Semliki Forest Virus (SFV) Self-Amplifying RNA Delivered to J774A.1 Macrophage Lineage by Its Association with a Purified Recombinant SFV Capsid Protein.

The Semliki Forest virus capsid protein (C) is an RNA binding protein which exhibits both specific and unspecific affinities to single-strand nucleic acids. The putative use of the self-amplifying RNAs (saRNAs) of alphaviruses for biotechnological purpose is one of the main studied strategies concerning RNA-based therapies or immunization. In this work, a recombinant C protein from SFV was expressed and purified from bacteria and used to associate in vitro with a saRNA derived from SFV. Results showed that the purified form of C protein can associate with the saRNA even after high temperature treatment. The C protein was associated with a modified saRNA coding for the green fluorescent protein (GFP) and delivered to murine macrophage cells which expressed the GFP, showing that the saRNA was functional after being associated with the recombinant purified C protein.

Production of Recombinant Zika Virus Envelope Protein by Airlift Bioreactor as a New Subunit Vaccine Platform.

The Zika Virus (ZIKV) is an emerging arbovirus of great public health concern, particularly in the Americas after its last outbreak in 2015. There are still major challenges regarding disease control, and there is no ZIKV vaccine currently approved for human use. Among many different vaccine platforms currently under study, the recombinant envelope protein from Zika Virus (rEZIKV) constitutes an alternative option for vaccine development and has great potential for monitoring ZIKV infection and antibody response. This study describes a method to obtain a bioactive and functional rEZIKV using an E. coli expression system, with the aid of a 5-L airlift bioreactor and following an automated fast protein liquid chromatography (FPLC) protocol, capable of obtaining high yields of approximately 20 mg of recombinant protein per liter of bacterium cultures. The purified rEZIKV presented preserved antigenicity and immunogenicity. Our results show that the use of an airlift bioreactor for the production of rEZIKV is ideal for establishing protocols and further research on ZIKV vaccines bioprocess, representing a promising system for the production of a ZIKV envelope recombinant protein-based vaccine candidate.

RBD and Spike DNA-Based Immunization in Rabbits Elicited IgG Avidity Maturation and High Neutralizing Antibody Responses against SARS-CoV-2.

Neutralizing antibodies (nAbs) are a critical part of coronavirus disease 2019 (COVID-19) research as they are used to gain insight into the immune response to severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infections. Among the technologies available for generating nAbs, DNA-based immunization methods are an alternative to conventional protocols. In this pilot study, we investigated whether DNA-based immunization by needle injection in rabbits was a viable approach to produce a functional antibody response. We demonstrated that three doses of DNA plasmid carrying the gene encoding the full-length spike protein (S) or the receptor binding domain (RBD) of SARS-CoV-2 induced a time-dependent increase in IgG antibody avidity maturation. Moreover, the IgG antibodies displayed high cross neutralization by live SARS-CoV-2 and pseudoviruses neutralization assays. Thus, we established a simple, low cost and feasible DNA-based immunization protocol in rabbits that elicited high IgG avidity maturation and nAbs production against SARS-CoV-2, highlighting the importance of DNA-based platforms for developing new immunization strategies against SARS-CoV-2 and future emerging epidemics.

Characterization of growth and metabolism of Drosophila melanogaster cells transfected with the rabies-virus glycoprotein gene.

In the present study, the growth and key metabolic features of a gene-transfected Drosophila melanogaster (fruitfly) S2 (Schneider 2) cell population (S2AcRVGP cells), cultured in Sf900-II medium, have been evaluated to provide substantial support for the development of a bioprocess to produce RVGP (rabies-virus glycoprotein). Experimental cultures were grown both in a 100 ml Schott flask incubated in a shaker at 28 degrees C and 100 rev./min and in a 3 litre stirred-tank bioreactor at 28 degrees C, with increasing agitation. In small-scale culture, S2AcRVGP cells reached a maximum cell concentration of 1.13 x 10(7) cell/ml, presented a mu(max) (maximum specific growth rate) of 0.037 h(-1) and the growth was limited by oxygen deprivation. An early and remarkably long stationary phase was observed under hypoxia. Cell cultures grown in the bioreactor without oxygen limitation exhibited a maximum cell concentration of 2.2 x 10(7) cells/ml and mu(max) values as high as 0.048 h(-1). The main substrate consumed in order to reach such a high growth rate was the amino acid proline, which seems to play an important role as a source of metabolic energy in the culture of S2AcRVGP cells. Under conditions of hypoxia, the cells were able to survive for 15 h without apparent damage, recovering their previous metabolic activity.

Production of Recombinant Rabies Virus Glycoprotein by Insect Cells in a Single-Use Fixed-Bed Bioreactor.

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.

Zika Virus Selectively Kills Aggressive Human Embryonal CNS Tumor Cells In Vitro and In Vivo.

Zika virus (ZIKV) is largely known for causing brain abnormalities due to its ability to infect neural progenitor stem cells during early development. Here, we show that ZIKV is also capable of infecting and destroying stem-like cancer cells from aggressive human embryonal tumors of the central nervous system (CNS). When evaluating the oncolytic properties of Brazilian Zika virus strain (ZIKVBR) against human breast, prostate, colorectal, and embryonal CNS tumor cell lines, we verified a selective infection of CNS tumor cells followed by massive tumor cell death. ZIKVBR was more efficient in destroying embryonal CNS tumorspheres than normal stem cell neurospheres. A single intracerebroventricular injection of ZIKVBR in BALB/c nude mice bearing orthotopic human embryonal CNS tumor xenografts resulted in a significantly longer survival, decreased tumor burden, fewer metastasis, and complete remission in some animals. Tumor cells closely resembling neural stem cells at the molecular level with activated Wnt signaling were more susceptible to the oncolytic effects of ZIKVBR Furthermore, modulation of Wnt signaling pathway significantly affected ZIKVBR-induced tumor cell death and viral shedding. Altogether, these preclinical findings indicate that ZIKVBR could be an efficient agent to treat aggressive forms of embryonal CNS tumors and could provide mechanistic insights regarding its oncolytic effects.Significance: Brazilian Zika virus strain kills aggressive metastatic forms of human CNS tumors and could be a potential oncolytic agent for cancer therapy. Cancer Res; 78(12); 3363-74. ©2018 AACR.

Impact of recombinant Drosophila S2 cell population enrichment on expression of rabies virus glycoprotein.

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.

Rabies virus glycoprotein and immune response pattern using recombinant protein or recombinant RNA viral vectors.

The present study shows the humoral and cellular aspects of immune response generated by a recombinant rabies virus glycoprotein (rRVGP) as compared to those generated by viral vector carrying the RNA coding for this protein (RVGP-RNA). The rRVGP was synthesized by stably transfected Drosophila melanogaster Schneider 2 (S2) cells and the RVGP-RNA was carried by a recombinant Semiliki Forest Virus (SFV-RVGP). The data show that protein as well as the RNA vaccine was capable of inducing reasonably acceptable levels of antibodies as compared to the optimized commercial whole virus vaccine. As expected, the RNA vaccine was clearly more effective than the protein vaccines in inducing a cellular immune response, as evaluated by the IgG2a/IgG1 ratio and synthesis of interferon gamma (IFNγ) and interleukin 2 (IL2). Our study supports the importance of vaccine designing taking into consideration the concept of DNA/RNA ability to induce an effective cell immune response.

Quantitative RT-PCR for titration of replication-defective recombinant Semliki Forest virus.

Virus titration may constitute a drawback in the development and use of replication-defective viral vectors like Semliki Forest virus (SFV). The standardization and validation of a reverse transcription quantitative PCR (qRT-PCR) method for SFV titration is presented here. The qRT-PCR target is located within the nsp1 gene of the non-structural polyprotein SFV region (SFV RNA), which allows the strategy to be used for several different recombinant SFV constructs. Titer determinations were carried out by performing virus titration and infection assays with SFVs containing an RNA coding region for the rabies virus glycoprotein (RVGP) or green fluorescent protein (GFP). Results showed that the standardized qRT-PCR is applicable for different SFV constructs, and showed good reproducibility. To evaluate the correlation between the amount of functional SFV RNA in a virus lot and its infectivity in BHK-21 cell cultures, a temperature mediated titer decrease was performed and successfully quantitated by qRT-PCR. When used for cell infection at the same multiplicity of infection (MOI), the temperature treated SFV-RVGP samples induced the same levels of RVGP expression. Similarly, when different SFV-GFP lots with different virus titers, as accessed by qRT-PCR, were used for cell infection at the same MOI, the cultures showed comparable amounts of fluorescent cells. The data demonstrate a good correlation between the amount of virus used for infection, as measured by its SFV RNA, and the protein synthesis in the cells. In conclusion, the qRT-PCR method developed here is accurate and enables the titration of replication-defective SFV vectors, an essential aid for viral vector development as well as for establishment of production bioprocesses.

Drosophila melanogaster S2 cells for expression of heterologous genes: From gene cloning to bioprocess development.

In the present review we discuss strategies that have been used for heterologous gene expression in Drosophila melanogaster Schneider 2 (S2) cells using plasmid vectors. Since the growth of S2 cells is not dependent on anchorage to solid substrates, these cells can be easily cultured in suspension in large volumes. The factors that most affect the growth and gene expression of S2 cells, namely cell line, cell passage, inoculum concentration, culture medium, temperature, dissolved oxygen concentration, pH, hydrodynamic forces and toxic metabolites, are discussed by comparison with other insect and mammalian cells. Gene expression, cell metabolism, culture medium formulation and parameters involved in cellular respiration are particularly emphasized. The experience of the authors with the successful expression of a biologically functional protein, the rabies virus glycoprotein (RVGP), by recombinant S2 cells is presented in the topics covered.

Behavior of wild-type and transfected S2 cells cultured in two different media.

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.

Study of kinetic parameters for the production of recombinant rabies virus glycoprotein.

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.

Analytical approach for the extraction of recombinant membrane viral glycoprotein from stably transfected Drosophila melanogaster cells.

Parameters for storage, lysis and concentration of Drosophila melanogaster Schneider 2 (S2AcRVGP) cells expressing the recombinant rabies virus glycoprotein (RVGP) were studied with regard to RVGP quantification by ELISA, for productivity evaluation and future purification. Lysis buffers were formulated with Tris, NaCl, glycerol, EDTA, KCl, Na(2)PO(4), MgCl(2), PMSF and NP-40 or CHAPS. S2AcRVGP cells (10(7) cells at the exponential growth phase) were frozen at -20 degrees C as a dry pellet, suspended in buffer (B) formulations or after treatment with lysis buffer (LB) formulations. They were then thawed as cell pellets or with B formulations or PBS at 4 degrees C or at room temperature and then lysed with LB formulations. For RVGP quantification by ELISA, a protocol was chosen of cell preparation including cell freezing as dry pellet, cell thawing at 4 degrees C with B4 (Tris, NaCl, MgCl(2), PMSF) and cell lysis with the LB4 (B4 + NP-40) since it fulfilled requirements of high RVGP detection, and was easily performed with mixtures freezing quickly, and a cost-saving LB formulation could be used. Using these established conditions, we examined the optimal cell concentration for RVGP quantification by ELISA. Results showed that an increase in the RVGP detection (from 62.5 to 1083 ng/10(7) cells) paralleled a decrease in the cell number (3 x 10(7) - 10(5) cells) used. The NP-40 concentration present in the LB4 was further investigated as a function of the cell number used for sample preparation. Previous results were confirmed indicating that higher NP-40 concentrations led to a decreased detection of RVGP. Altogether our data clearly pointed out the crucial effects of cell freeze, thaw, lysis and concentration on immune detection of recombinant membrane glycoproteins and can be useful as a guideline for sample preparation for this purpose.

Enhanced production of recombinant rabies virus glycoprotein (rRVGP) by Drosophila melanogaster S2 cells through control of culture conditions.

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).

Growth of recombinant Drosophila melanogaster Schneider 2 cells producing rabies virus glycoprotein in bioreactor employing serum-free medium.

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.

Enhancing effect of a protein from Lonomia obliqua hemolymph on recombinant protein production.

Gene expression in animal cells allows large scale production of proteins used for either structure and function studies or therapeutic purposes. Maximizing recombinant protein production is necessary to optimize cell growth and protein expression. Some studies have demonstrated the presence of pharmacologically active substances in insect hemolymph. In this work, we have identified and purified a protein from Lonomia obliqua hemolymph able to increase the production of the rabies virus glycoprotein, expressed in Drosophila melanogaster S2 cells, by about 59%.

Rabies virus glycoprotein expression in Drosophila S2 cells. I. Functional recombinant protein in stable co-transfected cell line.

Recombinant rabies virus glycoprotein (rRVGP) was expressed in Drosophila melanogaster Schneider 2 (S2) cells. The cDNA encoding the entire RVGP gene was cloned in an expression plasmid under the control of the constitutive actin promoter (Ac), which was co-transfected into S2 cells together with a hygromycin selection plasmid. Selected S2 cell populations (S2AcRVGP) had a decreased ability to grow and consume substrates, when compared to the non-transfected cells (S2). They were shown, by PCR, to express the RVGP gene and mRNA and, by immunoblotting, to synthesize the rRVGP in its expected molecular mass of 65 kDa. ELISA kinetic studies showed the rRVGP expression in cell lysates and supernatants attaining concentrations of 300 microg/L. By flow cytometry analysis, about 30% of the cells in the co-transfected populations were shown to express the rRVGP. Cell populations selected by limiting dilution expressed higher rRVGP yields. Mice immunized with rRVGP were shown to synthesize antibodies against rabies virus and be protected against experimental infection with rabies virus. The data presented here show that S2 cells can be suitable hosts for the rRVGP expression, allowing its synthesis in a high degree of physical and biological integrity.

Ocorrência de ocratoxina A e aflatoxinas em arroz / Occurrence of ochratoxin A and aflatoxin in rice

Conhecer as micotoxinas que possam ser encontradas no arroz, alimento básico da população brasileira, é importante para a saúde pública. Aflatoxinas e ocratoxina A foram determinadas pelo método de Soares e Rodriguez-Amaya acrescido de partição para ciclohexano. Os testes de recuperação foram realizados em arroz polido e em arroz integral, artificialmente contaminados com aflatoxina B1 (AFB1), com ocratoxina A (OTA) e, com AFB1 e OTA juntas. Recuperações entre 93 e 104 por cento e entre 87 a 103 por cento foram obtidas para AFB1 em arroz polido e em arroz integral, respectivamente. Para a OTA, as recuperações conseguidos para AFB1, AFB2, AFG1 e AFG2 foram de 1,1,3 e 1 ug/kg para o arroz polido, e de 3,1,4 e 3 ug/kg para arroz integral, respectivamente. Para a OTA foi de 4 ug/kg em arroz polido e de 10 ug/kg em arroz detectada em duas amostras de arroz polido tipo 1 (2,9 por cento), com teores de 9 e 6 ug/kg. Uma das amostras apresentou traços de aflatoxina B2

Study of kinetic parameters for the production of recombinant rabies virus glycoprotein

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 (ìrRVGP) was 30 ng rRVGP/107cell.day, higher than that observed in the non-enriched culture.

Enhanced production of recombinant rabies virus glycoprotein (rRVGP) by Drosophila melanogaster S2 cells through control of culture conditions

Abstract 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 °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 °C instead of the standard 28 °C. The maximum concentration of rRVGP reached was 591 ìg l−1. In bioreactor experiments, with control of pH at 6.20 and DO at 50%, the reduced culture temperature (22 °C) was the strategy that promoted the highest glycoprotein production, 928 ìg l−1. Keywords Drosophila melanogaster S2 cells - Dimethyl sulfoxide - Glycerol - Rabies virus glycoprotein - Recombinant protein production - Reduced temperature cultivation