Combinatorial pharmaceutical compositions for treatment of virus-related neurological diseases

Abstract

Provided herein are compositions and methods for treating a virus-related disease or disorder. In certain instances, the compositions and methods are useful for treating CMV infection.

Description

[0001] TITLE OF THE INVENTION Combinatorial Pharmaceutical Compositions for Treatment of Virus-related Neurological Diseases

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 661,992, filed June 20, 2024, which is hereby incorporated by reference herein in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under RO3-AH69437 awarded by the National Institute of Health. The government has certain rights in the invention.

[0006] BACKGROUND OF THE INVENTION

[0007] Human cytomegalovirus (HCMV) is a P-herpesvirus that causes a common congenital (in utero) infection, affecting approximately 6 in every 1000 births, making it the most common congenital infection in the United States (Kenneson et al., 2007, Rev Med Virol 17:253-276; Cheeran et al., 2009, Clin Microbiol Rev 22:99-126). Congenital HCMV (cHCMV) infection leads to long term neurological impairments, with approximately 20-30% of children developing hearing loss, vestibular defects, cognitive defects, and / or vision impairment (Cheeran et al., 2009, Clin Microbiol Rev 22:99-126). These neurological defects can persist throughout life (Boppana et al., 1992, Pediatr Infect Dis J 11 :93-98; Coats et al., 2000, Journal of American Association for Pediatric Ophthalmology and Strabismus 4: 110-116; Jin et al., 2017, Pediatric Infectious Disease Journal 36:877-882). Moreover, since HCMV persists for life after infection, a substantial number of asymptomatic newborns can develop symptoms later in development impairment (Cheeran et al., 2009, Clin Microbiol Rev 22:99-126; Boppana et al., 1994, Pediatr Infect Dis J 13: 1139—1142; Fowler et al., 1997, J Pediatr 130:624- 630). Despite the high prevalence of HCMV, little is known about the mechanisms of disease in affected tissues. Currently the only clinical intervention is anti-viral treatment, which is only effective during active viral replication and may not address the course of disease.

[0008] HCMV only infects humans and cannot be used in any animal models. However, investigators have modeled cHCMV infection of the brain by intracranial injections of murine (M)CMV into fetal, newborn or adult mice (Cheeran et al., 2009, Clin Microbiol Rev 22:99-126; Prasad et al., 2017, J Neuroinflammation 14:82; Chauhan et al., 2017, Sci Rep 7:41889; Lokensgard et al., 2016, J Neuroinflammation 13: 114; Lokensgard et al., 2015, Glia 63: 1982-1996; Schachtele et al., 2014, Glia 62: 1582-1594; Mutnal et al., 2012, PLoS One 7:e33143; Schachtele et al., 2011, J Neurovirol 17:201— 211; Mutnal et al., 2011, PLoS One 6:el6211; Mutnal et al., 2011, J Neurovirol 17:424- 437; Mutnal et al., 2010, J Neuroimmunol 227: 101-110; Cheeran et al., 2009, J Neurovirol 15:334-342; Cheeran et al., 2005, J Neurovirol 11 :274-280; Cheeran et al., 2004, J Neurovirol 10: 152-162; Zhou et al., 2022, JCI Insight 7) or intraperitoneal (i.p.) infections of newborn mice (<24 hrs old) (Lisnic et al., 2021, Humana Press Inc. 2244:365-401; Koontz et al., 2008, J Exp Med 205:423-435; Slavuljica et al., 2015, Cell Mol Immunol 12: 180-191). This latter model, originally developed by Jonjic and Britt, has two major advantages for studying congenital CMV disease: i) newborn mice are developmentally similar to humans in the late 2nd / early 3rdtrimester - a time when HCMV commonly infects the fetus, and ii) MCMV reaches neural tissues from the circulation, as would be expected from a placental transmission. Critically, these and other investigators have shown that MCMV infection of newborn mice recapitulates parts of a natural human infection with evidence of hearing loss and cognitive / developmental deficits (Koontz et al., 2008, J Exp Med 205:423-435; Bradford et al., 2015, PLoS Pathog 11 :el004774). Moreover, some data have suggested that disease in the brain may be driven by inflammation, possibly IFN-y and / or TNF-a and involves influx of innate immune cells and activation of microglia (Kvestak et al., 2021, Journal of Experimental Medicine 218; Bantug et al., 2008, The Journal of Immunology 181 :2111-2123; Seleme et al., 2017, J Virol 91; Brizic et al., 2019, Med Microbiol Immunol 208:487-494; Brizic et al., 2018, Eur J Immunol 48:950-964). While this newborn infection model has been used to study brain infections, it is unknown whether there is any involvement of the eye. Approximately, 20% of symptomatic children develop vision impairment after cHCMV infection (Boppana et al., 1992, Pediatr Infect Dis J 11 :93-98; Coats et al., 2000, Journal of American Association for Pediatric Ophthalmology and Strabismus 4: 110-116; Jin et al., 2017, Pediatric Infectious Disease Journal 36:877-882; Boppana et al., 1994, Pediatr Infect Dis J 13: 1139-1142). Typically, this is characterized by chorioretinitis with retinal lesions that occur randomly throughout the retina and can lead to permanent retinal scaring, and optic neuritis leading to optic nerve atrophy and cortical visual impairment (Coats et al., 2000, Journal of American Association for Pediatric Ophthalmology and Strabismus 4: 110-116; Ghekiere et al., 2012, J Pediatr Ophthalmol Strabismus 49:274- 282; Recchia, 2012, Archives of Ophthalmology 130:525). Additionally, inflammation in the retinal blood vessels has been described, manifesting as so called “frosted angiitis” (Recchia, 2012, Archives of Ophthalmology 130:525; Tawse et al., 2014, Journal of American Association for Pediatric Ophthalmology and Strabismus 18:78-80; Walker et al., 2004, Eye 18:527-533). However, the mechanisms behind these pathologies are unclear. The retina is a neural tissue, derived from the neural tube, separated from the blood supply by the blood-retinal barriers (BRBs) and considered to be part of the CNS (Campbell et al., 2012, Adv Exp Med Biol 763:70-84; Cunha-Vaz, 2009, European Ophthalmic Review 3: 105; O’Leary et al., 2023, FEBS J 290:878-891; Stenkamp, 2015, 397-414). However, while there are similarities between the brain and eye, there are also many fundamental differences including the existence of two distinct BRBs that form during the period of infection (Chow et al., 2017, Neuron 93: 1325-1333) and later than the blood brain barrier (Daneman et al., 2010, Nature 468:562-566), the presence of Muller glial cells, which are known to respond to infections (Reichenbach et al., 2020, Glia 68:768-796; Kumar et al., 2013, Crit Rev Immunol 33:119-135), the constant cellular damage caused by light, and multiple immune modulating mechanisms (Taylor, 2009, Eye 23: 1885-1889; Hunter et al., 2012, Prog Retin Eye Res 31 :28-42). Most significantly, unlike the immune pathology described for the brain, previous work on CMV-driven retinitis in adults has suggested that T cells are protective of the eye and that CMV induces retinal necrosis in the absence of an adequate T cell response (Atherton et al., 1992, Invest Ophthalmol Vis Sci 33:3353-60; Dix et al., 2003., J Virol 77:3402- 3408; Dix et al., 2004, Arch Virol 149:2235-2244; Lu et al., 1997, Invest Ophthalmol Vis Sci 38:301-10; Bigger et al., 1999, Invest Ophthalmol Vis Sci 40:2608-13; Zhang et al., 2002, Journal of Clinical Virology 25: 137-147; Chien et al., 2012, J Virol 86: 10961- 10978; Lu et al., 1997, Invest Ophthalmol Vis Sci 38:301-10; Mo et al., 2014, Investigative Opthalmology & Visual Science 55:7137; Dix et al., 1994, Curr Eye Res 13:587-595; Carter et al., 2021, Exp Eye Res 209: 108651). Thus, it is unclear how viral infection progresses or is controlled in the presence of an immature, but functional fetal or neonatal immune response, and how damage occurs during development of this critical CNS tissue.

[0009] There remains a need in the art for compositions and methods for treating or preventing virus-related diseases or disorders. The invention satisfies this unmet need.

[0010] SUMMARY OF THE INVENTION

[0011] The invention relates to compositions and methods for treating virus- related diseases or disorders, including neurological diseases and disorders and systemic diseases and disorders, such as hepatitis. The invention is based, in part, on the unexpected discovery that immunosuppressant treatment prevents immune cell infiltration and neurological pathology during viral infection. The invention is also based on the discovery that administration of immunosuppressants in combination with antiviral agents yielded synergistic effects.

[0012] In some embodiments, the at least one immunosuppressant is selected from the group consisting of prednisone, dexamethasone, fontolizumab, emapalumab, AMG 811, baricitinib, filgotinib, tofacitinib, upadacitinib, ruxolitnib, abrocitnib, itacitnib, fedratinib, pacritinib, gandotinib, momelotinib, pravastatin, ISS-840, PY*LKTK, PM- 73G, CJ-1383, resveratrol, catechins, PLX5622, adalafil, metformin, a CXCR3 inhibitor, a Integrin a4 inhibitor, a LFA-1 inhibitor, a VCAM inhibitor, and a ICAM inhibitor.

[0013] In some embodiments, the at least one antiviral agent is selected from the group consisting of ganciclovir, valganciclovir, letermovir, acyclovir, foscamet, cidofovir, rnaribavir, leflunomide, brincidofovir, cyclopropavir, BDCRB, TRCB, tomeglovir, quercetin, baicalein, artesunate, TF27, valnocatmide, and valproic acid. In some embodiments, the virus-related disease or disorder is a cytomegalovirus (CMV) infection. In some embodiments, the virus-related disease or disorder is a systemic disease or disorder. In some embodiments, the virus-related systemic disease or disorder is hepatitis. In some embodiments, the virus-related disease or disorder is a neurological disease or disorder. In some embodiments, the virus-related neurological disease or disorder is a cytomegalovirus (CMV) infection.

[0014] In some embodiments, the invention relates to methods of treating or preventing a virus-related disease or disorder in a subject comprising administering to the subject a therapeutically effective amount of a composition comprising at least one immunosuppressant and at least one antiviral agent.

[0015] In some embodiments, the at least one immunosuppressant is selected from the group consisting of prednisone, dexamethasone, fontolizumab, emapalumab, AMG 811, baricitinib, filgotinib, tofacitinib, upadacitinib, ruxolitnib, abrocitnib, itacitnib, fedratinib, pacritinib, gandotinib, momelotinib, pravastatin, ISS-840, PY*LKTK, PM- 73G, CJ-1383, resveratrol, catechins, PLX5622, adalafil, metformin, a CXCR3 inhibitor, a Integrin a4 inhibitor, a LFA-1 inhibitor, a VC AM inhibitor, and a ICAM inhibitor.

[0016] In some embodiments, the at least one antiviral agent is selected from the group consisting of ganciclovir, valganciclovir, letermovir, acyclovir, foscamet, cidofovir, maribavir, leflunomide, brincidofovir, cyclopropavir, BDCRB, TRCB, tomeglovir, quercetin, baicalein, artesunate, TF27, valnocatmide, and valproic acid.

[0017] In some embodiments, the at least one immunosuppressant and the at least one antiviral agent are administered at different times.

[0018] In some embodiments, the at least one immunosuppressant and at least one antiviral agent are administered simultaneously.

[0019] In some embodiments, the subject is an infant or child.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. Figure 1 A through Figure 1 J depict the results of exemplary experiments demonstrating MCMV infection disseminates to the eye and induces retinal pathologies. Figure 1A depicts representative weight gain measured in grams (g) overtime of mouse neonates infected with MCMV (n=3-15) or controls (n=3-21). Figure IB depicts representative (n> 24) survival at PND 15 of MCMV infected vs control pups based on the number injected on PND 0. Figure 1C depicts representative MCMV genome copies per 100 ng of DNA in ocular tissue over time (n=6 eyes from 6 individual mice per timepoint). Limit of detection (LOD) = 44 genomes / lOOng. Figure ID depicts representative MCMV titer in ocular tissue over time measured in PFU / g of tissue (n=3 eyes from 3 individual mice per timepoint). LOD = 150 plaques / g. Figure IE depicts representative H&E staining of eyes from mice injected with MCMV or PBS controls at PND 10, PND 15 and PND 28 (representative of n= 9 to 16 per group). Shown are cellular clustering in the GCL (doted circles) and cellular infdtration (arrows). Figure IE to Figure II depict specific pathologies of MCMV infected eyes. Figure IF depicts Rosette structures of the ONL and INL at PND 10. Figure 1G depicts cellular accumulation in GCL (dotted circle) and cellular infiltration (arrows) at PND 15. Figure 1H depicts breaks in RPE layer (star). White dotted line represents RPE layer. Figure II depicts cellular deposits in photoreceptor layer (white star) and cellular infiltration (arrows) at PND15. Figure 1J depicts representative cSLO IRAF images of MCMV and control eyes at PND 28 (representative of n=3 per group). White box highlighting representative area of photoreceptor infoldings (arrows). Scale bars = 100 pm Error bars represent standard deviation. Analyzed by one-way ANOVA with multiple comparisons; ***p<0.001.

[0022] Figure 2A through Figure 2H depict the results of exemplary experiments evaluating immune infiltration and cell death in the eyes of MCMV-infected neonates. Figure 2A depicts representative images showing areas of virus infected cells. MCMV infected eyes were stained for CD45 and MCMV-IE1 (pp89). White arrows denote MCMV positive cells and yellow arrows denote CD45 / MCMV double positive cells. Representative of n > 5 mice. Figure 2B depicts immune cell infiltration over time. MCMV and control mice at PND 10, PND 15 and PND 28 stained for CD45 and MCMV-IE1. MCMV-positive cells are indicated by the white arrows. Images on the right-hand side of the panels show higher magnification of selected regions. Representative of n > 5. Figure 2C depicts representative retinal flat mounts showing distribution of CD45+cell infiltration at the indicated timepoints. Tissue edges were outlined with white dotted lines. White arrows denote retinal vasculature. Representative of n=3-5 Figure 2D depicts representative retinal flat mount stained for CD45+and CD8+cells to identify overlap. Representative of n=3. Figure 2E depicts representative TUNEL staining at PND 15 of MCMV infected and control mice. Images on the right-hand side of the panels show higher magnification of selected regions. Representative of n=5 per group. Figure 2F depicts TUNEL+cells were calculated from 9 images per mouse in n=3- 4 infected mice or n=3 control mice. Error bars represent standard deviation. Figure 2G depicts retinal flat mounts of MCMV and control mice injected with 10,000 Da FITC- dextran. Vasculature breaks are indicated by the white arrows (n=3 mice per group). Figure 2H depicts representative RPE flat mounts of MCMV and control mice stained with occluding to identify RPE tight junctions. Representative images taken from mid retina. Breaks in occludin are indicated with white arrows (n=4 mice per group). Scale bars = 100 pm.

[0023] Figure 3 A through Figure 3C depict the results of exemplary experiments evaluating transcriptional profiling of dysregulated retinal homeostasis and immune activation caused by MCMV. Figure 3A depicts unsupervised co-expression clustering analysis was conducted on differentially expressed genes sets from MCMV-infected and control eyes at all time points. Line graphs of each gene from each cluster over time were plotted with respect to Z-score. Dotted lines separate control from infected conditions in each cluster. Figure 3B depicts representative differential expression analysis conducted between MCMV-infected and control eyes at different time points. Gene set enrichment analysis (GSEA) was used to generate enrichment for MSibDB C5 GO: gene ontology gene set at each timepoint. Some of the highest ranked, non-redundant enrichment was selected for each time point and plotted as lollipops by their normalized enrichment score (NES, denoted as line length) and significance (-logl0(FDR q-val), denoted as dot size). Figure 3C depicts representative differentially expressed genes from eyes at PND 10, PND 15 and PND 28 analyzed by GSEA to identify specific cellular identity signatures. Each time point and condition was compared to all differential expression data from all of the time points to identify unique cell signatures at a given time (MSibDB C8: Cell type signature gene set). Top enrichments for each cellular category for each time point were plotted as lollipops with NES denoted by line length and (-loglO(FDR q-val)) denoted as dot size. Open dots indicate control timepoints and closed dots are MCMV infected timepoints. n=3 samples per group with each sample pooled from 2 mice.

[0024] Figure 4A through Figure 4K depict the results of exemplary experiments demonstrating glial cell activation in response to MCMV infection in the eye. Figure 4A depicts representative microglia morphology over time. MCMV infected and control retinal flat mounts stained with Ibal (representative of n=5 per timepoint). Scale bars = 100 pm for main image and 50 pm for insets. Note that the zoom for each inset is scaled to the size of a single cell. Figure 4B depicts the average Ibal+ cells per 1 mm2 area of DAPI. Counts were averaged from 5 retinal flat mount images per mouse from n=5 mice. Figure 4C to Figure 4F depict representative morphological analysis of Ibal+ cells from MCMV and control retinas as assessed by: the total process endpoints / cell (Figure 4C), Total density of each cell (Figure 4D), Total number of branches per cell (Figure 4E) and Cell complexity measured by fractal dimensions per cell (Figure 4F). Each point shows an averaged value derived from n > 15 cells from an individual mouse and was graphed from n=3-5 individual mice per analysis. Significance was analyzed by unpaired t-test; *p < 0.05 **p <0.01 ***p < 0.001 ****p < 0.0001. Figure 4G depicts astrocyte distribution over time as assessed on retinal flat mounts stained with GFAP. Dotted white circle denotes an area of increased staining density. Figure 4H through Figure 41 depict activation of astrocytes as assessed by: intensity of GFAP staining in MCMV-infected and control retinas (Figure 4H) and percent coverage of GFAP cells per DAPI area of MCMV infected and control retinas (Figure 41). Data show an average derived from 5 images per animal and n=5 mice per group. Asterisks indicate statistical significance as in C-F. Figure 4J depicts representative Muller glial cell activation. Shown are sections from MCMV-infected and control samples stained with GFAP, (representative of n=3-5 mice per group). Figure 4K depicts representative differential expression analysis of genes associated with microglia and astrocyte activation. Genes indicating microglia and astrocyte activation were plotted. Color indicates Log2 fold change in infected mice compared to controls at the same timepoint. Dot size indicates significance (loglO(padj)). Figure 5A through Figure 5K depict the results of exemplary experiments demonstrating that glial nodules form in response to MCMV infection in the eye and recruit CD8+ T cells. Figure 5 A depicts representative nodules in the GCL of MCMV- infected eyes composed of Ibal+ microglia surrounded by GFAP+ astrocytes and Muller glial cells (representative of n=5 mice per group). Figure 5B depicts a representative image of MCMV+ cells in the center of the nodule in GCL. Figure 5C depicts a retinal flat mount of glial nodule stained for GFAP+ astrocytes, Ibal+ microglia and CD8+ T cells (representative of n=3 mice). Figure 5D and Figure 5E depict a representative 3D projection of retinal flat mounts from an MCMV infected eye stained with GFAP, Ibal, and CD8. Projections are shown with the GCL at the top and subretinal space at the bottom. Shown is an area with a glial nodule (Figure 5D) and area with no nodule formation (Figure 5E). Figure 5F depicts a representative contour graph of a retinal flat mount showing the distribution of Ibal+ staining intensity overlayed with the spatial distribution of CD8+ cells. See Figure 14E for the actual image. Figure 5G depicts a representative violin plot of the distance of each CD8+ T cell to highest intensity iba 1 + point on n=3 flat mount images, analyzed as in Figure 5F. GlialNodule+ denotes area with a formed nodule and Glial Nodule- denotes an area with no nodule formation in the same retina. Analyzed by unpaired t-test. Figure 5H depicts representative differential gene expression of the indicated chemokines at the indicated timepoints from the RNAseq analyses described in Figure 4. Analyzed by one-way ANOVA with multiple comparisons. Figure 51 depicts in situ hybridization (RNAScope) for expression of CXCL9 and CCL2 mRNA in glial nodules and co-stained with antibodies specific for Ibal. Dotted white circle indicates a glial nodule. Figure 5J depicts in situ hybridization for expression of CXCL10 and CXCL11. Dotted white circle indicates a glial nodule. Figure 5K depicts a representative image from an MCMV-infected eye stained with MHCII, iNOS and Ibal. White dotted circle denotes a glial nodule. White star indicates a blood vessel. Image is representative of n=3 mice. Scale bars indicate 100 pm throughout. *p < 0.05 **p <0.01 ***p < 0.001 ****p < 0.0001.

[0025] Figure 6A through Figure 61 depict the results of exemplary experiments demonstrating that blocking CXCR3 prevents retinal pathology and immune infiltration, while maintaining BRB integrity. Figure 6A depicts MCMV titer measured by plaque assay from lung tissue on PND15 from mice infected with MCMV and either treated with aCXCR3 or isotype control antibody. Data is pooled from n=12-17 mice per group. LOD = 150 plaques / g. Analyzed by unpaired t-test; *p < 0.05 **p <0.01 ***p < 0.001 ****p < 0.0001. Figure 6B depicts MCMV genome copies per 100 ng of DNA in ocular tissue of mice treated with or without aCXCR3 (n=5-6 mice per group, analyzed as in A). LOD = 44 genomes / g. Figure 6C depicts representative H&E stained sections from control (uninfected) mice or MCMV-infected mice treated with isotype control or and aCXCR3 antibodies. Images are representative of n=8 mice per group. Figure 6D through Figure 61 depict representative eyes from MCMV-infected mice treated with isotype-control or aCXCR3 antibodies (n=3-8 mice per group) showing: CD45+ and CD8+ cells on thin sections (Figure 6D). Images on the right-hands side show an enlarged region. GFAP, Ibal and CD45 staining on retinal flat mounts (Figure 6E). The white dotted line outlines the retinal vasculature in the bottom image. Figure 6F depicts representative TUNEL staining of thin sections. Images on the right-hands side show an enlarged region. Figure 6G depicts representative higher magnification images of GFAP, Ibal and CD45 staining on retinal flat mounts showing the presence of CD45+ cells in the vasculature. White line outline denotes vasculature. White box in top panels indicates site of vascular leakage. Figure 6H depicts FITC-dextran leakage from the vasculature in the absence of CXCR3 blockade visualized on retinal flat mounts. Sites of vasculature breaks and leakage are indicated by white arrows. (Figure 61 depicts representative occludin staining on RPE flat mounts. Representative images taken from mid retina. Breaks in occludin are indicated by white arrows. Scale bars indicate 100 pm, except in Figure 61 where scale bars indicate 50 pm.

[0026] Figure 7A through Figure 71 depict the results of exemplary experiments demonstrating that blocking CXCR3 reduces glial cell activation. Figure 7A depicts representative retinal flat mounts of control (uninfected) mice, and MCMV-infected mice treated with isotype control or aCXCR3 antibodies, were stained for Ibal+ cells (representative images from n=5-8 per group). Scale bars indicate 100 pm. Figure 7B depicts representative magnified images of microglia from the groups in A. Scale bars = 50pm. Figure 7C depicts representative 3D projections of single microglia from the groups in A. Figure 7D depicts the average Ibal+ cells per 1 mm2 area of DAPI. Counts were averaged from 5 retinal flat mount images per mouse from n=5 mice per group. Figure 7E through Figure 7G depict representative morphological analyses of Ibal+ microglia as assessed by: Total process endpoints / cell (Figure 7E), Summed processes length per cell (Figure 7F), Cell complexity measured by fractal dimensions per cell (Figure 7G). Each point shows an averaged value derived from n > 15 cells from an individual mouse and was graphed from n=3-5 individual mice per analysis. Significance was analyzed by unpaired t-test; *p < 0.05 **p <0.01 ***p < 0.001 ****p < 0.0001. Figure 7H and Figure 71 depict activation of astrocytes as assessed by: percent coverage of GFAP cells per DAPI area in each group (Figure 7H) and intensity of GFAP staining in each group (Figure 71). Data show an average derived from 5 images per animal and n=5 mice per group. Asterisks indicate statistical significance as in Figure 7D through Figure 7G.

[0027] Figure 8A through Figure 8H depict the results of exemplary experiments demonstrating that blocking CXCR3 reduces many infection-induced transcriptional changes in the retina and inhibits glial cell chemokine production. Figure 8A through Figure 8F depict representative RNA-seq performed on isolated retinas (n=5) and RPE / choroids (n=5) from control (uninfected) mice, and MCMV-infected mice treated with isotype control or aCXCR3 antibodies. Differential genes were defined for each infected group relative to uninfected control samples. Gene set enrichment analysis (GSEA) was used to identify enriched pathways for infected animals treated with isotype control or aCXCR3 antibodies. The highest relevant ranked enrichment was selected for each condition and plotted as lollipops by their normalized enrichment score (NES, denoted as line length) and significance (-logl0(FDR q-val), denoted as dot size). Differences between the enrichment of pathways were determined by GSVA analysis and p-values were determined by unpaired t-tests and are listed on each stem of the lollipop graphs. Figure 8A depicts enrichment for GO gene ontology for retinal tissue (MSibDB C5: GO gene ontology gene set). Figure 8B depicts enrichment for KEGG pathways for retinal tissue (MSibDB CP:KEGG). Figure 8C depicts enrichment for Hallmark genes for retinal tissue (MSibDB H: Hallmark gene set). Figure 8D depicts enrichment for GO gene ontology for RPE tissue (MSibDB C5: GO gene ontology gene set) (E) Enrichment for KEGG pathways for RPE tissue (MSibDB CP:KEGG). Figure 8F depicts enrichment for Hallmark genes for RPE tissue (MSibDB H: Hallmark gene set). Figure 8G and Figure 8H depict in situ hybridization (RNAScope) of chemokine expression in eyes from MCMV-infected mice treated with isotype control or aCXCR3 antibodies. Figure 8G depict sections probed for CXCL9 and CCL2 transcripts and co-stained for Ibal + cells. Figure 8H depicts sections probed for INF-y transcripts. Representative of n=3 mice per group. Scale bars indicate 100pm.

[0028] Figure 9A through Figure 9D depict the results of exemplary experiments. Figure 9A depicts MCMV titer in lung tissue of mice infected with MCMV over time measured by plaque assay and presented as PFU / g lung tissue (LOD = 150 plaques / g). Analyzed by one-way ANOVA with multiple comparisons *p < 0.05 **p <0.01 ***p < 0.001 ****p < 0.0001. n=5-13 mice per timepoint. Figure 9B depicts representative H&E staining of eyes from mice infected with MCMV or PBS control at PND5 (representative of n=5). Figure 9C depicts representative H&E staining of serial sections from one eye of one MCMV-infected mouse, demonstrating focal pathology at PND10. Figure 9D depicts a representative SLO image of MCMV infected mouse at PND28. OCT image was taken at the line on SLO. White box highlighting the same area as OCT zoom on all images. The arrows denote photoreceptor infoldings. Representative of n=3 mice. Scale bars indicate 100 pm.

[0029] Figure 10A through Figure 10C depict the results of exemplary experiments. Figure 10A depicts representative immunofluorescent staining of thin sections showing CD45+ and CD8+ cells in an MCMV infection mouse at PND15. Figure 10B depicts representative TUNEL staining at PND10 and PND28. Figure 10C depicts representative immunofluorescent staining of thin sections showing CD45+ and CD8+ cells in the optic nerve in an MCMV infection mouse at PND15. Scale bars indicate 100pm. Images are representative of n=3-5 mice.

[0030] Figure 11 A through Figure 11C depict the results of exemplary experiments. Figure 11 A depicts a heatmap of Z-scores determined by row gene expression of all differentially expressed genes across all time points. Samples named with C = control uninfected mice. Samples named with I = infected mice. D = Day. The first two digits of sample name indicate the post-natal day the sample was collected. The last digit is the mouse number. Therefore ID282 = Infected, Day 28, mouse 2. Figure 1 IB depicts individual volcano plots of all genes from MCMV-infected eyes vs PBS-treated control eyes at each time point. Plotted based on log2 fold change and log 10 of the adjusted p value. Red and green dots indicate significant positive and negative expression (significance = >.05 p value and > ±1 log2 fold change). Figure 11C depicts the top 10 go enrichment pathways on each cluster from co-expression cluster analysis (Figure 3A). Dot size denotes significant enrichment (log 10 of adjusted p value).

[0031] Figure 12A through Figure 121 depict the results of exemplary experiments. Figure 12A through Figure 12H depict differential expression analysis data from eyes at PND 5, PND 10, PND 15, and PND 28 analyzed by GSEA. Directional significance of enrichment was plotted over time. Figure 12A through Figure 12D depict enrichment for Hallmark genes (MSibDB H: Hallmark gene set). Figure 12E through Figure 12H depict enrichment for KEGG pathways (MSibDB CP: KEGG). Dotted lines represent calculated statistics for FDR q-val = 0.1 for enrichment; any point outside of dotted lines has an FDR q-val < 0.1. Size of dot at each time point denotes NES. Figure 121 depicts a heatmap of GSVA enrichment scores for cell signature gene sets (MSibDB C8: cell type signature gene sets) across all time points. Z-score based on row values. Sample names as in Figure 11.

[0032] Figure 13A through Figure 12E depict the results of exemplary experiments. Figure 13 A and Figure 13B depict the representative morphological analysis of Ibal+ microglia from MCMV and control retinas as assessed by: Summed processes length (pm / cell) (Figure 13 A), and Span Ratio. Each data point is an average of n>15 images (Figure 13B). n=3 mice per group. Analyzed by unpaired t-test; *p < 0.05 **p <0.01 ***p < 0.001 ****p < 0.0001. Figure 13C depicts RPE flat mounts from MCMV infected or control mice stained for Ibal+ cells. Magnified area showing microglia in subretinal space (Scale bars = 50 pm). Figure 13D depicts representative thin sections from MCMV infected and control eyes stained for Ibal+ and CD45+. White box denotes magnified area shown to the right. Scale bars indicate 100 pm. Figure 13E depicts autofluorescence images of MCMV and control eyes at PND28.

[0033] Figure 14A through Figure 14E depict the results of exemplary experiments. Figure 14A depicts a glial nodule from an MCMV-infected mouse at PND15, stained for CD45+ and Ibal+ cells. White circle denotes nodule. Representative of n=5 mice. Figure 14B depicts a representative retinal flat mount from an MCMV- infected mouse at PND15 stained with antibodies specific for GFAP, Ibal, and CD45. White circles outline glial nodules. Representative of n=5 mice. Figure 14C depicts the average size of glial nodules at each time point as measured on retinal flat mounts from MCMV-infected mice. Each data point represents the average size of nodules from a single mouse at the indicated timepoint n=3-10 mice per group. Figure 14D depicts the number of glial nodules per eye at each time point. Figure 14E depicts a representative retinal flat mount of an eye from an MCMV infected mouse at PND15 stained for CD8+ and Ibal+ cells and used to make the contour plot in Figure 5F. White circle denotes the nodule. Representative of n=3 mice. Scale bars throughout = 100 pm.

[0034] Figure 15A through Figure 15F depict the results of exemplary experiments. Figure 15 A and Figure 15B depict in situ hybridization (RNAScope) of mRNA expression in control (uninfected) eyes. Figure 15A depicts representative sections probed for CXCL9 and CCL2 transcripts and co-stained Ibal+ cells. Figure 15B depicts representative sections probed for CXCL11 and CXCL10 transcripts. Figure 15C depicts colocalization of CXCL9 transcripts and Ibal staining measured as the percent of CXCL9 that colocalized with Ibal (CXCL9 / Ibal+) and as the inverse, the percent of Ibal+ cells that colocalized with CXCL9 (Ibal / CXCL9+). Each data point represents an average co-localization in a section from n=3 mice per group. (D-F) In situ hybridization (RNAScope) for IFN-y transcripts in eyes from MCMV infected or uninfected control mice. Data are representative of n=3 mice per group. Shown are: An eye from an MCMV infected mouse at PND15 (Figure 15D). White circle denotes glial nodule. Figure 15E depicts a representative magnified image of the glial nodule from Figure 15D. Figure 15F depicts a representative eye from an uninfected control mouse at PND15.

[0035] Figure 16A through Figure 16H depict the results of exemplary experiments. Figure 16A depicts CXCR3 expression over time assessed by RNA-Seq (as in Figure 3). Data show the differential gene expression (Log2 fold change) from infected eyes compared to controls, n=3 samples per group. Figure 16B depicts a representative schematic of infection and antibody treatment timing for CXCR3 blocking experiments. Figure 16C through Figure 16E depict representative data from mice infected with MCMV on PND15 and treated with aCXCR3 antibody showing: a thin section stained for MCMV (pp89) and CD45 (Figure 16C), a thin section stained for GFAP and Ibal (white dotted circle denotes a nodule) (Figure 16D) or a flat mount stained for GFAP and Ibal (white dotted circle, nodule) (Figure 16E). Figure 16F depicts representative flat mounts from MCMV-infected mice on PND15 treated with isotype control antibody (left) or aCXCR3 antibody (right) and stained for CD45. Scale bars for Figure 16C through Figure 16F indicate 100pm. Figure 16G and Figure 16H depict the quantification of TUNEL staining of eyes from control (uninfected) mice, MCMV-infected mice or MCMV-infected mice treated with aCXCR3 antibodies. Each data point represents an average number of TUNEL+ cells per image as calculated from 9 images per mouse and n=2-4 mice per condition Significance was analyzed by unpaired t-test; *p < 0.05 **p <0.01 ***p < 0.001 ****p < 0.0001. Data shows: TUNEL+ cells in the retina (Figure 16G). TUNEL+ cells in the RPE / choroid (Figure 16H).

[0036] Figure 17A through Figure 17H depict the results of exemplary experiments. Figure 17A depicts a representative schematic of infection and prednisolone dose timing. Figure 17B depicts MCMV titer in lung tissue at PND10 of mice infected with MCMV and treated with prednisolone or PBS. Data was measured by plaque assay and is shown as PFU / g lung tissue (LOD = 150 plaques / g). Analyzed by unpaired t-test; *p < 0.05 **p <0.01 ***p < 0.001 ****p < 0.0001. Figure 17C depicts MCMV genome copies per 100 ng of DNA in ocular tissue at PND10 of mice infected with MCMV and treated with prednisolone or PBS (LOD = 44 genome copies / lOOng). Statistics analyzed as in Figure 17B. Figure 17D depicts representative H&E staining of eyes at PNDlO from control (uninfected) mice, MCMV-infected mice, and MCMV-infected treated with prednisolone. Figure 17E through Figure 17H depict representative thin eye sections of eyes at PND10 from mice infected with MCMV and treated with prednisolone or PBS. A magnified area is shown to the right of each image. Sections were stained for: CD45 and MCMV (pp89) (Figure 17E), TUNEL+ cells (Figure 17F), CD45 and Ibal (Figure 17G), or GFAP 9Figure 17H). Scale bars indicate 100 pm. Data are representative of n=3-6 mice.

[0037] Figure 18A through Figure 18E depict the results of exemplary experiments. Figure 18A through Figure 18C depict representative morphological analyses of Ibal + microglia from control (uninfected) and MCMV-infected mice treated with isotype control, or aCXCR3 antibodies. Each data point shows an averaged value derived from n>15 cells from an individual mouse and was graphed from n=3 individual mice per group. Significance was analyzed by one-way ANOVA with multiple comparisons *p < 0.05 **p <0.01 ***p < 0.001 ****p < 0.0001. Data show: Total branches per cell (Figure 18A), Cell density (Figure 18B), and Span ratio (Figure 18C). Figure 18D depicts a representative thin section of an eye at PND15 from an MCMV- infected mouse treated with aCXCR3 antibodies. Section was stained for GFAP+ and Ibal+ cells. Image on the right shows a magnified region (white box) to highlight the subretinal space. Data is representative of n=3 mice per group. Figure 18E depicts representative flat mounts of RPE from MCMV infected mice treated with isotype control or aCXCR3 antibodies. Flat mounts were stained with Ibal to show microglia in subretinal space. White arrows denote the only activated microglia found in the aCXCR3 treated mice (background Iba+ cells are tissue resident macrophages in choroid). Data is representative of n=8 mice per group. Scale bars throughout indicate 100pm.

[0038] Figure 19A through Figure 19E depict the results of exemplary experiments. Figure 19A depicts representative isolated retinas and RPE / choroids from uninfected mice (n=5, named CTR) or MCMV-infected mice treated with isotype control antibodies (n=5, named ISO) or aCXCR3 antibodies (n=5, named CX) were analyzed by RNA-Seq. Volcano plots show the differential gene expression between the indicated conditions. Data are plotted based on log2 fold change and log of the adjusted p-value. Red and green dots indicate significant positive and negative expression respectively (significance = >.05 p-value and > ±1 log2 fold change). Figure 19B and Figure 19C depict heatmaps of GSVA enrichment scores against GO gene sets for each individual sample (CTR1-5, ISO1-5, CX1-5) from Figure 19A. Individual pathway clusters determined by unsupervised clustering are labeled by number. Z-score is based on row values. Figure 19B depicts a heatmap of retina samples. Figure 19C depicts a heatmap of RPE samples. Figure 19D and Figure 19E depict pathways from GSEA analyses (GO, KEGG, and Hallmark gene sets) of MCMV+Isotype vs control uninfected and MCMV+ aCXCR3 vs control uninfected were selected for GSVA analysis. Heatmaps of GSVA pathway enrichment scores for each gene set were generated. Figure 19D depicts heatmaps for GO, KEGG, and HALLMARK selected pathways for retina samples. Figure 19E depicts heatmaps for GO, KEGG, and HALLMARK selected pathways for RPE samples.

[0039] Figure 20A and Figure 20B depict the results of exemplary experiments. Figure 20A depicts in situ hybridization (RNA-scope) for CXCL10 and CXCL11 transcripts in eyes from MCMV infected mice treated with isotype control antibodies (left) or aCXCR3 antibodies (right). Figure 20B depicts representative staining to identify activated microglia in an eye from an MCMV-infected mouse treated with aCXCR3. Section was stained for MHCII, iNOS and Ibal. Data are representative of n=3 mice per group.

[0040] Figure 21 depicts representative images of H&E-stained uninfected (control), isotype-treated (MCMV and control antibody), and CXCR3-treated (MCMV and anti-CXCR3 antibody) liver samples from mice on PND15.

[0041] Figure 22 A and Figure 22B show representative results demonstrating antiviral therapy reduces viral load but does not prevent immune infdtration. Figure 22A depicts representative lung viral titers of mice infected with MCMV <24 hours post-birth and treated with ganciclovir (50 mg / kg) or anti-CXCR3 antibody (25 mg / kg) daily on PND7-PND15. Figure 22B depicts a representative image of the retina of a mouse treated with ganciclovir as in Figure 22A stained for Glial Fibrillary Acidic Protein (GFAP) and Cluster of Differentiation 45 (CD45), demonstrating that antiviral treatment alone is insufficient to prevent immune infiltration.

[0042] DETAILED DESCRIPTION

[0043] The invention relates to compositions and methods for treating virus- related diseases or disorders, including neurologic diseases and disorders and systemic diseases and disorders. The invention is based, in part, on the unexpected discovery that immunosuppressant treatment prevents immune cell infiltration and pathology, including neurological and hepatic pathology, during and subsequent to viral infection. The invention is also based on the discovery that administration of immunosuppressants in combination with antiviral agents yielded synergistic effects. Accordingly, in some embodiments, the invention provides compositions comprising at least one immunosuppressant in combination with at least one antiviral agent, as well as methods of treating and preventing virus-related diseases or disorders by administering at least one immunosuppressant, alone or in combination with at least one antiviral agent.

[0044] Definitions

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, exemplary methods and materials are described.

[0046] As used herein, each of the following terms has the meaning associated with it in this section.

[0047] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0048] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0049] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.

[0050] In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health. As used herein, a disorder can also include impairments such as, but not limited to, hearing loss, vestibular defects, cognitive defects, and vision impairment.

[0051] A disease or disorder is “alleviated” if the severity of at least one sign or symptom of the disease or disorder, the frequency with which the at least one sign or symptom is experienced by a patient, or both, is reduced. The terms “patient,” “subject,” or “individual” are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In a non-limiting embodiment, the patient, subject or individual is a human.

[0052] As used herein, the term “pharmaceutical composition” refers to a mixture of at least one compound useful within the invention with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient or subject. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary, and topical administration.

[0053] A “therapeutic” treatment is a treatment administered to a subject who exhibits at least one sign or symptom of a disease or disorder, for the purpose of diminishing or eliminating the frequency or severity of at least one of those signs or symptoms.

[0054] As used herein, the term “treatment” or “treating” is defined as the application or administration of a therapeutic agent, i.e., a compound of the invention (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell from a patient (e.g., for diagnosis or ex vivo applications), who has a disease or disorder contemplated herein, at least one sign or symptom of a disease or disorder contemplated herein or the potential to develop a disease or disorder contemplated herein, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect a disease or disorder contemplated herein, at least one sign or symptom of a disease or disorder contemplated herein, or the potential to develop a disease or disorder contemplated herein. Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.

[0055] As used herein, the terms “effective amount,” “pharmaceutically effective amount," and “therapeutically effective amount,” refer to a sufficient amount of an agent to provide the desired biological or physiologic result. That result may be reduction and / or alleviation of at least one sign, symptom, or a cause of a disease or disorder, or any other desired alteration of a biological system. An appropriate effective amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

[0056] As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing an undesirable biological effect or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0057] As used herein, the term “potency” refers to the dose needed to produce half the maximal response (ED50).

[0058] As used herein, the term “efficacy” refers to the maximal effect (Emax) achieved within an assay.

[0059] “Measuring” or “measurement,” or alternatively “detecting” or “detection,” means assessing the presence, absence, quantity or amount (which can be an effective amount) of either a given substance within a sample, including the derivation of qualitative or quantitative concentration levels of such substances, or otherwise evaluating the values or categorization of the substance or the sample.

[0060] As used herein, “associated” refers to coincidence with the development or manifestation of a disease, condition, or phenotype. Association may be due to, but is not limited to, genes responsible for housekeeping functions, those that are part of a pathway that is involved in a specific disease, disorder, condition, or phenotype and those that indirectly contribute to the manifestation of a disease, disorder, condition or phenotype.

[0061] As used herein, “immunosuppressant” refers to any agent which generally or specifically inhibits, diminishes or decreases the intensity of the immune response in the body. Examples of immunosuppressants include, but are not limited to steroids, interferon-gamma antagonist antibodies, interferon-gamma receptor signaling antagonists such as Jakl / Jak2 inhibitors and Statl inhibitors, interferon-gamma partial agonists, glycolysis inhibitors, and inhibitors of targets such as CXCR3, Integrin a4, LFA-1, VCAM, or ICAM. As used herein, “antiviral agent” refers to any agent which inhibits or decreases production of a virus in the body. An antiviral agent can target specific viruses or can be broadly effective against a wide range of viruses. Examples of antiviral agents include, but are not limited to, nucleoside analogue-polymerase inhibitors, terminase complex inhibitors, pyrophosphate analogue-polymerase inhibitors, and COX-2 inhibitors.

[0062] The term “antibody” means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing (e.g., a glycoprotein), through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term “antibody” encompasses polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, and any other immunoglobulin molecule so long as the antibodies exhibit the desired biological activity. An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well known subunit structures and three- dimensional configurations. Antibodies can be naked, part of a fusion protein, or conjugated to other molecules such as toxins, radioisotopes, etc.

[0063] The term “antibody fragment” refers to a portion of an antibody. An “antigen-binding fragment,” “antigen-binding domain,” or “antigen-binding region,” refers to a portion of an antibody that binds to an antigen. An antigen-binding fragment can contain the antigenic determining regions of an antibody (e.g., the complementarity determining regions (CDR)). An antigen-binding fragment can contain some or all of the VH and / or VL chain polypeptides of an antibody. Examples of antigen-binding fragments of antibodies include, but are not limited to Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single chain antibodies. An antigen-binding fragment of an antibody can be derived from any animal species, such as rodents (e.g., mouse, rat, or hamster) and humans or can be artificially produced. Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0064] Description

[0065] The invention is based, in part, on the unexpected discovery of the protective activity, including neuroprotective activity, of the administration of an immunosuppressant, either alone, or in combination with an antiviral agent. Additionally, the invention is based on the unexpected discovery that administration of an immunosuppressant yields synergistic protective activity, including neuroprotective activity, when administered in combination with at least one antiviral agent. Thus, in one embodiment, the invention provides compositions comprising at least one immunosuppressant and at least one antiviral agent for the treatment of one or more virus-related diseases or disorders, including neurological diseases and disorders and systemic diseases and disorders.

[0066] In one embodiment, the invention provides a method of treating or preventing a virus-related neurological disease or disorder in a subject. In some embodiments, the virus-related neurological disease or disorder is an infection, or the consequence of an infection. In some embodiments, the infection is a cytomegalovirus (CMV) infection. In some embodiments, the infection is a congenital CMV infection.

[0067] In some embodiments, the invention provides a method of treating or preventing a systemic virus-related disease or disorder. In some embodiments, the systemic virus-related disease or disorder is hepatitis. In some embodiments, the method of treatment or prevention comprises administering at least one immunosuppressant to the subject. In other embodiments, the method of treatment or prevention comprises administering at least one immunosuppressant and at least one antiviral agent to the subject.

[0068] In some embodiments, the at least one immunosuppressant can include agents such as, but not limited to, steroids that suppress inflammation, inhibitors of integrins that facilitate lymphocyte diapedesis, inhibitors of chemokines and their receptors that attract immune cells, inhibitors of cytokines such as IFN-gamma and TNF- alpha, modulators of microglia, and inhibitors of reactive oxygen and nitrogen species. In some embodiments, an antiviral agent can include any agent which inhibits or decreases production of a virus in the body. An antiviral agent can target specific viruses or can be broadly effective against a wide range of viruses.

[0069] Compositions

[0070] In various embodiments, the invention provides compositions comprising at least one immunosuppressant. In some embodiments, an immunosuppressant is an agent that reduces or precent an immune response. Exemplary immunosuppressants include, but are not limited to, corticosteroids, calcineurin inhibitors, antiproliferative agents, kinase inhibitors, monoclonal or polyclonal antibodies, or serine protease inhibitors. In some embodiments, the at least one immunosuppressant suppresses molecular pathways such was, but not limited to, T-cell activation, lymphocyte trafficking, cytokine signaling, cell proliferation and survival, and immune cell depletion. In some embodiments, the at least one immunosuppressant targets an immune cell selected from the group consisting of: T cells, B cells, regulatory T cells, plasma cells, monocytes or macrophages, dendritic cells and natural killer (NK) cells. In some embodiments, the at least one immunosuppressant targets one or more proteins selected from the group consisting of: CXCR3, CXCR4, CXCR5, CCR5, CXC6, CXCR1 / 2, CCR6, CCR7, CD3, CD20, CD28, CD25, S1P1 receptor, TNF-a, B cell survival factor (BAFF), mTOR, Integrin a4, LFA-1, VCAM, and ICAM. In some embodiments, the at least one immunosuppressant is selected from the group consisting of: a chemical compound, a protein, a peptide, a peptidomimetic, a lipid, an antibody, an antibody fragment, an antibody mimetic, a chimeric antibody, a bispecific antibody, a ribozyme, a small molecule chemical compound, a monobody, a short hairpin RNA, RNAi, siRNA, miRNA, or a nucleic acid encoding an antisense nucleic acid molecule. Preferably, the at least immunosuppressant is a corticosteroid. Exemplary corticosteroids include, but are not limited to, prednisolone, hydrocortisone, prednisone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, fluticasone, or budesonide. In some embodiments, the at least one immunosuppressant is prednisolone. In another embodiment, the at least one immunosuppressant preferably inhibits one or more receptors selected from the group consisting of: CXCR3, CXCR4, CXCR5, CCR5, CXC6, CXCR1 / 2, CCR6, and CCR7. In some embodiments, the at least one immunosuppressant inhibits CXCR3. The CXCR3 inhibitor decreases the level or activity (e g., substrate binding activity, etc.) of CXCR3. In some embodiments, the inhibitor is an anti-CXCR3 antibody.

[0071] In some embodiments, the at least one immunosuppressant is at least one selected from the group consisting of a general immunosuppressant, such as, by way of non-limiting examples, prednisone or dexamethasone, an interferon-gamma antagonist antibody, such as, by way of non-limiting examples, fontolizumab, emapalumab, or AMG 811, a Jakl / Jak2 inhibitor, such as, by way of non-limiting examples, baricitinib, filgotinib, tofacitinib, upadacitinib, ruxolitnib, abrocitnib, itacitnib, fedratinib, pacritinib, gandotinib, or momelotinib, a Statl inhibitor, such as, by way of non-limiting examples, Pravastatin, ISS-840, PY*LKTK, PM-73G, CJ-1383, an oligodeoxynucleotide decoy, an anti-sense mRNAs, a natural product that inhibits JAK / STAT signaling, such as, by way of non-limiting examples, resveratrol or catechins, an interferon-gamma partial agonist, a microglia inhibitor, such as, by way of non-limiting examples, PLX5622, a glycolysis inhibitor, such as, by way of non-limiting examples, adalafil, metformin, MCT blockade, Glutl antagonism, HK blockade, or PKM2 blockade, and an inhibitor of a specific target molecule, such as, by way of non-limiting examples, CXCR3, Integrin a4, LFA-1, VCAM, or ICAM. In some embodiments, the at least one immunosuppressant is an inhibitor of CXCR3.

[0072] In some embodiments, the composition further comprises at least one antiviral agent. An anti-viral agent prevents viral propagation within a host organism, either by inhibiting viral entry into host cells or by disrupting viral replication processes. Exemplary embodiments can include fusion inhibitors, entry inhibitors, nucleoside / nucleotide analogs, RNA polymerase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, neuraminidase inhibitors, or interferons. In some embodiments, the at least one antiviral agent is selected from the group consisting of: ganciclovir, valganciclovir, letermovir, acyclovir, foscarnet, cidofovir, maribavir, leflunomide, brincidofovir, cyclopropavir, a benzimidazole analog, such as BDCRB, TRCB, or tomeglovir, a flavonoid, such as quercetin or baicalein, a COX-2 inhibitor, an artemisinin derivative, such as, by way of non-limiting examples, artesunate or TF27, valnocatmide, and valproic acid.

[0073] In some embodiments, the compounds possess one or more stereocenters, and each stereocenter may exist independently in either the R or S configuration. In one embodiment, compounds described herein are present in optically active or racemic forms. It is to be understood that the compounds described herein encompass racemic, optically active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein. Preparation of optically active forms is achieved in any suitable manner, including by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase. In one embodiment, a mixture of one or more isomer is utilized as the therapeutic compound described herein. In another embodiment, compounds described herein contain one or more chiral centers. These compounds are prepared by any means, including stereoselective synthesis, enantioselective synthesis and / or separation of a mixture of enantiomers and / or diastereomers. Resolution of compounds and isomers thereof is achieved by any means including, by way of nonlimiting example, chemical processes, enzymatic processes, fractional crystallization, distillation, and chromatography.

[0074] In some embodiments, compounds described herein are prepared as prodrugs. A “prodrug” refers to an agent that is converted into the parent drug in vivo. In one embodiment, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically, or therapeutically active form of the compound. In another embodiment, a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically, or therapeutically active form of the compound.

[0075] Methods

[0076] In various embodiments, the invention is a method of treating or preventing at least one virus-related disease or disorder, including neurological diseases and disorders and systemic diseases and disorders. In some embodiments, the virus- related disease or disorder is an infection, or a consequence of infection. In some embodiments, the infection is a cytomegalovirus (CMV). In some embodiments, the infection is a congenital CMV infection.

[0077] In some embodiments, the method of treating or prevention comprises administering to a subject in need thereof, an effective amount of at least one composition of the invention. In some embodiments, the composition of the invention comprises at least one immunosuppressant. In some embodiments, the composition of the invention comprises at least one antiviral agent. In some embodiments, the composition of the invention comprises at least one immunosuppressant and at least one antiviral agent. In some embodiments, the method comprises contacting at least one cell of the subject with a composition of the invention. In some embodiments, the method comprises contacting the at least one cell with a composition comprising at least one immunosuppressant. In some embodiments, the method comprises contacting the at least one cell with a composition comprising at least one antiviral agent. In some embodiments, the method comprises contacting the at least one cell with a composition comprising at least one immunosuppressant and at least one antiviral agent.

[0078] In some embodiments, the method comprises contacting the cells, separately, simultaneously, or sequentially, with a composition comprising at least one immunosuppressant and a composition comprising at least one antiviral agent. In some embodiments, the at least one immunosuppressant is selected from the group consisting of a general immunosuppressant, such as, by way of non-limiting examples, prednisone or dexamethasone, an interferon-gamma antagonist antibody, such as, by way of nonlimiting examples, fontolizumab, emapalumab, or AMG 811, a Jakl / Jak2 inhibitor, such as, by way of non-limiting examples, baricitinib, filgotinib, tofacitinib, upadacitinib, ruxolitnib, abrocitnib, itacitnib, fedratinib, pacritinib, gandotinib, or momelotinib, a Statl inhibitor, such as, by way of non-limiting examples, Pravastatin, ISS-840, PY*LKTK, PM-73G, CJ-1383, an oligodeoxynucleotide decoy, an anti-sense mRNAs, a natural product that inhibits JAK / STAT signaling, such as, by way of non-limiting examples, resveratrol or catechins, an interferon-gamma partial agonist, a microglia inhibitor, such as, by way of non-limiting examples, PLX5622, a glycolysis inhibitor, such as, by way of non-limiting examples, adalafil, metformin, MCT blockade, Glut! antagonism, HK blockade, or PKM2 blockade, and an inhibitor of a specific target molecule, such as, by way of non-limiting examples, CXCR3, Integrin a4, LFA-1, VCAM, or ICAM, In some embodiments, the at least one immunosuppressant is an inhibitor of CXCR3.

[0079] In various embodiments, the composition further comprises at least one antiviral agent. In some embodiments, the at least one antiviral agent is selected from the group consisting of, valganciclovir, letermovir, acyclovir, foscarnet, cidofovir, maribavir, leflunomide, brincidofovir, cyclopropavir, a benzimidazole analog, such as BDCRB, TRCB, or tomeglovir, a flavonoid, such as quercetin or baicalein, a COX-2 inhibitor, an artemisinin derivative, such as, by way of non-limiting examples, artesunate or TF27, valnocatmide, and valproic acid.

[0080] In some embodiments, the method comprises contacting at least one cell with a composition comprising at least one immunosuppressant before contacting the at least one cell with a composition comprising at least one antiviral agent. Examples of contacting the cells with a composition comprising at least one immunosuppressant before contacting the cells with a composition comprising at least one antiviral agent include, but are not limited to, contacting the cells with a composition comprising at least one immunosuppressant 4 weeks, 3 weeks, 2 weeks, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 36 hours, 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2.5 hours, 2 hours, 1.5 hours, 1 hour, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, and 30 seconds before contacting the cells with a composition comprising at least one antiviral agent. In some embodiments, the cells are contacted with a composition comprising at least one immunosuppressant multiple times before the cells are contacted with a composition comprising at least one antiviral agent.

[0081] In some embodiments, the method comprises contacting the at least one cell simultaneously with at least one immunosuppressant and a composition comprising at least one antiviral agent.

[0082] In some embodiments, the method comprises contacting the at least one cell with a composition comprising at least one immunosuppressant after contacting the at least one cell with a composition comprising at least one antiviral agent. Examples of contacting the at least one cell with a composition comprising at least one immunosuppressant after contacting the cells with a composition comprising at least one antiviral agent include, but are not limited to, contacting the cells with a composition comprising at least one immunosuppressant 4 weeks, 3 weeks, 2 weeks, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 36 hours, 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2.5 hours, 2 hours, 1.5 hours, 1 hour, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, and 30 seconds after contacting the cells with a composition comprising at least one antiviral agent. In some embodiments, the cells are contacted with a composition comprising at least one antiviral agent multiple times before the cells are contacted with a composition comprising at least one immunosuppressant.

[0083] In some embodiments at least one immunosuppressant is administered at a concentration of about 1 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, 32 mg / kg, 33 mg / kg, 34 mg / kg, 35 mg / kg, 36 mg / kg, 37 mg / kg, 38 mg / kg, 39 mg / kg, 40 mg / kg, 41 mg / kg, 42 mg / kg, 43 mg / kg, 44 mg / kg, 45 mg / kg, 46 mg / kg, 47 mg / kg, 48 mg / kg, 49 mg / kg, or 50 mg / kg. In some embodiments, the at least one immunosuppressant is a corticosteroid. In some embodiments, the at least one immunosuppressant is prednisolone. In some embodiments, prednisolone is administered at a concentration of about 7 mg / kg daily. In some embodiments, the at least one immunosuppressant is an anti-CXCR3 antibody. In some embodiments, the anti-CXCR3 antibody is administered at a concentration of about 25 mg / kg daily.

[0084] Methods of Treatment

[0085] In various embodiments, the invention is a method of treating or preventing a virus-related disease or disorder, such as a neurological disease or disorder or a systemic disease or disorder, in a subject. In some embodiments, the virus-related disease or disorder is an infection, or a consequence of infection. In one embodiment, the invention provides a method of treating a virus-related disease or disorder in a subject. In some embodiments, the virus-related disease or disorder is an infection. In some embodiments, the infection is a CMV infection. In some embodiments, the infection is a congenital CMV infection.

[0086] In some embodiments, the invention provides a method of treating or preventing a systemic virus-related disease or disorder. In some embodiments, the systemic disease or disorder is hepatitis.

[0087] In some embodiments, the method of treating or prevention comprises administering to a subject in need thereof, an effective amount of at least one composition of the invention. In some embodiments, the composition of the invention comprises at least one immunosuppressant. In some embodiments, the composition of the invention comprises at least one antiviral agent. In some embodiments, the composition of the invention comprises at least one immunosuppressant and at least one antiviral agent. In some embodiments, the method comprises administering to the subject, a composition comprising at least one immunosuppressant in combination with at least one antiviral agent. In certain embodiments, the method comprises administering to the subject, a first composition comprising at least one immunosuppressant and a second composition comprising at least one antiviral agent. The first and second compositions may be administered separately, simultaneously, or sequentially.

[0088] In some embodiments, the method comprises contacting the cells, separately, simultaneously, or sequentially, with a composition comprising at least one immunosuppressant and a composition comprising at least one antiviral agent. In some embodiments, the at least one immunosuppressant is selected from the group consisting of a general immunosuppressant, such as, by way of non-limiting examples, prednisone or dexamethasone, an interferon-gamma antagonist antibody, such as, by way of nonlimiting examples, fontolizumab, emapalumab, or AMG 811, a Jakl / Jak2 inhibitor, such as, by way of non-limiting examples, baricitinib, fdgotinib, tofacitinib, upadacitinib, ruxolitnib, abrocitnib, itacitnib, fedratinib, pacritinib, gandotinib, or momelotinib, a Statl inhibitor, such as, by way of non-limiting examples, Pravastatin, ISS-840, PY*LKTK, PM-73G, CJ-1383, an oligodeoxynucleotide decoy, an anti-sense mRNAs, a natural product that inhibits JAK / STAT signaling, such as, by way of non-limiting examples, resveratrol or catechins, an interferon-gamma partial agonist, a microglia inhibitor, such as, by way of non-limiting examples, PLX5622, a glycolysis inhibitor, such as, by way of non-limiting examples, adalafil, metformin, MCT blockade, Glut! antagonism, HK blockade, or PKV12 blockade, and an inhibitor of a specific target molecule, such as, by way of non-limiting examples, CXCR3, Integrin a4, LF -l , VC AM, or ICAM, In some embodiments, the at least one immunosuppressant is an inhibitor of CXCR3.

[0089] In various embodiments, the composition further comprises at least one antiviral agent. In some embodiments, the at least one antiviral agent is selected from the group consisting of, valganciclovir, letermovir, acyclovir, foscarnet, cidofovir, maribavir, leflunomide, brincidofovir, cyclopropavir, a benzimidazole analog, such as BDCRB, TRCB, or tomeglovir, a flavonoid, such as quercetin or baicalein, a COX-2 inhibitor, an artemisinin derivative, such as, by way of non-limiting examples, artesunate or TF27, valnocatniide, and valproic acid.

[0090] In some embodiments, the method comprises administering to the subject a composition comprising at least one immunosuppressant before administering a composition comprising at least one antiviral agent. Examples of administering a composition comprising immunosuppressant before administering a composition comprising at least one antiviral agent include, but are not limited to, administering a composition comprising at least one immunosuppressant 4 weeks, 3 weeks, 2 weeks, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 36 hours, 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2.5 hours, 2 hours, 1.5 hours, 1 hour, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, and 30 seconds before administering a composition comprising at least one antiviral agent. In some embodiments, a composition comprising at least one immunosuppressant is administered to the subject multiple times before a composition comprising at least one antiviral agent is administered.

[0091] In some embodiments, the method comprises administering to the subject a composition comprising at least one immunosuppressant and a composition comprising at least one antiviral agent simultaneously.

[0092] In some embodiments, the method comprises administering to the subject a composition comprising at least one immunosuppressant after administering a composition comprising at least one antiviral agent. Examples of administering a composition comprising at least one immunosuppressant after administering a composition comprising at least one antiviral agent include, but are not limited to, administering a composition comprising at least one immunosuppressant 4 weeks, 3 weeks, 2 weeks, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 36 hours, 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2.5 hours, 2 hours, 1.5 hours, 1 hour, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, and 30 seconds after administering a composition comprising at least one antiviral agent. In some embodiments, a composition comprising at least one antiviral agent is administered multiple times before a composition comprising at least one immunosuppressant is administered.

[0093] In some embodiments at least one immunosuppressant is administered at a concentration of about 1 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, 32 mg / kg, 33 mg / kg, 34 mg / kg, 35 mg / kg, 36 mg / kg, 37 mg / kg, 38 mg / kg, 39 mg / kg, 40 mg / kg, 41 mg / kg, 42 mg / kg, 43 mg / kg, 44 mg / kg, 45 mg / kg, 46 mg / kg, 47 mg / kg, 48 mg / kg, 49 mg / kg, or 50 mg / kg. In some embodiments, the at least one immunosuppressant is a corticosteroid. In some embodiments, the at least one immunosuppressant is prednisolone. In some embodiments, prednisolone is administered at a concentration of about 7 mg / kg daily. In some embodiments, the at least one immunosuppressant is an anti-CXCR3 antibody. In some embodiments, the anti-CXCR3 antibody is administered at a concentration of about 25 mg / kg daily.

[0094] In one embodiment, the method comprises administering to the subject a composition comprising at least one immunosuppressant, wherein the subject has previously been treated with an antiviral agent.

[0095] In some embodiments, the subject is an infant or child.

[0096] In some embodiments, the invention relates, in part, to methods of treating virus-related neurological impairments. In some embodiments, the virus-related neurological impairment is selected from the group consisting of hearing loss, vestibular defects, cognitive defects, and vision impairment. In some embodiments, the virus-related neurological impairment is vision impairment.

[0097] In some embodiments, the method comprises administering to the subject a composition of the invention. In some embodiments, the method comprises administering to the subject a composition comprising at least one immunosuppressant. In one embodiment, the subject has a virus-related neurological impairment. In some embodiments, the subject has a vision impairment.

[0098] In certain embodiments, the method comprises administering to the subject, a composition comprising at least one immunosuppressant in combination with at least one antiviral agent. In certain embodiments, the method comprises administering to the subject, a first composition comprising at least one immunosuppressant and a second composition comprising at least one antiviral agent. The first and second compositions may be administered separately, simultaneously, or sequentially.

[0099] Pharmaceutical compositions and formulations

[0100] The invention also encompasses the use of pharmaceutical compositions to practice the methods of the invention. Such a pharmaceutical composition may consist of at least one composition of the invention or a salt thereof in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one composition of the invention or a salt thereof, and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The compound or conjugate may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

[0101] Pharmaceutical compositions that are useful in the methods of the invention may be suitably developed for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, intravitreal, sub-retinal, intra-cochlear, intravenous, or another route of administration. Other contemplated formulations include liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically based formulations. The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the subject being treated, and the like.

[0102] Although the invention herein is principally directed to the ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist may design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

[0103] In one embodiment, the compositions utilized in the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In one embodiment, the pharmaceutical compositions comprise a therapeutically effective amount of a compound or conjugate of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that are useful, include, but are not limited to, glycerol, water, saline, ethanol, and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington’s Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

[0104] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In one embodiment isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, are included in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin. In one embodiment, the pharmaceutically acceptable carrier is not DM SO alone.

[0105] Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and / or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.

[0106] As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” that may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Genaro, ed. (1985, Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

[0107] The composition utilized in the invention may comprise a preservative from about 0.005% to 2.0% by total weight of the composition. The preservative is used to prevent spoilage in the case of exposure to contaminants in the environment. Examples of preservatives useful in accordance with the invention included but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea and combinations thereof. An exemplary preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

[0108] In one embodiment, the composition includes an antioxidant and a chelating agent that inhibits the degradation of the compound. Exemplary antioxidants for some compounds are BHT, BHA, alpha-tocopherol, and ascorbic acid in the range of about 0.01% to 0.3%. In one embodiment, the BHT is in the range of 0.03% to 0.1% by weight by total weight of the composition. In one embodiment, the chelating agent is present in an amount of from 0.01% to 0.5% by weight by total weight of the composition. Exemplary chelating agents include edetate salts (e.g., disodium edetate) and citric acid in the weight range of about 0.01% to 0.20%. In one embodiment, chelating agents may be in the range of 0.02% to 0.10% by weight by total weight of the composition. The chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are the exemplary antioxidant and chelating agent respectively for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.

[0109] Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water, and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e g., polyoxyethylene stearate, heptadecaethyleneoxy cetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin, and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n- propyl-para- hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.

[0110] Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. As used herein, an “oily” liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water. Liquid solutions of the pharmaceutical composition for use in the invention may comprise each of the components described regarding liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water, and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Powdered and granular formulations of a pharmaceutical preparation of the composition utilized in the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.

[0111] A pharmaceutical composition for use in the invention may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.

[0112] Methods for impregnating or coating a material with a chemical composition are known in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i.e., such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying.

[0113] The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the subject either prior to or after a diagnosis of disease. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0114] Administration of the compositions of the invention to a subject, such a mammal, including a human, may be carried out using known procedures, at dosages and for periods of time effective to prevent or treat disease. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A nonlimiting example of an effective dose range for a therapeutic compound for use in the invention is from about 1 and 5,000 mg / kg of body weight / per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0115] The invention may be practiced as frequently as several times daily, or it may be practiced less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc. Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.

[0116] A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0117] In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such a therapeutic compound for the treatment of a disease in a subject.

[0118] In certain embodiments, the composition of the invention provides for a controlled release of a therapeutic agent. In certain instances, controlled- or sustained- release formulations of a pharmaceutical composition of the invention may be made using conventional technology, using for example proteins equipped with pH sensitive domains or protease-cleavable fragments. In some cases, the dosage forms to be used can be provided as slow or controlled release of one or more active ingredients therein using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, micro-particles, liposomes, or microspheres or a combination thereof to provide the desired release profde in varying proportions. Suitable controlled-release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the pharmaceutical compositions of the invention. Thus, single unit dosage forms suitable for oral administration, such as tablets, capsules, gel-caps, lozenges, and caplets, which are adapted for controlled-release are encompassed by the invention.

[0119] Most controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include extended activity of the drug, reduced dosage frequency, and increased subject compliance. In addition, controlled-release formulations can be used to affect the time of onset of action or other characteristics, such as blood level of the drug, and thus can affect the occurrence of side effects.

[0120] Most controlled-release formulations are designed to initially release an amount of drug that promptly produces the desired therapeutic effect, and gradually and continually release of other amounts of drug to maintain this level of therapeutic effect over an extended period of time. In certain embodiments, the controlled-release formulation of the composition described herein allows for release of a therapeutic agent precisely when the agent is most needed. In another embodiment, the controlled-release formulation of the composition described herein allows for release of a therapeutic agent precisely in conditions in which the therapeutic agent is most active. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body.

[0121] In certain embodiments, the composition provides for an environmentdependent release, when and where the therapeutic agent is triggered for release. For example, in certain embodiments the composition invention releases at least one therapeutic agent when and where the at least one therapeutic agent is needed. The triggering of release may be accomplished by a variety of factors within the microenvironment of the treatment or prevention site, including, but not limited to, temperature, pH, the presence or activity of a specific molecule or biomolecule, and the like. Controlled release of an active ingredient can be stimulated by various inducers, for example pH, temperature, enzymes, water or other physiological conditions or compounds. The term “controlled-release component” in the context of the invention is defined herein as a compound or compounds, including, but not limited to, polymers, polymer matrices, gels, permeable membranes, liposomes, or microspheres or a combination thereof that facilitates the controlled-release of the active ingredient.

[0122] In certain embodiments, the formulations of the invention may be, but are not limited to, short-term, rapid offset, as well as controlled, for example, sustained release, delayed release, and pulsatile release formulations.

[0123] The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a month or more and should be a release that is longer that the same amount of agent administered in bolus form.

[0124] For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material that provides sustained release properties to the compounds. As such, the compounds for use the method of the invention may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.

[0125] In one embodiment of the invention, the compositions are administered to a subject, alone or in combination with another pharmaceutical agent, using a sustained release formulation.

[0126] The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.

[0127] The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration. The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.

[0128] As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.

[0129] As used herein, rapid offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.

[0130] In one embodiment, the invention is practiced in dosages that range from one to five times per day or more. In another embodiment, the invention is practiced in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It will be readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the invention will vary from subject to subject depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the invention should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any subject will be determined by the attending physical taking all other factors about the subject into account.

[0131] Routes of administration of include ocular, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, intravitreal, sub-retinal, intra-cochlear, and topical administration. In some embodiments, the preferred route of administration is intravenous, ocular, periocular, intraocular, oral, or topical. Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions, and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the invention are not limited to the particular formulations and compositions that are described herein.

[0132] EXPERIMENTAL EXAMPLES

[0133] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0134] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the invention and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure.

[0135] Example 1 : Immune Responses Drive Chorioretinitis and Retinal Pathology After Neonatal CMV Infection

[0136] Human cytomegalovirus (HCMV) causes a common congenital infection leading to long term neurological impairments. Approximately 20% of symptomatic cases develop visual pathology, yet the mechanisms are undefined and no animal model has been developed to explore the pathophysiology of disease in the neonatal retina. Infection of newborn mice with murine (M)CMV is an established model of congenital CMV infection, with recent work in this model suggesting that brain pathology could be traced to immune responses. In the eye however, it is generally accepted that CMV- retinitis is the result of virus-driven necrosis in the absence of T cell responses. Thus, children with congenital CMV retinopathies are treated with antiviral drugs. This study found that MCMV infection of newborn BALB / c mice recapitulates human eye disease after congenital CMV infection including focal chorioretinitis, inflamed vasculature and disrupted blood-retinal barriers (BRBs). Detailed analyses revealed extensive T cell infdtration, retinal cell death, disrupted retinal development and marked gliosis with glial nodules that attracted T cells to the ganglion cell layer of the retina. Remarkably, blocking immune responses generally, or specifically via targeting the chemokine receptor CXCR3, did not result in more severe retinal disease, despite viral infection of the eye, but rather completely preserved retinal development, prevented pathology and reduced glial cell activation. Thus, these data establish this MCMV model for novel studies of congenital retinal disease and show that the immune system is the driver of retinal disease in the neonatal eye.

[0137] This study utilized the MCMV model of congenital infection originally described by Jonjic and Britt and show here that MCMV drives a focal chorioretinitis with marked immune infiltration, glial cell activation, and pathology in the retina that closely mirrors what is observed in humans. Critically, these data show that the immune response drives pathology and can be targeted to prevent most disease manifestations, suggesting that therapies targeting immune function may be essential to reduce disease burden after congenital CMV infection.

[0138] The materials and methods used in the experiments are now described.

[0139] Study Design

[0140] The objective of this study was to determine the pathogenesis of cCMV infections of the retina. This study used the established cMCMV animal model and assessed eye development, pathology, viral loads and immune responses via: i) frozen section and flat mount histology, ii) live ocular imaging, iii) transcriptional analyses, and iv) viral plaque assays and qPCR. Additionally, this study determined whether interference with immune responses would alter the outcomes using blocking antibodies or prednisolone treatment of the mice. Breeder pairs were never reused after a litter was infected with MCMV. Each litter was used for a single experimental condition, without exclusions at least three independent liters were used for each experimental endpoint. For all analyses, this study examined at least 5 randomly chosen individual mice from 2-3 litters. Timepoints for analyses were chosen by pilot experiments to define the kinetics of retinal infection. All animals were housed in the same facility with 12-hour day / night cycles. Data was not blinded, and no data were excluded from this study.

[0141] Animals and Viruses

[0142] All experiments were approved by the Thomas Jefferson University Institutional Animal Care and Use Committee. For all experiments newborn BALB / c mice were derived from matings of 6-7-week-old BALB / c mice purchased from Jackson Laboratory. Stocks of the KI 81 strain of MCMV were produced from M2-10B4 cells and quantified by plaque assay as previously described (Zurbach et al., 2014, Virol J 11 :71). Newborn BALB / c pups were inoculated i.p. with 200 pfu in 20 pL PBS less than 24hrs after birth. Control groups were injected with an equal volume of PBS alone. To track weight gain, mice in a litter were weighed together as a group and the total weight divided by the number of pups to establish an average pup weight per litter. Mice were euthanized at post-natal day 5, 10, 15, and 28 as described in the figure legends, and the eye and lung tissues were collected for further analyses. Virus titers in lung and eye tissue were determined by plaque assay (Zurbach et al,. 2014, Virol J 11:71) by qPCR for the MCMV-E1 gene as described previously (Snyder et al., 2010, PLoS One 5 :e9681).

[0143] Prednisolone treatments

[0144] For prednisolone experiments, infected or control mice were treated with prednisolone sodium phosphate starting on PND4 by daily i.p. injection (7mg / kg) in 50pL PBS, or PBS as a control, as previously described (Recchia, 2012, Archives of Ophthalmology 130:525). Prednisolone treatment concluded on PND9, and all mice were sacrificed the following day (PND10).

[0145] CXCR3 blockade

[0146] To block CXCR3, mice were treated by i.p. injection with 25mg / kg (Kvestak, 2021, Journal of Experimental Medicine 218) anti-CXCR3 antibody (clone CXCR3-173) or an irrelevant isotype control antibody (clone PIP), both purchased from BioXCell. Antibody treatments began on PNDO (at the time of infection or PBS injection) and were repeated on PND5 and PND10. All mice were sacrificed on PND15.

[0147] Live Ocular Imaging

[0148] Confocal scanning laser ophthalmoscopy (cSLO) and spectral domain optical coherence tomography (SD-OCT) were used to visualize ocular structures of MCMV infected 1 month old mice. Mice were weighed individually and anesthetized with a mixture of ketamine (lOOmg / kg) and xylazine (10 mg / kg). To anesthetize the eyes, mice were given a drop of 0.5% proparacaine hydrochloride ophthalmic solution, followed by pupil dilation with a drop of 1% tropicamide solution. The Spectralis HRA + OCT (Heidelberg Engineering) was used to obtain infrared reflectance (IRAF), autofluorescence images of the retina and RPE and SD-OCT images of the ocular structures.

[0149] H&E, Immunofluorescence, TUNEL Staining and quantification

[0150] Eyes were enucleated and placed immediately in plastic molds, covered in NEG50 solution (Epredia), and then flash frozen in liquid nitrogen for cry opreservation. Serial cryosections (10 pm) of the eye were collected starting at ~l / 4thof the way into the globe and were continuously collected through -500 pm of tissue until passing the optic nerve. Sections were placed on slides and stored at -20°C. H&E staining was performed by the Thomas Jefferson University, Translational Research / Pathology core facility. To perform immunofluorescence on thin sections, the tissue was fixed with either cold acetone or 4% PFA for 10 minutes, and then blocked and permeabilized with 3% BSA and 0.1% Triton X-100 for 1 hour. After blocking, primary antibodies were diluted in blocking buffer and incubated for either 1 hr at room temperature or overnight at 4°C, followed by the appropriate secondary antibody for 30 min at room temperature. Finally, slides were stained with DAPI (1 : 100) for 10 minutes and mounted under cover glass with Flouromount-G mounting medium. Between each step described, slides were washed 3 times with lx PBS. For detection of MCMV-infected cells, sections were stained with pp89 (lEl)-specific antibody clone 6 / 58 / 1 (Reddehase et al., 1986, J Virol 60: 1125-1129). In brief, antibody was produced in a bioreactor and concentrated and purified by Protein A / G (NAB Protein A / G spin kit, Thermo Scientific) as per manufacturer’s protocol. Antibody titer was determined using Easy Titer Mouse IgG Assay Kit (Thermo Scientific) and labeled with Alexa Fluor 488 antibody labeling kit (Thermo Scientific), following the manufacturer’s protocol. TUNEL staining to detect cell death, was preformed using ApopTag Fluorescein In situ Apoptosis Detection Kit (Millipore sigma) following the manufacturer's protocol.

[0151] Whole Retina and RPE / Choroid Flat mounts

[0152] For whole mounts, eyes were enucleated and a small incision was made in cornea prior to immersion in 4% PFA for 8 min. After fixation, the cornea and lens were removed under a dissection microscope, and the retina was separated from the RPE / choroid. Both tissues were immersed in 4% PFA for an additional 8 min. Tissue was then washed with lx PBS, permeabilized with 0.3% Triton X-100, and blocked with 5% BSA + 0.1% Triton X-100 for 1.5 hours. For staining, the tissue was incubated in 96 well trays overnight with primary antibodies at 4°C, followed by secondary antibodies and DAPI. As above, tissues were washed 3 times with lx PBS between each step. After staining, the retina and RPE / choroid were placed on a glass slide and cut radially to flatten the tissue before adding Flouromount-G mounting medium and a coverslip.

[0153] Image capture and quantification

[0154] All H&E images were captured with Nikon Eclipse Ci. Immunofluorescent images were captured with either a Leica DM5000 B or Nikon Eclipse Ci for transmitted light, or a Nikon AIR confocal microscope for confocal images. Quantification of immunofluorescence was determined with ImageJ as described in the figure legends.

[0155] Assessment of blood-retinal -barrier integrity

[0156] To assess the integrity of the blood-retinal barrier, infected or control mice were injected with FITC-dextran (10,000 Da, Invitrogen). For this, mice were anesthetized with isoflurane and FITC-dextran was injected retro-orbitally. Mice were sacrificed 5 minutes after the injection and eyes were enucleated and immediately frozen in NEG50.

[0157] Microglia morphological analysis

[0158] Retinal flat mounts stained with Ibal were used for morphological analysis. All cell morphology analysis was preformed using ImageJ skeleton and fractal analyses as previously described (Morrison et al., 2017, Sci Rep 7:13211) using 5 images per animal for skeleton analyses and 15 cells per animal for fractal analyses.

[0159] RNA in situ hybridization (RNAscope)

[0160] RNAscope (ACD bio) in situ hybridization was performed using the multiplex Fluorescent Reagent Kit v2. Samples were prepared following the manufacturer’s fresh-frozen sample preparation and pretreatment protocol with some adjustments as follows: i) The PFA fixation time was increased from 15min to 30min, and ii) slides were baked at 37°C for 30min after the hydrogen peroxide step. Probes were purchased from ACD bio to visualize CXCL9, CXCL10, CXCL11, CCL2, and IFN- y. Opal dyes (Akoya Biosciences) were used as secondary staining. RNAscope was combined with Immunofluorescence using the protocol provided by ACD.

[0161] RNAseq

[0162] For analyses of whole eyes over time after infection, eyes were homogenized using a bead beater, and RNA was extracted using DNA / RNA extraction kit (Allprep DNA / RNA mini kit, Quiagen). Triplicate samples were used for analyses, each consisting of equal amounts of RNA combined from two eyes derived from two different pups from the same condition. For analyses of eyes after CXCR3 blockade, retinas and RPE / choroids were separated under a dissection microscope and homogenized using a pestle in a 1.5ml conical tube. RNA was extracted as above and sequencing was performed on matched retinas and RPE / choroids (i.e. derived from the same eyes) from 5 individual mice per group. In all cases, sequencing, alignment, quality control and differential analysis (DeSeq2) was performed by Novogene. Clustering of differentially expressed genes and plotting were performed as previously described (Abu- Jamous et al., 2018, Genome Biol 19: 172). Go enrichment analyses (g:Profiler) were performed against gene list derived from clustering analysis. Additional GSEA analyses were performed using GSEA 4.2.3 Mac App (Broad Institute) against GO: gene ontology gene sets, Hallmark gene sets, KEGG pathways, and curated C8 cell type signature gene sets (Molecular Signatures Database). Variance between all samples sequenced against curated gene sets was performed using GSVA(IOO). All analyses were visualized using ggplot R Packages. Further specifics on individual comparisons are described in figure legends.

[0163] Statistical analysis

[0164] Statistical analyses of RNA-seq data was conducted using DeSeq2. Differentially expressed genes were defined as genes with padj < 0.1. Significance of gene set enrichment was determined by unpaired t-test comparing individual variance scores from GSVA. All other statistical analyses were performed using GraphPad Prism version 10.0.0. Significance was determined using unpaired t-test and one way ANOVA with multiple comparisons as described in figure legends.

[0165] The results of the experiments are now described.

[0166] Neonatal infection with MCMV leads to eye infection and retinal pathology

[0167] Newborn BALB / c mice were infected with 200 plaque-forming unites (PFU) of MCMV strain K181 or an equal volume of PBS for controls, following the protocol developed by Jonjic and Britt to model cMCMV (Lisnic et al., 2021, Humana Press Inc. 2244:365-401; Koontz et al., 2008, J Exp Med 205:423-435; Slavuljica et al., 2015, Cell Mol Immunol 12:180-191). Infected pups had a significant reduction in weight gain compared to controls (Figure 1A) and a significant decrease in the survival of infected pups (Figure IB), as previously reported with this model (Bantug et al., 2008, The Journal of Immunology 181 :2111-2123). Interestingly, the pups had delayed eye opening compared to controls, with the majority of infected eyes remaining closed at post-natal day 15 (PND15) while all control eyes were open by PND12 (Table 1). Plaque assays measuring viral titers in the lungs demonstrated a robust systemic infection and confirmed the overall kinetics of viral growth (Figure 9A) similar to previous studies (Koontz et al., 2008, J Exp Med 205:423-435; Bantug et al., 2008, The Journal of Immunology 181 :2111-2123). Viral DNA in the eye was below the limit of detection on PND5, but by PND10, all eyes had significant levels of virus, which were sustained on PND15 and then reduced somewhat by PND28 (Figure 1C). Plaque assays of the whole eye showed active virus replication through the PND28 timepoint (Figure ID). Thus, MCMV robustly infected ocular tissue in this model.

[0168] Table 1 : Table of pathology observed in eyes of MCMV-infected mice over time.

[0169] To determine if MCMV infected eyes had any virally induced pathology, eyes from each timepoint were analyzed histologically by H&E staining (Figure IE and Figure 9B). Profound pathology was observed as early as PND10, was more wide-spread by PND15, and was only moderately resolved by PND28. Similar to what has been described in humans with congenital retinitis (Coats et al., 2000, Journal of American Association for Pediatric Ophthalmology and Strabismus 4: 110-116; Ghekiere et al., 2012, J Pediatr Ophthalmol Strabismus 49:274-282; Recchia, 2012, Archives of Ophthalmology 130:525; Tawse et al., 2014, Journal of American Association for Pediatric Ophthalmology and Strabismus 18:78-80), the affected areas appeared to be in random focal regions and were not evident in all sections from a given eye, requiring examination of multiple serial sections (Figure 9C, see methods). Nevertheless, every animal had evident pathology (Table 1). The most prominent pathology observed at PND10 was accumulation of cells in the ganglion cell layer (GCL, Figure IE, circle) and rosette-like structures in the outer nuclear layer (ONL) disrupting normal lamination (Figure IF). By PND15, there were increased frequencies of cellular clusters in the GCL (Figure IE and Figure 1G, circle), breaks in the retinal pigment epithelium (RPE) (Figure 1H, star), cellular infdtration in the inner retina (Figure II, arrows), and delamination and thinning of retinal layers with photoreceptor layer disruption (Figure IE). Although cellular infiltration had decreased by PND28, there was still noted retinal thinning, delamination, cell clusters in GCL, and some remaining cellular infiltration (Figure IE). Scanning laser ophthalmoscopy (SLO) of live animals at PND28 revealed infoldings in the photoreceptor layer determined by areas of less penetrating light (Figure 1J, arrows), which was confirmed by optical coherence tomography (OCT) of the affected areas showing disruption of layer lamination at these foci (Figure 9D).

[0170] Next, the regions of the eye with virally infected cells were identified by staining for viral pp89 (encoded by the MCMV IE1 gene). Infected cells were found predominantly in the choroid, and ciliary body but could also be found in the iris as well as in the neuroretina, including the outer and inner nuclear layers, inner plexiform layer, and importantly, the ganglion cell layer (GCL, Figure 2A). Virus-infected cells were evident at both PND10 and PND15 (Figure 2B) and infected CD45 immune cells in the choroid and ciliary body were frequently observed at these times (Figure 2B). No virally infected cells were observed by histology at PND28 (Figure 2B). Together, these data demonstrate that the cMCMV model enabled infection of the eye that induced severe, but focal, retinal pathology, including disrupted retinal lamination, infoldings in the photoreceptor layer, and cellular infiltrations.

[0171] MCMV infection drives focal immune infiltration

[0172] The pathology described above is consistent with focal chorioretinitis, which has been described in humans with cHCMV infection (Cheeran et al., 2009, Clin Microbiol Rev 22:99-126; Coats et al., 2000, Journal of American Association for Pediatric Ophthalmology and Strabismus 4: 110-116; Jin et al., 2017, Pediatric Infectious Disease Journal 36:877-882; Boppana et al., 1994, Pediatr Infect Dis J 13:1139-1142; Ghekiere et al., 2012, J Pediatr Ophthalmol Strabismus 49:274-282; Recchia, 2012, Archives of Ophthalmology 130:525; Frenkel et al., 1980, Pediatrics 66:763-6; Tagami et al., 2016, BMC Ophthalmol 16:81). In agreement with the H&E staining (Figure IE), infiltration of CD45+immune cells began in the outer retinal structures (choroid, iris, ciliary body) on PND10, but were not present in the neuroretina until PND15 (Figure 2B through Figure 2C). Notably, a significant accumulation of CD45+cells could be identified in the GCL on PND15, suggesting that the cell clustering observed by H&E (Figure IE, Figure 1G, and Figure II) included an accumulation of CD45+cells. By PND28, far fewer CD45+cells remained in the neuroretina, but residual clusters of cells were still evident in the GCL (Figure 2B). Importantly, whole retinal flat mounts showed CD45+cellular infiltration only in portions of the retina with evident involvement of the retinal vasculature (Figure 2C, arrows indicate CD45+cells in the vasculature), confirming the focal nature of these infiltrates. The majority of these infiltrating CD45+cells, over 90%, were CD8+T cells (Figure 2D and Figure 10A). Clinical observations from human patients described a similar random distribution of the affected areas in the retina, and inflammation that follows the retinal vasculature resulting in “frosted angiitis” (Recchia et al., 2012, Archives of Ophthalmology 130:525; Tawse et al., 2014, Journal of American Association for Pediatric Ophthalmology and Strabismus 18:78-80; Walker et al., 2004, Eye 18:527-533). Moreover, optic neuritis is a frequent complication of congenital CMV infection in humans and CD8+T cell infiltration of the optic nerve in the cMCMV model was routinely observed (Figure 10C), indicating optic neuritis. Thus, MCMV drove a focal chorioretinitis with involvement of the retinal vasculature and T cell infiltration of the retina and optic nerve.

[0173] Neonatal MCMV infection causes retinal cell death and damage to the blood-retinal barriers.

[0174] Next, the TUNEL assay was used to determine whether infection and immune infiltration was associated with increased cell death. As expected, TUNEL+cells were found in control eyes, as cell death is critical component of retinal development (Braunger et al., 2014, 9-13; Vecino et al., 2004, Int J Dev Biol 48:965-974). However, a marked increase in TUNEL+cells was evident at PND10, PND15 and PND28, which was most evident in the ONL and INL on PND10 but shifted to the choroid and GCL on PND15 and PND28, after the arrival of immune cells (Figure 2E through Figure 2F and Figure 10B).

[0175] Since there was an increased number of immune cells in the inner retina of infected mice on PND15, it was hypothesized that the blood retinal barriers (BRBs) were compromised. The eye is considered an immune-privileged site where the BRBs limit access to protect the homeostasis of the tissue (Campbell et al., 2012, Adv Exp Med Biol 763:70-84; Cunha-Vaz, 2009, European Ophthalmic Review 3: 105; O’Leary et al., 2023, FEBS J 290:878-891). In a healthy developing neonate, the BRBs are fully developed at approximately PND10 and closed by the time the neonates open their eyes on approximately PND12 (Chow et al., 2017, Neuron 93: 1325-1333. e3). To evaluate the BRB integrity on PND15, mice were injected with FITC-dextran and whole retina flat mounts were imaged to visualize any leakage of FITC-dextran into the retina. While the FITC-dextran was confined to retinal vasculature in control mice, there was considerable leakage of FITC-dextran in the infected eyes indicating a disrupted BRB (Figure 2G, arrows). The integrity of the outer BRB was also directly assessed by staining flat mounts of the RPE for occludin, a component of the tight junctions between RPE cells. Breaks in occludin staining were clearly evident in the infected eyes, but not control eyes, confirming that infection with MCMV disrupted the integrity of both inner and outer BRBs (Figure 2H, arrows). Collectively, these data show that cMCMV model recapitulates key features of human disease, with chorioretinitis and optic neuritis, focal disruptions of retinal lamination, focal immune infiltrations that trace the retinal vasculature and are associated with disruptions of the BRBs, and marked cell death in the retina, particularly in the GCL.

[0176] MCMV infection induced a gene signature consistent with immune response and dysregulation of retinal homeostasis

[0177] To further characterize the response to MCMV infection in a developing eye, bulk RNA sequencing (RNA-seq) of infected and control eyes was performed over time. These data identified approximately 2000 differentially expressed genes across all time points. Unsupervised clustering showed that MCMV infected groups clustered together at PND 10, 15 and 28 while eyes from infected mice at PND5 were most similar to PND5 controls (Figure 1 1 A). The most differentially expressed genes (DEGs) in infected mice compared to controls were detected on PND15, corresponding with the peak of pathology observed above (Figure 1 IB). Interestingly, there were very few significantly down regulated genes in infected eyes at any of the tested time points. To identify groups of genes that have similar kinetics over time, co-expression clustering of the differentially expressed genes was performed. This analysis identified 6 unique clusters that allowed visualization of gene expression changes overtime caused by MCMV infection (Figure 3A). Gene ontology (GO) enrichment analyses were then used to identify the biological processes represented by these clusters (Figure 11C). Together, these analyses revealed that MCMV accelerated the downregulation of pathways involved in tissue development (cluster Cl), impaired or delayed the expression of metabolic pathways (clusters C2 and C3), induced the abnormal expression of immune response pathways (clusters C4 and C5) and delayed the expression of normal immune response, immune regulation, stress response and cell adhesion pathways (cluster C6). Thus, MCMV disrupted normal eye developmental and metabolic processes while markedly inducing immune responses not normally observed in the eye. Next, each time point individually was compared using gene set enrichment analysis (GSEA) against GO gene sets, to identify specific biological differences at each stage of MCMV infection. At PND5, pathways involved with viral replication and the Type 1 interferon response were positively enriched, while pathways involved in protein synthesis were negatively enriched (Figure 3B). Thus, despite MCMV loads in the eye that were below the level of detection (Figure 1C) and no evidence of pathology (Figure 9B), the tissue is already initiating an innate immune response at PND5. By PND10, there was significant positive enrichment for pathways involved in innate immune responses and viral genome replication, further corroborating the histology and viral titer data (Figure 3B). On PND15, pathways involved with the adaptive immune response were strongly activated and some of these remained positively enriched at PND28. Interestingly, at PND28 results showed a positive enrichment in processes involved with lymphocyte apoptosis, indicating the resolution of the immune response. Extending these analyses to include the Hallmark gene ontology and KEGG pathways, which use larger gene sets and may therefore reveal additional features, confirmed the overall trends (Figure 12A, Figure 12B, Figure 12E, and Figure 12F), identified enrichment of cell death pathways at PND10 and PND15 and enrichment of cellular replication pathways at PND15, suggesting a transient increase cell proliferation that aligns with the peak of immune infiltration (Figure 12D & Figure 12H).

[0178] Although there was a very limited number of down regulated genes at any of the tested time points, there were negative trends in pathways associated with visual perception beginning at PND15 (Figure 3B). Although the negative enrichment of these pathway was not statistically significant, it should be noted that these analyses included the whole eye and not isolated retinas and suggest some level of impairment of visual processes as a result of MCMV infection. Thus, collectively, these data show that MCMV infection markedly disrupted normal retinal development and metabolism, with likely impacts on visual function, and drove profound innate and adaptive immune responses that were associated with cell death and were reduced, but not resolved even by PND28.

[0179] MCMV infection induces reactive glial cells

[0180] Next, GSEA and gene set variance analysis (GSVA) was used to identify differences in enrichment of pathways for specific cell signature gene sets (Figure 3C), with a focus on T cells, myeloid cells and microglia. Microglia are the tissue resident to the brain that respond to infection (Li et al., 2018, Nat Rev Immunol 18:225-242; Colonna et al., 2017, Annu Rev Immunol 35:441-468; Li et al., 2019, Neural Dev 14:12; Rathnasamy et al., 2019, Prog Neurobiol 173: 18-40; Mass et al., 2023, Nat Rev Immunol). GSEA analyses comparing each timepoint to all others enabled us to identify the relative kinetics of enrichment across the timepoints, clearly indicating that peak enrichment of T cell signatures occurred at PND15 (Figure 3C), in agreement with the histological data (Figure 2), while myeloid and microglia signatures were already enriched by PND10. Interestingly, GSVA analyses showed that microglia gene sets were the most significantly variant across the time course, with most microglia pathways showing statistically significant differences in the enrichment between all of the timepoints (Figure 121 and Table 2). In contrast, significant differences in myeloid cell gene sets were most evident between PND28 and the other timepoints while T cell gene sets showed the most significant differences between PND15 and PND28 (Table 2).

[0181] These data suggest that infection induced significant microglial activation across the time course.

[0182] Table 2: p-values of group comparisons generated from GSVA analyses on cell signature gene sets. Glial cells in the retina, including microglia, astrocytes, and Muller glia, are important for development, signal transduction, tissue homeostasis and regulating immune responses (Reichenbach et al., 2020, Glia 68:768-796; Kumar et al., 2013, Crit Rev Immunol 33: 119-135; Li et al., 2018, Nat Rev Immunol 18:225-242; Colonna et al., 2017, Annu Rev Immunol 35:441-468; Rathnasamy et al., 2019, Prog Neurobiol 173:18- 40; Giovannoni et al., 2020, Trends Immunol 41 :805-819). Activated microglia adopt a more compact shape, with shorter processes (Lull et al., 2010, Neurotherapeutics 7:354- 365; Woodbum et al., 2021, J Neuroinflammation 18:258) and indeed, microglia appeared more compact in MCMV infected retinas compared to controls at each of the timepoints (Figure 4A). Quantification of Ibal+microglia morphology at PND15 revealed significant increases in the numbers of microglia and their activation state as assessed by the numbers of processes, the cell density the number of branches per cell, and the complexity of the cells (Figure 4B through Figure 4F, Figure 13 A and Figure 13B). Activated microglia also move to sites of damage in the eye (Ng et al., 2001, Invest Ophthalmol Vis Sci 42:3301-10; Carbonell et al., 2005, The Journal of Neuroscience 25:7040-7047; Yu et al., 2022, CNS Neurosci Ther 28: 1279-1293). In the eyes of infected mice, there was a higher frequency of morphologically activated microglia in the subretinal space compared to controls (Figure 13C & Figure 13D), indicating a response to photoreceptor or RPE damage. This accumulation of activated microglia was also observed on PND28 by visualizing hyperfluorescent foci by autofluorescent SLO imaging (Bell et al., 2015, Exp Eye Res 135: 192-205) (Figure 13E).

[0183] Other glial cells in the eye are also known to respond to damage or infection, as well as contribute to neurotoxic pathologies (Ridet et al., 1997, Trends Neurosci 20:570-577; Dyer et al., 2000, Nat Neurosci 3:873-880; MacLaren, 1996, British Journal of Ophthalmology 80:458-464; Liddelow et al., 2017, Nature 541 :481- 487; Guttenplan et al., 2020, Cell Rep 31; Block et al., 2007, Nat Rev Neurosci 8:57-69). The glial fibrillary acidic protein (GFAP) is expressed by astrocytes and expression is strongly increased on reactive astrocytes, while Muller glial cells express GFAP when activated. MCMV infection induced the strong upregulation of GFAP on astrocytes in the GCL and disrupted their normal patterning across all time points (Figure 4G, Figure 4H and Figure 41), suggesting reactive astrocytes were induced early in the infection and sustained throughout the time course. GFAP expression was also induced on Muller glial cells, which traverse the retinal layers, with a significant increase in expression evident at PND15, consistent with peak of cellular infiltration (Figure 4J). Additionally, results showed significant differential expression of genes involved in microglia and astrocyte activation in MCMV-infected mice (Figure 4K), most of which were upregulated at PND10 and PND15. Notably, some of these genes have been linked with neurotoxic glia, including MHC-IT (H2-A / E genes and CD74 invariant chain), Aifl (encodes Ibal), nos2 (encodes iNos) and C3, or with ZFN-y stimulation, including iigpl, igtp, gbp2, serpin l, CXCL9 and CXCL10 (Woodburn et al., 2021, J Neuroinflammation 18:258; Liddelow et al., 2017, Nature 541 :481-487; Liddelow et al., 2017, Nature 541 :481-487; Block et al., 2007, Nat Rev Neurosci 8:57-69; Kann et al., 2022, Trends Neurosci 45:913-927; Papageorgiou et al., 2016, Proc Natl Acad Sci U S A 1 13:212-217). Overall, these data show retinal glial cells are activated in response to MCMV infection in the eye and may be skewed toward a pathologic activation state.

[0184] MCMV infection induces the formation of glial nodules in the ganglion cell layer, which attract CD8+T cells and express a neurotoxic phenotype.

[0185] Nodules of activated glial cells were previously observed in studies of MCMV infection in the brain, where focal glial nodules were observed around areas of infected cells (Lokensgard et al., 2015, Glia 63:982-1996). Similarly, nodules of nuclei were observed focally in the GCL in MCMV-infected mice (Figure IE and Figure 1G), and frequently contained CD45+immune cells (Figure 2B). These nodules in the retina were composed of Ibal+CD45dimmicroglia and surrounded by GFAP+astrocytes and Muller glial cells (Figure 5A and Figure 14A). Occasionally, pp89+virally infected cells were observed in the center of these nodules (Figure 5B), although it was rare to observe this. These nodules were clearly focal and randomly distributed when visualized by whole retinal flat mounts (Figure 14B) and ranged in size with the most abundant and largest nodules observed at PND15 (Figure 14C and Figure 14D). Astrocytes were cleared from the nodule center and accumulated around the edges where GFAP+Muller glial processes were also evident (Figure 5C and Figure 5D). Confocal imaging of these nodules showed that accumulation of cells was mostly confined to the GCL but that the Muller glial processes around the nodule extended through all layers of the retina while areas with no nodules tended to lack activated GFAP+Muller glia but still had microglia in the subretinal space (Figure 5E). Microglia and CD8+T cells were most abundant in the center of the nodules (Figure 5C and Figure 5D) and CD8+T cells appeared to be particularly enriched within the nodules compared to areas with no nodule formation (Figure 5E). To quantify this, the distance of each T cell to the location of the nodule was measured as defined by the highest intensity of Ibal staining in the image. These analyses demonstrated that CD8+T cells accumulated significantly closer to microglia where glial nodules formed, compared to areas of the retina with no nodules (Figure 5F, Figure 5G and Figure 14E). Thus, MCMV caused the formation of glial nodules and T cells in the retina were being recruited to these nodules.

[0186] The lymphocyte chemoattractants CXCL9, CXCL10, and CXCL11 are major drivers of T cell migration and were significantly upregulated in the eye as assessed by RNAseq (Figure 5H). RNA scope in situ hybridization showed that CXCL9, CXCL10, and CXCL11 were highly expressed in the choroid and in the inner retina of infected mice (Figure 51 and Figure 5 J) but were absent from control uninfected animals (Figure 15A and Figure 15B). Importantly, in the neuroretina on PND15 after infection, expression of these chemokines was almost exclusively localized within glial nodules in the GCL (Figure 51 and Figure 5J, white doted circle). In contrast, CCL2, which is known to be expressed by activated astrocytes (Cherry et al., 2020, J Neuroinflammation 17:370; He et al., 2016, Cellular Physiology and Biochemistry 38:859-870), was expressed along the GCL where astrocytes reside, and not exclusively in the nodules (Figure 51). Expression of CXCL9 is a marker of activated and potentially neurotoxic microglia (Strack et al., 2002, Acta Neuropathol 103:458-468; Capuccini et al., 2016, Sci Rep 6:39258), and CXCL9 transcript expression colocalized with Ibal staining in the GCL (Figure 15C). Likewise, MHC-II and iNOS, which are also associated with neurotoxic microglia, were both observed on Ibal+cells in glial nodules (Figure 5K). These molecules are known to be induced by interferon-gamma (IFN-y) and most of the IFN-y expression was localized to glial nodules of MCMV infected eyes (Figure 15D through Figure 15F). Overall, these data suggest that glial nodules were forming around MCMV- infected cells leading to the recruitment of CD8+T cells into the neuroretina and the expression of IFN-y-stimulated genes by microglia.

[0187] Blocking CXCR3 prevents immune infiltration and retinal pathology

[0188] The receptor for CXCL9, CXCL10, and CXCL11 is CXCR3, and gene expression of CXCR3 was strongly upregulated in the eye with kinetics that mirrored T cell recruitment (Figure 16A). To determine whether CXCR3 was guiding T cells to the glial nodules, infected mice were treated with a CXCR3 blocking antibody or an irrelevant antibody and assessed outcomes at PND15 (Figure 16B). Blockade of CXCR3 did not increase the amount of MCMV found in the eyes but did result in significantly more virus replicating in the lungs of mice (Figure 6A, Figure 6B, and Figure 16C). Surprisingly however, the pathology in the eyes of the aCXCR3 treated mice was almost completely rescued by CXCR3 blockade: there were no observed disruptions in the lamination of the retinal layers (Figure 6C), nodules in the ganglion cell layer were less frequent and smaller (Figure 16D and Figure 16E), cellular infiltration of the neuroretina was essentially absent (Figure 6D and Figure 6E) and cell death in the GCL was prevented (Figure 6F and Figure 16G). In contrast, the choroids of mice treated with aCXCR3 still had an abundance of CD45+immune cells, similarly to eyes of infected mice treated with an irrelevant antibody (Figure 6D, Figure 6E, and Figure 16F), and exhibited elevated cell death (Figure 16H). Moreover, immune cells were clearly observed lining the retinal vasculature via flat mounts (outlined by GFAP1astrocytes end feet) in aCXCR3 -treated mice (Figure 6E, dotted lines). Thus, CXCR3 blockade completely prevented immune infiltration of the retina and any resulting retinal pathology but did not prevent immune cells from accumulating in the retinal vasculature and choroid.

[0189] Prednisolone treatment also prevents retinal pathology after MCMV infection

[0190] These results from the CXCR3 blockade suggest that the immune response is the primary driver of retinal pathology after neonatal MCMV infection. As a second approach to test this idea, the immune response was dampened more generally by treating with the corticosteroid prednisolone (Figure 17A). Previous studies using this model showed that treatment with steroids rescued early MCMV -induced pathology in the brain (Kosmac et al., 2013, PLoS Pathog 9). In this study, prednisolone treatment led to increased viral titers in the lungs and eyes (Figure 17B and Figure 17C). Most infected mice that were treated with prednisolone died by PND15, but retinal pathology was clearly reduced by prednisolone treatment on PND10. Indeed, prednisolone treatment of infected mice led to normal lamination of retinal layers, the complete absence of rosettelike structures in the outer nuclear layer, and a reduction in the nodules in the ganglion cell layer (Figure 17D). Moreover, while an increased frequency of infected cells was detected in the predni sol one-treated eyes, the retinas were nearly devoid of CD45+immune cells (Figure S17E) and cell death was reduced to baseline levels as assessed by TUNEL-staining (Figure 17F). Finally, glial cells appeared less activated: there was decreased microglia accumulation in the GCL (Figure 17G), decreased GFAP expression on astrocytes (Figure 17H), and no Muller glial cell activation (Figure 17H). Thus, immune suppression profoundly reverses retinal damage after neonatal infection by MCMV.

[0191] Blood retinal barrier integrity is preserved by CXCR3 blockade

[0192] Since CXCR3 blockade prevented cellular infiltration of the retina after neonatal MCMV infection, the BRB integrity was assessed in these mice. Astrocytes are integral for BRB maintenance and their end feet wrap around endothelial cells forming tight junctions that are involved in the formation of the blood-brain barrier (Campbell et al., 2012, Adv Exp Med Biol 763:70-84; Fresta et al., 2020, Int J Mol Sci 21 : 1636; Abbott et al., 2006, Nat Rev Neurosci 7:41-53). For infected mice treated with irrelevant antibody, staining for GFAP+astrocytes showed gaps in areas with cellular infiltration, suggesting loss of astrocyte end feet and BRB leakage at the affected area (Figure 6G, box). Strikingly, CXCR3 blockade completely prevented the formation of these astrocyte gaps and hematopoietic cells remained trapped in the vasculature (outlined in dotted lines) contained by the astrocyte end-feet. Likewise, no leakage of FITC Dextran from the blood into the retina was observed in any of the CXCR3-blocked mice (Figure 6H), and occludin staining of the outer BRB showed no loss of tight junction integrity, in contrast to the breakages observed in infected mice treated with an irrelevant antibody (Figure 61). These data show that CXCR3 -blockade prevented the breakdown of BRB, thus preventing immune infiltration and subsequent retinal damage.

[0193] CXCR3 blockade reduces glial activation after MCMV infection

[0194] Microglia activation by MCMV was also altered by CXCR3 blockade. Morphologically, aCXCR3 treatment led to microglia that had more, and thinner cell processes and appeared larger in size compared to microglia in infected mice not treated with the blocking antibody (Figure 7A through Figure 7C). Quantifying the microglial numbers and morphology revealed that aCXCR3 treatment resulted in microglia with an intermediate activation phenotype. The CXCR3 blockade reversed the expansion of microglia numbers (Figure 7G) and the increase in process endpoints per cell (Figure 7H). Total branches and overall density of microglia from aCXCR3 treated mice were also significantly reduced but were still increased compared to uninfected control microglia (Figure 18A and Figure 18B), indicating some intermediate activation. Further, the summed process length and span ratio were still reduced regardless of CXCR3 blockade, indicating a more amoeboid shape (Figure 71 and Figure 18C) and while the overall complexity of the cells measured by fractal dimensions was reduced by aCXCR3 treatment, the microglia in these mice were still significantly more complex than in uninfected controls (Figure 7J). Importantly, microglia were mostly absent from the subretinal space in aCXCR3 treated eyes (Figure 18D and Figure 18E), suggesting less damage to the photoreceptors or RPE during aCXCR3 treatment. Similarly, while CXCR3 blockade prevented the expansion of astrocytes (assessed by the percent coverage of GFAP-staining, Figure 7K), the intensity of GFAP expression by astrocytes was unaffected by CXCR3 blockade (Figure 7L). Notably, upregulation of GFAP on astrocytes in the ganglion cell layer was also reduced in predni sol one-treated animals (Figure 17H). Together, these data suggest that CXCR3 blockade reduced and altered, but did not prevent, the activation of glial cells after MCMV infection.

[0195] Blocking CXCR3 changes the expression of IFN-y-stimulated genes in glial nodules.

[0196] To assess transcriptional changes in response to CXCR3 blockade RNAseq was performed on isolated retinas and RPE / choroids from mice infected by MCMV (PND15) and treated with the aCXCR3 blocking antibody or irrelevant control antibody. Unsurprisingly, MCMV infection drove significant differential gene expression in the retina and RPE / choroid compared to uninfected control tissues, regardless of CXCR3 blockade (Figure 19A). However, transcriptional differences between conditions were evident. To assess variations between samples and conditions and relate them to biological processes, GSVA for GO gene sets was utilized. Clustering analyses were performed to visualize sample variance and to cluster similar pathway enrichment into modules (Figure 19B and Figure 19C). For both retina and RPE / choroid samples, each condition clustered together. Importantly, in the retina, aCXCR3 -treated samples appeared to be generally more similar to uninfected controls than samples from infected mice treated with an irrelevant antibody (Figure 19B). However, there was some notable mouse-to-mouse variability, with two retina samples from mice treated with aCXCR3 (CX2 and CX3) that appeared more similar to infected mice treated with the irrelevant antibody. Even with this variability, aCXCR3-treatment strongly reverted genes in modules 4, 6 & 7 to a pattern that closely approximated uninfected retinas across all 5 samples in this group (Figure 19B). Module 4 pathways are mostly involved with metabolism while modules 6 and 7 are involved with development. Furthermore, module 7 includes pathways involved in neuronal patterning. Thus, aCXCR3 -treatment partially repairs the metabolic and developmental damage in the retina caused by MCMV infection. In contrast, aCXCR3 -treatment had less of an impact on pathways in the RPE / choroid with the exception of module 3, which includes pathways involved with cell cycle and development, (Figure 19C).

[0197] To determine specific pathways that were affected by aCXCR3 treatment during MCMV infection, GSEA analyses of these samples were used. Immune response pathways were still significantly enriched in retinas from aCXCR3 -treated mice, but to a significantly reduced degree when compared to infected mice treated with irrelevant antibody (Figure 8A through Figure 8C and Figure 19D). Specifically, the aCXCR3- treatment significantly reduced the pathways involved in leukocyte transendothelial migration, T cell receptor signaling, focal adhesion, natural killer mediated cytotoxicity (Figure 8B, Figure 8C, and Figure 19D). Notably, based on the flat mount data (Figure 6E), the residual enrichment of immune signatures is likely attributable to immune cells contained in the inner retinal vasculature and ciliary body, which could not be removed. Additionally, CXCR3 -blockade led to significantly reduced enrichment of cell cycle and metabolic pathways compared to retinas treated with an irrelevant antibody, together supporting the conclusion that aCXCR3 treatment partially restored normal retinal development (Figure 8C and Figure 19D). Interestingly, the isolation of the retina in these experiments allowed us to detect significant negative enrichment of pathways involved with visual perception (Figure 8A), indicating that visual function pathways were indeed disrupted by MCMV infection as was suggested after whole-eye RNA-seq (Figure 3B). However, CXCR3-blockade did not significantly change these pathways suggesting that viral infection may still cause some retinal function defects.

[0198] In the RPE / choroid, immune related pathways were similarly enriched regardless of aCXCR3 treatment with the exception of some small, but significant differences were detected in cytokine, chemokine and T cell response pathways (Figure 8D through Figure 8F, and Figure 19E). Together these data suggest that an immune response is still occurring in the RPE / choroid regardless of treatment but to a lesser degree in mice treated with aCXCR3.

[0199] Finally, retinal glial nodules were specifically assessed via RNAscope in situ hybridization and immune fluorescence. Strikingly, glial nodules, and indeed the majority of the neuroretina, expressed no CXCL9, CXCL10, CXCL11, iNOS or MHC-II (Figure 8G, Figure 20A and Figure 20B). Likewise, INF-y was absent from the neuroretina in aCXCR3 -treated mice while CCL2, which can be produced by astrocytes in direct response to CMV (Lokensgard et al., 2015, Glia 63: 1982-1996), was unaffected (Figure 8G and Figure 8H). Together, these data suggest blocking CXCR3 prevented the expression of INF -y stimulated genes in retinal glial nodules, presumably as a result of preventing lymphocyte infiltration, altering the outcomes of immune response to MCMV infection in the retina. These data show that immune interventions were able to preserve the BRBs and prevent retinal pathology, while partially reverting glial cell activation and restoring retinal development during neonatal MCMV infection.

[0200] Approximately 20% of children with symptomatic congenital CMV infection develop severe vision impairment (Boppana et al., 1992, Pediatr Infect Dis J 11 :93-98; Coats et al., 2000, Journal of American Association for Pediatric Ophthalmology and Strabismus 4: 110-116; Jin et al., 2017, Pediatric Infectious Disease Journal 36:877-882; Boppana et al., 1994, Pediatr Infect Dis J 13:1139-1142; Ghekiere, et al., 2012, J Pediatr Ophthalmol Strabismus 49:274-282). However, the mechanisms of disease are unknown and no prior animal models have been developed. These data demonstrate that MCMV infection of newborn mice drove profound, focal pathology in developing retinas, similar to what is observed in humans. Strikingly, using this model, results showed that by targeting the immune system, most disease manifestations were reduced. HCMV and MCMV are both well-known to cause severe retinitis in immune- suppressed hosts. Indeed, HCMV retinitis is a common complication of T cell decline in patients with AIDS, and extensive work in the MCMV model has clearly demonstrated that T cells are protective against severe retinitis in adults (Bigger et al., 1999, Invest Ophthalmol Vis Sci 40:2608-13; Lu et al., 1997, Invest Ophthalmol Vis Sci 38:301-10; Munro et al., 2019, Microorganisms 8:55). In contrast, while newborn mice are immune immature, they do mount MCMV-specific T cell responses with slightly delayed kinetics (Venturi et al., 2016, The Journal of Immunology 196: 1604-1616), and T cells are critical for survival of infected pups (Bantug et al., 2008, The Journal of Immunology 181:2111-2123; Brizic et al., 2019, Med Microbiol Immunol 208:487-494; Brizic et al., 2019, Med Microbiol Immunol 208:487-494; Brizic et al., 2018, Eur J Immunol 48:950- 964). Moreover, the arrival of T cells in the eyes of mice corresponded to a reduction in detectable MCMV-infected cells in the eye. Thus, it might have been expected that blocking T cell responses with prednisolone, or T cell migration with ocCXCR3 would lead to more severe glial nodules, more virus in the eye and more pathology, mirroring an immune deficient state. Surprisingly however, the opposite was true: immune suppression preserved the BRB integrity, inhibited retinal cell death, reduced the size of glial nodules, prevented glial cells from expressing IFN-y-stimulated genes that are normally associated with neurotoxicity, and restored many features of normal retinal development and metabolism. Thus, at least transiently, immune suppression blocked MCMV-driven retinitis during eye development.

[0201] This is the first description of retinal pathology in the cMCMV model. Previous work has used this model to assess aging-related damage to the retina (Xu et al., 2021, Am J Pathol 191 : 1787-1804) but these investigators did not describe any notable neonatal pathology or investigate early life immune responses. The focal nature of the observed pathology in the eye meant that multiple serial sections or whole retinal flat mounts were necessary to find the affected regions in every mouse. Nevertheless, pathology was evident in all infected mice and these data, with and without immune suppression, allow us to develop a temporal model of this neonatal MCMV retinitis. MCMV loads were below the limit of detection on PND5, but the mice were beginning to make an innate immune response as assessed by RNA-Seq, suggesting that viral amplification elsewhere in the body led to viremia in the first week that seeded the eye with MCMV. This had the effect of strongly activating retinal glial cells, including marked activation of astrocytes and microglia by PND10, before the T cells arrived in the retina, and the development of nascent glial nodules in the GCL. Microglia normally reside in the inner and outer plexiform areas, but migrate towards damage (Lull et al., 2010, Neurotherapeutics 7:354-365; Ng et al., 2001, Invest Ophthalmol Vis Sci 42:3301- 10; Carbonell et al., 2005, The Journal of Neuroscience 25:7040-7047; Yu et al., 2022, CNS Neurosci Ther 28: 1279-1293; Karlstetter et al., 2015, Prog Retin Eye Res 45:30- 57). Based on these data, and prior work from Lokensgard and colleagues, this suggests that activated microglia migrated towards the CCL2 expressed by activated astrocytes in the GCL to initiate nodule formation. Such nodules may be a common feature of CNS infections by CMV and have been seen in the brains of CMV-infected mice (Lokensgard, et al., 2015, Glia 63: 1982-1996) and humans (Nobuloni et al., 2000, J Neurovirol 6:46- 50).

[0202] Next, T cells were recruited to the eye and accumulated in the growing glial nodules. Together these cells formed a unique aberration in the GCL composed of astrocytes, microglia, infiltrating CD8+T cells, and activated Muller glial cells. Interestingly, these nodules appeared to create a barrier of astrocytes around an area filled with CD8+T cells and microglia, with the microglia expressing T cell attracting chemokines and a neurotoxic phenotype. Of note, cell death was particularly enriched in these nodules, suggesting this as a potential site for the killing of virally infected cells. Previous studies have described this phenomenon in other models of retinal disease and have suggested that their purpose is to encircle an area of damage, shielding the surrounding area from further destruction (Silver et al., 2004, Nat Rev Neurosci 5: 146- 156; Faulkner et al., 2004, The Journal of Neuroscience 24:2143-2155).

[0203] Based on the timing of these events, it was hypothesized that the CXCR3 blockade would limit the recruitment of CD8 T cells to the glial nodules within the retina, but not prevent T cells from gaining access to the retina per se. Strikingly however, blocking this receptor completely prevented T cells from accessing the retina, trapping them in the retinal vasculature, and prevented most of the pathological outcomes. Although further work will be needed, these data strongly suggest that CXCR3 is used by T cells first within the vasculature, resulting in the breakdown of the BRBs. Moreover, blockade of CXCR3 also prevented the microglia and astrocytes from progressing beyond the initial activation observed at PND10. Microglia were smaller and more complex than resting microglia and they were still found in the GCL in infected mice treated with CXCR3 blockade, but their morphology was not the same as in MCMV- infected mice treated with isotype control antibodies and their numbers were restored to baseline (uninfected) levels by the CXCR3 blockade. Similarly, astrocytes still appeared activated based on the intensity of GFAP staining and their expression of CCL2, but their numbers were restored to baseline levels by CXCR3 blockade. Thus, MCMV likely directly activated these cells, but the arrival of immune cells caused the nodules to progress to the enlarged, destructive state with the central, T cell-rich killing zone containing neurotoxic microglia, surrounded by activated astrocytes and Muller glia. Notably, the activation of Muller glia was prevented by prednisolone and CXCR3 blockade, suggesting that Muller glial cells are not directly activated by MCMV at this stage, but depend on the progression of the immune response.

[0204] Remarkably, CXCR3 blockade also resulted in a distinct absence of the chemokines CXCL9, CXCL10, CXCL11, as well as iNOS and MHC-II in the glial nodules, indicating an absence of neurotoxic glial cells. These genes are known to be induced by IFN-y (Kann et al., 2022, Trends Neurosci 45:913-927; Papageorgiou et al., 2016, Proc Natl Acad Sci U S A 113:212-217; Strack et al., 2002, Acta Neuropathol 103:458-468; Capuccini et al., 2016, Sci Rep 6:39258). Thus, loss of BRB integrity and secretion of IFN-y in the retina likely plays a central role in the progression of the glial nodules and the neurotoxic phenotype. Recent work has suggested that NK cells are a critical early source of IFN-y in the brain in the cMCMV model (Kvestak et al., 2021, Journal of Experimental Medicine 218). In this work, the NK cells arrived by PND6 and peaked on PND8, driving early IFN-y-dependent microglial activation. NK cells have been stained for but rarely are these cells found in the retina. Moreover, immune infiltration of any kind was still absent from the retina as late as PND10, suggesting that these early NK cell responses are not involved in retinal pathology. Thus, it is suggested that T cell-derived IFN-y may be more critical in the retina, although this requires future study. In this study, depleting NK cells in MCMV-infected pups resulted in mortality before PND15, precluding a direct test of the role of NK cells on late retinal pathology and glial activation. Overall, these data describe a step-wise induction of immune- pathology during MCMV retinal infection in neonatal mice, and show that targeting immune response mechanisms can ameliorate disease, at least transiently.

[0205] Although there are limitations in comparing a mouse pathogen to its human counterpart, these data suggest many parallels between this mouse model and the human disease. In humans, congenital infection leads to focal chorioretinitis and optic neuritis (Coats et al., 2000, Journal of American Association for Pediatric Ophthalmology and Strabismus 4: 110-116; Ghekiere et al., 2012, J Pediatr Ophthalmol Strabismus 49:274-282; Recchia et al., 2012, Archives of Ophthalmology 130:525). In this study, results showed profound focal immune infiltration of the retina and optic nerve. Importantly, these focal patterns observed during human infection tended to follow the vasculature and have been described as “frosted angiitis” of inflamed blood vessels (Recchia, 2012, Archives of Ophthalmology 130:525; Tawse et al., 2014, Journal of American Association for Pediatric Ophthalmology and Strabismus 18:78-80; Walker et al., 2004, Eye 18:527-533). Likewise, in this mouse model, the immune cells tended to cluster around blood vessels in areas with disrupted BRBs. Thus, this cMCMV model appears to replicate the human congenital disease.

[0206] The current standard for treatment for congenital CMV infection is the antiviral drug ganciclovir. While this treatment can rapidly resolve active viral replication, it would have only an indirect impact on immune-mediated disease. Thus, targeting the immune system could be a critical adjunct therapy to any virus-targeted interventions. These data using aCXCR3 as a treatment demonstrated that blocking lymphocyte recruitment prevented the observed pathology and restored retinal development. This is unlikely to be a viable clinical approach as it would depend on catching the disease before T cells have reached the eye. However, these data further suggest that IFN-y-stimulated genes were absent from glial cells when lymphocyte entry was blocked. Microglia are known to be driven toward a neurotoxic state by JFN-y (Kann et al., 2022, Trends Neurosci 45:913-927) and indeed results showed iNOS and MHC-II, along with CXCL9, 10 and 11 expression in microglia from infected eyes, but not when CXCR3 was blocked. Thus, IFN-y could be a critical central mediator of retinal pathology (Koontz et al., 2008, J Exp Med 205:423-435), as has also been proposed for the brain (Kvestak et al., 2021, Journal of Experimental Medicine 218). Importantly however, T cell and NK cell responses are vital for MCMV control, including in this neonatal infection model (Bantug et al., 2008, The Journal of Immunology 181 :2111— 2123). Therefore, therapies targeting T cell or NK cell effector functions will almost certainly inhibit viral control and promote disease over time. Indeed, blocking CXCR3 resulted in increased viral loads in the lungs. It is suggested that immune targeting would have to be combined with antiviral therapy to limit both viral growth and immunopathology.

[0207] Transcriptional evaluation allowed us to begin defining how CMV and the associated immune response disrupted normal eye development with data suggesting a particular impact on metabolism, cell migration, differentiation and neuronal patterning. Likewise, assessing isolated retinas at PND15 showed a negative enrichment for pathways involved in visual function. Further work will be needed to determine how these developmental and metabolic changes impact visual perception. Interestingly, while it was apparent that aCXCR3 treatment markedly improved the retinal inflammation and normalized many of the developmental and metabolic pathways in the retina, it was less clear that aCXCR3 treatment significantly restored visual processes that were impaired during infection. Thus, further work will be needed to determine what effect these early- life disruptions, or the immune interventions, have on long-term visual function, as the mice grow to adults.

[0208] In summary, this study has characterized MCMV infection of the eye during development and implicated the immune response in disease manifestations. Since newborn mice are at a similar developmental stage as a late second trimester human fetus-a time when CMV is known to cross the placenta-this model allowed us to explore potential developmental changes caused by the CMV in the eye. By blocking lymphocyte recruitment via chemokine receptor CXCR3, many of the pathological outcomes of MCMV infection were prevented. Thus, this study is the first to describe MCMV induced retinal pathology during early life development and to use this model to reveal mechanisms of disease that may suggest novel therapeutic targets. Example 2: Hepatic Pathology After Neonatal CMV

[0209] Given the positive impact of immunosuppressants on retinal pathology observed after infection with CMV, the systemic impacts of CMV, and therapeutic benefits of immunosuppressants to treat the infection, were examined. Newborn mice were infected with MCMV within 24 hours of birth, or left uninfected as a control. MCMV-infected animals were then treated with an anti-CXCR3 antibody or, as a negative control, an isotype antibody. On PND15, liver samples were harvested, and stained with hematoxylin and eosin (H&E) stain. While significant hepatic injury was observed in the liver of isotype treated antibody, the liver of mice treated with anti- CXCR3 antibody showed no significant damage (Figure 21).

[0210] Example 3: Retinal Pathology With Antiviral Treatment

[0211] To further examine the impact of CMV on the retina, standard-of-care ganciclovir was compared to immunosuppressants. Newborn mice were infected with MCMV within 24 hours of birth and left untreated (control) or treated with either anti- CXCR3 antibody or ganciclovir beginning on PND7 through PND15. On PND15 lung viral titers were assessed by plaque assay, confirming significant reduction in viral load in mice treated with ganciclovir, but not with anti-CXCR3 antibody (Figure 22A). Contrary to the alteration in viral load, however, retinal biopsies stained for Glial Fibrillary Acidic Protein (GFAP) and CD45, revealed that immune infiltration still occurred in animals treated with ganciclovir (Figure 22B), unlike in mice treated with anti-CXCR3 antibody.

[0212] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMSWhat is claimed is:

1. A composition for treating or preventing a virus-related neurological disease or disorder comprising at least one immunosuppressant and at least one antiviral agent.

2. The composition of claim 1, wherein the at least one immunosuppressant is selected from the group consisting of prednisone, dexamethasone, fontolizumab, emapalumab, AMG 811, baricitinib, filgotinib, tofacitinib, upadacitinib, ruxolitnib, abrocitnib, itacitnib, fedratinib, pacritinib, gandotinib, momelotinib, pravastatin, ISS-840, PY*LKTK, PM-73G, CJ-1383, resveratrol, catechins, PLX5622, adalafil, metformin, a CXCR3 inhibitor, a Integrin a.4 inhibitor, a LFA-1 inhibitor, a VCAM inhibitor, and a ICAM inhibitor.

3. The composition of claim 1, wherein the at least one antiviral agent is selected from the group consisting of ganciclovir, valganciclovir, letermovir, acyclovir, foscarnet, cidofovir, maribavir, leflunomide, brincidofovir, cyclopropavir, BDCRB, TR.CB, tomeglovir, quercetin, baicalein, artesunate, TF27, valnocatmide, and valproic acid.

4. The composition of claim 1, wherein the virus-related neurological disease and disorder is a cytomegalovirus (CMV) infection.

5. A method of treating or preventing a virus-related neurological disease or disorder in a subject comprising administering to the subject a therapeutically effective amount of a composition comprising at least one immunosuppressant and at least one antiviral agent.

6. The method of claim 5, wherein the at least one immunosuppressant is selected from the group consisting of prednisone, dexamethasone, fontolizumab, emapalumab, AMG 811, baricitinib, filgotinib, tofacitinib, upadacitinib, ruxolitnib,abrocitnib, itacitnib, fedratinib, pacritinib, gandotinib, momelotinib, pravastatin, ISS-840, PY*LKTK, PM-73G, CJ-1383, resveratrol, catechins, PLX5622, adalafil, metformin, a CXCR3 inhibitor, a Integrin a4 inhibitor, a LFA-1 inhibitor, a VCAM inhibitor, and a ICAM inhibitor.

7. The method of claim 5, wherein the at least one antiviral agent is selected from the group consisting of ganciclovir, valganciclovir, letermovir, acyclovir, foscamet, cidofovir, maribavir, leflunomide, brincidofovir, cyclopropavir, BDCRB, TRCB, tomeglovir, quercetin, baicalein, artesunate, TF27, valnocatmide, and valproic acid.

8. The method of claim 5, wherein the at least one immunosuppressant and the at least one antiviral agent are administered at different times.

9. The method of claim 5, wherein the at least one immunosuppressant and at least one antiviral agent are administered simultaneously.

10. The method of claim 5, wherein the virus-related neurological disease or disorder is a cytomegalovirus (CMV) infection.

11. The method of claim 10, wherein the subject is an infant or child.

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