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1. WO2016115092 - PROCÉDÉS DE CIBLAGE DE MÉCANISME VASCULAIRE HÔTE À DES FINS DE PROTECTION THÉRAPEUTIQUE CONTRE LA FIÈVRE HÉMORRAGIQUE

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[ EN ]

METHODS FOR TARGETING HOST VASCULAR MECHANISM FOR THERAPEUTIC PROTECTION AGAINST HEMORRHAGIC FEVER

Related Patent Applications

[0001 ] This patent application claims the benefit of U.S. Provisional Patent Application No. 62/102,490 filed on January 12, 2015, entitled METHODS AND COMPOSITIONS FOR THE TREATMENT OF HEMORRHAGIC FEVER, and naming Sujan Shresta as inventor; and claims the benefit of U.S. Provisional Patent Application No. 62/157,464 filed on May 6, 2015, entitled METHODS FOR TARGETING HOST VASCULAR MECHANISM FOR THERAPEUTIC PROTECTION AGAINST HEMORRHAGIC FEVER, and naming Sujan Shresta as inventor. The entire content of the foregoing applications is incorporated herein by reference, including all text, tables and drawings.

Field

[0002] Certain embodiments of the invention relate to compositions, and methods of using the same, for the treatment, prevention, reduction of severity of, or delay of the onset of, a viral infection, or a symptom thereof, caused by a virus selected from the family of Arenaviridae, Filoviridae, Bunyaviridae, Flaviviridae, and Rhabdoviridae. Some

embodiments of the invention relate to methods of treatment, prevention, reduction of severity of, or delay of the onset of, hemorrhagic fever.

Background

[0003] One major pathophysiologic feature of hemorrhagic fever is an acute increase in vascular permeability that leads to leakage of fluid into tissues and severe hypovolemia. Hemorrhagic fever can be caused by five distinct families of RNA viruses, and by many different strains within any one species. For example, dengue virus (DENV), an NIAID Category A pathogen, is a member of the virus family Flaviviridae. There are at least four DENV serotypes (DENV1 -4) identified in more than 100 countries, resulting in an estimated 390 million infections per year. One of the disease manifestations is the acute dengue fever (DF). In roughly 1 .5 % of the cases, DF evolves into to a life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). DHF/DSS often requires hospitalization in intensive care units for symptomatic management and represents a significant economic burden for countries where DENV is endemic. Despite extensive experience and training of physicians in these countries, complex physiological changes in DHF/DSS patients can result in major complications, and mortality remains around 4%.

Uncontrolled urbanization, globalization, and the spread of the DENV-transmitting mosquitoes have resulted in co-circulation of different DENV serotypes and increased frequency of epidemics and disease severity. Although the major disease burden is in South-East Asia, Latin America, and the Western Pacific, both travel-associated DENV infections and limited endemic outbreaks now occur world-wide.

Brief Summary Of The Invention

[0004] The invention is based, in part, on a role of the vascular endothelial growth factor (VEGF) pathway, the angiopoietin (Ang-1 and Ang-2 in particular) pathway, tight junction proteins and adherens junction proteins in the pathogenesis of viral hemorrhagic fever, for example dengue hemorrhagic fever / dengue shock syndrome (DHF/DSS). DENV impacts 3.6 billion people worldwide, but no antivirals or vaccines are available to treat and prevent DENV infections or DENV induced hemorrhagic fever. Thus in some aspects, presented herein are methods for treating hemorrhagic fever.

[0005] In some aspects, a method comprises modulating an activity or expression of an adherens junction protein. In some aspects, a method for treating hemorrhagic fever comprises administering to a subject, an agonist or antagonist of an adherens junction protein in an amount sufficient to treat a hemorrhagic fever. In some aspects an adherens junction protein is a cadherin, VE-cadherin, p120, gamma-catenin, or alpha-catenin.

[0006] In some aspects, a method for treating hemorrhagic fever comprises modulating an activity or expression of a tight junction protein in a subject. In some aspects a method for treating hemorrhagic fever comprises administering to a subject an agonist or antagonist of a tight junction protein in an amount sufficient to treat a hemorrhagic fever. In certain aspects, a tight junction protein is ZO-1 , ZO-2, ZO-3, an occluden, a claudin, or JAM-1 .

[0007] In some aspects, a method for treating hemorrhagic fever comprises modulating a VEGF pathway in a subject. In some aspects a method for treating hemorrhagic fever comprises administering to a subject an agonist or antagonist of a VEGF pathway in an amount sufficient to treat a hemorrhagic fever. In certain embodiments a method for treating hemorrhagic fever in a subject, comprising administering to the subject an effective amount of a VEGF antagonist sufficient to treat the hemorrhagic fever. In certain embodiments a VEGF antagonist comprises one of apatinib, bevacizumab, pazopanib, sunitinib, sorafenib, axitinib, vandetanib, cabozantinib, ramucirumab, ponatinib, regorafenib or ziv-aflibercept, ZM 323881 HCI.

[0008] In some aspects, a method for treating hemorrhagic fever in a subject comprises modulating an Ang-1/Ang-2 pathway in a subject. In some aspects the method comprises administering to a subject an agonist or antagonist of the Ang-1/Ang-2 pathway in an amount sufficient to treat a hemorrhagic fever. In certain embodiments an Ang-1 antagonist comprises ml_4-3. In certain embodiments an Ang-2 antagonist comprises L1 -7(N) or Angy-2-1 .

[0009] In some embodiments a method for treating hemorrhagic fever in a subject comprises administering an agonist or antagonist of a vascular endothelial protein tyrosine phosphatase (VE-PTP) in an amount sufficient to treat the hemorrhagic fever.

[0010] In certain embodiments a method for treating hemorrhagic fever in a subject comprises administration of an effective amount of a Tie-2 agonist or antagonist. In some embodiments a method for treating hemorrhagic fever in a subject comprises administering to the subject an effective amount of a Tie-2 agonist or antagonist sufficient to treat a hemorrhagic fever. In certain embodiments a Tie-2 antagonist comprises one of SB-203580, 4-(6-Methoxy-2-naphthyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1 H-imidazole; Disodium succinate; 5-[4-[[[2-[[(1 S)-1 -Cyclohexylethyl]amino]-2-oxoethyl][(4-methylphenoxy)carbonyl]amino]methyl]phenyl]-3-pyridinecarboxylic acid, MGCD-265, 4-(4-pyrrolidinylphenyl)-1 ,3-thiazole-2-ylamine; or 2-Amino-4-(4-pyrrolidin-1 -ylphenyl)-1 ,3-thiazole.

[001 1 ] In certain embodiments a method for treating hemorrhagic fever in a subject, comprising administering to the subject an effective amount of a TNFa antagonist sufficient to treat the hemorrhagic fever. In certain embodiments, a TNFa antagonist comprises one of Etanercept, Infliximab, Adalimumab or Golimumab.

[0012] In certain aspects a method for treating hemorrhagic fever in a subject comprises modulating two or more of a VEGF pathway, an Ang-1/Ang-2 pathway and a TNF pathway in combination. In some embodiments a method for treating hemorrhagic fever in a subject, comprises administering to a subject a TNFa agonist or antagonist, and a Tie-2 agonist or antagonist in an amount sufficient to treat a hemorrhagic fever. In some embodiments a method for treating hemorrhagic fever in a subject comprises administering a VEGF agonist or antagonist, and a Tie-2 agonist or antagonist in an amount sufficient to treat the hemorrhagic fever. In some embodiments a method for treating hemorrhagic fever in a subject comprises administering a TNFa agonist or antagonist, and a VEGF agonist or antagonist in amounts sufficient to treat a hemorrhagic fever. In some embodiments a method for treating hemorrhagic fever in a subject comprises administering to a subject a TNFa agonist or antagonist, and an Ang-1 agonist or antagonist in an amount sufficient treat the hemorrhagic fever. In some embodiments a method for treating hemorrhagic fever in a subject comprises administering to the subject a TNFa agonist or antagonist, and an Ang-2 agonist or antagonist in an amount sufficient to treat the hemorrhagic fever. In some aspects a method for treating hemorrhagic fever in subject comprises administering to the subject a VEGF agonist or antagonist, and an Ang-1 agonist or antagonist in amounts sufficient treat the hemorrhagic fever. In certain embodiments a method for treating hemorrhagic fever in a subject comprises administering to the subject, a VEGF agonist or antagonist, and an Ang-2 agonist or antagonist in amounts sufficient to treat the hemorrhagic fever.

[0013] In some embodiments, a hemorrhagic fever comprises a viral hemorrhagic fever caused by one or more of Arenaviridae (Lassa virus, Lujo virus, Junin virus, Machupo virus, Sabia virus or Guanarito virus), Bunyaviridae (Hantavirus, Nairovirus, Garissa virus, llesha virus, Orthobunyavirus or Phlebovirus), Filoviradae (Ebola virus, Marburg virus), Flaviviridae (Dengue virus, Yellow fever virus, Omsk hemorrhagic fever virus or Kyasanur Forest disease virus) or Rhabdoviridae. In some embodiments a hemorrhagic fever comprises Lassa fever, South American hemorrhagic fevers including Argentine hemorrhagic fever, Bolivian hemorrhagic fever, Venezuelan hemorrhagic fever or Brazilian hemorrhagic fever, Whitwater Arroyo virus fever, Flexal virus fever, Ebola hemorrhagic fever, Marburg hemorrhagic fever, Crimean-Congo hemorrhagic fever, Rift Valley fever, hemorrhagic fevers with renal syndrome including Hantaan virus hemorrhagic fever, Seoul virus hemorrhagic fever, Dobrava virus hemorrhagic fever, Puumala virus hemorrhagic fever, hantavirus pulmonary syndrome-associated hemorrhagic fevers, including Bayou virus hemorrhagic fever, Black Creek Canal virus hemorrhagic fever, New York virus hemorrhagic fever, Sin Nombre virus hemorrhagic fever, Andes virus hemorrhagic fever, Oran virus hemorrhagic fever, Juquitiba virus hemorrhagic fever, Laguna Negra virus hemorrhagic fever, Lechiguanas virus hemorrhagic fever, dengue hemorrhagic fever, dengue shock syndrome, Kyasanur Forest disease, Omsk hemorrhagic fever or yellow fever. In certain embodiments a hemorrhagic fever comprises Dengue fever. In some embodiments the hemorrhagic fever is caused by antibody dependent enhancement of Dengue fever. In some aspects the treatment reduces, decreases, inhibits, delays, eliminates or prevents the probability, severity, frequency, or duration of one or more symptoms associated with or caused by the hemorrhagic fever. In certain embodiments the symptoms comprise one or more of pain, disseminated

intravascular coagulation, bone marrow dysfunction, bleeding, headache, muscle or joint pain, nausea, vomiting, rash, vascular leakage, plasma leakage, fluid accumulation, respiratory distress, organ impairment or damage, fatigue or restlessness. In certain

embodiments an agonists and/or antagonists is combined into a single molecule. In some embodiments, the single molecule comprises a bi-specific or tri-specific antibody or aptamer.

[0014] In some aspects a method for treating hemorrhagic fever in a subject comprises modulating the inflammatory response in a subject in combination with reducing, decreasing, inhibiting, delaying, eliminating or preventing vascular leakage in the subject.

[0015] In certain embodiments a method for treating hemorrhagic fever in a subject comprising modulating the inflammatory response in the subject in combination with modulating angiogenesis in the subject.

[0016] In certain embodiments a method for treating hemorrhagic fever in a subject comprises treating antibody-dependent-enhancement of infection in the subject. In certain embodiments a subject has vascular leakage resulting from antibody-dependent-enhancement of infection.

[0017] In certain embodiments a subject has cytokine storm. In certain embodiments a subject a subject has secondary viral infection.

[0018] In certain embodiments, presented herein is a composition comprising one or more of a TNFa agonist or antagonist, an Ang-1 agonist or antagonist, an Ang-2 agonist or antagonist, a VEGF agonist or antagonist and a Tie-2 agonist or antagonist. In certain embodiments a composition comprises a TNFa agonist or antagonist and an Ang-1 agonist or antagonist. In certain embodiments a composition comprises a TNFa agonist or antagonist and an Ang-2 agonist or antagonist. In certain embodiments a composition further comprises a VEGF agonist or antagonist. In certain embodiments a composition comprises a VEGF agonist or antagonist and an Ang-1 agonist or antagonist. In certain embodiments a composition comprises a VEGF agonist or antagonist and an Ang-2 agonist or antagonist. In certain embodiments a composition comprises a Tie-2 agonist or antagonist. In certain embodiments a composition comprises a TNFa agonist or antagonist and a Tie-2 agonist or antagonist. In certain embodiments a composition comprises a VEGF agonist or antagonist and a Tie-2 agonist or antagonist. In certain embodiments a composition comprises a VEGF agonist or antagonist and a TNFa agonist or antagonist.

[0019] In certain embodiments a composition herein is a pharmaceutical composition.

[0020] In certain aspects, presented herein is a method of treating, preventing, or slowing one or more adverse symptoms and/or complications associated with a hemorrhagic fever pathology comprising administering to a subject having hemorrhagic fever, or at risk of having hemorrhagic fever, one or more Tie-2 antagonists in an amount sufficient to treat, prevent or slow the one or more adverse symptoms and/or complications. In some embodiments a hemorrhagic fever is caused by a virus of the family Flaviviridae. In some embodiments a hemorrhagic fever is caused by a virus of the genus Flavivirus. In some embodiments a hemorrhagic fever is caused by DENV.

[0021 ] In certain embodiments of the present invention an agonist or antagonist is an antibody, a small molecule, a protein, a peptide, an antisense nucleic acid or an aptamer, including an antibody-small molecule conjugate, a bispecific antibody or bispecific molecule.

Brief Description Of The Drawings

[0022] Figure 1. Schematic diagram of the VEGF and Ang-1/Ang-2 signaling pathways in endothelial cells. VEGF binding to the VEGF receptor activates SRC, which in turn causes the phosphorylation and internalization of VE-cadherin after dissociation from vascular endothelial protein tyrosine phosphatase (VE-PTP), ultimately resulting in increased vascular permeability and cell-cell junction destabilization. Ang-1 acts as a Tie-2 receptor agonist and promotes endothelial survival via PI3K/AKT signaling while inhibiting N F-KB mediated inflammation and SRC-dependent vascular leakage. Ang-2 functions as an Ang-1 antagonist and mediates increases in vascular permeability.

[0023] Figure 2. Elevated levels of VEGF and Ang-2 in DENV-infected mice. AG129 mice were infected with 2 x 104 PFU of DENV2 strain, S221 i.v., in the presence of 5 μg of anti-DENV monoclonal antibody 2H2. Levels of VEGF (panel A) and Ang-2 (panel B) in serum collected from na'ive and infected mice at 2, 3, and 4 days following infection were detected by ELISA. *p < 0.05, **p < 0.01 . ***p < 0.001 . Each symbol represents an individual mouse.

[0024] Figure 3. Absence of ZO-1 expression in DENV-infected mice. AG129 mice were infected with DENV via the ADE mode as described in Fig. 2. The small intestine from na'ive control and DENV-infected mice were harvested on day 3 p.i. and processed for staining with anti-ZO-1 antibody (red). Phalloidin was used to stain F-actin (green), and nuclei were stained with Hoechst. A single blood vessel from the dotted rectangle in the Merged panel was magnified from the original multi-panel stitch mega image using the ZEN software. Isosurface rendered Images in the last two panels on the right are 3D

reconstructions of the confocal z-stacks that were created using Imaris. Scale bars are 100 μιη in the Merged panels and 15 μιη in the 3D panels.

[0025] Figure 4. Sunitinib treatment can protect mice from lethal DENV infection.

AG129 mice were infected with DENV2 via the ADE route as described in Fig. 2. One group was administered 1200 μg sunitinib intraperitoneally (i.p.) once daily on days 1 and 2 (left panel), while two other groups received the same amount of sunitinib once on either day 2 or 3 after infection (right panel). Control group was injected with the drug carrier (mixture of kolliphor, ethanol, DMSO, and water) once daily on days 1 and 2 after infection. Survival was assessed. **p < 0.05; **p < 0.01 . The number of mice (n) is indicated for each group

[0026] Figure 5A. Sunitinib treatment can protect mice from DENV-induced vascular leakage. AG129 mice were infected with DENV as described in Fig. 2. Infected mice were treated with 1200 μg sunitinib or vehicle only (i.p.; once per day) on 1 and 2 days after infection. Evans Blue was injected on day 3.5 p.i., followed by harvesting of the small intestine. Evans Blue in the small intestine was extracted with formamide and quantified spectrophotometrically. *p < 0.05.

[0027] Figure 5B. Sunitinib treatment does not impact virus levels in the liver of DENV-infected mice. AG129 mice were infected with DENV as described in Fig. 2. One group of the infected mice were treated with 1200 μg sunitinib i.p. once daily on 1 and 2 days after infection. The other group of infected mice was similarly injected with the drug carrier. Viral RNA levels in the liver on day 3 p.i. were quantitated by qRT-PCR

[0028] Figure 6. Treatment with a Tie-2 kinase inhibitor can protect the host against DENV. AG129 mice were infected with DENV as described in Fig. 2. Mice were treated with 1200, 900, or 600 μg Tie-2 inhibitor (Selleckchem) i.p. once daily on 1 , 2, and 3 days after infection. Control group was similarly injected with the drug carrier (mixture of kolliphor, ethanol, DMSO, and water). Survival was monitored. *p < 0.05

[0029] Figure 7. Elevated levels of Ang-2 in mice with lethal DENV challenge.

AG129 mice were infected with DENV as described in Fig. 2. Mice were administered 30 g or 100 g of anti-TNF monoclonal antibody i.p. once on day 2 after infection. Both treatment groups survive the DENV challenge, whereas all untreated mice succumb to infection by day 5 (data not shown). Mice were bled retrorbitally on days 2 and 3 after infection to collect serum samples. Ang-2 in the sera was detected by ELISA. *p < 0.05, **p < 0.01 , ***p < 0.001

[0030] Figure 8. The LysM-Cre+lfnar1f f mouse model of DENV infection. LysM-Cre+lfnar1f/f (lack type I I FN receptor in macrophages and neutrophils) were infected with 106 PFU DENV2 strain, S221 i.v., alone (primary infection) or in the presence of 10 μΙ of

DENV-immune mouse serum (ADE infection). Immune serum was administered via i.p. injection one hour before viral infection. Survival was assessed, n = 5 mice per group.

[0031 ] Figure 9. A Schematic of the experimental design for testing the effects of the VEGF pathway inhibitors on DENV disease pathogenesis.

[0032] Figure 10. A Schematic of the experimental design for testing the effects of the Ang-1/Ang-2 pathway modulators on DENV disease pathogenesis in mice and cell cultures.

[0033] Figure 11. A Schematic of the experimental design for testing the efficacy of anti-TNF, in combination with the VEGF and/or Ang1-Ang-2 pathway modulator.

[0034] Figure 12. Dual treatment of DENV-infected AG 129 mice with anti-TNF antibody and sunitinib. AG129 mice were infected with DENV as described in Fig. 2. Top panel: To define minimal therapeutic and subtherapeutic doses of anti-TNF treatment against DENV, mice were treated with 20, 10, or 5 μg of neutralizing anti-TNF (clone XT3.1 1 , BioXcell) i.p. once on day 2 after infection. Control group was similarly injected with isotype control antibody (anti-horseradish peroxidase rat lgG1 ; clone HRPN from BioXcell). Bottom panel: To explore whether combination therapy targeting the inflammatory and

angiogenesis pathways may be feasible against DENV, mice were treated once on day 2 p.i. with subtherapeutic doses of anti-TNF antibody (5 μg) and sunitinib (600 μg). Survival was assessed. *p < 0.05, ***p < 0.001 . n = 15 mice/group.

Detailed Description Of The Invention

[0035] In some embodiments described herein is a broad-spectrum treatment that is active against all DENV serotypes and multiple genotypes, and avoids the emergence of drug resistant DENV strains by targeting host pathways instead of the virus.

[0036] The invention is based, at least in part, on a role for the pathways of angiogenic factors VEGF pathway, the angiopoietin (Ang-1 and Ang-2 in particular) pathway, tight junction proteins and adherens junction proteins in DHF/DSS. Angiopoietins are part of a family of vascular growth factors. In some aspects, angiopoietins are involved with controlling microvascular permeability, vasodilation, and vasoconstriction by signaling smooth muscle cells surrounding vessels. Angiopoietins comprise ANGPT1 (Ang-1 ), ANGPT2 (Ang-2), ANGPT4, and in some aspects, related proteins such as ANGPTL1 , ANGPTL2, ANGPTL3, ANGPTL4, ANGPTL5, ANGPTL6, and ANGPTL7. The inventors discovered a role for VEGF, Ang-1 and Ang-2 in DHF/DSS using small molecule inhibitors

and a model of ADE-mediated dengue disease in AG129 mice lacking type I and II IFN receptors.

[0037] The present inventors have identified regulators of endothelial integrity that can be targeted for development of a novel host-based therapeutic approach against hemorrhagic fever including from dengue virus infection. The present inventors have found that DENV-infected mice have elevated levels of vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) relative to uninfected mice, and treatment of infected mice with sunitinib, a small molecule inhibitor of the VEGF receptor (VEGFR), or a small molecule inhibitor of Tie-2, a tyrosine kinase receptor for Ang-1 and 2, can protect against the lethal DHF/DSS-like disease. Without being limited to any particular theory, targeting the VEGF and/or Ang-1 /Ang-2 pathway may improve vascular integrity in DENV-infected host.

[0038] The present inventors have demonstrated that TNFa blockade prevents lethal DHF/DSS-like disease in mice, and that treatment of DENV-infected mice with neutralizing anti-TNFa antibodies results in decreased Ang-2 levels. Thus there are presently provided methods comprising blockade of both angiogenic and inflammatory pathways to provide therapeutic activity against DENV. Therapeutics that block vascular leakage and permit DHF/DSS patients to recover without complex fluid replacement are urgently needed. In certain embodiments, the present invention provides the use of inexpensive small molecules (some of which have already been tested in humans) that modulated the angiogenic and/or inflammatory pathways as therapeutics for treatment of DHF/DSS.

[0039] In certain embodiments, the present methods, by focusing on a host-targeted approach, circumvent the challenges associated with developing virus-targeted therapeutics and vaccines that must protect against all four DENV serotypes and multiple genotypes within each serotype of an RNA virus, including drug resistance and partial immune responses.

[0040] The present inventors have found that tight junction proteins or adherens junction proteins can be targeted for treatment of hemorrhagic fever and that modulating the expression or activity of tight junction proteins or adherens junction proteins may protect against hemorrhagic fever. Non-limiting examples of tight junction proteins include Zo-1 (TJP1 ), Zo-2 (TJP2), Zo-3 (TJP3), an occluden, occludin (OCLN), Junctional Adhesion Molecules (JAM-A, JAM-B and JAM-C), Jam1 (F1 1 R), a claudin (e.g., CLDN1 -CLDN25), Coxsackie Virus And Adenovirus Receptor (CAR), Par-3, Par-6, MAGI, MUPP1 , and PATJ. Non-limiting examples of adherens junction proteins include a cadherin (e.g., Cadherin 1 (E-cadherin), Cadherin 2 (N-cadherin)), Cadherin 4 (R-cadherin), Cadherin 5 (VE-cadherin),

Cadherin 13 (T-cadherin), Cadherin 3 (P-cadherin), Cadherin 1 1 (OB-cadherin), Cadherin 15 (M-cadherin), Cadherin 12 (N-cadherin 2), Cadherin 10 (T2-cadherin), Cadherin 16 (KSP-cadherin), Cadherin 18 Type 2, Cadherin 8 Type 2, Cadherin 9 Type 2, Cadherin 7 Type 2, Cadherin 19 Type 2, Cadherin 20 Type 2, Cadherin 22 Type 2, p120 (Catenin (Cadherin-Associated Protein, Delta 1 ), gamma-catenin (Catenin (Cadherin-Associated Protein), Beta 1 ), and alpha-catenin (Catenin (Cadherin-Associated Protein), Alpha 1 ).

[0041 ] In some embodiments a composition comprises a VE-PTP agonist or antagonist. In some embodiments a method comprises administering a VE-PTP agonist or antagonist to a subject. In some embodiments, the present invention provides modulating expression or activity of VE-PTP to treat hemorrhagic fever including methods for treating hemorrhagic fever by administering an agonist or antagonist of VE-PTP in an amount sufficient to treat the hemorrhagic fever. A non-limiting examples of a VE-PTP antagonist include AKB-9778.

[0042] In some embodiments a composition comprises a TNF antagonist. In some embodiments a method comprises administering a TNF antagonist (e.g., a TNFa antagonist) to a subject. In certain embodiments, a TNF antagonist is a drug (e.g., a pharmaceutical drug, compound, molecule, reagent or protein) that inhibits or suppresses a physiologic response to tumor necrosis factor (TNF, TNFa). Non-limiting examples of TNF antagonist include etanercept, infliximab, adalimumab, golimumab, certolizumab pegol, anti-TNFa antibodies (e.g., TNFa neutralizing antibodies), certain xanthine derivatives, (e.g.

pentoxifylline), bupropion, derivatives thereof or combinations thereof.

[0043] In some embodiments a composition comprises a TNF agonist. In some embodiments a method comprises administering a TNF agonist to a subject. In certain embodiments, a TNF agonist is a drug (e.g., a pharmaceutical drug, compound, molecule, reagent or protein) that stimulates a pro-inflammatory response by stimulating signaling through a TNF receptor. Non-limiting examples of a TNF agonist includes TNFa, LTa, homotrimers thereof, heterotrimers thereof, agonist antibodies that bind to and signal through TNFR80 or TNFR60, derivatives thereof, binding fragments thereof, and

combinations thereof.

[0044] In some embodiments a composition comprises a Tie-2 agonist. In some embodiments a method comprises administering a Tie-2 agonist to a subject. In certain embodiments, a Tie-2 agonist is a drug (e.g., a pharmaceutical drug, compound, molecule, reagent or protein) that binds a Tie-2 receptor (e.g., as a ligand) that stimulates tyrosine phosphorylation of Tie-2. Non-limiting examples of a Tie-2 agonist include Ang-1 ,

Vasculotide, agonist antibodies, derivatives thereof or combinations thereof. In some

embodiments a Tie-2 agonist comprises Ang-1 . In some embodiments a Tie-2 agonist comprises Ang-2 (e.g., at high doses with corresponding low levels of Ang-1 ).

[0045] In some embodiments a composition comprises an Ang-2 antagonist. In some embodiments a method comprises administering an Ang-2 antagonist to a subject. In certain embodiments, a Tie-2 agonist comprises an Ang-2 antagonist. In certain

embodiments, an Ang-2 antagonist can bind to a Tie-2 ligand (e.g., a Tie-2 antagonist, e.g., Ang-2) and blocks binding of the ligand to a membrane bound Tie-2 receptor. In some embodiments an Ang-2 antagonist comprises an Ang-2 binding protein (e.g., a soluble receptor, fc-fusion protein or antibody). In certain embodiments, an Ang-2 antagonist comprises the soluble Fc fusion protein L1 -7(N) (Oliner J, Min H, Leal J, et al. (2004) Cancer Cell. 6:507-16). In certain embodiments, an Ang-2 antagonist comprises Angy-2-1

(Adipogen) or Angy-1 -4 (Adipogen).

[0046] In some embodiments a composition comprises a Tie-2 antagonist. In some embodiments a method comprises administering a Tie-2 antagonist to a subject. In certain embodiments, a Tie-2 antagonist is a drug (e.g., a pharmaceutical drug, compound, molecule, reagent or protein (e.g., ligand or antibody)) that inhibits tyrosine phosphorylation of Tie-2 by a Tie-2 receptor agonist (e.g., Ang-1 ). In some embodiments a Tie-2 antagonist is a ligand or antibody that binds a Tie-2 receptor (e.g., binds an extracellular portion of Tie-2). In certain embodiments, a Tie-2 antagonist is a drug (e.g., a pharmaceutical drug, compound, molecule, reagent or protein) that inhibits or blocks Ang-1 binding to Tie-2. In some embodiments, a Tie-2 antagonist does not inhibit or compete with binding of Ang-1 to Tie-2. Non-limiting examples of a Tie-2 antagonist that binds to a Tie-2 receptor include Ang-2, L1 -7[N], humanized or chimeric derivatives of a Tie-2 blocking antibody, binding fragments of a Tie-2 blocking antibody, derivatives thereof or combinations thereof. In some embodiments a Tie-2 antagonist comprises Ang-2, a derivative or binding fragment thereof. In some embodiments a Tie-2 antagonist comprises a small compound Tie-2 inhibitor. In certain embodiments, a Tie-2 antagonist is a drug (e.g., a pharmaceutical drug, small compound) that inhibits or blocks tyrosine kinase activity of a Tie-2 receptor stimulated by a Tie-2 receptor agonist (e.g., Ang-1 ). Any suitable tyrosine kinase inhibitor that inhibits Tie-2 induced tyrosine kinase activity can be used for a composition or method herein. Non-limiting examples of a small compound Tie-2 antagonist include SB-203580, S1577

(Selleckchem catalog S1577, CAS 948557-43-5), (Santa Cruz Biotechnology catalog No. CAS 1020412-97-8), 4-(6-Methoxy-2-naphthyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1 H-imidazole; Disodium succinate; 5-[4-[[[2-[[(1 S)-1 -Cyclohexylethyl]amino]-2-oxoethyl][(4-methylphenoxy)carbonyl]amino]methyl]phenyl]-3-pyridinecarboxylic acid, MGCD-265, 4-(4-

pyrrolidinylphenyl)-1 ,3-thiazole-2-ylamine; 2-Amino-4-(4-pyrrolidin-1 -ylphenyl)-1 ,3-thiazole, derivatives thereof and combinations thereof.

[0047] In some embodiments a composition comprises an Ang-1 antagonist. In some embodiments a method comprises administering an Ang-1 antagonist to a subject. In certain embodiments, a Tie-2 antagonist comprises an Ang-1 antagonist. In some embodiments an Ang-1 antagonist comprises an Ang-1 binding protein (e.g., a soluble receptor, fc-fusion protein or antibody). In certain embodiments, an Ang-1 antagonist can bind to Ang-1 and blocks binding of Ang-1 to a membrane bound Tie-2 receptor. In certain embodiments, an Ang-1 antagonist comprises ml_4-3, an Ang-1 neutralizing peptibody (Oliner J, Min H, Leal J, et al. (2004) Cancer Cell. 6:507-16).

[0048] In some embodiments a composition comprises a VEGF agonist. In some embodiments a method comprises administering a VEGF agonist to a subject. In certain embodiments, a VEGF agonist stimulates signaling through a VEGF receptor (e.g., VEGFR-1 , VEGFR-2 and/or VEGFR-3). In some embodiments, a VEGF agonist binds to a VEGF receptor and induces vasculogenesis and/or angiogenesis (e.g., in vascular endothelium). Non-limiting examples of a VEGF agonist include natural and modified functional variants of VEGF ligands (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, and VEGF-E), agonistic antibodies that bind to and signal through a VEGF receptor, derivatives thereof and combinations thereof.

[0049] In some embodiments a composition comprises a VEGF antagonist. In some embodiments a method comprises administering a VEGF antagonist to a subject. In certain embodiments, a VEGF antagonist inhibits and/or blocks signaling through a VEGF receptor (e.g., VEGFR-1 , VEGFR-2 and/or VEGFR-3). In some embodiments, a VEGF antagonist inhibits or blocks binding of a VEGF ligand (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, and VEGF-E) to a VEGF receptor. In some embodiments, a VEGF antagonist binds to a VEGF receptor. In some embodiments, a VEGF antagonist inhibits and/or blocks vasculogenesis and/or angiogenesis (e.g., in vascular endothelium) induced by a VEGF ligand. Non-limiting examples of a VEGF antagonist include soluble VEGF receptors and modified variants thereof that bind a VEGF ligand, antagonistic antibodies that bind to and/or block signaling through a VEGF receptor, antagonistic antibodies that bind to a VEGF ligand and/or block binding of a VEGF ligand to a VEGF receptor, derivatives thereof and combinations thereof. Additional non-limiting examples of a VEGF antagonist include apatinib, bevacizumab, pazopanib, sunitinib, sorafenib, axitinib, vandetanib, cabozantinib, ramucirumab, ponatinib, regorafenib or ziv-aflibercept, ZM 323881 HCI, lenvatinib, motesanib, pazopanib, nintedanib (BIBF 1 120), afatinib, derivatives thereof and combinations thereof.

[0050] An antibody, as referred to herein, can be a polyclonal or monoclonal antibody, or binding fragment thereof. Antibodies sometimes are IgG, IgM, IgA, IgE, or an isotype thereof (e.g., lgG1 , lgG2a, lgG2b or lgG3), sometimes are polyclonal or monoclonal, and sometimes are chimeric, humanized or bispecific versions of an antibody. In some embodiments an antibody or portion thereof, comprises a chimeric antibody, Fab, Fab', F(ab')2, Fv fragment, scFv, diabody, aptamer, synbody, camelid, the like and/or a combination thereof.

[0051 ] Methods of the invention include treatment methods, which result in any therapeutic or beneficial effect. In various methods embodiments, "treatment" or "treating" refers to decreasing, reducing inhibiting, suppressing, preventing, controlling or limiting one or more adverse (e.g., physical) symptoms, disorders, illnesses, diseases or complications caused by or associated with hemorrhagic fever pathology (e.g., pain, disseminated intravascular coagulation, bone marrow dysfunction, bleeding, headache, muscle or joint pain, nausea, vomiting, rash, vascular leakage, plasma leakage, fluid accumulation, respiratory distress, organ impairment or damage, fatigue or restlessness, fever, rash, headache, pain behind the eyes, muscle or joint pain, nausea, vomiting, loss of appetite). In additional various particular embodiments, treatment methods include reducing, decreasing, inhibiting, delaying or preventing onset, progression, frequency, duration, severity, probability or susceptibility of one or more adverse symptoms, disorders, illnesses, diseases or complications caused by or associated with hemorrhagic fever pathology (e.g., pain, disseminated intravascular coagulation, bone marrow dysfunction, bleeding, headache, muscle or joint pain, nausea, vomiting, rash, vascular leakage, plasma leakage, fluid accumulation, respiratory distress, organ impairment or damage, fatigue or restlessness, fever, rash, headache, pain behind the eyes, muscle or joint pain, nausea, vomiting, loss of appetite). In further various particular embodiments, treatment methods include improving, accelerating, facilitating, enhancing, augmenting, or hastening recovery of a subject from a hemorrhagic fever pathogenesis, or one or more adverse symptoms, disorders, illnesses, diseases or complications caused by or associated with hemorrhagic fever pathology (e.g., pain, disseminated intravascular coagulation, bone marrow dysfunction, bleeding, headache, muscle or joint pain, nausea, vomiting, rash, vascular leakage, plasma leakage, fluid accumulation, respiratory distress, organ impairment or damage, fatigue or restlessness, fever, rash, headache, pain behind the eyes, muscle or joint pain, nausea, vomiting, loss of appetite). In yet additional various embodiments, treatment methods include stabilizing pathogenesis, or an adverse symptom, disorder, illness, disease or complication caused by or associated with hemorrhagic fever pathology.

[0052] The term "hemorrhagic fever" as used herein refers to hemorrhagic pathology which is caused by infection of a subject by a virus of the family Arenaviridae, Filoviridae, Bunyaviridae, Flaviviridae, or Rhabdoviridae. In certain embodiments a hemorrhagic fever is a viral hemorrhagic fever. Non-limiting examples of a virus that can cause hemorrhagic fever include Arenaviridae (Lassa virus, Lujo virus, Junin virus, Machupo virus, Sabia virus or Guanarito virus), Bunyaviridae (Hantavirus, Nairovirus, Garissa virus, llesha virus, Orthobunyavirus or Phlebovirus), Filoviradae (Ebola virus, Marburg virus), Flaviviridae (Dengue virus, Yellow fever virus, Omsk hemorrhagic fever virus or Kyasanur Forest disease virus) and Rhabdoviridae. In some embodiments, a virus that causes hemorrhagic fever is a virus of the the family Flaviviridae; genus Flavivirus. In some embodiments, a virus that causes hemorrhagic fever is a DENV.

[0053] In some embodiments presented herein is a method of preventing or treating hemorrhagic fever caused by a virus of the family Arenaviridae, Filoviridae, Bunyaviridae, Flaviviridae, and/or Rhabdoviridae. In some embodiments presented herein is a method of preventing or treating hemorrhagic fever caused by a virus of the genus Flavivirus. In some embodiments presented herein is a method of preventing or treating hemorrhagic fever caused by DENV.

[0054] Non-limiting examples of hemorrhagic fever include Lassa fever, South American hemorrhagic fevers including Argentine hemorrhagic fever, Bolivian hemorrhagic fever, Venezuelan hemorrhagic fever or Brazilian hemorrhagic fever, Whitwater Arroyo virus fever, Flexal virus fever, Ebola hemorrhagic fever, Marburg hemorrhagic fever, Crimean-Congo hemorrhagic fever, Rift Valley fever, hemorrhagic fevers with renal syndrome including Hantaan virus hemorrhagic fever, Seoul virus hemorrhagic fever, Dobrava virus hemorrhagic fever, Puumala virus hemorrhagic fever, hantavirus pulmonary syndrome-associated hemorrhagic fevers, including Bayou virus hemorrhagic fever, Black Creek Canal virus hemorrhagic fever, New York virus hemorrhagic fever, Sin Nombre virus hemorrhagic fever, Andes virus hemorrhagic fever, Oran virus hemorrhagic fever, Juquitiba virus hemorrhagic fever, Laguna Negra virus hemorrhagic fever, Lechiguanas virus hemorrhagic fever, dengue hemorrhagic fever, dengue fever, dengue shock syndrome, Kyasanur Forest disease, Omsk hemorrhagic fever or yellow fever.

[0055] A therapeutic or beneficial effect of treatment is therefore any objective or subjective measurable or detectable improvement or benefit provided to a particular subject. A therapeutic or beneficial effect can, but need not be, complete ablation of all or any particular adverse symptom, disorder, illness, disease or complication caused by or associated with hemorrhagic fever pathology (e.g., pain, disseminated intravascular

coagulation, bone marrow dysfunction, bleeding, headache, muscle or joint pain, nausea, vomiting, rash, vascular leakage, plasma leakage, fluid accumulation, respiratory distress, organ impairment or damage, fatigue or restlessness, fever, rash, headache, pain behind the eyes, muscle or joint pain, nausea, vomiting, loss of appetite). Thus, treatment may be achieved when there is an incremental improvement or a partial reduction in an adverse symptom, disorder, illness, disease or complication caused by or associated with hemorrhagic fever pathology, or an inhibition, decrease, reduction, suppression, prevention, limit or control of worsening or progression of one or more adverse symptoms, disorders, illnesses, diseases or complications caused by or associated with hemorrhagic fever pathology, over a short or long duration (hours, days, weeks, months, etc.).

[0056] A therapeutic or beneficial effect also includes reducing or eliminating the need, dosage frequency or amount of a second active treatment such as another drug or other agent (e.g., anti-viral) used for treating a subject having or at risk of having a hemorrhagic fever pathology. For example, reducing an amount of an adjunct therapy, for example, a reduction or decrease of a treatment for a hemorrhagic fever.

[0057] Adverse symptoms and complications associated with hemorrhagic fever pathology include, for example, pain, disseminated intravascular coagulation, bone marrow dysfunction, bleeding, headache, muscle or joint pain, nausea, vomiting, rash, vascular leakage, plasma leakage, fluid accumulation, respiratory distress, organ impairment or damage, fatigue or restlessness, fever, rash, headache, pain behind the eyes, muscle or joint pain, nausea, vomiting, loss of appetite, etc., and the like. Other symptoms of hemorrhagic fever pathogenesis are known to one of skill in the art and treatment thereof in accordance with the invention is provided. Thus, the aforementioned symptoms and complications are treatable in accordance with the invention. In some embodiments a method comprises preventing and/or treating an adverse symptom and/or complication associated with hemorrhagic fever pathology. In some embodiments a method comprises reducing, decreasing, inhibiting, delaying, eliminating, and/or preventing the severity, frequency, and/or duration of one or more symptoms associated with, or caused by, a hemorrhagic fever.

[0058] In invention methods in which there is a desired outcome, such as a therapeutic or prophylactic method that provides a benefit from treatment, agonists or antagonists can be administered in a sufficient or effective amount. As used herein, a "sufficient amount" or "effective amount" or an "amount sufficient" or an "amount effective" refers to an amount that provides, in single (e.g., primary) or multiple (e.g., booster) doses, alone or in combination with one or more other compounds, treatments, therapeutic regimens or agents (e.g., a

drug), a long term or a short term detectable or measurable improvement in a given subject or any objective or subjective benefit to a given subject of any degree or for any time period or duration (e.g., for minutes, hours, days, months, years, or cured).

[0059] In some embodiments, an amount sufficient, or an amount effective, is provided in a single administration. In some embodiments, an amount sufficient, or an amount effective, is provided in multiple administrations. In some embodiments, an amount sufficient, or an amount effective, is achieved by agonists or antagonists alone, or in a composition or method that comprises a second active component. In addition, an amount sufficient or an amount effective need not be sufficient or effective if given in single or multiple doses without a second or additional administration or dosage, since additional doses, amounts or duration above and beyond such doses, or additional antigens, compounds, drugs, agents, treatment or therapeutic regimens may be included in order to provide a given subject with a detectable or measurable improvement or benefit to the subject.

[0060] An amount sufficient or an amount effective need not be therapeutically or prophylactically effective in each and every subject treated, nor a majority of subjects treated in a given group or population. An amount sufficient or an amount effective means sufficiency or effectiveness in a particular subject, not a group of subjects or the general population. As is typical for such methods, different subjects will exhibit varied responses to treatment.

[0061 ] The term "subject" refers to an animal, typically a mammalian animal (mammal), such as a nonhuman primate (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), a domestic animal (dogs and cats), a farm animal (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), experimental animal (mouse, rat, rabbit, guinea pig) and humans.

[0062] Any suitable mammal can be treated by a method described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). Subjects include animal disease models, for example, a mouse model, and other animal models of pathogen (e.g., Dengue Virus) infection known in the art. In some embodiments a mammal is a human. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. A mammal can be a pregnant female. In certain

embodiments a mammal can be an animal disease model, for example, animal models used for the study of viral infections (e.g., DENV infection).

[0063] In some embodiments a subject or mammal is "at risk" of a virus infection (e.g., an infection by a virus of the family Flaviviridae, an infection by a virus of the genus

Flavivirus, or infection by a DENV). A mammal that is at risk may have increased risk factors for a virus infection, non-limiting examples of which include immunocompromised individuals or immune deficient subjects (e.g., bone marrow transplant recipients, irradiated individuals, subjects having certain types of cancers, particularly those of the bone marrow and blood cells (e.g., leukemia, lymphoma, multiple myeloma), subjects with certain types of chronic infections (e.g., HIV, e.g., AIDS), subjects treated with immunosuppressive agents, subjects suffering from malnutrition and aging, subjects taking certain medications (e.g. disease-modifying anti-rheumatic drugs, immunosuppressive drugs, glucocorticoids) and subjects undergoing chemotherapy), the like or combinations thereof). In some

embodiments a subject at risk is, will be, or has been in a location or environment suspected of containing a virus. For example, a subject at risk can be a medical professional that is providing care to another who is suspected of being infected with, or known to be infected with a virus. In certain embodiments, a subject at risk is any subject that has been exposed to a virus.

[0064] In some embodiments a subject in need of a treatment or composition described herein is a subject at risk of a virus infection. In some embodiments a subject in need of a treatment or composition described herein is a subject infected with, or suspected of being infected with, a virus. In some embodiments a subject in need of a treatment or composition described herein is a subject experiencing one or more symptoms associated with a viral infection (e.g., a hemorrhagic pathology). Non-limiting examples of symptoms associated with a virus infection include fever (e.g., a body temperature greater than 38.6 °C or

101 .5 °F), hemorrhagic fever pathology, severe headache, muscle pain, weakness, diarrhea, vomiting, abdominal (stomach) pain, unexplained hemorrhage (bleeding or bruising), or combinations thereof. In certain embodiments a composition described herein is used to treat a symptom of a viral infection.

[0065] In some embodiments, subjects appropriate for treatment include those having or at risk of having hemorrhagic fever pathology. Target subjects therefore include subjects that have been exposed to or contacted with a viral hemorrhagic fever (e.g., a DENV), or that have an ongoing infection or have developed one or more adverse symptoms caused by or associated with hemorrhagic fever pathology, regardless of the type, timing or degree of onset, progression, severity, frequency, duration of the symptoms.

[0066] Treatment of an infection can be at any time during the hemorrhagic fever.

Agonists or antagonists can be administered as a combination (e.g., with a second active), or separately, concurrently or in sequence (sequentially) in accordance with the methods as a single or multiple dose e.g., one or more times hourly, daily, weekly, monthly or annually or between about 1 to 10 weeks, or for as long as appropriate, for example, to achieve a reduction in the onset, progression, severity, frequency, duration of one or more symptoms or complications associated with or caused by hemorrhagic fever pathology, or an adverse symptom, condition or complication associated with or caused by a hemorrhagic fever. Thus, a method can be practiced one or more times (e.g., 1 -10, 1 -5 or 1 -3 times) an hour, day, week, month, or year. The skilled artisan will know when it is appropriate to delay or discontinue administration. A non-limiting dosage schedule is 1 -7 times per week, for 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more weeks, and any numerical value or range or value within such ranges.

[0067] The exact formulation and route of administration for a composition for use according to the methods of the invention described herein can be chosen by a caregiver (e.g., a medical professional, a physician) in view of the patient's condition. See e.g., Fingl et al. 1975, in "The Pharmacological Basis of Therapeutics," Ch. 1 , p. 1 ; which is

incorporated herein by reference in its entirety. Any suitable route of administration can be used for administration of a compound described herein. Methods of the invention may be practiced by any mode of administration or delivery, or by any route, systemic, regional and local administration or delivery. Exemplary administration and delivery routes include intravenous (i.v.), intraperitoneal (i.p.), intrarterial, intramuscular, parenteral, subcutaneous, intra-pleural, topical, dermal, intradermal, transdermal, transmucosal, intra-cranial, intraspinal, rectal, oral (alimentary), mucosal, inhalation, respiration, intranasal, intubation, intrapulmonary, intrapulmonary instillation, buccal, sublingual, intravascular, intrathecal, intracavity, iontophoretic, intraocular, ophthalmic, optical, intraglandular, intraorgan, or intralymphatic. Other non-limiting examples of routes of administration include topical or local (e.g., transdermal^ or cutaneously, (e.g., on the skin or epidermus), in or on the eye, intranasally, transmucosally, in the ear, inside the ear (e.g., behind the ear drum)), enteral (e.g., delivered through the gastrointestinal tract, e.g., orally (e.g., as a tablet, capsule, granule, liquid, emulsification, lozenge, or combination thereof), sublingual, by gastric feeding tube, and the like), by parenteral administration (e.g., parenterally, e.g.,

intravenously, intra-arterially, intramuscularly, intraperitoneally, intradermal^,

subcutaneously, intracavity, intracranially, intraarticular, into a joint space, intracardiac (into the heart), intracavernous injection, intralesional (into a skin lesion), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intrauterine, intravaginal,

intravesical infusion, intravitreal), the like or combinations thereof.

[0068] In some embodiments a composition herein is provided to a subject. A composition that is provided to a subject can be provided to a subject for self-administration or to another (e.g., a caregiver, a medical professional) for administration to a subject. For example a composition described herein can be provided as an instruction written by a medical practitioner that authorizes a patient to be provided a composition or treatment described herein (e.g., a prescription). In another example, a composition can be provided to a subject wherein the subject self-administers a composition orally, intravenously or by way of an inhaler, for example.

[0069] A dose can be administered in an effective amount or an amount sufficient to treat, prevent or slow a virus infection or to treat, prevent or slow one or more adverse symptoms and/or complications. An exact dose can be determined by a caregiver or medical professional by methods known in the art (e.g., by analyzing data and/or the results of a clinical trial).

[0070] Doses can be based upon current existing protocols, empirically determined, using animal disease models or optionally in human clinical trials. Initial study doses can be based upon animal studies set forth herein, for a mouse, which weighs about 30 grams, and the amount of agonist or antagonist administered that is determined to be effective.

Exemplary non-limiting amounts (doses) are in a range of about 0.1 mg/kg to about 100 mg/kg, and any numerical value or range or value within such ranges. Greater or lesser amounts (doses) can be administered, for example, 0.01 -500 mg/kg, and any numerical value or range or value within such ranges. The dose can be adjusted according to the mass of a subject, and will generally be in a range from about 1 μg/kg-500 mg/kg, 1 -10 μg/kg, 10-25 μg/kg, 25-50 μg/kg, 50-100 μg/kg, 100-500 μg/kg, 500-1 ,000 μg/kg, 1 -5 mg/kg, 5-10 mg/kg, 10-20 mg/kg, 20-50 mg/kg, 50-100 mg/kg, 100-250 mg/kg, 250-500 mg/kg, or more, two, three, four, or more times per hour, day, week, month or annually. A typical range will be from about 0.3 mg/kg to about 50 mg/kg, 0-25 mg/kg, or 1 .0-10 mg/kg, or any numerical value or range or value within such ranges.

[0071 ] Doses can vary and depend upon whether the treatment is prophylactic or therapeutic, whether a subject has been previously exposed to, infected with our suffered from a hemorrhagic fever, the onset, progression, severity, frequency, duration probability of or susceptibility of the symptom, condition, pathology or complication, or vaccination or immunization to which treatment is directed, the clinical endpoint desired, previous or simultaneous treatments, the general health, age, gender, race or immunological

competency of the subject and other factors that will be appreciated by the skilled artisan. The skilled artisan will appreciate the factors that may influence the dosage and timing required to provide an amount sufficient for providing a therapeutic or prophylactic benefit.

[0072] Typically, for therapeutic treatment, compositions, agonists or antagonists disclosed herein will be administered as soon as practical, typically within 1 -2, 2-4, 4-12, 12-24 or 24-72 hours after a subject is suspected of having a hemorrhagic fever, or within 1 -2, 2-4, 4-12, 12-24 or 24-48 hours after onset or development of one or more adverse symptoms, conditions, pathologies, complications, etc., associated with or caused by a hemorrhagic fever pathology.

[0073] The dose amount, number, frequency or duration may be proportionally increased or reduced, as indicated by the status of the subject. For example, whether the subject has a pathogen infection, whether the subject has been exposed to, contacted or infected with pathogen or is merely at risk of pathogen contact, exposure or infection, whether the subject is a candidate for or will be vaccinated or immunized. The dose amount, number, frequency or duration may be proportionally increased or reduced, as indicated by any adverse side effects, complications or other risk factors of the treatment or therapy.

[0074] Agonists and antagonists can be incorporated into compositions, including pharmaceutical compositions, e.g., a pharmaceutically acceptable carrier or excipient. Such pharmaceutical compositions are useful for, among other things, administration to a subject in vivo or ex vivo.

[0075] As used herein the term "pharmaceutically acceptable" and "physiologically acceptable" mean a biologically acceptable formulation, gaseous, liquid or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery or contact. Such formulations include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration or in vivo contact or delivery.

Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powder, granules and crystals. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral and antifungal agents) can also be incorporated into the compositions.

[0076] Pharmaceutical compositions can be formulated to be compatible with a

particular route of administration. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes. Exemplary routes of administration for contact or in vivo delivery which a composition can optionally be formulated include inhalation, respiration, intranasal, intubation, intrapulmonary instillation, oral, buccal, intrapulmonary, intradermal, topical, dermal, parenteral, sublingual,

subcutaneous, intravascular, intrathecal, intraarticular, intracavity, transdermal,

iontophoretic, intraocular, ophthalmic, optical, intravenous (i.v.), intramuscular,

intraglandular, intraorgan, or intralymphatic.

[0077] Formulations suitable for parenteral administration comprise aqueous and nonaqueous solutions, suspensions or emulsions of the active compound, which preparations are typically sterile and can be isotonic with the blood of the intended recipient. Non-limiting illustrative examples include water, saline, dextrose, fructose, ethanol, animal, vegetable or synthetic oils.

[0078] Co-solvents may be added to an agonist or antagonist composition or formulation. Non-limiting examples of co-solvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethylene glycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters. Non-limiting examples of co-solvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethylene glycol, polypropylene glycol, glycol ether; glycerol;

polyoxyethylene alcohols and polyoxyethylene fatty acid esters.

[0079] Supplementary compounds (e.g., preservatives, antioxidants, antimicrobial agents including biocides and biostats such as antibacterial, antiviral and antifungal agents) can also be incorporated into the compositions. Pharmaceutical compositions may therefore include preservatives, anti-oxidants and antimicrobial agents.

[0080] Preservatives can be used to inhibit microbial growth or increase stability of ingredients thereby prolonging the shelf life of the pharmaceutical formulation. Suitable preservatives are known in the art and include, for example, EDTA, EGTA, benzalkonium chloride or benzoic acid or benzoates, such as sodium benzoate. Antioxidants include, for example, ascorbic acid, vitamin A, vitamin E, tocopherols, and similar vitamins or provitamins.

[0081 ] An antimicrobial agent or compound directly or indirectly inhibits, reduces, delays, halts, eliminates, arrests, suppresses or prevents contamination by or growth, infectivity, replication, proliferation, reproduction, of a pathogenic or non- pathogenic microbial organism. Classes of antimicrobials include antibacterial, antiviral, antifungal and antiparasitics. Antimicrobials include agents and compounds that kill or destroy (-cidal) or inhibit (-static) contamination by or growth, infectivity, replication, proliferation, reproduction of the microbial organism.

[0082] Exemplary anti-bacterials (antibiotics) include penicillins (e.g., penicillin G, ampicillin, methicillin, oxacillin, and amoxicillin), cephalosporins (e.g., cefadroxil, ceforanid, cefotaxime, and ceftriaxone), tetracyclines (e.g., doxycycline, chlortetracycline, minocycline, and tetracycline), aminoglycosides (e.g., amikacin, gentamycin, kanamycin, neomycin, streptomycin, netilmicin, paromomycin and tobramycin), macrolides (e.g., azithromycin, clarithromycin, and erythromycin), fluoroquinolones (e.g., ciprofloxacin, lomefloxacin, and norfloxacin), and other antibiotics including chloramphenicol, clindamycin, cycloserine, isoniazid, rifampin, vancomycin, aztreonam, clavulanic acid, imipenem, polymyxin, bacitracin, amphotericin and nystatin.

[0083] Particular non-limiting classes of anti-virals include reverse transcriptase inhibitors; protease inhibitors; thymidine kinase inhibitors; sugar or glycoprotein synthesis inhibitors; structural protein synthesis inhibitors; nucleoside analogues; and viral maturation inhibitors. Specific non-limiting examples of anti-virals include nevirapine, delavirdine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, zidovudine (AZT), stavudine (d4T), larnivudine (3TC), didanosine (DDI), zalcitabine (ddC), abacavir, acyclovir, penciclovir, ribavirin, valacyclovir, ganciclovir, 1 ,-D-ribofuranosyl-1 ,2,4-triazole-3

carboxamide, 9->2-hydroxy-ethoxy methylguanine, adamantanamine, 5-iodo-2'-deoxyuridine, trifluorothymidine, interferon and adenine arabinoside.

[0084] Pharmaceutical formulations and delivery systems appropriate for the

compositions and methods of the invention are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20th ed., Mack Publishing Co., Easton, PA;

Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, PA; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, NJ ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa. ; Ansel ad Soklosa, Pharmaceutical Calculations (2001 ) 1 1 th ed., Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al., Drug Delivery Systems (1980), R. L. Juliano, ed., Oxford, N.Y., pp. 253-315).

[0085] Unless otherwise defined, 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 methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0086] All applications, publications, patents and other references, GenBank citations and ATCC citations cited herein are incorporated by reference in their entirety. In case of conflict, the specification, including definitions, will control.

[0087] As used herein, the singular forms "a," "and," and "the" include plural referents unless the context clearly indicates otherwise.

[0088] As used herein, numerical values are often presented in a range format throughout this document. The use of a 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 use of a range expressly includes all possible subranges, all individual numerical values within that range, and all numerical values or numerical ranges include integers within such ranges and fractions of the values or the integers within ranges unless the context clearly indicates otherwise. This construction applies regardless of the breadth of the range and in all contexts throughout this patent document. Thus, to illustrate, reference to a range of 90-100% includes 91 -99%, 92-98%, 93-95%, 91 -98%, 91 -97%, 91 -96%, 91 -95%, 91 -94%, 91 -93%, and so forth. Reference to a range of 90-100%, includes 91 %, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91 .1 %, 91 .2%, 91 .3%, 91 .4%, 91 .5%, etc., 92.1 %, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. Reference to a range of 1 -5 fold therefore includes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, fold, etc., as well as 1 .1 , 1 .2, 1 .3, 1 .4, 1 .5, fold, etc., 2.1 , 2.2, 2.3, 2.4, 2.5, fold, etc., and so forth. Further, for example, reference to a series of ranges of 2-72 hours, 2-48 hours, 4-24 hours, 4-18 hours and 6-12 hours, includes ranges of 2-6 hours, 2, 12 hours, 2-18 hours, 2-24 hours, etc., and 4-27 hours, 4-48 hours, 4-6 hours, etc.

[0089] As also used herein a series of range formats are used throughout this document. The use of a series of ranges includes combinations of the upper and lower ranges to provide a range. Accordingly, a series of ranges include ranges which combine the values of the boundaries of different ranges within the series. This construction applies regardless of the breadth of the range and in all contexts throughout this patent document. Thus, for example, reference to a series of ranges such as 5-10, 10-20, 20-30, 30-40, 40-50, 50-75, 75-100, 100-150, and 150-171 , includes ranges such as 5-20, 5-30, 5-40, 5-50, 5-75, 5-100, 5-150, 5-171 , and 10-30, 10-40, 10-50, 10-75, 10-100, 10-150, 10-171 , and 20-40, 20-50, 20-75, 20-100, 20-150, 20-171 , and so forth.

[0090] The invention is generally disclosed herein using affirmative language to describe the numerous embodiments and aspects. The invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, or procedures. For example, in certain embodiments or aspects of the invention, materials and/or method steps are excluded. Thus, even though the invention is generally not expressed herein in terms of what the invention does not include aspects that are not expressly excluded in the invention are nevertheless disclosed herein.

[0091 ] A number of embodiments of the invention have been described. Nevertheless, one skilled in the art, without departing from the spirit and scope of the invention, can make various changes and modifications of the invention to adapt it to various usages and conditions. Accordingly, the following examples are intended to illustrate but not limit the scope of the invention claimed.

Examples

[0092] Example 1

[0093] Vascular leakage is a hallmark of DHF/DSS; however, the precise nature and pathogenic mechanisms of DENV-induced vascular leakage are poorly understood. The angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietins (Ang-1 and Ang-2 in particular) are important mediators of vascular integrity and remodelling (Fig. 1 ).

[0094] AG129 mice were passively administered 5 μg of DENV-specific mouse monoclonal antibody (clone 2H2; DENV1 -4 cross- reactive lgG2a) before infection with 2 x 104 PFU of the DENV2 strain S221 . Mice treated with 2H2 succumb early to S221 infection (day 4-6) and feature the hallmarks of DHF/DSS in humans (high viral load, elevated hematocrit, cytokine storm, low platelet count, increased vascular permeability, hemorrhagic manifestations, and shock-induced death). This mouse model of ADE-mediated DHF/DSS was more clinically relevant than mice inoculated with a high viral challenge dose in the absence of enhancing antibodies, as severe dengue disease was mostly observed in individuals with secondary DENV infection and infants born to DENV-immune mothers (i.e. patients with DENV-specific antibodies). To determine whether this mouse model can be used to explore the role of the VEGF and Ang-1 /Ang-2 pathways in response to DENV infection, levels of VEGF and Ang-2 were measured in the serum of mice on days 2, 3, and 4 after infection (Fig. 2). Both VEGF and Ang-2 levels were higher in DENV-infected mice relative to uninfected mice on day 2 post-infection (p.i.) and the levels varied widely in the infected mice on day 4, when some of the mice had already died and all were severely ill.

[0095] Example 2

[0096] To validate this mouse model for evaluating endothelial cell integrity during DENV infection, the expression of ZO-1 , a tight junction protein that has been studied in the context of human DENV infection, was examined. In particular, incubation of human umbilical vein endothelial cells (HUVECs) with sera from DHF/DSS patients resulted in downregulation of ZO-1 expression. Focus was on the small intestine, which is a major site of vascular leakage in our mouse model, as determined by the Evans blue assay. ZO-1 expression was detectable in the small intestine capillaries of na'rve but not DENV-infected mice (Fig. 3).

[0097] Example 3

[0098] To evaluate a role for the VEGF pathway in impacting manifestations of

DHF/DSS, AG129 mice were treated with 1200 μg of sunitinib (LC Laboratories), a small molecule inhibitor of at least eight protein tyrosine kinases including VEGF-R1/R2/R3, platelet-derived growth factors (PDGFRa and PDGFRp), stem cell factor receptor (kit), Flt-3, and colony stimulating factor-1 receptor (CSF-1 R), administered intraperitoneal^ once daily on days 1 and 2 or only on day 2 or day 3 after DENV infection. As expected, almost all of the infected mice injected with carrier only died by day 5 (Fig. 4). Days 1 and 2 treatment with sunitinib protected 80% of mice (Fig. 4, left panel), while day 2 only and day 3 only treatment decreased protection to 50% and 40% of mice, respectively (Fig. 4, right panel). These results demonstrated that sunitinib can protect a host against DENV, and that treatment with sunitinib can be delayed at least until day 2 after infection. Whether sunitinib treatment can impact a DENV-induced increase in vascular permeability was assessed. Sunitinib-treated mice with DENV infection exhibited lower levels of vascular leakage than vehicle-treated control animals in the small intestine, as determined by the Evans Blue extravasation assay (Fig. 5A), indicating that sunitinib treatment can prevent DENV-induced vascular leakage. Next it was assessed whether sunitinib treatment influenced DENV infection. The potentially therapeutic effect of sunitinib administration at late time points after infection suggested that the VEGF pathway does not influence DENV levels. In this mouse model, viral load in the liver on day 3 after infection with DENV correlated with survival. The inventors measured DENV RNA levels in sunitinib-treated vs. untreated mice, and observed that sunitinib treatment did not impact the liver viral titer on day 3 p.i. (Fig. 5B). Although sunitinib affects multiple kinases, these results suggested that the VEGF pathway likely regulates endothelial barrier function during DENV infection, and therefore is involved in the host response to DENV infection and that inhibiting this pathway prevents vascular leakage

and protect against DENV-induced disease.

[0099] Example 4

[00100] To investigate whether the Ang-1 /Ang-2 signaling pathway is also involved in the regulation of endothelial barrier function during DENV infection, AG129 mice were treated with a Tie-2 kinase inhibitor (Selleckchem catalog S1577, CAS 948557-43-5) once daily on 1 , 2, and 3 days after DENV infection. Almost all control mice injected with carrier only and all mice administered 600 μg of the Tie-2 inhibitor succumbed to DENV infection by day 5 (Fig. 6). In contrast, 44% and 40% of mice treated with 900 μg and 1200 μg of the Tie-2 inhibitor, respectively, survived the lethal DENV challenge, suggesting that Ang-1 /Ang-2 signaling contributed to DENV-induced vascular leakage. This result supports that modulation of the Ang-1 /Ang-2 pathway benefited the host by improving endothelial cell integrity and by preventing increases in vascular permeability. Taken together, the present data with sunitinib and Tie-2 inhibitor implicated a role for both the VEGF and Ang-1 /Ang-2 pathway in DENV pathogenesis, and that blockade or agonism of each pathway individually or together contributed to host protection against DENV.

[00101 ] Example s

[00102] To determine a potential relationship between the TNF and Ang-1/Ang-2 pathway, AG129 mice were treated with neutralizing anti-TNF monoclonal antibody once on day 2 after infection with DENV, followed by measurement of Ang-2 levels in the mouse sera 30 minutes after anti-TNF injection on day 2 and on days 3 and 4 after infection. DENV-infected mice that were treated with anti-TNF had lower levels of Ang-2 than untreated mice with DENV infection (Fig. 7), demonstrating that an interplay between the TNF and Ang-1 /Ang-2 pathway contributed to DENV pathogenesis. The presence of higher levels of Ang-2 in anti-TNF-treated mice than uninfected na'ive mice further supported that simultaneously targeting both an inflammatory (i.e. TNF) and an angiogenic pathway (either Ang-1/Ang-2 or VEGF) provides an additional benefit over anti-TNF alone in protecting the host against DENV-induced vascular leakage.

[00103] Example 6

[00104] Wild-type mice are resistant to parenteral infection with DENV, as the virus is able to block type I and type II interferon (IFN) receptor signaling in human but not murine cells. The antiviral IFN response must be disrupted in mice to make them susceptible to DENV infection and manifest signs of severe disease. Therefore, the inventor has developed a model of DHF/DSS-like disease first in 129/Sv mice lacking the type I and II IFN receptors (also known as AG129) upon infection with DENV alone or via the ADE route. This mouse model reproduces key pathophysiological features of DHF/DSS, including similar cellular and tissue tropism and lethal vascular leakage, cytokine storm, low platelet count, elevated hematocrit, and hemorrhage. To increase pathogenesis relevance of results obtained using AG129 mice, the inventor created a similar dengue disease model in single-deficient IFNAR" _ mice. The relevance of the IFNAR" " mouse model was further increased by crossing them with HLA transgenic mice expressing human MHC class I or class II molecule. Most recently, LysM-Cre+lfna^' mice have been used to develop the most immunocompetent animal model of DHF/DSS developed to date (Fig. 8). These mice were made by crossing C57BL/6 LysM-Cre+ mice to C57BL/6 Ifna^' mice. These mice lack the type I IFN receptor on

macrophages, a small subset of DCs, and neutrophils, and have substantial deletion of the receptor on monocytes and splenic macrophages. As macrophages are the main targets of DENV in mice, the lack of type I IFN receptor on these cells allowed for DENV replication. Thus, the lack of the type I IFN receptor on only a subset of cells rendered the LysM-Cre+lfnaiil mice susceptible to DENV-induced DHF/DSS-like disease; the rest of their immune system, including T, B, and dendritic cells, was remarkably intact. LysM-Cre^ "Ifna^ mice uniformly succumb to infection with 106 PFU of the DENV2 strain S221 in the presence of enhancing levels of exogenously injected mouse DENV-immune sera (Fig. 8). In the absence of DENV-immune sera, a high viral challenge dose (107 PFU of DENV2 strain S221 ) was required to induce a lethal disease in these mice (data not shown).

[00105] To test the efficacy of the various pathway modulators in protecting against DENV-induced disease, the ADE model of DENV infection is used in LysM-Cre+lfnar1^ m\ce. The ADE model is clinically relevant (i.e. DENV alone), as DHF/DSS was observed mostly after sequential infections in DENV-endemic countries. Additionally, the ADE model required a lower viral challenge dose that may be more physiologically relevant to human infection than the primary infection model. Key results based on the LysM-Cre+ Ifnat1" mouse model are confirmed using another recently generated model of DENV infection in the Itgax-Cre+lfnaiil mice, which lack the type I IFN receptor in dendritic cells.

[00106] Experiments using serotypes other than DENV 2 are conducted. For example, at AG129 mouse model of DENV4-induced lethal disease are generated using the DENV4 strain H241 (Philippino human isolate from 1956), and DENV3 strain C0360/94 (Thai human isolate from 1994) that causes a lethal DHF/DSS-like disease in AG129 mice is obtained. Further, a mouse model of DENV1 infection is developed by passaging of clinical DENV1 strains in AG129 mice. Each of these DENV strains and DENV-specific antibodies are

titrated to generate models of ADE-mediated DENV1 -, DENV3-, or DENV4-induced lethal disease in LysM-Cre+H 'na^' mice. These DENV strains are used to infect tissue culture cells to validate mechanism of action of each pathway modulator at a cellular level. Cell culture models of DENV infection include both macrophage and endothelial cells, and represent both mouse and human cells, and primary and transformed cells. Both direct (virus alone) and ADE (virus + DENV-specific antibody) routes of infection are tested in studies with macrophages and liver sinusoidal endothelial cells (LSECs) (which express Fey receptors) where the ADE mode impacts the angiogenesis pathways. Where DENV exposure leads to expression of Fey receptors on these cells (as determined via flow cytometric analysis), the ADE route is included in studies with other endothelial cell types.

[00107] Example 7

[00108] To modulate the VEGF signaling pathway, small molecule compounds and monoclonal antibodies are used. Multiple inhibitors that target either the VEGF ligand or receptor are tested. Apatinib (MedChem Express) is a tyrosine kinase inhibitor that inhibits VEGF-R2 with IC50 of 1 nM, and ZM 323881 HCI (MedChem Express) is another VEGF-R2 inhibitor with IC50 of <2 nM. Unlike sunitinib, which affects at least eight tyrosine kinase receptors, apatinib and ZM 323881 HCI, may be more selective in blocking VEGF-R2 than sunitinib. Apatinib, ZM 323881 HCI, sunitinib, and anti-mouse VEGF-R2 antibody (clone DC101 ; BioXcell) or anti-mouse VEGF-A antibody (clone 2G1 1 -2A05; BioLegend) that inhibits mouse VEGF binding to mouse VEGF-R2 are administered to block the VEGF pathway in mice with DENV infection. Anti-human VEGF-R2 (clone 89106; R&D Systems) or anti-human VEGF (clone MAB293; R&D Systems) in human cell culture studies.

[00109] Example 8

[001 10] To validate the effects of the VEGF pathway inhibitors on DENV disease pathogenesis (Fig. 9), groups of 5-6 week old, sex-matched (both males and females) LysM-Cre+lfna^' mice (n = 5 to 10 mice per group) are infected with DENV2 strain S221 via the ADE mode as described in Fig. 8. To evaluate the efficacy of drug treatment on protection from DENV-induced lethality, mice are injected i.p. with each drug at -1 , 1 , 24, 48, 72, and 96 hours after DENV infection and monitored for survival. Different doses of each drug are tested based on the data (Fig. 4). A control group is injected with appropriate vehicle or isotype control antibody. As a part of these survival studies, body weight is measured, and health of mice evaluated on a scale of 1 -5 for appearance of coat (smooth coat to very ruffled coat), mobility (active/scurrying to no movement/spastic movement) and attitude (alert to extremely lethargic). These are recorded once a day at 3 p.m. by the same staff involved in the experiment. Mice in control groups are expected to become sick by day 3 after viral challenge and succumb to infection by day 5-6. Experiments are terminated on day 30 p.i.

[001 1 1 ] Due to longer half-life of antibodies relative to small molecule compounds, it is expected that treatment with anti-VEGF or anti-VEGF-R2 antibody show both prophylactic and therapeutic efficacy. The dosing regimen (in terms of amount, frequency, interval, and timing of drug administration) for each drug is optimized. The maximum tolerated dose (MTD) and the treatment dose at which half of the animals survive to day 30 under day 1 , 2, or 3 p.i. treatment regimen (ED50) is determined. MTD is selected based on the results of a dose-escalation study in which groups of mice receive vehicle control or increasing doses of a drug, followed by evaluation of the mice for morbidity, mortality, and weight loss on a daily basis, clinical pathology (hematology and clinical chemistry) on days 3 and 7, and gross necropsy on all animals, whether they die on-study or survive to day 7. The therapeutic index (Tl) (calculated as the nominal MTD divided by the ED50) of each drug is then defined.

[001 12] To validate the mechanisms by which each VEGF pathway inhibitor modulates survival of the infected mice, studies examining viral burden, cytokine storm, and vascular leakage are performed using the optimal dosing protocol that protects 100% of DENV-infected mice in the above studies. These three aspects of DENV infection are selected because viremia is a defining feature of human DENV infection, and vascular leakage with cytokine storm is a hallmark of severe dengue disease in humans. Viral burden in several tissues, including serum, spleen, liver, and the intestine, is quantitated via qRT-PCR on days 1 and 3 p.i., when viral titers in lymphoid and nonlymphoid tissues, respectively, are expected to peak. Additionally between day 3 and 6 p.i., moribund mice are euthanized and their organs and sera harvested to measure viral titers in both lymphoid and non-lymphoid tissues. Cytokine storm is assessed by quantifying serum levels of cytokines associated with DHF/DSS in humans and mouse models (TNF, IL-6, IL-8, and IL-10) by ELISA.

Vascular permeability in key organs such as the liver and intestine is quantitated by the Evans Blue assay and confocal imaging of tight junction and adherens junction protein expression in endothelial cells (Fig. 3). For quantitation of vascular leakage via imaging, co-localization of CD31 (an endothelial cell marker) with ZO-1 and other tight junction (occludin, ZO-2, ZO-3, claudin-1 , and JAM-1 ) and adherens junction (VE-cadherin) proteins can be examined. The assays for assessing vascular leakage and cytokine storm, which are most readily evident 12 to 24 hours prior to death, are performed using day 3 p.i. and moribund samples.

[001 13] As the VEGF pathway antagonists prevent/restore the endothelial barrier function, drug-treated animals should exhibit little or no vascular leakage. The expression pattern of various tight and adherens junction proteins in untreated vs. drug-treated mice is used to provide insights into particular junctional protein pathways that are targeted by each drug. Similarly, serum levels of key cytokines in untreated vs. drug-treated mice are used to indicate whether cytokine release is affected by the action of the VEGF pathway blocker. Where cytokine production is influenced by the drug treatment, tissues of mice are evaluated for inflammation/recruitment of immune cells and identification of cell types producing key cytokines via histopathologic evaluation and immunohistochemistry- and flow cytometry-based approaches. Differences in the patterns of inflammation and cytokine storm in drug-treated vs. vehicle-treated mice is used to provide important insights into interaction between angiogenesis and inflammatory pathways during DENV infection in vivo.

[001 14] Example 9

[001 15] To validate the effects of the VEGF pathway inhibitors at a cellular level in the context of DENV infection (Fig. 10) three different in vitro models of DENV infection are used. First, HUVECs (primary human macrovascular endothelial cells), HMEC-1 (a human dermal microvascular endothelial cell line), and HPMEC-ST1 .6R (a human pulmonary microvascular endothelial cell line), HUVEC and HMEC-1 or HPMEC-ST1 .6R endothelial cell culture models of DENV infection are used. The drug effects on viral replication, cellular activation/differentiation (i.e. expression of E-selectin, VCAM-1 , ICAM-1 , procoagulant molecules such as von Willebrand factor, tissue factor, PAI-1 and PAF that are found at elevated levels in DHF/DSS patients relative to DF cases, and markers of apoptosis and necrosis), production of cytokines/angiogenesis factors (i.e. TNF, IL-6, IL-8, IL-10, VEGF, Ang-1 , and Ang-2), and barrier function is assessed. The endothelial cell barrier function is assessed via assays that measure transwell permeability of fluorescently labeled 10-kDa dextran and transendothelial electrical resistance (TEER) and expression of key endothelial cell junction proteins (e.g., ZO-1 and VE-cadherin) by immunohistochemistry. Confocal microscopy-based data on the integrity of endothelial cell junctions can be confirmed via electron microscopy. Finally, the mouse and cell culture data are linked by comparing the effects (in terms of endothelial barrier function) of treating the endothelial cell cultures with serum from untreated vs. drug-treated mice with DENV infection and culture supernatant from untreated vs. drug-treated macrophage cultures.

[001 16] Studies with our mouse models and autopsy samples implicated liver sinusoidal endothelial cells (LSEC) as the endothelial cell type that supports DENV replication.

Therefore as a second tissue culture model of DENV infection, mouse LSECs are isolated from LysM-Cre+lfna^' mice and SK Hep1 (an immortalized human cell line that may be of LSEC origin and that has been reported to support DENV infection used to develop a

primary and transformed LSEC model of DENV infection, respectively). Where SK Hep1 cells do not support significant levels of DENV infection, human LSECs are immortalized. Mechanisms of drug affect on viral infection, production of cytokines/angiogenesis factors, cellular activation/differentiation, and barrier function in LSECs is assessed.

[001 17] Third, the model herein described of DENV infection is used in mouse bone marrow-derived macrophages (BMM), and DENV infection, and to validate production of cytokines/angiogenesis factors, and cellular activation/differentiation compared in untreated vs. drug-treated BMM. BMM are chosen because macrophages are the primary targets of DENV in our mouse model, and cells of the monocyte/macrophage/dendritic cell lineage are likely the major cell type supporting DENV replication in. As macrophages can express VEGF-R2 under certain conditions, significant effects of drug treatment in the BMM model of DENV infection may be observed. In this case, similar studies are performed using primary human macrophage model of DENV infection (e.g. models of DENV infection using human blood monocyte-derived MO and M2 macrophages)

[001 18] In these cell culture studies, the VEGF pathway antagonists transduce VEGF-R2 signaling and thus abrogate/minimize DENV-induced disruption of endothelial barrier function.

[001 19] Example 10

[00120] Experiments to validate modulation of the Ang-1 /Ang-2 pathway to protect a host against DENV are provided. DHF/DSS is associated with low levels of Ang-1 and high levels of Ang-2 in circulation, and the present data show that mice with DHF/DSS-like disease have higher levels of Ang-2 than uninfected mice (Fig. 2). Consistent with these observations, DENV can decrease the expression of Ang-1 and increase expression of Ang-2 in HUVEC, implying the imbalance of Ang-1 and Ang-2 plays a role in vascular leakage during DENV infection. Treatment of mice with a Tie-2 kinase inhibitor provided some protection against lethal DHF/DSS-like disease (Fig. 7), further implicating a role for the Ang-1 /Ang-2 pathway in mediating vascular leakage during DENV infection.

[00121 ] The protective effect of the Tie-2 antagonist against DENV-induced lethality in mice is counterintuitive: Ang-1 binds Tie-2 and stimulated phosphorylation and downstream signaling to stabilize blood vessels, while Ang-2 competed with Ang-1 for Tie-2 binding and reduced Tie-2 phosphorylation. Thus, Ang-1 and Ang-2 act as endogenous Tie-2 agonists and antagonists, respectively, and therapeutic agents that agonize (but not antagonize) Tie-2 function are generally expected to promote endothelial cell integrity and barrier function.

Data here suggested that Tie-2 antagonism (instead of Tie-2 agonism) protects against DENV-induced lethality in mice highlights the complexity and recently recognized actions of the Ang-1 /Ang-2 pathway that appear to be context-dependent. In particular, Ang-2 can also function as a Tie-2 agonist especially when Ang-2 levels are high and Ang-1 levels are low/absent, and Tie-1 , which is structurally homologous to Tie-2 but is considered an orphan receptor that does not bind either Ang-1 or Ang-2, can inhibit Tie-2 signaling by forming a heterodimeric complex with Tie-2. Expression of both the receptors and ligands of the Ang-1/Ang-2 pathway can be dynamic; reduced Tie-2 levels are associated with vascular complications induced by Ebola virus infection in mice and septic shock in rats and mice. Additionally, studies in the arthritis and atherosclerosis field have suggested that Tie-1 can mediate a proinflammatory response in endothelial cells. Thus in the current studies inhibition of Tie-1 activity (via treatment with the Tie-2 kinase inhibitor that might also block Tie-1 ) could result in the activation of Tie-2 signaling and/or blunted inflammatory responses during DENV infection of mice. Investigating the expression and function of both Tie-1 and Tie-2 receptors and Ang-1 and Ang-2 ligands in untreated vs. mice/cells treated with known Tie-2 agonist or antagonist defines both the role of the Ang-1 /Ang-2 pathway in DENV disease pathogenesis and the mechanism of action of potential drug candidates for treatment of DHF/DSS.

[00122] Example 11

[00123] To modulate the Ang-1 /Ang-2 pathway, agonists and antagonists of Tie-2 signaling are used. Two Tie-2 inhibitors and blocking anti-Ang-2 antibody are used as antagonists. The Tie-2 kinase inhibitor (Selleckchem Catalog No. S1577) is an optimized compound of SB-203580, blocks Tie-2 activity with an IC50 of 0.25 μΜ and >10 fold selectivity over VEGF-R2 and PDGFR, and was used in the studies in Figure 7. Another small molecule Tie-2 inhibitor (Santa Cruz Biotechnology catalog No. CAS 1020412-97-8) has an IC50 of 1 μΜ, and it was tested to be selective against 17 different tyrosine kinases (including the receptor tyrosine kinases VEGF-R2, EGFR, and c-MET) up to 20 μΜ compound concentrations. Anti-mouse Ang-2 (clone Angy-2-1 ; AdipoGen) and anti-human Ang-2 (clone Angy-1 -4; AdipoGen) are used in mouse and human studies, respectively. Vasculotide (GenScript or custom synthesis), a synthetic PEGylated peptide that binds to Tie-2 without displacing Ang-1 or Ang-2, and recombinant human Ang-1 (R&D Systems) that can stabilize endothelial barrier function in a mouse model of sepsis are used as agonists.

[00124] Example 12

[00125] To validate the effects of the Ang-1 /Ang-2 pathway modulators on DENV disease

pathogenesis in mice and cell cultures (Fig. 10) and evaluate the effect of treatment with each Ang-1 /Ang-2 pathway modulator in vivo and in vitro, studies with LysM-Cre+lfna^' mice and various cell cultures are performed. The Tie-1 /Tie-2 signaling are investigated. The ability of the drugs to provide significant therapeutic protection from survival, cytokine storm, and vascular leakage in mice are evaluated followed by evaluation of treatment of mice with blocking anti-Ang-2 antibody and recombinant Ang-1 protein and initiation of the cell culture studies to dissect the drug mechanism of action. Due to the unexpected results obtained with a Tie-2 kinase inhibitor (Fig. 7) and the complexities of the Ang-1 /Ang-2 pathway, it may be evaluated how each treatment impacts the expression of both receptors (Tie-1 and Tie-2) and ligands (Ang-1 and Ang-2) and activation of Tie-1 and Tie-2 in mice and cell cultures. To assess Tie-1 and Tie-2 activation, the phosphorylation status of Tie-1 , Tie-2, and the downstream components, including AKT, ERK, and eNOS are determined.

[00126] Example 13

[00127] Experiments to evaluate whether targeting TNF in combination with the VEGF and/or Ang-1/Ang-2 pathway provides better protection against DENV than TNF alone are provided. Clinical studies have reported that (i) DHF/DSS patients have higher circulating levels of TNF than DF cases, (ii) polymorphisms in the 77VFgene are associated with dengue disease severity and (iii) individuals on anti-TNF antibody therapy do not develop DHF/DSS. Consistent with these findings, the present inventor has found the anti-TNF treatment can prevent DHF/DSS-like disease in AG129 mice. The present data

demonstrates an intersection of the TNF and Ang-1/Ang-2 pathways during DENV infection in mice (Fig. 8) and the present inventor has discovered that simultaneously targeting both TNF and one of the angiogenic pathways (either VEGF or Ang-1 /Ang-2) provided a benefit over anti-TNF alone in protecting the host against DHF/DSS.

[00128] To validate the efficacy of anti-TNF, in combination with the VEGF and/or Ang-1 /Ang-2 pathway modulator (Fig. 1 1 ), the minimal therapeutic and subtherapeutic doses of neutralizing anti-TNF antibody treatment in Lys M-Cre+ If nar1^ m\ce with DENV infection are determined, as done in AG129 mice. Treatment of AG129 mice with 20 μg, 10 μg, and 5 μg of neutralizing anti-TNF antibody on day 2 after lethal DENV infection protects 100%, 50%, and 15% of mice, respectively, thereby identifying the minimal effective and sub-therapeutic doses of anti-TNF antibody treatment against DENV in this mouse model (Fig. 12, top panel). To evaluate additive or synergistic effects of the combination therapy, LysM-Cre+lfna^' mice are treated with 10 μg or 5 μg of anti-TNF antibody, together with partially protective doses of the VEGF or Ang-1 /Ang-2 pathway modulators and evaluated for survival, weight loss, and health. Anti-TNF treatment reduces Ang-2 levels in DENV-infected AG129 mice (Fig. 8) and combination therapy of anti-TNF and sunitinib can provide protection against DENV-induced lethality in AG129 mice (Fig. 12, bottom panel).

[00129] To define the therapeutic window of the combination therapy, the drugs can be administered at different time points after infection. Based on results showing that day 2 p.i. is the latest time point for an effective anti-TNF or sunitinib treatment, the combination therapy may prolong the dosing window to at least day 3 p.i. Once a combination therapy protocol that protects LysM-Cre+lfnar1^ m\ce from lethal DENV challenge is identified, the mechanism of action of the combined drugs in these mice can be dissected, as described above. Based on the present data on sunitinib-treatment of DENV-infected AG129 mice (Figs. 5-6), the combination treatment may protect against DENV-induced lethality by improving endothelial barrier function with minimal impact on viral replication. The activation status of VEGF-R2, Tie-1 , and Tie-2 in tissues also indicate how the combination treatment impacts the VEGF and Ang-1 /Ang-2 signaling.