Test and control samples were plated in triplicate in ELISAs and in neutralization assays
Test and control samples were plated in triplicate in ELISAs and in neutralization assays. that expresses RSV F in an unconstrained, soluble form can induce humoral and cellular immunity that protects against contamination with RSV. == Introduction == Respiratory syncytial computer virus (RSV) can cause close to 200000 deaths in a single year worldwide (16). The most vulnerable individuals are premature infants, children UNC-2025 with congenital heart/lung disease and immunocompromized patients. Currently, there is only one form of successful RSV prophylaxis, which involves the injection of preformed anti-RSV antibodies into vulnerable infants (7). However, because of the high cost and logistical difficulty associated with treatments, preformed antibody prophylaxis is usually rarely available to the children who need it most (8,9). RSV vaccines have been studied for decades, but there remains no licensed product. Clearly, the development of a successful RSV vaccine is usually a critical, unmet need in the pediatric healthcare industry (10,11). Here we describe the testing of a Sendai computer virus (SeV)-based vaccine that expresses an unconstrained, soluble, RSV F protein. SeV was chosen as a vector, because it induces quick and durable B cell and T cell responses in blood and in mucosal tissues of the respiratory tract. The computer virus has already been tested in adults and 36-year-old children, and shown to be immunogenic and well tolerated. This study addresses a question pertinent to recent literature: can an unconstrained, soluble RSV F protein, truncated to remove transmembrane and intracellular protein regions, suffice as a recombinant vaccine target? The question is based on an understanding that in the absence of its transmembrane region, the prefusion form of RSV F protein is usually metastable and often converts to a post-fusion form, a low-energy six helix bundle that lacks certain antibody neutralizing determinants (12,13). Our data show that even though the RSV F protein expressed by SeVRSV-Fs is usually unconstrained, the vaccine elicits RSV-specific binding and neutralizing antibodies and T cell responses in cotton rats. The vaccine fully protects against RSV challenge without enhanced immunopathology. == Methods == == Construct design == To make SeVRSV-Fs cDNA, we inserted a His tag sequence and a stop codon after the codon for residue 524 in the RSV F gene, using QuickChange Site-directed Mutagenesis Kit (Stratagene). The gene encodes a protein truncated by 50 amino acids at its C-terminus and therefore lacking transmembrane and intracellular regions The mutated gene was inserted into theNotIsite of pSV(E),Fig. 1A(14,15). == Fig. 1. == Design and characterization of UNC-2025 recombinant SeV expressing RSV F as a secreted protein. (A) The diagram illustrates the design of SeVRSV-Fs. A unique NotI restriction enzyme site was created in the non-coding region of the HN gene of the full genome SeV cDNA for insertion of the RSV Fs gene. The RSV gene was isolated from RSV-A2 with RT-PCR, digested with NotI and cloned into the NotI site of pSV(E). T7, T7 promoter; ribo, hepatitis delta computer virus ribozyme sequence. (B) In a preliminary experiment, cotton rats were vaccinated i.n. with 2 x 106PFU SeVRSV-Fs and examined throughout a one week period for vaccine computer virus amplification in the lower respiratory tract, by screening homogenized lungs in plaque assays on LLC-MK2cells. Each sign represents ZNF346 computer virus measurements from a different animal. Reverse genetics rescue was performed as explained previously (16). The 293T UNC-2025 cell collection was infected with a UV-inactivated, T7 RNA polymerase-expressing recombinant vaccinia computer virus (vTF7.3) for 1h at 37C. Cells were then cotransfected with plasmids made up of recombinant SeV cDNA plasmid and with supporting T7-driven plasmids respectively expressing the NP, P and L genes of SeV (pTF1SVNP, pTF1SVP and pTF1SVL) in the presence of Lipofectamine (Life Technologies, Grand Island, NY, USA)..