Identification of a neutralizing linear epitope within the VP1 protein of coxsackievirus A10.

BACKGROUND: Coxsackievirus A10 (CV-A10) is a leading cause of hand, foot, and mouth disease (HFMD). It is necessary to identify neutralizing epitopes to investigate and develop an epitope-based vaccine against CV-A10. The viral protein VP1 is the immunodominant capsid protein and contains the critical neutralizing epitope. However, neutralizing epitopes within VP1 protein of CV-A10 have not been well characterized. METHODS: Bioinformatics techniques were applied to predict linear epitopes on the CV-A10 VP1 protein. The advanced structural features of epitopes were analyzed by three-dimensional (3D) modeling. The anticipated epitope peptides were synthesized and used to immunize mice as antigens. ELISA and micro-neutralization assay were used to determine the specific IgG antibody and neutralizing antibody titers. The protective efficacy of the epitope peptides in vivo was evaluated using a passive immunization/challenge assay. RESULTS: Three linear epitopes (EP3, EP4, and EP5) were predicted on CV-A10 VP1, all spatially exposed on the capsid surface, and exhibited adequate immunogenicity. However, only EP4, corresponding to residues 162-176 of VP1, demonstrated potent neutralization against CV-A10. To determine the neutralizing capacity of EP4 further, EP4 double-peptide was synthesized and injected into mice. The mean neutralizing antibody titer of the anti-EP4 double-peptide sera was 1:50.79, which provided 40% protection against lethal infection with CV-A10 in neonatal mice. In addition, sequence and advanced structural analysis revealed that EP4 was highly conserved among representative strains of CV-A10 and localized in the EF loop region of VP1, like EV-A71 SP55 or CV-A16 PEP55. CONCLUSIONS: These data demonstrate that EP4 is a specific linear neutralizing epitope on CV-A10 VP1. Its protective efficacy can be enhanced by increasing its copy number, which will be the foundation for developing a CV-A10 epitope-based vaccine.

Construction and characterization of an infectious cDNA clone of coxsackievirus A 10.

BACKGROUND: Coxsackievirus A10 (CA10) constitutes one of the four major pathogens causing hand, foot and mouth disease in infants. Infectious clones are of great importance for studying viral gene functions and pathogenic mechanism. However, there is no report on the construction of CA10 infectious clones. METHODS: The whole genome of CA10 derived from a clinical isolate was amplified into two fragments and ligated into a linearized plasmid vector in one step by In-Fusion Cloning. The obtained CA10 cDNA clones and plasmids encoding T7 RNA polymerase were co-transfected into 293 T cells to rescue CA10 virus. The rescued virus was identified by SDS-PAGE, Western blotting and transmission electron microscopic. One-day-old ICR mice were intracerebrally inoculated with the CA10 virus and clinical symptoms were observed. Multiple tissues of moribund mice were harvested for analysis of pathogenic changes and viral distribution by using H&E staining, real-time PCR and immunohistochemical staining. RESULTS: CA10 viruses were rescued from the constructed cDNA clone and reached a maximum titer of 108.125TCID50/mL after one generation in RD cells. The virus exhibited similar physical and chemical properties to those of the parental virus. It also showed high virulence and the ability to induce death of neonatal ICR mice. Severe necrotizing myositis, intestinal villus interstitial edema and severe alveolar shrinkage were observed in infected mice. The viral antigen and the maximum amount of viral RNA were detected in limb skeletal muscles, which suggested that the limb skeletal muscles were the most likely site of viral replication. CONCLUSION: Infectious clones of CA10 were successfully constructed for the first time, which will facilitate the establishment of standardized neonatal mouse models infected with CA10 for the evaluation of vaccines and antiviral drugs, as well as preservation and sharing of model strains.

An enterovirus A71 virus-like particle with replaced loops confers partial cross-protection in mice.

Enterovirus A71 (EV-A71), coxsackievirus A16 (CV-A16), and CV-A10 belong to the main prevailing types causing hand-foot-and-mouth disease. Since EV-A71 monovalent vaccine does not confer cross-protection, developing a multivalent vaccine is essential. In this study, a trivalent chimeric virus-like particle of EV-A71 (EV-A71-VLPCHI3) was constructed based on EV-A71-VLP backbone by replacing the corresponding surface loops with CV-A16 VP1 G-H, CV-A10 VP1 B-C and E-F loops, which are critical for immunogenic neutralization. The baculovirus-insect cell expression system was employed for EV-A71-VLPCHI3 production. EV-A71-VLPCHI3 was purified by sucrose density gradient and observed by transmission electron microscopy. The immunogenicity and protective efficacy of EV-A71-VLPCHI3 were evaluated in mice. Our results revealed that EV-A71-VLPCHI3 had a similar morphology to inactivated EV-A71 particles and could induce specific IgG antibodies against EV-A71, CV-A16 and CV-A10 in mice. More importantly, EV-A71-VLPCHI3 enhanced cross-reactive protection against CV-A16 and CV-A10, by 20 % and 40 %, compared to inactivated EV-A71 counterparts, respectively. In conclusion, the successful construction of EV-A71-VLPCHI3 suggested that loop-dependent heterologous protection could be transferred by loops replacement on the surface of viral capsid. This strategy may also supplement the development of multivalent vaccines against other infectious viral diseases.

Coxsackievirus B5 virus-like particle vaccine exhibits greater immunogenicity and immunoprotection than its inactivated counterpart in mice.

Coxsackievirus B group 5 (CVB5) represents one of the major pathogens that cause diseases such as hand, foot and mouth disease (HFMD) and aseptic meningitis et al. Currently, no specific drugs and vaccines are available, and a safe and effective CVB5 vaccine is of great value for control of the diseases. In this study, CVB5 P1 precursor and 3CD protease were co-expressed in Sf9 cells by using a baculovirus expression system. The P1 was processed by 3CD and self-assembled into CVB5 virus-like particles (VLPs). VP1 and VP3 capsid proteins of CVB5 could be detected by SDS-PAGE and Western blotting. Transmission electron microscopy revealed that the CVB5 VLPs were spherical particles with a diameter of about 30 nm, mimicking wild-type CVB5 virus. Our study showed that the total IgG and neutralizing antibodies induced by CVB5 VLPs were higher than those induced by inactivated vaccine. More importantly, the CVB5 VLPs conferred full protection to the CVB5-challenged suckling mice via passive immunity while protection efficiency of the inactivated vaccine was only 80%. The CVB5 VLPs vaccine could protect the limb muscles, brain, and heart tissues of suckling mice from CVB5-induced damage. These results demonstrated that the CVB5 VLPs vaccine possessed stronger immunogenicity and provided more robust immunoprotection than the inactivated CVB5 vaccine, suggesting that the CVB5 VLPs promise to be a CVB5 vaccine candidate in future.