Yasushi Uematsu

T cell receptor (TCR) gene segments begin to rearrange in CD4-8-thymic lymphoblasts. In severe combined immune deficiency (scid) mice the development of T cells is arrested at this early stage as the scid thymus does not contain any CD4+ or CD8+ lymphocytes. This block in T cell development can be overcome by introducing productively rearranged TCR genes into the scid strain which results in the formation of CD4+8+ lymphocytes. While this early differentiation step requires TCR's of any specificity, later developmental stages depend on the specificity of the TCR: in scid mice, a transgenic TCR restricted by Db class I major histocompatibility complex (MHC) antigens allows the formation of CD4-8+ but not CD4+8- lymphocytes in Db positive but not Db negative animals. Thus, a TCR-MHC interaction in the absence of nominal antigen is required for the generation of mature T cells, and this interaction determines the CD4/CD8 phenotype. If both nominal antigen and presenting MHC antigen...

Exclusion and inclusion of T cell receptor (TCR) genes were analyzed in alpha beta TCR transgenic mice. Both transgenes are expressed unusually early on the surface of CD4-8-, HSA+, IL-2R- thymocytes. These progenitor cells give rise to progeny, which at the single-cell level contains endogenous alpha but not beta TCR-RNA as well as protein, in addition to products encoded by the transgenes. Thus, the surface expression of an alpha beta TCR does not prevent further alpha TCR rearrangement in immature thymocytes that still transcribe RAG-1 and RAG-2 genes. Reduced levels of RAG-1 and RAG-2 RNA are detectable only in CD4+8+ TCR high cells, which result from positive selection in the thymus. The results suggest that a developing T cell may try different alpha beta TCRs for binding to thymic MHC ligands, and that recombination at the alpha locus ceases only after positive selection.

This chapter provides a brief introduction to nucleic acid-based vaccines and recent research in developing self-amplifying mRNA vaccines. These vaccines promise the flexibility of plasmid DNA vaccines with enhanced immunogenicity and safety. The key to realizing the full potential of these vaccines is efficient delivery of nucleic acid to the cytoplasm of a cell, where it can amplify and express the encoded antigenic protein. The hydrophilicity and strong net negative charge of RNA impedes cellular uptake. To overcome this limitation, electrostatic complexation with cationic lipids or polymers and physical delivery using electroporation or ballistic particles to improve cellular uptake has been evaluated. This chapter highlights the rapid progress made in using nonviral delivery systems for RNA-based vaccines. Initial preclinical testing of self-amplifying mRNA vaccines has shown nonviral delivery to be capable of producing potent and robust innate and adaptive immune responses in small animals and nonhuman primates. Historically, the prospect of developing mRNA vaccines was uncertain due to concerns of mRNA instability and the feasibility of large-scale manufacturing. Today, these issues are no longer perceived as barriers in the widespread implementation of the technology. Currently, nonamplifying mRNA vaccines are under investigation in human clinical trials and can be produced at a sufficient quantity and quality to meet regulatory requirements. If the encouraging preclinical data with self-amplifying mRNA vaccines are matched by equivalently positive immunogenicity, potency, and tolerability in human trials, this platform could establish nucleic acid vaccines as a versatile new tool for human immunization.

Parainfluenza virus type 3 (PIV3) infections continue to be a significant health risk for infants, young children, and immunocompromised adults. We describe a gene-based vaccine strategy against PIV3 using replication-defective alphavirus vectors. These RNA replicon vectors, delivered as virus-like particles and expressing the PIV3 hemagglutinin-neuraminidase glycoprotein, were shown to be highly immunogenic in mice and hamsters, inducing PIV3-specific neutralizing antibody

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We have expressed and characterized the severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein in cDNA-transfected mammalian cells. The full-length spike protein (S) was newly synthesized as an endoglycosidase H (endo H)-sensitive glycoprotein (gp170) that is further modified into an endo H-resistant glycoprotein (gp180) in the Golgi apparatus. No substantial proteolytic cleavage of S was observed, suggesting that S is not processed into head (S1) and stalk (S2) domains as observed for certain other coronaviruses. While the expressed full-length S glycoprotein was exclusively cell associated, a truncation of S by excluding the C-terminal transmembrane and cytoplasmic tail domains resulted in the expression of an endoplasmic reticulum-localized glycoprotein (gp160) as well as a Golgi-specific form (gp170) which was ultimately secreted into the cell culture medium. Chemical cross-linking, thermal denaturation, and size fractionation analyses suggested that the full-length S glycoprotein of SARS-CoV forms a higher order structure of approximately 500 kDa, which is consistent with it being an S homotrimer. The latter was also observed in purified virions. The intracellular form of the C-terminally truncated S protein (but not the secreted form) also forms trimers, but with much less efficiency than full-length S. Deglycosylation of the full-length homotrimer with peptide N-glycosidase-F under native conditions abolished recognition of the protein by virus-neutralizing antisera raised against purified virions, suggesting the importance of the carbohydrate in the correct folding of the S protein. These data should aid in the design of recombinant vaccine antigens to prevent the spread of this emerging pathogen.

Four recombinational breakpoints were mapped in the K-A interval of the mouse major histocompatibility complex (MHC) by Southern blot analysis. The breakpoint in B10.SBR, containing a b/s recombinant MHC haplotype, is located about 45 kb upstream of the A beta 2 gene close to the breakpoint in B10.AQR. Crossover in two cas3/cas4 and one cas4/cas3 recombinant haplotypes has taken place in the previously identified K/A beta 3 and A beta 3/A beta 2 recombinational hot spots. The same hot spots are thus active in crossover between two Mus musculus castaneus MHC haplotypes and in crossover between a laboratory and a M. m. castaneus MHC haplotype.

Transgenic mice have been obtained with genes coding for an alpha beta T-cell receptor that recognizes the male-specific antigen H-Y in association with the Db class I major histocompatibility complex molecule. Most if not all of the T-cells express the beta chain encoded by the transgene and show allelic exclusion of endogenous beta genes. In contrast, the expression of the alpha transgene does not completely block rearrangement and formation of functional endogenous alpha genes. In H-2b transgenic female mice the transgenic T-cell receptor is functionally expressed on at least 30% of CD8+ peripheral T-lymphocytes as indicated by their ability to lyse male target cells. Also in transgenic H-2b male mice a large proportion of peripheral T-cells appear to express the transgenic receptor. However, these cells do not react with male target cells because they show only low level or no expression of CD8 cell interaction molecules. Tolerance is established in the male transgenic thymus through deletion of CD4+CD8+ immature thymocytes.

... Clonal analysis of y6 cells from CSF in MS patients 355 Sabine Nick, Piero Pileri, Stefania Tongiani, Yasushi Uematsu, Ludwig KapposA and Gennaro De Libero Experimental Immunology, Department of Research and Neurological ClinicA, University Hospital, Basel ...

Antivector immunity has been recognized as a potential caveat of using virus-based vaccines. In the present study, an alphavirus-based replicon particle vaccine platform, which has demonstrated robust immunogenicity in animal models, was tested for effects of antivector immunity on immunogenicity against hemagglutinin of influenza virus as a target antigen and efficacy for protection against lethal challenge with the virus. Chimeric alphavirus-based replicon particles, comprising Venezuelan equine encephalitis virus nonstructural and Sindbis virus structural components, induced efficient protective antibody responses, which were not adversely influenced after multiple immunizations with the same vector expressing various antigens.

Transgenic mice were constructed with a functional T cell receptor beta gene. Transcription of the introduced gene is largely confined to T cells, but low levels of transcripts are also seen in B cells and in other tissues. Serological analyses show that most, if not all, of the T lymphocytes express the transgenic beta chain on the cell surface and lack beta chains encoded by endogenous beta genes. Molecular genetic analyses of uncloned and cloned T lymphocytes demonstrate that rearrangement of endogenous beta genes is incomplete. Partial D beta 1-J beta 1 rearrangements are found preferentially, while complete VDJ rearrangements are not seen. These findings show that expression of the transgene regulates the rearrangement of endogenous beta genes. Although the alpha beta T cell receptors of the transgenic mice are homogeneous with respect to the beta chain, they are fully functional, at least in a variety of allogeneic responses.