Immunogenicity and protective efficacy of inactivated coxsackievirus B4 viral particles

Abstract

Coxsackievirus B4 (CVB4) is associated with a range of acute and chronic diseases such as hand, foot, and mouth disease, myocarditis, meningitis, pancreatitis, and type 1 diabetes, affecting millions of young children annually around the world. However, no vaccine is currently available for preventing CVB4 infection. Here, we report the development of inactivated viral particle vaccines for CVB4. Two types of inactivated CVB4 particles were prepared from CVB4-infected cell cultures as vaccine antigens, including F-particle (also called mature virion) consisting of VP1, VP3, VP2, and VP4 subunit proteins, and E-particle (also called empty capsid) which is made of VP1, VP3, and uncleaved VP0. Both the inactivated CVB4 F-particle and E-particle were able to potently elicit neutralizing antibodies in mice, despite slightly lower neutralizing antibody titres seen with the E-particle vaccine after the third immunization. Importantly, we demonstrated that passive transfer of either anti-F-particle or anti-E-particle sera could completely protect the recipient mice from lethal CVB4 challenge. Our study not only defines the immunogenicity and protective efficacy of inactivated CVB4 F-particle and E-particle but also reveals the central role of neutralizing antibodies in anti-CVB4 protective immunity, thus providing important information that may accelerate the development of inactivated CVB4 vaccines.

Keywords: Coxsackievirus B4; E-particle; F-particle; inactivated vaccine; neutralizing antibody; virus challenge.

Figure 1.

Sucrose gradient analysis of inactivated CVB4 viral particles. The lysates of mock – or CVB4-infected RD cells were inactivated and then sedimented on a 20% sucrose cushion. The resulting pellets were resuspended in PBS and then subjected to ultracentrifugation on 10%−50% sucrose gradient. Twenty-four gradient fractions were collected and then assayed. (A-D) SDS-PAGE and western blot analysis of the inactivated CVB4 samples. (E-F) SDS-PAGE and western blot analysis of the mock-infected control samples. Western blots were detected with anti-VP0 (B and F), anti-VP1 (C), or anti-VP3 (D) polyclonal antibodies, respectively.

Figure 2.

Characterization of purified CVB4 E-particle and F-particle. (A) SDS-PAGE and western blot analysis of purified antigens. Lane M, protein marker; lane 1, the control antigen prepared from uninfected cells; lane 2, the CVB4 E-particle preparation; lane 3, the CVB4 F-particle preparation. The detecting antibodies used in western blotting were indicated. (B) Negative stain electron microscopy of the CVB4 E-particle and F-particle preparations. Scale bar =100 nm.

Figure 3.

Both E-particle and F-particle experimental vaccines potently elicited neutralizing antibodies in mice. (A) Mouse immunization schedule. Three groups of BALB/C mice (n = 8) were injected i.p. with alum-formulated inactivated CVB4 E-particle, F particle, or the control antigen at weeks 0, 2, and 4. Serum samples were collected from individual mice at weeks 4 and 6. (B) The week-4 antisera were diluted 1:100 and analyzed for CVB4 VP1-binding activity by ELISA. Each symbol represents one mouse. (C) CVB4 VP1-binding titres of individual antisera collected at weeks 4 and 6. Each symbol represents one mouse. Sera that did not exhibit any binding activity at the lowest serum dilution (1:100) were assigned a titre of 50 for computation of geometric mean titre (GMT). GMTs ± SD for each group were shown. (D) Neutralization titres of the antisera against CVB4 strain JVB. Each symbol represents an individual mouse. Sera that did not exhibit neutralization at the lowest sera dilution (1:32) were assigned a titre of 16 for GMT computation. Neutralizing GMTs ± SD for each group were shown. p values were analyzed with t-test and indicated as follows: ns, no significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 4.

Passive transfer of anti-E-particle or anti-F-particle sera effectively protected mice from lethal CVB4 challenge. (A) Schedule of antisera transfer and virus challenge. groups of naive ICR mice (1-day-old) were injected i.p. with 30μl of the pooled anti-E-particle, anti-F-particle, or the control sera, respectively. One day later, the mice were inoculated i.p. with live CVB4 (3.15 × 10⁶ TCID₅₀). Then, the mice were monitored daily for survival and clinical signs for a period of 14 days. (B) Survival rates of the mouse groups. The numbers of mice in each group were shown in brackets. Survival curves were compared using Logrank test and statistical significance was indicated as follows: ns, no significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. (C) Clinical scores of the mouse groups. Clinical scores were graded as follows: 0, healthy; 1, lethargy and reduced mobility; 2, limb weakness; 3, limb paralysis; 4, death. (D-E) Virus loads in the limb muscle (D) and brain (E) of the challenged mice at 4 days post-infection. Three mice from each treatment groups were randomly selected and their limb muscle and brain were collected and examined for CVB4 virus loads by the TCID₅₀ assay. Each symbol represents an individual mouse. Geometric mean virus titres for each group were shown. Statistical analysis was performed using the t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.