Intein-mediated backbone cyclization of VP1 protein enhanced protection of CVB3-induced viral myocarditis

Abstract

CVB3 is a common human pathogen to be highly lethal to newborns and causes viral myocarditis and pancreatitis in adults. However, there is no vaccine available for clinical use. CVB3 capsid protein VP1 is an immunodominant structural protein, containing several B- and T-cell epitopes. However, immunization of mice with VP1 protein is ineffective. Cyclization of peptide is commonly used to improve their in vivo stability and biological activity. Here, we designed and synthesizd cyclic VP1 protein by using engineered split Rma DnaB intein and the cyclization efficiency was 100% in E. coli. As a result, the cyclic VP1 was significantly more stable against irreversible aggregation upon heating and against carboxypeptidase in vitro and the degradation rate was more slowly in vivo. Compared with linear VP1, immunization mice with circular VP1 significantly increased CVB3-specific serum IgG level and augmented CVB3-specific cellular immune responses, consequently afforded better protection against CVB3-induced viral myocarditis. The cyclic VP1 may be a novel candidate protein vaccine for preventing CVB3 infection and similar approaches could be employed to a variety of protein vaccines to enhance their protection effect.

Figure 1. Protein cyclization in vivo using intramolecular trans-splicing activity of Rma DnaB intein.

(a) VP1 is sandwiched between the C-terminal fragment (RB_C) and N-terminal fragment (RB_N) of Rma DnaB intein. Splicing mediates the ligation of the N and C termini of VP1 through a native peptide bond. (b) Schematic representation of expression vector pERB_C-RB_N and pERB_C-VP1-RB_N. (c) Amino acid sequence of the fusion protein RBc-VP1-RB_N. The C-terminal 43-residue segment (RB_C) and the N-terminal 104-residue segment (RB_N) of the Rma DnaB intein are enclosed with a rectangle. The linker sequence of SA and H₆-GG is bold and the thrombin-specific cleavage sites marked with black arrow. (d) Models of the 3-D structure of linear (left) and circular form of VP1. The models were created based on the coordinates of the PDB code 1COV. N and C indicate N- and C-termini in the linear form.

Figure 2. Protein expression, purification and characterization.

(a) SDS-PAGE and Western blotting analysis of VP1 protein expression. Protein bands were visualized by Coomassie Blue staining (lane 1, 2, 4, 5) or by Western blotting (lane 3). Lane M, protein marker; Lane 1, total cellular proteins of E. coli before IPTG-induced expression; lane 2, total cellular proteins of E. coli after IPTG-induced expression at 37 for 4 h; lane 3, protein bands were visualized by Western blotting using anti-VP1 antibody; lane 4, purified cyclic VP1 (C-VP1); lane 5, purified linear VP1 (L-VP1). (b) Diagram illustrating the proteolytic treatment on C-VP1 by thrombin and time course of the digestion. 0 h: before addition of thrombin; 2 h, 4 h, O/N: time after addition of thrombin. The positions of the circular and linear form of VP1 are indicated. (c) Proteolysis resistance of C-VP1. Lane 1, L-VP1 and C-VP1 mix proteins before addition of carboxypeptidase Y; Lane 2, incubation of L-VP1 and C-VP1 mix proteins with carboxypeptidase Y at 25 °C for 10 h. (d) Effect of heat treatment at different temperatures on C-VP1 and L-VP1. C-VP1 stands for splicing product of cyclic VP1 protein, I_N stands for N-terminal fragment of Rma DnaB, L-VP1 stands for linear VP1 protein, L′-VP1 stands for cyclic VP1 digestion by thrombin. (e) CD spectra of L-VP1 and C-VP1 protein.

Figure 3. CVB3-specific systermic responses were elicited by immunization with groups of C-VP1, L-VP1 or PBS administration.

(a) CVB3-specific IgG levels were measured by ELISA 2 weeks after the last immunization, (b) antibody avidity. (c) CVB3-specific T cell proliferation was assessed by Roche BrdU-Kit after stimulation with 20 ug/ml VP1_237–249 peptide in the culture of 20 U/ml IL-2 for 72 h. (d) CVB3-specific cytokine-secreting lymphocytes were quantified by ELISPOT assay in response to VP1_237–249 peptide. (e) CVB3-specific CTL activity of splenic cells was evaluated by lactate dehydrogenase assays using pcDNA3.1-VP1 stable transfected autologous SP2/0 cells as target cells. Individual experiments were repeated 3 times with similar results, *P < 0.05, **P < 0.01. ND, not detected.

Figure 4. Validation of C-VP1 stability in vivo and spleen DCs maturation.

Mice were intramuscularly immunized with 25 ug of purified C-VP1or L-VP1 protein, four days later (a) muscles were collected and subjected to Immunohistochemical analysis to detect protein stability in vivo and (b) spleens were collected and stained with DC maturation markers CD80, CD86 and MHCII to determine by flow cytometry, **P < 0.01, ***P < 0.001.

Figure 5. Enhanced resistance to CVB3-induced acute myocarditis by immunization with C-VP1.

Seven days after 3LD50 CVB3 challenge. (a) Body weight loss. (b) Cardiac function was detected by echocardiography using a 2-dimensional guided M-mode ultrasound system for each group. (c) The representative heart HE-stained sections were shown for each group (magnification: 20 X). (d) Myocardial histopathological scores. (e) The survival rate of mice was observed for 28 days following a lethal dose of CVB3 (5LD50) infection. (f) Myocardial viral load. Individual experiment was repeated 3 times with 8 mice per group. **P < 0.01.