A Genetic Immunization Adjuvant System Based on BVP22–Antigen Fusion

Oliveira, Sérgio C.; Harms, Jerome S.; Afonso, R.R.; Splitter, Gary A.

doi:10.1089/104303401750271002

Cited by 32 publications

(19 citation statements)

References 21 publications

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“…Others have reported that, following expression of a GFP marker protein fused to the HSV VP22 translocation protein, an intensity gradient of GFP fluorescence is present in the fixed tissue culture cells, with individual brighter cells being surrounded by cells showing less intense signal. 3,[7][8][9] This appearance has been interpreted as showing VP22-eGFP transfer from cells expressing the protein (the brighter cells), into nonexpressing cells (the less bright). Therefore, we examined both of our live cell populations for such fluorescence gradients, but were unable to detect them by fluorescence microscopy.…”

Section: Studies In Live Cells Show That the Hiv Tat Ptd Fails To Dirmentioning

confidence: 99%

Full-length proteins attached to the HIV tat protein transduction domain are neither transduced between cells, nor exhibit enhanced immunogenicity

2002

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Section: Studies In Live Cells Show That the Hiv Tat Ptd Fails To Dirmentioning

confidence: 99%

Full-length proteins attached to the HIV tat protein transduction domain are neither transduced between cells, nor exhibit enhanced immunogenicity

2002

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“…Such sequences, termed 'protein transduction domains' (PTDs), have been identified in the human immunodeficiency virus (HIV) tat protein, the herpes simplex virus (HSV) VP22 protein, the Drosophila melanogaster homeoproteins, and elsewhere (reviewed in Lindgren et al 9 and Prochiantz 10 ). These sequences have been successfully used as immunoadjuvants in several models, [11][12][13][14] and it has been postulated that the immune enhancement results from the improved delivery of these translocatory proteins to APCs. We have found that fusion of the HIV-1 tat PTD to an epitope enhanced its ability to stimulate CD8 þ T cells in vitro and in vivo, and improved antiviral protection when administered as a plasmid vaccine.…”

Section: Introductionmentioning

confidence: 99%

The cationic region from HIV tat enhances the cell-surface expression of epitope/MHC class I complexes

et al. 2003

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The potential of genetic immunization has been acknowledged for almost a decade, but disappointing immunogenicity in humans has delayed its introduction into the clinical arena. To try to increase the potency of genetic immunization, we and others have evaluated 'translocatory' proteins, which are thought to exit living cells by an uncharacterized pathway, and enter neighboring cells in an energy-independent manner. Several laboratories, including our own, have begun to question these remarkable properties. Our previous studies showed that the ability of an epitope to induce major histocompatibility complex (MHC) class I restricted CD8 þ T cells was, indeed, enhanced by its being attached to the proposed translocatory sequence of the HIV-1 tat protein. However, we found little evidence that the increased immunogenicity resulted from transfer of the fusion peptide between living cells, and we proposed that it resulted instead from an increased epitope/MHC expression on the surface of transfected cells. Here, we directly test this hypothesis. We show that cells cotransfected with plasmids encoding an epitope, and the relevant MHC class I allele, can stimulate epitope-specific T cells, and that attachment of the epitope to a putative translocatory sequence -which we term herein an 'integral cationic region' (ICR) -leads to a marked increase in stimulatory activity. This elevated stimulatory capacity does not result from a nonspecific increase in MHC class I expression. We use a high-affinity T-cell receptor (TcR) specific for the epitope/MHC combination to quantitate directly the cell-surface expression of the immunogenic complex, and we show that the attachment of the tat ICR to an epitope results in a substantial enhancement of its cellsurface presentation. These data suggest an alternative explanation for the immune enhancement seen with ICRs.

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“…BHV-1 VP22 has a marked capacity for intercellular trafficking, and it can improve the immune response to a DNA vaccine both in mice (25) and in cattle (32). Because of its potential importance both in virus infection and as a therapeutic transporter, we investigated the existence of nuclear transport sequences in VP22 and identified a functional NLS and NES of VP22.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the Nuclear Localization and Nuclear Export Signals of Bovine Herpesvirus 1 VP22

Zheng

Brownlie

Babiuk

et al. 2005

J Virol

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The bovine herpesvirus 1 (BHV-1) tegument protein VP22 is predominantly localized in the nucleus after viral infection. To analyze subcellular localization in the absence of other viral proteins, a plasmid expressing BHV-1 VP22 fused to enhanced yellow fluorescent protein (EYFP) was constructed. The transient expression of VP22 fused to EYFP in COS-7 cells confirmed the predominant nuclear localization of VP22. Analysis of the amino acid sequence of VP22 revealed that it does not have a classical nuclear localization signal (NLS). However, by constructing a series of deletion derivatives, we mapped the nuclear targeting domain of BHV-1 VP22 to amino acids (aa) 121 to 139. Furthermore, a 4-aa motif, 130 PRPR 133 , was able to direct EYFP and an EYFP dimer (dEYFP) or trimer (tEYFP) predominantly into the nucleus, whereas a deletion or mutation of this arginine-rich motif abrogated the nuclear localization property of VP22. Thus, 130 PRPR 133 is a functional nonclassical NLS. Since we observed that the C-terminal 68 aa of VP22 mediated the cytoplasmic localization of EYFP, an analysis was performed on these C-terminal amino acid sequences, and a leucine-rich motif, 204 LDRMLKSAAIRIL 216 , was detected. Replacement of the leucines in this putative nuclear export signal (NES) with neutral amino acids resulted in an exclusive nuclear localization of VP22. Furthermore, this motif was able to localize EYFP and dEYFP in the cytoplasm, and the nuclear export function of this NES could be blocked by leptomycin B. This demonstrates that this leucine-rich motif is a functional NES. These data represent the first identification of a functional NLS and NES in a herpesvirus VP22 homologue.Bovine herpesvirus 1 (BHV-1) is composed of four concentric compartments, the nucleoprotein core surrounding the double-stranded DNA, the capsid, the tegument, and the envelope (18). The tegument of alpha herpesviruses is defined as the amorphous region located between the virion capsid and the envelope and contains at least 15 viral gene products (20,31). Not only are tegument proteins important viral structural proteins, but they also may play significant roles at several stages during virus infection. They are the first to interact with the intracellular environment and could exert their functions prior to viral gene expression and thus subjugate the host cell (22). At a later stage, the tegument proteins must associate to create the tegument of new virions (20).BHV-1 VP22 is a 258-amino-acid (aa) tegument protein encoded by UL49 which has been shown to be dispensable for BHV-1 replication (16), although a BHV-1 VP22 deletion mutant yielded a lower titer than the wild-type virus in cell culture. Interestingly, this VP22 deletion mutant was asymptomatic and avirulent in cattle (15). Thus, VP22 might play an important role during BHV-1 infection. Previous studies have indicated that BHV-1 VP22 is predominantly localized in the nuclei of BHV-1-infected cells (16), which suggests that VP22 may have regulatory functions (16). However, the exact ...

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A Genetic Immunization Adjuvant System Based on BVP22–Antigen Fusion

Cited by 32 publications

References 21 publications

Full-length proteins attached to the HIV tat protein transduction domain are neither transduced between cells, nor exhibit enhanced immunogenicity

Full-length proteins attached to the HIV tat protein transduction domain are neither transduced between cells, nor exhibit enhanced immunogenicity

The cationic region from HIV tat enhances the cell-surface expression of epitope/MHC class I complexes

Characterization of the Nuclear Localization and Nuclear Export Signals of Bovine Herpesvirus 1 VP22

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