High-titer packaging cells producing recombinant retroviruses resistant to human serum.
Ontology highlight
ABSTRACT:
SUBMITTER: Cosset FL
PROVIDER: S-EPMC189680 | biostudies-other | 1995 Dec
REPOSITORIES: biostudies-other
Publications
High-titer packaging cells producing recombinant retroviruses resistant to human serum.
Cosset F L FL Takeuchi Y Y Battini J L JL Weiss R A RA Collins M K MK
Journal of virology 19951201 12
Novel retroviral protein expression constructs were designed to retain minimal retroviral sequences and to express dominant selectable markers by reinitiation of translation after expression of the viral genes. HT1080 cells were selected as producer cells for their ability to release high-titer viruses that are resistant to inactivation by human serum. Two HT1080-based packaging cell lines which produce Moloney murine leukemia virus cores with envelope glycoproteins of either amphotropic murine ...[more]
Similar Datasets
Project description:Porcine endogenous retroviruses (PERVs) pose a potential stumbling block for therapeutic xenotransplantation, with the greatest threat coming from viruses generated by recombination between members of the PERV subgroup A (PERV-A) and PERV-C families (PERV-A/C recombinants). PERV-A and PERV-B have been shown to infect human cells in culture, albeit with low titers. PERV-C has a more restricted host range and cannot infect human cells. A recombinant PERV-A/C virus (PERV-A14/220) contains the PERV-A sequence between the end of pol and the middle of the SU region in env. The remaining sequence is derived from PERV-C. PERV-A14/220 is approximately 500-fold more infectious than PERV-A. To determine the molecular basis for the increased infectivity of PERV-A14/220, we have made a series of vector constructs. The primary determinant for the enhanced replicative potential of the recombinant virus appeared to be the env gene. Using a series of chimeric env genes, we could identify two determinants of high infectivity; one was an isoleucine to valine substitution at position 140 between variable regions A and B, and the other lies within the proline rich region. Taken together, these results show that the novel juxtaposition of env gene sequences enhanced the infectivity of PERV-A14/220 for human cells, perhaps by stabilization of the envelope glycoprotein or increased receptor binding.
| S-EPMC533952 | biostudies-literature
Project description:Although recombinant adeno-associated virus serotype 3 (AAV3) vectors were largely ignored previously, owing to their poor transduction efficiency in most cells and tissues examined, our initial observation of the selective tropism of AAV3 serotype vectors for human liver cancer cell lines and primary human hepatocytes has led to renewed interest in this serotype. AAV3 vectors and their variants have recently proven to be extremely efficient in targeting human and nonhuman primate hepatocytes in vitro as well as in vivo. In the present studies, we wished to evaluate the relative contributions of the cis-acting inverted terminal repeats (ITRs) from AAV3 (ITR3), as well as the trans-acting Rep proteins from AAV3 (Rep3) in the AAV3 vector production and transduction. To this end, we utilized two helper plasmids: pAAVr2c3, which carries rep2 and cap3 genes, and pAAVr3c3, which carries rep3 and cap3 genes. The combined use of AAV3 ITRs, AAV3 Rep proteins, and AAV3 capsids led to the production of recombinant vectors, AAV3-Rep3/ITR3, with up to approximately two to fourfold higher titers than AAV3-Rep2/ITR2 vectors produced using AAV2 ITRs, AAV2 Rep proteins, and AAV3 capsids. We also observed that the transduction efficiency of Rep3/ITR3 AAV3 vectors was approximately fourfold higher than that of Rep2/ITR2 AAV3 vectors in human hepatocellular carcinoma cell lines in vitro. The transduction efficiency of Rep3/ITR3 vectors was increased by ~10-fold, when AAV3 capsids containing mutations in two surface-exposed residues (serine 663 and threonine 492) were used to generate a S663V+T492V double-mutant AAV3 vector. The Rep3/ITR3 AAV3 vectors also transduced human liver tumors in vivo approximately twofold more efficiently than those generated with Rep2/ITR2. Our data suggest that the transduction efficiency of AAV3 vectors can be significantly improved both using homologous Rep proteins and ITRs as well as by capsid optimization. Thus, the combined use of homologous Rep proteins, ITRs, and capsids should also lead to more efficacious other AAV serotype vectors for their optimal use in human gene therapy.
| S-EPMC4856060 | biostudies-literature
Project description:BackgroundThe structural and enzymatic proteins of the human immunodeficiency virus (HIV) are initially generated as two long polyproteins encoded from overlapping reading frames, one producing the structural proteins (Gag) and the second producing both structural and enzymatic proteins (Gag-Pol). The Gag to Gag-Pol ratio is critical for the proper assembly and maturation of viral particles. To minimize the risk of producing a replication competent lentivirus (RCL), we developed a "super-split" lentiviral packaging system in which Gag was separated from Pol with minimal loss of transducibility by supplying protease (PR) in trans independently of both Gag and Pol.ResultsIn developing this "super-split" packaging system, we incorporated several new safety features that include removing the Gag/Gag-Pol frameshift, splitting the Gag, PR, and reverse transcriptase/integrase (RT/IN) functions onto separate plasmids, and greatly reducing the nucleotide sequence overlap between vector and Gag and between Gag and Pol. As part of the construction of this novel system, we used a truncated form of the accessory protein Vpr, which binds the P6 region of Gag, as a vehicle to deliver both PR and RT/IN as fusion proteins to the site of viral assembly and budding. We also replaced wt PR with a slightly less active T26S PR mutant in an effort to prevent premature processing and cytoxicity associated with wt PR. This novel "super-split" packaging system yielded lentiviral titers comparable to those generated by conventional lentiviral packaging where Gag-Pol is supplied intact (1.0 x 106 TU/ml, unconcentrated).ConclusionHere, we were able to create a true "split-function" lentiviral packaging system that has the potential to be used for gene therapy applications. This novel system incorporates many new safety features while maintaining high titers. In addition, because PR is supplied in trans, this unique system may also provide opportunities to examine viral protein processing and maturation.
| S-EPMC2259377 | biostudies-literature
Project description:In order to study the impact of zinc and copper on the titer levels of mAb and recombinant protein in CHO cells, the IgG-expressing (DP12) and EPO-expressing (SK15) cell lines were cultured in chemically defined media with increasing concentrations of either metal. Supplementation with 25 mg/l in CDM media resulted in a significant increase in EPO (1.7-fold) and IgG (2.6-fold) titers compared to control (no added zinc). Titers at this Zn concentration in CDM containing the insulin replacing agent aurintricarboxylic acid (ATA) (CDM?+?A) showed a 1.8-fold (EPO) and 1.2-fold (IgG) titers increase compared to control. ATA appeared to also reduce the specific productivity (Qp) enhancement induced by Zn-25, with up to 4.9-fold (DP12) and 1.9-fold (SK15) Qp increase in CDM compared to the 1.6-fold (DP12) and 1.5-fold (SK15) Qp increase observed in CDM?+?A. A 31% reduced Viable Cell Density (VCD) in DP12 was observed in both Zn-supplemented media (3?×?106 cells/ml vs 4.2?×?106 cells/ml, day 5), whereas SK15 Zn-25 cultures displayed a 24% lower peak only in CDM?+?A (2.2?×?106 cells/ml vs 3.2?×?106 cells/ml, day 5). Supplementation with copper at 13.7-20 mg/l resulted in less significant cell line/product-type dependent effects on titer, VCD and Viability. Analysis of the energetic phenotype of both cell lines in 25 mg/l Zn-supplemented CDM media revealed a twofold increase in the oxygen consumption rate (OCR) compared to non-supplemented cells. Together, these data suggest that high zinc supplementation may induce an increase in oxidative respiration metabolism that results in increased Qp and titers in suspension CHO cultures.
| S-EPMC6787129 | biostudies-literature
Project description:Vaccines that generate robust and long-lived protective immunity against SARS-CoV-2 infection are urgently required. We assessed the potential of vaccine candidates based on the SARS-CoV-2 spike in cynomolgus macaques (M. fascicularis) by examining their ability to generate spike binding antibodies with neutralizing activity. Antigens were derived from two distinct regions of the spike S1 subunit, either the N-terminal domain (NTD) or an extended C-terminal domain containing the receptor-binding domain (RBD) and were fused to the human IgG1 Fc domain. Three groups of 2 animals each were immunized with either each antigen, alone or in combination. The development of antibody responses was evaluated through 20 weeks post-immunization. A robust IgG response to the spike protein was detected as early as 2 weeks after immunization with either protein and was maintained for over 20 weeks. Sera from animals immunized with antigens derived from the RBD were able to prevent binding of soluble spike proteins to the ACE2 receptor, shown by in vitro binding assays, while sera from animals immunized with the NTD alone lacked this activity. Crucially, sera from animals immunized with the RBD but not the NTD had potent neutralizing activity against SARS-CoV-2 pseudotyped virus, with titers in excess of 10,000, greatly exceeding that typically found in convalescent humans. Neutralizing activity persisted for more than 20 weeks. These data support the utility of spike subunit-based antigens as a vaccine for use in humans.
| S-EPMC7781324 | biostudies-literature
Project description:BackgroundVaccines that generate robust and long-lived protective immunity against SARS-CoV-2 infection are urgently required.MethodsWe assessed the potential of vaccine candidates based on the SARS-CoV-2 spike in cynomolgus macaques (M. fascicularis) by examining their ability to generate spike binding antibodies with neutralizing activity. Antigens were derived from two distinct regions of the spike S1 subunit, either the N-terminal domain or an extended C-terminal domain containing the receptor-binding domain and were fused to the human IgG1 Fc domain. Three groups of 2 animals each were immunized with either antigen, alone or in combination. The development of antibody responses was evaluated through 20 weeks post-immunization.ResultsA robust IgG response to the spike protein was detected as early as 2 weeks after immunization with either protein and maintained for over 20 weeks. Sera from animals immunized with antigens derived from the RBD were able to prevent binding of soluble spike proteins to the ACE2 receptor, shown by in vitro binding assays, while sera from animals immunized with the N-terminal domain alone lacked this activity. Crucially, sera from animals immunized with the extended receptor binding domain but not the N-terminal domain had potent neutralizing activity against SARS-CoV-2 pseudotyped virus, with titers in excess of 10,000, greatly exceeding that typically found in convalescent humans. Neutralizing activity persisted for more than 20 weeks.ConclusionsThese data support the utility of spike subunit-based antigens as a vaccine for use in humans.
| S-EPMC7805460 | biostudies-literature
Project description:Human serum albumin (HSA) is extensively used in clinics to treat a variety of diseases, such as hypoproteinemia, hemorrhagic shock, serious burn injuries, cirrhotic ascites and fetal erythroblastosis. To address supply shortages and high safety risks from limited human donors, we recently developed recombinant technology to produce HSA from rice endosperm. To assess the risk potential of HSA derived from Oryza sativa (OsrHSA) before a First-in-human (FIH) trial, we compared OsrHSA and plasma-derived HSA (pHSA), evaluating the potential for an immune reaction and toxicity using human peripheral blood mononuclear cells (PBMCs). The results indicated that neither OsrHSA nor pHSA stimulated T cell proliferation at 1x and 5x dosages. We also found no significant differences in the profiles of the CD4(+) and CD8(+) T cell subsets between OsrHSA- and pHSA-treated cells. Furthermore, the results showed that there were no significant differences between OsrHSA and pHSA in the production of cytokines such as interferon-gamma (IFN-?), tumor necrosis factor-alpha (TNF-?), interleukin (IL)-10 and IL-4. Our results demonstrated that OsrHSA has equivalent immunotoxicity to pHSA when using the PBMC model. Moreover, this ex vivo system could provide an alternative approach to predict potential risks in novel biopharmaceutical development.
| S-EPMC4123919 | biostudies-literature
Project description:Drosophila melanogaster Schneider 2 (S2) cells have been increasingly used as a suitable expression system for the production of different recombinant proteins, and the employment of bioreactors for large-scale culture is an important tool for this purpose. In this work, Drosophila S2 cells producing the rabies virus glycoprotein RVGP were cultivated in bioreactor, employing a serum-free medium, aiming an improvement in cell growth and in glycoprotein production. To overcome cell growth limitation commonly observed in stirred flasks, different experiments in bioreactor were performed, in which some system modifications were carried out to attain the desired goal. The study showed that this cell line is considerably sensitive to hydrodynamic forces, and a high cell density (about 16.0 x 10(6) cells mL(-1)) was only obtained when Pluronic F68((R)) percentage was increased to 0.6% (w/v). Despite ammonium concentration affected RVGP production, and also cell growth, an elevated amount of the target protein was obtained, attaining 563 ng 10(-7) cells.
| S-EPMC2553640 | biostudies-other
Project description:ObjectiveWe aimed to investigate the effects of special packaging (child-resistant, adult-friendly) and tamper-resistant packaging on health and behavioral outcomes in order to identify research gaps and implications for packaging standards for tobacco products.MethodsWe searched seven databases for keywords related to special and tamper-resistant packaging, consulted experts, and reviewed citations of potentially relevant studies. 733 unique papers were identified. Two coders independently screened each title and abstract for eligibility. They then reviewed the full text of the remaining papers for a second round of eligibility screening. Included studies investigated a causal relationship between type of packaging or packaging regulation and behavioral or health outcomes and had a study population composed of consumers. Studies were excluded on the basis of publication type, if they were not peer-reviewed, and if they had low external validity. Two reviewers independently coded each paper for study and methodological characteristics and limitations. Discrepancies were discussed and resolved.ResultsThe review included eight studies: four assessing people's ability to access the contents of different packaging types and four evaluating the impact of packaging requirements on health-related outcomes. Child-resistant packaging was generally more difficult to open than non-child-resistant packaging. Child-resistant packaging requirements have been associated with reductions in child mortality.ConclusionsChild-resistant packaging holds the expectation to reduce tobacco product poisonings among children under six.
| S-EPMC5299541 | biostudies-literature
Project description:Foamy virus (FV) vectors are promising tools for gene therapy, but low titer is a major challenge for large-scale clinical trials. Here, we increased FV vector titer 50-fold by constructing novel vector plasmids and using polyethylenimine-mediated transfection. FV and lentiviral (LV) vectors were used separately to transduce human CD34(+) cells at multiplicities of infection of 25, and those cells were transplanted into immunodeficient mice. FV vector transduction frequencies of repopulating human cells were 37.1 ± 1.9% in unstimulated cells and 36.9 ± 2.2% in prestimulated cells, and engraftment frequencies were 40.9 ± 4.9% in unstimulated cells and 47.1 ± 3.3% in prestimulated cells. Engraftment frequencies of FV vector-transduced cells were significantly higher than those of LV vector-transduced cells. Linear amplification-mediated PCR with Illumina paired-end runs showed that all human chromosomes contained FV provirus. FV had an integration preference near transcriptional start sites and CpG islands of RefSeq genes but not within genes. Repopulating lymphoid and myeloid cells contained common integration sites, suggesting that FV vector could transduce multilineage hematopoietic stem/progenitor populations. Our new FV vector backbone may be a suitable candidate for developing therapeutic FV vectors for use in clinical trials.
| S-EPMC4441046 | biostudies-literature