Rotavirus nonstructural protein 4 (NSP4) is a protein with pleiotropic properties. strain, and SA11 NSP4 localized withina 2/6 -virus like particle (VLP) also exhibited adjuvant effects. These studies suggest that the rotavirus enterotoxin NSP4 can function as an adjuvant to enhance immune responses for a P005672 HCl co-administered antigen. Introduction A wide range of infectious pathogens come into contact with the host at mucosal surfaces. Conventional parenteral vaccines are generally ineffective at eliciting mucosal immunity [1C3]. Recent efforts have focused on the POLR2H development of mucosal vaccines in an attempt to combat invading pathogens at the site of contact by efficiently inducing both mucosal and systemic immune responses. However, one P005672 HCl major drawback is the intrinsic low immunogenicity of many protein antigens when administered mucosally. Therefore, the need for mucosal adjuvants is pivotal for development of effective and safe mucosal vaccines. The most widely studied mucosal adjuvants are the cholera toxin (CT) from and induce dose- and age-dependent diarrhea (Sf9) insect cells infected with baculovirus recombinants as described previously [9, 12, 13]. Briefly, NSP4-encoding rotavirus gene 10 sequences were cloned in the TOPO TA vector (Invitrogen Life Technologies, Chicago, IL) and subcloned into the baculovirus transfer vector pFastBAC1 (Invitrogen). Recombinant baculoviruses expressing NSP4 were generated as described by the manufacturer, and recombinant virus stocks were plaque purified. NSP4 was first semi-purified by fast protein liquid chromatography using a quaternary methylamine anion exchange column pre-equilibrated with buffer (20 mM Glycine-HCl, pH 8.1). The NSP4-rich fractions were pooled and further purified using an agarose immunoaffinity column onto which purified anti-NSP4 (114-135) rabbit IgG had been immobilized . The bound NSP4 was eluted with 0.1 M Tris-HCl buffer at pH 2.8. The eluate was dialyzed against 50 mM NH4HCO3, lyophilized, and stored at 4C. Prior to use, NSP4 proteins were reconstituted in PBS. Expression and Purification of NSP4-containing 2/6 Virus-Like Particle (VLP) Rotavirus 2/6 virus like particles were expressed using complementary DNA sequences (cDNA) for simian rotavirus SAl1 gene segment 2, which codes VP2, and gene segment 6, which codes VP6 were made from mRNA and sub-cloned into pCRII TOPO TA vectors (Invitrogen). The rotavirus genes were inserted into a baculovirus transfer vector capable of co-expressing up to four different proteins (see below). The plasmid, pBAC4X (Novagen, San Diego, CA), contains two polyhedron promoters and two p10 promoters with the homologous promoters orientated in opposite directions, one of each in the left hand direction, and the others, in the right-hand direction. Each newly inserted sequence was subsequently confirmed by restriction digestion and the cloned gene was sequenced to confirm its integrity. (i) Insertion of VP6 into the pBAC4X Baculovirus Transfer Plasmid ( pB4X/VP6) The VP6 gene segment was PCR amplified from the full length clone pSP65/SA11-6 using the sense primer 5-TCTAGAGGCCGGCCTTTTAAACG (amebocyte assay (Associates of Cape Cod, Inc., Woods Hole, Mass.). Electron microscopy was performed on each of the VLP preparations just prior to inoculation to confirm the integrity of the VLPs. Inoculation of Animals Groups of five BALB/c mice were used to test each antigen. All experiments included a group of mice co-administered 10 g of the mucosal adjuvant, mutant heat-labile enterotoxin [LT(R192G)]( mLT) as a immunostimulatory control . The animals were anaesthetized by intraperitoneal administration of ketamine (3.75 mg/mouse), xylazine (0.19 mg/mouse), and acepromazine (0.037 mg/mouse) before immunization. Two doses of intranasal immunization of 100 g of KLH or OVA alone or with full length NSP4 (6g) or the truncated NSP4(112-175) (10 or20 g) were carried out three weeks apart. T etanus toxoidused for immunization was kindly provided by Dr. Jerry McGhee (University of Alabama, Birmingham)or from the Statens Serum Institute(Copenhagen, Denmark). Animalswere immunized intranasally with 10 g of TT alone or P005672 HCl co-administered with10 g of either full-length NSP4 or NSP4 internalized in VLPs (NSP4-2/6 VLP ) three times, two weeks apart. Sample Collection Serum and fecal samples were collected before vaccination (0 DPI) and at 14 days post second or third immunization. Blood samples were collected by tail bleed for separation of serum. Fecal samples were collected with a fecal collection cage as.