Cytokine

#This application is a US national stage under 35 U.S.C. §371 of PCT/GB2006/050121 filed May 25, 2006, which in turn claims priority to British application number GB0510559.8 filed May 25, 2005. These applications are herein incorporated by reference.

#The present invention relates to a cytokine. Aspects of the invention relate to forms of the cytokine having agonistic effects on a range of cytokine receptors, while further aspects of the invention relate to forms of the cytokine having antagonistic effects on a range of cytokine receptors. Other aspects of the invention relate to treatment of various disorders characterised by aberrant cytokine activity; and to use of the cytokine as an adjuvant or diagnostic; or as screens for inhibitors of function or for processing of the cytokine gene.

#Human herpesvirus 6, HHV-6, exists in at least two strain groups: variant A and variant B, HHV-6A and HHV-6B. They are closely related, averaging 5% in herpesvirus conserved genes with greater divergence at the ends of the genomes and selected sites between conserved gene blocks (1-3). These variants are related to a smaller genome of human herpesvirus 7 (HHV-7) forming the Roseoloviruses and together with the more distant human cytomegalovirus form the betaherpesvirus subgroup of the herpesvirus family, maintaining a conserved gene order and similarity in sites of latency including monocytic/macrophage cell types. HHV-6A and HHV-6B have differing geographic prevalence with HHV-6B dominant in USA, Europe and Japan, while HHV-6A appears only a minor variant except in African countries where it appears equally prevalent to HHV-6B (4, 5). However exhaustive surveys have not been conducted using serological specific reagents given the close relation between these viruses. There are hotspots for variation between representatives of these virus genomes and these may contribute to some cellular tropism and pathological differences which have been anecdotally reported. For example, only HHV-6A has been detected in skin biopsies and HHV-6A has been increasingly implicated in cases of multiple sclerosis where careful genotyping and identification of active infections have been carried out (5-10). These studies either implicate immune abnormalities in clearance of the virus or possible complications of rare primary adult infection with this variant, since in countries where this has been studied HHV-6B is the predominant variant identified.

#The present invention will now be described by way of example only and with reference to the accompanying drawings, in which:

#FIG. 1 shows SDS-PAGE and HPLC data of purification of a number of U83A variations;

#FIG. 2 shows identification of the purified U83A variations by western blot

#FIG. 1. Purification of U83A. A, Shown are traces (A280 nm) from three separate purifications using reversed phase high pressure liquid chromatography. B, Silver-stained SDS-PAGE of peak fractions show the separation of native U83A in fractions 49 and 50, from uncleaved U83GST fusion protein and cleaved GST in fractions 56-60.

#FIG. 2. Identification of purified U83A and variants by Western blot. A, Silver stained SDS-PAGE showing HPLC purified U83ANpep, U83A purified after EK cleavage, U83A(EK) (EK-U83-GST construct), or after Xa cleavage U83A(Xa) (Xa-U83A-GST construct), or N-terminal variants of U83A as indicated (GS-U83A, GSRIEGR-U83A, GSRDDDDK-U83A) purified after thrombin digestion of constructs U83A-GST, Xa-U83-GST (factor Xa site N-terminal modified U83A), and EK-U83A-GST (enterokinase site N-terminal modified U83A). MW are molecular weight markers as indicated and GST is the HPLC purified GST moiety. B, Western blot results using anti-U83A intron peptide sera identified native full-length U83A and N-terminal variants. C, Control Western blot results using anti-GST sera confirm specificity and purity, identifying only the GST.

#FIG. 3. Induction of intracellular calcium mobilisation using U83A and human endogenous chemokine ligands. CHO cell lines stably transfected with CCR1, CCR4, CCR6 and CCR8 and labelled with Fluo-3 are treated after 20 secs with 50 nM U83A, U83A-Npep or endogenous human chemokine ligands CCL3, CCL20, CCL17 and CCL1 respectively. RFU are relative fluorescent unit, indicating relative levels of induction of calcium mobilisation. EC50s were calculated as indicated from measuring peak induction levels after stimulation with 100, 50, 10, 7.5, 2.5, 1 nM concentrations of indicated U83A or human chemokines. Negative controls were the vehicle buffer without added chemokine.