EBV structural antigens, gp350 and gp85, as targets for ex vivo virus-specific CTL during acute infectious mononucleosis: potential use of gp350/gp85 CTL epitopes for vaccine design.
For many years, EBV vaccine development efforts have concentrated on the use of structural Ag, gp350, and have been directed toward Ab-mediated blocking virus attachment to the target cell. There is increasing evidence to suggest that the development of neutralizing Abs in vaccinated animals does not always correlate with protection; nevertheless, it has been postulated that gp350-specific T cell-mediated immune responses may have an effector role in protection. This hypothesis has largely remained untested. In the present study, we demonstrate that CTL from acute infectious mononucleosis patients display strong ex vivo reactivity against the EBV structural Ags, gp85 and gp350. Moreover, long-term follow up studies on infectious mononucleosis-recovered individuals showed that these individuals maintain gp350- and gp85-specific memory CTL, albeit at low levels, in the peripheral blood. These results strongly suggest that CTL specific for EBV structural proteins may play an important role in the control of EBV infection during acute infection. More importantly, we also show that prior immunization of HLA A2/Kb transgenic mice with gp350 and gp85 CTL epitopes induced a strong epitope-specific CTL response and afforded protection against gp85- or gp350-expressing vaccinia virus challenge. These results have important implications for future EBV vaccine design and provides evidence, for the first time, that CTL epitopes from EBV structural proteins may be used for establishing strong antiviral immunity against EBV infection. (+info)
Adaptation of very virulent infectious bursal disease virus to chicken embryonic fibroblasts by site-directed mutagenesis of residues 279 and 284 of viral coat protein VP2.
The full-length RNA genomes of a chicken embryonic fibroblast (CEF)-nonpermissive, very virulent infectious bursal disease virus (IBDV) (strain HK46) were amplified into cDNAs by reverse transcription-PCR. The full-length cDNAs were sequenced and subcloned into a eukaryotic expression vector, from which point mutations were introduced into the VP2 region by site-directed mutagenesis. The wild-type and mutated plasmids were transfected directly into CEFs to examine their ability to generate CEF-permissive recombinant viruses. Substitution of amino acid residues 279 (Asp-->Asn) and 284 (Ala-->Thr) of the VP2 protein yielded a recombinant virus which was able to be passaged in CEFs, whereas the wild-type cDNAs and an amino acid substitution at residue 330 (Ser-->Arg) of the VP2 protein alone did not yield viable virus. The results indicated that mutation of other viral proteins, including VP1, VP3, VP4, and VP5, was not required for CEF adaptation of the virus. The same approach may be used to produce CEF-adapted strains from newly evolved IBDVs or to manipulate the antigenicity of the virus. (+info)
Activation of baculovirus very late promoters by interaction with very late factor 1.
Very late factor 1 (VLF-1) of Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) activates the transcription of two genes, polyhedrin (polh) and p10, during the final, occlusion-specific phase of infection. Using transient expression assays responsive to VLF-1, we identified linker scan mutations in the polh and p10 promoters which abolished or weakened the ability of the promoters to respond to stimulation by VLF-1. These mutations were located between the transcriptional and translational initiation sites, a region previously shown to be essential for the burst of expression during the very late phase. Addition of partially purified, epitope-tagged VLF-1 to DNA encompassing this "burst sequence" resulted in a shift in the gel electrophoretic mobility of the DNA, indicating that VLF-1 forms a complex with DNA. Addition of an antibody specific for the epitope tag of VLF-1 decreased the mobility of the DNA further, confirming the presence of VLF-1 in the complex. DNase I footprint assays revealed that VLF-1 partially purified from either insect cells or bacterial cells interacted with the burst sequences of both the polh and p10 very-late promoters. Linker scan mutations within the burst sequences severely impaired interaction between VLF-1 and the promoters. We propose that VLF-1 transactivates the polh and p10 promoters by interacting with the burst sequences. (+info)
Critical relationship between glycosylation of recombinant lutropin receptor ectodomain and its secretion from baculovirus-infected insect cells.
The lutropin receptor ectodomain overexpressed under the control of the powerful polyhedrin promoter in baculovirus-infected Sf9 insect cells, is mainly found in an inactive, intracellularly-aggregated form. It is secreted in an active form under the control of the P10 promoter, a somewhat weaker and earlier promoter, at the price of a lower production. The apparent molecular masses of the two species encoded by the same cDNA are 48 kDa and 60-68 kDa, respectively. The relationship between the extent and type of glycosylation and the extracellular targeting for the recombinant lutropin receptor ectodomains was investigated precisely with endoglycosidases, lectins of various specificities, and a glycosylation inhibitor, and tested with monoclonal and polyclonal antibodies. The results indicate that the strong polyhedrin promoter probably overwhelms the processing capacity of the ER in Sf9 cells, so that only a high-mannose precursor is expressed in large amounts. Only a minute amount of protein is secreted, which has been processed by Sf9 exoglycosidases/glycosyltransferases and bears complex/hybrid oligosaccharides. The weaker P10 promoter allows secretion of a mature and active receptor ectodomain, bearing complex glycosylation. An important O-linked glycosylation is also added post-translationally on this species. In particular, beta-galactose and sialic acid residues were specifically detected in the secreted species, evidence of the induction of the corresponding glycosyltransferases or of their genes. These results suggest that Sf9 cells should eventually be engineered with chaperones and glycosyltransferases in order to improve the production of demanding glycoproteins such as the porcine lutropin ectodomain, so as to open the way to resolution of the three-dimensional structures of these receptors. (+info)
Live-cell analysis of a green fluorescent protein-tagged herpes simplex virus infection.
Many stages of the herpes simplex virus maturation pathway have not yet been defined. In particular, little is known about the assembly of the virion tegument compartment and its subsequent incorporation into maturing virus particles. Here we describe the construction of a herpes simplex virus type 1 (HSV-1) recombinant in which we have replaced the gene encoding a major tegument protein, VP22, with a gene expressing a green fluorescent protein (GFP)-VP22 fusion protein (GFP-22). We show that this virus has growth properties identical to those of the parental virus and that newly synthesized GFP-22 is detectable in live cells as early as 3 h postinfection. Moreover, we show that GFP-22 is incorporated into the HSV-1 virion as efficiently as VP22, resulting in particles which are visible by fluorescence microscopy. Consequently, we have used time lapse confocal microscopy to monitor GFP-22 in live-cell infection, and we present time lapse animations of GFP-22 localization throughout the virus life cycle. These animations demonstrate that GFP-22 is present in a diffuse cytoplasmic location when it is initially expressed but evolves into particulate material which travels through an exclusively cytoplasmic pathway to the cell periphery. In this way, we have for the first time visualized the trafficking of a herpesvirus structural component within live, infected cells. (+info)
Immunization with potato plants expressing VP60 protein protects against rabbit hemorrhagic disease virus.
The major structural protein VP60 of rabbit hemorrhagic disease virus (RHDV) has been produced in transgenic potato plants under the control of a cauliflower mosaic virus 35S promoter or a modified 35S promoter that included two copies of a strong transcriptional enhancer. Both types of promoters allowed the production of specific mRNAs and detectable levels of recombinant VP60, which were higher for the constructs carrying the modified 35S promoter. Rabbits immunized with leaf extracts from plants carrying this modified 35S promoter showed high anti-VP60 antibody titers and were fully protected against the hemorrhagic disease. (+info)
Packaging cell lines for simian foamy virus type 1 vectors.
Foamy viruses are nonpathogenic retroviruses that offer several unique opportunities for gene transfer in various cell types from different species. We have previously demonstrated the utility of simian foamy virus type 1 (SFV-1) as a vector system by transient expression assay (M. Wu et al., J. Virol. 72:3451-3454, 1998). In this report, we describe the first stable packaging cell lines for foamy virus vectors based on SFV-1. We developed two packaging cell lines in which the helper DNA is placed under the control of either a constitutive cytomegalovirus (CMV) immediate-early gene or inducible tetracycline promoter for expression. Although the constitutive packaging expressing cell line had a higher copy number of packaging DNA, the inducible packaging cell line produced four times more vector particles. This result suggested that the structural gene products in the constitutively expressing packaging cell line were expressed at a level that is not toxic to the cells, and thus vector production was reduced. The SFV-1 vector in the presence of vesicular stomatitis virus envelope protein G (VSV-G) produced an insignificant level of transduction, indicating that foamy viruses could not be pseudotyped with VSV-G to generate high-titer vectors. The availability of stable packaging cell lines represents a step toward the use of an SFV-1 vector delivery system that will allow scaled-up production of vector stocks for gene therapy. (+info)
Stable alphavirus packaging cell lines for Sindbis virus and Semliki Forest virus-derived vectors.
Alphavirus vectors are being developed for possible human vaccine and gene therapy applications. We have sought to advance this field by devising DNA-based vectors and approaches for the production of recombinant vector particles. In this work, we generated a panel of alphavirus vector packaging cell lines (PCLs). These cell lines were stably transformed with expression cassettes that constitutively produced RNA transcripts encoding the Sindbis virus structural proteins under the regulation of their native subgenomic RNA promoter. As such, translation of the structural proteins was highly inducible and was detected only after synthesis of an authentic subgenomic mRNA by the vector-encoded replicase proteins. Efficient production of biologically active vector particles occurred after introduction of Sindbis virus vectors into the PCLs. In one configuration, the capsid and envelope glycoproteins were separated into distinct cassettes, resulting in vector packaging levels of 10(7) infectious units/ml, but reducing the generation of contaminating replication-competent virus below the limit of detection. Vector particle seed stocks could be amplified after low multiplicity of infection of PCLs, again without generating replication-competent virus, suggesting utility for production of large-scale vector preparations. Furthermore, both Sindbis virus-based and Semliki Forest virus-based vectors could be packaged with similar efficiency, indicating the possibility of developing a single PCL for use with multiple alphavirus-derived vectors. (+info)