Identification of amino acids within norovirus polymerase involved in RNA binding and viral replication.

Until recently, molecular studies on human norovirus (HuNoV), a major causative agent of gastroenteritis, have been hampered by the lack of an efficient cell culture system. Murine norovirus-1 (MNV-1) has served as a surrogate model system for norovirus research, due to the availability of robust cell culture systems and reverse genetics. To identify amino acids involved in RNA synthesis by the viral RNA-dependent RNA polymerase (NS7), we constructed NS7 mutants in which basic amino acids surrounding the catalytic site were substituted with alanine. Electrophoretic mobility shift assay revealed that these residues are important for RNA binding, particularly R396. Furthermore, in vitro RNA synthesis and reverse genetics were used to identify conserved amino acids essential for RNA synthesis and viral replication. These results provide additional functional insights into highly conserved amino acids in NS7 and provide potential methods of rational attenuation of norovirus replication.

Protection against lethal vaccinia virus infection in mice using an siRNA targeting the A5R gene.

BACKGROUND: Although the World Health Organization has declared the eradication of smallpox in 1980, the fear of its potential use in bioterrorism has become a reality. Since the effectiveness of current vaccines and antiviral drugs is limited, development of new therapeutic strategies is needed. In this study, we investigated small interfering RNA (siRNA) as a therapeutic approach for preventing and treating smallpox infection. METHODS: Eight siRNA sequences were designed and evaluated for antiviral activity against vaccinia virus (VACV) in vitro and in vivo. RESULTS: Of eight siRNAs, A5R1 siRNA targeted the A5R gene and reduced VACV replication in cell culture by up to 85% at 100 nM concentration without inducing cytotoxicity. A prolonged prophylactic as well as therapeutic effect of siRNA was observed. In addition, real-time PCR analysis showed that A5R1 siRNA can especially reduce the target mRNA. Finally, intraperitoneal delivery of A5R1 siRNA in Balb/c mice significantly protected these animals from lethal challenge with VACV. CONCLUSIONS: This study suggests the potential of A5R1 siRNA as a therapeutic antiviral agent against smallpox.

Enhanced Immune Response to DNA Vaccine Encoding Bacillus anthracis PA-D4 Protects Mice against Anthrax Spore Challenge.

Anthrax has long been considered the most probable bioweapon-induced disease. The protective antigen (PA) of Bacillus anthracis plays a crucial role in the pathogenesis of anthrax. In the current study, we evaluated the efficiency of a genetic vaccination with the fourth domain (D4) of PA, which is responsible for initial binding of the anthrax toxin to the cellular receptor. The eukaryotic expression vector was designed with the immunoglobulin M (IgM) signal sequence encoding for PA-D4, which contains codon-optimized genes. The expression and secretion of recombinant protein was confirmed in vitro in 293T cells transfected with plasmid and detected by western blotting, confocal microscopy, and enzyme-linked immunosorbent assay (ELISA). The results revealed that PA-D4 protein can be efficiently expressed and secreted at high levels into the culture medium. When plasmid DNA was given intramuscularly to mice, a significant PA-D4-specific antibody response was induced. Importantly, high titers of antibodies were maintained for nearly 1 year. Furthermore, incorporation of the SV40 enhancer in the plasmid DNA resulted in approximately a 15-fold increase in serum antibody levels in comparison with the plasmid without enhancer. The antibodies produced were predominantly the immunoglobulin G2 (IgG2) type, indicating the predominance of the Th1 response. In addition, splenocytes collected from immunized mice produced PA-D4-specific interferon gamma (IFN-γ). The biodistribution study showed that plasmid DNA was detected in most organs and it rapidly cleared from the injection site. Finally, DNA vaccination with electroporation induced a significant increase in immunogenicity and successfully protected the mice against anthrax spore challenge. Our approach to enhancing the immune response contributes to the development of DNA vaccines against anthrax and other biothreats.

Small interfering (Si) RNA mediated baculovirus replication reduction without affecting target gene expression.

The baculovirus expression vector system (BEVS) is widely used to produce large quantities of recombinant protein with posttranslational modification. Recombinant baculoviruses (such as Autographa californica multiple nuclear polyhedrosis virus) are especially useful in producing recombinant proteins and virus-like particles (VLPs) as biodrugs or candidate vaccines for the prevention of serious infectious diseases. However, during the bioprocessing of recombinant proteins in insect cells, baculovirus replication and viral budding are coincident. In some cases, residual baculovirus contaminants remain in the recombinant protein products, even though various purification processes are applied such as ion-exchange chromatography, ultracentrifugation, or gel filtration. To reduce unexpected contamination caused by replication and budding-out of the baculovirus, we designed short interfering (si) RNAs targeting glycoprotein 64 (GP64) or single-stranded DNA-binding protein (DBP) to inhibit baculovirus replication during overexpression of recombinant foreign genes. GP64 is known to be critical both for the entry of virions into cells and for the assembly of the budded virion at the cell surface. DBP is also essential for virus assembly by regulation of the capsid protein P39 and the polyhedrin protein. This study showed that GP64 expression was suppressed by GP64 siRNAs in Western blot experiments, while the expression of recombinant proteins was unaffected. In addition, transfection of GP64 siRNAs and DBP siRNAs reduced the level of baculovirus replication, compared with the treatment with scrambled siRNAs. However, DBP siRNA also suppressed the expression of recombinant proteins. In conclusion, our GP64 siRNAs showed that an interfering RNA system, such as siRNAs and short hairpin (sh) RNAs, can be applicable to reduce baculovirus contaminants during the bioprocessing of recombinant proteins in insect cells. Further investigation should be carried out to establish transformed insect cell lines with stable expression of corresponding interfering RNAs.

Simultaneous detection of major enteric viruses using a combimatrix microarray.

Various enteric viruses including norovirus, rotavirus, adenovirus, and astrovirus are the major etiological agents of food-borne and water-borne disease outbreaks and frequently cause non-bacterial gastroenteritis worldwide. Sensitive and high-throughput detection methods for these viral pathogens are compulsory for diagnosing viral pathogens and subsequently improving public health. Hence, we developed a sensitive, specific, and high-throughput analytical assay to detect most major enteric viral pathogens using "Combimatrix" platform oligonucleotide probes. In order to detect four different enteric viral pathogens in a sensitive and simultaneous manner, we first developed a multiplex RT-PCR assay targeting partial gene sequences of these viruses with fluorescent labeling for the subsequent microarray. Then, five olignonucleotides specific to each of the four major enteric viruses were selected for the microarray from the oligonulceotide pools targeting the specific genes obtained by multiplex PCR of these viruses. The oligonucleotide microarray was evaluated against stool specimens containing single or mixed viral species. As a result, we demonstrated that the multiplex RT-PCR assay specifically amplified partial sequences of four enteric viruses and the subsequent microarray assay was capable of sensitive and simultaneous detection of those viruses. The developed method could be useful for diagnosing enteric viruses in both clinical and environmental specimens.

cDNA cloning of Korean human norovirus and nucleotidylylation of VPg by norovirus RNA-dependent RNA polymerase.

Norovirus, a member of the Caliciviridae family, is a major causative agent of gastroenteritis worldwide. The cDNA of the entire genome of human norovirus (HuNV) was cloned using the RNA extracted from the stool sample of a Korean patient. The RNA genome consists of 7,559 nucleotides, carries 3 open reading frames (ORFs), 5 and 3 noncoding regions, and a poly(A) tail at the 3 end. Phylogenic analysis of the nucleotide sequence indicated that it belongs to GII.4, the most dominant genogroup. To analyze RNA synthesis and nucleotidylylation of VPg by RNA-dependent RNA polymerase (RdRp), recombinant RdRp and VPg were expressed in Escherichia coli as His-tagged forms. The HuNV RdRp exhibited template and divalent cation-dependent RNA synthesis in vitro. The HuNV RdRp nucleotidylylated HuNV VPg but not murine norovirus (MNV) VPg, whereas MNV RdRp nucleotidylylated both MNV and HuNV VPg more efficiently than HuNV RdRp.

Role of chondroitin sulfate C in the action of anthrax toxin.

Anthrax toxin is produced by Bacillus anthracis, the causative agent of anthrax, and is responsible for the majority of disease symptoms. The toxin consists of 3 proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), which combine to form lethal and edema toxin. Glycosaminoglycans, which are present on the surface of cells, were investigated with regard to their role in toxicity resulting from anthrax toxin exposure. Lethal toxin-induced cytotoxicity of the RAW 264.7 cells was significantly inhibited by the addition of chondroitin sulfate C as determined by the MTT assay. By contrast, several other glycosaminoglycans, including heparin, heparan sulfate, and dermatan sulfate did not show significant levels of inhibition. Studies utilizing fluorescence-labeled PA demonstrated decreased PA binding to RAW 264.7 cells with the addition of chondroitin sulfate C. Formation of PA oligomers at the surface of cells after binding was also inhibited by chondroitin sulfate C. Interestingly, enzymatic degradation of endogenous chondroitin sulfate C from the cell surface with chondroitinase ABC was accompanied by increased sensitivity to the toxin. These findings were further confirmed by pretreating cells with sodium chlorate to reduce the degree of cell surface glycosaminoglycans sulfation. In addition, chondroitin sulfate C effectively inhibits edema toxin-induced cAMP accumulation in cells. Our results indicate that chondroitin sulfate C may play an important role in the toxicity of anthrax toxin.

Short-hairpin RNA-mediated gene expression interference in Trichoplusia ni cells.

RNA interference (RNAi) is rapidly becoming a valuable tool in biological studies, as it allows the selective and transient knockdown of protein expression. The short-interfering RNAs (siRNAs) transiently silence gene expression. By contrast, the expressed short-hairpin RNAs induce long-term, stable knockdown of their target gene. Trichoplusia ni (T. ni) cells are widely used for mammalian cell-derived glycoprotein expression using the baculovirus system. However, a suitable shRNA expression system has not been developed yet. We investigated the potency of shRNA-mediated gene expression inhibition using human and Drosophila U6 promoters in T. ni cells. Luciferase, EGFP, and beta-N-acetylglucosaminidase (GlcNAcase) were employed as targets to investigate knockdown of specific genes in T. ni cells. Introduction of the shRNA expression vector under the control of human U6 or Drosophila U6 promoter into T. ni cells exhibited the reduced level of luciferase, EGFP, and beta-N-acetylglucosaminidase compared with that of untransfected cells. The shRNA was expressed and processed to siRNA in our vector-transfected T. ni cells. GlcNAcase mRNA levels were down-regulated in T. ni cells transfected with shRNA vectors-targeted GlcNAcase as compared with the control vector-treated cells. It implied that our shRNA expression vectors using human and Drosophila U6 promoters were applied in T. ni cells for the specific gene knockdown.

Crystal structures of murine norovirus-1 RNA-dependent RNA polymerase in complex with 2-thiouridine or ribavirin.

Murine norovirus-1 (MNV-1) shares many features with human norovirus (HuNoV) and both are classified within the norovirus genus of Caliciviridae family. MNV-1 is used as the surrogate for HuNoV research since it is the only form that can be grown in cell culture. HuNoV and MNV-1 RNA dependent RNA polymerase (RdRp) proteins with the sequence identity of 59% show essentially identical conformations. Here we report the first structural evidence of 2-thiouridine (2TU) or ribavirin binding to MNV-1 RdRp, based on the crystal structures determined at 2.2Å and 2.5Å resolutions, respectively. Cellular and biochemical studies revealed stronger inhibitory effect of 2TU on the replication of MNV-1 in RAW 264.7 cells, compared to that of ribavirin. Our complex structures highlight the key interactions involved in recognition of the nucleoside analogs which block the active site of the viral RNA polymerase.

Genetic analysis of hepatitis A virus strains that induced epidemics in Korea during 2007-2009.

Hepatitis A virus is one of the most prominent causes of fecally transmitted acute hepatitis worldwide. In order to characterize the viral agents causing an outbreak in Korea (comprising North and South Korea) from June 2007 to May 2009, we collected specimens and performed genotyping of the VP1/P2A and VP3/VP1 regions of hepatitis A virus. We then used a multiple-alignment algorithm to compare the nucleotide sequences of the 2 regions with those of reference strains. Hepatitis A virus antibodies were detected in 64 patients from 5 reported outbreaks (North Korea, June 2007 [n = 11]; Jeonnam, April 2008 [n = 15]; Daegu, May 2008 [n = 13]; Seoul, May 2009 [n = 22]; and Incheon, May 2009 [n = 3]). We found 100% homology between strains isolated from the Kaesong Industrial Region and Jeonnam. While those strains were classified as genotype IA strains, strains from Seoul and Incheon were identified as genotype IIIA strains and showed 98.9 to 100% homology. Genotype IIIA was also dominant in Daegu, where strains were 95.7 to 100% homologous. All hepatitis A virus strains isolated from the Kaesong Industrial Region, Jeonnam, Seoul, and Incheon belonged to a single cluster. However, strains from Daegu could be classified into 2 clusters, suggesting that the outbreak had multiple sources. This study indicates that hepatitis A virus strains of 2 different genotypes are currently cocirculating in Korea. Moreover, it documents an increasing prevalence of genotype IIIA strains in the country.

Biodistribution and blood clearance of plasmid DNA administered in arginine peptide complexes.

BACKGROUND: Peptide/DNA complexes have great potential as non-viral methods for gene delivery. Despite promising results for peptide-mediated gene delivery technology, an effective systemic peptide-based gene delivery system has not yet been developed. METHODS: This study used pCMV-Luc as a model gene to investigate the biodistribution and the in vivo efficacy of arginine peptide-mediated gene delivery by polymerase chain reaction (PCR). RESULTS: Plasmid DNA was detected in all organs tested 1 h after intraperitoneal administration of arginine/DNA complexes, indicating that the arginine/DNA complexes disseminated widely through the body. The plasmid was primarily detected in the spleen, kidney, and diaphragm 24 h post administration. The mRNA expression of plasmid DNA was noted in the spleen, kidney, and diaphragm for up to 2 weeks, and in the other major organs, for at least 1 week. Blood clearance studies showed that injected DNA was found in the blood as long as 6 h after injection. CONCLUSIONS: Taken together, our results demonstrated that arginine/DNA complexes are stable in blood and are effective for in vivo gene delivery. These findings suggest that intraperitoneal administration of arginine/DNA complexes is a promising tool in gene therapy.

Crystal structures of murine norovirus-1 RNA-dependent RNA polymerase.

Norovirus is one of the leading agents of gastroenteritis and is a major public health concern. In this study, the crystal structures of recombinant RNA-dependent RNA polymerase (RdRp) from murine norovirus-1 (MNV-1) and its complex with 5-fluorouracil (5FU) were determined at 2.5 Å resolution. Crystals with C2 symmetry revealed a dimer with half a dimer in the asymmetrical unit, and the protein exists predominantly as a monomer in solution, in equilibrium with a smaller population of dimers, trimers and hexamers. MNV-1 RdRp exhibited polymerization activity with a right-hand fold typical of polynucleotide polymerases. The metal ion modelled in close proximity to the active site was found to be coordinated tetrahedrally to the carboxyl groups of aspartate clusters. The orientation of 5FU observed in three molecules in the asymmetrical unit was found to be slightly different, but it was stabilized by a network of favourable interactions with the conserved active-site residues Arg185, Asp245, Asp346, Asp347 and Arg395. The information gained on the structural and functional features of MNV-1 RdRp will be helpful in understanding replication of norovirus and in designing novel therapeutic agents against this important pathogen.

Evaluation of process efficiency and bioequivalence of biosimilar recombinant human chorionic gonadotropin (rhCG).

BACKGROUND: Human chorionic gonadotropin (hCG) is a therapeutic protein used for ovulation induction in women with infertility. Dong-A Pharm. Co. has developed recombinant hCG (rhCG) [product code DA-3803] produced in Chinese hamster ovary cells and evaluated its biologic properties, such as biologic potency, efficacy, and pharmacokinetic profile, compared with a reference product, Ovidrel®. OBJECTIVE: The purpose of this study was to evaluate the efficiency of the purification process of Dong-A rhCG (DA-3803) and its bioequivalence from a biosimilar perspective. METHODS: The efficiency of the purification process was estimated through scale-down clearance studies for viruses, endotoxins, host cell DNAs (HCDs) and host cell proteins (HCPs). To confirm bioequivalence, the in vivo/in vitro biologic potency, ovulation induction rate, and pharmacokinetic profile of DA-3803 were compared with those of Ovidrel®. RESULTS: In the clearance studies, the lowest log reduction value (LRV) for model viruses was 8.43. LRVs for endotoxins, HCDs, and HCPs were greater than 5.27, 16.36, and 3.37, respectively. DA-3803 showed equivalent potency with Ovidrel®, and similarity between DA-3803 and Ovidrel® was observed in an efficacy evaluation that measured ovulation induction. The bioequivalence was also confirmed in a rat pharmacokinetic study, which compared pharmacokinetic parameters such as maximum serum concentration, area under the concentration-time curve, time to reach maximum serum concentration, and half-life. In a comparison of different isoform groups of DA-3803, it was shown that the potency and pharmacokinetic profile depend on the sialic acid content. CONCLUSION: The purification process of DA-3803 was effective in removing the major process impurities, and DA-3803 showed similar biologic properties to the reference drug, Ovidrel®.

An hydrophobically modified arginine peptide vector system effectively delivers DNA into human mesenchymal stem cells and maintains transgene expression with differentiation.

BACKGROUND: The ability of human mesenchymal stem cells (hMSCs) to differentiate into specific cells holds promise for therapeutic use in cell- or gene-based therapy. Genetic modification of hMSCs by introduction of therapeutic genes is an important tool for successful cell-mediated gene therapy. Similar to most primary cells, hMSCs are difficult to transfect with currently available gene delivery methods. Viral vectors are the most efficient DNA delivery system in stem cells. However, the use of viral vectors has disadvantages involving safety. To overcome these problems, nonviral gene delivery has been studied as an alternative strategy, and the cationic peptide was an efficient vector for transfecting various mammalian cell types. However, little is known about the capacity of this delivery method to transfect to hMSCs. In the present study, we examined the use of a short arginine peptide as a carrier for DNA delivery in hMSCs. METHODS: Plasmids containing the enhanced green fluorescent protein (EGFP) were transfected into hMSCs by arginine peptide in vitro. pEGFP delivery and EGFP expression were assessed by the fluorescence-activated cell sorting technique. RESULTS: The hydrophobically modified arginine peptide, palmitic acid-R15 (PA-R15), formed a condensed complex with DNA and successfully delivered the gene into hMSCs without compromising cell survival rate. Complexes readily internalized into hMSCs after a 4 h incubation, and PA-R15 showed higher cellular uptake compared to the unmodified arginine peptide (R15). In addition, transfected cells retained their stem cell properties, including the ability to differentiate into adipogenic and osteogenic lineages. Moreover, the erythropoietin (EPO) gene was successfully transfected into hMSCs and the cells produced EPO for 4 weeks. CONCLUSIONS: Hydrophobically modified arginine peptides are promising candidates with low toxicity. They comprise efficient nonviral gene delivery vectors for hMSCs and these may be used as a potential tool for basic research and the therapeutic application of hMSCs.

Human papillomavirus type 16 E6-specific antitumor immunity is induced by oral administration of HPV16 E6-expressing Lactobacillus casei in C57BL/6 mice.

Given that local cell-mediated immunity (CMI) against the human papillomavirus type 16 E6 (HPV16 E6) protein is important for eradication of HPV16 E6-expressing cancer cells in the cervical mucosa, the HPV16 E6 protein may be a target for the mucosal immunotherapy of cervical cancer. Here, we expressed the HPV16 E6 antigen on Lactobacillus casei (L. casei) and investigated E6-specific CMI following oral administration of the L. casei-PgsA-E6 to mice. Surface expression of HPV16 E6 antigens was confirmed and mice were orally inoculated with the L. casei-PgsA or the L. casei-PgsA-E6. Compared to the L. casei-PgsA-treated mice, significantly higher levels of serum IgG and mucosal IgA were observed in L. casei-PgsA-E6-immunized mice; these differences were significantly enhanced after boost. Consistent with this, systemic and local CMI were significantly increased after the boost, as shown by increased counts of IFN-gamma-secreting cells in splenocytes, mesenteric lymph nodes (MLN), and vaginal samples. Furthermore, in the TC-1 tumor model, animals receiving the orally administered L. casei-PgsA-E6 showed reduced tumor size and increased survival rate versus mice receiving control (L. casei-PgsA) immunization. We also found that L. casei-PgsA-E6-induced antitumor effect was decreased by in vivo depletion of CD4(+) or CD8(+) T cells. Collectively, these results indicate that the oral administration of lactobacilli bearing the surface-displayed E6 protein induces T cell-mediated cellular immunity and antitumor effects in mice.

Murine norovirus-1 3Dpol exhibits RNA-dependent RNA polymerase activity and nucleotidylylates on Tyr of the VPg.

We investigated the roles and biochemical properties of recombinant murine norovirus-1 (MNV-1) 3D(pol) in RNA synthesis and virus genome-linked protein (VPg) nucleotidylylation. We therefore expressed VPg and 3D(pol) of MNV-1 in Escherichia coli. MNV-1 3D(pol) exhibited RNA-dependent RNA polymerase (RdRp) activity in vitro with poly(A) RNA as a template and MnCl(2) as a cofactor. MNV-1 3D(pol) demonstrated optimum RNA-synthesis activity at pH 7.4 and 37 degrees C in the absence of a primer. Further, VPg was guanylylated by MNV-1 3D(pol) in the presence of MnCl(2) in a template-independent manner. The guanylylation reaction conducted with VPg substitution mutants (Y26F, Y40F, Y45F and Y117F) and a deletion mutant (Delta117-124) indicated that Tyr(117) was the probable target site of guanylylation. Homopolymeric RNAs did not enhance VPg guanylylation, whereas in vitro-transcribed (-) subgenomic (SG) and (+)SG RNA enhanced VPg guanylylation by 9.2 and 3.2 times, respectively. Within (-)SG RNA, the (-)ORF3 region played a critical role in enhancing VPg guanylylation, suggesting that the MNV-1 ORF3 region of negative-strand RNA contains a cis-acting element that stimulates 3D(pol)-mediated VPg guanylylation.

RNA interference in vitro and in vivo using an arginine peptide/siRNA complex system.

Efficient delivery systems are required to exploit the enormous potential of RNA interference. We introduced an arginine peptide-based small-interference RNA (siRNA) delivery system for in vitro and in vivo RNA interference. Arginine peptides formed stable complexes with siRNA and transduced siRNA into COS-7 cells in vitro, resulting in efficient gene silencing. The intracellular path of the peptide/siRNA complex was investigated in live cells using fluorescent labeling and confocal microscopy. At 24h after transfection, most of the siRNA signals were observed in the perinuclear region, indicating that siRNA was targeted to the perinuclear region for interactions with RNA-induced silencing complex (RISC). Effective in vivo RNA interference was achieved in a mouse model bearing a subcutaneous tumor. Intratumoral administration of HER-2-specific siRNA/peptide complexes resulted in a marked reduction of tumor growth. Body weight monitoring during treatment showed that our delivery system was nontoxic. Our approach offers the potential for siRNA delivery in various in vitro and in vivo applications.

Expression and In vitro activity of recombinant canstatin in stably transformed bombyx mori cells.

We describe the expression of recombinant canstatin from stably transformed Bombyx mori Bm5 (Bm5) cells. Recombinant canstatin was secreted into a culture medium with a molecular mass of approximately 29 kDa. Densitometric scanning showed that the secreted canstatin accounted for approximately 91% of the total canstatin production. Recombinant canstatin was also purified to homogeneity using a simple one-step Ni-NTA affinity fractionation. The identity of the purified protein was confirmed as human canstatin by nano-LC-MS/MS analysis. Purified recombinant canstatin inhibited human endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition (ED50) for recombinant canstatin expressed in stably transformed Bm5 cells was approximately 0.64 mg/ml. A maximum production level of 11 mg/l recombinant canstatin was obtained in a T-flask culture of Bm5 cells after 6 days of incubation.

Characterization of the replication-competent porcine endogenous retrovirus class B molecular clone originated from Korean domestic pig.

Xenotransplantation from pigs offers an opportunity to resolve the shortage of human organs. The porcine endogenous retrovirus (PERV) cannot be eliminated because of its presence in the germline DNA. Three subgroups of the replication-competent PERV (PERV-A, PERV-B, and PERV-C) have been identified in pigs. We constructed a molecular clone of PERV-B from a Korean domestic pig BAC clone containing PERV genomes, and its replication competency was characterized in human cells. The pol region of PERV-B was detected in the genomic DNA of 293T cells transfected with PERV-B (465D1) and in the genomic DNA of 293T cells infected with PERV-B (465D1) viruses. The 293T cells transfected with PERV-B (465D1) were maintained for 140 days. PERV-B (465D1) showed the stationary replication competence with no toxicity in the cell growth.

Oral administration of poly-gamma-glutamate induces TLR4- and dendritic cell-dependent antitumor effect.

Previously, we reported that the oral administration of high molecular mass poly-gamma-glutamate (gamma-PGA) induced antitumor immunity but the mechanism underlying this antitumor activity was not understood. In the present study, we found that application of high molecular mass gamma-PGA induced secretion of tumor necrosis factor (TNF)-alpha from the bone-marrow-derived macrophages of wild type (C57BL/6 and C3H/HeN) and Toll-like receptor 2 knockout (TLR2(-/-)) mice, but not those of myeloid differentiation factor 88 knockout (MyD88(-/-)) and TLR4-defective mice (C3H/HeJ). Production of interferon (IFN)-gamma-inducible protein 10 (IP-10) in response to treatment with gamma-PGA was almost abolished in C3H/HeJ mice. In contrast to LPS, gamma-PGA induced productions of TNF-alpha and IP-10 could not be blocked by polymyxin B. Furthermore, gamma-PGA-induced interleukin-12 production was also impaired in immature dendritic cells (iDCs) from MyD88(-/-) and C3H/HeJ mice. Downregulation of MyD88 and TLR4 expression using small interfering RNA (siRNA) significantly inhibited gamma-PGA-induced TNF-alpha secretion from the RAW264.7 cells. Gamma-PGA-mediated intracellular signaling was markedly inhibited in C3H/HeJ cells. The antitumor effect of gamma-PGA was completely abrogated in C3H/HeJ mice compared with control mice (C3H/HeN) but significant antitumor effect was generated by the intratumoral administration of C3H/HeN mice-derived iDCs followed by 2,000 kDa gamma-PGA in C3H/HeJ. These findings strongly suggest that the antitumor activity of gamma-PGA is mediated by TLR4.