Ecdysone-inducible foreign gene expression in stably-transformed lepidopteran insect cells.

Cultured cell lines that can be stably transformed with inducible gene constructs could prove extremely valuable for the continuous and economical production of recombinant proteins. Toward this goal, we have established 11 clones (designated NISES-BoMo-DK1 to 11) from a previously reported silkworm cell line, NISES-BoMo-DZ. Nine of these clonal lines showed a distinct morphological change. i.e., cell aggregation, in response to treatment with 1 microM 20-hydroxyecdysone (20E). DK10 cells transfected with various reporter assay plasmids under optimal conditions (i.e., 20-30% transfection efficiency) showed inducibility of gene expression by 20E. The 20E treatment of the prototypical DK10 cells resulted in a simultaneous, transient increase of the nuclear ecdysone (E) receptor levels. Further, this inducibility was also observed in a DK10 cell line stably transformed with the reporter plasmid that carries the hygromycin-resistance gene. This offers an opportunity to achieve efficient, continuous production of recombinant proteins. It could also allow high throughput screening for potential E agonists.

Rapid detection of multiple nucleopolyhedroviruses using polymerase chain reaction.

A technique using the polymerase chain reaction (PCR) was developed for detection of the nucleopolyhedrovirus (NPV) polyhedrin gene. The amino acid sequences of the polyhedrin gene were compared in twenty-six NPVs. A highly conserved DNA sequence within the coding region of the polyhedrin gene was targeted for amplification. One pair of degenerate PCR primers was designed to produce fragments of about 430 bp. The NPVs detected by this technique were Autographa californica NPV, Bombyx mori NPV, Hyphantria cunea NPV, Spodoptera exigua NPV, S. litura NPV, and Lymantria dispar NPV. This technique would be useful in monitoring the distribution of NPVs and release of the wild type and recombinant NPVs.

High-level expression of canine parvovirus VP2 using Bombyx mori nucleopolyhedrovirus vector.

For the potential use as recombinant vaccine, canine parvovirus (CPV) major capsid protein VP2 was expressed using Bombyx mori nucleopolyhedrovirus (BmNPV) vector. CPV VP2 gene was introduced into polyhedrin-based BmNPV transfer vector pBmKSK3, and recombinant virus BmK1-Parvo was prepared. When anti-CPV.VP2 monoclonal antibody was employed in immunofluorescence staining, an intense signal was observed within BmK1-Parvo-infected Bm5 cells but not within uninfected cells or cells infected with a wild-type BmNPV-K1. In hemagglutination assay, the expression level of VP2 were 3.2 x 10(3) HA units/ml from infected Bm5 cells, 2.1x 10(5) HA units/larvae from infected larval fat body, and 1.6x 10(6) HA units/ml from infected larval hemolymph. These results suggested that BmNPV vector system using B. mori larva as host could be applied to efficient mass-production of recombinant vaccines.

Characterization of four isolates of Bombyx mori nucleopolyhedrovirus.

Bombyx mori nucleopolyhedroviruses (BmNPVs), isolated from a sericultural Korean farm, were purified and characterized by their DNA restriction pattern, virus replication, polyhedra production and gene structures. The EcoR I and Sal I fragments showed similar overall patterns with minor difference but distinguishable patterns in each isolate. There was no significant difference in the virus replication pattern, yield of total polyhedra production and polyhedra morphology, but the yield of released polyhedra by BmNPV-K1 in Bm5 cells was 2 to 5 times higher than that of other isolates. In comparative studies of p10 gene, BmNPV-K1 and K3 had same structure and they encoded a protein consisting of 94 amino acids. Although BmNPV-K2 encoded the same length of amino acids with BmNPV-K1 and K3, it had different structure, and BmNPV-K4 had the p10 gene encoding 70 amino acids.

High-level expression of a foreign gene by a recombinant baculovirus with an expanded host range.

The usefulness of host range expanded viruses as an expressionvector system was investigated by following the expression ofthe E. coli lacZ gene. The host range expanded recombinantviruses were obtained from Sf-21 or BmN-4 cells coinfected withAutographa californica and Bombyx mori nuclearpolyhedrosis viruses. Among the host range expanded viruses,RecB-8 and RecS-B6 have similar enzyme digestion profiles butdifferent infection characteristics in cells. Therefore, tostudy the foreign gene expression efficiency of these twoviruses, we constructed recombinant viruses RecB8-LacZ andRecSB6-LacZ containing the lacZ gene instead of the polyhedringene. Also, the host range expanded recombinant AcNPV, Bac-BH,containing lacZ gene in the polyhedrin gene locus was constructedby substitution of the 0.6 kb region within the helicase gene ofBacPAK6 with that of BmNPV. beta-Galactosidase expressionefficiency by these viruses were determined and compared in Sf-21and BmN-4 cells. The result showed that Bac-BH has highexpression efficiency only in Sf-21 cells, whereas RecB8-LacZhas high expression efficiency both in Sf-21 and BmN-4 cells.Also, in BmN-4 cells, beta-galactosidase expressionefficiency of RecB8-LacZ was higher than that of recombinantBmNPV (BmK1-LacZ containing lacZ gene in polyhedrin gene locus).In addition, the expression efficiency was not correlated withvirus titer.

Development of a novel expression vector system using Spodoptera exigua nucleopolyhedrovirus.

To develop a novel Spodoptera exigua nucleopolyhedrovirus (SeNPV) expression vector system, we examined characteristics of the SeNPV polyhedrin expression in S. exigua cells (Se301). While the extracellular virus titer of SeNPV was 100-fold lower than that of Autographa californica nucleopolyhedrovirus (AcNPV), the levels of polyhedral inclusion body (PIB) formation and polyhedrin expression were higher in SeNPV. To investigate foreign gene expression under the control of the polyhedrin promoter, polyhedrin-based transfer vector pSeKSK2 was constructed, and then recombinant virus SeK1-LacZ was constructed by inserting E. coli lacZ gene as a reporter gene into a genomic DNA of SeNPV using this transfer vector. The beta-Galactosidase activity of SeK1-LacZ in Se301 was about 1.3 times higher than that of BacPAK6. Thus, the SeNPV expression vector system constructed in this study would be very useful in the expression of foreign proteins, specifically for the enhancement of the pesticidal properties of SeNPV by inserting pesticidal genes.

The morphology of the polyhedra of a host range-expanded recombinant baculovirus and its parents.

The host range-expanded recombinant baculovirus, RecB-8 was isolated from BmN-4 cells coinfected with Autographa californica and Bombyx mori nuclear polyhedrosis viruses. Its genome was compared with those of its parents by restriction endonuclease digestion and their polyhedra compared in an electron microscope. Interestingly, the polyhedra of RecB-8 were tetrahedral although the polyhedrin gene was the same as that of the BmNPV parent which has icosahedral polyhedra. Thus the morphology of the RecB-8 polyhedra resulted from host cell factors and/or another viral genome in the host cells.

Distribution, serological identification, and PCR analysis of Bacillus thuringiensis isolated from soils of Korea.

A total, 58 strains of Bacillus thuringiensis were isolated from soils of various regions in Korea. Serological tests showed that B. thuringiensis isolates represented 10 H serotypes, indicating a varied flora of B. thuringiensis. But the H serotypes did not have a significantly uneven distribution, ranging from 1 to 11 isolates. In toxicity tests, 35% of all isolates were toxic to lepidoptera, 20% were toxic to diptera, and 9% were non-toxic isolates. Especially, a large number of lepidopteran/dipteran-active isolates (36%) were found. Forty all lepidopteran-active isolates produced typical rhomboidial inclusions, and the remainder, which belong to dipteran-active and non-toxic isolates, were spherical in shape. In addition, lepidopteran/dipteran-active isolates produced rhomboidal or spherical inclusions. PCR analysis using cryI, II, III, IV, and V gene-specific primers showed that the frequency of the cryIC gene (57%) predominated, followed by the cryIA(b) (45%) and cryIIA genes (34%). But, the cryIE, cryIF, cryIII, cryIVC and cryV genes were not reactive. Several isolates had unusual PCR products and multiple insecticidal crystal protein genes. PCR results showed varied distribution of the cry-type gene. Seven isolates were selected for evaluation of novel activity according to the following criteria: flagellar serotypes, parasporal inclusion morphology, SDS-PAGE, plasmid DNA patterns, toxicity, and the cry-type gene in PCR analysis. Two isolates, named S333 (H7) and S225 (H7), among them synthesized PCR products of the cryIC gene, but the S333 isolate producing rhomboidal inclusion was toxic to both Plutella xylostella and Culex pipiens, whereas the S225 isolate having toxicity to only C. pipiens produced spherical inclusion.

Isolation of a strain of Bacillus thuringiensis ssp. kurstaki HD-1 encoding delta-endotoxin Cry1E.

A strain of Bacillus thuringiensis, STB-1, toxic against Spodoptera exigua, was isolated. Bacillus thuringiensis STB-1 produced bipyramidal inclusions and reacted with the H antiserum of B. thuringiensis ssp. kurstaki. The plasmid and protein profiles of B. thuringiensis STB-1 were compared with those of its reference strains, ssp. kurstaki and ssp. kenyae. To verify the gene type of B. thuringiensis STB-1, PCR analysis was performed with Spodoptera-specific cry gene primers. The result showed that B. thuringiensis STB-1, unlike its reference strains, had crylAa, crylAb, crylAc and crylE, suggesting that B. thuringiensis STB-1 was a unique strain with respect to gene type. In addition, B. thuringiensis STB-1 showed a high level of toxicity against both S. exigua and Bombyx mori, whereas B. thuringiensis ssp. kurstaki HD-1 or ssp. kenyae showed a high level of toxicity against only Bombyx mori or S. exigua, respectively.

Cloning and expression of a novel gene encoding a new antibacterial peptide from silkworm, Bombyx mori.

We differentially screened a novel gene encoding a new antibacterial peptide from the immunized Bombyx mori cDNA library. The gene showed a similar structure to that of cecropin-family, encoding 59 amino acids including a putative leader peptide and mature peptide. The deduced peptide, named Enbocin, had conserved amino acid residues which have been known to play an important role in the antibacterial activities. Enbocin genomic sequence revealed that the transcription unit of Enbocin gene was about 1.2 kb, and the coding sequence was interrupted by an intron of 660 bases. Recombinant Enbocin, expressed under the control of the baculovirus polyhedrin promoter, demonstrated a broad range of antibacterial activities against gram positive and gram negative bacteria.

Biological, immunological, and genetic analysis of Bacillus thuringiensis isolated from granary in Korea.

To isolate a naturally occurring novel Bacillus thuringiensis strain, we investigated the distribution, toxicity, morphology, H serotype, and gene type of B. thuringiensis from residue samples of granary in Korea. A total of 163 B. thuringiensis isolates out of 411 samples producing spore and crystal were obtained. In toxicity tests, 80% of all isolates were toxic to lepidoptera, and 12% were not toxic to any of tested insects. And dipteran-active and lepidopteran/dipteran-active isolates were rare (2% and 6%, respectively). 152 B. thuringiensis isolates produced typical rhomboidal crystals, and the remainder produced parasporal inclusions with various morphologies. Serological test showed that B. thuringiensis isolates in granary represented 12 H serotypes, indicating varied distribution of B. thuringiensis. Of these, the serotype 3ab predominated, followed by the serotype 7 and 4ac. B. thuringiensis isolates of the serotype 3ab, 4ac, 5ab, 7, 8ab, 9, and 23 were toxic to lepidoptera, and the serotype 8bd, 12, 18, and 20ac were nontoxic, while 14 isolates were untypable by 33 B. thuringiensis H antisera. The frequency of toxicity against lepidoptera and diptera was primarily highly toxic. PCR analysis using cryI gene type-specific primers showed that cryIA(b) genes are frequently found and cryIE gene exists in only one isolate. Analysis of B. thuringiensis crystals and plasmid DNAs indicated a diversity of crystal and gene types.

Effect of silkworm hemolymph on the expression of E. coli beta-galactosidase in insect cell lines infected with recombinant baculoviruses.

The effects of silkworm hemolymph on the expression of foreign genes by recombinant baculoviruses in cell lines were studied. The expression efficiency of beta-galactosidase by recombinant virus containing the E. coli lacZ gene at various concentrations of hemolymph and FBS was determined in BmN and Sf cell lines. The addition of hemolymph to the medium containing FBS accelerated the expression of beta-galactosidase by recombinant viruses in both cells. It was more effective in BmN cells than in Sf cells. Hemolymph was most effective in enhancing virus multiplicity under conditions of 5% FBS.

Detection by polymerase chain reaction of residual cells with the bcl-2 translocation is associated with increased risk of relapse after autologous bone marrow transplantation for B-cell lymphoma.

Although molecular biologic techniques can now detect minimal numbers of residual cancer cells in patients in complete clinical remission, the clinical significance of minimal residual disease has never been conclusively established. If the detection of minimal residual disease predicts which patients will relapse, then therapy could be altered based upon the detection of these cells. The t(14;18) can be detected by polymerase chain reaction (PCR) amplification in 50% of patients with B-cell non-Hodgkin's lymphoma and allows detection of one lymphoma cell in up to 1 million normal cells. To determine the clinical significance of the detection of minimal residual lymphoma cells in the bone marrow (BM) PCR amplification was used to detect the presence of residual lymphoma cells after autologous BM transplantation (ABMT) in serial BM samples from 134 patients with B-cell lymphoma in whom a bcl-2 translocation could be detected. PCR analysis was performed on a total of 542 BM samples obtained while these patients were in complete remission. Disease-free survival was markedly increased in patients with no PCR-detectable lymphoma cells in the marrow compared with those in whom residual lymphoma cells were detected (P < .00001), and the presence of detectable lymphoma cells was associated with a 48-fold increase in the risk of relapse. Of the 77 patients (57%) with no PCR-detectable lymphoma cells in their most recent BM sample, none have relapsed. In contrast, all 33 patients (25%) who have relapsed had PCR-detectable lymphoma cells detected in their BM before clinical relapse occurred. In 19 patients (14%), residual lymphoma cells in the BM were detected early following transplantation and subsequently were no longer detectable, although these patients received no further therapy. In these patients, residual lymphoma cells may already have been irreversibly damaged by the high-dose therapy or an endogenous immune mechanism may be capable of eliminating residual lymphoma cells in some patients. Therefore, although the detection of minimal residual disease by PCR following ABMT in patients with lymphoma identifies those patients at high risk of relapse, the presence of residual minimal disease early after transplantation may not be associated with poor prognosis in a small subset of patients. Confirmatory studies will be required to determine more definitively the role of minimal disease detection to identify which patients require additional therapy.

All advanced stage non-Hodgkin's lymphomas with a polymerase chain reaction amplifiable breakpoint of bcl-2 have residual cells containing the bcl-2 rearrangement at evaluation and after treatment.

Polymerase chain reaction (PCR) of bcl-2 provides an extremely sensitive method to detect minimal disease in approximately 50% of patients with non-Hodgkin's lymphomas (NHL). In an attempt to determine the clinical usefulness of this technique, we examined the bone marrow (BM) of 152 patients with advanced-stage NHL at the time of evaluation and after induction or salvage chemotherapy before autologous BM transplantation. The BM proved to be an accessible and reproducible tissue source to determine PCR positivity because all of the 102 patients examined had the same PCR-amplifiable breakpoint in their BM and lymph node. At the time of evaluation, PCR analysis in advanced-stage NHL patients added little additional information to morphologic analysis because each technique identified BM infiltration in approximately 70% of patients. PCR was significantly more useful in determining BM infiltration after induction or salvage therapy. At that time, approximately 50% of patients had morphologically normal BM, whereas PCR analysis remained positive in 100% of those with an amplifiable breakpoint. These observations were confirmed in a clinical trial attempting to induce remission in previously untreated low-grade advanced-stage NHL patients. In this series, PCR was positive in all patients after treatment although the BM was histologically uninvolved in 50% of cases, showing that conventional therapy did not eradicate bcl-2-positive cells.

Immunologic purging of marrow assessed by PCR before autologous bone marrow transplantation for B-cell lymphoma.

BACKGROUND: The use of autologous bone marrow transplantation is increasing in the management of advanced cancers. Many investigators have attempted to "purge" autologous marrow of residual tumor cells because of concern that reinfused tumor cells might contribute to relapse. The efficacy of purging remains unproved. METHODS: We performed clonogenic assays in a tumor cell line in culture to determine the efficiency of immunologic purging. Amplification by the polymerase chain reaction (PCR) was used to detect residual lymphoma cells before and after purging of bone marrow from 114 patients with B-cell non-Hodgkin's lymphoma in whom a translocation (t(14;18] that could be amplified by PCR was detected at the time of their initial evaluation. RESULTS: Immunologic purging in vitro resulted in a 3-to-6-log destruction of cells in the tumor cell line. Residual lymphoma cells were detected by PCR in the bone marrow of all patients before purging. No lymphoma cells could be detected in the marrow of 57 patients after purging. Disease-free survival was increased in these 57 patients as compared with those whose marrow contained detectable residual lymphoma (P less than 0.00001). The ability to purge residual lymphoma cells was not associated with the degree of bone marrow involvement (P = 0.4494) or the previous response to therapy (P = 0.1298). CONCLUSIONS: The inability to purge residual lymphoma cells was the most important prognostic indicator in predicting relapse. These results provide evidence of the clinical usefulness of ex vivo purging of autologous bone marrow in the treatment of patients with lymphoma and suggest that the reinfusion of malignant cells in autologous marrow contributes to relapse