Immunogenicity studies of bivalent inactivated virions of EV71/CVA16 formulated with submicron emulsion systems

Abstract

We assessed two strategies for preparing candidate vaccines against hand, foot, and mouth disease (HFMD) caused mainly by infections of enterovirus (EV) 71 and coxsackievirus (CV) A16. We firstly design and optimize the potency of adjuvant combinations of emulsion-based delivery systems, using EV71 candidate vaccine as a model. We then perform immunogenicity studies in mice of EV71/CVA16 antigen combinations formulated with PELC/CpG. A single dose of inactivated EV71 virion (0.2 μg) emulsified in submicron particles was found (i) to induce potent antigen-specific neutralizing antibody responses and (ii) consistently to elicit broad antibody responses against EV71 neutralization epitopes. A single dose immunogenicity study of bivalent activated EV71/CVA16 virion formulated with either Alum or PELC/CpG adjuvant showed that CVA16 antigen failed to elicit CVA16 neutralizing antibody responses and did not affect EV71-specific neutralizing antibody responses. A boosting dose of emulsified EV71/CVA16 bivalent vaccine candidate was found to be necessary to achieve high seroconversion of CVA16-specific neutralizing antibody responses. The current results are important for the design and development of prophylactic vaccines against HFMD and other emerging infectious diseases.

Figure 1

EV71 antigen-specific (a) IgG, (b) VN antibody responses, and (c) seroconversion rate in BALB/c mice vaccinated with a single dose of EV71 inactivated virus vaccine. Mice (N = 8) were injected i.m. with 0.2 μg of inactivated EV71 virus alone or formulated with different emulsions: (-x-) no adjuvant, (-o-) PELC, (-□-) PELC(T), and (-∆-) PELC(A). Serum samples were collected from the vaccinated mice and the antibodies were measured by ELISA and VN. The data were presented as GMTs with standard errors of eight mice per group. The dotted horizontal line represents a VN titer of 40. The seroconversion rate is the percentage of mice achieving a postvaccination titer ≥40.

Figure 2

Antigen-specific IgG antibody responses in BALB/c mice vaccinated with a single dose of EV71 inactivated virus formulated with different adjuvant. BALB/c mice (N = 6) were i.m. vaccinated with 0.2 μg of inactivated EV71 virus alone or formulated with Alum or PELC emulsified particles. At week 8, serum samples were collected from blood and the antibodies were measured by ELISA; the reactivity of neutralization epitope VP1_211–225 or VP2_136–150 was determined by peptide ELISA. The data were presented as GMTs with standard errors of six mice per group. *P < 0.05: comparison with the group without adjuvant. ^# P < 0.05: comparison with the group of Alum adjuvant.

Figure 3

EV71-specific VN antibody responses in mice vaccinated with single dose of inactivated EV71 virus formulated with different adjuvant. BALB/c mice (N = 6) were vaccinated i.m. once with the candidate vaccine formulations: (-x-) no adjuvant, (-o-) PELC, and (-∆-) PELC/CpG. Blood samples were collected from vaccinated mice at different weeks and the antibody titers were determined by VN assays. Data are presented as mean titers with standard errors of six mice per group; the dotted horizontal line represents a VN titer of 40.

Figure 4

(a) EV71-specific and (b) CVA16-specific antibody responses in mice vaccinated with inactivated EV71/CVA16 combination vaccine. BALB/c mice (N = 6) were vaccinated i.m. once with different candidate formulations containing 0.2 μg of EV71 and 0.2 μg of CVA16. Serum samples were collected from vaccinated mice and the antibody titers were determined by immunoassays. Data are presented as geometric mean titers with standard errors of six mice per group. The dotted horizontal line represents a VN titer of 40. *P < 0.05: comparison with the groups without adjuvant at the same time point. ^# P < 0.05: comparison with the group of Alum adjuvant at the same time point.

Figure 5

CVA16-specific VN antibody responses in vaccinated BALB/c mice. Three groups of mice (N = 8) were primed i.m. with 0.2 μg of EV71 and 0.2 μg of CVA16 combination vaccine, alone or formulated either with Alum or PELC/CpG. At week 14, mice were boosted i.m. with the same vaccine formulations. Serum samples were collected from vaccinated mice and the antibody titers were determined by VN immunoassay. The dotted horizontal line represents a VN titer of 40.