Percent body weight per group was calculated compared to the body weight at the time of challenge. shown to be transmitted through blood transfusion, organ transplantation, and breast-feeding , , , , , . JEV is the single-most important cause of viral encephalitis in Asia, with case fatality rates averaging 30% , , . JEV is usually a major problem in South-East Asia, India, and China, where the P300/CBP-IN-3 virus is usually endemic. In recent years, JEV has spread to other geographic areas such as Australia and Pakistan, and has thus become an important emerging computer virus contamination in these areas. Vaccination against JEV using a mouse brain-derived, inactivated P300/CBP-IN-3 vaccine has been shown to be very effective and P300/CBP-IN-3 has led to a decreased disease burden , , . However, there are concerns about the immunogenicity and the safety of this vaccine , . A live-attenuated (SA14-14-2 strain) and a cell culture-based JEV vaccine that are produced on primary hamster kidney (PHK) cells have Igfbp1 been licensed for use in China and have been shown to be safe and effective . However, since PHK is P300/CBP-IN-3 not an approved cell line for production of human vaccine, many countries will not use this JEV vaccine. Therefore, a recombinant protein-based vaccine represents a stylish alternative. The E protein of flaviviruses is the most immunogenic and suitable for the purpose of vaccine development. The protein E consists of three structural domains (DICDIII) , , of which DIII contains predominantly sub-complex- and type-specific epitopes, many of which induce neutralizing antibodies , , , , , , . Several vaccines based on DIII have been shown to be immunogenic and effective under certain conditions , , . The 315 nucleotides of WNV-NY99 E protein (accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”AF196835″,”term_id”:”11597239″,”term_text”:”AF196835″AF196835, passage 5, obtained from the Health Protection Agency, Porton Down, UK) that constitute the ectodomain of DIII were BamH1 ( em caggatccaGGAACAACCTATGGCGTCT /em ) and Sal1 ( em atgtcgacTTTGCCAATGCTGCTTCCA /em ) cloned into the pTRHis2A bacterial vector (Invitrogen), upstream of the histidine repeat string. Recombinant DIII (rDIII) protein expression was induced with isopropyl–d-thiogalactopyranoside. Purification of rDIII was performed with nickel-affinity chromatography as previously described . Purification of the solubilised rDIII resulted in 80C90% real fractions of rDIII (not shown), which were used in subsequent vaccination experiments. The authenticity of the purified recombinant WNV E DIII protein was confirmed by western blot analyses using a WNV monoclonal antibody (7H2; Bioreliance Corp., Rockville, USA). The rDIII protein had a molecular weight of 13?kDa, which corresponds to what has been described previously . To produce an inactivated whole computer virus vaccine, WNV was propagated in Vero E6 cells, P300/CBP-IN-3 concentrated and purified by sucrose gradient centrifugation and subsequently inactivated with -propiolactone (BPL) in a biosafety-level 3 laboratory, using the same method as previously described for inactivation of the SARS coronavirus . All vaccine preparations used for primary immunization were adjuvanted with synthetic CpG oligonucleotide (ODN: TCCATGACGTTCCTGACGTT, Sigma, Haverhill, UK), which was mouse optimized . For booster immunizations, vaccine candidates were adjuvanted with oil (Stimune?; Cedi-Diagnostics, Lelystad, The Netherlands) as recommended by the manufacturer. Immunization experiments were performed as follows: six groups of 10 mice each were immunized subcutaneously with 25?g of the purified rDIII (groups A and B), 15?g BPL-inactivated WNV (WNV-BPL; groups C and D), and a control vaccine (15?g BPL-inactivated rabies; groups E and F). Animals were immunized first with 25?g of ODN adjuvanted vaccine on day 0 and boosted with the same oil-adjuvanted vaccine on day 14. To determine the virus-specific IgG response upon vaccination, an enzyme-linked immunosorbent assay (ELISA) was developed using WNV-NY99 or JEV-Beijing-1 strains as antigens. To this end, purified computer virus was solubilised (end-concentration 250?g/ml) in PBS containing 4% (w/v) Mega-10 (Sigma, Zwiijndrecht, The Netherlands) and microtitre plates (Costar, Cambridge, MA, USA) were coated with 2?g total protein (diluted in PBS) per well. Twofold dilutions of sera (1:25C1:3200) were prepared in PBS made up of 0.2% (w/v) bovine serum albumin, 0.1% (w/v), dry milk powder and 3% (w/v) sodium chloride (Sigma, Zwijndrecht, The Netherlands). Virus specific IgG was measured using a HRPO labeled.