Engineering a New IFN-ApoA-I Fusion Protein with Low Toxicity and Prolonged Action

Abstract

Recombinant human interferon alpha-2b (rIFN) is widely used in antiviral and anticancer immunotherapy. However, the high efficiency of interferon therapy is accompanied by a number of side effects; this problem requires the design of a new class of interferon molecules with reduced cytotoxicity. In this work, IFN was modified via genetic engineering methods by merging it with the blood plasma protein apolipoprotein A-I in order to reduce acute toxicity and improve the pharmacokinetics of IFN. The chimeric protein was obtained via biosynthesis in the yeast P. pastoris. The yield of ryIFN-ApoA-I protein when cultivated on a shaker in flasks was 30 mg/L; protein purification was carried out using reverse-phase chromatography to a purity of 95-97%. The chimeric protein demonstrated complete preservation of the biological activity of IFN in the model of vesicular stomatitis virus and SARS-CoV-2. In addition, the chimeric form had reduced cytotoxicity towards Vero cells and increased cell viability under viral load conditions compared with commercial IFN-a2b preparations. Analysis of the pharmacokinetic profile of ryIFN-ApoA-I after a single subcutaneous injection in mice showed a 1.8-fold increased half-life of the chimeric protein compared with ryIFN.

Keywords: Pichia pastoris; SARS-CoV-2; Tween 20; antiviral activity; apolipoprotein A-I (ApoA-I); apoptosis; cytotoxicity; fusion protein; prolonged half-life; yeast-derived IFNα2b (ryIFN).

Figure 1

Biosynthesis of the genetically engineered protein IFN-ApoA-I. I. Genetic map of pPICZα-A/IFN-link-ApoA-I recombinant plasmid (a) and representation a hypothetical tertiary structure of the chimeric protein ryIFN-ApoA-I (b). II. SDS-PAGE analysis of TCA precipitated proteins from culture supernatants of P. pastoris clones expressing ryIFN (a) and ryIFN-ApoA-I (b) when cultured in a 96-deep-square-well plate. Lane M—standard protein molecular weight marker (14–116 kDa) (Thermo Fisher Scientific); lanes 1–9—TCA precipitated proteins from culture supernatants of nine clones after 96 h induction with 1.0% (v/v) methanol. III. Western blot analysis of proteins from the supernatant of the P. pastoris culture using clone #6. Lane M—the pre-stained standard molecular weight marker (15–250 kDa) (Bio-Rad). Lane 1—ryIFN-ApoA-I expressed by P. pastoris. IV. SDS-PAGE analysis of TCA-precipitated proteins from supernatants of yeast cultures after 4 days of fermentation in conical baffled flasks in the presence or absence of 0.2% v/v Tween 20. Proteins in the samples of supernatant were concentrated 15 times. Lane M—standard molecular weight marker (10–200 kDa); lanes 1,2—proteins from the supernatants of yeast cultures carrying the plasmid pPICZαA/IFN growing in the absence (1) or presence (2) of 0.2% v/v Tween 20; lanes 3,4—proteins from the supernatants of yeast cultures carrying the plasmid pPICZα-A/IFN-link-ApoA-I growing in the absence (3) or presence (4) of 0.2% v/v Tween 20. V. SDS-PAGE analysis of purified ryIFN (1) and ryIFN-ApoA-I (2). Lane M—standard molecular weight marker (Sib Enzyme) (10–200 kDa); VI. SDS-PAGE analysis of purified ryIFN (a) and ryIFN-ApoA-I (b) under reducing (1) and non-reducing (2) conditions. Lane M—standard molecular weight marker (10–200 kDa) (Sib Enzyme).

Figure 2

Apoptotic and necrotic patterns of the death of Vero cells infected with SARS-CoV-2. Zoomed-in image of each panel. (A) Control samples and virus titers of 5 × 10⁶, 5 × 10⁴, and 5 × 10 CPE/mL. (B) The fluorescence signals of Hoechst 33258 and PI in the infected Vero cells were counted 5 days post-infection using 5 × 10⁶ CPE/mL SARS-CoV-2. Zoomed-in image of “Altevir”-preserved monolayer.

Figure 3

Cytotoxic effect of recombinant IFN-α2b on Vero cells. On the left is the determination of the IC50s for the obtained recombinant interferons (ryIFN and ryIFN-ApoA-I) and commercial preparations of IFN-α2b (“Altevir” and “Lifeferon”). On the right are representative images obtained using a live imaging system demonstrating cell monolayer disruption in the presence of interferon preparations containing non-ionic detergents (“Altevir” containing Tween 80 and ryIFN-ApoA-I containing Tween 20) and ryIFN without Tween 20 (40×).

Figure 4

Pharmacokinetic curves of the ryIFN and ryIFN-ApoA-I concentrations in blood plasma after a single subcutaneous injection of them into male CD-1 mice at doses equivalent to their equal molar doses, i.e., 10 μg/kg and 25 μg/kg, respectively. Numerical data are presented as mean ± standard deviation (n = 5).