Generation of Lymphocytic Choriomeningitis Virus Based Vaccine Vectors
Vaccination with a recombinant LCMV based vector expressing tumor-associated or viral antigens is a safe and versatile method to induce an immune response against tumors or viral infections. Here, we describe the generation of recombinant LCMV vectors in
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Introduction Exposure of mice to the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is one of the best infection models to study T cell immunity. Important key concepts in immunology and viral pathogenesis such as MHC restriction, T cell exhaustion, and persistent viral infections were developed studying LCMV [1, 2]. In 1933, LCMV was first isolated by Armstrong and Lillie from a patient who was suspected to suffer from an infection with the St. Louis Encephalitis Virus [3]. Persistently LCMV infected rodents spread the virus with the urine, feces or saliva. LCMV can also be transmitted to humans through contact with infected murine excretions. In immunocompetent individuals an infection with LCMV often remains asymptomatic or shows mild flu-like symptoms. However, immunosuppressed individuals particularly organ-recipients can develop life threatening severe aseptic meningitis or meningoencephalitis [4]. Furthermore, LCMV is a particular concern for pregnant women as it was shown that congenital infection can lead to severe and permanent brain injury in children [5, 6]. LCMV is a non-cytopathic enveloped negative-strand RNA virus with a spherical shape and a diameter between 50 and 300 nm. Its genome consists of two single-strand RNA segments both encoding for two viral genes in ambisense orientation. The viral genes on each segment are separated by an intergenic region (IGR) which forms a stable hair pin loop in the RNA sequence (Fig. 1a). The short segment (S segment) is 3.4 kb long and contains the glycoprotein (GP) precursor (GPC) genes, GP-1 and GP-2, and the nucleoprotein (NP) gene [7]. The nucleoproteins build complexes with the viral RNA segments. The glycoprotein of LCMV forms the spikes on the viral envelope and mediates the interaction with host cell surface receptors and the entry of the virus into the
Sunil Thomas (ed.), Vaccine Design: Methods and Protocols, Volume 2: Vaccines for Veterinary Diseases, Methods in Molecular Biology, vol. 1404, DOI 10.1007/978-1-4939-3389-1_24, © Springer Science+Business Media New York 2016
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Sandra Ring and Lukas Flatz
Fig. 1 Schematic overview of the generation of recombinant LCMV vectors. (a) The wild-type LCMV genome is composed of two single-strand RNA segments (S and L segment). The S segment encodes for the GP and NP and the L segment encodes for Z and L. In the rLCMV vectors the gene encoding the LCMV-GP is substituted with a gene of interest (vaccine antigen). (b) The RNA genome, composed of the S and L segment, is intracellularly expressed under the control of a rodent-specific Pol-I-promoter and a Pol-I-terminator (Pol-I-Sv and Pol-I-L). The plasmids Pol-II-NP and Pol-II-L contain the ORFs for the trans-acting proteins NP and L with a poly-A tail under the control of an actin promoter. (c) rLCMV vectors are created with a four-plasmid transfection system in BHK21-GP producer cells (virus rescue). For the generation of viral vectors, 293T-GP cells are used (virus production). Both cell lines stably express the viral GP to comple
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