Renato Mancini Astray

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.

Rabies virus glycoprotein expression in Drosophila S2 cells. I: design of expression/selection vectors, subpopulations selection and influence of sodium butyrate and culture medium on protein expression.

The cDNA encoding the rabies virus glycoprotein (RVGP) gene was cloned in expression plasmids under the control of the inductive metallothionein promoter. They were designed in order to bear or not a secretion signal (i) and a cDNA coding for the selection hygromycin. These vectors were transfected into S2 cells, cell populations selected and subpopulations were then obtained by reselection with hygromycin. Cell cultures were examined for kinetics of cell growth, detection of RVGP mRNA and expression of RVGP. All cell populations were shown to express the RVGP mRNA upon induction. S2MtRVGPHy cell population, transfected with one vector that contains RGPV gene and selection gene, was shown to express higher amounts of RVGP as evaluated by flow cytometry ( approximately 52%) and ELISA (0.64 microg/10(7)cells at day 7). Subpopulation selection allowed a higher RVGP expression, specially for the S2MtRVGPHy(+) (5.5 microg/10(7)cells at day 7). NaBu treatment leading to lower cell growth and higher RVGP expression allowed an even higher RVGP synthesis by S2MtRVGPHy(+) (8.4 microg/10(7)cells at day 7). SF900II medium leading to a higher S2MtRVGPHy(+)cell growth allowed a higher final RVGP synthesis in this cell culture. RVGP synthesis may be optimized by the expression/selection vectors design, cell subpopulations selection, chromatin exposure and culture medium employed.

Recombinant rabies virus glycoprotein synthesis in bioreactor by transfected Drosophila melanogaster S2 cells carrying a constitutive or an inducible promoter

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 (<0.2 microg/mL) indicating good integrity of cells in culture. Overall the RVGP productivity was of 1.5-3mg/L. Our data showed an important influence of dissolved oxygen on RVGP synthesis allowing a higher and sustained productivity by S2MtRVGP-Hy cells when cultivated with a DO of 10% air saturation. The RVGP productivity in bioreactors shown here mirrors those previously observed for T-flasks and shaker bottles and allow the preparation of the large RVGP quantities required for studies of structure and function.

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 °C and 100 rev./min and in a 3 litre stirred‐tank bioreactor at 28 °C, with increasing agitation. In small‐scale culture, S2AcRVGP cells reached a maximum cell concentration of 1.13×107 cell/ml, presented a μmax (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 2.2×107 cells/ml and μmax 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.

High-level expression of rabies virus glycoprotein with the RNA-based Semliki Forest Virus expression vector

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.

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%.

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.

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.

Effect of hypothermic temperatures on production of rabies virus glycoprotein by recombinant Drosophila melanogaster S2 cells cultured in suspension

Aiming at maximizing the production of transmembrane rabies virus glycoprotein (rRVGP), the influence of hypothermic temperature on a recombinant Drosophila melanogaster S2 cell culture in Sf-900II medium was investigated. Cell growth and rRVGP production were assessed at 4 culture temperatures in Schott flasks: 16, 20, 24 and 28 °C. The maximum specific growth rates μ(max) were, respectively: 0.009, 0.019, 0.038 and 0.035 h(-1), while the maximum rRVGP levels C(max)(rRVGP) were: 0.075, 2.973, 0.480 and 1.404 mg L(-1). The best production temperature (20 °C) was then tested in a bioreactor with control of pH and dissolved oxygen in batch and fed-batch modes. In the batch culture, μ(max) and C(max)(rRVGP) were 0.060 h(-1) and 0.149 mg L(-1) at 28 °C and 0.026 h(-1) and 0.354 mg L(-1) at 20 °C, respectively. One batch-culture experiment was carried out with adaptation of the cells by the temperature falling in steps from 20 °C to 16 °C, so that μ(max) fell from 0.023 to 0.013 h(-1), while C(max)(rRVGP) was improved to 0.567 mg L(-1). In the fed-batch mode at 20 °C, μ(max) was 0.025 h(-1) and C(max)(rRVGP) was 1.155 mg L(-1). Taken together, these results indicate that the best strategy for optimized rRVGP production is the culture at hypothermic temperature of 20 °C, when μ(max) is kept low and with feeding of limitant aminoacids.

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.