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This work aimed to assess, following upstream optimization in Schott flasks, the scalability from this culture platform to a stirred-tank bioreactor in order to yield rabies-recombinant baculovirus, bearing genes of G (BVG) and M (BVM) proteins, and to obtain rabies virus-like particles (VLP) from them, using Sf9 insect cells as a host. Equivalent assays in Schott flasks and a bioreactor were performed to compare both systems and a multivariate statistical approach was also carried out to maximize VLP production as a function of BVG and BVM’s multiplicity of infection (MOI) and harvest time (HT). Viable cell density, cell viability, virus titer, BVG and BVM quantification by dot-blot, and BVG quantification by Enzyme-Linked Immunosorbent Assay (ELISA) were monitored throughout the assays. Furthermore, transmission electron microscopy was used to characterize rabies VLP. The optimal combination for maximum VLP expression was BVG and BVM MOI of 2.3 pfu/cell and 5.1 pfu/cell, respectively, and 108 h of harvest time. The current study confirmed that the utilization of Schott flasks and a benchtop bioreactor under the conditions applied herein are equivalent regarding the cell death kinetics corresponding to the recombinant baculovirus infection process in Sf9 cells. According to the results, the hydrodynamic and chemical differences in both systems seem to greatly affect the virus and VLP integrity after release.

This work aimed to quantify growth and biochemical parameters (viable cell density, Xv; cell viability, CV; glucose, lactate, glutamine, glutamate, ammonium, and potassium concentrations) in upstream stages to obtain rabies virus-like particles (rabies VLP) from insect cell-baculovirus system using on-line and off-line Raman spectra to calibrate global models with minimal experimental data. Five cultivations in bioreactor were performed. The first one comprised the growth of uninfected Spodoptera frugiperda (Sf9) cells, the second and third runs to obtain recombinant baculovirus (rBV) bearing Rabies G glycoprotein and matrix protein. The fourth one involved the generation of rabies VLP from rBVs and the last one was a repetition of the third one with cell inoculum infected by rBV. The spectra were acquired through a Raman spectrometer with a 785-nm laser source. The fitted Partial Least Square models for nutrients and metabolites were comparable with those previously reported for mammalian cell lines (Relative error15%). However, the use of this chemometrics approach for Xv and CV was not as accurate as it was for other parameters. The findings from this work established the basis for bioprocess Raman spectroscopical monitoring using insect cells for VLP manufacturing, which are gaining ground in the pharmaceutical industry.

This work aimed to describe the dynamics of the Sf9 insect cells death and primary metabolism when this host is infected simultaneously by two recombinant baculoviruses (BV) expressing rabies glycoprotein (BVG) and matrix protein (BVM) genes to produce rabies virus-like particles (VLP) at different multiplicities of infection (MOI). Schott flasks essays covering a wide range of MOI for both BV were performed. Viable cell density, cell viability, glucose, glutamine, glutamate, lactate, ammonium, and rabies proteins concentrations were monitored over the infection phase. The expression of both recombinant proteins was not limited by glucose, glutamine, and glutamate in a broad MOI (pfu/cell) range of BVG (0.15-12.5) and BVM (0.1-5.0) using SF900 III serum free culture medium. Death phase initiation and the specific death rate depend on BV MOI. The wave pattern of nutrient/metabolite profiles throughout the viral infection phase is related to the baculovirus lytic cycle. The optimal MOIs ratio between BVG (2.5-4.5) and BVM (1.0-3.0) for maximum protein expression was defined. The produced rabies VLP sizes are close to 78 nm. In general, these work outputs bring a better understanding of the metabolic performance of Sf9 cells when infected by BV for producing VLP, and specifically, for progressing in a rabies VLP vaccine development.

Rabies is an ancient zoonotic disease that still causes the death of over 59,000 people worldwide each year. The rabies lyssavirus encodes five proteins, including the envelope glycoprotein and the matrix protein. RVGP is the only protein exposed on the surface of viral particle, and it can induce immune response with neutralizing antibody formation. RVM has the ability to assist with production process of virus-like particles. VLPs were produced in recombinant baculovirus system. In this work, two recombinant baculoviruses carrying the RVGP and RVM genes were constructed. From the infection and coinfection assays, we standardized the best multiplicity of infection and the best harvest time. Cell supernatants were collected, concentrated, and purified by sucrose gradient. Each step was used for protein detection through immunoassays. Sucrose gradient analysis enabled to verify the separation of VLPs from rBV. Through the negative contrast technique, we visualized structures resembling rabies VLPs produced in insect cells and rBV in the different fractions of the sucrose gradient. Using ELISA to measure total RVGP, the recovery efficiency of VLPs at each stage of the purification process was verified. Thus, these results encourage further studies to confirm whether rabies VLPs are a promising candidate for a veterinary rabies vaccine.

Caecilians (order Gymnophiona) are apodan, snake-like amphibians, usually with fossorial habits, constituting one of the most unknown groups of terrestrial vertebrates. As in orders Anura (frogs, tree frogs and toads) and Caudata (salamanders and newts), the caecilian skin is rich in mucous glands, responsible for body lubrication, and poison glands, producing varied toxins used in defence against predators and microorganisms. Whereas in anurans and caudatans skin gland morphology has been well studied, caecilian poison glands remain poorly elucidated. Here we characterised the skin gland morphology of the caecilian Siphonops annulatus, emphasising the poison glands in comparison to those of anurans and salamanders. We showed that S. annulatus glands are similar to those of salamanders, consisting of several syncytial compartments full of granules composed of protein material but showing some differentiated apical compartments containing mucus. An unusual structure resembling a mucous gland is frequently observed in lateral/apical position, apparently connected to the main duct. We conclude that the morphology of skin poison glands in caecilians is more similar to salamander glands when compared to anuran glands that show a much-simplified structure.

The new outbreak of coronavirus disease 2019 (COVID-19) has infected and caused the death of millions of people worldwide. Intensive efforts are underway around the world to establish effective treatments. Immunoglobulin from immunized animals or plasma from convalescent patients might constitute a specific treatment to guarantee the neutralization of the virus in the early stages of infection, especially in patients with risk factors and a high probability of progressing to severe disease. Worldwide, a few clinical trials using anti-SARS-CoV-2 immunoglobulins from horses immunized with the entire spike protein or fragments of it in the treatment of patients with COVID-19 are underway. Here, we describe the development of an anti-SARS-CoV-2 equine F(ab')

The production of biopharmaceuticals as vaccines in serum-free media results in reduced risk of contamination and simpler downstream processing. The production of enveloped viruses and viral vectors such as Semliki Forest Virus (SFV) typically requires lipids that are provided by supplementation with animal serum, so production under serum-free conditions is challenging. In this work, the capacity to deliver genetic material of SFV-viral replicon particles (SFV-VRPs) produced in BHK-21 cells adapted to serum-free medium (BHK/SFM) was evaluated. Three transgenes were evaluated: GFP used as a model protein, while hepatitis C virus nonstructural protein 3 protease domain (HCV-NS3p) and rabies virus glycoprotein (RVGP) were selected based on their distinct nature (enzyme and glycoprotein, respectively). BHK/SFM cells produced a sevenfold higher number of SFV-VRPs, as determined by qRT-PCR. These particles showed similar capacities of infecting BHK/FBS or BHK/SFM cells. GFP expression was evaluated by flow cytometry, HCV-NS3p activity by enzymatic assay, and RVGP expression by ELISA and Western Blot. Expression analysis revealed higher levels of GFP and HCV-NS3p in BHK/SFM, while the levels of RVGP were similar for BHK/SFM and BHK/FBS. In conclusion, the BHK/SFM cells showed increased SFV-VRP production yields, without affecting vector infectivity or heterologous gene expression, hence validating the use of BHK/SFM for industrial applications.

Made available in DSpace on 2020-12-12T01:32:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Most rabies vaccines are based on inactivated virus, which production process demands a high level of biosafety structures. In the past decades, recombinant rabies virus glycoprotein (RVGP) produced in several expression systems has been extensively studied to be used as an alternative vaccine. The immunogenic characteristics of this protein depend on its correct conformation, which is present only after the correct post-translational modifications, typically performed by animal cells. The main challenge of using this protein as a vaccine candidate is to keep its trimeric conformation after the purification process. We describe here a new immunoaffinity chromatography method using a monoclonal antibody for RVGP Site II for purification of recombinant rabies virus glycoprotein expressed on the membrane of Drosophila melanogaster S2 cells. RVGP recovery achieved at least 93%, and characterization analysis showed that the main antigenic proprieties were preserved after purification. Science and Technology Institute Federal University of São Paulo (UNIFESP) Viral Immunology Laboratory Butantan Institute Medical School São Paulo State University (UNESP) National Institute for Biotechnology Research and Training (NIBRT) Medical School São Paulo State University (UNESP)

The recent outbreaks of Zika virus (ZIKV) infection and the potential association with Guillain-Barre syndrome in adults and with congenital abnormalities have highlighted the urgency for an effective vaccine. The ZIKV Envelope glycoprotein (EZIKV) is the most abundant protein on the virus surface, and has been evaluated together with the pre-membrane protein (prM) of the viral coat as a vaccine candidate in clinical trials. In this study, we performed a head-to-head comparison of the immune response induced by different EZIKV-based vaccine candidates in mice. We compared different platforms (DNA, recombinant protein), adjuvants (poly (I:C), CpG ODN 1826) and immunization strategies (homologous, heterologous). The hierarchy of adjuvant potency showed that poly (I:C) was a superior adjuvant than CpG ODN. While poly (I:C) assisted immunization reached a plateau in antibody titers after two doses, the CpG ODN group required an extra immunization dose. Besides, the administration of poly (I:C) induced higher EZIKV-specific cellular immune responses than CpG ODN. We also show that immunization with homologous prime-boost EZIKV protein + poly (I:C) regimen induced a more robust humoral response than homologous DNA (pVAX-EZIKV) or heterologous regimens (DNA/protein or protein/DNA). A detailed analysis of cellular immune responses revealed that homologous (EZIKV + poly (I:C)) and heterologous (pVAX-EZIKV/EZIKV + poly (I:C)) prime-boost regimens induced the highest magnitude of IFN-γ secreting cells and cytokine-producing CD4+ T cells. Overall, our data demonstrate that homologous EZIKV + poly (I:C) prime-boost immunization is sufficient to induce more robust specific-EZIKV humoral and cellular immune responses than the other strategies that contemplate homologous DNA (pVAX-EZIKV) or heterologous (pVAX-EZIKV/EZIKV + poly (I:C), and vice-versa) immunizations.

Recombinant Drosophila S2 cells have been used for the expression of many proteins of medical interest. However, membrane-attached glycoproteins, which commonly exhibit lower expression levels compared to soluble proteins, may require special procedures in order to attain high levels of expression. In this study, two S2 cell population enrichment methods (antibiotic and immunomagnetic selection) were evaluated for their ability to enhance expression of the membrane-anchored rabies virus glycoprotein (RVGP). Quantification of RVGP production and determination of its cDNA copy number in transformed cells showed that both enrichment methods increased RVGP expression without significantly affecting its gene copy number. More interestingly, RVGP mRNA levels measured after cycloheximide treatment were poorly correlated with glycoprotein levels. Both enrichment methods enhanced expression of RVGP by recombinant S2 cells, with the highest level of expression achieved using immunomagnetic selection.

Insect cells are largely used for industrial production of vaccines, viral vectors and recombinant proteins as well as in research and development as an important tool for biology and bioprocess studies. They grow in suspension and are semi-adherent cells. Among the cell culture systems enabling scalable bioprocess the single-use fixed-bed iCELLis(®) bioreactors offer great advantages. We have established the conditions for Drosophila melanogaster Schneider 2 (S2) and Spodoptera frugiperda (Sf9) cells entrapment into the fixed-bed, cell growth and recover from the fixed-bed once high cell densities were attained. Our established protocol allowed these cells, at a cell seeding of 2×1E5 cells/microfiber carriers (MC) (3.5×1E6cells/mL; 1.7×1E4cells/cm(2)), to grow inside a 4m(2)/200mL fixed-bed attaining a concentration of 5.3×1E6 cells/MC (9.5×1E7cells/mL; 4.7×1E5 cells/cm(2)) for S2 cells or 4.6×1E6 cells/MC (8×1E7cells/mL; 4.1×1E5cells/cm(2)) for Sf9 cells. By washing the fixed-bed, entrapped cells could then be recovered from the fixed-bed at a high rate (>85%) with high viability (>95%) by increasing the agitation to 1200/1500rpm. Although the cell yields in the fixed-bed bioreactor were comparable to those obtained in a stirred tank (respectively, 1.3×1E10 and 2.5×1E10 total cells), S2 cells stably transfected with a cDNA coding for the rabies virus glycoprotein (RVGP) showed a 30% higher preserved rRVGP production (2.5±0.1 and 1.9±0.1μg/1E7 cells), as evidenced by a conformational ELISA evaluation. These findings demonstrate not only the possibility to entrap, cultivate to high densities and recover insect cells using a single-use fixed-bed bioreactor, but also that this system provides suitable physiological conditions for the entrapped cells to produce a cell membrane associated recombinant protein with higher specific biological activity as compared to classical suspension cell cultures.

Zika virus (ZIKV) causes congenital Zika syndrome (CZS), which is characterized by fetal demise, microcephaly and other abnormalities. ZIKV in the pregnant woman circulation must cross the placental barrier that includes fetal endothelial cells and trophoblasts, in order to reach the fetus. CZS occurs in ~1–40% of cases of pregnant women infected by ZIKV, suggesting that mothers’ infection by ZIKV during pregnancy is not deterministic for CZS phenotype in the fetus. Therefore, other susceptibility factors might be involved, including the host genetic background. We have previously shown that in three pairs of dizygotic twins discordant for CZS, neural progenitor cells (NPCs) from the CZS-affected twins presented differential in vitro ZIKV susceptibility compared with NPCs from the non-affected. Here, we analyzed human-induced-pluripotent-stem-cell-derived (hiPSC-derived) trophoblasts from these twins and compared by RNA-Seq the trophoblasts from CZS-affected and non-affected twins. Following in vitro exposure to a Brazilian ZIKV strain (ZIKVBR), trophoblasts from CZS-affected twins were significantly more susceptible to ZIKVBR infection when compared with trophoblasts from the non-affected. Transcriptome profiling revealed no differences in gene expression levels of ZIKV candidate attachment factors, IFN receptors and IFN in the trophoblasts, either before or after ZIKVBR infection. Most importantly, ZIKVBR infection caused, only in the trophoblasts from CZS-affected twins, the downregulation of genes related to extracellular matrix organization and to leukocyte activation, which are important for trophoblast adhesion and immune response activation. In addition, only trophoblasts from non-affected twins secreted significantly increased amounts of chemokines RANTES/CCL5 and IP10 after infection with ZIKVBR. Overall, our results showed that trophoblasts from non-affected twins have the ability to more efficiently activate genes that are known to play important roles in cell adhesion and in triggering the immune response to ZIKV infection in the placenta, and this may contribute to predict protection from ZIKV dissemination into fetuses’ tissues., Author summary The Zika virus (ZIKV) infection in adults is usually characterized by mild flu-like symptoms, with most cases remaining asymptomatic. However, in the last years, widespread ZIKV infection was shown for the first time to be associated with congenital Zika syndrome (CZS) and death of neonates. It is estimated that CZS occurs in ~1–40% of cases of pregnant women infected by ZIKV, which suggests that different susceptibility factors might be involved, including the host genetic background. Here, by analyzing trophoblast cells that recapitulate the placenta from three pairs of dizygotic twins discordant for CZS, we were able to show that trophoblasts from CZS-affected twins were significantly more susceptible to ZIKV infection when compared with trophoblasts from the non-affected twins. We also provide a detailed picture of genes differentially expressed by trophoblasts from the discordant twins after infection with ZIKV. These genes can be further investigated as possible therapeutic targets to avoid viral dissemination into developing fetus’ tissues. Our results suggest that CZS might be caused, among other factors, by a decreased ability of the placenta to respond to ZIKV infection in CZS-affected neonates, concomitant with a previously known deregulation of neural development genes in ZIKV-infected neuroprogenitor cells of these CZS-affected babies.

Congenital Zika syndrome (CZS) causes early brain development impairment by affecting neural progenitor cells (NPCs). Here, we analyze NPCs from three pairs of dizygotic twins discordant for CZS. We compare by RNA-Seq the NPCs derived from CZS-affected and CZS-unaffected twins. Prior to Zika virus (ZIKV) infection the NPCs from CZS babies show a significantly different gene expression signature of mTOR and Wnt pathway regulators, key to a neurodevelopmental program. Following ZIKV in vitro infection, cells from affected individuals have significantly higher ZIKV replication and reduced cell growth. Whole-exome analysis in 18 affected CZS babies as compared to 5 unaffected twins and 609 controls excludes a monogenic model to explain resistance or increased susceptibility to CZS development. Overall, our results indicate that CZS is not a stochastic event and depends on NPC intrinsic susceptibility, possibly related to oligogenic and/or epigenetic mechanisms., Zika virus (ZIKV) infection can cause congenital Zika syndrome (CZS), but the underlying mechanisms are poorly understood. Here, the authors generate neural progenitor cells from dizygotic twins with a discordant phenotype regarding CZS and study their response to ZIKV infection.

Viral hepatitis caused by the hepatitis C virus (HCV) affects millions of people worldwide. The non-structural protein 3 (NS3), one of the most conserved proteins in HCV, is the target of many therapeutic studies. The NS3 protease domain (NS3p) has a range of cytotoxic T lymphocyte (CTL) epitopes, and synthesizing the protein inside the cells is the most appropriate way to present it to the immune system. We developed a tool to study this kind of presentation, using two vectored particle (VP) systems, one based on the Semliki Forest virus (SFV) and the other on HCV pseudoparticles (HCVpp), both carrying the protease domain of the NS3 gene. In addition to producing the particles, we developed a method to quantify these VPs using qRT-PCR. We produced batches of approximately 2.4 × 104 SFV-NS3p/μL and 4.0 × 102 HCVpp-NS3p/μL. BHK-21 and HuH-7 cells treated with the VPs expressed the NS3 protein, thus showing the functionality of this system.

Congenital Zika syndrome (CZS) causes early brain development impairment by affecting neural progenitor cells (NPCs). Here, we analyze NPCs from three pairs of dizygotic twins discordant for CZS. We compare by RNA-Seq the NPCs derived from CZS-affected and CZS-unaffected twins. Prior to Zika virus (ZIKV) infection the NPCs from CZS babies show a significantly different gene expression signature of mTOR and Wnt pathway regulators, key to a neurodevelopmental program. Following ZIKV in vitro infection, cells from affected individuals have significantly higher ZIKV replication and reduced cell growth. Whole-exome analysis in 18 affected CZS babies as compared to 5 unaffected twins and 609 controls excludes a monogenic model to explain resistance or increased susceptibility to CZS development. Overall, our results indicate that CZS is not a stochastic event and depends on NPC intrinsic susceptibility, possibly related to oligogenic and/or epigenetic mechanisms.

Made available in DSpace on 2018-11-26T17:51:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-06-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The transmembrane rabies virus glycoprotein (RVGP) is the main antigen of vaccine formulations used around the world to prevent rabies, the most lethal preventable infectious disease known. The objective of this work was to evaluate the potential of a bioreactor using wave-induced agitation in the initial steps of scaling up the rRVGP production process by a Drosophila melanogaster S2 cell line to produce rRVGP in sufficient quantities for immunization and characterization studies. Taking advantage of some remarkable features recognized in Drosophila S2 cells for scaling the culture process, a robust recombinant lineage (S2MtRVGPH-His) engineered by our group for the expression of rRVGP using a copper-inducible promoter was used in the bioreactor cultures. The WAVE Bioreactor was chosen because it represents an innovative approach to the cultivation of animal cells using single-use technology. For that purpose, we firstly established a procedure for culturing the S2MtRVGPH-His lineage in 100 mL Schott flasks. Using an inoculum of 5 x 10(5) cells/mL in culture medium (Sf900-III) induced with solution of CuSO4 (0.7 mM) and a convenient pH range (6.2-7.0), optimal parameter values such as time of induction (72 h) and temperature (28 degrees C) to increase rRVGP production could be defined. This procedure was reproduced in culture experiments conducted in a WAVE Bioreactor (TM) 2/10 using a 2 L Cellbag. The results in Schott flasks and inWAVE Bioreactor (TM) were very similar, yielding a maximum titer of rRVGP above of 1 mg.L-1. The immunization study showed that the rRVGP produced in the bioreactor was of high immunogenic quality. Univ Fed Sao Carlos, Dept Chem Engn, BR-13565905 Sao Carlos, SP, Brazil Butantan Inst, Lab Viral Immunol, BR-05503900 Sao Paulo, SP, Brazil Sao Paulo State Univ, Dept Vet Hyg & Publ Hlth, BR-18618970 Botucatu, SP, Brazil Sao Paulo State Univ, Dept Vet Hyg & Publ Hlth, BR-18618970 Botucatu, SP, Brazil CNPq: 402439/2013-9

A single-use fixed-bed bioreactor (iCELLis nano) can be used for cultivating non adherent insect cells, which can be then recovered for scaling up or for harvesting a membrane-associated viral glycoprotein with high quality in terms of preserved protein structure and biological function. Here, we describe the procedures for establishing genetically modified Drosophila melanogaster Schneider 2 (S2) cell cultures in the iCELLis nano bioreactor and for quantifying by ELISA the recombinant rabies virus glycoprotein (rRVGP) synthesized. By using the described protocol of production, the following performance can be regularly achieved: 1.7 ± 0.6 × 1E10 total cells; 2.4 ± 0.8 × 1E7 cells/mL and 1.2 ± 0.9 μg of rRVGP/1E7 cells; 1.5 ± 0.8 mg of total rRVGP.

Made available in DSpace on 2015-08-21T17:53:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2014 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Fundação para o Desenvolvimento Tecnológico da Engenharia (FDTE) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Mammalian cells are the most frequently used hosts for biopharmaceutical proteins manufacturing. Inoculum quality is a key element for establishing an efficient bioconversion process. The main objective in inoculation expansion process is to generate large volume of viable cells in the shortest time. The aim of this paper was to optimize the inoculum preparation stage of baby hamster kidney (BHK)-21 cells for suspension cultures in benchtop bioreactors, by means of a combination of static and agitated culture systems. Critical parameters for static (liquid column height: 5, 10, 15 mm) and agitated (working volume: 35, 50, 65 mL, inoculum volume percentage: 10, 30 % and agitation speed: 25, 60 rpm) cultures were study in T-flask and spinner flask, respectively. The optimal liquid column height was 5 mm for static culture. The maximum viable cell concentration in spinner flask cultures was reached with 50 mL working volume and the inoculum volume percentage was not significant in the range under study (10–30 %) at 25 rpm agitation. Agitation speed at 60 rpm did not change the main kinetic parameters with respect to those observed for 25 rpm. These results allowed for a schedule to produce more than 4 × 109 BHK-21 cells from 4 × 106 cells in 13 day with 1,051 mL culture medium. Universidade Estadual Paulista Júlio de Mesquita Filho, Assis, Unesp - Campus Assis, Parque Universitário, CEP 19806900, SP, Brasil Universidade Estadual Paulista Júlio de Mesquita Filho, Assis, Unesp - Campus Assis, Parque Universitário, CEP 19806900, SP, Brasil FAPESP: 2010/52521-6 CNPq: 483009/2010-5

Ependymoma is a metastatic CNS tumor responsible for 10% of all pediatric brain tumors. Displaying a poor survival rate of only 32.5%, the conventional ependymoma treatment is surgery and radiation therapy associated to significant morbidity. Previous studies found significantly increased expression of NESTIN, a neural progenitor cell (NPC) marker, in aggressive ependymoma. Zika virus (ZIKV) has recently emerged as an oncolytic therapy against adult and pediatric CNS tumors due to its ability to infect NPCs and neural stem-like tumor cells. We showed for the first time that Brazilian Zika virus strain efficiently destroyed pediatric CNS tumor cells in vitro and in vivo, resulting in significantly mice longer survival and fewer metastasis. Once tumor cells overexpressing NPCs markers are more susceptible to ZIKV infection, here we evaluated the oncolytic properties of Brazilian ZIKV against human ependymoma. A patient-derived ependymoma cell line (USP21-EPE) has been established and found to abnormally express the pluripotency markers CD133, SOX2 and NESTIN. USP21-EPE monolayer culture showed massive cell death three days after ZIKV infection in four tested MOIs: 0.01, 0.1, 1 and 2. At MOI 0,1 50% of cell population decreased viability while at higher MOIs was observed 100% of cell death 72 hours post ZIKV infection. Moreover, ZIKV significantly disrupted CNS ependymoma tumorspheres reinforcing its oncolytic effect against the ependymoma tumor cells. Considering the poor effectiveness and severe side effects of available treatments for ependymoma and that most ZIKV infections are associated to very mild or no symptoms, our findings open new avenues for novel therapies against this aggressive pediatric tumor. Citation Format: Carolini Kaid, Patrícia Semedo-Kuriki, Ernesto Goulart, Luiz Caires-Júnior, Renato Astray, Oswaldo Keith Okamoto, Mayana Zatz. Zika virus is a potent oncolytic agent against aggressive human ependymoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2869.

Made available in DSpace on 2014-12-03T13:11:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Fundacao para o Desenvolvimento Tecnologico da Engenharia (FDTE) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Monitoring mammalian cell culture with UV-vis spectroscopy has not been widely explored. The aim of this work was to calibrate Partial Least Squares (PLS) models from off-line UV-vis spectral data in order to predict some nutrients and metabolites, as well as viable cell concentrations for mammalian cell bioprocess using phenol red in culture medium. The BHK-21 cell line was used as a mammalian cell model. Spectra of samples taken from batches performed at different dissolved oxygen concentrations (10, 30, 50, and 70% air saturation), in two bioreactor configurations and with two strategies to control pH were used to calibrate and validate PLS models. Glutamine, glutamate, glucose, and lactate concentrations were suitably predicted by means of this strategy. Especially for glutamine and glucose concentrations, the prediction error averages were lower than 0.50 +/- 0.10 mM and 2.21 +/- 0.16 mM, respectively. These values are comparable with those previously reported using near infrared and Raman spectroscopy in conjunction with PLS. However, viable cell concentration models need to be improved. The present work allows for UV-vis at-line sensor development, decrease cost related to nutrients and metabolite quantifications and establishment of fed-batch feeding schemes. (c) 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:241-248, 2014 Inst Butantan, Lab Especial Pesquisa & Desenvolvimento Imunol V, BR-05503900 Sao Paulo, Brazil Univ Sao Paulo, Escola Politecn, Dept Engn Quim, Lab Celulas Anim, BR-05508900 Sao Paulo, Brazil Univ Estadual Paulista, Dept Ciencias Biol, BR-19806900 Assis, SP, Brazil Inst Butantan, Lab Imunol Viral, BR-05503900 Sao Paulo, Brazil Inst Pesquisas Tecnol Estado Sao Paulo SA, Nucleo Bionanomanufatura, Lab Biotecnol Ind, BR-05508901 Sao Paulo, Brazil Univ Estadual Paulista, Dept Ciencias Biol, BR-19806900 Assis, SP, Brazil FAPESP: 10/52521-6 CNPq: CNPq: 483009/2010-5

Recombinant rabies virus glycoprotein (RVGP) was expressed in cell membranes of stably transfected Drosophila S2 cells using constitutive and inducible promoters. Although with quantitative differences of RVGP expression in both systems, the cDNA transcription, as evaluated by relative RVGP mRNA levels measured by qRT-PCR, sustained the amount of RVGP producing cells and the RVGP volumetric (ΠRVGP) productivity. At the transition to the stationary cell growth phase, once the cell culture slowed down its rate of multiplication, an accumulation of RVGP mRNA and RVGP was clearly observed in both cell populations. Nevertheless, cell cultures performed under sub-optimal temperatures indicated that an envisaged increase in the RVGP production is not only dependent on cell growth rate, but essentially on optimal cell metabolic state.

The expression of recombinant viral envelope glycoproteins in S2 (Drosophila melanogaster) has been performed with good results. This chapter contains protocols for the utilization of this system for the expression and analysis of proteins presented in cell plasma membrane.

The expression of recombinant viral envelope glycoproteins in S2 (Drosophila melanogaster) has been performed with good results. This chapter contains protocols for the utilization of this system for the expression and analysis of proteins presented in cell plasma membrane.

An animal protein-free medium composed of IPL-41 containing 6 g L(-1) yeastolate ultrafiltrate, 10 g L(-1) glucose, 2 g L(-1) lactose, 5 g L(-1) glutamine, 1% lipid emulsion, and 0.1% Pluronic F-68 was used for producing recombinant proteins in batch mode employing two cell lines, S2AcRVGP2k expressing the G glycoprotein from rabies virus (RVGP) and S2AcHBsAgHy-9C expressing the surface antigen of hepatitis B virus (HBsAg), both obtained from Drosophila melanogaster S2 cells. Growth of wild-type S2 cells was also evaluated in the same medium. Cell behavior in the tested medium was compared to that verified in Sf900 II®. The results show that in shake flasks, S2AcRVGP2k and S2AcHBsAgHy-9C cells reached around 2 × 10(7) cells mL(-1) in both media. In supplemented IPL-41 and Sf900 II® media, S2AcRVGP2k cells produced 367 ng RVGP mL(-1) and 638 ng RVGP mL(-1), respectively, while S2AcHBsAgHy-9C cells correspondently produced 573 ng HBsAg mL(-1) and 322 ng HBsAg mL(-1) in the mentioned media. In stirred tanks, S2AcRVGP2k cells reached 3 × 10(7) cells mL(-1) and produced up to 758 ng RVGP mL(-1). In general, glucose was consumed by cells, while lactate and ammonia were produced.

S2 cell populations (S2AcRVGP2K and S2MtRVGP-Hy) were selected after transfection of gene expression vectors carrying the cDNA encoding the rabies virus glycoprotein (RVGP) gene under the control of the constitutive (actin) or inductive (metallothionein) promoters. These cell populations were cultivated in a 1L bioreactor mimicking a large scale bioprocess. Cell cultures were carried out at 90 rpm and monitored/controlled for temperature (28 degrees C) and dissolved oxygen (10 or 50% air saturation). Cell growth attained approximately 1.5-3 x 10(7)cells/mL after 3-4 days of cultivation. The constitutive synthesis of RVGP in S2AcRVGP2K cells led to values of 0.76 microg/10(7) cells at day 4 of culture. The RVGP synthesis in S2MtRVGP-Hy cell fraction increased upon CuSO(4) induction attaining specific productivities of 1.5-2 microg/10(7) cells at days 4-5. RVGP values in supernatant as a result of cell lysis were always very low (

Gene expression in insect cells is an advantageous system for recombinant protein production, mainly because of its capacity to produce complex proteins with correct post-translational modifications. Recently, we identified and purified a protein from Lonomia obliqua hemolymph able to increase the production of rabies virus glycoprotein, expressed in Drosophila melanogaster cells, by about 60%. In this work, the kinetic parameters for cell growth and recombinant rabies virus glycoprotein production were determined in cultures of transfected Drosophila melanogaster Schneider 2 (S2) cells expressing recombinant rabies virus glycoprotein (rRVGP), enriched and non-enriched with the hemolymph of Lonomia obliqua (Hb). The highest concentration of rRVGP was achieved at the beginning of the culture enriched with Hb, indicating that the cells produce greater amounts of rRVGP per cell (specific rRVGP concentration) at the early exponential growth phase. After day 8, a decrease in the concentration of rRVGP (ng/mL) was observed, probably due to protein decomposition. The average specific rRVGP production rate (mu(rRVGP)) was 30 ng rRVGP/10(7)cell.day, higher than that observed in the non-enriched culture.

Rabies is to this date one of the most important death causing zoonotic viral diseases, with 98% of deaths reported in developing countries, where access to modern vaccines and tools for efficient diagnostic remain unaffordable. In this paper, we describe a newly engineered RNA-based rabies virus glycoprotein (RVGP) expression vector based on the Semliki Forest Virus (SFV) system. A recombinant SFV carrying an RNA coding for RVGP (SFV-RVGP) was constructed and the RVGP expression was evaluated in animal cell cultures. The mRNA coding for RVGP and the RVGP itself were assessed by qPCR, Western-blotting, confocal microscopy, flow cytometry and ELISA. Moreover, SFV-RVGP was proven to be highly efficient in expressing the functionally trimeric RVGP. SFV-RVGP is easy to produce and efficient in different cell lines, making it an interesting candidate for efficient and functional viral glycoprotein expression.

Culture conditions that affect product quality are important to the successful operation and optimization of recombinant protein production. The objective of this study was to optimize culture conditions for growth of recombinant Drosophila melanogaster S2 cells (S2AcRVGP) in order to enhance the production of rRVGP. The addition of DMSO and glycerol to the medium and growth at a reduced temperature (22 degrees C) were the culture condition variations selected to be tested. Experimental cultures were first performed in serum-free Sf900 II medium in 250 ml Schott flasks. The most promising conditions identified in these experiments were also tested on a higher scale in a 3l bioreactor. In the Schott flasks experiments, all the changes in culture conditions resulted in an increase of rRVGP production. The protein concentration was 3.6-fold higher with addition of 1% DMSO and 1% glycerol and 9.3-fold higher when the cells were cultured at 22 degrees C instead of the standard 28 degrees C. The maximum concentration of rRVGP reached was 591 mug l(-1). In bioreactor experiments, with control of pH at 6.20 and DO at 50%, the reduced culture temperature (22 degrees C) was the strategy that promoted the highest glycoprotein production, 928 mug l(-1).

Although several reports have been published on recombinant protein expression using Drosophila cells, information on their metabolism and growth in vitro is relatively scarce. In the present study, we have analyzed the growth and metabolism of transfected S2 cells (S2AcRVGP) in bioreactor cultures with serum-free medium Sf900 II, to evaluate its potential for mass production of a rabies virus glycoprotein (RVGP). Cells were cultured in a 3 l-stirred-tank bioreactor at 28 degrees C with pH controlled at 6.2 and dissolved oxygen at 50% air saturation. The cells attained a specific growth rate and maximum cell density as high as 0.084 h(-1) and 2.3 x 10(7 )cell ml(-1), respectively. The main substrates consumed during this rapid growth phase were glucose, glutamine and proline. An atypical accumulation of ammonia and alanine was observed in the culture medium, up to 62 mM and 47 mM, respectively, but lactate was produced in low levels. After exhaustion of glutamine and proline as energy sources, alanine was consumed and production of ammonia increased. The production of recombinant RVGP reached concentrations as high as 178 mug l(-1). Premature exhaustion of glutamine, serine and cysteine could be related to degradation of the recombinant glycoprotein. In general, the results demonstrated that S2AcRVGP can be considered an effective vehicle for large-scale recombinant glycoprotein expression and that several critical factors of the bioprocess could be optimized to increase the quality and productivity of the RVGP.