Recovering Immunogenic Orthohantavirus puumalaense N Protein from Pellets of Recombinant Escherichia coli

Abstract

(1) Background: Hemorrhagic fever with renal syndrome (HFRS) remains a prevalent zoonosis in Eurasia. Orthohantavirus puumalaense (PUUV), carried by bank voles (Myodes glareolus), is the principal zoonotic pathogen of HFRS in this region. Despite ongoing efforts to develop effective drugs and vaccines against PUUV, this challenge remains. (2) Aim: In this study, we aimed to express a large quantity of the PUUV recombinant N (rN) protein using E. coli. We also sought to develop a protocol for extracting the rN protein from pellets, solubilizing, and refolding it to restore its native form. This protocol is crucial for producing a large quantity of rN protein to develop vaccines and diagnostic tools for HFRS. (3) Methods; PUUV S segment open reading frame (ORF) coding for N protein was synthesized and cloned into the plasmid vector pET-28 (A+). The ORF was transformed, expressed and induced in BL21(DE3) pLysS E. coli strain. Subsequently, rN protein was purified using immobilized metal affinity and ion chromatography. Immune reactivity of rN protein was tested by employing in house and commercial VektoHanta-IgG kit ELISA methods (both in vitro and in vivo). (4) Results: The best conditions for scaling up the expression of the PUUV rN protein were an incubation temperature of 20 °C during a 20 h incubation period, followed by induction with 0.5 mM IPTG. The most significant protein yield was achieved when the pellets were incubated in denaturing buffer with 8M urea. The highest yield of refolded proteins was attained using non-denaturing buffer (50 mM Tris-HCl) supplemented with arginine. A final 50 μL of PUUV rN protein solution with a concentration of 7 mg/mL was recovered from 1 L of culture. The rN protein elicited an antibody response in vivo and reacted with serum taken from patients with HFRS by ELISA in vitro. (5) Conclusion: Therefore, the orthohantavirus N protein's ability to elicit immune response in vivo suggests that it can be used to develop vaccines against PUUV after conducting in vitro and in vivo studies to ascertain neutralising antibodies.

Keywords: N protein; Orthohantavirus puumalaense; hemorrhagic fever with renal syndrome; immunization; immunogenicity; vaccine.

Figure 1

Immunogenicity assessment of recombinant N protein in vivo schematic experiments. Serum samples were collected from mice. Then, mice were inoculated subcutaneously with N protein (50 µg and 100 µg in Freunds adjuvant) at day 0. After one week (at day 7) post immunization serum, samples were collected and mice were again given a booster injection with the same dose of N protein mixed with PBS. Two weeks later (at day 14), serum samples were collected and animals were euthanized according to the ethical international principles. All collected serum samples were kept at −80 °C until analysis.

Figure 2

SDS-PAGE analyses of recombinant N protein expressions in E. coli (BL21 (DES) pLysS) strain using different culture conditions. Supernatant and pellet obtained after centrifugation were analyzed by SDS-PAGE using 12% gel. Two fractions obtained are (A)—supernatant analyzed by SDS-PAGE; (B)—pellet analysed by SDS-PAGE and showing the recombinant N protein (approximately 50 kDa). The best expression condition marked with a black star was selected for large expression of rN protein.

Figure 3

Released protein from pellets. Buffer 4, effect of denaturing (8 M urea), Buffer 5, mild denaturing (2 M urea and 6 M propanol) and Buffer 6, non-denaturing (5% (v/v) DMSO) solubilizing agents in Tris-HCl buffer (pH 8.0) on released proteins for 24 h. All experiments were carried out in triplicates. The released N protein concentration was measured at 280. The results are presented as the mean and standard deviations. Blue asterisks represent statistical significance between Buffer 4 and 6, and pink is between Buffer 4 and 5, p < 0.05.

Figure 4

Recovery of denatured PUUV N protein using various dialysis buffers. Buffer 7 and 8 contain 150 mM NaCl and 5 mM EDTA, and in addition, Buffer 8 was supplemented with 0.3 M glycerol, whilst Buffer 9 had only 0.5 M arginine. All dialysis agents were dissolved in 50 mMTris-HCl buffer at pH 8.0. All experiments were carried out in triplicates. The released N protein concentration was determined spectrophotometrically at 280 nm using DeNovix DS11. The results are presented as the mean and standard deviations. For all statistical analyses, one-way ANOVAs with multiple comparisons were performed using log-transformed data. All data were significant with ** p < 0.005 and **** p < 0.0001.

Figure 5

Immune reactivity of serum from HFRS patients. Shows the results of ELISA using N protein as an antigen coated on the plate and ELISA results using commercial kit (VektoHanta IgG kit). Twenty-seven (27) HFRS serum and twenty-seven control serum samples were tested for the presence of IgG antibodies. All experiments were carried out in triplicates. The results are presented as the mean and standard deviations of OD450 sample values. For all statistical analyses, one-way ANOVAs with multiple comparisons were performed using log-transformed data. All data were significant with **** p < 0.0001.

Figure 6

Reactivity of serum from HFRS patients and mice immunized with purified recombinant N Protein. (A) Immunoreactivity of serum from mice immunized with N protein using N protein as an antigen coated on the plate. Control 1 represents serum collected from 10 mice before immunization. Control 2 refers to serum collected from two (2) unimmunized mice after 1 week of the experiment, whilst Control 3 implies serum collected from 2 mice after two weeks of immunization. Additionally, 50 µg indicates the dose of N protein used to immunize four (4) mice and 100 µg is the dose of N protein used to immunize other four (4) mice; serum was taken after 1 week. 50 µg is the dose of N protein used to immunize four (4) mice and 100 µg indicates the dose of N protein used to immunize other four (4) mice; at the same time serum was taken after 2 weeks. (B) Reactivity of serum from immunized mice using VektoHanta-IgG kit, conducted following the manufacturer’s recommendations (with explanations same as Figure (A)). Positive control on Figure (B) represents the control provided in VektoHanta-IgG kit and was only used with the kit. For all statistical analyses, one-way ANOVAs with multiple comparisons were performed using log-transformed data. All data were significant with * p < 0.05.