An inactivated hand-foot-and-mouth disease vaccine using the enterovirus 71 (C4a) strain isolated from a Korean patient induces a strong immunogenic response in mice

<div><p>Enterovirus 71 (EV71) is a major causative agent of hand-foot-and-mouth disease (HFMD) frequently occurring in children. HFMD induced by EV71 can cause serious health problems and has been reported worldwide, particularly in the Asia-Pacific region. In this study, we assessed the immunogenicity of a formalin-inactivated HFMD vaccine using an EV71 strain (FI-EV71 C4a) isolated from a Korean patient. The vaccine candidate was evaluated in mice to determine the vaccination doses and vaccine schedules. BALB/c mice were intramuscularly administered 5, 10, or 20 μg FI-EV71 vaccine, followed by a booster 2 weeks later. EV71-specific antibodies and neutralizing antibodies were induced and maintained until the end of the experimental period in all vaccinated groups. To determine the effectiveness of adjuvant for the EV71 vaccine, three adjuvants, i.e., aluminium hydroxide gel, monophosphoryl lipid A, and polyinosinic-polycytidylic acid, were administered separately with the FI-EV71 vaccine to mice via the intramuscular route. Mice administered the FI-EV71 vaccine formulated with all three adjuvants induced a significantly increased antibody response compared with that of the single adjuvant groups. The vaccinated group with triple adjuvants exhibited more rapid induction of EV71-specific and neutralizing antibodies than the other groups. These results suggested that the role of adjuvant in inactivated vaccine was important for eliciting effective immune responses against EV71. In conclusion, our results showed that FI-EV71 was a potential candidate vaccine for prevention of EV71 infection.</p></div

In Situ Synthesized La_0.6Sr_0.4Co_0.2Fe_0.8O_3−δ–Gd_0.1Ce_0.9O_1.95 Nanocomposite Cathodes via a Modified Sol–Gel Process for Intermediate Temperature Solid Oxide Fuel Cells

Composite cathodes comprising nanoscale powders are expected to impart with high specific surface area and triple phase boundary (TPB) density, which will lead to better performance. However, uniformly mixing nanosized heterophase powders remains a challenge due to their high surface energy and thus ease with which they agglomerate into their individual phases during the mixing and sintering processes. In this study, we successfully synthesized La_0.6Sr_0.4Co_0.2Fe_0.8O_3−δ (LSCF)–Gd_0.1Ce_0.9O_1.95 (GDC) composite cathode nanoscale powders via an in situ sol–gel process. High-angle annular dark field scanning transmission electron microscopy analysis of in situ prepared LSCF–GDC composite powders revealed that both the LSCF and GDC phases were uniformly distributed with a particle size of ∼90 nm without cation intermixing. The in situ LSCF–GDC cathode sintered on a GDC electrolyte showed a low polarization resistance of 0.044 Ω cm² at 750 °C. The active TPB density and the specific two phase (LSCF/pore) boundary area of the in situ LSCF–GDC cathode were quantified via a 3D reconstruction technique, resulting in 12.7 μm^–2 and 2.9 μm^–1, respectively. These values are significantly higher as compared to reported values of other LSCF–GDC cathodes, demonstrating highly well-distributed LSCF and GDC at the nanoscale. A solid oxide fuel cell employing the in situ LSCF–GDC cathode yielded excellent power output of ∼1.2 W cm^–2 at 750 °C and high stability up to 500 h

EV71-specific immune responses in serum samples from mice vaccinated with the formalin-inactivated EV71 vaccine combined with diverse adjuvants.

<p>Mice were administered the inactivated EV71 vaccine mixed with 500 μg for alum, 2 μg for MPLA, and 10 μg for poly I:C adjuvant. (A) The lgG antibody titer against EV71 was measured by ELISA at 4, 8, 12 and 16 weeks. (B) IgG isotype analysis of EV71-specific IgGs in sera of mice at 8 weeks postvaccination. The IgG isotype graph shows means + SEMs. ***<i>p</i> < 0.0001 using one-way ANOVA with Tukey’s post-hoc test.</p

EV71-specific humoral immune responses induced by a formalin-inactivated EV71 vaccine in mice.

<p>Mice were administered inactivated EV71 vaccine mixed with 500 μg for alum, 2 μg for MPLA, and 10 μg for poly I:C adjuvant or PBS as a control. (A) Titers of total IgG antibodies against EV71 viral particles were determined by ELISAs. (B) The levels of neutralization antibodies against EV71 were measured by endpoint dilution of serum in neutralization assays.</p

EV71-specific cell mediated immune responses induced by a formalin-inactivated EV71 vaccine in mice.

<p>Mice were administered inactivated EV71 vaccine mixed with 500 μg for alum, 2 μg for MPLA, and 10 μg for poly I:C adjuvant or PBS as a control. Splenocytes from vaccinated mice were isolated and stimulated with the EV71 vaccine. The supernatants of splenocytes were harvested at 48 h after stimulation and analyzed for cytokines using a multiplex system. Graphs show means + SEMs. *<i>p</i> < 0.01, using one-way ANOVA with Tukey’s post-hoc test.</p

EV71-specific cell mediated immune responses induced by the formalin-inactivated EV71 vaccine with diverse adjuvants.

<p>Splenocytes from vaccinated mice were isolated and stimulated with the EV71 vaccine strain. Supernatants of splenocytes were harvested at 48 h after stimulation and analyzed for cytokine levels using a multiplex system. Graphs show means + SEMs. *<i>p</i> < 0.01, using one-way ANOVA with Tukey’s post-hoc test.</p

Virus titers in the lungs of vaccinated mice.

<p>All vaccinated mice were intranasally (i.n.) challenged with 30 µl 10⁵ TCID₅₀/ml of the CA/04/09 virus 2 weeks after the last immunization. Lung tissue samples were obtained at 2, 4, 6, and 8 days of virus challenge. Virus titers were measured in 11-day-old embryonated chicken eggs expressed as log₁₀ EID₅₀/g. The limit of virus detection was set to 0.7 log₁₀ EID₅₀/g.</p>*<p>Standard deviation titers.</p

Hemagglutinin inhibition (HI) titers against the pandemic (H1N1) 2009 or Korean swine H1N1 virus in mini-pigs vaccinated with two doses of a-CAN01/04 or RgCA/04/09xPR8.

<p>Groups of two eight-week-old specific pathogen-free mini-pigs were vaccinated intramuscularly with one or two doses each of inactivated vaccines containing 7.5 µl/dose of HA with 2% of aluminum hydroxide adjuvant, administered 2 weeks apart. Sera were collected from recipients after 2 weeks the last vaccine was administered and mean antibody titers against CAN01/04 or CA/04/09 virus were determined by HI assays expressed as the reciprocal of the highest dilution of serum that inhibits 8 HA units of virus (e.g., as 80 versus 1∶80) with <20 HI units as the limit of detection. Data are mean ± standard deviation titers.</p

Gross pathological examination of lung tissue samples from infected and contact mini-pigs after challenge with the CA/04/09 virus.

<p>Lungs of 2-dose vaccinated [7.5 µg HA of either a-CAN01/04 (a) or RgCA/04/09xPR8 (b)] and subsequently challenged mini-pigs, including naïve contact hosts, were harvested at 5 dpi to examine gross tissue morphological features after infection with the pandemic (H1N1) 2009 virus.</p

Nasal excretion of CA/04/09 in vaccinated ferrets.

<p>Ferrets administered twice with 7.5 µl/dose HA of a-Brisbane/59/07, a-CAN01/04 or RgCA/04/09xPR8 were experimentally instillated i.n. with 10⁵ TCID₅₀ CA/04/09 virus challenge in a 1.0 ml volume. Aerosol transmission of the test virus was monitored by adding seronegative contacts in an isolator with 5 cm perforated separation barrier. Nasal wash specimens were obtained at 2, 4, 5, 6, 7, and 8 dpi. Virus titrations were done in embryonated chicken eggs (log₁₀ EID₅₀/ml) where the limit of virus detection was set to 0.7 log₁₀ EID₅₀/ml. Dash marks indicate no virus detection.</p>*<p>Standard deviation titers.</p