WO2010071610A1

WO2010071610A1 - Severe chikungunya biomarkers

Info

Publication number: WO2010071610A1
Application number: PCT/SG2009/000487
Authority: WO
Inventors: Lisa Fong Poh Ng; Yee Sin Leo; Angela Chow
Original assignee: Agency For Science, Technology And Research (A*Star)
Priority date: 2008-12-19
Filing date: 2009-12-21
Publication date: 2010-06-24

Abstract

We describe a method of distinguishing a severe chikungunya infection from a non- severe chikungunya infection in an individual, in which the method comprises detecting, in a sample in or of an individual an increased expression or activity of IL- 1β, an increased level of IL-6 and a decreased level of RANTES, in which such levels indicate severe chikungunya; an increased expression or activity of all of IL-1α, IL-1β, IL-2, IL-6, IL-7, IL-8, IL-12, IL-15, IFN-a, IP-10, Eotaxin, MCP-1, MIG and a decreased level of RANTES in which such levels indicate severe acute chikungunya; or an increased expression or activity of all of IL-1α, IL- 1β, IL-2R, IL-4, IL-6, IL-7, IL-8, IFN-α, IL-12, IL-15, MCP-I, MIP-1α, Eotaxin, RANTES, IP-10, MIG, EGF, FGF-b, G-CSF, HGF, in which such levels indicate severe chronic chikungunya.

Description

SEVERE CHIKUNGUNYA BIOMARKERS

FIELD

The present invention relates to the fields of medicine, cell biology, molecular biology and genetics. More particular, the invention relates to methods of nucleic acid sequences suitable for use in detecting a pathogen or products thereof in a sample.

BACKGROUND

In recent years, emerging and re-emerging tropical infectious diseases have been shown to cause high social and economic impact. Vector-borne infectious diseases such as Dengue, West Nile have been resurging largely due to the spread of insecticide resistance, to socio-demographic changes, and to genetic mutations in the pathogens.

More recently, chikungungya fever (CHIKF) has now emerged as the next important infection in South-East Asia, the Pacific region and Europe [1-5], making it a major threat that requires immediate attention. Recent epidemic resurgence of CHIKF in several African and Asian countries demonstrated that infection can spread alarmingly rapidly [6-9] from limited early transmission that then developed into an unprecedented and unexpected epidemic, infecting 38% of the population as occurred in Reunion island [6,8].

The appearance of cases in Europe, the United States and other countries by travelers returning from known outbreak areas underscores the contributory factors of increased human mobility, tourism, global climate change, and increases in insecticide resistance [10-15]. In this era of globalization, the threat of such disease epidemics should not be underestimated as such public health events could cripple public health systems and economies.

Given the expanding geographic range of CHIKV and its potential to rapidly cause large scale epidemics, a particular problem has been the provision of tools to guide the development of targeted and effective control of chikungunya. Another problem is to provie effective treatment strategies for individuals infected with chikungunya, including distinguishing between severe and mild forms of chikungunya, in order to provide appropriate treatments depending on disease severity. SUMMARY

According to a 1^st aspect of the present invention, we provide a method of distinguishing a severe chikungunya infection from a non-severe chikungunya infection in an individual.

The method may comprise detecting, in a sample in or of an individual, an increased expression or activity of IL-I β, an increased level of IL-6 and a decreased level of RANTES, in which such levels indicate severe chikungunya.

The method may comprise detecting, in a sample in or of an individual, an increased expression or activity of all of IL-I α, IL-lβ, IL-2, IL-6, IL-7, IL-8, IL-12, IL-15, IFN-a, IP-IO, Eotaxin, MCP-I, MIG and a decreased level of RANTES, in which such levels indicate severe acute chikungunya.

The method may comprise detecting, in a sample in or of an individual, an increased expression or activity of all of IL-lα, IL-lβ, IL-2R, IL-4, IL-6, IL-7, IL-8, IFN-α, IL-12, IL- 15, MCP-I, MIP-Ia, Eotaxin, RANTES, IP-IO, MIG, EGF, FGF-b, G-CSF, HGF, in which such levels indicate severe chronic chikungunya.

The method may comprise detecting a variant, homologue, derivative or fragment of any of the above sequences such as a sequence having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology thereto.

IL-lβ may comprise a polypeptide sequence having GenBank Accession Number NP_000567 or a nucleic acid sequence having GenBank Accession number NM_000576 or a variant, homologue, derivative or fragment thereof such as a sequence having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology thereto and comprising (or encoding a sequence comprising) interleukin-lβ activity.

IL-6 may comprise a polypeptide sequence having GenBank Accession Number NP_000591 or a nucleic acid sequence having GenBank Accession number NM_000600 or a variant, homologue, derivative or fragment thereof such as a sequence having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology thereto and comprising (or encoding a sequence comprising) interleukin-6 activity; or RANTES (syn. CCL5) may comprise a polypeptide sequence having GenBank Accession Number NP_002976 or a nucleic acid sequence having GenBank Accession number NM_002985 or a variant, homologue, derivative or fragment thereof such as a sequence having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology thereto and comprising (or encoding a sequence comprising) RANTES/CCL5 activity.

The level of the biomarker in the sample may be compared to a reference level being the level of the biomarker in an individual who is not suffering from severe chikungunya. For example, the level of the biomarker in the sample may be compared to a reference level as set out in column 6 of Table Dl .

An individual with severe chikungunya infection may exhibit, or may be expected to exhibit any one or more, such as all, of the following symptoms: (a) a temperature of > 38.5 ⁰C ; (b) pulse rate > 100/min, and (c) platelet count < 100x10⁹ g/L.

Detection of acute chikungunya may indicate that the disease is within day 2 to day 19 of infection, or in which detection of chronic chikungunya indicates that the disease is after day 19 of infection.

The detection may comprise polymerase chain reaction, such as real-time polymerase chain reaction (RT-PCR), Northern Blot, immunological detection such as ELISA, RNAse protection or microarray hybridisation.

There is provided, according to a 2^nd aspect of the present invention, a combination of two or more nucleic acids or polypeptides specified above or probes or antibodies capable of binding specifically thereto, such as a combination of nucleic acids immobilised on a substrate, preferably in the form of a microarray.

We provide, according to a 3^rd aspect of the present invention, a nucleic acid or polypeptide as specified above, a combination as specified above, or an agonist or antagonist thereof for use in a method of detecting, determining the severity of or treating chikungunya.

As a 4^th aspect of the present invention, there is provided a pharmaceutical composition comprising a nucleic acid or polypeptide as specified above, a combination as specified above, or an agonist or antagonist thereof. We provide, according to a 5^th aspect of the present invention, a diagnostic kit for cliikungunya or the severity thereof or susceptibility thereto, the kit comprising any one or more of the following: (a) a polypeptide as specified above or a variant, homologue, derivative or fragment thereof such as a sequence having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology thereto; (b) a molecule capable of binding to such a polypeptide, such as an antibody; (c) a nucleic acid capable of encoding such a polypeptide, such as a nucleic acid as specified above or a variant, homologue, derivative or fragment thereof such as a sequence having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology thereto; and (d) a molecule capable of binding to such a nucleic acid sequence, such as a nucleic acid, for example probe preferably comprising a complementary nucleic acid; optionally together with instructions for use.

The present invention, in a 6^th aspect, provides a method of identifying a molecule suitable for the treatment, prophylaxis or alleviation of chikungunya, the method comprising determining if a candidate molecule is an agonist or antagonist of a polypeptide specified above.

In a 7^th aspect of the present invention, there is provided a polypeptide as specified above or a nucleic acid capable of encoding such a polypeptide, for example a sequence having an accession number shown in Table Dl, for use in a method of treating, preventing or diagnosing severe chikungunya.

According to an 8^th aspect of the present invention, we provide a method of treatment or prevention of chikungunya in an individual, the method comprising detecting chikungunya or determining the severity thereof, or both in an individual by a method as described and administering a suitable treatment or prophylactic, such as a drug known or suspected to be useful for treating chikungunya, to the individual.

We provide, according to a 9^th aspect of the invention, a method for the treatment or prevention of chikungunya in an individual, in which the method comprises modulating the expression of a nucleic acid or polypeptide specified above, or in which the method comprises administering an agonist or antagonist of such a nucleic acid or polypeptide.

The method may be such that the expression or activity of one or more such as a subset of the genes set out in (a), (b) and (c) is detected. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N. Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford University Press; M. J. Gait (Editor), 1984,

Oligonucleotide Synthesis: A Practical Approach, IrI Press; D. M. J. Lilley and J. E. Dahlberg, 1992, Methods ofEnzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; Using Antibodies : A Laboratory Manual : Portable Protocol NO. I by Edward Harlow, David Lane, Ed Harlow (1999, Cold Spring Harbor Laboratory Press, ISBN 0-87969-544-7); Antibodies : A Laboratory Manual by Ed Harlow (Editor), David Lane (Editor) (1988, Cold Spring Harbor Laboratory Press, ISBN 0- 87969-314-2), 1855. Handbook of Drug Screening, edited by Ramakrishna Seethala, Prabhavathi B. Fernandes (2001, New York, NY, Marcel Dekker, ISBN 0-8247-0562-9); and Lab Ref: A Handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench, Edited Jane Roskams and Linda Rodgers, 2002, Cold Spring Harbor Laboratory, ISBN 0- 87969-630-3. Each of these general texts is herein incorporated by reference.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Two-way hierarchical clustering analysis. Each cell in the 2-dimensional graph indicates the measure of a single mediator in 1 sample, with standardized levels indicated by color according to the scale on the top. Sample clustering resulting from the algorithm described is shown at the right side of the graph, with an indication of the group to which each individual sample belongs. Mediator clustering is depicted on the top of the graph, and detailed at the bottom.

Figure 2. Differences in plasma mediator levels in CHIKF patients and healthy controls, a. Levels of cytokines (pg/ml) were determined as described and only those with a P value of < 0.05 are illustrated. Horizontal bars indicate the respective groupwise medians, b. Levels of chemokines (pg/ml) were determined, c. Levels of growth factors (pg/ml) were determined.

Figure 3. Differences in cytokines, chemokines and growth factors levels determine disease severity, a. Box-and-whisker plots illustrating the significant differences of IL-I β, IL- 6 and RANTES in patients with non-severe and severe CHIKF. b. Diagrammatic representation of the mediator profiles in CHIKV-infected patients and healthy control subjects, and the distinction between non-severe and severe CHIKI⁷.

Figure 4. Complete profile of the levels (pg/ml) of cytokines, chemokines and growth factors determined by multiplex-bead arrays from blood samples collected from CHIKV- infected patients and healthy control subjects. This figure may also be labelled Figure Sl.

Figure 5. Box-and-whisker graphs of 14 immune mediators (cytokines and chemokines) determined from blood samples collected from healthy control subjects and CHIKV-infected patients. This figure may also be labelled Figure S2a.

Figure 6. Box-and-whisker graphs of 13 Chemokines and growth factors determined from blood samples collected from healthy control subjects and CHIKV-infected patients. This figure may also be labelled Figure S2b.

Figure 7. Two-way hierarchical clustering analysis of 30 patients taken longitudinally. Each cell in the 2-dimensional graph indicates the measure of a single mediator in 1 sample, with standardized levels indicated by color according to the scale on the top. Sample clustering resulting from the algorithm described is shown at the right side of the graph, with an indication of the group to which each individual sample belongs. Mediator clustering is depicted on the top of the graph and detailed at the bottom.

Figure 8. Differences in plasma inflammatory cytokine levels (pg/ml) in CHIKF patients and healthy controls for all 4 different collections. Levels of inflammatory cytokines were determined as described. Horizontal bars indicate the respective groupwise medians. Only those with a P value of < 0.05 and less are illustrated and considered to be statistically significant. *P values of <0.05; *P values of <0.01; *P values <0.001. Figure 9. Differences in other cytokine levels (pg/ml) in CHIKF patients and healthy controls for all 4 different collections. *P values of <0.05; *P values of <0.01; *P values <0.001 were all considered to be statistically significant.

Figure 10. Differences in chemokine levels (pg/ml) in CHIKF patients and healthy controls for all 4 different collections. *P values of <0.05; *P values of <0.01; *P values <0.001 were all considered to be statistically significant.

Figure 11. Differences in growth factor levels (pg/ml) in CHIKF patients and healthy controls for all 4 different collections. *P values of < 0.05; *P values of <0.01; *P values <0.001 were all considered to be statistically significant.

Figure 12. Illustration of all 19 immune mediators as indicators for biomarkers of determining disease progression of CHIKF from acute to chronic phase after onset of disease.

DETAILED DESCRIPTION

SEVERE CHIKUNGUNYA BIOMARKERS

We disclose a number of biomarkers for severe chikungunya infection. In particular, we disclose biomarkers for severe chikungunya, including acute severe chikungunya and chronic severe chikungunya.

We further for methods and assays which examine expression of one or more biomarkers in a tissue or cell sample, in which the expression of one or more such biomarkers is predictive of whether an individual from which the tissue or cell sample is derived is suffering from severe chikungunya infection.

The severe chikungunya biomarkers are shown in Table Dl below.

Table Dl. Production levels of severe chikungunya biomarkers in affected individuals. Legend: + high production; - low production

We therefore disclose the use of any one or more, such as a combination of, for example all of IL-lα, IL-I β, IL-2R, IL-4, IL-6, IL-7, IL-8, IFN-α, IL-12, IL-15, MCP-I, MIP- 1 α, Eotaxin, RANTES, IP- 10, MIG, EGF, FGF-b, G-CSF, HGF as biomarkers for severe chikungunya infection. These biomarkers may be referred to as "severe chikungunya biomarkers".

The severe chikungunya biomarkers listed above may be used as biomarkers for different phases of infection by chikungunya. These include the acute phase of infection and the chronic phase of infection.

The term "biomarker" as used in the present application refers generally to a molecule, including a gene, protein, carbohydrate structure, or glycolipid, the expression of which in or on or by a tissue or cell can be detected by standard methods (or methods disclosed in this document) and is predictive for a individual suffering from severe chikungunya infection, such as acute severe chikungunya and chronic severe chikungunya. The expression of such a biomarker may be determined to be higher or lower than that observed for a control tissue or cell sample. For example, the expression of such a biomarker may be determined in a PCR or FACS or immunoassay to be at least 1.1 -fold, such as at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or more higher in the test tissue or cell sample than that observed for a control tissue or cell sample.

It will be appreciated that not all of biomarkers listed above need be tested, and any subset of this set of proteins may be tested for the same purpose. Furthermore, it will be appreciated that expression profiles of one or more of the proteins within the set of biomarkers may be established, such as by microarray hybridisation or microbead immunoassays. Such profiles may be monitored to track the progression of the disease through different phases, for example from acute to chronic infection, and from mild to severe infection, or both.

We therefore disclose a method of determining the phase or severity (or both) of chikungunya infection in an individual, the method comprising establishing an expression profile comprising the level or activity or both of any one or more of the severe chikungunya biomarkers disclosed here, and comparing the expression profile thus obtained to an expression profile from an individual known to be suffering from a particular phase (e.g., acute or chronic) or severity (e.g., mild or severe) of chikungunya infection. If the expression profile is similar, then the individual concerned can be established as suffering from the relevant grade or severity of chikungunya infection.

We further describe the use of such expression profiles for tracking the progress of infection of chikungunya in an individual. The method may comprise establishing a plurality of expression profiles of an infected individual, by a method as described above. Such expression profiles may then be compared to expression profiles from individuals known to be suffering from different phases or severities of infection.

For example, we disclose the use of IL-I α, IL- lβ, IL-2R, IL-6, IL-7, IL-8, IFN-α, IL- 12, IL-15, MCP-I, Eotaxin, RANTES, IP-IO, MIG as biomarkers for severe acute chikungunya infection. Such biomarkers may be referred to as "severe acute chikungunya biomarkers".

We demonstrate that certain of these biomarkers display increased expression or protein levels in patients with severe acute chikungunya.

Accordingly, we disclose a method of detecting severe acute severe chikungunya in an individual by detecting an increased expression or protein level in any one or more, such as a combination, for example, all of IL-lα, IL-I β, IL-2R, IL-6, JL-I, IL-8, IFN-α, IL-12, IL-15, MCP-I, Eotaxin, RANTES, IP-10, MIG in individual.

As a particular example, we disclose the use of IL-I β, IL-6 and RANTES as biomarkers for severe acute severe chikungunya. Thus, detection of increased expression or levels of IL-I β or IL-6, or both, may be used to detect acute severe chikungunya.

We further demonstrate that certain of these biomarkers display reduced expression or protein levels in patients in severe chikungunya. Accordingly, we disclose a method of detecting severe acute severe chikungunya in an individual by detecting a reduced expression or protein level of RANTES in an individual.

Thus, detection of decreased expression or levels of RANTES may be used to detect acute severe chikungunya.

The above may be combined; thus, a method for detection of severe acute severe chikungunya may comprise detection of any one or more, such as all of: (a) increased expression or levels of IL-I β; (b) increased expression or levels of IL-6; (c) decreased expression or levels of RANTES.

IL-I β

IL-I β may comprise a polypeptide sequence having GenBank Accession Number NP_000567. It may comprise a nucleic acid sequence having GenBank Accession number NM_000576. It may comprise a variant, homologue, derivative or fragment of either such as a sequence having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology thereto and comprising (or encoding a sequence comprising) interleukin-lβ activity. IL-6

IL-6 may comprise a polypeptide sequence having GenBank Accession Number NP_000591. It may comprise a nucleic acid sequence having GenBank Accession number NM_000600. It may comprise a variant, homologue, derivative or fragment thereof such as a sequence having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology thereto and comprising (or encoding a sequence comprising) interleukin-6 activity.

RANTES

RANTES (syn. CCL5) may comprise a polypeptide sequence having GenBank Accession Number NP_002976. It may comprise a nucleic acid sequence having GenBank Accession number NM_002985. It may comprise a variant, homologue, derivative or fragment thereof such as a sequence having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology thereto and comprising (or encoding a sequence comprising) RANTES/CCL5 activity.

Other Severe Chikungunya Biomarkers Severe chikungunya biomarkers other than IL- 1 β, IL-6 and RANTES are described in this document generally and may comprise the polypeptide sequences having GenBank Accession Numbers as shown in Table Dl. They may comprise nucleic acid sequences capable of encoding such polypeptide sequences. They may comprise a variant, homologue, derivative or fragment of any such sequence such as a sequence having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology thereto. Such sequences may comprise (or encode a sequence comprising) activity of the severe chikungunya biomarker.

As noted above, for the detection of chronic severe chikungunya, we disclose the use of IL-lα, IL-lβ, IL-2R, IL-4, IL-6, IL-7, IL-8, IFN-α, IL-12, IL-15, MCP-I, MIP-Ia, Eotaxin, RANTES, IP-10, MIG, EGF, FGF-b, G-CSF, HGF as biomarkers for severe acute chikungunya infection. Such biomarkers may be referred to as "severe chronic chikungunya biomarkers".

The biomarkers disclosed here enable stratification of disease severity and early identification of patients with poor prognosis. The biomarkers can be used for diagnosis, prognosis and monitoring of chikungunya disease. In particular, we provide a method of prognosis of an individual with chikungunya infection, the method comprising detecting whether the individual has severe chikungunya by a method as described.

Each of the sets of biomarkers may be used for a specific purpose. For example, the expression levels of one or more of the biomarkers in a set may be tested in an individual. The individual may be suffering or suspected to be suffering from chikungunya. The expression level of the biomarker may be quantitated, for example, by a number of methods.

The level of expression of the biomarker may be compared to that of a reference, norm or standard. Such a reference, etc may comprise the level of expression of that biomarker in a control, which may comprise a cognate individual, group or population. The control may comprise one or more individuals who are not suffering from the condition to be tested, such as individuals not infected with chikungunya. It will be appreciated that where severe infection is being tested for, the controls may comprise non-severe individual(s).

It will also be appreciated that the reference need not be established at the time of testing, or be repeated for every instance of testing. Rather, the reference may be established beforehand and data relating to levels of expression stored for future use, for example in a database or table.

For example, referring to Table Dl above, column 3 and column 4 indicate whether the level of expression of the particular biomarker is increased or decreased in acute severe chikungunya, while column 5 indicates whether the level of expression of the particular biomarker is increased or decreased in chronic severe chikungunya. Column 6 indicates the minimum serum protein level of biomarker expected in severe chikungunya. Sampling an individual and measuring the level of one or more of the severe chikungunya biomarkers disclosed here, with reference to Table Dl, therefore enables the detection of severe chikungunya, severe acute severe chikungunya and servere chronic severe chikungunya.

The biomarkers demonstrated for use in severe chikungunya infection are disclosed in the following paragraphs. They will be referred to in this document as "severe chikungunya biomarkers". In other words, severe chikungunya biomarkers may include any one or more, such as any combination, for example all of IL-I α, IL-I β, IL-2, IL-6, IL-7, IL-8, IL- 12, IL-15, IFN-a, IP-IO, Eotaxin, MCP-I, MIG and RANTES. Severe chikungunya biomarkers may also include any one or more, such as any combination, for example all of IL-I a, IL- lβ, IL2R, IL- 4, IL-6, IL-7, IL-8, IL- 12, IL-15, IFN-a, IP-10, Eotaxin, MCP-I, MIG, MIP-Ia, Rantes, EGF, HGF, FGF-b and G-CSF.

Any combinations and subsets of any of the above are also possible and may be used for the detection of severe chikungunya.

Acute Infection - Initial Study In an initial study, we took the opportunity to conduct a detailed study on the patients from the first outbreak of CHIKF in Singapore [22,23].

We measured circulating levels of a wide range of cytokines, chemokines, and growth factors in 10 laboratory confirmed cases of CHIKF, and compared them with healthy individuals. We next determined which biomarker was associated with infection and/or severity.

We show for the first time that CHIKV infection induces a wide range of cytokines, chemokines, and growth factors. We subsequently found that 3 specific biomarkers, namely IL- lβ, IL-6, and RANTES levels, are associated with severe CHIKF.

We find that levels of IL-I β and IL-6 are higher in patients with severe acute chikungunya compared to control non-infected individuals. We also find that the levels of RANTES are lower in patients with severe acute chikungunya compared to control non- infected individuals.

We therefore provide a method of determining the severity of chikungunya, such as acute chikungunya infection, in an individual, through use of these markers.

Thus, for example, the level of expression of IL-I β may be determined in an individual known or suspected to be suffering from chikungunya. If the level of IL-I β is higher in that individual than a control non-infected individual, then that individual may be classed as suffering from acute severe chikungunya. Alternatively or in addition, the level of expression of IL-6 may be determined in an individual known or suspected to be suffering from chikungunya. If the level of IL-6 is higher in that individual than a control non-infected individual, then that individual may be classed as suffering from acute severe chikungunya. Alternatively or in addition, the level of expression of RANTES may be determined in an individual known or suspected to be suffering from chikungunya. If the level of IL-I β is lower in that individual than a control non-infected individual, then that individual may be classed as suffering from acute severe chikungunya.

The severe acute chikungunya biomarkers IL- lβ, IL-6, and RANTES may also be used for other purposes, such as prognosis, as described in further detail below.

Acute Infection - Subsequent Study A subsequent, more detailed study was conducted on a larger number patients for subsequent outbreaks of CHIKF in Singapore.

The longitudinal study was conducted in a similar manner to the initial study of acute infection described above, with the exception that a larger cohort of 30 patients were included in the longitudinal study. The study lasted 6 months, and four different collections were taken, representing four distinct phases of infection, i.e., acute phase (2 to 4 days after disease onset), late acute phase (7 to 10 days after disease onset), 14 days after disease onset and chronic phase (1 month to 3 months after disease onset).

We demonstrate using the data from the subsequent study that the protein level of each of IL-I α, IL- lβ, IL-2, IL-6, IL-7, IL-8, IL- 12, IL- 15, IFN-a, IP-IO, Eotaxin, MCP-I, MIG is higher in individuals suffering from acute severe chikungunya compared to control non- infected individuals. We show that the protein level of RANTES is lower in individuals suffering from acute severe chikungunya compared to control non-infected individuals.

Thus, the levels of expression of each or any combination of IL- lα, IL-I β, IL-2, IL-6,

IL-7, IL-8, IL-12, IL-15, IFN-a, IP-10, Eotaxin, MCP-I and MIG may be determined in an individual suffering or suspected to be suffering from chikungunya. Establishment of a higher level of any such protein or combination compared to that of control non-infected individuals indicates that the individual is suffering from severe acute chikungunya. It will be appreciated that not all of IL-lα, IL-I β, IL-2, IL-6, IL-7, IL-8, IL-12, IL-15, IFN-a, IP-IO, Eotaxin, MCP-I, MIG need be tested for detecting severe acute chikungunya, and any subset of this set of proteins may be tested for the same purpose. For example, levels of any one or more, such as all, of IL-lα, IL-lβ, IL-6, IFN-a, IL-7, IL-12. IL-15, IP-IO, MCP- 1 , MIG may be tested for the purpose of establishing acute severe chikungunya in an individual.

Chronic Infection - Subsequent Study

We demonstrate using the data from the subsequent study that the protein level of each of IL-lα, IL-lβ, IL2R, IL-4, IL-6, IL-7, IL-8, IL-12, IL-15, IFN-a, IP-IO, Eotaxin, MCP-I, MIG, MIP-Ia, Rantes, EGF, HGF, FGF-b, G-CSF is higher in individuals suffering from chronic severe chikungunya compared to control non-infected individuals.

We therefore provide a method of determining the severity of chikungunya, such as chronic chikungunya infection, in an individual, through use of these markers.

Thus, the levels of expression of each or any combination of IL-lα, IL-lβ, IL2R, IL-4, IL-6, IL-7, IL-8, IL-12, IL-15, IFN-a, IP-10, Eotaxin, MCP-I, MIG, MIP-Ia, Rantes, EGF, HGF, FGF-b, G-CSF may be determined in an individual suffering or suspected to be suffering from chikungunya. Establishment of a higher level of any such protein or combination compared to that of control non- infected individuals indicates that the individual is suffering from severe chronic chikungunya. It will be appreciated that not all of IL-lα, IL- lβ, IL2R, IL-4, IL-6, IL-7, IL-8, IL-12, IL-15, IFN-a, IP-10, Eotaxin, MCP-I, MIG, MIP-Ia, Rantes, EGF, HGF, FGF-b, G-CSF need be tested, and any subset of this set of proteins may be tested for the same purpose. For example, levels of any one or more, such as all, IL-lα, IL- lβ, IL2R, IL-4, IL-6, IL-7, IL-8, IL-12, IL-15, IFN-a, IP-10, Eotaxin, MCP-I, MIG, MIP-Ia, Rantes, EGF, HGF, FGF-b, G-CSF may be tested for the purpose of establishing chronic severe chikungunya in an individual.

CHIKUNGUNYA INFECTION

Severe and Non-Severe Chikungunya Infection

For the purposes of this document, a patient is described as having "severe" chikungunya infection if he or she exhibits (or is expected to exhibit) any one or more, such as all, of the following symptoms: (a) a temperature of > 38.5 ⁰C ; (b) pulse rate > 100/min, and (c) platelet count < 100x10⁹ g/L.

Similarly, for the purposes of this document, a patient has "mild" or "non-severe" chikungunya infection (or is decsribed as not having severe chikungunya infection) if he or she exhibits or is expected to exhibit any one or more, such as all, of the following symptoms: (a) a temperature of < 38.5 ⁰C ; (b) pulse rate < 100/min, and (c) platelet count > lOOxlO⁹ g/L.

Acute and Chronic Chikungunya Infection

For the purposes of this document, a patient is described as being in the "acute" phase of chikungunya infection if the chikungunya infection is in its early phases, such as within the first week, the first two weeks or the first three weeks of infection. For example, acute chikungunya may be used to refer to chikungunya infection within day 2 to day 19 of infection.

For the purposes of this document, a patient is described as being in the "chronic" phase of chikungunya infection if the chikungunya infection is in its later post-chronic phases, such as after the first week, after the first two weeks or after the first three weeks of infection. For example, acute chikungunya may be used to refer to chikungunya infection after day 19 of infection.

Chronic infection could refer to the infection phase after the first month of infection, after the first month and a half of infection, after the second month of infection, after the second month and a half of infection, after the third month of infection, after the third month and a half of infection, after the fourth month of infection, after the fouth month and a half of infection, after the fifth month of infection, after the fifth month and a half of infection, or after the sixth month of infection.

BiOMARKER COMBINATIONS We further provide for combinations of severe chikungunya biomarkers disclosed in this document. Such combinations may comprise mixtures of biomarker genes or corresponding probes, such as in a form which is suitable for detection of expression. For example, the combination may be provided in the form of DNA in solution. As used herein, a "probe" is an oligonucleotide that hybridizes specifically to a target sequence in a nucleic acid, preferably in an amplified nucleic acid, under conditions that promote hybridization, to form a detectable hybrid. A probe optionally may contain a detectable moiety which either may be attached to the end(s) of the probe or may be internal. The nucleotides of the probe which combine with the target polynucleotide need not be strictly contiguous, as may be the case with a detectable moiety internal to the sequence of the probe. Detection may either be direct (i.e., resulting from a probe hybridizing directly to the target sequence or amplified nucleic acid) or indirect (i.e., resulting from a probe hybridizing to an intermediate molecular structure that links the probe to the target sequence or amplified nucleic acid). The "target" of a probe generally refers to a sequence contained within an amplified nucleic acid sequence which hybridizes specifically to at least a portion of a probe oligonucleotide using standard hydrogen bonding (i.e., base pairing). A probe may comprise target-specific sequences and optionally other sequences that are non-complementary to the target sequence that is to be detected. These non-complementary sequences may comprise a promoter sequence, a restriction endonuclease recognition site, or sequences that contribute to three-dimensional conformation of the probe (e.g., as described in Lizardi et al., U.S. Pat. Nos. 5,118,801 and 5,312,728). Sequences that are "sufficiently complementary" allow stable hybridization of a probe oligonucleotide to a target sequence that is not completely complementary to the probe's target-specific sequence.

By "substantially complementary" is meant that the subject oligonucleotide has a base sequence containing an at least 10 contiguous base region that is at least 70% complementary, preferably at least 80% complementary, more preferably at least 90% complementary, and most preferably 100% complementary to an at least 10 contiguous base region present in a target nucleic acid sequence (excluding RNA and DNA equivalents). (Those skilled in the art will readily appreciate modifications that could be made to the hybridization assay conditions at various percentages of complementarity to permit hybridization of the oligonucleotide to the target sequence while preventing unacceptable levels of non-specific hybridization.) The degree of complementarity is determined by comparing the order of nucleobases making up the two sequences and does not take into consideration other structural differences which may exist between the two sequences, provided the structural differences do not prevent hydrogen bonding with complementary bases. The degree of complementarity between two sequences can also be expressed in terms of the number of base mismatches present in each set of at least 10 contiguous bases being compared, which may range from 0-2 base mismatches.

By "sufficiently complementary" is meant a contiguous nucleic acid base sequence that is capable of hybridizing to another base sequence by hydrogen bonding between a series of complementary bases. Complementary base sequences may be complementary at each position in the base sequence of an oligonucleotide using standard base pairing (e.g., G:C, A:T or A:U pairing) or may contain one or more residues that are not complementary using standard hydrogen bonding (including abasic "nucleotides"), but in which the entire complementary base sequence is capable of specifically hybridizing with another base sequence under appropriate hybridization conditions. Contiguous bases are preferably at least about 80%, more preferably at least about 90%, and most preferably about 100% complementary to a sequence to which an oligonucleotide is intended to specifically hybridize. Appropriate hybridization conditions are well known to those skilled in the art, can be predicted readily based on base sequence composition, or can be determined empirically by using routine testing (e.g., See Sambrook et al., Molecular Cloning, A Laboratory Manual, 2.sup.nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989) at .sctn..sctn. 1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57 particularly at 9.50-9.51, 11.12- 11.13, 11.45-11.47 and 11.55-11.57).

In other embodiments, a microarray or chip is provided which comprises any combination of biomarker genes or probes, in the form of cDNA, genomic DNA, or RNA, within the classifiers. In some embodiments, the microarray or chip comprises all the genes or probes in Table Dl, such as severe acute chikungunya biomarkers or severe chronic chikungunya biomarkers. The genes may be synthesised or obtained by means known in the art, and attached on the microarray or chip by conventional means, as known in the art. Such microarrays or chips are useful in monitoring gene expression of any one or more of the genes comprised therein, and may be used for chikungunya grading or detection as described here.

The probes or probe sets are suitably synthesised or made by means known in the art, for example by oligonucleotide synthesis, and may be attached to a microarray for easier carriage and storage. They may be used in a method of assigning a grade to a chikungunya infection as described herein. SAMPLE

The methods and compositions described here may be used for detecting severe chikungunya from an individual. This is conveniently done in the context of a sample from the individual. The sample may comprise a biological sample. The sample may be taken from an individual, which may be a human or animal.

The sample may comprise any number of things, including, but not limited to, bodily fluids (including, but not limited to, blood, nasopharyngeal secretions, urine, serum, lymph, saliva, anal and vaginal secretions, perspiration and semen, of virtually any organism, for example mammalian samples, environmental samples (including, but not limited to, air, agricultural, water and soil samples); biological warfare agent samples; research samples; purified samples, such as purified genomic DNA, RNA, proteins, etc.; raw samples (bacteria, virus, genomic DNA, etc. As will be appreciated by those in the art, virtually any, or no, experimental manipulation may have been done on the sample.

The sample may comprise a tissue or cell sample. By "tissue or cell sample" is meant a collection of similar cells obtained from a tissue of a subject or patient. The source of the tissue or cell sample may be solid tissue as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood constituents; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from any time in gestation or development of the subject. The tissue sample may also be primary or cultured cells or cell lines. The tissue sample may contain compounds which are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.

USE OF BIOMARKERS

Detection of expression of any one or more biomarkers (e.g., genes and proteins) as described in this document can be used to diagnose, or provide an indication useful in the diagnosis of, severe chikungunya infection, such as severe acute chikungunya infection or severe chronic chikungunya infection. Where the term "diagnosis" is used in this document, it should be taken to include prognosis as well as diagnosis.

In particular, detection of expression of any one or more biomarkers as described in this document can be used to detect severe chikungunya infection, such as severe acute chikungunya infection or severe chronic chikungunya infection, to distinguish severe chikungunya infection, such as severe acute chikungunya infection or severe chronic chikungunya infection from other diseases which may have the same or similar symptoms, and to predict the severity of disease.

We have established that it is possible to distinguish patients displaying the symptoms of chikungunya as suffering from severe chikungunya infection, such as severe acute chikungunya infection or severe chronic chikungunya infection, from patients displaying the symptoms of chikungunya, but who do not have severe chikungunya infection, such as severe acute chikungunya infection or severe chronic chikungunya infection.

For example, the level of expression or activity of a severe chikungunya biomarker may be detected to detect severe chikungunya infection, such as severe acute chikungunya infection or severe chronic chikungunya infection. The level of expression or activity of the severe chikungunya biomarker may be compared against an absolute level, such as disclosed at the column marked "Lowest Limit (pg/ml)" in Table Dl above.

For example, with regard to IL-lα and IL-lβ, a serum level of 17.57 pg/ml of IL-lα and IL-lβ may be used for example as a cut-off point. Thus, if a patient has a serum level of more than about 17.57 pg/ml of IL-lα/IL-lβ, then he is likely be actually suffering from severe chikungunya infection. It will be appreciated that the level of 17.57 pg/ml of IL-lα/IL- lβ is not an absolute figure, and that it is possible to use other levels, e.g, in the range of 16- 18 pg/ml of IL- 1 α, IL- 1 β as cut-offs, but with perhaps a lower level of accuracy.

As we have demonstrated that the biomarkers may be used as markers for the severity of chikungunya disease, they may be used for example to assess whether it is likely that a patient needs further treatment, e.g., hospitalisation.

The expression or activity of the genes and proteins described here may be detected together with other indicators of chikungunya disease, for example, body temperature, pulse, blood pressure, blood cell count, haematocrit, haemoglobin levels, etc.

Furthermore, as these genes are activated in patients with chikungunya disease, inhibiting these genes using compounds is likely to reduce virus production and relieve or treat the disease. We therefore provide for the use of any of these genes or corresponding proteins in the treatment or prevention of chikungunya. For example, activity or expression of the genes may be regulated for treating or preventing chikungunya. The genes or proteins may also be used as targets for drug development. We therefore provide for screens for molecules which bind to, agonise or antagonise any of these genes in the three pathways. Such screens may be used to identify molecules suitable for the treatment or prevention of chikungunya.

The term "agonist" is used in the broadest sense, and includes any molecule that partially or fully enhances, stimulates or activates one or more biological activities of severe chikungunya biomarker proteins, in vitro, in situ, or in vivo. Examples of biological activities of chikungunya biomarker proteins are known in the literature. An agonist may function in a direct or indirect manner. For instance, the agonist may function to partially or fully enhance, stimulate or activate one or more biological activities of chikungunya biomarker proteins, in vitro, in situ, or in vivo as a result of its direct binding to chikungunya biomarker proteins. The agonist may also function indirectly to partially or fully enhance, stimulate or activate one or more biological activities of chikungunya biomarker proteins, in vitro, in situ, or in vivo as a result of, e.g., stimulating another effector molecule which then causes chikungunya biomarker protein activation. It is contemplated that an agonist may act as an enhancer molecule which functions indirectly to enhance or increase chikungunya biomarker protein activity.

Similarly, the term "antagonist" is also used in the broadest sense, and includes any molecule that partially or fully reduces, down-regulates or inactivates one or more biological activities of severe chikungunya biomarker proteins, in vitro, in situ, or in vivo. An antagonist may function in a direct or indirect manner. For instance, the antagonist may function to partially or fully reduce, down-regulate or inactivate one or more biological activities of chikungunya biomarker proteins, in vitro, in situ, or in vivo as a result of its direct binding to chikungunya biomarker proteins. The antagonist may also function indirectly to partially or fully reduce, down-regulate or inactivate one or more biological activities of chikungunya biomarker proteins, in vitro, in situ, or in vivo as a result of, e.g., reducing, down-regulating or inactivating another effector molecule which then causes chikungunya biomarker protein inactivation, etc. Therefore, accurately targeting the genes described here as biomarkers of severe chikungunya using small molecules, or other drug mechanisms, to reduce their activation is likely to provide a therapy for chikungunya disease.

This invention shows the mechanisms to enable early, rapid and easy diagnosis and prognosis of severe chikungunya infection; together with novel human drug targets that would prevent chikungunya disease.

According to the methods and compositions described here, any protein or activity in the following group IL-lα, IL-I β, IL-2, IL-6, IL-7, IL-8, IL-12, IL-15, IFN-a, IP-IO, Eotaxin, MCP-I, MIG, may be modulated in order to reduce chikungunya viral function, which may be for the treatment or alleviation of acute severe chikungunya in an individual. Similarly, any protein or activity in the following group IL-lα, IL-I β, IL2R, IL-4, IL-6, IL-7, IL-8, IL-12, IL-15, IFN-a, IP-IO, Eotaxin, MCP-I, MIG, MIP-Ia, Rantes, EGF, HGF, FGF-b, G-CSF may be modulated (such as down-regulated) in order to reduce chikungunya viral function, which may be for the treatment or alleviation of acute severe chikungunya in an individual.

Specifically, chikungunya may be treated or prevented by modulating the level of expression of, or the activity of, or both of any of these proteins for the relevant purpose. Thus, for example, a compound capable of modulating IL-lα/IL-lβ may be used as a treatment for acute or chronic severe chikungunya. For example, the expression or activity or both of any of these proteins may be down-regulated for this purpose. Alternatively or in addition, the expression or activity of RANTES may be modulated, such as down-regulated, for this purpose.

Any component of the pathway that leads to the relevant protein activity in a cell may be modulated. Thus, the component may be modulated at the protein level, at the niRNA level, in translation, transcription, post-translational modification, etc so long as it modulates protein activity or in expression. In some embodiments, such as for RANTES, the activity of a relevant protein is down-regulated to disrupt viral function or to treat chikungunya infection.

The inhibitor or inhibitors of these genes may be used in combination with any agent which is known or suspected to be efficacious in treating or alleviating chikungunya. Examples include the compounds disclosed in United States Patent 7,514,436 to Gschwend, et al. The inhibitors, agonists, antagonists, etc for the treatment, prevention, alleviation and/or diagnosis of chikungunya may be packaged in a kit.

Such a kit may comprise means for the detection of a change in the expression pattern or level of any one or more of the following: (a) IL-lα, IL-lβ, IL-2, IL-6, IL-7, IL-8, IL-12, IL-15, IFN-a, IP-IO, Eotaxin, MCP-I, MIG, RANTES; or (b) IL-lα, IL-lβ, IL2R, IL-4, IL-6, IL-7, IL-8, IL-12, IL-15, IFN-a, IP-10, Eotaxin, MCP-I, MIG, MIP-Ia, Rantes, EGF, HGF, FGF-b, G-CSF, or both (a) and (b), together with instructions for use and may be suitable for detection, diagnosis or prognosis of chikungunya.

A further example of a kit may comprise means for modulating the level of expression of: (a) IL-lα, IL-lβ, IL-2, IL-6, IL-7, IL-8, IL-12, IL-15, IFN-a, IP-10, Eotaxin, MCP-I,

MIG, RANTES; or (b) IL-lα, IL-lβ, IL2R, IL-4, IL-6, IL-7, IL-8, IL-12, IL-15, IFN-a, IP-10, Eotaxin, MCP-I, MIG, MIP-Ia, Rantes, EGF, HGF, FGF-b, G-CSF, or both (a) and (b), together with instructions for use. Such a kit may be useful for treatment or prevention of chikungunya in an individual.

CHIKUNGUNYA

Chikungunya virus (CHIKV), which causes CHIKF, is an alphavirus of the Togaviridae family, with a 12,000-nucleotides linear, positive-sense, single-stranded RNA genome containing two large open reading frames (ORF).

The first, ORFl, encodes 4 non-structural proteins (nsPl, nsP2, nsP3 and nsP4) while ORF2 encodes structural proteins that include 1 capsid protein (C), 2 major envelope surface glycoproteins (El, E2) and 2 small proteins (E3, 6K) [8,9].

CHIKV is transmitted by Aedes mosquitoes (mainly A. albopictus and A aegypti).

CHIKF is an acute illness with abrupt fever, skin rash, arthralgia, and occasional involvement of the nervous system, heart and liver. Prolonged incapacitating arthralgia has sometimes been reported to persist for years [8,9,13].

It is of concern that the re-emerged CHIKV has caused considerable morbidity and some fatalities, whereas previously CHIKF was considered as relatively benign. Despite the fact that the clinical features of recent acute CHIKV infections from several countries have been described [16-18], little is known about the long-term sequelae or the pathogenesis of arthropathy, and the acquisition of protective immunity remains unexplored. It has been proposed that CHIKV-induced arthritis or arthralgia is of immunopathologic origin [19,20].

At present, there is no specific or effective treatment for CHIKF, and patient management is largely symptomatic relief and primarily anti-inflammatory drugs [8].

SEVERE CHIKUNGUNYA BIOMARKER POLYPEPTIDES

The methods and compositions described here make use of severe chikungunya biomarker polypeptides, which are described in detail below.

As used here, the term "severe chikungunya biomarker polypeptide" is intended to refer to a sequence having GenBank Accession number as shown in Table Dl .

Homologues variants and derivatives thereof of any , some or all of these polypeptides are also included.

A "polypeptide" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.

"Polypeptides" include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications.

Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-inking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-inks, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. See, for instance, Proteins - Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993 and Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al., "Analysis for protein modifications and nonprotein cofactors", Meth Enzymol (1990) 182:626-646 and Rattan et aL, "Protein Synthesis: Posttranslational Modifications and Aging", Ann NYAcadSci (1992) 663:48-62.

The term "polypeptide" includes the various synthetic peptide variations known in the art, such as a retroinverso D peptides. The peptide may be an antigenic determinant and/or a T-cell epitope. The peptide may be immunogenic in vivo. The peptide may be capable of inducing neutralising antibodies in vivo.

As applied to a severe chikungunya biomarker polypeptide, the resultant amino acid sequence may have one or more activities, such as biological activities in common with a severe chikungunya biomarker polypeptide polypeptide. In particular, the term "homologue" covers identity with respect to structure and/or function. With respect to sequence identity (i.e. similarity), there may be at least 70%, such as at least 75%, such as at least 85%, such as at least 90% sequence identity. There may be at least 95%, such as at least 98%, sequence identity. These terms also encompass polypeptides derived from amino acids which are allelic variations of the severe chikungunya biomarker nucleic acid sequence.

Where reference is made to the "activity" or "biological activity" of a polypeptide such as severe chikungunya biomarker, these terms are intended to refer to the metabolic or physiological function of severe chikungunya biomarker, including similar activities or improved activities or these activities with decreased undesirable side effects. Also included are antigenic and immunogenic activities of severe chikungunya biomarker. Examples of such activities, and methods of assaying and quantifying these activities, are known in the art, and are described in detail elsewhere in this document.

Other Severe Chikungunya Biomarker Polypeptides

Severe chikungunya biomarker variants, homologues, derivatives and fragments are also of use in the methods and compositions described here.

The terms "variant", "homologue", "derivative" or "fragment" in relation to severe chikungunya biomarker include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acid from or to a sequence. Unless the context admits otherwise, references to "severe chikungunya biomarker" includes references to such variants, homologues, derivatives and fragments of severe chikungunya biomarker having accession numbers shown in Table Dl.

As used herein a "deletion" is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent. As used herein an "insertion" or "addition" is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring substance. As used herein "substitution" results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.

Severe chikungunya biomarker polypeptides as described here may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent amino acid sequence. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

Conservative substitutions may be made, for example according to the table below. Amino acids in the same block in the second column and in the same line in the third column may be substituted for each other:

Severe chikungunya biomarker polypeptides may further comprise heterologous amino acid sequences, typically at the N-terminus or C-terminus, such as the N-terminus. Heterologous sequences may include sequences that affect intra or extracellular protein targeting (such as leader sequences). Heterologous sequences may also include sequences that increase the immunogenicity of the severe chikungunya biomarker polypeptide and/or which facilitate identification, extraction and/or purification of the polypeptides. Another heterologous sequence that may be used is a polyamino acid sequence such as polyhistidine which may be N-terminal. A polyhistidine sequence of at least 10 amino acids, such as at least 17 amino acids but fewer than 50 amino acids may be employed.

The severe chikungunya biomarker polypeptides may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro- sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production.

Severe chikungunya biomarker polypeptides as described here are advantageously made by recombinant means, using known techniques. However they may also be made by synthetic means using techniques well known to skilled persons such as solid phase synthesis. Such polypeptides may also be produced as fusion proteins, for example to aid in extraction and purification. Examples of fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and β-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences, such as a thrombin cleavage site. The fusion protein may be one which does not hinder the function of the protein of interest sequence.

The severe chikungunya biomarker polypeptides may be in a substantially isolated form. This term is intended to refer to alteration by the hand of man from the natural state. If an "isolated" composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide, nucleic acid or a polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide, nucleic acid or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein.

It will however be understood that the severe chikungunya biomarker protein may be mixed with carriers or diluents which will not interfere with the intended purpose of the protein and still be regarded as substantially isolated. A severe chikungunya biomarker polypeptide may also be in a substantially purified form, in which case it will generally comprise the protein in a preparation in which more than 90%, for example, 95%, 98% or 99% of the protein in the preparation is a severe chikungunya biomarker polypeptide.

By aligning severe chikungunya biomarker sequences from different species, it is possible to determine which regions of the amino acid sequence are conserved between different species ("homologous regions"), and which regions vary between the different species ("heterologous regions").

The severe chikungunya biomarker polypeptides may therefore comprise a sequence which corresponds to at least part of a homologous region. A homologous region shows a high degree of homology between at least two species. For example, the homologous region may show at least 70%, at least 80%, at least 90% or at least 95% identity at the amino acid level using the tests described above. Peptides which comprise a sequence which corresponds to a homologous region may be used in therapeutic strategies as explained in further detail below. Alternatively, the severe chikungunya biomarker peptide may comprise a sequence which corresponds to at least part of a heterologous region. A heterologous region shows a low degree of homology between at least two species. Severe Chikungunya Biomarker Homologues

The severe chikungunya biomarker polypeptides disclosed for use include homologous sequences obtained from any source, for example related viral/bacterial proteins, cellular homologues and synthetic peptides, as well as variants or derivatives thereof. Thus polypeptides also include those encoding homologues of severe chikungunya biomarker from other species including animals such as mammals (e.g. mice, rats or rabbits), especially primates, more especially humans. More specifically, homologues include human homologues.

In the context of this document, a homologous sequence is taken to include an amino acid sequence which is at least 15, 20, 25, 30, 40, 50, 60, 70, 80 or 90% identical, such as at least 95 or 98% identical at the amino acid level, for example over at least 50 or 100, 110, 115, 120, 125, 130, 135, 140, 141, 142, 143, 144, 145, 146, 147, 148 or 149 or more amino acids with the sequence of a relevant severe chikungunya biomarker sequence.

In particular, homology should typically be considered with respect to those regions of the sequence known to be essential for protein function rather than non-essential neighbouring sequences. This is especially important when considering homologous sequences from distantly related organisms.

Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present document homology may be expressed in terms of sequence identity.

Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These publicly and commercially available computer programs can calculate % identity between two or more sequences.

% identity may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues (for example less than 50 contiguous amino acids). Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local identity or similarity.

However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps.

Most alignment programs allow the gap penalties to be modified. However, the default values may be used when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package (see below) the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A; Devereux et al, 1984, Nucleic Acids Research 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al, 1999 ibid - Chapter 18), FASTA (Altschul et al, 1990, J. MoI. Biol., 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al, 1999 ibid, pages 7-58 to 7- 60). The GCG Bestfit program may be used.

Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). The public default values for the GCG package may be used, or in the case of other software, the default matrix, such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible to calculate % homology, such as % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

The terms "variant" or "derivative" in relation to amino acid sequences includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acids from or to the sequence providing the resultant amino acid sequence retains substantially the same activity as the unmodified sequence, such as having at least the same activity as the severe chikungunya biomarker polypeptides.

Polypeptides having the severe chikungunya biomarker amino acid sequence disclosed here, or fragments or homologues thereof may be modified for use in the methods and compositions described here. Typically, modifications are made that maintain the biological activity of the sequence. Amino acid substitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30 substitutions provided that the modified sequence retains the biological activity of the unmodified sequence. Alternatively, modifications may be made to deliberately inactivate one or more functional domains of the polypeptides described here. Amino acid substitutions may include the use of non-naturally occurring analogues, for example to increase blood plasma half-life of a therapeutically administered polypeptide.

Severe Chikungunya Biomarker Polypeptide Fragments Polypeptides for use in the methods and compositions described here also include fragments of the full length sequence of any of severe chikungunya biomarker polypeptides identified above. Fragments may comprise at least one epitope. Methods of identifying epitopes are well known in the art. Fragments will typically comprise at least 6 amino acids, such as at least 10, 20, 30, 50 or 100 amino acids. Included are fragments comprising or consisting of, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or more residues from a relevant severe chikungunya biomarker amino acid sequence.

We further describe peptides comprising a portion of a severe chikungunya biomarker polypeptide as described here. Thus, fragments of severe chikungunya biomarker and its homologues, variants or derivatives are included. The peptides may be between 2 and 200 amino acids, such as between 4 and 40 amino acids in length. The peptide may be derived from a severe chikungunya biomarker polypeptide as disclosed here, for example by digestion with a suitable enzyme, such as trypsin. Alternatively the peptide, fragment, etc may be made by recombinant means, or synthesised synthetically.

Such severe chikungunya biomarker fragments may be used to generate probes to preferentially detect severe chikungunya biomarker expression, for example, through antibodies generated against such fragments. These antibodies would be expected to bind specifically to severe chikungunya biomarker, and are useful in the methods of diagnosis and treatment disclosed here.

Severe chikungunya biomarkers and their fragments, homologues, variants and derivatives, may be made by recombinant means. However they may also be made by synthetic means using techniques well known to skilled persons such as solid phase synthesis. The proteins may also be produced as fusion proteins, for example to aid in extraction and purification. Examples of fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and β-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. The fusion protein may be one which will not hinder the function of the protein of interest sequence. Proteins may also be obtained by purification of cell extracts from animal cells.

The severe chikungunya biomarker polypeptides, variants, homologues, fragments and derivatives disclosed here may be in a substantially isolated form. It will be understood that such polypeptides may be mixed with carriers or diluents which will not interfere with the intended purpose of the protein and still be regarded as substantially isolated. A severe chikungunya biomarker variant, homologue, fragment or derivative may also be in a substantially purified form, in which case it will generally comprise the protein in a preparation in which more than 90%, e.g. 95%, 98% or 99% of the protein in the preparation is a protein.

The severe chikungunya biomarker polypeptides, variants, homologues, fragments and derivatives disclosed here may be labelled with a revealing label. The revealing label may be any suitable label which allows the polypeptide, etc to be detected. Suitable labels include radioisotopes, e.g. ¹²⁵I, enzymes, antibodies, polynucleotides and linkers such as biotin. Labelled polypeptides may be used in diagnostic procedures such as immunoassays to determine the amount of a polypeptide in a sample. Polypeptides or labelled polypeptides may also be used in serological or cell-mediated immune assays for the detection of immune reactivity to said polypeptides in animals and humans using standard protocols.

A severe chikungunya biomarker polypeptides, variants, homologues, fragments and derivatives disclosed here, optionally labelled, may also be fixed to a solid phase, for example the surface of an immunoassay well or dipstick. Such labelled and/or immobilised polypeptides may be packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like. Such polypeptides and kits may be used in methods of detection of antibodies to the polypeptides or their allelic or species variants by immunoassay.

Immunoassay methods are well known in the art and will generally comprise: (a) providing a polypeptide comprising an epitope bindable by an antibody against said protein; (b) incubating a biological sample with said polypeptide under conditions which allow for the formation of an antibody-antigen complex; and (c) determining whether antibody-antigen complex comprising said polypeptide is formed.

The severe chikungunya biomarker polypeptides, variants, homologues, fragments and derivatives disclosed here may be used in in vitro or in vivo cell culture systems to study the role of their corresponding genes and homologues thereof in cell function, including their function in disease. For example, truncated or modified polypeptides may be introduced into a cell to disrupt the normal functions which occur in the cell. The polypeptides may be introduced into the cell by in situ expression of the polypeptide from a recombinant expression vector (see below). The expression vector optionally carries an inducible promoter to control the expression of the polypeptide.

The use of appropriate host cells, such as insect cells or mammalian cells, is expected to provide for such post-translational modifications (e.g. myristolation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products. Such cell culture systems in which the severe chikungunya biomarker polypeptides, variants, homologues, fragments and derivatives disclosed here are expressed may be used in assay systems to identify candidate substances which interfere with or enhance the functions of the polypeptides in the cell.

SEVERE CHIKUNGUNYA BIOMARKER NUCLEIC ACIDS

The methods and compositions described here may employ, as a means for detecting expression levels of severe chikungunya biomarker, severe chikungunya biomarker polynucleotides, severe chikungunya biomarker nucleotides and severe chikungunya biomarker nucleic acids, as well as variants, homologues, derivatives and fragments of any of these. In addition, we disclose particular severe chikungunya biomarker fragments useful for the methods of diagnosis described here. The severe chikungunya biomarker nucleic acids may also be used for the methods of treatment or prophylaxis described.

The terms "severe chikungunya biomarker polynucleotide", "severe chikungunya biomarker nucleotide" and "severe chikungunya biomarker nucleic acid" may be used interchangeably, and should be understood to specifically include both cDNA and genomic severe chikungunya biomarker sequences. These terms are also intended to include a nucleic acid sequence capable of encoding a severe chikungunya biomarker polypeptide and/or a fragment, derivative, homologue or variant of this.

Where reference is made to a severe chikungunya biomarker nucleic acid, this should be taken as a reference to any severe chikungunya biomarker nucleic acids, for example those having accession numbers shown in Table Dl, or capable of encoding the sequences having accession numbers shown in Table Dl. Also included are any one or more of the nucleic acid sequences set out as "Other severe chikungunya biomarker nucleic acid sequences" below.

"Polynucleotide" generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides" include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications has been made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.

It will be understood by the skilled person that numerous nucleotide sequences can encode the same polypeptide as a result of the degeneracy of the genetic code.

As used herein, the term "nucleotide sequence" refers to nucleotide sequences, oligonucleotide sequences, polynucleotide sequences and variants, homologues, fragments and derivatives thereof (such as portions thereof). The nucleotide sequence may be DNA or RNA of genomic or synthetic or recombinant origin which may be double-stranded or single- stranded whether representing the sense or antisense strand or combinations thereof. The term nucleotide sequence may be prepared by use of recombinant DNA techniques (for example, recombinant DNA).

The term "nucleotide sequence" may means DNA.

Other Nucleic Acids

We also provide nucleic acids which are fragments, homologues, variants or derivatives of severe chikungunya biomarker nucleic acids. The terms "variant", "homologue", "derivative" or "fragment" in relation to severe chikungunya biomarker nucleic acid include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acids from or to the sequence of a severe chikungunya biomarker nucleotide sequence. Unless the context admits otherwise, references to "severe chikungunya biomarker" include references to such variants, homologues, derivatives and fragments of severe chikungunya biomarker.

The resultant nucleotide sequence may encode a polypeptide having any one or more severe chikungunya biomarker activity. The term "homologue" may be intended to cover identity with respect to structure and/or function such that the resultant nucleotide sequence encodes a polypeptide which has severe chikungunya biomarker activity. For example, a homologue etc of severe chikungunya biomarker may have a increased expression level in an individual suffering from severe chikungunya compared to a normal individual. With respect to sequence identity (i.e. similarity), there may be at least 70%, at least 75%, at least 85% or at least 90% sequence identity. There may be at least 95%, such as at least 98%, sequence identity to a relevant sequence (e.g., a severe chikungunya biomarker sequence having a GenBank accession number shown in Table Dl or capable of encoding such a sequence). These terms also encompass allelic variations of the sequences.

Variants, Derivatives and Homologues

Severe chikungunya biomarker nucleic acid variants, fragments, derivatives and homologues may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of this document, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest.

Where the polynucleotide is double-stranded, both strands of the duplex, either individually or in combination, are encompassed by the methods and compositions described here. Where the polynucleotide is single-stranded, it is to be understood that the complementary sequence of that polynucleotide is also included. The terms "variant", "homologue" or "derivative" in relation to a nucleotide sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence. Said variant, homologues or derivatives may code for a polypeptide having biological activity. Such fragments, homologues, variants and derivatives of severe chikungunya biomarker may comprise modulated activity, as set out above.

As indicated above, with respect to sequence identity, a "homologue" may have at least 5% identity, at least 10% identity, at least 15% identity, at least 20% identity, at least 25% identity, at least 30% identity, at least 35% identity, at least 40% identity, at least 45% identity, at least 50% identity, at least 55% identity, at least 60% identity, at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to the relevant sequence (e.g., a severe chikungunya biomarker sequence having GenBank accession number shown in Table Dl or capable of encoding such a sequence).

There may be at least 95% identity, at least 96% identity, at least 97% identity, at least

98% identity or at least 99% identity. Nucleotide identity comparisons may be conducted as described above. A sequence comparison program which may be used is the GCG Wisconsin Bestfit program described above. The default scoring matrix has a match value of 10 for each identical nucleotide and -9 for each mismatch. The default gap creation penalty is -50 and the default gap extension penalty is -3 for each nucleotide.

Hybridisation

We further describe nucleotide sequences that are capable of hybridising selectively to any of the sequences presented herein, or any variant, fragment or derivative thereof, or to the complement of any of the above. Nucleotide sequences may be at least 15 nucleotides in length, such as at least 20, 30, 40 or 50 nucleotides in length.

The term "hybridization" as used herein shall include "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" as well as the process of amplification as carried out in polymerase chain reaction technologies.

Polynucleotides capable of selectively hybridising to the nucleotide sequences presented herein, or to their complement, may be at least 40% homologous, at least 45% homologous, at least 50% homologous, at least 55% homologous, at least 60% homologous, at least 65% homologous, at least 70% homologous, at least 75% homologous, at least 80% homologous, at least 85% homologous, at least 90% homologous, or at least 95% homologous to the corresponding nucleotide sequences presented herein (e.g., a severe chikungunya biomarker sequence having GenBank accession number shown in Table Dl or capable of encoding such a sequence). Such polynucleotides may be generally at least 70%, at least 80 or 90% or at least 95% or 98% homologous to the corresponding nucleotide sequences over a region of at least 20, such as at least 25 or 30, for instance at least 40, 60 or 100 or more contiguous nucleotides.

The term "selectively hybridizable" means that the polynucleotide used as a probe is used under conditions where a target polynucleotide is found to hybridize to the probe at a level significantly above background. The background hybridization may occur because of other polynucleotides present, for example, in the cDNA or genomic DNA library being screening. In this event, background implies a level of signal generated by interaction between the probe and a non-specific DNA member of the library which is less than 10 fold, such as less than 100 fold as intense as the specific interaction observed with the target DNA. The intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with ³ P or ³³P or with non-radioactive probes (e.g., fluorescent dyes, biotin or digoxigenin).

Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning

Techniques, Methods in Enzymology, VoI 152, Academic Press, San Diego CA), and confer a defined "stringency" as explained below.

Maximum stringency typically occurs at about Tm-5°C (5⁰C below the Tm of the probe); high stringency at about 5⁰C to 10⁰C below Tm; intermediate stringency at about 10⁰C to 2O⁰C below Tm; and low stringency at about 20⁰C to 25°C below Tm. As will be understood by those of skill in the art, a maximum stringency hybridization can be used to identify or detect identical polynucleotide sequences while an intermediate (or low) stringency hybridization can be used to identify or detect similar or related polynucleotide sequences.

We provide nucleotide sequences that may be able to hybridise to the severe chikungunya biomarker nucleic acids, fragments, variants, homologues or derivatives under stringent conditions (e.g. 65°C and O.lxSSC (IxSSC = 0.15 M NaCl, 0.015 M Na₃ Citrate pH 7.0)).

Generation ofHomologues, Variants and Derivatives

Polynucleotides which are not 100% identical to the relevant sequences (e.g., a severe chikungunya biomarker sequence having GenBank accession number shown in Table Dl or capable encoding such a sequencw) but which are also included, as well as homologues, variants and derivatives of severe chikungunya biomarker can be obtained in a number of ways. Other variants of the sequences may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations. For example, severe chikungunya biomarker homologues may be identified from other individuals, or other species. Further recombinant severe chikungunya biomarker nucleic acids and polypeptides may be produced by identifying corresponding positions in the homologues, and synthesising or producing the molecule as described elsewhere in this document.

In addition, other viral/bacterial, or cellular homologues of severe chikungunya biomarker, particularly cellular homologues found in mammalian cells (e.g. rat, mouse, bovine and primate cells), may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to human severe chikungunya biomarker. Such homologues may be used to design non-human severe chikungunya biomarker nucleic acids, fragments, variants and homologues. Mutagenesis may be carried out by means known in the art to produce further variety.

Sequences of severe chikungunya biomarker homologues may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of any of the severe chikungunya biomarker nucleic acids, fragments, variants and homologues, or other fragments of severe chikungunya biomarker under conditions of medium to high stringency.

Similar considerations apply to obtaining species homologues and allelic variants of the polypeptide or nucleotide sequences disclosed here.

Variants and strain/species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the severe chikungunya biomarker nucleic acids. Conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example the GCG Wisconsin PiIeUp program is widely used.

The primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences. It will be appreciated by the skilled person that overall nucleotide homology between sequences from distantly related organisms is likely to be very low and thus in these situations degenerate PCR may be the method of choice rather than screening libraries with labelled fragments the severe chikungunya biomarker sequences.

In addition, homologous sequences may be identified by searching nucleotide and/or protein databases using search algorithms such as the BLAST suite of programs.

Alternatively, such polynucleotides may be obtained by site directed mutagenesis of characterised sequences, for example, severe chikungunya biomarker nucleic acids, or variants, homologues, derivatives or fragments thereof. This may be useful where for example silent codon changes are required to sequences to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.

The polynucleotides described here may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors. Such primers, probes and other fragments will be at least 8, 9, 10, or 15, such as at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term "polynucleotides" as used herein.

Polynucleotides such as a DNA polynucleotides and probes may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques. In general, primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.

Primers comprising fragments of severe chikungunya biomarker are particularly useful in the methods of detection of severe chikungunya biomarker expression, such as up- regulation of severe chikungunya biomarker expression, for example, as associated with severe chikungunya infection. Suitable primers for amplification of severe chikungunya biomarker may be generated from any suitable stretch of severe chikungunya biomarker sequence. Primers which may be used include those capable of amplifying a sequence of severe chikungunya biomarker which is specific.

Although severe chikungunya biomarker primers may be provided on their own, they are most usefully provided as primer pairs, comprising a forward primer and a reverse primer.

Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides), bringing the primers into contact with mRN A or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector

Polynucleotides or primers may carry a revealing label. Suitable labels include radioisotopes such as ³²P or ³⁵S, digoxigenin, fluorescent dyes, enzyme labels, or other protein labels such as biotin. Such labels may be added to polynucleotides or primers and may be detected using by techniques known per se. Polynucleotides or primers or fragments thereof labelled or unlabeled may be used by a person skilled in the art in nucleic acid-based tests for detecting or sequencing polynucleotides in the human or animal body.

Such tests for detecting generally comprise bringing a biological sample containing DNA or RNA into contact with a probe comprising a polynucleotide or primer under hybridising conditions and detecting any duplex formed between the probe and nucleic acid in the sample. Such detection may be achieved using techniques such as PCR or by immobilising the probe on a solid support, removing nucleic acid in the sample which is not hybridised to the probe, and then detecting nucleic acid which has hybridised to the probe. Alternatively, the sample nucleic acid may be immobilised on a solid support, and the amount of probe bound to such a support can be detected. Suitable assay methods of this and other formats can be found in for example WO89/03891 and WO90/13667.

Tests for sequencing nucleotides, for example, the severe chikungunya biomarker nucleic acids, involve bringing a biological sample containing target DNA or RNA into contact with a probe comprising a polynucleotide or primer under hybridising conditions and determining the sequence by, for example the Sanger dideoxy chain termination method (see Sambrook et ah).

Such a method generally comprises elongating, in the presence of suitable reagents, the primer by synthesis of a strand complementary to the target DNA or RNA and selectively terminating the elongation reaction at one or more of an A, C, G or TYU residue; allowing strand elongation and termination reaction to occur; separating out according to size the elongated products to determine the sequence of the nucleotides at which selective termination has occurred. Suitable reagents include a DNA polymerase enzyme, the deoxynucleotides dATP, dCTP, dGTP and dTTP, a buffer and ATP. Dideoxynucleotides are used for selective termination.

SCREENING ASSAYS Any one or more of the severe chikungunya biomarkers disclosed here, including homologues, variants, and derivatives, whether natural or recombinant, may be employed in a screening process for compounds which bind the protein and which activate (agonists) or inhibit activation of (antagonists) of the protein. Antagonists of the biomarker may be referred to as "anti-severe chikungunya biomarker agents". Such agonists and antagonists may be used in the treatment, prevention or alleviation of chikungunya.

Thus, the severe chikungunya biomarkers may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See Coligan et al., Current Protocols in Immunology l(2):Chapter 5 (1991). As described herein, inhibitors of any of the severe chikungunya biomarkers proteins may be used to target chikungunya viral function, and for the treatment or alleviation of symptoms of chikungunya fever.

Accordingly, it is desirous to find compounds and drugs which can stimulate (agonists) or inhibit (antagonists) the function of severe chikungunya biomarker proteins. In general, agonists and antagonists are employed for therapeutic and prophylactic purposes for chikungunya infection. Thus, antagonists of all the severe chikungunya biomarker proteins described here (with the exception of RANTES) may be used to treat severe chikungunya. Similarly, agonists of RANTES may be used to treat severe chikungunya.

An agonist may activate the severe chikungunya biomarker (except for RANTES) to any degree. Similarly, an antagonist may deactivate, or inhibit the activation of RANTES protein to any degree. The protein may therefore be deactivated partially to any degree to its inherent, basal or background level of activity by an antagonist (partial antagonist) or fully to such a level (antagonist or full antagonist). The antagonist may deactivate the protein even further, for example to zero activity (inverse agonist). The term "antagonist" therefore specifically includes both full antagonists, partial antagonists and inverse agonists.

Also included within the terms "agonist" and "antagonist" are those molecules which modulate the expression of a severe chikungunya biomarker protein, as the case may be, at the transcriptional level and / the translational level, as well as those which modulate its activity.

Rational design of candidate compounds likely to be able to interact with a severe chikungunya biomarker may be based upon structural studies of the molecular shapes of a polypeptide. One means for determining which sites interact with specific other proteins is a physical structure determination, e.g., X-ray crystallography or two-dimensional NMR techniques. These will provide guidance as to which amino acid residues form molecular contact regions. For a detailed description of protein structural determination, see, e.g., Blundell and Johnson (1976) Protein Crystallography, Academic Press, New York.

An alternative to rational design uses a screening procedure which involves in general allowing a severe chikungunya biomarker protein to contact a candidate modulator and detecting an effect thereof. In general, such a method comprises producing appropriate cells which express the relevant protein or polypeptide on the surface thereof, optionally together with a partner protein, and contacting the protein or the cell or both with a candidate modulator, and detecting a change in the intracellular level of a relevant molecule.

A candidate compound may be tested for the ability to inhibit an activity of a severe chikungunya biomarker protein. For example, a candidate molecule may be assayed by assaying its effect on proteasome activity. Such assays may make use of specific substrates, for example, the substrates provided in Proteasome Inhibitor Set (Calbiochem Cat. No. 539164), which contains 1 mg Proteasome Inhibitor I (Cat. No. 539160), 1 mg MG-132 (Cat. No. 474790), and 200 μg Lactacystin (Cat. No. 426100). A kit for assaying activity of the 26S proteasome is available as Cat. No. 539159 from Calbiochem (San Diego, USA).

ANTIBODY BASED ASSAY

Molecules whose concentrations are affected by activity of severe chikungunya biomarker proteins, and which may be used as markers for detecting protein activity, are known in the art. These are referred to for convenience as "protein sensitive markers", and these may be detected as a means of detecting activity of the relevant protein.

Cells which may be used for the screen may be of various types. Such cells include cells from animals, yeast, Drosophila or E. coli. Cells expressing the severe chikungunya biomarker protein are then contacted with a test compound to observe binding, or stimulation or inhibition of a functional response.

Instead of testing each candidate compound individually with the severe chikungunya biomarker protein, a library or bank of candidate molecules may advantageously be produced and screened.

Where the candidate compounds are proteins, in particular antibodies or peptides, libraries of candidate compounds may be screened using phage display techniques. Phage display is a protocol of molecular screening which utilises recombinant bacteriophage. The technology involves transforming bacteriophage with a gene that encodes one compound from the library of candidate compounds, such that each phage or phagemid expresses a particular candidate compound. The transformed bacteriophage (which may be tethered to a solid support) expresses the appropriate candidate compound and displays it on their phage coat. Specific candidate compounds which are capable of binding to a severe chikungunya biomarker protein, polypeptide or peptide are enriched by selection strategies based on affinity interaction. The successful candidate agents are then characterised. Phage display has advantages over standard affinity screening technologies. The phage surface displays the candidate agent in a three dimensional configuration, more closely resembling its naturally occurring conformation. This allows for more specific and higher affinity binding for screening purposes.

Another method of screening a library of compounds utilises eukaryotic or prokaryotic host cells which are stably transformed with recombinant DNA molecules expressing a library of compounds. Such cells, either in viable or fixed form, can be used for standard binding- partner assays. See also Parce et al. (1989) Science 246:243-247; and Owicki et al. (1990) Proc. Nat'l Acad. Sci. USA 87;4007-4011 , which describe sensitive methods to detect cellular responses. Competitive assays are particularly useful, where the cells expressing the library of compounds are contacted or incubated with a labelled antibody known to bind to a severe chikungunya biomarker protein, such as ¹²⁵I-antibody, and a test sample such as a candidate compound whose binding affinity to the binding composition is being measured. The bound and free labelled binding partners for the polypeptide are then separated to assess the degree of binding. The amount of test sample bound is inversely proportional to the amount of labelled antibody binding to the polypeptide.

Any one of numerous techniques can be used to separate bound from free binding partners to assess the degree of binding. This separation step could typically involve a procedure such as adhesion to filters followed by washing, adhesion to plastic following by washing, or centrifugation of the cell membranes.

Still another approach is to use solubilized, unpurified or solubilized purified polypeptide or peptides, for example extracted from transformed eukaryotic or prokaryotic host cells. This allows for a "molecular" binding assay with the advantages of increased specificity, the ability to automate, and high drug test throughput.

Another technique for candidate compound screening involves an approach which provides high throughput screening for new compounds having suitable binding affinity, e.g., to a severe chikungunya biomarker protein, and is described in detail in International Patent application no. WO 84/03564 (Commonwealth Serum Labs.), published on September 13 1984. First, large numbers of different small peptide test compounds are synthesized on a solid substrate, e.g., plastic pins or some other appropriate surface; see Fodor et al. (1991). Then all the pins are reacted with solubilized polypeptide and washed. The next step involves detecting bound polypeptide. Compounds which interact specifically with the polypeptide will thus be identified.

The assays may simply test binding of a candidate compound wherein adherence to the cells bearing the relevant protein is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor. Further, these assays may test whether the candidate compound results in a signal generated by activation of the protein, using detection systems appropriate to the cells bearing the protein. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed.

Further, the assays may simply comprise the steps of mixing a candidate compound with a solution containing a severe chikungunya biomarker protein to form a mixture, measuring severe chikungunya biomarker protein activity in the mixture, and comparing the protein activity of the mixture to a standard.

The severe chikungunya biomarker protein cDNA, protein and antibodies to the protein may also be used to configure assays for detecting the effect of added compounds on the production of the relevant mRNA and protein in cells. For example, an ELISA may be constructed for measuring secreted or cell associated levels of severe chikungunya biomarker protein using monoclonal and polyclonal antibodies by standard methods known in the art, and this can be used to discover agents which may inhibit or enhance the production of severe chikungunya biomarker protein (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues. Standard methods for conducting screening assays are well understood in the art.

The screening assays may be conducted in vitro, as described above, or in vivo. In vivo assays may in particular be conducted using cells or suitable animal models for chikungunya.

Any of the suitable animal models described in these documents may be used for the screening assays described here. An example of such an assay employs administering a candidate molecule to an animal suffering from chikungunya, and detecting a change in a parameter indicative of chikungunya infection or progression, such as viral load, viral replication, any symptom of chikungunya etc. Candidate molecules which alleviate or reduce chikungunya symptoms, including viral replication, may be useful as drugs for the prevention, alleviation or treatment of chikungunya.

Examples of potential severe chikungunya biomarker protein antagonists include small molecules, antibodies or, in some cases, nucleotides and their analogues, including purines and purine analogues, oligonucleotides or proteins.

We therefore also provide a compound capable of binding specifically to a severe chikungunya biomarker protein and/or peptide.

The term "compound" or "agent" may be used to refer to a chemical compound (naturally occurring or synthesised), such as a biological macromolecule (e.g., nucleic acid, protein, non-peptide, or organic molecule), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues, or even an inorganic element or molecule. The compound may be an antibody.

The materials necessary for such screening to be conducted may be packaged into a screening kit. Such a screening kit is useful for identifying agonists and antagonists, for severe chikungunya biomarker proteins or compounds which decrease or enhance the production of such polypeptides. The screening kit comprises one or more severe chikungunya biomarker proteins. The screening kit may optionally comprise instructions for use.

DETECTION OF EXPRESSION OF SEVERE CHIKUNGUNYA BIOMARKERS

Expression of severe chikungunya biomarkers in individuals affected by severe chikungunya disease is modulated (i.e., up-regulated or modulated, e.g., RANTES) when compared to unaffected individuals.

Accordingly, we provide for a method of diagnosis of severe chikungunya disease, including acute or chornic severe chikungunya, comprising detecting modulation of expression of any one or more of the severe chikungunya biomarkers, in a sample of an individual. The method may comprise use of the anti-severe chikungunya biomarker antibodies described in this document. The anti- severe chikungunya biomarker antibodies may be used in immunoassays to detect and assay the quantity of severe chikungunya biomarker in a biological sample, and hence provide an indication of the level of expression of severe chikungunya biomarker in a cell, tissue, organ or individual from which the sample is derived. Immunoassays include ELISA, Western Blot, etc, and methods of employing these to assess severe chikungunya biomarker expression are known to the skilled reader.

It will be appreciated that as the level of severe chikungunya biomarker varies with the severity of the disease, that detection of severe chikungunya biomarker expression, amount or activity may also be used to predict a survival rate of an individual with severe chikungunya, i.e., high levels of severe chikungunya biomarker indicating a lower survival rate or probability and low levels of severe chikungunya biomarker indicating a higher survival rate or probability, both as compared to individuals or cognate populations with normal levels of severe chikungunya biomarker. Detection of expression, amount or activity of severe chikungunya biomarker may therefore be used as a method of prognosis of an individual with severe chikungunya disease.

Detection of severe chikungunya biomarker expression, amount or level may be used to determine the likelihood of success of a particular therapy in an individual with a severe chikungunya disease. It may be used in a method of determining whether a chikungunya infection in an individual is, or is likely to be, a severe chikungunya infection. Appropriate therapy may then be chosen for that individual (e.g., a more aggressive therapy may be indicated for a person suffering from a severe chikungunya infection, while a less aggressive or milder therapy may be indicated for a person suffering from a non-severe chikungunya infection).

The diagnostic methods described in this document may be combined with the therapeutic methods described. Thus, we provide for a method of treatment, prophylaxis or alleviation of chikungunya in an individual, the method comprising detecting modulation of expression, amount or activity of severe chikungunya biomarker in a cell or sample of the individual and administering an appropriate therapy to the individual based on the severity of the chikungunya. Typically, physical examination of the patient and laboratory analysis are used for the detection of chikungunya.

Symptoms of the disease include a fever up to 40 ⁰C (104 ⁰F), a petechial or maculopapular rash of the trunk and occasionally the limbs, and arthralgia or arthritis affecting multiple joints. Other nonspecific symptoms can include headache, conjunctival infection, and slight photophobia. Typically, the fever lasts for two days and then ends abruptly. However, other symptoms — namely joint pain, intense headache, insomnia and an extreme degree of prostration — last for a variable period; usually for about 5 to 7 days.

Common laboratory tests for chikungunya include RT-PCR, virus isolation, and serological tests. Virus isolation provides the most definitive diagnosis but takes 1-2 weeks for completion and must be carried out in Biosafety level 3 laboratories. The technique involves exposing specific cell lines to samples from whole blood and identifying chikungunya virus-specific responses. RT-PCR using nested primer pairs to amplify several Chikungunya-specifϊc genes from whole blood. Results can be determined in 1-2 days. Serological diagnosis requires a larger amount of blood than the other methods and uses an ELISA assay to measure Chikungunya-specifϊc IgM levels. Results require 2-3 days and false positives can occur with infection via other related viruses such as O'nyong'nyong virus and Semliki Forest Virus.

Detection of severe chikungunya biomarker expression, amount or activity can be used to diagnose, or further confirm the diagnosis of, chikungunya, along with the standard procedures described above. This may be especially useful when the analysis does not yield a clear result.

The presence and quantity of severe chikungunya biomarker polypeptides and nucleic acids may be detected in a sample as described in further detail below. Thus, severe chikungunya, such as acute severe chikungunya and chronic severe chikungunya, can be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased expression, amount or activity, such as a increased expression, amount or activity, of the severe chikungunya biomarker polypeptide or severe chikungunya biomarker mRNA. The sample may comprise a cell or tissue sample from an organism or individual suffering or suspected to be suffering from a disease associated with increased, reduced or otherwise abnormal severe chikungunya biomarker expression, amount or activity, including spatial or temporal changes in level or pattern of expression, amount or activity. The level or pattern of expression, amount or activity of severe chikungunya biomarker in an organism suffering from or suspected to be suffering from such a disease may be usefully compared with the level or pattern of expression, amount or activity in a normal organism as a means of diagnosis of disease.

The sample may comprise a cell or tissue sample from an individual suffering or suspected to be suffering from chikungunya, such as a relevant tissue or cell sample.

In some embodiments, an increased level of expression, amount or activity of severe chikungunya biomarker is detected in the sample. The level of severe chikungunya biomarker may be increased to a significant extent when compared to normal cells, or cells known not to be from individuals infected with chikungunya. Such cells may be obtained from the individual being tested, or another individual, such as those matched to the tested individual by age, weight, lifestyle, etc.

In some embodiments, the level of expression, amount or activity of severe chikungunya biomarker is increased by 10%, 20%, 30% or 40% or more. In some embodiments, the level of expression, amount or activity of severe chikungunya biomarker is increased by 45% or more, such as 50% or more, as judged by immunoassay or cDNA hybridisation.

The expression, amount or activity of severe chikungunya biomarker may be detected in a number of ways, as known in the art, and as described in further detail below. Typically, the amount of severe chikungunya biomarker in a sample of tissue from an individual is measured, and compared with a sample from an unaffected individual. Both severe chikungunya biomarker nucleic acid, as well as severe chikungunya biomarker polypeptide levels may be measured.

Detection of the amount, activity or expression of severe chikungunya biomarker may be used to grade the chikungunya. For example, a high level of amount, activity or expression of severe chikungunya biomarker (not RANTES) may indicate an severe chikungunya. Similarly, a low level of amount, activity or expression of RANTES may indicate a non- severe chikungunya. Such a grading system may be used in conjunction with established grading systems.

Levels of severe chikungunya biomarker gene expression may be determined using a number of different techniques.

MEASURING EXPRESSION OF SEVERE CHIKUNGUNYA BIOMARKER AT THE RNA LEVEL

Severe chikungunya biomarker gene expression can be detected at the RNA level.

In one embodiment therefore, we disclose a method of detecting the presence of a nucleic acid comprising a severe chikungunya biomarker nucleic acid in a sample, by contacting the sample with at least one nucleic acid probe which is specific for the severe chikungunya biomarker nucleic acid and monitoring said sample for the presence of the severe chikungunya biomarker nucleic acid. For example, the nucleic acid probe may specifically bind to the severe chikungunya biomarker nucleic acid, or a portion of it, and binding between the two detected; the presence of the complex itself may also be detected.

RNA detection of expression of severe chikungunya biomarker may be used to supplement polypeptide expression assays, as described below, which may employ the anti- severe chikungunya biomarker agents such as antibodies described here.

Thus, in one embodiment, the amount of severe chikungunya biomarker nucleic acid in the form of severe chikungunya biomarker mRNA may be measured in a sample. Severe chikungunya biomarker mRNA may be assayed by in situ hybridization, Northern blotting and reverse transcriptase-polymerase chain reaction. Nucleic acid sequences may be identified by in situ hybridization, Southern blotting, single strand conformational polymorphism, PCR amplification and DNA-chip analysis using specific primers. (Kawasaki, 1990; Sambrook, 1992; Lichter et al, 1990; Orita et al, 1989; Fodor et al, 1993; Pease et al., 1994).

Severe chikungunya biomarker RNA may be extracted from cells using RNA extraction techniques including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), or RNeasy RNA preparation kits (Qiagen).Typical assay formats utilising ribonucleic acid hybridisation include nuclear run-on assays, RT-PCR and RNase protection assays (Melton et al. , Nuc. Acids Res. 12:7035. Methods for detection which can be employed include radioactive labels, enzyme labels, chemilurninescent labels, fluorescent labels and other suitable labels.

Each of these methods allows quantitative determinations to be made, and are well known in the art. Decreased or increased severe chikungunya biomarker expression, amount or activity can therefore be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides. Any suitable probe from a severe chikungunya biomarker sequence, for example, any portion of a suitable human severe chikungunya biomarker sequence may be used as a probe.

Sequences for designing severe chikungunya biomarker probes may include a sequence having accession number AAH08678.1 (EL-I α), AAB92270.1 (BL-I β),

CAA25525.1 (IL-2R), AAH70123.1 (IL-4), AAHl 5511.1 (IL-6), AAH47698.1 (IL-7), AAH13615.1 (IL-8), AAA52724.1 (IFN-α), AAD56386.1 (IL-12), AAH18149.1 (IL-15), AAB29926.1 (MCP-I), NP_002974.1 (MEP-Ia)₅ CAB07027.1 (Eotaxin), BAA76939.1 (RANTES), AAHl 0954.1 (IP-IO), AAH95396.1 (MIG), AAS83395.1 (EGF), AAA52448.1 (FGF-b), CAA27290.1 (G-CSF), AAA52649.1 (HGF), or a portion thereof.

Typically, RT-PCR is used to amplify RNA targets. In this process, the reverse transcriptase enzyme is used to convert RNA to complementary DNA (cDNA) which can then be amplified to facilitate detection.

Many DNA amplification methods are known, most of which rely on an enzymatic chain reaction (such as a polymerase chain reaction, a ligase chain reaction, or a self-sustained sequence replication) or from the replication of all or part of the vector into which it has been cloned.

Many target and signal amplification methods have been described in the literature, for example, general reviews of these methods in Landegren, U. et at, Science 242:229-237 (1988) and Lewis, R., Genetic Engineering News 10:1, 54-55 (1990).

For example, the polymerase chain reaction may be employed to detect severe chikungunya biomarker mRNA.

The "polymerase chain reaction" or "PCR" is a nucleic acid amplification method described inter alia in U.S. Pat. Nos. 4,683,195 and 4,683,202. PCR can be used to amplify any known nucleic acid in a diagnostic context (Mok et al, 1994, Gynaecologic Oncology 52:247-252). Self-sustained sequence replication (3SR) is a variation of TAS, which involves the isothermal amplification of a nucleic acid template via sequential rounds of reverse transcriptase (RT), polymerase and nuclease activities that are mediated by an enzyme cocktail and appropriate oligonucleotide primers (Guatelli et al, 1990, Proc. Natl. Acad. Sci. USA 87:1874). Ligation amplification reaction or ligation amplification system uses DNA ligase and four oligonucleotides, two per target strand. This technique is described by Wu, D. Y. and Wallace, R. B., 1989, Genomics 4:560. In the Qβ Replicase technique, RNA replicase for the bacteriophage Qβ, which replicates single-stranded RNA, is used to amplify the target DNA, as described by Lizardi et al, 1988, Bio/Technology 6:1197.

A PCR procedure basically involves: (1) treating extracted DNA to form single- stranded complementary strands; (2) adding a pair of oligonucleotide primers, wherein one primer of the pair is substantially complementary to part of the sequence in the sense strand and the other primer of each pair is substantially complementary to a different part of the same sequence in the complementary antisense strand; (3) annealing the paired primers to the complementary sequence; (4) simultaneously extending the annealed primers from a 3' terminus of each primer to synthesize an extension product complementary to the strands annealed to each primer wherein said extension products after separation from the complement serve as templates for the synthesis of an extension product for the other primer of each pair; (5) separating said extension products from said templates to produce single- stranded molecules; and (6) amplifying said single-stranded molecules by repeating at least once said annealing, extending and separating steps.

Reverse transcription-polymerase chain reaction (RT-PCR) may be employed. Quantitative RT-PCR may also be used. Such PCR techniques are well known in the art, and may employ any suitable primer from a severe chikungunya biomarker sequence.

Alternative amplification technology can also be exploited. For example, rolling circle amplification (Lizardi et al, 1998, Nat Genet 19:225) is an amplification technology available commercially (RCAT™) which is driven by DNA polymerase and can replicate circular oligonucleotide probes with either linear or geometric kinetics under isothermal conditions. A further technique, strand displacement amplification (SDA; Walker et al, 1992, Proc. Natl. Acad. Sci. USA 80:392) begins with a specifically defined sequence unique to a specific target. MEASURING EXPRESSION OF SEVERE CHIKUNGUNYA BIOMARKER AT THE POLYPEPTIDE LEVEL

Severe chikungunya biomarker expression can be detected at the polypeptide level.

In a further embodiment, therefore, severe chikungunya biomarker expression, amount or activity may be detected by detecting the presence or amount of severe chikungunya biomarker polypeptide in a sample. This may be achieved by using molecules which bind to severe chikungunya biomarker polypeptide. Suitable molecules/agents which bind either directly or indirectly to the severe chikungunya biomarker polypeptide in order to detect its presence include naturally occurring molecules such as peptides and proteins, for example antibodies, or they may be synthetic molecules.

Thus, we disclose a method of detecting the presence of a severe chikungunya biomarker polypeptide by contacting a cell sample with an antibody capable of binding the polypeptide and monitoring said sample for the presence of the polypeptide.

For example, the severe chikungunya biomarker polypeptide may be detected using an anti-severe chikungunya biomarker antibody as described here. Such antibodies may be made by means described in detail in this document.

In a specific example, an anti-IL-lα, antibody may comprise an antibody with catalogue number 11-7118, obtainable from eBioscience (San Diego, USA).

The assay may conveniently be achieved by monitoring the presence of a complex formed between the antibody and the polypeptide, or monitoring the binding between the polypeptide and the antibody. Methods of detecting binding between two entities are known in the art, and include FRET (fluorescence resonance energy transfer), surface plasmon resonance, etc.

Standard laboratory techniques such as immunoblotting as described above can be used to detect altered levels of severe chikungunya biomarker protein, as compared with untreated cells in the same cell population.

Gene expression may also be determined by detecting changes in post-translational processing of severe chikungunya biomarker polypeptides or post-transcriptional modification of severe chikungunya biomarker nucleic acids. For example, differential phosphorylation of severe chikungunya biomarker polypeptides, the cleavage of severe chikungunya biomarker polypeptides or alternative splicing of severe chikungunya biomarker RNA, and the like may be measured. Levels of expression of gene products such as severe chikungunya biomarker polypeptides, as well as their post-translational modification, may be detected using proprietary protein assays or techniques such as 2D polyacrylamide gel electrophoresis.

Assay techniques that can be used to determine levels of severe chikungunya biomarker protein in a sample derived from a host are well-known to those of skill in the art. Antibodies can be assayed for immunospecific binding by any method known in the art.

The immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA, sandwich immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement- fixation assays, immunoradiometric assays, fluorescent immunoassays and protein A immunoassays. Such assays are routine in the art (see, for example, Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety).

The specimen may be assayed for polypeptides/proteins by immunohistochemical and immunocytochemical staining (see generally Stites and Terr, Basic and Clinical Immunology, Appleton and Lange, 1994), ELISA, RIA, immunoblots, Western blotting, immunoprecipitation, functional assays and protein truncation test. Other assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.

ELISA assays are well known to those skilled in the art. Both polyclonal and monoclonal antibodies may be used in the assays. Where appropriate other immunoassays, such as radioimmunoassays (RIA) may be used as are known to those in the art. Available immunoassays are extensively described in the patent and scientific literature. See, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521 as well as Sambrook et al, 1992. DIAGNOSTIC KITS

We also provide diagnostic kits for detecting severe chikungunya infection, including acute severe chikungunya infection and chronic severe chikungunya infection in an individual, or susceptibility to such in an individual.

The diagnostic kit may comprise means for detecting expression, amount or activity of any of the severe chikungunya biomarkers disclosed above, by any means as described in this document. The diagnostic kit may therefore comprise an anti-chikungunya biomarker antibody, which may comprise a monoclonal antibody. It may comprise a probe capable of specifically binding to a chikungunya biomarker. The antibody or probe may be labelled for detection.

The diagnostic kit may comprise instructions for use, or other indicia. The diagnostic kit may further comprise means for treatment or prophylaxis of severe chikungunya infection, such as any of the compositions described in this document, or any means known in the art for treating severe chikungunya infection.

PROPHYLACTIC AND THERAPEUTIC METHODS

We disclose methods of treating severe chikungunya infection, including chronic severe chikungunya infection and acute severe chikungunya infection. Methods of preventing severe chikungunya infection (i.e., prophylaxis) also suitably employ the same or similar approaches.

Accordingly, we provide for the use of anti-severe chikungunya biomarker agents, such as antibodies against any of the severe chikungunya biomarkers disclosed in this document in the treatment or prevention of severe chikungunya disease. The severe chikungunya disease may comprise acute severe chikungunya or chronic severe chikungunya infection. The anti-severe chikungunya biomarker agents, such as antibodies may be used as drugs or therapies to treat severe chikungunya. They may be used to prevent such infection or progress of the disease.

In general terms, our methods involve manipulation of cells, by modulating (such as down-regulating) the expression, amount or activity of a severe chikungunya biomarker. The treatment may comprise generally contacting an chikungunya infected cell, or a cell suspected of being a chikungunya infected cell, with an anti-severe chikungunya biomarker anti-severe chikungunya biomarker agent, such as antibody. The methods may involve exposing a patient to an anti-chikungunya biomarker agent, such as an antibody or variant thereof as described here.

It may or in addition be exposed to an anti-chikungunya agent such as an antibody or other molecule known to have effect in preventing or treating chikungunya such as severe chikungunya. Where this is so, the cell may be exposed to both the anti-chikungunya biomarker agent and the anti-chikungunya agent together, or individually in sequence. The exposure may be repeated a number of times. Any combination of anti-chikungunya biomarker agent and an other agent or antibody in whatever amount or relative amount, in whatever timing of exposure, may be used.

We therefore provide for the use of combinations of anti-chikungunya biomarker agent and anti-chikungunya agents, as described above, in the treatment of a severe chikungunya disease such as acute severe chikungunya or chronic severe chikungunya infection.

The cell may be an individual cell, or it may be in a cell mass. The cell may be inside the body of an organism. The organism may be one which is known to be suffering from severe chikungunya infection, or it could be one in which severe chikungunya infection is suspected, or it could be one which is susceptible to severe chikungunya infection. The treatment may comprise administering the antibody or antibodies to the organism. As above, a single antibody may be administered, or a combination of anti-chikungunya biomarker antibody and an anti-chikungunya agent may be administered. The administration may be simultaneous or sequential, as described above. Thus, the treatment may comprise administering an anti-chikungunya biomarker antibody simultaneously or sequentially with an anti-chikungunya agent to the individual.

The term "treating" refers to any indicia of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; improving a patient's physical or mental well-being; or, in some situations, preventing the onset of dementia. For this purpose, a number of criteria may be designated, which reflect the progress of treatment or prophylaxis or the well-being of the patient. Useful criteria in the case of chikungunya may include fever up to 40 ⁰C (104 ⁰F), a petechial or maculopapular rash of the trunk and occasionally the limbs, and arthralgia or arthritis affecting multiple joints. Other nonspecific symptoms can include headache, conjunctival infection, and slight photophobia. Symptoms of severe disease include a maximum temperature of more than 38.5 ⁰C, or a maximum pulse rate of more than 100 beats/minute, or a nadir platelet count of less than 100 x 10⁹/L, and these may be used as criteria.

Thus, as an example, a treated individual may show a decrease in such a symptom as measured by an appropriate assay or test. A treated individual may for example show a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more decrease in one or more symptoms, compared to an individual who has not been treated.

For example, a patient disease may be defined as being "treated" if a condition associated with the disease is significantly inhibited (z. e. , by 50% or more) relative to controls. The inhibition may be by at least 75% relative to controls, such as by 90%, by 95% or 100% relative to controls. By the term "treatment" we mean to also include prophylaxis or alleviation of flaviviral infection.

An antibody approach to therapy involving use of anti-chikungunya biomarker antibodies may be combined with other approaches for therapy of such disorders including conventional drug based approaches.

ANTI-SEVERE CHIKUNGUNYA BIOMARKER ANTIBODY PRODUCTION

The anti-severe chikungunya biomarker antibody can be produced by recombinant DNA methods or synthetic peptide chemical methods that are well known to those of ordinary skill in the art.

By way of example, the anti-severe chikungunya biomarker antibody may be synthesized by techniques well known in the art, as exemplified by "Solid Phase Peptide Synthesis: A Practical Approach" E. Atherton and R. C. Sheppard, IRL Press, Oxford England. Similarly, multiple fragments can be synthesized which are subsequently linked together to form larger fragments. These synthetic peptide fragments can also be made with amino acid substitutions at specific locations in order to test for activity in vitro and in vivo.

The anti-severe chikungunya biomarker antibody can be synthesized in a standard microchemical facility and purity checked with HPLC and mass spectrophotometry. Methods of peptide synthesis, HPLC purification and mass spectrophotometry are commonly known to those skilled in these arts.

The anti-severe chikungunya biomarker antibody may also be expressed under in vitro and in vivo conditions in a transformed host cell into which has been incorporated the DNA sequences described here (such as variable sequences) or allelic variations thereof and which can be used in the prevention and/or treatment of severe chikungunya infection.

The term "vector" includes expression vectors and transformation vectors. The term "expression vector" means a construct capable of in vivo or in vitro expression. The term "transformation vector" means a construct capable of being transferred from one species to another.

Vectors which may be used for expression include recombinant viral vectors, in particular recombinant retroviral vectors (RRV) such as lentiviral vectors, adenoviral vectors including a combination of retroviral vectors.

The term 'recombinant retroviral vector" (RRV) refers to a vector with sufficient retroviral genetic information to allow packaging of an RNA genome, in the presence of packaging components, into a viral particle capable of infecting a target cell. Infection of the target cell includes reverse transcription and integration into the target cell genome. The RRV carries non- viral coding sequences which are to be delivered by the vector to the target cell. An RRV is incapable of independent replication to produce infectious retroviral particles within the final target cell. Usually the RRV lacks a functional gag pol and/or env gene and/or other genes essential for replication. Vectors which may be used include recombinant pox viral vectors such as fowl pox virus (FPV), entomopox virus, vaccinia virus such as NYVAC, canarypox virus, MVA or other non-replicating viral vector systems such as those described for example in WO9530018. Pox viruses may be engineered for recombinant gene expression and for the use as recombinant live vaccines in a dual immunotherapeutic approach. The principal rationale for using live attenuated viruses, such as viruses, as delivery vehicles and/or vector based vaccine candidates, stems from their ability to elicit cell mediated immune responses. The viral vectors, as outlined above, are capable of being employed as delivery vehicles and as vector based vaccine candidates because of the irnmunogenicity of their constitutive proteins, which act as adjuvants to enhance the immune response, thus rendering a nucleotide sequence of interest (NOI) such as a nucleotide sequence encoding an anti-severe chikungunya biomarker antibody more immunogenic.

The pox virus vaccination strategies have used recombinant techniques to introduce

NOIs into the genome of the pox virus. If the NOI is integrated at a site in the viral DNA which is non-essential for the life cycle of the virus, it is possible for the newly produced recombinant pox virus to be infectious, that is to say to infect foreign cells and thus to express the integrated NOI. The recombinant pox virus prepared in this way can be used as live vaccines for the prophylaxis and/or treatment of severe chikungunya infection.

Other requirements for pox viral vector delivery systems include good immunogenicity and safety. MVA is a replication-impaired vaccinia strain with a good safety record. In most cell types and normal human tissue, MVA does not replicate. Limited replication of MVA is observed in a few transformed cell types such as BHK21 cells. Carroll et al (1997 Vaccine 15 : 387-394) have shown that the recombinant MVA is equally as good as conventional recombinant vaccinia vectors at generating a protective CD8+T cell response and is an efficacious alternative to the more commonly used replication competent vaccinia virus. The vaccinia virus strains derived from MVA, or independently developed strains having the features of MVA which make MVA particularly suitable for use in a vaccine, are also suitable for use as a delivery vehicle.

The nucleotide sequence of interest, and of which expression is desired, may operably linked to a transcription unit. The term "transcription unit" as described herein are regions of nucleic acid containing coding sequences and the signals for achieving expression of those coding sequences independently of any other coding sequences. Thus, each transcription unit generally comprises at least a promoter, an optional enhancer and a polyadenylation signal. The term "promoter" is used in the normal sense of the art, e. g. an RNA polymerase binding site. The promoter may contain an enhancer element. The term "enhancer" includes a DNA sequence which binds to other protein components of the transcription initiation complex and thus facilitates the initiation of transcription directed by its associated promoter. The term "cell" includes any suitable organism. The cell may comprise a mammalian cell, such as a human cell.

The term "transformed cell" means a cell having a modified genetic structure. For example, as described here, a cell has a modified genetic structure when a vector such as an expression vector has been introduced into the cell. The term "organism" includes any suitable organism. The organism may comprise a mammal such as a human.

Here the term "transgenic organism" means an organism comprising a modified genetic structure. For example, the organism may have a modified genetic structure if a vector such as an expression vector has been introduced into the organism.

ANTIBODY EXPRESSION

We further describe a method comprising transforming a host cell with a nucleic acid sequence capable of expressing an anti-severe chikungunya biomarker antibody.

We also provide a method comprising culturing a transformed host cell-which cell has been transformed with a or the such nucleotide sequences under conditions suitable for the expression of the anti-severe chikungunya biomarker antibody encoded by said nucleotide sequences.

We further provide a method comprising culturing a transformed host cell-which cell has been transformed with a or the such nucleotide sequences under conditions suitable for the expression of the anti-severe chikungunya biomarker antibody encoded by said nucleotide sequences; and then recovering said anti-severe chikungunya biomarker antibody from the transformed host cell culture.

Thus, anti-severe chikungunya biomarker antibody encoding nucleotide sequences, fusion proteins or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression thereof in appropriate host cells. By way of example, anti-severe chikungunya biomarker antibody may be produced in recombinant E. coli, yeast or mammalian expression systems, and purified with column chromatography.

In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. The smaller size of the fragments allows for rapid clearance, and may lead to improve tumour to non-tumour ratios. Fab, Fv, ScFv antibody fragments can all be expressed in and secreted from E. coli, thus allowing the production of large amounts of the such fragments.

The nucleotide sequences encoding the anti-severe chikungunya biomarker antibody may be operably linked to a promoter sequence capable of directing expression of the anti- severe chikungunya biomarker antibody encoding nucleotide sequences in a suitable host cell. When inserted into the host cell, the transformed host cell may be cultured under suitable conditions until sufficient levels of the anti-severe chikungunya biomarker antibody are achieved after which the cells may be lysed and the anti-severe chikungunya biomarker antibody is isolated.

Host cells transformed with the anti-severe chikungunya biomarker antibody encoding nucleotide sequences may be cultured under conditions suitable for the expression and recovery of the anti-severe chikungunya biomarker antibody from cell culture. The protein produced by a recombinant cell may be secreted or may be contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing the

Anti-severe chikungunya biomarker antibody encoding nucleotide sequences can be designed with signal sequences which direct secretion of the anti-severe chikungunya biomarker antibody encoding nucleotide sequences through a particular prokaryotic or eukaryotic cell membrane. Other recombinant constructions may join the anti-severe chikungunya biomarker antibody encoding nucleotide sequence to a nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins (Kroll DJ et al(1993) DNA Cell Biol 12:441- 5 3', see also the discussion below on vectors containing fusion proteins). The anti-severe chikungunya biomarker antibody may also be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals (Porath J (1992) Protein Expr Purif 3-26328 1), protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle, WA). The inclusion of a cleavable linker sequence such as Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the anti-severe chikungunya biomarker antibody is useful to facilitate purification.

The nucleotide sequences described here may be engineered in order to alter a the anti- severe chikungunya biomarker antibody encoding sequences for a variety of reasons, including but not limited to alterations which modify the cloning, processing and/or expression of the gene product. For example, mutations may be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis to insert new restriction sites, to alter glycosylation patterns or to change codon preference.

In another embodiment, a or the natural, modified or recombinant anti-severe chikungunya biomarker antibody encoding nucleotide sequences may be ligated to a heterologous sequence to encode a fusion protein. By way of example, fusion proteins comprising the anti-severe chikungunya biomarker antibody or an enzymatically active fragment or derivative thereof linked to an affinity tag such as glutathione-S-transferase (GST), biotin, His6, ac-myc tag (see Emrich etal 1993 BiocemBiophys Res Commun 197(1): 21220), hemagglutinin (HA) (as described in Wilson et al (1984 Cell 37 767) or a FLAG epitope (Ford etal 1991 Protein Expr Purif Apr; 2 (2):95-107). May be produced

The fused recombinant protein may comprise an antigenic coprotein such as GST, beta-galactosidase or the lipoprotein D from Haemophilus influenzae which are relatively large co-proteins, which solubilise and facilitate production and purification thereof. Alternatively, the fused protein may comprise a carrier protein such as bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH). In certain embodiments, the marker sequence may comprise a hexa-histidine peptide, as provided in the pQE vector (Qiagen Inc) and described in Gentz et al (1989 PNAS 86: 821-824). Such fusion proteins are readily expressable in yeast culture (as described in Mitchell et al 1993 Yeast 5:715-723) and are easily purified by affinity chromatography. A fusion protein may also be engineered to contain a cleavage site located between the nucleotide sequence encoding the anti-severe chikungunya biomarker antibody and the heterologous protein sequence, so that the anti- severe chikungunya biomarker antibody may be cleaved and purified away from the heterologous moiety. In another embodiment, an assay for the target protein may be conducted using the entire, bound fusion protein. Alternatively, the co-protein may act as an adjuvant in the sense of providing a generalised stimulation of the immune system. The co-protein may be attached to either the amino or carboxy terminus of the first protein.

Although the presence/absence of marker gene expression suggests that the nucleotide sequence for anti-severe chikungunya biomarker antibody is also present, its presence and expression should be confirmed. For example, if the anti-severe chikungunya biomarker antibody encoding nucleotide sequence is inserted within a marker gene sequence, recombinant cells containing the anti-severe chikungunya biomarker antibody coding regions may be identified by the absence of the marker gene function. Alternatively, a marker gene may be placed in tandem with a anti-severe chikungunya biomarker antibody encoding nucleotide sequence under the control of a single promoter.

Expression of the marker gene in response to induction or selection usually indicates expression of the anti-severe chikungunya biomarker antibody as well.

Additional methods to quantitate the expression of a particular molecule include radiolabeling (Melby PC etal 1993 J Immunol Methods 159:235-44) or biotinylating (Duplaa C et al 1993 Anal Biochem229-36) nucleotides, co amplification of a control nucleic acid, and standard curves onto which the experimental results are interpolated.

Quantitation of multiple samples may be speeded up by running the assay in an ELISA format where the anti-severe chikungunya biomarker antibody of interest is presented in various dilutions and a spectrophotometric or calorimetric response gives rapid quantitation.

Altered anti-severe chikungunya biomarker antibody nucleotide sequences which may be made or used include deletions, insertions or substitutions of different nucleotide residues resulting in a nucleotide sequence that encodes the same or a functionally equivalent anti- severe chikungunya biomarker antibody. By way of example, the expressed anti-severe chikungunya biomarker antibody may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent anti-severe chikungunya biomarker antibody. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity. and/or the amphipathic nature of the residues as long as the binding affinity of the anti-severe chikungunya biomarker antibody is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid: positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

Gene therapy whereby the anti-severe chikungunya biomarker antibody encoding nucleotide sequences as described here is regulated in vivo may also be employed. For example, expression regulation may be accomplished by administering compounds that bind to the anti-severe chikungunya biomarker antibody encoding nucleotide sequences, or control regions associated with the anti-severe chikungunya biomarker antibody encoding nucleotide sequence or its corresponding RNA transcript to modify the rate of transcription or translation.

By way of example, the anti-severe chikungunya biomarker antibody encoding nucleotide sequences described here may be under the expression control of an expression regulatory element, usually a promoter or a promoter and enhancer. The enhancer and/or promoter may be preferentially active in a hypoxic or ischaemic or low glucose environment, such that the anti-severe chikungunya biomarker antibody encoding nucleotide sequences is preferentially expressed in the particular tissues of interest, such as in the environment of a tumour cell or mass. Thus, any significant biological effect or deleterious effect of the anti- severe chikungunya biomarker antibody encoding nucleotide sequences on the individual being treated may be reduced or eliminated. The enhancer element or other elements conferring regulated expression may be present in multiple copies.

The promoter and/or enhancer may be constitutively efficient, or may be tissue or temporally restricted in their activity. Examples of suitable tissue restricted promoters/enhancers are those which are highly active in tumour cells such as a promoter/enhancer from a MUCl gene, a CEA gene or a STV antigen gene. Examples of temporally restricted promoters/enhancers are those which are responsive to ischaemia and/or hypoxia, such as hypoxia response elements or the promoter/enhancer of agrp78 or agrp94 gene. The alpha fetoprotein (AFP) promoter is also a tumour-specific promoter. Another promoter-enhancer combination is a human cytomegalovirus (hCMV) major immediate early (MIE) promoter/enhancer combination.

The promoters may be tissue specific. That is, they may be capable of driving transcription of a anti-severe chikungunya biomarker antibody encoding nucleotide sequences in one tissue while remaining largely "silent" in other tissue types.

The term "tissue specific" means a promoter which is not restricted in activity to a single tissue type but which nevertheless shows selectivity in that they may be active in one group of tissues and less active or silent in another group. A desirable characteristic of such promoters is that they possess a relatively low activity in the absence of activated hypoxia- regulated enhancer elements, even in the target tissue. One means of achieving this is to use "silencer" elements which suppress the activity of a selected promoter in the absence of hypoxia.

The term "hypoxia" means a condition under which a particular organ or tissue receives an inadequate supply of oxygen.

The level of expression of a or the anti-severe chikungunya biomarker antibody encoding nucleotide sequences under the control of a particular promoter may be modulated by manipulating the promoter region. For example, different domains within a promoter region may possess different gene regulatory activities. The roles of these different regions are typically assessed using vector constructs having different variants of the promoter with specific regions deleted (that is, deletion analysis). This approach may be used to identify, for example, the smallest region capable of conferring tissue specificity or the smallest region conferring hypoxia sensitivity.

A number of tissue specific promoters, described above, may be used. In most instances, these promoters may be isolated as convenient restriction digestion fragments suitable for cloning in a selected vector. Alternatively, promoter fragments may be isolated using the polymerase chain reaction. Cloning of the amplified fragments may be facilitated by incorporating restriction sites at the 5' end of the primers. PHARMACEUTICAL COMPOSITIONS

As disclosed herein, anti-severe chikungunya biomarker agents, including severe chikungunya biomarker agonists, severe chikungunya biomarker antagonists and anti-severe chikungunya biomarker antibodies, may be used to treat or prevent severe chikungunya disease and infection, including severe acute chikungunya infection and severe chronic chikungunya infection.

Anti- antibodies can be administered in a variety of ways including enteral, parenteral and topical routes of administration. For example, suitable modes of administration include oral, subcutaneous, transdermal, transmucosal, iontophoretic, intravenous, intramuscular, intraperitoneal, intranasal, subdural, rectal, and the like.

In accordance with other embodiments, there is provided a composition comprising an anti-severe chikungunya biomarker agent, together with a pharmaceutically acceptable carrier or excipient for the treatment or prevention of severe chikungunya disease and infection, including severe acute chikungunya infection and severe chronic chikungunya infection.

Suitable pharmaceutically acceptable excipients include processing agents and drug delivery modifiers and enhancers, such as, for example, calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-p-cyclodextrin, poly vinylpyrrolidinone, low melting waxes, ion exchange resins, and the like, as well as combinations of any two or more thereof. Other suitable pharmaceutically acceptable excipients are described in "Remington's Pharmaceutical Sciences," Mack Pub. Co., New Jersey (1991), incorporated herein by reference.

Pharmaceutical compositions containing an anti-severe chikungunya biomarker agent may be in any form suitable for the intended method of administration, including, for example, a solution, a suspension, or an emulsion. Liquid carriers are typically used in preparing solutions, suspensions, and emulsions. Liquid carriers contemplated for use in the practice include, for example, water, saline, pharmaceutically acceptable organic solvent (s), pharmaceutically acceptable oils or fats, and the like, as well as mixtures of two or more thereof. The liquid carrier may contain other suitable pharmaceutically acceptable additives such as solubilizers, emulsifiers, nutrients, buffers, preservatives, suspending agents, thickening agents, viscosity regulators, stabilizers, and the like. Suitable organic solvents include, for example, monohydric alcohols, such as ethanol, and polyhydric alcohols, such as glycols.

Suitable oils include, for example, soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, and the like. For parenteral administration, the carrier can also be an oily ester such as ethyl oleate, isopropyl myristate, and the like. Compositions may also be in the form of microparticles, microcapsules, liposomal encapsulates, and the like, as well as combinations of any two or more thereof.

The anti-severe chikungunya biomarker agent may be administered orally, parenterally, sublingually, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. Topical administration may also involve the use of transdermal administration such as transdermal patches or ionophoresis devices. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrastemal injection, or infusion techniques.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 -propanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

Suppositories for rectal administration of the drug can be prepared by mixing the drug with a suitable nonirritating excipient such as cocoa butter and polyethylene glycols that are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug. Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound may be admixed with at least one inert diluent such as sucrose lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e. g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, cyclodextrins, and sweetening, flavoring, and perfuming agents.

In accordance with yet other embodiments, we provide methods for inhibiting any activity of envelope glycoprotein (E) domain III, in a human or animal subject, the method comprising administering to a subject an amount of an anti-severe chikungunya biomarker agent (or composition comprising such agent) effective to inhibit the relevant activity in the subject. Other embodiments provide methods for treating severe chikungunya infection, including severe acute chikungunya infection and severe chronic chikungunya infection, in a human or animal subject, comprising administering to the cell or to the human or animal subject an amount of a agent or composition as described here effective to inhibit a severe chikungunya biomarker activity in the cell or subject. The subject may be a human or non- human animal subject. Inhibition of protein activity includes detectable suppression of the relevant protein activity either as compared to a control or as compared to expected protein activity.

Effective amounts of the anti-severe chikungunya biomarker agent generally include any amount sufficient to detectably inhibit the relevant protein activity by any of the assays described herein, by other assays known to those having ordinary skill in the art or by detecting an alleviation of symptoms in a subject afflicted with severe chikungunya infection, including severe acute chikungunya infection and severe chronic chikungunya infection. Successful treatment of a subject in accordance may result in the inducement of a reduction or alleviation of symptoms in a subject afflicted with a medical or biological disorder to, for example, halt the further progression of the disorder, or the prevention of the disorder. Thus, for example, treatment of severe chikungunya disease and infection, including severe acute chikungunya infection and severe chronic chikungunya infection can result in a reduction in symptoms such as fever, petechial or maculopapular rash of the trunk and occasionally the limbs, arthralgia or arthritis affecting multiple joints, headache, conjunctival infection, photophobia, joint pain, intense headache, insomnia and an extreme degree of prostration.

The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific agent employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease undergoing therapy. The therapeutically effective amount for a given situation can be readily determined by routine experimentation and is within the skill and judgment of the ordinary clinician.

A therapeutically effective dose will generally be from about lOμg/kg/day to 100mg/kg/day, for example from about 25μg/kg/day to about 20 mg/kg/day or from about

50μg/kg/day to about 2mg/kg/day of an anti-severe chikungunya biomarker agent, which may be administered in one or multiple doses.

The anti-severe chikungunya biomarker agent can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono-or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a agent, stabilizers, preservatives, excipients, and the like. Lipids which may be used include the phospholipids and phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N. W., p. 33 et seq (1976).

While the anti-severe chikungunya biomarker agent can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more other agents used in the treatment of disorders.

When additional active agents are used in combination with the anti-severe chikungunya biomarker agent, the additional active agents may generally be employed in therapeutic amounts as indicated in the PHYSICIANS 'DESK REFERENCE (PDR) 53rd Edition (1999), which is incorporated herein by reference, or such therapeutically useful amounts as would be known to one of ordinary skill in the art.

The anti-severe chikungunya biomarker agent and the other therapeutically active agents can be administered at the recommended maximum clinical dosage or at lower doses. Dosage levels of the active anti-severe chikungunya biomarker agent in the compositions may be varied so as to obtain a desired therapeutic response depending on the route of administration, severity of the disease and the response of the patient. The combination can be administered as separate compositions or as a single dosage form containing both agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition. Bioavailability

The anti-severe chikungunya biomarker agents disclosed here (and combinations) are in some embodiments orally bioavailable. Oral bioavailablity refers to the proportion of an orally administered drug that reaches the systemic circulation. The factors that determine oral bioavailability of a drug are dissolution, membrane permeability and metabolic stability. Typically, a screening cascade of firstly in vitro and then in vivo techniques is used to determine oral bioavailablity.

Dissolution, the solubilisation of the drug by the aqueous contents of the gastrointestinal tract (GIT), can be predicted from in vitro solubility experiments conducted at appropriate pH to mimic the GIT. The anti-severe chikungunya biomarker agent may in some embodiments have a minimum solubility of 50 mg/ml. Solubility can be determined by standard procedures known in the art such as described in Adv. Drug Deliv. Rev. 23, 3-25, 1997.

Membrane permeability refers to the passage of the agent through the cells of the GIT. Lipophilicity is a key property in predicting this and is defined by in vitro Log D₇.₄ measurements using organic solvents and buffer. The anti-severe chikungunya biomarker agent may have a Log D_7.4 of -2 to +4 or -1 to +2. The log D can be determined by standard procedures known in the art such as described in J. Pharm. Pharmacol. 1990, 42:144.

Cell monolayer assays such as CaCO₂ add substantially to prediction of favourable membrane permeability in the presence of efflux transporters such as p-glycoprotein, so-called caco-2 flux. The anti-severe chikungunya biomarker agent may have a caco-2 flux of greater than 2XlO^-6CmS^"1, for example greater than 5XlO^-6CmS^"1. The caco flux value can be determined by standard procedures known in the art such as described in J. Pharm. Sci, 1990, 79, 595-600.

Metabolic stability addresses the ability of the GIT or the liver to metabolise agents during the absorption process: the first pass effect. Assay systems such as microsomes, hepatocytes etc are predictive of metabolic liability. The compounds described here may in some embodiments show metabolic stability in the assay system that is commensurate with an hepatic extraction of less than 0.5. Examples of assay systems and data manipulation are described in Curr. Opin. Drug Disc. Devel., 201, 4, 36-44, Drug Met. Disp.,2000, 28, 1518- 1523.

Because of the interplay of the above processes further support that a drug will be orally bioavailable in humans can be gained by in vivo experiments in animals. Absolute bioavailability is determined in these studies by administering the agent separately or in mixtures by the oral route. For absolute determinations (% absorbed) the intravenous route is also employed. Examples of the assessment of oral bioavailability in animals can be found in Drug Met. Disp.,2001, 29, 82-87; J. Med Chem , 1997, 40, 827-829, Drag Met. Disp.,1999, 27, 221-226.

The term "pharmaceutically acceptable carrier" as used herein generally refers to organic or inorganic materials, which cannot react with active ingredients. The carriers include but are not limited to sugars, such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethycellulose, ethylcellulose and cellulose acetates; powdered tragancanth; malt; gelatin; talc; stearic acids; magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cotton seed oil, sesame oil, olive oil, corn oil and oil of theobroma; polyols such as propylene glycol, glycerine, sorbitol, mannitol, and polyethylene glycol; agar; alginic acids; pyrogen-free water; isotonic saline; and phosphate buffer solution; skim milk powder; as well as other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, lubricants, excipients, tabletting agents, stabilizers, anti-oxidants and preservatives, can also be present.

The term "therapeutically effective amount" as used herein generally refers to an amount of an agent, for example the amount of an anti-severe chikungunya biomarker agent as an active ingredient, that is sufficient to effect treatment as defined herein when administered to a subject in need of such treatment. A therapeutically effective amount of a agent, salt, derivative, isomer or enantiomer of the present invention will depend upon a number of factors including, for example, the age and weight of the subject, the precise condition requiring treatment and its severity, the nature of the formulation, and the route of administration, and will ultimately be at the discretion of the attendant physician or veterinarian. However, an effective amount of an anti-severe chikungunya biomarker agent described here for the treatment of disorders associated with bacterial or viral infection, in particular bacterial meningitis, will generally be in the range of about 10 to about 40 nig/kg body weight of recipient (mammal) per day and more usually about 40 mg/kg body weight per day. Thus, for a 70 kg adult subject, the actual amount per day would typically be about 2,800 mg, and this amount may be given in a single dose per day or more usually in a number (such as two, three, four, five or six) of sub-doses per day such that the total daily dose is the same. An effective amount of a salt of the present invention may be determined as a proportion of the effective amount of the compound per se.

The term "treatment" as used herein refers to any treatment of a condition or disease in an animal, particularly a mammal, more particularly a human, and includes: preventing the disease or condition from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; inhibiting the disease or condition, i.e. arresting its development; relieving the disease or condition, i.e. causing regression of the condition; or relieving the conditions caused by the disease, i.e. symptoms of the disease.

Chemical derivative

The term "derivative" or "derivatised" as used herein includes chemical modification of a compound. Illustrative of such chemical modifications would be replacement of hydrogen by a halo group, an alkyl group, an acyl group or an amino group.

Chemical modification

In one embodiment, the compound may be a chemically modified compound.

The chemical modification of a compound may either enhance or reduce hydrogen bonding interaction, charge interaction, hydrophobic interaction, Van Der Waals interaction or dipole interaction between the compound and the target.

In one aspect, the identified compound may act as a model (for example, a template) for the development of other compounds.

Individual The compounds are delivered to individuals. As used herein, the term "individual" refers to vertebrates, particularly members of the mammalian species. The term includes but is not limited to domestic animals, sports animals, primates and humans.

METHOD OF DETERMINING THE SEVERITY OF CHIKUNGUNYA

We describe a method of determining the severity of chikungunya, such as acute chikungunya infection, in an individual, the method comprising detecting the expression or activity, or a change in either, of one or more of IL-I β, IL-6 and RANTES or a variant, homologue, derivative or fragment thereof such as a sequence having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology thereto in a sample in or of the individual.

The IL- lβ may comprise a polypeptide sequence having GenBank Accession Number NP_000567. It may comprise a nucleic acid sequence having GenBank Accession number

NM_000576. It may comprise a variant, homologue, derivative or fragment of either such as a sequence having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology thereto and comprising (or encoding a sequence comprising) interleukin-lβ activity. The IL-6 may comprise a polypeptide sequence having GenBank Accession Number NP_000591. It may comprise a nucleic acid sequence having GenBank Accession number NM_000600. It may comprise a variant, homologue, derivative or fragment thereof such as a sequence having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology thereto and comprising (or encoding a sequence comprising) interleukin-6 activity; or

The RANTES (syn. CCL5) may comprise a polypeptide sequence having GenBank Accession Number NP_002976. It may comprise a nucleic acid sequence having GenBank Accession number NM_002985. It may comprise a variant, homologue, derivative or fragment thereof such as a sequence having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology thereto and comprising (or encoding a sequence comprising) RANTES/CCL5 activity.

The method may comprise detecting increased expression or activity of IL-I β as specified as an indicator of severe chikungunya in the individual. It may comprise detecting increased expression or activity of IL-6 as specified as an indicator of severe chikungunya in the individual. It may comprise detecting reduced expression or activity of RANTES as specified as an indicator of severe chikungunya in the individual. It may comprise detecting as specified the expression of a variant, homologue, derivative or fragment thereof.

The method may comprise detecting reduced expression or activity of IL-I β as specified as an indicator of mild chikungunya in the individual. It may comprise detecting reduced expression or activity of IL-6 as specified as an indicator of mild chikungunya in the individual. It may comprise detecting increased expression or activity of RANTES as specified as an indicator of mild chikungunya in the individual. It may comprise detecting as specified the expression of a variant, homologue, derivative or fragment thereof.

The method may comprise detecting severe chikungunya. Severe chikungunya may be characterised by a temperature of 38.5⁰C or higher. It may be characterised by a pulse rate 100/min or higher. It may be characterised by a platelet count of 100x10⁹ g/L or less. It may be characterised by any one or more of these symptoms.

The method may comprise detecting mild chikungunya. Mild chikungunya may be characterised by a temperature of lower than 38.5⁰C. It may be characterised by a pulse rate of lower than 100/min. It may be characterised by a platelet count of higher than 100x10 g/L. It may be characterised by any one or more of these symptoms.

We also describe a method of detecting chikungunya in an individual, the method comprising detecting the expression or activity, or a change in either, of one or more of: (a) a cytokine, preferably comprising one or more of IL-2R, IL5, IL-6, IL-7, IL-8, IL-10, IL-15, IFNα and IL8; (b) a chemokine, preferably comprising one or more of: IP-10, MIG and Eotoxin; (c) a growth factor, preferably comprising one or more of: HGF, FGF-basic, VEGF, EGF, GM-CSF and G-CSF; or a variant, homologue, derivative or fragment thereof, such as a sequence having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology thereto and comprising (or encoding a sequence comprising) the relevant activity, in a sample in or of the individual.

The method of detecting chikungunya may comprise detecting an increase in expression or activity of any one or more of IL-2R, IL5, IL-6, IL-7, IL-8, IL-10, IL-15, IFNα, IP-10, MIG, HGF, FGF-basic and VEGF or a variant, homologue, derivative or fragment thereof such as a sequence having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology thereto as compared to an individual not suffering from chikungunya. It may comprise detecting a decrease in expression or activity of any one or more of IL 8, Eotoxin, EGF, GM-CSF or G-CSF or a variant, homologue, derivative or fragment thereof such as a sequence having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology thereto as compared to an individual not suffering from chikungunya. It may comprise detecting both (a) and (b), as an indicator of chikungunya.

The detection may comprise polymerase chain reaction, such as real-time polymerase chain reaction (RT-PCR), Northern Blot, immunological detection such as ELISA, RNAse protection, microarray hybridisation, etc.

We further describe a method of treatment or prevention of chikungunya in an individual, the method comprising detecting chikungunya or determining the severity thereof, or both in an individual by a method as set out above, and administering a suitable treatment or prophylactic, such as a drug known or suspected to be useful for treating chikungunya, to the individual. We describe a combination of two or more nucleic acids or polypeptides as specified above. The combination may comprise a combination of nucleic acids immobilised on a substrate. The nucleic acid combination may comprise be in the form of a microarray .

We describe a nucleic acid or polypeptide as specified above, a combination as set out above, or an agonist or antagonist thereof for use in a method of detecting, determining the severity of or treating chikungunya.

We describe a pharmaceutical composition comprising a nucleic acid or polypeptide as specified above, a combination as set out above, or an agonist or antagonist thereof.

We describe a diagnostic kit for chikungunya or the severity thereof or susceptibility thereto, the kit comprising any one or more of the following: (a) a polypeptide as specified in above or a variant, homologue, derivative or fragment thereof such as a sequence having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology thereto; (b) a molecule capable of binding to such a polypeptide, such as an antibody; (c) a nucleic acid capable of encoding such a polypeptide, such as a nucleic acid as specified in any of Claims 1 to 9 or a variant, homologue, derivative or fragment thereof such as a sequence having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology thereto; and (d) a molecule capable of binding to such a nucleic acid sequence, such as a nucleic acid, for example probe preferably comprising a complementary nucleic acid; optionally together with instructions for use.

We describe a method for the treatment or prevention of chikungunya in an individual, in which the method comprises modulating the expression of a nucleic acid or polypeptide specified above, or in which the method comprises administering an agonist or antagonist of such a nucleic acid or polypeptide.

We describe a method of identifying a molecule suitable for the treatment, prophylaxis or alleviation of chikungunya, the method comprising determining if a candidate molecule is an agonist or antagonist of a polypeptide specified above.

EXAMPLES

Example 1. Methods - Patients and Clinical Samples

Ten patients who presented with acute CHIKF to the Communicable Disease Centre at Tan Tock Seng Hospital (CDC/TTSH), the national infectious disease referral centre in Singapore, during the outbreak period from January to February 2008, are included in this study.

An acute case of CHIKF is defined as any case with clinical features consistent with CHIKF, and had CHIKV infection confirmed by either reverse transcription-polymerase chain reaction (RT-PCR) or virus isolation [53,54]. The study is approved by the institution's domain-specific ethics review board (DSRB Reference No. B/08/026). Written consent is obtained from each patient and healthy control subject.

Plasma samples are obtained from patients during the acute phase of their illness. Data on demographic characteristics, pre-morbid conditions, clinical features, and routine hematological and biochemical laboratory test findings (i.e. full blood count, renal and liver function tests, C-reactive protein) are also collected. All symptomatic patients are isolated at CDC/TTSH until the febrile illness resolved and a negative CHIKV RT-PCR test is obtained. During the hospital stay, daily monitoring of body temperature, vital parameters, and blood counts are carried out. A patient is defined as having severe illness, if he had either a maximum temperature of more than 38.5 ⁰C, or a maximum pulse rate of more than 100 beats/minute, or a nadir platelet count of less than 100 x 10⁹/L. Laboratory results are expressed as mean ± SD.

In addition, plasma or serum samples from 9 healthy volunteers (who did not have a febrile illness in the preceding week and are not epidemiologically-linked to the outbreak) are also included as controls in our study.

Example 2. Methods - Multiplex Microbead Immunoassay

Plasma samples collected as described above, are aliquoted and stored at -80 ⁰C until analyses are done. A multiplex biometric immunoassay, containing fluorescent dyed microspheres conjugated with a monoclonal antibody specific for a target protein, is used for cytokine measurement according to manufacturer's instructions (Biosource Human Cytokine 30-plex Assay, Invitrogen).

The following groups of cytokines are: inflammatory (GM-CSF, IL-lbeta, IL-IRA, IL-6, IL-8, TNF-alpha); Thl/Th2 (IFN-gamma, IL-2, IL-2R, IL-4, IL-5, IL-10) ; Cytokine II (IFN-alpha, IL-7, IL-12p40/p70, IL-13, IL-15, IL-17); Chemokines (Eotaxin, IP-10, MCP-I, MIG, MlP-lalpha, MIP-lbeta, RANTES); and Growth Factors (EGF, HGF, FGF-basic, G- CSF, VEGF). Briefly, 25 ul of plasma samples are diluted in 1 :2 and incubated with antibody coupled beads for 2 h at RT ⁰C.

Complexes are ished twice with the use of a vacuum manifold before incubation with biotinylated detector antibody for 1 h at RT ⁰C. Complexes are then ished twice followed by incubation with Streptavidin-phycoeryrhrin (RPE) for 30 min at RT ⁰C. Complexes are ished thrice and incubated with ish buffer for another 3 min before detection in the Luminex 200™ instrument. Results are acquired by the IS 2.3 software and the standard curves are plotted through a five-parameter logistic curve setting.

Example 3. Methods - Algorithms for Data Analyses

To compare and analyze the expression profiles across the results, the raw cytokine values are normalized using z-score conversion based on the formula:

x - μ z = σ

where x is the raw value to be converted, μ is the mean of the population and σ is the standard deviation (SD) of the population. The transformed value is denoted by z and exhibits positive value when the raw value is above mean and vice versa. Further, to examine the nuances and correlations masked in the full set of data, the z values are subjected to cluster analysis [55] to yield an ordered NumOfRows.x NumOfCols expression level matrix,

Hierarchical clustering is applied on the columns which represent the myriad of cytokine levels measurements (e.g.) based on McQuitty's or WPGMA method [56] where the distance between a pair of groups A and B is measured using the weighted arithmetic mean of all the pairwise distances between the data points in A and B. The rows which represent the suspected CHIKF patients and healthy individuals are left untouched. Seriation [57] is performed following the clustering approach to re-order the clustered data points using the minimum path length algorithm to minimize the sum of all the distances between adjacent columns. π-l

S = ∑d(i,i + l)

M

Euclidean distance is used in both the clustering and seriation phases to measure the difference or dissimilarity, d, between data points (X₁, y^ and (x₂, y₂) given by the equation:

Example 4. Methods - Statistical Analyses

Comparisons between groups are calculated by Mann- Whitney rank sum. Further statistical analyses are done by Kruskal-Wallis test followed by Dunn's multiple comparison tests. P values of <0.05 are considered to be statistically significant.

Example 5. Initial Study - Acute Disease

All 10 patients included in this study are males. Their age ranged from 22 to 65 years

(median, 35 years). All except one are foreign nationals. Half of our patients are classified as severe CHIKF.

We defined severe CHIKF as having a temperature of > 38.5 ⁰C or pulse rate > 100/min, or platelet count < 100x10⁹ g/L based on studies defining severe diseases [24-28].

Except for the Singapore resident, none had any pre-existing medical condition.

Despite being previously healthy, four non-residents developed severe illness. Fever lasted 2- 10 days, and fever duration is not significantly different between those who had more severe illness and those who had not (mean, 6.6 days vs. mean, 3.8 days; P>0.05). Two patients reported persistent arthralgia lasting more than two weeks. Demographic and clinical details of the 10 CHIKF patients are summarized in Table El . Table 1. Demographic and epidemiologic data on 10 patients with PCR-confirmed chikungunya infection.

Patient No, Age (years) Gender Nationality fever (days) Illness severity⁹ Prβ-morbid condition Clinical Outcome

1 45

2 32

Not Severe None

^'3 33 Complete recovery

4 45 Not Severe

5 28 Not Severe

6 65 Singapore resident 10 Severe atrial fibrillation, anaemia Complete recovery

J 34 4 Severe δ 39 6 Severe

9 37 9 Severe None Complete recovery

10 22 Malaysian 4 Severe None

^a Severity was defined as having a temperature >38.5degC or pulse rate >100/min or platelet count <100x10^Λ9/L b Glinical outcome at 2 weeks post-illness onset

Table El. Demographic and Epidemiologic Data on 10 Patients with PCR-confirmed Chikungunya Infection

Among our patients, the most common clinical features are fever (100%), arthralgia (90%), rash (50%), and conjunctivitis (40%) (Table E2). Gastrointestinal and constitutional symptoms are less prominent. Arthritis is observed in only one patient, who had an effusion on the right knee. None had neurologic involvement or hemorrhagic manifestation. Table E2. Clinical Features

Table 2. Clinical features.

Sign/Symptom No. (%) of patients

Fever 10 (100)

Arthralgia 9 (90)

Rash 5 (50)

Conjunctivitis 4 (40)

Gastrointestinal symptom^* 3 (30)

Headache 3 (30)

Eye pain 2 (20)

Back pain 2 (20)

Mylagia 1 (10)

Arthritis 1 (10) a Nausea, vomiting, diarrhoea, or abdominal pain

Table E2 presents a summary of the key laboratory findings among our patients throughout the course of their illness.

White cell count, hemoglobin, hematocrit, platelet count, erythrocyte sedimentation rate for most patients are within the normal range. The mean nadir platelet count (± SD) is 199 ± 115 x 10⁹/L. Only one patient had severe thrombocytopenia (nadir platelet count, <100 x 10⁹/L) during the course of his illness. Elevated C-reactive protein levels (CRP, >10.0 mg/L) are observed in 60% of patients, but the peak C-reactive protein level is not significantly different between those who classified as severely ill and those who are not (mean, 40.3 mg/L vs. mean, 9.9 mg/L; P=O.195). The mean peak alanine and aspartate transaminases (ALT and AST) (±SD) are 58 ± 36 LVL and 50 ± 25 U/L respectively. Both ALT and AST are 2-fold greater than the upper limit of normal in one patient, who had pre-existing liver cirrhosis. None of the patients had a clinically abnormal total protein, urea or creatinine level. Among our patients, the mean nadir protein level (±SD) is 67 ± 5 g/dL, and the mean peak urea and creatinine levels (±SD) are 5.1 ± 1.7 mmol/L and 101 ± 16 mmol/L respectively. Lactate dehydrogenase level is the only laboratory parameter that is significantly higher in severely ill patients, compared to those who are not (mean, 732 U/L vs. mean, 525 U/L; P=O.047). Table E3. Laboratory Parameters in Chikungunya Confirmed Patients

Table 3. Laboratory parameters in chikungunya confirmed patients.

Variable Normal range Mean ± SD

Nadir white cell count, x10^Λ9/L 3.6-9.3 4.5 + 1.1

Nadir hemoglobin, g/dL 13.0-17.0 14.6 + 1.3

Peak hematocrit, % 41.0-51.0 46.6 ± 3.4

Nadir platelet count, x10^Λ9/L 170-420 199 + 115

Peak erythrocyte sedimentation rate (ESR), mm/hr 1-10 7 ± 8

Peak C-reactive protein (CRP), mg/L 0.0-5.0 26.8 ± 33.7

Peak alanine transaminase (ALT), U/L 17-63 58 ± 36

Peak aspartate transaminase (AST), U/L 15-41 50 ± 25

Nadir total protein, g/dL 63-81 67 + 5

Peak urea, mmol/L 2.9-9.3 5.1 ± 1.7

Peak creatinine, umol/L 60-110 101 + 16

Peak lactate dehydrogenase (LDH), U/L 250-580 654 + 150

Example 6. Initial Study - Acute Disease - Cytokine, Chemokine and Growth Factor Profiles

Profiles of 30 cytokines, chemokines and growth factors are determined by a multiplex-microbead immunoassay on acute blood samples collected upon hospitalization. The samples collected ranged from day 2 to day 19 of illness (median, day 4.5).

To characterize the overall patterns, a two-way hierarchical clustering analysis is done to allow the classification of individuals according to disease severity based on the clinical features (Figure 1). Evidently, this had the power to discriminate the clinical forms of CHIKF in the samples in this study from the healthy controls, with patients classified as non-severe and severe disease segregating perfectly.

The levels of 8 plasma cytokines (IL-2R, IL-5, IL-6, IL-7, IL-8, IL-IO, IL-15 and IFN- α) are observed to be most significantly elevated (Figure 2A) in CHIKF patients compared to uninfected subjects (P<0.05). Among these is proinflammatory cytokine, IL-6 which is very significant. Interestingly, another proinflammatory cytokine, IL-8 is down-regulated in these patients. Anti-inflammatory cytokine, IL-10 is found to be significantly raised in most of the patients (P<0.05). The plasma concentrations of IL-2R and IL-5 are found to be increased in all patients. Levels of IFNα and IL-7 are elevated in all patients. The levels of other cytokines such as IL-2, IL-4, IL- 12, IL- 13, IL- 17, IFN-γ, and TNF-α, are only marginally increased in the CHIKF group compared with those in the uninfected group (Figure 4).

Profiles of chemokines, IP-10 and MIG are shown to be significantly elevated, while Eotaxin is suppressed (Figure 2B). There is no difference in the levels of other chemokines namely, MCP-I, MIP-Ia, MlP-lβ and RANTES (Figure 4).

Interestingly, the levels of 4 growth factors are found to be significant in the patients, with up-regulation of HGF, FGF-basic and VEGF, with the exception of EGF which is almost totally suppressed (Figure 2C). It is observed that the CHIKF patients exhibited low levels of GM-CSF and G-CSF (Figure 4).

Example 7. Initial Study - Acute Disease - Gene Expression Related to Severity of Chikungunya

Finally, in an effort to identify cytokine, chemokine, and growth factor plasmatic levels associated with severity, statistical analyses are performed after stratification of the CHIKF patients according to severity.

It is observed that an increase in levels of IL-I β and IL-6, and a decrease in RANTES respectively are associated with disease severity (Figure 3A). The levels of all other markers are not significantly different (Figure 5 and Figure 6).

Example 8. Initial Study - Acute Disease - Discussion

CHIKF, an emerging arboviral infection, which induces high fever, has only been recently reported in Singapore. Up to Dec 2007, all CHIKF patients had contracted the infection overseas [22]. The first local outbreak of CHIKF occurred in Jan 2008. More than 2,500 people who lived or worked in the outbreak area were screened and a total of 13 PCR- confirmed cases were identified [22,24]. All confirmed CHIKF cases were referred to the CDC/TTSH. Our report included 10 patients who participated in this study.

Phylogenetic analysis of the viral sequences of our patients has revealed that the circulating strains were of the Indian Ocean genotype and closely related to those from the 2006 outbreak in India [29] but without the A226V mutation, further emphasizing how remarkably rapid the disease could spread with the right environmental conditions. The attack rate in our outbreak was 0.5%, much lower than the 34% reported in Reunion Island and the 5.4% observed in Italy [13,30]. This could be attributed to the rapid removal of human reservoirs through isolation, enhanced vector control, or the circulation of a virus strain of lower epidemic potential. Clinical features of our patients were similar to those reported in recent outbreaks [6-9,11-14,16-18,21,30], indicating that although people are genetically diverse response to diseases is homogeneous across people in non-homogeneous populations. The majority of our patients was ≤ 45 years and had no pre-morbid condition. Unlike patients reported in the Reunion Island outbreak [6], where the patients' underlying medical conditions could have contributed to the observed morbidities, our patients were younger and healthier. Furthermore, none of our patients was co-infected with dengue, as confirmed by RT-PCR and dengue enzyme-linked irnmunoabsorbent assay (ELISA)-IgM and IgG [31]. Hence, our immunologic observations can be largely attributed to acute CHIKV infection itself.

In recent years, most of the studies on CHIKF have been addressed with the clinical description of the disease [4-9,11,17,18,21], the molecular nature of the virus [9,10,19] and diagnostics methods [8-10,24], and the interactions of the virus with its mosquito vector, Aedes [1-3,11-15]. Here, we describe for the first time the comprehensive systemic production of cytokines, chemokines, and growth factors during acute CHIKV infection which may light the path ahead in understanding the innate response to the infection. We first showed that a wide range of cytokines such as IFN-α,, IL-5, IL-6, IL-7, IL-10, IL-15 were produced in response to CHIKV infection. IFN-α is a potent anti- viral cytokine and has been shown to strongly inhibit CHIKV in vitro [32]. The high levels of IFN-α that we detected provide a logical explanation for how the body rapidly brings CHIKV viremia under control [8,11]. It has been shown that the main producers of IFN-α are plasmocytoid dendritic cells [33] and monocytes [34].

The profile of circulating cytokines revealed a predominance of Type 2 cytokines. Mainly IL-5, IL-6 and IL-10 levels were increased and those of IFN-γ or TNF-α were unchanged as compared to non-infected controls. This suggests that acute CHIKV infection tilts the cytokine profile to anti-inflammatory response, which would argue against the common understanding of CHIKV infection which does not really support the common description of the CHIKV infection as an inflammatory disease [8]. Alternatively, it is possible that an inflammatory response might occur earlier when the virus is actively replicating, and then gets down-regulated by a counter-anti-inflammatory response when the virus is being eliminated from circulation. High levels of anti-inflammatory IL-IO and the presence of high levels of chemokines IP-IO and MIG (ligands of CXCR3 associated with Thl-type reactions) [35], detected here would support this hypothesis. Further studies would be needed to clarify this issue. The Type 2 cytokines detected are also important mediators of B cell growth and maturation, and thus may allow the production of high levels of persisting anti-CHIKV IgG [5].

The detection of high levels of circulating IL- 15 is of interest, since this cytokine has been shown to be a major stimulator of NK cells [36] and T cells [37]. Thus our data suggest that these lymphocytes population might be activated during acute infection and may also contribute to viral control during the acute phase of CHIKV infection. Detection of soluble IL- 2R in the plasma suggests T cell activation since this molecule is secreted by activated T cells [38]. Experiments are planned to study the activation phenotype of T and NK cell subsets in acutely infected patients.

The detection of IL-7 and IL- 15 is significantly interesting with regards to the immunopathology of CHIKF since CHIKV infection has been shown to induce rapidly developing and persisting arthralgia [6]. Here, 9 of the 10 patients manifested this pathology. IL-7 is known to have an important role in the development of rheumatoid arthritis [39], while IL- 15 has been associated with the development of joint inflammation [40]. It has been proposed that expansion of a particular IL15-induced NK cell subsets was responsible for this phenomenon [41]. The role of IL- 15 and NK cells in the development of CHIKV arthralgia would definitively be worth investigating. We did not detect TNF-α in the plasma of the patients with acute CHIKV infection. This is surprising since this cytokine has been detected repeatedly in the blood of patients suffering from other arthritides such as rheumatoid arthritis and is known to be involved in the pathogenesis of these entities [42]. Thus, it is possible that CHIKV-induced arthralgia does not depend on TNF-α. Alternatively, TNF-α might be produced only locally. Analysis of synovial fluid or joint tissue immunohistochemistry would be necessary to provide important information on the role of TNF-α and other mediators.

Chemokines are crucial mediators of innate and adaptive immunity against various viral infections [43]. IP-10, and MIG had increased plasma levels during CHIKV infection. These two chemokines signal through the same receptor CXCR3 and thus might activate and direct migration of this T cell subset [35]. IL-8 and Eotaxin levels were lower than those of naive controls. Defining the exact contributions of these different chemokines will require further studies.

We also tested the presence of growth factors in the plasma of CHIKV-infected patients. HGF₅ FGF-basic and VEGF were produced at high levels and may reflect a physiological response to tissue destruction resulting for the viral infection. Interestingly, EGF levels were lower than in healthy controls. The low levels of EGF might be due to the concomitant decrease of platelets observed in infected patients since previous studies have shown that plasma levels of EGF are associated with circulating platelets [44].

Although limited, we had access to sufficient patients to perform data analysis in relation to the severity of the disease (severe illness was defined as fever > 38.5 ⁰C, or maximum pulse rate > 100 beats/minute, or nadir platelet count < 100 x 1O⁹ZL). Using this definition, we observed that higher disease severity was associated with increased plasma levels of IL-I β and IL-6 and a decreased level in RANTES (Figure 3b). IL-lβ and IL-6, whose levels are already high in the CHIKV infected patients, are potent endogenous pyrogens [45-48]. Therefore, elevations of IL-lβ and IL-6 might account for the high fever in the severe cases. The increase production of IL-lβ might also mediate the development of abrupt and persistent arthralgia since this cytokine is involved in the immunopathogenesis of many arthritic pathologies such as rheumatoid arthritis [49]. On the contrary, T cell chemokine RANTES levels were significantly suppressed in severe CHIKF patients. Platelets are a major reservoir of RANTES in the peripheral circulation [50], and severe CHIKF was characterized by thrombocytopenia. Thus, as mentioned above for EGF, thrombocytopenia can also reduce levels of circulating RANTES. Low levels of RANTES correlate with disease severity and mortality in individuals with severe malaria, who were also correspondingly thrombocytopenic [51]. Interestingly, it was observed in other studies that RANTES levels were up-regulated in dengue [48], except for one single report from Cuba [52]. Since the symptoms of CHIKF mimic those of dengue fever, results obtained from this study strongly suggest that RANTES could be a potential biomarker that differentiates between these 2 clinically very similar diseases. One limitation of this study is in the classification of disease severity as none of our patients developed neurologic or hemorrhagic complications previously reported in CHIKF patients. Nonetheless, our definition of severe illness would have included patients with sepsis, a serious form of infection commonly associated with a temperature of >38°C and heart rate of >90/minute [24]. Furthermore, we included thrombocytopenia of <10Ox 10⁹/L as a criteria for severe CHIKlF. Marked thrombocytopenia is a common feature of sepsis [25] and has been identified as a predictor of mortality [26, 27]. The degree of thrombocytopenia is a determinant of survival and once the platelet count decreases below 100xl0⁹/L, mortality continues to increase, even though the risk of bleeding does not [28]. A wide spectrum of disease has been reported in CHIKF ranging from asymptomatic infections, to self-limiting febrile illness [8], to neurologic complications, and death [17]. The "severe illness" cohort in our study possibly represents a more severe form of self-limiting febrile illness, an intermediate group with higher levels of viremia (data not shown) and distinctly more severe clinical features (i.e. high temperature, tachycardia, and severe thrombocytopenia). Using this clinical phenotype, we have shown in this study that immune mediators are able to distinguish very mild disease from more severe forms of CHIKF disease at the acute stage. Follow-up studies will be required to determine if long-term sequelae are indeed different between non- severe and severe clinical presentations. Elucidating the association of disease severity with two cytokines and one chemokine can be useful in order to provide early identification and monitoring of patients with severe disease. Although this study is limited by the size of the outbreak, nevertheless, based on these observations, measurement of immune mediators could be helpful for the management of future outbreaks. This study strongly suggests these biomarkers be used for measuring disease severity and be tested in outbreaks in different populations and different strains. Once confirmed, they will be useful for follow-up studies, association studies, and prognosis for public health management. More importantly, these biomarkers can potentially lead to the development of modulators to reduce disease severity and to halt disease progression. Example 9. Longitudinal Study - Acute and Chronic - Longitudinal Study of Immune Responses from CHIKV patients

The new manifestation of an old disease like CHIKF posed new challenges in diagnosis, monitoring, control and treatment in many countries already struggling to cope with a rising incidence of Dengue cases in urban centres.

The current knowledge on the possible interactions between the immune system and the different stages of the virus life cycle remains poorly defined. Understanding to what extent protective and pathogenic immune mechanisms are present in CHIKV disease is critical to have a better insight into the pathology and possible therapeutic routes targeting the viral niches and the chronic inflammation.

Therefore, an important challenge in understanding the pathogenesis of CHIKV infections is the elucidation of specific host responses in acute as well as chronic conditions.

Therefore, longitudinal studies involving patients is important as this will give insights on how the host responds to viral infection, which represents a complex orchestration of divergent pathways designed to eradicate the virus and benefit the host.

Example 10. Longitudinal Study - Acute and Chronic - Introduction

The longitudinal study was conducted in a similar manner to the initial study of acute infection as described in Examples 1 and 5, with the exception that a larger cohort of 30 patients were included in the longitudinal study. The study lasted 6 months, and samples were taken from July 2008 to January 2009.

Four different collections were made in the manner described in Example 1 :

1st Collection: Acute - 2 to 4 days after disease onset

2nd Collection: Late Acute - 7 to 10 days after disease onset

3rd Collection: 14 days after disease onset

4th Collection: Chronic - 1 month to 3 months after disease onset Analysis of inflammatory cytokine, other cytokine, chemokine and growth factor protein levels was conducted according to Examples 2 and 6. Data analysis, including statistical analysis, was conducted according to Examples 3, 4 and 7.

Example 11. Longitudinal Study - Acute and Chronic - Results

Results of the longitudinal study are shown below in Table E4.

Table E4. Production levels of severe chikungunya biomarkers in affected individuals. Legend: + high production; - low production

Example 12. Longitudinal Study - Discussion - Insights to Disease Chronicity and Immuno-pathogenesis of CHIKF

In the earlier study (Examples 5 to 8), it was shown that global analyses on the specific involvement of cytokines and chemokines have showed that IL- lβ, IL-6, and RANTES were associated with disease severity (defined as having a temperature of > 38.5 ⁰C or pulse rate > 100/min, or platelet count < 100x10⁹ g/L) thus enabling the identification of patients with poor prognosis and monitoring of the disease. More importantly, these biomarkers can potentially lead to the development of modulators to reduce disease severity and halt disease progression.

Fever experienced by all CHIKF patients could be attributed to cytokines such as IL- lβ, IL-6 and TNF-α, which are known pyretics. In the new study, we demonstrated that cytokines were detected at high levels in acutely infected patients and the levels returned to normal after fever and viremia has disappeared. Arthralgia experienced by CHIKF patients closely resembles the symptoms induced by other viruses like RRV and Barmah Forest virus (BFV). Such alphavirus-induced arthralgia mirrors rheumatoid arthritis, a condition which is characterised by severe joint pains due to inflammation and tissue destruction caused by inflammatory cytokines such as IL- lβ, IL-6 and TNF-α as was observed in the 2^nd study (Examples 9 and 10). It is thus plausible that CHIKV infection and/or other arthritis-causing alphaviruses induce similar pro-inflammatory cytokines that cause arthralgia, explaining why joint pains are constant ailments of many patients infected with CHIKV even years after recovery from the initial febrile phase.

It has been shown that the sequestration of macrophages and their associated pro- inflammatory soluble mediators significantly improved joint and muscular tissue inflammation. Since high concentrations of these pro-inflammatory factors were found in the joints of humans afflicted with RRV-induced polyarthritis, they probably have a causative role in chronic joint and muscle pains that plague patients; more specifically it was found that TNF-α, IFN-γ and macrophage chemoattractant protein (MCP)-I were implicated in RRV- induced inflammatory diseases . The finding that aberrant Type-I interferon signalling in mice led to severe forms of CHIKF further highlighted important roles cytokines play in the pathology of CHIKV infection. In a separate study, it was found that in CHIKV+ biopsies (synovial tissue and fluid), gene expression of IFN-α, ILlO but not of proinflammatory cytokines (TNF-α, ILl β, IFN-γ). These findings are in agreement with a canonical chronic immune response reminiscent of but distinct from rheumatoid arthritis. The absence of neutrophils in the synovial fluid and the paucity of proinflammatory cytokines such as TNF-α and ILl-β are important components to consider while selecting the best treatment regime for chronic CHIKV disease.

The 2^nd study (Examples 9 and 10) has shown the presence of the above immune mediators in significant levels from plasma collections of CHIKF patients, further establishing the importance in using such approaches to determine disease progression and irnmuno- pathology.

Example 13. Detection of Severity and Prognostic Tests in Chikungunya Patients

Plasma samples are obtained from patients during the acute phase of their illness (upon 1^st medical contact).

Clinical features, and routine hematological and biochemical laboratory test findings

(i.e. full blood count, renal and liver function tests, C-reactive protein) are collected.

To confirm the presence of CHIKV-infection, routine laboratory tests are conducted (PCR and serology tests).

Plasma samples are collected as described above in Example 1. They are aliquoted and stored at -80 ⁰C until analyses are done.

A multiplex biometric immunoassay, containing fluorescent dyed microspheres conjugated with a monoclonal antibody specific for a target protein, is used for cytokine measurement according to manufacturer's instructions (Biosource Human Cytokine 30-plex Assay, Invitrogen), as described in Example 2.

50 μl of plasma samples are incubated with antibody coupled beads for 2 h at RT ⁰C.

Complexes are washed twice with the use of a vacuum manifold before incubation with biotinylated detector antibody for 1 h at RT ⁰C. Complexes are then washed twice followed by incubation with Streptavidin-phycoeryrhrin (RPE) for 30 min at RT ⁰C. Complexes are washed thrice and incubated with wash buffer for another 3 min before detection in the Luminex 200™ instrument.

Results are acquired by the IS 2.3 software and the standard curves were plotted through a five-parameter logistic curve setting.

Expression levels of one or more of the factors shown in Table Dl or Table E4 above are compared to control (non-infected) individuals. Where a factor is annotated "+" (i.e., high production) in the column corresponding to a particular condition, and the actual protein level of that factor is higher in the individual concerned than that of a control individual, then it is likely that the individual has that condition. Likewise, where a factor is annotated "-" (i.e., low production) in the column corresponding to a particular condition, and the actual protein level of that factor is lower in the individual concerned than that of a control individual, then it is likely that the individual has that condition.

Thus, for example, where an individual exhibits an over-expressed level of IL-lα/IL- 1 β and an under-expressed level of RANTES, compared to a control individual, then he is likely to be suffering from severe acute chikungunya infection. Similarly, where an individual is found to exhibit a higher level of expression of IL-4 protein compared to a control individual, then he is likely to be suffering from severe chronic chikungunya infection.

A patient is defined as having severe illness, if he had either a maximum temperature of more than 38.5 ⁰C, or a maximum pulse rate of more than 100 beats/minute, or a nadir platelet count of less than 100 x 10⁹/L. Laboratory results are expressed as mean ± SD.

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Each of the applications and patents mentioned in this document, and each document cited or referenced in each of the above applications and patents, including during the prosecution of each of the applications and patents ("application cited documents") and any manufacturer's instructions or catalogues for any products cited or mentioned in each of the applications and patents and in any of the application cited documents, are hereby incorporated herein by reference. Furthermore, all documents cited in this text, and all documents cited or referenced in documents cited in this text, and any manufacturer's instructions or catalogues for any products cited or mentioned in this text, are hereby incorporated herein by reference.

Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the claims.

Claims

1. A method of distinguishing a severe chikungunya infection from a non-severe chikungunya infection in an individual, in which the method comprises detecting, in a sample in or of an individual:

(a) an increased expression or activity of IL-I β, an increased level of IL-6 and a decreased level of RANTES, in which such levels indicate severe chikungunya;

(b) an increased expression or activity of all of IL-I α, IL- lβ, IL-2, IL-6, IL-7, IL-8, IL- 12, IL-15, IFN-a, IP-10, Eotaxin, MCP-I, MIG and a decreased level of RANTES, in which such levels indicate severe acute chikungunya; or (c) an increased expression or activity of all of IL-I α, IL- lβ, IL-2R, IL-4, IL-6, IL-7,

IL-8, IFN-α, IL-12, IL- 15, MCP-I, MJP-I α, Eotaxin, RANTES, IP-IO, MIG, EGF, FGF-b, G-CSF, HGF, in which such levels indicate severe chronic chikungunya; or a variant, homologue, derivative or fragment thereof such as a sequence having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology thereto.

2. A method according to Claim 1 , in which:

(a) IL- lβ comprises a polypeptide sequence having GenBank Accession Number NP_000567 or a nucleic acid sequence having GenBank Accession number NM_000576 or a variant, homologue, derivative or fragment thereof such as a sequence having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology thereto and comprising (or encoding a sequence comprising) interleukin-lβ activity;

(b) IL-6 comprises a polypeptide sequence having GenBank Accession Number NP_000591 or a nucleic acid sequence having GenBank Accession number NM_000600 or a variant, homologue, derivative or fragment thereof such as a sequence having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology thereto and comprising (or encoding a sequence comprising) interleukin-6 activity; or

(c) RANTES (syn. CCL5) comprises a polypeptide sequence having GenBank Accession Number NP_002976 or a nucleic acid sequence having GenBank Accession number NM_002985 or a variant, homologue, derivative or fragment thereof such as a sequence having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology thereto and comprising (or encoding a sequence comprising) RANTES/CCL5 activity.

3. A method according to Claim 1 or 2, in which the level of the biomarker in the sample is compared to a reference level being the level of the biomarker in an individual who is not suffering from severe chikungunya, for example in which the level of the biomarker in the sample is compared to a reference level as set out in column 6 of Table Dl.

4. A method according to Claim 1 , 2 or 3, in which an individual with severe chikungunya infection exhibits, or is expected to exhibit any one or more, such as all, of the following symptoms: (a) a temperature of > 38.5 ⁰C ; (b) pulse rate > 100/min, and (c) platelet count < 100x10⁹ g/L.

5. A method according to any preceding claim, in which detection of acute chikungunya indicates that the disease is within day 2 to day 19 of infection, or in which detection of chronic chikungunya indicates that the disease is after day 19 of infection.

6. A method according to any preceding claim, in which the detection comprises polymerase chain reaction, such as real-time polymerase chain reaction (RT-PCR), Northern Blot, immunological detection such as ELISA, RNAse protection or microarray hybridisation.

7. A combination of two or more nucleic acids or polypeptides specified in any of Claims 1 to 6 or probes or antibodies capable of binding specifically thereto, such as a combination of nucleic acids immobilised on a substrate, preferably in the form of a microarray.

8. A nucleic acid or polypeptide as specified in any of Claims 1 to 6, a combination according to Claim 7, or an agonist or antagonist thereof for use in a method of detecting, determining the severity of or treating chikungunya.

9. A pharmaceutical composition comprising a nucleic acid or polypeptide as specified in any of Claims 1 to 6, a combination according to Claim 7, or an agonist or antagonist thereof.

10. A diagnostic kit for chikungunya or the severity thereof or susceptibility thereto, the kit comprising any one or more of the following: (a) a polypeptide as specified in any of Claims 1 to 6 or a variant, homologue, derivative or fragment thereof such as a sequence having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology thereto;

(b) a molecule capable of binding to such a polypeptide, such as an antibody; (c) a nucleic acid capable of encoding such a polypeptide, such as a nucleic acid as specified in any of Claims 1 to 6 or a variant, homologue, derivative or fragment thereof such as a sequence having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology thereto; and

(d) a molecule capable of binding to such a nucleic acid sequence, such as a nucleic acid, for example probe preferably comprising a complementary nucleic acid;

optionally together with instructions for use.

11. A method of identifying a molecule suitable for the treatment, prophylaxis or alleviation of chikungunya, the method comprising determining if a candidate molecule is an agonist or antagonist of a polypeptide specified in any of Claims 1 to 6.

12. A polypeptide as specified in any of Claims 1 to 6 or a nucleic acid capable of encoding such a polypeptide, for example a sequence having an accession number shown in Table Dl, for use in a method of treating, preventing or diagnosing severe chikungunya.

13. A method of treatment or prevention of chikungunya in an individual, the method comprising detecting chikungunya or determining the severity thereof, or both in an individual by a method according to any preceding claim and administering a suitable treatment or prophylactic, such as a drug known or suspected to be useful for treating chikungunya, to the individual.

14. A method for the treatment or prevention of chikungunya in an individual, in which the method comprises modulating the expression of a nucleic acid or polypeptide specified in any of Claims 1 to 6, or in which the method comprises administering an agonist or antagonist of such a nucleic acid or polypeptide.

15. A method according to any preceding claim, in which the expression or activity of one or more such as a subset of the genes set out in (a), (b) and (c) is detected.

16. A method, combination, nucleic acid, polypeptide or pharmaceutical composition substantially as hereinbefore described with reference to and as shown in Figures 1 to 12 of the accompanying drawings.