This plasmid encodes a chimeric protein from the interferon regulatory factor family, which can enhance both the Th1 and Th2 responses in T cells, leading to the activation of cytotoxic T cells and/or the production of antibodies

This plasmid encodes a chimeric protein from the interferon regulatory factor family, which can enhance both the Th1 and Th2 responses in T cells, leading to the activation of cytotoxic T cells and/or the production of antibodies.37,38 In our study, this adjuvant showed no significant positive effect on antibody production and spleen cell proliferation. the disease incidence has increased due to environmental changes and increased outdoor activity leading to frequent exposure to chigger mites.6 Although scrub typhus can be treated with antibiotics Nicotinuric acid such as doxycycline, tetracycline, and azithromycin, reinfection occurs frequently because of poor cross-reactive immunity and the short duration of protective immunity.7 Therefore, continuous efforts have been invested in the development of an effective prophylactic vaccine.8C14 In addition, vaccination would overcome troubles in early clinical diagnosis and high mortality, and could limit the potential for developing antibiotic resistance.15C18 Several recombinant proteins have been tested for protection against homologous and/or heterologous strains in mice and nonhuman primates.4,19C22 However, no vaccine is available to date. In the present study, we studied the feasibility of the 47-kDa outer membrane protein (OMP), a major OMP of contamination. In this study, we used intranasal and intramuscular immunization with recombinant 47-kDa OMP (rec47) and 47-kDa OMP-expressing DNA (p47) in mice. Immunization efficacy including humoral and cellular immune responses, as well as protection against a homologous challenge, were assessed. MATERIALS AND METHODS Ethics statement. All animal care and experimental procedure conformed to the Korea National Institutes of Health (KNIH) guidelines (KNIH publication no. 11-1352173-000133-01, 2013) in accordance with National Guideline for the Care and Use of Laboratory Animals; the animal protocol used in the present study was reviewed and approved by the Institutional Animal Care and Use Committee of Korea Centers for Disease Control and Prevention (approval number: KCDC-040-14-2A). Bacterial strains and generation of recombinant plasmids. The pathogenic Boryong strain, an endemic isolate from Korea, was used in this study. The pathogen was propagated in L929 cells (ATCC CLC-1) as described previously.26 The infected cells were incubated at 34C in 5% CO2. At 3C4 days postinfection, infectivity was decided using an indirect immunofluorescence assay. Genomic DNA was extracted using the QIAamp genomic DNA kit (QIAGEN, Hilden, Germany). TOP10 and BL21 DE3 strains (Invitrogen, Carlsbad, CA) were used for cloning and prokaryotic expression, respectively. The primers OTBS1837-31-F (5-GTGGATCCATGGTATTACCTCAACAAAAATC-3) and OTBS1837-466-R (5-GTCTCGAGTTACTTATTAATATTAGGTAAAGC-3) were used for amplification of a truncated form (amino acids 31C466) of the 47-kDa OMP gene (GenBank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”NC_009488.1″,”term_id”:”148283997″,”term_text”:”NC_009488.1″NC_009488.1). The polymerase chain reaction product was digested with BL21 (DE3) for overexpression. The bacterial culture conditions and the procedures for detection and purification of rec47 were as described previously.27 The nucleotides for the 47-kDa OMP DNA vaccine were synthesized with codon optimization for mammalian expression (Bioneer, Daejeon, Korea). The synthetic nucleotides were cloned into pVAX1 (Invitrogen) to generate the recombinant plasmid for mammalian expression p47. Protein expression was confirmed by transient expression of p47 in BHK-21 cells, using Lipofectamine 2000 Reagent (Invitrogen) according to the manufacturer’s instructions. Immunization and challenge of mice. Ninety-one 6-week-old female BALB/c mice (Charles River Laboratories, MA) were randomly divided into seven groups of 13 mice and used for immunization. In protein administration groups, mice were immunized intranasally with 10 g of rec47 with or without 10 g of heat-labile enterotoxin B subunit (LTB; Sigma Aldrich Co., Saint Louis, MO) from or 10 g of cholera toxin (CT; List Rabbit polyclonal to CD146 Biological Laboratories Inc., CA, USA) adjuvants. In DNA administration groups, mice were intramuscularly injected with 100 g of p47 plasmid with or without 100 g pBOOST2-samIRF7/3 (pB; InvivoGen, San Diego, CA). All immunizations were performed three times with 2-week intervals. At day 10 after each immunization, blood was collected from the mice by tail bleeding. Blood sera were recovered by centrifugation and stored at ?20C until further use. At day 10 after the third immunization, three mice of each group were euthanized and Nicotinuric acid spleens were isolated for cell proliferation analysis. For the protection study, 10 immunized mice of each group were challenged by intraperitoneal injection of 100 occasions the median Nicotinuric acid lethal dose of the homologous Boryong strain (4.3 .