FIELD OF INVENTION

The invention relates to a pharmaceutical composition for use in the treatment or inhibition of dengue virus infection and/or dengue shock syndrome.

BACKGROUND OF INVENTION The number of cases of dengue fever has increased dramatically since the 1960s, with between 50 and 528 million people infected yearly. Dengue has become a global problem and is endemic in more than 1 10 countries. Because of the great disease burden associated with dengue, the World Health Organization considers dengue as a major public health problem and has issued a mandate to develop strat- egies to prevent and treat this disease. Treatment of acute dengue is supportive, using either oral or intravenous rehydration for mild or moderate disease, and intravenous fluids and blood transfusion for more severe cases. Apart from eliminating the mosquitoes, work is ongoing on vaccines, as well as medication targeted directly at the virus.

Dengue virus (DENV) is an RNA virus, a Flavivirus, and is transmitted to humans by the bite of mosquitoes. The dengue virus genome contains about 1 1,000 nucleotide bases, which code for the three different types of protein molecules (C, prM and E) that form the virus particle and seven other types of protein molecules (NSl , NS2a, NS2b, NS3, NS4a, NS4b, NS5) that are only found in infected host cells and are required for replication of the virus. There are five serotypes, of which the first four are referred to as DENV-1, DENV-2, DENV-3 and DENV-4. The infection can either be asymptomatic or manifest in three clinical forms of increasing severity: dengue fever, dengue hemorrhagic fever, and dengue shock syndrome (DSS). In severe cases, circulatory failure and death can occur.

Clinical observations have revealed significant abnormalities in coagulation and inflammation systems, with increased levels of tissue factor (TF) and the chemokine IL-8, correlating with the severity of the disease and implicating damage to endo- thelial vascular cells (EVC).

Lipopolysaccharide (LPS) levels are elevated in dengue virus infected patients and correlate with disease severity. Interestingly, microbial translocation in the gut lead to immune activation in dengue virus infected patients. Moreover, significantly in- creased LPS levels were found in patients with a pronounced proinflammatory cytokine profile, which was correlated with patient mortality. There is a crosstalk between the regulatory signaling pathways of the coagulation- inflammation processes, during dengue virus infection of EVC. Dengue virus up- regulates protease activated receptor type-1 (controlling inflammation) and TF (controlling coagulation), via the phosphorylation of p38 and ERKl/2 mitogen-activated protein kinases (MAPK), which induce the activation of NF-κΒ transcription factor.

The activation of coagulation and fibrinolysis during the acute stage of dengue virus infection is offset by the increase of platelet and PAI-1 during convalescent stage. Taken together, these results suggest that the degree of coagulation and fibrinolysis activation induced by dengue virus infection is associated with the disease severity. The dengue protein NS1 is a conserved nonstructural glycoprotein (~48 kDa) with six invariant intramolecular disulfide bonds. NS1 is synthesized as a monomer, di- merizes after posttranslational modification in the lumen of the ER, is processed in the trans-Golgi network, and secreted into the extracellular space as a hexameric lipoprotein particle. NS1 hexamers have a central lipid-rich core and are held to- gether by weak hydrophobic interactions that dissociate into dimers in the presence of nonionic detergents. NS1 can be secreted at high levels into the extracellular environment, with accumulation of up to 50 μg/mL in the serum of some dengue virus-infected patients. NS1 may act as a target for antibodies, and generation of this has been seen during dengue infection. Furthermore, NS1 also has immune evasive functions in the extracellular space, on the surface of cells, and possibly within cells. NS1 binds several complement proteins (Clq, Cls, and C4) and regulators (factor H, C4 binding protein, and clusterin) and antagonizes their functions. There is a correlation between levels of circulating NS 1 and dengue disease severity. Dengue virus is detected in peripheral monocytes during acute disease and in in vitro infection, leading to cytokine production, indicating that virus-target cell interactions are relevant to pathogenesis. It is also known that dengue fever has im- munopathological patterns, with up-regulation of inflammatory molecules and immunological cells playing a role in coagulation disorders. A sequential monocyte - activation model has been proposed in which dengue virus infection triggers TLR2/4 expression and inflammatory cytokine production, leading eventually to hemorrhagic manifestations, thrombocytopenia, coagulation disorders, plasmatic leakage and shock development. Recent results indicate that MAPKs are activated during dengue infections. Using human PBMCs, stable cell lines, as well as mouse model, it was shown p38 MAPK inhibitor SB203580 suppressed inflammation and limited disease development during dengue virus infection. In vitro infection of human myeloid cells with dengue virus induces secretion of various chemokines, including IL-8, MCP-1, and ANTES. It was observed that viral infection induced cytokine and chemokine production in PBMCs, THP-1 cells as well as KU812 cells and that SB203580 inhibited p38 MAPK-mediated TNF-a secretion from humans. However, several adverse effects have been reported for p38 MAPK inhibitors in pre-clinical and clinical studies. As part of the innate immune system, monocytes and macrophages play important roles in response to invading pathogens. Upon pathogen recognition, a plethora of inflammatory responses is rapidly induced, including the production of cytokines such as tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), and IL-12, but also various chemokines and other biologically active substances, which subsequently contribute to eradication of the pathogen. The recognition of pathogens and their pathogen-associated molecular patterns (PAMPs) relies on a diverse set of pattern- recognition receptors (PRRs). One example is the Toll-like receptor 4 (TLR4), which recognizes LPS, a cell surface component of Gram-negative bacteria. LPS binds to the acute phase plasma LPS-binding protein (LBP) and is delivered to CD 14 at the cell surface of monocytes/macrophages, leading to interaction with the TLR4/MD-2 (myeloid differentiation protein-2) complex. This interaction induces downstream signaling, leading to activation of MAPK, such as ERK, JNK p38a, and NF-KB, finally resulting in the production of cytokines and other inflammatory molecules. However, undue cell stimulation might result in an uncontrolled host response leading to tissue damage and organ dysfunction as seen in DSS.

SUMMARY OF THE INVENTION

Cationic host defense peptides (HDP) constitute an important part of the innate im- mune system by facilitating the defense against invading pathogens. They have attracted significant attention as novel anti-infectives due to their ability to directly kill bacteria, but also to modulate a variety of immune responses. For example, studies using various cationic HDPs have shown that peptide -binding to LPS may block the subsequent LPS-LBP interaction, resulting in reduction of TNF- a produc- tion by macrophages. By neutralizing circulating endotoxins, HDPs can reduce proinflammatory responses, hence preventing the cytokine storm and organ damage seen in endotoxin shock and bacterial infections. Although LPS scavenging is a well-established effect of some HDPs, less is known about other possible functions mediating specific inhibitory effects.

The present invention is based on the novel and unexpected finding that the peptide GKY25 binds directly to inflammatory cells, including monocytes and macrophages, and inhibits down-stream inflammatory pathways involving MAPK, resulting in reduced NF-κΒ activation. Thus, in a previously undisclosed manner, GKY25 reduces such cell responses that are typical for dengue virus infection.

In addition, and as described above, dengue infection and particularly DSS, involves over-activation of multiple inflammatory pathways, including coagulation. Furthermore, dengue infection leads to bacterial translocation in the gut and excessive LPS. Apart from inhibiting excessive coagulation, GKY25 has the ability to block the excessive endotoxin responses during dengue infection, via its inhibition of TL 4 dimerization and subsequent MAPK activation.

A HDP, which in this unique way targets several pathologies during a viral infection, and particularly during dengue infection, has not been previously described. The HDP thus meets an urgent clinical need and is of clear interest for the treatment or inhibition of dengue virus infection and/or dengue shock syndrome.

Since the peptide GKY25 (SEQ ID NO:2) have that particular characteristic it is also presumed that the structure as defined by SEQ ID NO: l and following criteria, have the same effects since that formula have been developed using a combination of in silico analyses and evaluation of results from experimental studies. Thus it is expected that all peptides being derived from that particular formula will show the same effect (Kasetty et al., J Innate Immun 201 1 :3,471-482). The invention relates in a first aspect to a pharmaceutical composition comprising a peptide comprising or consisting of the peptide having the general formula (I)

Xi X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X8 X ₉ Xio Xi l X12 X13 X14 W X ₁₆ X ₁₇ X ₁₈ X19 X20 X21 X22 X23 X ₂₄ X ₂₅ (SEQ ID NO: 1) wherein

X ₃ is P, F or Y

X ₅ is A, F, I, L or V

X ₆ is A, F or Y

X ₇ is A, I, T or V

X ₉ is I, L or V,

X _i6 is I or L and Xi, X ₂ , X _4; X ₈ , Xio-15 and Xi _7-25 independently represent any amino acid residue or parts thereof, wherein the peptide has a relative hydrophobicity (μΗΓβΙ) of >0.4 and net charge >2. for use in the treatment or inhibition of dengue virus infection and/or dengue shock syndrome. Thus it will for the first time be possible to inhibit or treat the global infection problem caused by the dengue virus.

The invented pharmaceutical composition is suitable for the inhibition of the dengue virus mediated inflammatory responses during dengue infection and/or inhibition of NS1 mediated inflammatory responses during dengue infection, and/or inhi- bition of combined dengue virus and LPS induced inflammatory responses during dengue infection and/of inhibition of combined NS 1 and LPS induced inflammatory responses during dengue infection.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings,

figure 1 shows GKY25 binding to mouse macrophages,

figure 2 shows electron microscopy of peptide binding to macrophages, figure 3 shows activation of monocytes by LPS and the protein NS 1 , and effects of GKY25,

figure 4 shows effects of GKY25 on NS 1 stimulated human blood monocytes, figure 5 shows effects of PEGylated GKY25 on LPS-mediated activation, and figure 6 shows effects of GKY25 on NS 1 -mediated induction of inflammation in vivo. DETAILED DESCRIPTION OF THE INVENTION

Definitions inhibition, identity

In the context of the present application and invention the following definitions ap- ply.

The term "identity", meaning that amino acid residues are identical, i.e., 95 % identity of a peptide having 25 amino acid residues meaning that 23 amino acid residues are identical and 2 are different. The difference may that that the amino acid residue has been substituted.

The term "substituted" is intended to mean that an amino acid residue is replaced by another amino acid residue.

Description The present invention relates to the unexpected finding that a peptide GKY25 (SEQ ID NO:2 was found to interact directly with monocytes and macrophages, thereby mediating anti-inflammatory effects. Thus, this activity was separate from the previously disclosed effects based on scavenging of LPS in the extracellular space.

As mentioned above, NS1 is the major secreted protein during dengue infections, correlated to outcome. No previous data exist showing that NS1 is proinflammatory. The inventors have found that NS1 affects human monocytes by activating NF- KB. GKY25 reduced NF-κΒ activation in response to NS1 protein. Also, cells pre- treated with GKY25 showed diminished NS1 -responses. Confocal microscopy and electron microscopy analysis demonstrated peptide binding to monocytes.

The unexpected results demonstrated a previously undisclosed activity of the host defense peptide GKY25 (SEQ ID NO:2), based on reduction of NF-κΒ activity and pro-inflammatory cytokine production in response to NS 1 protein. GKY25 (SEQ ID NO:2) functions by preventing TLR dimerization at the surface of macrophages and monocytes, leading to inhibition of activation of MAPK and NF-κΒ, and finally, reduced cytokine production. This unexpected new activity of GKY25 (SEQ ID NO:2) enables its use in conditions where inhibition of unwanted NF-κΒ is preferred.

Thus GKY25 (SEQ ID NO:2) is suitable to be used in the treatment or inhibition of dengue virus infection and/or dengue shock syndrome.

In a second aspect the invention relates to a pharmaceutical composition, wherein

X ₂ is K

X ₃ is Y

X ₄ is G

X ₅ is F

X ₆ is Y

X ₇ is T

X ₉ is V,

X _i6 is I and

Xi, X ₈ , Xio-i5 and Xi _7-25 independently represent any amino acid residue or parts thereof, , wherein the resulting peptide has a relative hydrophobicity (μΗΓβΙ) of >0.4 and net charge >2 for use in the treatment or inhibition of dengue virus infection and/or dengue shock syndrome.

In a third aspect the invention relates to a pharmaceutical composition, comprising or consisting of the peptide of SEQ ID NO:2:

GKYGFYTHVF LKKWIQKVIDQFGE (SEQ ID NO:2) or a peptide having 15 amino acids identity to SEQ IDNO:2 or parts thereof for use in the treatment or inhibition of dengue virus infection and/or dengue shock syndrome.

However, the identity may be 16, 17, 18, 19, 20 amino acid residues or more, such as 21, 22, 23, 24, or 25 amino acid residues.

In a fourth aspect the invention relates to a pharmaceutical composition, comprising or consisting of a fragment of SEQ ID NO: l or 2 for use in the treatment or inhibition of dengue virus infection and/or dengue shock syndrome. The peptide in the pharmaceutical composition comprising or consisting of from 20 to 30 amino acid residues, such as 21, 22, 23, 24,25, 26, 27, 28 or 29 amino acid residues for use in the treatment or inhibition of dengue virus infection and/or dengue shock syndrome

Accordingly the peptide present within the pharmaceutical composition may be modified/altered in a way that one or more amino acid residues in said peptide is modified by PEGylation (PEG = polyethylene glycol)

How to PEGylate a peptide is common general knowledge for a person skilled in the art. The size of PEG may vary and being determined by a person skilled in the art, such as less than PEG50, such as between 6-24. For example PEGylation of GKY25 may be performed utilizing PEG6, 12, or 24, wherein retained antiinflammatory effects is still maintained. Thus, PEG 12-24 modifications may be the best choice for therapeutic uses. PEG6, 12, and 24 means that polyethylene glycol particles have 6, 12, and 24 discrete ethylene glycol units.

In a fifth aspect the invention relates to a pharmaceutical composition according to any of preceding claims, wherein the composition comprises an antiviral agent for use in the treatment or inhibition of dengue virus infection and/or dengue shock syndrome. The antiviral agent may be any known antiviral agent well known for a person skilled in the art, such as all those that could be useful in combatting the disease of interest within this particular disclosure The pharmaceutical composition according will normally comprise a pharmaceutically acceptable excipient, diluent, carrier or buffer for use in the treatment or inhibition of dengue virus infection and/or dengue shock syndrome.

"Pharmaceutically acceptable" means a non-toxic material that does not decrease the effectiveness of the biological activity of the active ingredients, i.e., the antimicrobial peptide(s). Such pharmaceutically acceptable buffers, carriers or excipients are well-known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A.R Gennaro, Ed., Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed ., Pharmaceutical Press (2000).

The term "buffer" is intended to mean an aqueous solution containing an acid-base mixture with the purpose of stabilising pH. Examples of buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazolelactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO and TES.

The term "diluent" is intended to mean an aqueous or non- aqueous solution with the purpose of diluting the peptide in the pharmaceutical preparation. The diluent may be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil).

The term "adjuvant" is intended to mean any compound added to the formulation to increase the biological effect of the peptide. The adjuvant may be one or more of zinc, copper or silver salts with different anions, for example, but not limited to fluoride, chloride, bromide, iodide, tiocyanate, sulfite, hydroxide, phosphate, car- bonate, lactate, glycolate, citrate, borate, tartrate, and acetates of different acyl composition.

The excipient may be one or more of carbohydrates, polymers, lipids and minerals. Examples of carbohydrates include lactose, sucrose, mannitol, and cyclodextrines, which are added to the composition, e.g., for facilitating lyophilisation. Examples of polymers are starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxy- propylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginates, carageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, poly- sulphonate, polyethylenglycol/polyethylene oxide, polyethyleneox- ide/polypropylene oxide copolymers, polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, and polyvinylpyrrolidone, all of different molecular weight, which are added to the composition, e.g., for viscosity control, for achieving bioad- hesion, or for protecting the lipid from chemical and proteolytic degradation. Examples of lipids are fatty acids, phospholipids, mono-, di-, and triglycerides, ceramides, sphingolipids and glycolipids, all of different acyl chain lenght and satu- ration, egg lecithin, soy lecithin, hydrogenated egg and soy lecithin, which are added to the composition for reasons similar to those for polymers. Examples of minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition to obtain benefits such as reduction of liquid accumulation or advantageous pigment properties.

The invented formulation may also contain one or more mono- or di-sacharides such as xylitol, sorbitol, mannitol, lactitiol, isomalt, maltitol or xylosides, and/or monoacylglycerols, such as monolaurin. The characteristics of the carrier are dependent on the route of administration. One route of administration is topical ad- ministration. For example, for topical administrations, a preferred carrier is an emulsified cream comprising the active peptide, but other common carriers such as certain petrolatum/mineral-based and vegetable-based ointments can be used, as well as polymer gels, liquid crystalline phases and microemulsions. The peptide as a salt may be an acid adduct with inorganic acids, such as hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, phosphoric acid, perchloric acid, thiocyanic acid, boric acid etc. or with organic acid such as formic acid, acetic acid, haloacetic acid, propionic acid, glycolic acid, citric acid, tartaric acid, succinic acid, gluconic acid, lactic acid, malonic acid, fumaric acid, anthranilic acid, benzoic acid, cinnamic acid, p-toluenesulfonic acid, naphthalenesulfomc acid, sulfanilic acid etc.

The pharmaceutical compositions may be subjected to conventional pharmaceutical operations such as sterilisation and/or may contain conventional adjuvants such as preservatives, stabilisers, wetting agents, emulsifiers, buffers, fillers, etc., e.g., as disclosed elsewhere herein.

The pharmaceutical compositions according to the invention may be administered locally or systemically. Routes of administration include parenteral (intravenous, subcutaneous, and intramuscular

The pharmaceutical compositions will be administered to a patient in a pharmaceutically effective dose. By "pharmaceutically effective dose" is meant a dose that is sufficient to produce the desired effects in relation to the condition for which it is administered. The exact dose is dependent on the activity of the compound, manner of administration, nature and severity of the disorder, age and body weight of the patient different doses may be needed. The administration of the dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals

The pharmaceutical compositions of the invention may be administered alone or in combination with other therapeutic agents, such as antiviral agents, or therapeutic antibodies or peptides.

The present invention concerns humans. Thus the methods are applicable to human therapy applications. The objects, suitable for such a treatment may be identified by well-established hallmarks of an infection, such as fever, pulse, blood pressure, blood parameters including serology, measurement of NSl in blood, identification of organisms, and the like. The following examples are intended to illustrate, but not to limit, the invention in any manner, shape, or form, either explicitly or implicitly.

EXAMPLES EXAMPLE 1 - GKY25 binding to mouse macrophages.

RAW 264.7 cells were incubated for 30 min at 37°C with the indicated concentrations of T-GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE) or T-WFF25 ( WFFF YYLIIGGGVVTHQQRKKKKDE) . Cell binding was analyzed by flow cytometry (mean±SEM, n=3-4) as shown in fig. 1A. Binding of 1 μΜ T-GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE) and T-WFF25 to RAW 264.7 cells at 37°C after indicated incubation times was visualized by confocal microscopy as shown in fig. IB. The nuclear DNA was stained in blue with DAPI and the T- peptide by red fluorescence. (One representative image out of 3 experiments is shown, scale bar: 5 μηι). RAW 264.7 cells were incubated with of 10 μΜ T-GKY25 and T-WFF25 at 37°C, results shown in fig. 1C, or 4°C, results shown in fig. ID and binding was determined at indicated time points (mean±SEM, n=3) (MFI - median fluorescence intensity). Binding of 1 μΜ T-GKY25 and T-WFF25 to RAW 264.7 cells after 15 min of incubation at indicated temperatures was analyzed by confocal microscopy (One representative image out of 3-4 experiments is shown, scale bar: 10 μηι), see fig. IE.

EXAMPLE 2 - Electron microscopy of peptide binding to macrophages.

For transmission electron microscopy RAW cells were incubated with GKY25 (20 μΜ). Figure 2 shows gold-labeled antibodies against the VFR17 epitope of GKY25 (small gold particles) and TLR4 (large gold particles). Scale bar represents 200 nm. EXAMPLE 3 - Activation of monocytes by LPS and the protein NS 1 , and effects of GKY25.

THP-1 cells were stimulated as indicated in figure 3 with LPS (100 ng/ml) or NS l protein (1 and 2 ug/ml) and the NF-KB/AP-1 activity was determined 20 h after addition of stimuli, see figure 3. NF-KB/AP-1 activation induces production of secreted embryonic alkaline phosphatase (SEAP). SEAP in cell supernatants was mixed with Quanti-Blue reagent and absorbance was measured at 600 nm. GKY25 at 10 and 20 μΜ inhibited NF-KB/AP-1 activation after stimulation with LPS and NS 1 , respectively.

EXAMPLE 4 - Effects of GKY25 on NSl stimulated human blood monocytes. PBMCs were used for the stimulation experiments with various types of NSl protein. Cytokines, including IL-6 and TNF-a were analysed. In short human PBMC were isolated from blood by gradient centrifugation and immediately cultured in DMEM media with addition of 10 ug/ml of NSl proteins and with various doses of GKY25 peptide. Supernatants were collected at 12 h and analysed for presence of the secreted inflammatory cytokines IL-6 and TNF-a by ELISA, see figures 4A and 4B. EXAMPLE 5 - Effects of PEGylated GKY25 on LPS-mediated activation.

THP-1 cells were stimulated as indicated in figure 5A-5D with LPS (100 ng/ml) and GKY25 and its PEGylated variants were added at the indicated concentrations. LPS induces NF-KB/AP-1 activity, which is blocked by GKY25 and its PEGylated forms. NF-KB/AP-1 activity was determined 20 h after addition of stimuli, see fig- ure 5A - 5D.

EXAMPLE 6 - Effects of GKY25 on NSl -mediated induction of inflammation in vivo.

Reporter mice carrying luciferase gene under an NF-κΒ promoter were injected i.p. with 50 μg of purified NS l protein followed by i.p. injection of 100 μg GKY25 peptide or buffer only. Induction of NF-κΒ driven inflammation was monitored by in vivo light emission analysis of anesthetized mice 10 min after D-luciferin (luciferase substrate) injection in an IVIS Spectrum system. The heat map is a two- dimensional representation of light emitted ventrally from the mice. Figure 6A and 6B each show the results from one mouse, the left image being the result of the NSl protein + buffer only, and the right image being the result of NSl protein + GKY25. The graph in figure 6C shows light intensity emission (photons/sec/cm * 10 ^A 6) measured for each animal. EXAMPLE 7 - Dengue virus infection in vivo. AG 129 mice were housed under specific pathogen free conditions in individual ventilated cages. Mice were administered with 10 ² to 10 ⁷ plaque forming units (PFU) of a Dengue virus strain via the subcutaneous (sc) or intraperitoneal (ip) route. After periods of time, GKY25 (50-500 ug/mouse) was administrated sc or ip. Blood sam- pies were collected and centrifuged for 5 min at 6,000 g to obtain plasma. The presence of infectious viral particles was determined by plaque assay. For histology, mice were euthanized, and tissues were harvested and immediately fixed in 10% formalin in PBS. Fixed tissues were paraffin embedded, sectioned and stained with Hematoxylin and Eosin. Cytokine levels (including IFN-γ, TNF-a and IL-6) were measured in individual blood samples using detection kits according to the manufacturer' instructions.

EXAMPLE 8 - NS1 and/or LPS in vivo.

Mice were housed in individual ventilated cages. Mice were administered with NS 1 protein (with or without added LPS) via the intraperitoneal (ip) or subcutaneous (sc) route. After periods of time, GKY25 (50-500 ug/mouse) was administrated via the ip or sc route. Blood samples were collected. Cytokine levels (including IFN-γ, TNF-a and IL-6) were measured in individual blood samples using individual detection kits according to the manufacturer' instructions. In some experiments, an IVIS Spectrum Bioimaging system was used to determine activation of NF-kB in the mice.

EXAMPLE 9 - Effects on viral infection in vitro.

Cells were infected with Dengue virus under different conditions; pre-treatment with GKY25 at various concentrations, co-incubation of DV with peptide, or Dengue virus infection followed by addition of peptide. After incubation for 4 days, determination of Dengue virus by plaque assay was performed.