Development a novel multiepitope DNA vaccine against human SARS coronavirus-2: an immunoinformatic designing study.

Human SARS coronavirus 2 (SARS-CoV-2) causes the current global COVID-19 pandemic. The production of an efficient vaccine against COVID-19 is under heavy investigation. In this study, we have designed a novel multiepitope DNA vaccine against SARS-CoV-2 using reverse vaccinology and DNA vaccine approaches. Applying these strategies led to reduce the time and costs of vaccine development and also improve the immune protective characteristics of the vaccine. For this purpose, epitopes of nucleocapsid, membrane glycoprotein, and ORF8 proteins of SARS-CoV-2 chose as targets for B and T-cell receptors. Accordingly, DNA sequences of selected epitopes have optimized for protein expression in the eukaryotic system. To this end, the Kozak and tissue plasminogen activator sequences were added into the epitope sequences for proper protein expression and secretion, respectively. Furthermore, interleukin-2 and beta-defensin 1 preproprotein sequences were incorporated to the designed DNA vaccine as an adjuvant. Modeling and refinement of fused protein composed of SARS-CoV-2 multiepitope antigens (fuspMA) have performed based on homology modeling of orthologous peptides, then constructed 3D model of fuspMA was more investigated during 50 ns of molecular dynamics simulation. Further bioinformatics predictions demonstrated that fuspMA is a stable protein with acceptable antigenic features and no allergenicity or toxicity characteristics. Finally, the affinity of fuspMA to the MHC I and II and TLRs molecules validated by the molecular docking procedure. In conclusion, it seems the designed multiepitope DNA vaccine could have a chance to be introduced as an efficient vaccine against COVID-19 after more in vivo evaluations.

Production, purification, and chemical stability of recombinant human interferon-γ in low oxygen tension condition: a formulation approach.

The low stability of recombinant human interferon-γ (rhIFN-γ) therapeutic protein imposes some restrictions in its medical applications. In the current study, the effect of oxygen tension on the stability of purified rhIFN-γ was investigated. The rhIFN-γ was purified (>99%) by a two-step chromatographic process. Storage vials were filled by purified formulated product under normal atmospheric oxygen and low oxygen tension conditions. At different time intervals, the amounts of rhIFN-γ covalent dimers and deamidated forms were analyzed using analytical high-performance liquid chromatography (HPLC; size exclusion and cation exchange) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) methods. To determine the biological activity of purified rhIFN-γ, an antiviral activity assay against vesicular stomatitis virus (VSV) was performed. Upon rhIFN-γ long-term storage in a low oxygen tension condition, the amounts of rhIFN-γ covalent dimers and deamidated forms and also the biological activity of rhIFN-γ changed a little. In contrast, by 9 months of storage of rhIFN-γ preparations under normal atmospheric condition, the amount of covalent dimers and deamidated forms increased with time and reached to approximately 3.5% and 11.5% of the initial amount, respectively. The antiviral specific activity of 9-month-old rhIFN-γ preparations decreased to 41% of the initial amount at normal storage condition, while no significant reduction was seen at the low oxygen tension condition. In conclusion, oxygen tension during storage could have a significant impact on rhIFN-γ stability and finally on the quality of pharmaceutical rhIFN-γ product.

Prevention of human granulocyte colony-stimulating factor protein aggregation in recombinant Pichia pastoris fed-batch fermentation using additives.

rhG-CSF (recombinant human granulocyte colony-stimulating factor) was expressed in the yeast Pichia pastoris under the control of the AOX1 (alcohol oxidase 1) promoter. The production of rhG-CSF was induced by switching from growth on glycerol to growth on methanol. In the induction phase, the methanol feed rate had a significant effect on the specific expression rate of rhG-CSF. A constant feed rate of 16 ml.h(-1).l(-1) was found to be optimal for a high specific expression rate of rhG-CSF (0.058 mg(-1).h(-1).g DCW(-1); DCW is dry cell weight). Under this condition, a maximum concentration of 300 mg/l of rhG-CSF and the expression yield of 0.6 mg of rhG-CSF/g of methanol were attained. However, the secreted rhG-CSF was shown to exist as aggregates in the culture broth, owing to hydrophobic interactions. To prevent undesirable protein aggregation, the presence of additional additives in the P. pastoris culture medium was investigated. Among seven additives tested, Tween 20, Tween 80 and betaine exhibited the best results in respect of preventing the formation of rhG-CSF protein aggregates.

Improving purification of recombinant human interferon gamma expressed in Escherichia coli; effect of removal of impurity on the process yield.

Process development and optimization studies were performed in order to improve the purification process of (rhIFN-gamma). The objective was to generate material with higher purity and quantity. An in-process control screening was developed to obtain the optimal condition for column chromatographic purification by measuring LPS, nucleic acids, rhIFN- gamma, monomer and its covalent dimers. A new resin screening method was applied to select optimal resin for each of the chromatographic columns. The resulting process used Butyl and Q-Sepharose, refolding and SP-Sepharose for purification of IFN-gamma. Effects of different process conditions such as cell lysis, removal of impurity and oxygen concentration were evaluated. Removal of impurities was evaluated by washing of inclusion bodies with 1% Triton X-100 and 3M urea and different chromatography steps. The results reveal that Triton removed about 43% of the LPS but urea had no effect on removal of nucleic acids and LPS. Further analysis show that removal of impurities by column chromatography decreases aggregation and increases the process yield. Oxygen concentration was identified as parameter that could have a significant impact on covalent dimers formation, as an unacceptable pharmaceutical form of rhIFN-gamma. On the basis of small-scale studies, optimum operating conditions were chosen and the purification process was successfully scaled-up to a pilot scale process with step yield and product quality that were better than previous reports.