Viral Protein VP1 Virus-like Particles (VLP) of CVB4 Induces Protective Immunity against Lethal Challenges with Diabetogenic E2 and Wild Type JBV Strains in Mice Model

Abstract

Several epidemiological studies demonstrated that coxsackievirus B4 (CVB4) causes viral pancreatitis and can ultimately result in type 1 diabetes mellitus (T1D). Prevention of CVB4 infection is therefore highly desirable. There is currently no vaccine or antiviral therapeutic reagent in clinical use. VLP are structurally similar to native virus particles and therefore are far better immunogens than any other subunit vaccines. Many studies have shown the potential of capsid protein VP1 on providing protective effects from different viral strains. In this study, we contributed towards the development of a CVB4 VLP-based vaccine from the total protein VP1 of the diabetogenic CVB4E2 strain and assessed whether it could induce a protective immunity against both the wild-type CVB4JBV and the diabetogenic CVB4E2 strains in mice model. Serum samples, taken from mice immunized with VLP, were assayed in vitro for their anti-CVB4 neutralizing activity and in vivo for protective activity. We show that VLP vaccine generates robust immune responses that protect mice from lethal challenges. Results demonstrate that CVB4 VP1 capsid proteins expressed in insect cells have the intrinsic capacity to assemble into non-infectious VLP, which afforded protection from CVB4 infection to mice when used as a vaccine.

Keywords: coxsackievirus B4; immunization; lethal challenge; type 1 diabetes; vaccine; viral protein VP1; virus-like particles.

Figure 1

Mice immunization and challenge schedule. Mice Group 1 were intraperitoneally (ip) immunized with a prime-boost regimen at days 0 and 15. Immunized mice Group 2 and Group 3 were immunized with a prime-boost regimen and then challenged at day 22 successively with CVB4E2 and CVB4JBV strains. Mice Group 4 and Group 5 received PBS at days 0 and 15 and challenged successively with CVB4E2 and CVB4JBV strains. Group 6 mice represent the naive control group receiving only PBS at days 0 and 15. Neutralizing antibody and cytokine titers were measured in serum collected from mice tails at days 7, 14, 21, 28, 35, and 42.

Figure 2

Characterization of the ultracentrifugation-purified VP1-VLP vaccine particles. (A) SDS-PAGE analysis of the three different fractions (Lanes 1–3) of ultracentrifugation-purified VP1-VLP. MW: molecular weight marker (10–250 kD). (B) Western blot analysis of purified VP1-VLP revealed that the VP1-VLP were recognized by an anti-enterovirus VP1 5D-8/1 clone monoclonal antibody (mAb). Lanes 1–3: fractions 1–3 of ultracentrifugation-purified VP1-VLP stock produced by Bac-VP1. (C) Immunofluorescence (IF) microscopy photo analysis of High Five infected cells at 2 days post infection revealed the presence of VP1-VLP particles in the cytoplasm of cells stained (fluorescence color) by the 5-D8/1 mAb with the goat anti-mouse coupled to AlexaFluor 488. Cell nuclei were stained with DAPI (blue color). Zoom 3X STED. (D) Transmission electron micrographs (TEM) of ultracentrifugation-purified VLPs. Scale bars, 20 nm.

Figure 3

Production and titers of specific neutralizing antibodies in mice vaccinated with VP1-VLP. VP1-VLP anti-E2 represents mice group vaccinated with a prime-boost regimen at days 0 and 15, then challenged at day 22 with CVB4E2 strain. VP1-VLP anti-JBV represents mice group vaccinated with a prime-boost regimen at days 0 and 15, then challenged at day 22 with CVB4JBV strain. Titers of neutralizing antibodies produced in mice sera were measured at different times post immunization. Naïve control mice represent the negative control.

Figure 4

Antibody response. Serum IgG recognizing VP1-VLP in immunized mice. Titers of total IgG in the three immunized groups of mice. Mice (n = 5) receiving CVB4E2 VP1-VLP vaccine were immunized intraperitoneally by prime-boost regimen at days 0 and 15 with 0.5 μg of VP1-VLP. Mice were then challenged at day 22 with CVB4E2 or CVB4JBV pathogenic strains. PBS naïve mice group represents the control group. The results represent the mean of three replicate experiments.

Figure 5

Antibody subtyping. Isotypes of the specific IgG antibodies in serum of the three VP1-VLP immunized groups. Sera from each mouse was obtained at day 35 post the first immunization and tested by ELISA method. PBS naïve mice group represents the control group.

Figure 6

Measuring of IFN-γ by ELISA in the sera of the three different immunized groups. Sera of the mice group were collected at different days post the first immunization (as described in Section 2). The concentrations of IFN-γ were evaluated and measured by ELISA method. PBS naïve mice group represents the control group.

Figure 7

Body weight changes observed in all mice groups at different days post lethal challenge with pathogenic E2 and JBV strains. Mice groups not immunized and challenged by pathogenic strains showed substantial decrease in body weight percentage. PBS naïve mice group represents the control group.

Figure 8

Survival percentage of all mice groups at different days after lethal challenge with pathogenic E2 and JBV strains. Mice groups not immunized and challenged by pathogenic strains showed the most elevated percentage of mortality. PBS naïve mice group represents the control group.

Figure 9

Viral load in the pancreas tissues of naive not challenged, naive challenged, and VP1-VLP vaccinated mice groups at day 20 after lethal CVB4E2 or CVB4JBV challenges (42 days post first immunization). Viral load (TCID₅₀/mg) was determined in HeLa cells using Reed–Muench method [21]. VP1-VLP vaccinated and challenged mice groups showed a 10-fold lower viral load than the naive challenged mice groups.