Alexandre Gonçalves de Rezende

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.

A multivariate calibration procedure for UV/VIS spectrometric monitoring of BHK-21 cell metabolism and growth

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.

Adaptation to serum-free culture of HEK 293T and Huh7.0 cells.

Background The fetal bovine serum (FBS) is a component of higher value added to the medium, which may cause difficulty in the process of recovery and purification of bioproduct. Considering all these reasons, it is essential to develop a simple cell adaptation process for the culture of cells in serum-free, animal component-free, or protein-free medium conditions [1,2]. The adaptation of cellular lineages to culture FBS or animal protein free medium can allow optimization of cell growth and expression of heterologous genes, facilitating recovery of recombinant proteins expressed in these cells. Mammalian cell growth can be adapted to serum-free media through progressive reductions of serum concentrations [3]. This gradual reduction over time increases the probability of successful adaptation to low-serum or serum-free mediaby allowing the self-adjustment of the cells to the environment, with the time required depending on the cell line and the composition of the media [4]. Our aim was to establish and to study mammalian cell lines adapted to serum free medium for the expression of recombinant proteins. Thus, we assessed cell growth, nutrient consumption and metabolite production kinetics in the cell culture media tested.

Evaluation of Lawsonia inermis (henna) pigment as a carrier of antigens inoculated by transcutaneous immunization pigment in mice [Abstract in English]

AIMS: To evaluate the role of pigment Lawsonia inermis (henna) as a carrier of antigens in transcutaneous vaccination in mice. METHODS: Mice were immunized transcutaneously into the skin of the abdomen and the ear’s skin with crude antigen of Paracoccidioides brasiliensis and with bovine serum albumin at concentrations of 1, 10 and 50 mg/mL, three times at one week intervals in the presence or absence of pigments L. inermis . One week after last immunization, the animals were evaluated for the presence of serum antibodies, interleucin-4 and interferon-gamma cytokines, and nitric oxide in culture supernatants. RESULTS: No specific antibodies were detected to P.brasiliensis or bovine serum albumin antigens. There were no significant differences in the production of nitric oxide, interleucin-4 and interferon-gamma in supernatants of cell culture. CONCLUSIONS: Transcutaneous immunization with crude antigen of P. brasiliensis or bovine serum albumin suspended in pigment of L. inermis produced no detectable antigenic response in mice.Aims: To evaluate the role of pigment Lawsonia inermis (henna) as a carrier of antigens in transcutaneous vaccination in mice. Methods: Mice were immunized transcutaneously into the skin of the abdomen and the ear’s skin with crude antigen of Paracoccidioides brasiliensis and with bovine serum albumin at concentrations of 1, 10 and 50 mg/mL, three times at one week intervals in the presence or absence of pigments L. inermis. One week after last immunization, the animals were evaluated for the presence of serum antibodies, interleucin-4 and interferon-γ cytokines, and nitric oxide in culture supernatants. Results: No specific antibodies were detected to P. brasiliensis or bovine serum albumin antigens. There were no significant differences in the production of nitric oxide, interleucin-4 and interferon- γ in supernatants of cell culture. Conclusions: Transcutaneous immunization with crude antigen of P. brasiliensis or bovine serum albumin suspended in pigment of L. inermis produced no detectable antigenic response in mice.

Semliki forest virus as a vector: pros and cons for its use in biopharmaceuticals production

The number of biopharmaceuticals for medical and veterinarian use produced in mammalian cells is increasing year after year. All of them are obtained by stable recombinant cell lines. However, it is recognized that transient gene expression produces high level expression in a short time. In that sense, viral vectors have been extensively used for producing recombinant proteins on lab-scale. Among them, Semliki Forest virus is commonly employed for this purpose. This review discusses the main aspects related to the use of Semliki Forest virus technology as well as its advantages and drawbacks which limit currently its utilization in biopharmaceutical industry on large-scale.