excipient or the composition of the present disclosure.

In an embodiment of the present disclosure, the modulating viral reactivation involves suppression, inhibition or diminishing of viral reactivation; the modulating viral replication involves suppression, inhibition or diminishing of viral replication; and the modulating viral latency involves maintenance of the latency of the cell and blocking it from reactivation.

In an embodiment of the present disclosure, the virus is selected from a group comprising human immunodeficiency virus (HIV), coronavirus, influenza virus and combinations thereof.

In an embodiment of the present disclosure, the HIV may be HIV 1 or HIV 2.

In an embodiment of the present disclosure, the H2S donor is a chemically synthesized H2S donor, a naturally occurring H2S donor, or a combination thereof.

In an embodiment of the present disclosure, the chemically synthesized H2S donor is selected from a group comprising morpholin-4-ium 4-methoxyphenyl(morpholino)phosphinodithioate (GYY4137), 5-amino-2-hydroxy-benzoic acid 4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenyl ester hydrochloride (ATB-429), Diallyl disulfide, Na-morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate (Na-GYY4137), Otenaproxesul (ATB-346), ACS-15, Na2S, and combinations thereof; and wherein the naturally occurring H2S donor is selected from a group comprising S-allyl-L-cysteine, Diallyl trisulfide, S-propyl-L-cysteine, Sulphoraphane and combinations thereof.

In a preferred non-limiting embodiment of the present disclosure, the H2S donor is morpholin-4-ium 4-methoxyphenyl(morpholino)phosphinodithioate (GYY4137) or Na-morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate (Na-GYY4137).

The present disclosure also relates to an invitro method of modulating HIV reactivation, HIV replication and/or HIV latency in a cell, said method comprising contacting the cell with an H2S donor optionally along with a pharmaceutically acceptable excipient or the composition of the present disclosure.

In a preferred non-limiting embodiment of the present disclosure, the method is carried out using GYY4137 or Na-GYY4137 optionally along with a pharmaceutically acceptable excipient.

In another preferred non-limiting embodiment of the present disclosure, the method is carried out using a composition comprising GYY4137 or Na-GYY4137 and at least one antiretroviral drug optionally along with a pharmaceutically acceptable excipient.

The present disclosure also relates to a method of managing a viral infection or a viral disease, said method comprising administering an H2S donor optionally along with a pharmaceutically acceptable excipient or the composition of the present disclosure to a subject in need thereof.

In an embodiment of the present disclosure, the H2S donor optionally along with a pharmaceutically acceptable excipient or the composition of the present disclosure is formulated into a dosage form selected from a group comprising liquid, powder, capsule, tablet, injectable, patch, ointment, gel, emulsion, cream, lotion, dentifrice, spray, drop and combinations thereof.

In an embodiment of the present disclosure, the H2S donor optionally along with a pharmaceutically acceptable excipient or the composition of the present disclosure is administered orally, intranasally, intravenously, intraperitoneally and combinations thereof.

In an embodiment of the present disclosure, the H2S donor optionally along with a pharmaceutically acceptable excipient or the composition of the present disclosure is administered at a dosage ranging from about 2 mg/kg to about 50 mg/kg body weight of the subject.

In an embodiment of the present disclosure, the H2S donor is a chemically synthesized H2S donor, a naturally occurring H2S donor, or a combination thereof.

In an embodiment of the present disclosure, the chemically synthesized H2S donor is selected from a group comprising morpholin-4-ium 4-methoxyphenyl(morpholino)phosphinodithioate (GYY4137), 5-amino-2-hydroxy-benzoic acid 4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenyl ester hydrochloride (ATB-429), Diallyl disulfide, Na-morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate (Na-GYY4137), Otenaproxesul (ATB-346), ACS-15, Na2S, and combinations thereof; and wherein the naturally occurring H2S donor is selected from a group comprising S-allyl-L-cysteine, Diallyl trisulfide, S-propyl-L-cysteine, Sulphoraphane and combinations thereof.

In a preferred non-limiting embodiment of the present disclosure, the H2S donor is morpholin-4-ium 4-methoxyphenyl(morpholino)phosphinodithioate (GYY4137) or Na-morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate (Na-GYY4137).

In an embodiment of the present disclosure, managing the viral infection or the viral disease comprises modulating viral reactivation, modulating viral replication, modulating viral latency, treating the viral infection or the viral disease with or without co-infection with opportunistic pathogen and combinations thereof.

In an embodiment of the present disclosure, the modulating viral reactivation involves suppression, inhibition or diminishing of viral reactivation; the modulating viral replication involves suppression, inhibition or diminishing of viral replication; and the modulating viral latency involves maintenance of the latency of the cell and blocking it from reactivation.

In an embodiment of the present disclosure, the viral infection or the viral disease is selected from a group comprising human immunodeficiency virus (HIV), coronavirus, influenza virus, and combinations thereof.

In an embodiment of the present disclosure, the HIV is HIV 1 or HIV 2.

In an embodiment of the present disclosure, the viral infection or viral disease is Acquired Immune Deficiency Syndrome (AIDS).

In an embodiment of the present disclosure, the viral infection or viral disease is caused by coronovirus.

In an embodiment of the present disclosure, the viral infection or viral disease is caused by influenza virus.

In an embodiment of the present disclosure, the viral infection or viral disease is selected from group comprising HIV infection, AIDS, HIV infection/AIDS co-infected with opportunistic pathogens such as but not limiting to Mycobacterium tuberculosis, coronavirus, influenza virus and combinations thereof.

In an embodiment of the present disclosure, the subject is a mammal, including human being.

The present disclosure also relates to a method of managing HIV infection/AIDS or HIV infection/AIDS, said method comprising administering an H2S donor optionally along with a pharmaceutically acceptable excipient or the composition of the present disclosure to a subject in need thereof.

The present disclosure also relates to a method of managing HIV infection/AIDS or HIV infection/AIDS co-infected with opportunistic pathogens such as but not limiting to Mycobacterium tuberculosis, said method comprising administering an H2S donor optionally along with a pharmaceutically acceptable excipient or the composition of the present disclosure to a subject in need thereof.

In a preferred non-limiting embodiment of the present disclosure, the method is carried out using GYY4137 or Na-GYY4137 optionally along with a pharmaceutically acceptable excipient.

In another preferred non-limiting embodiment of the present disclosure, the method is carried out using a composition comprising GYY4137 or Na-GYY4137 and at least one antiretroviral drug optionally along with a pharmaceutically acceptable excipient.

The present disclosure also relates to a method of managing Coronavirus, said method comprising administering an H2S donor optionally along with a pharmaceutically acceptable excipient or the composition of the present disclosure to a subject in need thereof.

In a preferred non-limiting embodiment of the present disclosure, the method is carried out using GYY4137 or Na-GYY4137 optionally along with a pharmaceutically acceptable excipient.

In another non-limiting embodiment of the present disclosure, the method is carried out using a composition comprising GYY4137 or Na-GYY4137 and at least one antiviral agent optionally along with a pharmaceutically acceptable excipient.

The present disclosure also relates to use of an H2S donor optionally along with a pharmaceutically acceptable excipient or the composition of the present disclosure for managing a viral infection or viral disease in a subject in need thereof.

In an embodiment of the present disclosure, the H2S donor optionally along with a pharmaceutically acceptable excipient or the composition of the present disclosure is formulated into a dosage form selected from a group comprising liquid, powder, capsule, tablet, injectable, patch, ointment, gel, emulsion, cream, lotion, dentifrice, spray, drop and combinations thereof.

In an embodiment of the present disclosure, the H2S donor optionally along with a pharmaceutically acceptable excipient or the composition of the present disclosure is administered orally, intranasally, intravenously, intraperitoneally and combinations thereof.

In an embodiment of the present disclosure, the H2S donor optionally along with a pharmaceutically acceptable excipient or the composition of the present disclosure is administered at a dosage ranging from about 2 mg/kg to about 50 mg/kg body weight of the subject.

In an embodiment of the present disclosure, the H2S donor is a chemically synthesized H2S donor, a naturally occurring H2S donor, or a combination thereof.

In an embodiment of the present disclosure, the chemically synthesized H2S donor is selected from a group comprising morpholin-4-ium 4-methoxyphenyl(morpholino)phosphinodithioate (GYY4137), 5-amino-2-hydroxy-benzoic acid 4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenyl ester hydrochloride (ATB-429), Diallyl disulfide, Na-morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate (Na-GYY4137), Otenaproxesul (ATB-346), ACS-15, Na2S, and combinations thereof; and wherein the naturally occurring H2S donor is selected from a group comprising S-allyl-L-cysteine, Diallyl trisulfide, S-propyl-L-cysteine, Sulphoraphane and combinations thereof.

In a preferred non-limiting embodiment of the present disclosure, the H2S donor is morpholin-4-ium 4-methoxyphenyl(morpholino)phosphinodithioate (GYY4137) or Na-morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate (Na-GYY4137).

In an embodiment of the present disclosure, managing the viral infection or the viral disease comprises modulating viral reactivation, modulating viral replication, modulating viral latency, treating the viral infection or the viral disease with or without co-infection with opportunistic pathogen and combinations thereof.

In an embodiment of the present disclosure, the modulating viral reactivation involves suppression, inhibition or diminishing of viral reactivation; the modulating viral replication involves suppression, inhibition or diminishing of viral replication; and the modulating viral latency involves maintenance of the latency of the cell and blocking it from reactivation.

In an embodiment of the present disclosure, the viral infection or the viral disease is selected from a group comprising human immunodeficiency virus (HIV), coronavirus, influenza virus, and combinations thereof.

In an embodiment of the present disclosure, the HIV is HIV 1 or HIV 2.

In an embodiment of the present disclosure, the viral infection or viral diseases is Acquired Immune Deficiency Syndrome (AIDS).

In an embodiment of the present disclosure, the viral infection or viral disease is selected from a group comprising HIV infection, AIDS, HIV infection/AIDS co-infected with opportunistic pathogens such as but not limiting to Mycobacterium tuberculosis, coronavirus, influenza virus and combinations thereof.

In an embodiment of the present disclosure, the subject is a mammal, including human being.

The present disclosure also relates to use of GYY4137 or Na-GYY4137 optionally along with a pharmaceutically acceptable excipient in managing a viral infection or viral disease selected from a group comprising HIV infection, AIDS and HIV infection/AIDS co-infected with opportunistic pathogens such as but not limiting to Mycobacterium tuberculosis.

The present disclosure also relates to use of a composition comprising GYY4137 or Na-GYY4137 and an antiretroviral drug optionally along with a pharmaceutically acceptable excipient in managing a viral infection or viral disease selected from a group comprising HIV infection, AIDS and HIV infection/AIDS co-infected with opportunistic pathogens such as but not limiting to Mycobacterium tuberculosis.

The present disclosure also relates to use of GYY4137 or Na-GYY4137 optionally along with a pharmaceutically acceptable excipient in managing coronavirus infection.

The present disclosure also relates to use of a composition comprising GYY4137 or Na-GYY4137 and an antiviral agent optionally along with a pharmaceutically acceptable excipient in managing coronavirus infection.

In an embodiment of the present disclosure, the composition and methods are useful in treating HIV-infected patients on suppressive ART, which harbors the latent but replication-competent virus. The composition and methods mediate HIV-1 persistence by regulating gene-expression, redox signaling, mitochondrial bioenergetics, etc.

In an embodiment, the present disclosure elucidates an unexpected role of hydrogen sulfide (H2S) in locking HIV in a latent state and blocking viral reactivation. It was found that the reactivation of HIV-1 is associated with down-regulation of the key H2S producing enzyme cystathionine-?-lyase (CTH) and reduction in endogenous H2S. Genetic silencing of CTH disrupts redox homeostasis, impairs mitochondrial function, and remodels the transcriptome of latent cells to trigger HIV reactivation. Accordingly, chemical complementation of CTH activity using a slow-releasing H2S donor, GYY4137, suppressed HIV reactivation and diminished virus replication. Importantly, a combination of GYY4137 with clinically-relevant anti-retrovirals (ARVs) blocked reactivation of HIV-1 from latently infected CD4+ T cells isolated from HIV patients.

In a preferred embodiment, H2S donor, GYY4137, has potency to suppress HIV replication and reactivation from latently infected CD4+ T cells. In combination with ARV’s, GYY4137 significantly suppresses HIV replication and reactivation from latently infected CD4+ T cells.

In another embodiment, the present disclosure provides for more effectively treating HIV infection and preventing transmission.

In another embodiment, H2S donor, GYY4137 acts as an effector molecule. While H2S deficiency reactivates HIV-1, the H2S donor GYY4137 can potently inhibit residual levels of HIV-1 transcription during suppressive ART and block virus reactivation upon stimulation. Hence, the present disclosure can be exploited to lock HIV in a state of persistent latency by impairing the ability to reactivate. Several of the biological dysfunctions such as loss of mitochondrial functions, oxidative stress, inflammation, and apoptosis associated with HIV-1 reactivation are corrected by H2S. Collectively, the results suggest that H2S reduces HIV-1 transcriptional activity, promotes silencing of its promoter, and reduces its potential for reactivation. The virus-suppressing effects of H2S were observed alongside its beneficial consequences on cellular physiology (e.g., mitochondrial function and redox balance). H2S in combination with ART can push transcriptional repression beyond a certain threshold where it will be impossible to reactivate HIV, thereby blocking and locking virus in a state of persistent latency. The results of the present disclosure provide a proof-of-concept of a gasotransmitter H2S that can be explored for a functional cure of HIV. Blocking HIV rebound by inhibitors of viral factors (e.g., Tat) invariably results in the emergence of resistant mutants. In this context, blocking HIV-1 rebound via H2S-directed modulation of host pathways helps in overcoming the problem of evolution of escape variants. Finally, therapy non-compliance and frequent blips contributes to continuous replenishment of latent reservoir in vivo. Combining H2S donors with ART regiments potentially prevents reservoir replenishment during infection.

It is to be understood that the foregoing description is illustrative not a limitation. While considerable emphasis has been placed herein on particular features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. Those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein. Similarly, additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon description provided herein.

Descriptions of well-known/conventional methods/steps and techniques are omitted so as to not unnecessarily obscure the embodiments herein. Further, the disclosure herein provides for examples illustrating the above-described embodiments, and in order to illustrate the embodiments of the present disclosure, certain aspects have been employed. The examples used herein for such illustration are intended merely to facilitate an understanding of ways in which the embodiments may be practiced and to further enable those of skill in the art to practice the embodiments. Accordingly, following examples should not be construed as limiting the scope of the embodiments herein.

EXAMPLES
Management of HIV
Example 1: HIV Reactivation Suppresses Endogenous H2S Levels
In this study, it was observed that reactivation of HIV from latency leads to depletion in levels of transcripts and proteins of H2S biogenesis enzymes. Moreover, the endogenous levels of H2S are reduced upon HIV reactivation. Next, it was found that a key H2S producing enzyme (CTH) is essential to sustain HIV in a latent form by RNAi approach. Moreover, the pathways central for maintenance of HIV-1 latency including cellular antioxidant response, NF-?B signaling, apoptotic response, antiviral/repressive factors and mitochondrial bioenergetics are affected upon depletion of endogenous H2S levels.

Material and methods

Mammalian cell lines and culture conditions
The human pro-monocytic cell line U937, CD4+ T lymphocytic cell line Jurkat, HEK293T were procured from ATCC, Manassas, VA. The chronically infected ACH-2, J-Lat 6.3, U1, J1.1 and TZM-bl cell lines were obtained through AIDS Research and Reference Reagent Program, NIH, USA. The cell lines were cultured in RPMI1640 (Cell Clone) supplemented with L-glutamine (2 mM), 10% fetal bovine serum (MP biomedicals), penicillin (100 units/mL), streptomycin (100 ?g/mL) at 37?C and 5% CO2. HEK293T and TZM-bl cells were cultured in DMEM (Cell Clone) supplemented with 10% FBS.

Chemical reagents
Sodium hydrosulfide (NaHS), morpholin-4-ium 4-methoxphenyl(morpholino) phosphinodithioate dichloromethane complex (GYY4137), Phorbol-12-myristate-13-acetate (PMA), N-acetyl cysteine (NAC), prostratin and L-Cysteine were purchased from Sigma-Aldrich. Recombinant human TNF-? was purchased from InvivoGen. The antiretroviral drugs efavirenz, zidovudine, raltegravir, and lamivudine were obtained through the NIH AIDS Reagent Program.

Latent viral reactivation
Latently infected U1 and J1.1 (2 ? 105 cells/ml) were stimulated with PMA (5 ng/ml) or TNF-? (100 ng/ml) for the time indicated in the figure legends. HIV-1 reactivation was determined by intracellular gag RT-qPCR or p24 estimation in supernatant by HIV-1 p24 ELISA (J. Mitra and Co. Pvt. Ltd., India). J-Lat 6.3 cells were stimulated with Prostratin (2.5 ?M) for 24 h and HIV-1 reactivation was assessed by estimating GFP+ cells (excitation: 488 nm; emission: 510 nm) using BD FACSVerse flow cytometer (BD Biosciences). The data were analysed using FACSuite software (BD Biosciences).

Reverse transcription quantitative PCR (RT-qPCR)
Total cellular RNA was isolated by RNeasy mini kit (Qiagen), according to the manufacturer’s protocol. RNA (500 ng) was reverse transcribed to cDNA (iScript? cDNA synthesis kit, Bio-Rad), subjected to quantitative real-time PCR (iQ? SYBR® Green Supermix, Bio-Rad), and performed using the Bio-Rad C1000? real time PCR system. HIV-1 reactivation was assessed using gene-specific primers (Table S6). The expression level of each gene is normalized to human ß-actin as an internal reference gene.

Western blot analysis
Total cell lysates of PMA-treated and untreated U1, J1.1, U937, and Jurkat cell lines were prepared using radioimmunoprecipitation (RIPA) lysis buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 1% Triton X-100, 1 % sodium deoxycholate, 0.1% SDS (sodium dodecyl sulfate), 1? protease inhibitor cocktail (Sigma-Aldrich), 1? phosphatase inhibitor cocktail (Sigma-Aldrich). After incubation on ice for 20 min, the lysates were centrifuged at 12,000 rpm, 4?C for 15 mins. Clarified supernatant was taken and total protein concentration was determined by Bicinchoninic acid assay (Pierce?, Thermo Fisher Scientific). Total protein extracts were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Membranes were probed with an anti-CBS (EPR8579), CTH (ab151769), MPST (ab154514), and anti-HIV-1 p24 (ab9071) from abcam; Nrf2 (CST-12721), Keap1 (CST-4678), NF-?B p65 (CST-6956), phospho-NF-?B p65 (Ser536) (CST-3033), YY1 (CST-63227) and GAPDH (CST-97166) from Cell Signaling Technologies, Inc. Anti-rabbit IgG (CST-7074) and anti-mouse IgG (CST-7076) were used a secondary antibodies. Proteins were detected by ECL and visualized by chemiluminescence (Perkin Elmer, Waltham, MA) using the Bio-Rad Chemidoc Imaging system (Hercules, CA). For membrane reprobing, stripping buffer was used (2% SDS [w/v], 62 mM Tris-Cl buffer (0.5 M, pH 6.7) and 100 mM ?-mercaptoethanol) for 20 min at 55°C. After extensive washing with PBS containing 0.1 % Tween 20 (Sigma-Aldrich), membrane was blocked and reincubated with desired antibodies.

H2S detection assays
Endogenous H2S levels of U1 and U937 cells were detected using H2S specific fluorescent probe as described. Briefly, cells were treated with PMA (5 ng/ml) for 24 h, washed with 1? PBS, and stained with 200 ?M 7-Azido-4-Methylcoumarin (AzMC) (Sigma). The stained cells were mounted on a glass slide and visualized using Leica TCS SP5 confocal microscope (excitation: 405 nm; emission: 450 nm). Images obtained were analyzed using LAS AF Lite software (Leica Microsystems) and semi-quantification of 50 cells was performed using ImageJ software.

H2S generation was also measured using methylene blue assay. The supernatant of U1 cells treated with NaHS or GYY4137 was incubated with Zinc acetate (1%) and NaOH (3%) (1:1 ratio) to trap H2S for 30 min. The reaction was terminated using 10% trichloroacetic acid solution. Following this, reactants were incubated with 20 mM N,N-dimethylphenylendiamine (NNDPD) in 7.2 M HCl and 30 mM FeCl3 in 1.2 M HCl for 30 mins and absorbance was measured at 670 nm. The concentration of H2S was determined by plotting absorbance on a standard curve generated using NaHS (0-400 µM; R2=0.9982).

Stable cell line generation
For generating CBS and CTH knockdown in U1 and J1.1 cells, we used validated pooled gene specific shRNAs from the RNAi Consortium (TRC) library (Sigma Aldrich, USA; shRNA sequences given in Table S1). The lentiviral particles were generated in HEK293T cells using the packaging vectors, psPAX2 and pMD2.G. The pLKO.1-puro vector encoding a non-mammalian targeting shRNA (shNT) was used as a control. The U1 cells were transduced with lentiviral particles in opti-MEM containing polybrene (10 ?g/ml) for 6 hours. Cells were washed and stable clones were selected in culture medium containing 250 ng/ml of puromycin. Total RNA or cell lysates were prepared to validate knockdown of CBS and CTH.

Intracellular HIV-1 p24 staining
For intracellular p24 staining, U1-shCTH and U1-shNT cells were stimulated with PMA (5 ng/ml), washed with PBS followed by fixation and permeabilization using a fixation and permeabilization kit (eBiosciences). Permeabilized cells were then incubated with 50 ?l of 1:100 dilution of phycoerythrin (PE)-conjugated mouse anti-p24 monoclonal antibody (KC57-RD1; Beckman Coulter, Inc.) for 30 min at room temperature. After incubation, the cells were washed twice and the fluorescence of stained samples were acquired using BD FACSVerse flow cytometer (BD Biosciences). The data were analysed using FACSuite software (BD Biosciences).

NanoString nCounter assay
Total RNA was isolated using an RNeasy mini kit (Qiagen) according to manufacturer’s instructions. RNA concentration and purity were measured using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA), bioanalyzers systems (Agilent Technologies, Inc.), and Qubit Assays (Thermo Fisher Scientific). An nCounter gene expression assay was performed according to the manufacturer’s protocol. The assay utilized a custom-made NanoString codeset designed to measure 185 genes, including 6 house-keeping genes (Table S2). This custom-made panel included genes associated with oxidative stress and HIV-1 infection (Table S2). All genes were assayed simultaneously in multiplexed reactions and analyzed by fully automated nCounter Prep Station and digital analyzer (NanoString Technologies). The data were normalized to B2M, used as a housekeeping gene due to its minimum % CV across the samples, and analysis was done using nSolver 4.0 software.

Measurement of Oxygen Consumption Rates
Oxygen consumption rates (OCR) were measured using a Seahorse XFp extracellular flux analyzer (Agilent Technologies) as per manufacturer’s instructions. Briefly, cells (U1 or Primary CD4+ T cells) were seeded at a density of 104-105 per well in a Seahorse flux analyzer plate precoated with Cell-Tak (Corning). Cells were incubated for 1 h in a non-CO2 incubator at 37?C before loading the plate in the seahorse analyzer. To assess mitochondrial respiration, three OCR measurements were performed without any inhibitor in XF assay media to measure basal respiration, followed by sequential addition of oligomycin (1 ?M), an ATP synthase inhibitor (complex V) and three OCR measurements to determine ATP-linked OCR and proton leakage. Next, cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP; 0.25 ?M), was injected to determine the maximal respiration rate and the spare respiratory capacity (SRC). Finally, rotenone (0.5 ?M) and antimycin A (0.5 ?M), inhibitors of NADH dehydrogenase (complex I) and cytochrome c - oxidoreductase (complex III), respectively, were injected to completely shut down the electron transport chain (ETC) to analyze non-mitochondrial oxygen consumption rate (nmOCR). Seahorse data were normalized to total amount of protein (?g) and mitochondrial respiration parameters were analyzed using Wave Desktop 2.6 software (Agilent Technologies).

Estimation of intracellular glutathione content and ROS
Total cell lysate was prepared from 107 cells using sonication in MES (2-(N-morpholino) ethanesulfonic acid) buffer. Lysates were clarified by centrifugation and total protein concentration was estimated using BCA assay. Total glutathione, reduced glutathione (GSH), and oxidized glutathione (GSSG) were measured using the glutathione assay kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions. To measure ROS, cells were loaded with 10 ?M of CM-H2DCFDA (Excitation: 492 nm; Emission; 517 nm) or 5 ?M of MitoSOX? Red (Excitation: 510 nm; Emission; 580 nm) for 30 min at 37°C and exposed to H2O2 (100 ?M) or antimycin A (2 ?M) or PMA (5 ng/ml) or left untreated at 37°C and 5% CO2. The fluorescence of stained samples was acquired using BD FACSVerse flow cytometer (BD Biosciences). Data were analyzed using FlowJo software (BD Biosciences).

Results
Diminished biogenesis of endogenous H2S during HIV-1 reactivation
To investigate the link between HIV-1 latency and H2S, we measured changes in the expression of genes encoding H2S-generating enzymes CBS, CTH, and MPST in monocytic (U1) and lymphocytic (J1.1) models of HIV-1 latency (Figure 1A). The U1 and J1.1 cell lines are well-studied models of post-integration latency and were derived from chronically infected clones of a promonocytic (U937) and a T-lymphoid (Jurkat) cell line, respectively. Both U1 and J1.1 show very low basal expression of the HIV-1 genome, which can be induced by several latency reversal agents (LRAs) such as PMA, TNF-?, and prostratin (Figure 1B). First, virus reactivation was confirmed by measuring HIV-1 gag transcript in U1 with a low concentration of PMA (5 ng/ml). Treatment of PMA induces detectable expression of gag at 12 h, which continued to increase for the entire 36 h duration of the experiment (Figure 1C). Next, mRNA and protein levels of CBS, CTH, and MPST in U1 during virus latency (untreated) and reactivation (PMA-treated) was assessed. The mRNA and protein expression levels of CTH showed a significant reduction at 24 and 36 h post-PMA treatment as compared to the untreated control, whereas the expression of CBS was not detected and MPST remained unaffected (Figure 1D-E). As an uninfected control, the expression of CBS, CTH, and MPST in U937 cells was measured. In contrast to U1, PMA treatment stimulated the mRNA and protein levels of CTH in U937 (Figure 1F-G), while CBS was barely detectable. Similar to U1 monocytic cells, PMA treatment reactivated HIV-1 and reduced the expression of CTH in J1.1 lymphocytic cells but not in the uninfected Jurkat cells in a time-dependent manner (Figure 2). The expression of CBS was reduced in both J1.1 and Jurkat upon PMA treatment, indicating that only CTH specifically down-regulates in response to HIV-1 reactivation (Figure 2). Finally, endogenous H2S levels was directly measured using a fluorescent probe 7-azido-4-methylcoumarin (AzMc) that quantitatively detects H2S in living cells. Consistent with the expression data, reactivation of HIV-1 by PMA reduced H2S generation in U1, whereas H2S levels were significantly increased by PMA in the uninfected U937 (Figure 3). Taken together, these data indicate that HIV-1 reactivation is associated with diminished biogenesis of endogenous H2S.

CTH-mediated reactivation of HIV-1 from latency
Results suggested that H2S biogenesis via CTH is associated with HIV-1 latency. Therefore, it was assessed whether CTH-derived H2S regulates reactivation of HIV-1 from latency. To test this, endogenous CTH levels in U1 using RNA interference (RNAi) was depleted. The short hairpin RNA specific for CTH (U1-shCTH) silenced the expression of CTH mRNA and protein by 90% as compared to non-targeting shRNA (U1-shNT) (Figure 4A-B). Moreover, using AzMC probe, reduction in endogenous H2S levels was confirmed in U1-shCTH as compared to U1-shNT (Figure 4C-D). Next, the effect of H2S depletion via CTH suppression on HIV-1 latency was investigated by measuring viral transcription (gag transcript), translation (HIV-1 p24 capsid protein), and release (HIV-1 p24 abundance in the cell supernatant). It was ound that the low basal expression of HIV-1 gag in U1-shCTH was stimulated by 16-fold upon depletion of CTH (p = 0.0018) (Figure 4E). Furthermore, while PMA induced expression of gag transcript by 73-fold in U1-shNT, a further enhancement to 200-fold was observed in U1-shCTH (Figure 4E). Consistent with this, levels of p24 capsid protein inside cells or released in the supernatant were significantly elevated in U1-shCTH as compared to U1-shNT with (p = 0.028) or without PMA-treatment (p = 0.015) (Figure 4F-G). CTH levels in J1.1 cells was also depleted using RNAi and HIV-1 reactivation was monitored by assessing intracellular p24 levels under basal conditions. The depletion of CTH triggered HIV-1 reactivation from latency as evident from a 7-fold increase in p24 levels as compared to shNT (Figure 5). A minor increase in p24 levels (3.6-fold) was also apparent upon the depletion of CBS in J1.1 (Figure 5). These data indicate that CTH and likely CTH-derived H2S supports latency and impedes the reactivation of HIV-1.

Altered expression of genes involved in HIV-1 reactivation upon CTH depletion
The above results indicate that CTH is a target controlling HIV-1 reactivation from latency, prompting to investigate the mechanism. Several pathways that induce HIV-1 reactivation from latency are also influenced by H2S. These include redox signaling, NF-?B pathway, inflammatory response, and mitochondrial bioenergetics. Hence, a focused expression profiling of 185 human genes intrinsically linked to HIV-1 reactivation using the NanoString nCounter technology (see supplementary Table S2 for the gene list) to measure absolute amounts of multiple transcripts without reverse transcription was conducted. Because depletion of CTH promoted reactivation of HIV-1 in U1, the expression profile of U1-shCTH with U1-shNT was compared to further understand the link between H2S biogenesis and HIV-1 latency. Examination of 84 genes related to oxidative stress response revealed differential regulation of 22 genes in U1-shCTH compared with U1-shNT (Figure 6 and supplementary Table S3). Interestingly, more than 90 % of genes showing altered expression were down-regulated in U1-shCTH. Of these, genes encoding key cellular antioxidant enzymes and buffers such as catalase (CAT), superoxide dismutase (SOD1), peroxiredoxin family, thioredoxin family (TXN, TXNRD1 and TXNRD2), sulfiredoxin (SRXN1), glutathione metabolism (GCLM, GSS, and GSR), and sulfur metabolism (MPST, AHCY, and MAT2A) were significantly less expressed in U1-shCTH as compared to U1-shNT (Figure 6). Since oxidative stress elicits HIV-1 reactivation, these findings indicate that HIV-1 reactivation through CTH depletion could be a consequence of an altered redox balance. Further, pathways involved in promoting HIV-1 reactivation such as NF-?B signaling and apoptosis were also induced in U1-shCTH as compared to U1-shNT (Figure 6). Notably, multiple HIV-1 restriction factors important for viral latency including type I interferon signaling (IRF2, STAT-1), APOBEC3G, CDK9-CCNT1, SLP-1, and chromatin remodelers (SMARCB1, BANF1, YY1) were down-regulated in U1-shCTH. HIV-1 proteins are known to target mitochondria to induce mitochondrial depolarization, elevate mitochondrial ROS, and apoptosis for replication. Several genes involved in sustaining mitochondrial function and membrane potential (e.g., BAX, UCP2, LHPP, MPV17) were repressed in U1-shCTH (Figure 6), highlighting a potential association between unrestricted virus replication, mitochondrial dysfunction, and CTH depletion.

Since PMA is known to reactivate HIV-1, the gene expression in U1-shNT upon reactivation of HIV-1 by PMA was examined. It was found that 80 % of genes affected by the depletion of CTH were similarly perturbed in response to PMA. Signature of transcripts associated with oxidative stress response, sulfur metabolism, anti-viral factors, and mitochondrial function was comparable in U1-shCTH and PMA-treated U1-shNT (Figure 6). Based on these similarities, it was hypothesized that combining PMA with CTH depletion would have an additive effect on the expression of genes linked to HIV-1 reactivation. Indeed, exposure of U1-shCTH to PMA induced gene expression changes which surpassed those produced by PMA treatment or CTH-depletion alone (Figure 6). Notably, significant expression changes in U1-shCTH upon PMA treatment are consistent with the data showing maximal HIV-1 activation under these conditions (see Figure 4). Overall, these results indicate that CTH contributes to HIV-1 latency by modulating multiple pathways coordinating cellular homeostasis (e.g., redox balance, mitochondrial function) and the anti-viral response.

CTH is required to maintain redox homeostasis and mitochondrial function
Physiological levels of H2S support redox balance and mitochondrial function by maintaining GSH balance, protecting against ROS, and acting as a substrate for the electron transport chain. On this basis, it was reasoned that the depletion of CTH could contribute to redox imbalance and mitochondrial dysfunction to promote HIV-1 reactivation. Hence, total glutathione content (GSH+GSSG) and GSSG concentration in U1-shNT and U1-shCTH using chemical-enzymatic analysis of whole-cell extract was first measured. Whole-cell glutathione content was not significantly different in U1-shNT and U1-shCTH (p = 0.2801) (Figure 7A). However, the GSSG concentration was elevated in U1-shCTH resulting in a concomitant decrease in the GSH/GSSG ratio of U1-shCTH as compared to U1-shNT (p = 0.0032) (Figure 7A). The increased GSSG pool and reduced GSH/GSSG poise confirm that cells are experiencing oxidative stress upon CTH depletion. Total ROS was measured using a fluorescent probe, 5,6-chloromethyl-2?,7?-dicholrodihydrofluorescein diacetate (CM-H2DCFDA), which non-specifically responds to any type of ROS within the cells. Both U1-shNT and U1-shCTH showed comparable levels of cytoplasmic ROS (Figure 7B), which remained unchanged after stimulation with PMA. In addition to cytoplasmic ROS, mitochondrial ROS (mitoROS) was also measured using the red fluorescent dye MitoSOX, which selectively stains mitoROS. Lowering the levels of CTH severely increased mitoROS in U1, which was further accentuated after PMA stimulation (Figure 7C). The effect of CTH depletion was then studied on mitochondrial functions using a Seahorse XF Extracellular Flux Analyzer (Agilent) (Figure 7D). Both basal and ATP-coupled respiration was significantly decreased in U1-shCTH as compared to U1-shNT (Figure 7E-F), consistent with the role of endogenous H2S in reducing cytochrome C oxidase for respiration. The maximal respiratory capacity, attained by the dissipation of the mitochondrial proton gradient with the uncoupler FCCP, was markedly diminished in U1-shCTH (Figure 7F). The maximal respiration also facilitated the estimation of the spare respiratory capacity, which was nearly exhausted in U1-shCTH. Additional hallmarks of HIV-1 reactivation and dysfunctional mitochondria such as coupling efficiency and non-mitochondrial oxygen consumption rate (nmOCR) (30) were also adversely affected upon depletion of CTH (Figure 7F). These results indicate that CTH depletion decelerates respiration, diminishes the capacity of macrophages to maximally respire, and promotes nmOCR. All of these parameters are important features of mitochondrial health and are likely to be crucial for maintaining HIV-1 latency.

Example 2: Potent Suppression of HIV Reactivation by H2S Donor-GYY4137
In this study, it is reported that slow-releasing H2S donor molecule, GYY4137, subverts HIV reactivation in monocytic and T cell line model of viral latency. Mechanistically, GYY4137 induces cellular antioxidant response by upregulating Nrf2 activity, reduces activity of proviral factor (NF-?B), and induces viral suppressive factor (YY1) occupancy on HIV-LTR. Additionally, using primary CD4+ T cells isolated from ART-treated HIV infected patient which harbors latent reservoirs of HIV, treatment with GYY4137 lead to suppression of HIV reactivation in five human subject samples. Moreover, in primary cell model of HIV, it is shown that long-term treatment with GYY4137 is able to suppress HIV transcription without affecting pro-viral content, global T-cell activation, frequency to T cell subsets, and maintains mitochondrial bioenergetics when used in combination with current ART regimen.

Material and methods

Subject samples
Peripheral blood mononuclear cells (PBMCs) were collected from five HIV-1 seropositive subjects on stable suppressive ART (Table S5). All subject signed informed consent forms approved by Indian Institute of Science, Bangalore and Bangalore Medical College and Research Institute (BMCRI) review boards (Institute human ethics committee [IHEC] No-3-14012020).

Virus Production
HIV-1 particle production was carried out using Lipofectamine 2000 transfection reagent (Invitrogen, Life Technologies), according to the manufacturer’s protocol, in HEK293T cells using HIV-1 NL4-3 DNA (NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH). The medium was replaced with fresh medium at 6 h post-transfection, and supernatants were collected after 60 h, centrifuged (10 min, 200 ?g, room temperature), and filtered through a 0.45 ?m-pore-size membrane filter (MDI; Membrane Technologies) to clear cell debris. Virus was concentrated using 5? PEG-it? (System Biosciences) as per manufacturer’s protocol and virus pellet obtained was aliquoted in opti-MEM and stored at -80°C. Viral titration was done using HIV-1 reporter cell line, TZM-bl (NIH AIDS reagent program) as described earlier.

Assessing the effect of GYY4137 on HIV-1 replication
Briefly, 0.5?106 Jurkat cells were infected with HIV-1 NL4.3 at 0.2 M.O.I for 4 h at 37°C. Post-infection, cells were washed with 1? PBS and cultured in presence or absence of 300 µM GYY4137. Cells and culture supernatants were harvested to isolate total RNA for gag RT-qPCR and p24 estimation, respectively. The p24 concentration was determined in the culture supernatant by sandwich HIV-1 p24 ELISA (J. Mitra and Co. Pvt. Ltd., India) according to the manufacturer’s instruction.

Chromatin immunoprecipitation and quantitative genomic PCR
The chromatin immunoprecipitation (ChIP) assays was performed using SimpleChIP? Enzymatic Chromatin IP Kit (Magnetic Beads) (Cell Signaling Technologies, Inc., MA, US) according to the manufacturer’s instructions. Briefly, 107 U1 cells were pre-treated with GYY4137 (5 mM) for 6 h or left untreated and then stimulated with PMA (30 ng/ml) for 4 h. Cells were fixed using 1% formaldehyde, neutralized with glycine, and harvested in ice-cold PBS. Cells were lysed and nuclei were sheared by sonication using Bioruptor? Pico (Diagenode Inc.) to obtain DNA fragments of 200 to 500 nucleotides. The clarified lysates were immunoprecipitated using ChIP-grade anti-NF-?B p65 (CST-6956) or anti-YY1 (CST-63227) or normal rabbit IgG (as a negative control) for overnight at 4°C followed by incubation with ChIP-grade protein G magnetic beads at 4°C for 4 h. Chromatin was eluted from protein G beads, reverse-cross linked, and DNA was column purified. Quantitative genomic PCR was done by SYBR green-based real-time PCR using primers spanning the ER, RBE I, and RBE III regions on HIV-1 LTR. The GAPDH gene was used as the reference gene to see non-specific binding. Total input (10 %) was used to normalize equal amount of chromatin taken across the samples. The relative proportion of co-immunoprecipitated DNA fragments were determined with the help of threshold cycle (CT) values for each qPCR product using the equation (100 ? 2[CT(input-3.32)- CT(IP)]). The data obtained were represented as fold enrichment normalized to IgG background for each IP reaction.

Generation of expanded primary CD4+ T cells from aviremic subjects
The PBMCs isolated from blood samples using Histopaque-1077 (Sigma-Aldrich) density gradient centrifugation were used for CD4+ T cells purification. The CD4+ T cells were purified from 50 × 106 PBMCs using EasySep? Human CD4+ T cell isolation kit (STEMCELL Technologies). Primary CD4+ T cells were cultured at 37?C in a 5 % (v/v) CO2 humidified atmosphere in Gibco? RPMI 1640 medium (Life Technologies) with GlutaMAX, HEPES, 100 U/ml interleukin-2 (IL-2; Peprotech, London, United Kingdom), 1 ?g/mL phytohemagglutinin (PHA) (Thermo Fisher Scientific), gamma-irradiated feeder PBMCs (healthy control) and either ART alone (100 nM efavirenz, 180 nM zidovudine, and 200 nM raltegravir) or ART + 100 ?M GYY4137. After 7 days, CD4+ T cells were cultured only with ART alone or ART + GYY4137 and 100 U/ml IL-2. For stimulation experiments on day 28, ART and ART + GYY4137 were washed off and 1 × 106 cells were treated with 1 µM prostratin for 24 h.

HIV-1 RNA and DNA isolation from primary CD4+ T cells
CD4+ T cells (1 × 106) from ART or ART + GYY4137 treated groups were harvested to isolate total RNA using Qiagen RNAeasy isolation kit and 200 ng of total RNA was reverse transcribed (iScript? cDNA synthesis kit, Bio-Rad). Reverse transcribed cDNA was diluted 10-fold and amplified using primers against HIV LTRs and seminested – PCR was performed using primers and probe listed in Table S6. Serially diluted pNL4.3 plasmid was used to obtain the standard curve. Isolation and RT-qPCR of total HIV DNA was performed as described earlier. Briefly, 1 × 106 cells were lysed (10 mM Tris-HCl, 50 nM KCl, 400 mg/ml proteinase K) at 55°C for 16 h followed by inactivation at 95°C for 5 min. Digested product was used as a template to set up first PCR with Taq polymerase (NEB), 1X Taq buffer, dNTPs, HIV and CD3 primers for 12 cycles. The second round amplification was done using seminested PCR strategy wherein 10 fold dilution of first round PCR product was used as a template, HIV and CD3 primers/probes, Taqman™ Fast Advance master mix (Applied Biosystems?) using SetupOnePlus? Real-time PCR system (Applied Biosystems?). DNA isolated from ACH2 cells that contain single copy of HIV per cell was used to obtain standard curve.

Surface marker analysis of primary CD4+ T cells derived from HIV patients
CD4+ T cells derived from HIV-1 infected patients were stained for surface markers using monoclonal antibodies: CD4-BUV395 (SK3), CD45RA-APC-H7 (HI100), CD27-BV785, CCR7-Alexa 647 (G043H7), CD38-PE-Cy5 and CD127-PerCP-Cy5.5. Additionally, cells were stained with Live/Dead fixable Aqua dead cell stain or AviD (Invitrogen) to exclude dead cells from the analysis as per manufacturer’s instructions. Stained samples were run on BD FACSAria? Fusion flow cytometer (BD Biosciences, San Jose, CA) and data was analyzed with FlowJo version 9.9.6 software (Treestar, Ashland, OR).

Statistical analysis
All statistical analyses were performed using GraphPad Prism software for Macintosh (version 9.0.0). The data values are indicated as mean ± S.D. For statistical analysis, Student t-test (in which two groups are compared) and one-way or two-way ANOVA (for analysis involving multiple groups) were used. Analysis of NanoString data was performed using the nSolver platform. Differences in P values <0.05 and fold change >1.5 were considered significant.

Results

A small-molecule H2S donor diminished HIV-1 reactivation and viral replication
One of the aims of the HIV-1 functional cure is to identify small molecules that can be combined with ART to delay or halt virus replication, reactivation, and replenishment of the latent reservoir (60,61). Having shown that diminished levels of endogenous H2S is associated with HIV-1 reactivation, it was examined if elevating H2S levels using a small-molecule H2S donor- GYY4137 sustains HIV-1 latency. The GYY4137 is a widely used H2S donor that releases a low amount of H2S over a prolonged period to mimic physiological production (Figure 8A). Because H2S discharge is unusually sluggish by GYY4137, the final concentration of H2S released is likely to be significantly lower than the initial concentration of GYY4137. This was confirmed by measuring H2S release in U1 cells treated with low (0.3 mM) or high (5 mM) concentrations of GYY4137 using methylene blue colorimetric assay. As a control, U1 cells were treated with 0.3 mM NaHS, which rapidly releases a high amount of H2S. As expected, NaHS treatment rapidly released 90 % of H2S within 5 min of treatment (Figure 8B). In contrast, GYY4137 uniformly released a low amount of H2S for the entire 120 h duration of the experiment (Figure 8B).

The effect of GYY4137 on HIV-1 reactivation systematically tested using multiple models of HIV-1 latency and replication. As a control, N-acetyl cysteine (NAC) was used that is known to block HIV-1 reactivation. Pretreatment of U1 with a non-toxic dose of GYY4137 (5 mM) (Figure 8C) diminished the expression of gag transcript by 2-fold at 24 h and 10-fold at 48 h post-PMA treatment (Figure 8D). The effect of GYY4137 on p24 levels in the supernatant was even more striking as it completely abolished the time-dependent increase in p24 concentration post-PMA treatment (Figure 8E). As expected, pretreatment with NAC similarly prevented PMA-triggered reactivation of HIV-1 in U1 (Figure 8E). Pretreatment of U1 with spent GYY4137, which comprises the decomposed backbone, showed no effect on PMA induced gag transcript (Figure 8F). Because TNF-? is a physiologically relevant cytokine that reactivates HIV-1 from latency, the effect of GYY4137 was tested on TNF-?-mediated virus reactivation. Treatment of U1 with TNF-? stimulated the expression of gag transcript by 42- and 169-fold at 24 and 48 h, post-treatment, respectively (Figure 9A). Addition of GYY4137 or NAC nearly abolished the reactivation of HIV-1 in response to TNF-? treatment (Figure 9A). Earlier, it was shown that depletion of CTH stimulated HIV-1 reactivation in U1 (Figure 4E). Therefore, it was tested to see if GYY4137 could complement this genetic deficiency and subvert HIV-1 reactivation. The U1-shCTH cells were pre-treated with 5 mM GYY4137 and p24 levels in the supernatant were measured. The elevated levels of p24 in U1-shCTH were reduced by 2-fold under basal conditions and 3.2-fold upon PMA stimulation in response to GYY4137 (Figure 9B). Both RNAi and chemical complementation data provide evidence that H2S is one of the factors regulating HIV-1 latency and reactivation in U1.

Similar to U1, it was next examined whether GYY4137 subverts HIV-1 reactivation in the J1.1 T-cell line model. Treatment of J1.1 with PMA for 12 h resulted in a 38-fold increase in gag transcript as compared to untreated J1.1 (Figure 10A), indicating efficient reactivation of HIV-1. Importantly, pretreatment with various non-toxic concentrations of GYY4137 (Figure 10B) reduced the stimulation of gag transcription by 2-fold upon subsequent exposure to PMA (Figure 10A). The inhibitory effect of GYY4137 on HIV-1 reactivation was relatively greater compared to NAC in J1.1 (Figure 10A). Another well-established T cell-based model of HIV-1 latency (J-Lat) was used to examine the influence of H2S. In J-Lat, the integrated HIV-1 genome encodes green-fluorescent protein (GFP), which allows precise quantification of HIV-1 reactivation from latency in response to LRAs such as PMA, TNF-?, and prostratin. Consistent with this, treatment of J-Lat with 5 ?M prostratin for 24 h induced significant HIV-1 reactivation, which was translated as 100 % increase in GFP+ cells (Figure 11). Pretreatment with GYY4137 significantly reduced HIV-1 reactivation in a dose-dependent manner (Figure 11).

It was also examined if an endogenous increase in H2S by GYY4137 halts the replication of HIV-1. To this end, CD4+ T cell (Jurkat) was infected with CXCR4-using HIV-1 (pNL4.3) and monitored HIV-1 replication by measuring the expression of gag transcript and the levels of p24 in the supernatant. A progressive increase in gag transcription and p24 release was detected for the entire 96 h duration of the experiment, indicating efficient viral replication (Figure 12A-C). Pretreatment with a non-toxic concentration of GYY4137 (0.3 mM) (Figure 12A) resulted in a 3 to 3.4-fold inhibition of HIV-1 replication at 72 and 96h post-infection (Figure 12B-C). Overall, these data establish that elevated levels of endogenous H2S efficiently suppress HIV-1 reactivation and replication.

GYY4137 reduced the expression of host genes involved in HIV-1 reactivation
To dissect the mechanism of GYY4137-mediated inhibition on HIV-1 reactivation, the expression of 185 genes associated with HIV-1 reactivation was examined using the NanoString nCounter technology as described above (Figure 13A and supplementary Table S4). Expression was analyzed for viral latency (unstimulated U1), reactivation (PMA-stimulated treated U1; PMA), and H2S-mediated suppression (U1-GYY+PMA). Consistent with the role of PMA-mediated oxidative stress in preceding HIV-1 reactivation, expression of genes encoding ROS, and RNS generating enzymes (e.g., NADPH oxidase [NCF1, CYBB] and Nitric oxide synthase [NOS2]) were up-regulated (Figure 13A). Also, the expression of major antioxidant enzymes (e.g., GPXs, PRDXs, and CAT) and redox buffers (GSH and TRX pathways) remained repressed in U1-PMA, indicative of elevated oxidative stress. Additionally, pro-inflammatory signatures (e.g., TGFB1 and SERPINA1) and trans-activators (e.g., FOS) were induced, whereas host factors involved in HIV-1 restriction (e.g., IRF1 and YY1) were repressed in U1-PMA as compared to U1. In agreement with the reduction in endogenous H2S levels during HIV-1 reactivation, expression of CTH involved in H2S anabolism was down-regulated and H2S catabolism (SQRDL) was up-regulated by PMA.

It was noticed that the treatment with GYY4137 reversed the effect of PMA on the expression of genes associated with oxidative stress, inflammation, anti-viral response, apoptosis, and trans-activators (Figure 13A). For example, GYY4137 elicited a robust induction of genes regulated by nuclear factor erythroid 2-related factor 2 (Nrf2) in U1-GYY+PMA compared to U1 or U1-PMA (Figure 13A). Nrf2 acts as a master regulator of redox metabolism by binding to the antioxidant response element (ARE) and initiating transcription of major antioxidant genes (e.g., GSH pathway, TXNRD1, HMOX1, CTH, GPX4, and SRXN1). The Nrf2 activity has been shown to pause HIV-1 infection by inhibiting the insertion of reverse-transcribed viral cDNA into the host chromosome. Furthermore, sustained activation of Nrf2-dependent antioxidant response is essential for the establishment of viral latency. A few genes encoding H2O2 detoxifying enzymes (e.g., CAT, GPX1, and PRDXs) were repressed in U1-GYY+PMA, indicating that the expression of these enzymes was likely counterbalanced by the elevated expression of other antioxidant systems by H2S. In line with the antagonistic effect of GYY4137 on HIV-1 transcription, the expression of HIV-1 trans-activator (FOS) was down-regulated, and an inhibitor of NF-?B signaling (NFKBIA) was induced in U1-GYY+PMA as compared to U1 or U1-PMA. Lastly, GYY4137 stimulated the expression of several anti-HIV (YY1, STAT1, STAT3, and IRF1) and pro-survival factors (CDKN1A and LTBR) that were repressed by PMA (Figure 13A). Altogether, H2S supplementation induces a major realignment of redox metabolism and immune pathways associated with HIV-1 reactivation.

GYY4137-mediated modulation of the Keap1-Nrf2 axis, NF-?B signaling, and activity of epigenetic factor YY1
Expression data indicate activation of the Nrf2 pathway and modulation of transcription factors such as NF-?B and YY1 upon treatment with GYY4137. It was tested if the mechanism of H2S-mediated subversion of HIV-1 reactivation involves these pathways. H2S has recently been shown to prevent cellular senescence by activation of Nrf2 via S-persulfidation of its negative regulator Keap-1. Under unstimulated conditions, Nrf2 binds to Keap1, and the latter promotes Nrf2 degradation via the proteasomal machinery. Nrf2 disassociates from the S-persulfidated form of Keap1, accumulates in the cytoplasm, and translocates to nuclei where it induces transcription of antioxidant genes upon oxidative stress. It was first examined if GYY4137 treatment accumulates Nrf2 in the cytoplasm. As expected, Nrf2 was not detected in U1 owing to its association with Keap1 under unstimulated conditions. However, a noticeable accumulation of Nrf2 was observed in U1-GYY+PMA compared to U1 or U1-PMA (Figure 13B). As an additional verification, the levels of an Nrf2-dependent protein HMOX-1was quantified. Similar to Nrf2, the levels of HMOX-1 were also induced in U1-GYY+PMA (Figure 13B). These findings are consistent with NanoString data showing activation of Nrf2-specific oxidative stress responsive genes in GYY4137 treated U1.

Next, it was determined if GYY4137 treatment targets NF-?B, which is a major regulator of HIV-1 reactivation. The cellular level of phosphorylated serine-536 in p65, a major subunit of NF-?B, is commonly measured to assess NF-?B activation. Since PMA reactivates HIV-1, an increase in p65 ser-536 phosphorylation in U1-PMA (Figure 13C) was observed. In contrast, pretreatment with GYY4137 significantly decreased PMA-induced p65 ser-536 phosphorylation (Figure 13C). These findings suggest that GYY4137 is likely to affect the DNA binding and transcriptional activity of NF-?B. Consistent with this, a two-step chromatin immunoprecipitation and genomic qPCR (ChIP-qPCR) assay showed that GYY4137-treatment significantly reduced the occupancy of p65 at its binding site (ER, enhancer region) on the HIV-1 LTR (Figure 14A-B).

GYY4137 induces the expression of another transcription factor YY1, which binds to HIV-1 LTR at RBEI and RBEIII and recruits histone deacetylase (HDACs) to facilitate repressive chromatin modifications. Overexpression of YY1 is known to promote HIV-1 latency. It was tested if GYY4137 promotes the binding of YY1 at RBEI and RBEIII sites on HIV-1 LTR by ChIP-qPCR. The occupancy of YY1 at RBEI and RBEIII was significantly enriched in the case of U1-GYY+PMA as compared to U1-PMA or U1 (Figure 14C). Taken together, these results indicate that increasing endogenous H2S levels by using a slow-releasing donor effectively modulate the Keap1-Nrf2 axis and activity of transcription factors to maintain redox balance and control HIV-1 reactivation.

GYY4137 blocks HIV-1 rebound from latent CD4+ T cells isolated from infected individuals on suppressive ART
Having shown that H2S suppresses HIV-1 reactivation in multiple cell line models of latency, the ability of GYY4137 was studied to limit virus transcription in primary CD4+ T cells derived from virally suppressed patients. A previously established methodology of maintaining CD4+ T cells was used from infected individuals on ART for a few weeks without any loss of phenotypic characteristics associated with HIV reservoirs (60). CD4+ T cells was isolated from peripheral blood mononuclear cells (PBMCs) of five HIV-infected subjects on suppressive ART. The CD4+ T cells were expanded in the presence of interleukin-2 (IL-2), phytohemagglutinin (PHA), and feeder cells (Figure 15A). The expanded cells were cultured for 4 weeks in a medium containing IL-2 with ART (100 nM efavirenz, 180 nM zidovudine, and 200 nM raltegravir) either in the presence or absence of GYY4137 (100 ?M) (Figure 15A). In this model, the virus reactivates by day 14 followed by progressive suppression at day 21 and latency establishment by day 35. The virus can be reactivated from this latent phase using well-known LRAs (60). Temporal expression of viral RNA was quantified using RT-qPCR periodically at 7 days interval. The levels of viral RNA increased initially (day 7 to 14), followed by low to undetectable levels by day 28 (Figure 15B). By day 28, virus RNA was uniformly untraceable in cells derived from GYY4137 treated and untreated groups, indicating the establishment of latency (Figure 15B).

The ability of GYY4137 was next assessed to efficiently block viral reactivation. On day 28, CD4+ T cells were stimulated with the protein kinase C (PKC) activator, prostratin, in the absence of any treatment. The activation of viral transcription was measured 24 h later by RT-qPCR. The removal of ART uniformly resulted in a viral reactivation by prostratin in CD4+ T cells of HIV-patients (Figure 15C). In contrast, upon ART+GYY4137 removal followed by prostratin stimulation, viral reactivation was attenuated by 90 % (N = 5, mean) for all 5 patient samples, and individual inhibition ranged from 78.9 % to 100 % (Figure 15C). Overall, pretreatment with ART+GYY4137 significantly reduced prostratin-mediated HIV-reactivation when compared to ART alone (p < 0.01) (Figure 15D).

The immune-phenotype of CD4+ T cells treated with ART or ART+GYY4137 was also examined by monitoring the expression of activation (CD38) and quiescence (CD127) marker. As expected, CD38 expression increased, and CD127 expression decreased at day 7 and 14 during the activation phase, followed by a gradual reversal of the pattern during the resting phase at day 21 and 28 (Figure 15E-F). Interestingly, GYY4137 treatment did not alter the temporal changes in the expression of activation and quiescence markers on CD4+ T cells (Figure 15E-F). Since memory CD4+ T cells are preferentially targeted by HIV, it was further analyzed if the status of naive (TN), central memory (TCM), transitional memory (TTM), and effector memory (TEM) is affected by the GYY4137 in ex vivo expansion model. It was observed that the CD4+ T cells that responded to ex vivo activation were mainly composed of TTM and TEM in patient samples (Figure 16A-E). This is consistent with the study reporting the presence of translation-competent genomes mainly in the TTM and TEM of ART-suppressed individuals. Interestingly, the fraction of TTM shows a progressive decline, whereas TEM displays gradual increase during transition from activation (7-14 days) to quiescence phase (21-28 days) (Figure 16D-E). The addition of GYY4137 did not affect dynamic changes in the frequency of TTM and TEM subpopulations (Figure 16D-E).

Finally, it was tested if the reduction in viral RNA upon GYY4137 treatment is due to the loss of cells with ability to reactivate virus or selection of cells subset that is non-responsive to reactivating stimuli. We did not find significant differences in total HIV DNA content between the CD4+ T cells immediately isolated from the patient’s PBMCs (ex vivo) and the expanded cells treated with ART+GYY4137 or ART alone for the entire duration of the experiment (Figure 15G). The viability of cells remained comparable between ART+GYY4137 and ART treatment groups overtime (Figure 16F). Altogether, using a range of cellular and immunological assays, it was confirmed that the characteristics of an individual’s viral reservoir remain preserved, and the suppression of viral RNA upon GYY4137 treatment is the result of H2S-mediated inhibition of HIV-1 transcription rather than a reduction in proviral content or an altered CD4+ T cell subsets. In sum, prolonged exposure to GYY4137 results in potent inhibition of viral reactivation, suggesting a new H2S-based mechanism to neutralize bursts of virus reactivation under suppressive ART in vivo.

GYY4137 prevents mitochondrial dysfunction in CD4+ T cells of HIV-patients during viral rebound
Because virus reactivation upon depletion of endogenous H2S resulted in mitochondrial dysfunction and redox imbalance in U1, it was tested if the elevation of H2S levels by GYY4137 improves mitochondrial health of primary CD4+ T cells during virus reactivation ex vivo. As described earlier, CD4+ T cells harboring latent virus upon prolonged (28 days) treatment of ART and ART+GYY4137 were stimulated by prostratin or left unstimulated and subjected to mitochondrial flux analysis. The unstimulated cells from both ART and ART+GYY4147 cultures did not show any difference in OCR (Figure 17A). In contrast, several features reflecting efficient mitochondrial activity such as basal respiration and ATP-coupled respiration were significantly higher in prostratin stimulated CD4+ T cells in case of ART+GYY4137 treatment than ART alone (Figure 17B-C). Consistent with this, mitoROS generation upon stimulation with prostratin or other LRAs such as PMA/ionomycin was reduced in ART+GYY4137 treated CD4+ T cells than ART alone (p = 0.005 and p = 0.014), and was nearly comparable to unstimulated cells (Figure 17D-E). The reduction in mitoROS could be a consequence of GYY4137-mediated increase in the expression of Nrf2-dependent antioxidant systems. RT-qPCR analysis of a selected set of Nrf2-dependent genes on CD4+ T cells treated with GYY4137 for 28 days was directly tested. Consistent with the findings in U1, treatment with ART+GYY4137 uniformly induced the expression of antioxidant genes in the latently infected CD4+ T cells compared to cell treated with ART alone (Figure 17F).

Altogether, these data suggest that H2S not only prevents virus reactivation but also improves mitochondrial bioenergetics and maintains redox homeostasis, which could be important for in vivo suppression of viral rebound and replenishment of the reservoir.

Management of Coronavirus

Measurement of levels of H2S metabolizing genes (cbs, cth, mpst, sqrdl) in different cell lines (VeroE6, HEK-ACE2, Calu-3) during SARS-CoV-2 infection
Cells were seeded in 24 or 12 well plates such that the density at the time of infection is 90-95%. Cells were infected with SARS-CoV-2 at an MOI of 0.01 and 0.1 for 1 h. Virus inoculum was removed after 1 h and infection medium (DMEM with 2% FBS) was added and incubated further. Cells were harvested at multiple time points post infection (8h, 24h, 48 h) and processed for total cellular RNA isolation (for qRT-PCR). Total cellular RNA or viral RNA was converted to cDNA as per manufacturer protocol (Bio-rad iScript cDNA synthesis kit). Reverse transcribed cDNA was subjected to SYBR based real time PCR. The expression level of each gene was normalized to 18S. It was found that there was significant downregulation of cbs, cth and mpst and upregulation of sqrdl.

Measurement of Oxidative stress upon SARS-CoV-2 infection
To measure the induction of oxidative stress during SARS-CoV-2 infection, a genetically encoded biosensor Grx1roGFP2 was utilized. The Grx1-roGFP2 is a ratiometric redox biosensor, which shows higher 405/488 fluorescence excitation ratio at a uniform emission of 510 nm upon oxidative stress, whereas a decrease in ratio indicates reductive stress.
Vero E6 cells stably expressing cytosolic and mitochondrial localized Grx1-roGFP2 were prepared. These cell lines were used for SARS-CoV-2 infection. Cells were seeded in 24 well plates and infected with SARS-CoV-2 MOI of 0.01 and 0.1 for 1 h. Virus inoculum was removed after 1 h and infection medium was added and incubated further. Cells were harvested at multiple time points (8h, 24h, and 48 h) post infection and blocked with 1 mM NEM and fixed with 4% PFA before analyzing in FACS. The readings were normalized and intracellular sensor redox potential EroGFP2 was calculated. The same protocol was followed for a second group of cells. However, these were further treated with the H2S donor GYY-4137. As shown in Figure 18A, infected cells untreated with GYY-4137 showed high oxidative stress. However, infected cells treated with GYY-4137 showed significant decrease in oxidative stress.
H2S inhibits SARS-CoV-2 infection in cell line models
Vero E6 cells were pretreated with 5 mM GYY4137 for 4 h and subsequently infected with SARS-CoV-2 virus at an MOI of 0.01 for 1 h. Post this, viral inoculum was removed and fresh media restoring the compound was added, and incubated further for 48 h. After 48 h, supernatant was processed for determining infectious viral titre by plaque assay and for viral RNA isolation. The same protocol was followed for the untreated/control cells, but without the pretreatment with GYY4137. As shown in Figure 18B, significant reduction in viral load was obtained in cells treated with GYY4137 both by qRT-PCR and plaque assay as compared to the untreated cells. The data was also validated in different cell lines (HEK-ACE2 and Calu-3).
H2S inhibits SARS-CoV-2 infection in Gold Syrian hamster models
Gold Syrian Hamsters were intranasally inoculated with 105 PFU SARS-CoV-2 in 100 µL PBS. Treatment strategy involved administration of Na-GYY4137 (50 mg/kg) 1 h before and 6 h after infection. Na-GYY4137 was not administered to the control group. Total body weight was recorded each day during the entire course of the experiment until the animals were sacrificed at 4 dpi. Viral RNA load in lung tissue specimens was detected by Plaque Assay. As shown in Figure 18C, significant reduction in viral load was observed in Na-GYY4137 treated animals compared to the control (untreated) group.
The above data clearly demonstrates downregulation of H2S in SARS-CoV-2 infected condition. The data also shows that H2S donors such as GYY-4137/Na-GYY4137 help in mitigating SARS-CoV-2 replication both in vitro and in vivo.
,CLAIMS:
1. A composition comprising at least one H2S donor and at least one antiviral agent, optionally along with pharmaceutically acceptable excipient.
2. The composition as claimed in claim 1, wherein the at least one H2S donor and the at least one antiviral agent are provided in a single composition.
3. The composition as claimed in claim 1, wherein the composition is in the form of a combination or a kit.
4. The composition as claimed in claim 3, wherein the at least one H2S donor is provided as a separate component from the at least one antiviral agent.
5. The composition as claimed in claim 1, wherein the H2S donor is present at a concentration ranging from about 100 ?M to about 5 mM and the antiviral agent is present at a concentration ranging from about 100 nM to about 500 nM.
6. The composition as claimed in claim 1, wherein the H2S donor is a chemically synthesized H2S donor, a naturally occurring H2S donor, or a combination thereof.
7. The composition as claimed in claim 6, wherein the chemically synthesized H2S donor is selected from a group comprising morpholin-4-ium 4-methoxyphenyl(morpholino)phosphinodithioate, 5-amino-2-hydroxy-benzoic acid 4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenyl ester hydrochloride , Diallyl disulfide, Na-morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate, Otenaproxesul, ACS-15, Na2S, and combinations thereof.
8. The composition as claimed in claim 6, wherein the naturally occurring H2S donor is selected from a group comprising S-allyl-L-cysteine, Diallyl trisulfide, S-propyl-L-cysteine, Sulphoraphane and combinations thereof.
9. The composition as claimed in claim 7, wherein the H2S donor is morpholin-4-ium 4-methoxyphenyl(morpholino)phosphinodithioate or Na-morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate.
10. The composition as claimed in claim 1, wherein the antiviral agent is an antiviral drug.
11. The composition as claimed in claim 10, wherein the antiviral drug is an antiretroviral drug for HIV and is selected from a group comprising Efavirenz, Zidovudine, Raltegravir, Lamivudine, Tenofovir disoproxil and combinations thereof.
12. The composition as claimed in claim 1, wherein the pharmaceutically acceptable excipient is selected from a group comprising additives, gums, sweeteners, coatings, binders, disintegrants, lubricants, disintegration agents, suspending agents, solvents, colorants, glidants, anti-adherents, anti-static agents, surfactants, plasticizers, emulsifying agents, flavors, viscocity enhancers, antioxidants, and combination thereof.
13. The composition as claimed in claim 1, wherein the composition is in a form selected from a group comprising liquid, powder, capsule, tablet, injectable, patch, ointment, gel, emulsion, cream, lotion, dentifrice, spray, drop and combinations thereof.
14. A method of preparing the composition as claimed in claim 1, said method comprising:
(b) obtaining at least one H2S donor, obtaining at least one antiviral agent and
combining the at least one H2S donor and the at least one antiviral agent optionally along with pharmaceutically acceptable excipient to obtain the composition; or (b) assembling at least one H2S donor, at least one antiviral agent, optionally along with pharmaceutically acceptable excipient and an instruction manual.
15. An invitro method of modulating viral reactivation, viral replication and/or viral latency in a cell, said method comprising contacting the cell with an H2S donor optionally along with a pharmaceutically acceptable excipient or the composition as claimed in claim 1.
16. The invitro method as claimed in claim 15, wherein the virus is selected from a group comprising human immunodeficiency virus, coronavirus, influenza virus and combinations thereof.
17. The invitro method as claimed in claim 15, wherein the H2S donor is a chemically synthesized H2S donor, a naturally occurring H2S donor, or a combination thereof.
18. The invitro method as claimed in claim 17, wherein the chemically synthesized H2S donor is selected from a group comprising morpholin-4-ium 4-methoxyphenyl(morpholino)phosphinodithioate, 5-amino-2-hydroxy-benzoic acid 4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenyl ester hydrochloride , Diallyl disulfide, Na-morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate, Otenaproxesul , ACS-15, Na2S, and combinations thereof; and wherein the naturally occurring H2S donor is selected from a group comprising S-allyl-L-cysteine, Diallyl trisulfide, S-propyl-L-cysteine, Sulphoraphane and combinations thereof.
19. The invitro method as claimed in claim 18, wherein the H2S donor is morpholin-4-ium 4-methoxyphenyl(morpholino)phosphinodithioate or Na-morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate.