Comparative analysis of the biological characteristics of three CV-A10 clones adaptively cultured on Vero cells.

Coxsackievirus A10 (CV-A10) is a major pathogen that causes hand, foot, and mouth disease. There are no effective therapeutic drugs for CV-A10 infection; therefore, CV-A10 vaccines should be developed. Previously, we isolated a CV-A10 strain (N25) that can be cultured on Vero cells. In this study, the N25 strain was plaque-purified three times from Vero cells, and three clones were selected for adaptive culture. The three clones of the 5th, 12th, and 19th generations were compared and analyzed in terms of viral titers, plaque morphology, pathogenicity in suckling mice, and nucleotide and amino acid sequences of the complete genome. The infectivity titers of the three clones (P2-P22) were maintained at 6.5-7.0 lgCCID50 /ml. The three clones began to proliferate at 6 h and peaked at 36 h; the corresponding CCID50 was in the range of 106.5 -106.875 /ml, which gradually decreased. The suckling mice in the challenged group exhibited clinical symptoms such as paralysis of the limbs, which gradually worsened until death. The inactivated vaccines prepared using the three clones efficiently induced antigen-specific serum antibodies in mice. There were eight nucleotide mutations in the three clones, which resulted in two and four amino acid substitutions in the VP3 and VP1 coding regions, respectively. The nucleotide and amino acid sequence homology between the three clones and N25 were 99.92%-100% and 99.78%-100%, respectively, indicating high genetic stability. Our findings provide a theoretical basis for screening CV-A10 vaccine candidate clones.

An inactivated enterovirus 71 vaccine in healthy children.

BACKGROUND: Enterovirus 71 (EV71) is a major cause of hand, foot, and mouth disease in children and may be fatal. A vaccine against EV71 is needed. METHODS: We conducted a randomized, double-blind, placebo-controlled phase 3 trial involving healthy children 6 to 71 months of age in Guangxi Zhuang Autonomous Region, China. Two doses of an inactivated EV71 vaccine or placebo were administered intramuscularly, with a 4-week interval between doses, and children were monitored for up to 11 months. The primary end point was protection against hand, foot, and mouth disease caused by EV71. RESULTS: A total of 12,000 children were randomly assigned to receive vaccine or placebo. Serum neutralizing antibodies were assessed in 549 children who received the vaccine. The seroconversion rate was 100% 4 weeks after the two vaccinations, with a geometric mean titer of 170.6. Over the course of two epidemic seasons, the vaccine efficacy was 97.4% (95% confidence interval [CI], 92.9 to 99.0) according to the intention-to-treat analysis and 97.3% (95% CI, 92.6 to 99.0) according to the per-protocol analysis. Adverse events, such as fever (which occurred in 41.6% of the participants who received vaccine vs. 35.2% of those who received placebo), were significantly more common in the week after vaccination among children who received the vaccine than among those who received placebo. CONCLUSIONS: The inactivated EV71 vaccine elicited EV71-specific immune responses and protection against EV71-associated hand, foot, and mouth disease. (Funded by the National Basic Research Program and others; ClinicalTrials.gov number, NCT01569581.).

A comparative study of multiple clinical enterovirus 71 isolates and evaluation of cross protection of inactivated vaccine strain FY-23 K-B in vitro.

BACKGROUND: Enterovirus 71 (EV71) is one of the causative agents of hand, foot and mouth disease, which mostly affects infants and children and leads to severe neurological diseases. Vaccination offers the best option for disease control. We have screened the virus strain FY-23 K-B, which is used as an inactivated vaccine strain. An important issue in the development of vaccines is whether they provide cross protection against all other strains. METHODS: We collected and identified 19 clinical EV71 isolates from mainland China, which all belong to the C4 genotype. We established growth curves of the strains in Vero cells, performed genetic analysis, and evaluated the cross protection efficacy through neutralizing assays using antisera from a rabbit, monkey and adult human immunized with the FY-23 K-B vaccine strain. RESULTS: The antisera showed broad cross protection among the C4 subgroup strains and homotype strain. Neutralizing indexes (NIs) among the isolates and homotype strain of antisera varied between 56.2-1995.3 for rabbit, 17.8-42,169.7 for monkey and 31.6-17,782.8 for human, whereas NIs against Coxsackievirus A16 or other enteroviruses were below 10. CONCLUSIONS: These results suggested that FY-23 K-B used as an antigen could elicit broad spectrum neutralizing antibodies with cross protective efficacy among C4 genotype strains.

In vitro bioactivity, cytocompatibility, and antibiotic release profile of gentamicin sulfate-loaded borate bioactive glass/chitosan composites.

Borate bioactive glass-based composites have been attracting interest recently as an osteoconductive carrier material for local antibiotic delivery. In the present study, composites composed of borate bioactive glass particles bonded with a chitosan matrix were prepared and evaluated in vitro as a carrier for gentamicin sulfate. The bioactivity, degradation, drug release profile, and compressive strength of the composite carrier system were studied as a function of immersion time in phosphate-buffered saline at 37 °C. The cytocompatibility of the gentamicin sulfate-loaded composite carrier was evaluated using assays of cell proliferation and alkaline phosphatase activity of osteogenic MC3T3-E1 cells. Sustained release of gentamicin sulfate occurred over ~28 days in PBS, while the bioactive glass converted continuously to hydroxyapatite. The compressive strength of the composite loaded with gentamicin sulfate decreased from the as-fabricated value of 24 ± 3 MPa to ~8 MPa after immersion for 14 days in PBS. Extracts of the soluble ionic products of the borate glass/chitosan composites enhanced the proliferation and alkaline phosphatase activity of MC3T3-E1 cells. These results indicate that the gentamicin sulfate-loaded composite composed of chitosan-bonded borate bioactive glass particles could be useful clinically as an osteoconductive carrier material for treating bone infection.

Pathogenesis study of enterovirus 71 infection in rhesus monkeys.

Enterovirus 71 (EV71), a major pathogen that is responsible for causing hand-foot-and-mouth disease (HFMD) worldwide, is a member of the Human Enterovirus species A, family Picornaviridae. HFMD that is caused by EV71 is usually characterized by vesicular lesions on the skin and oral mucosa and high morbidity rates in children; additionally, occasional fatal cases have been reported involving brainstem encephalitis and myelitis associated with cardiopulmonary collapse. Although viral pathogenesis in humans is unclear, previous animal studies have indicated that EV71, inoculated via various routes, is capable of targeting and injuring the central nervous system (CNS). We report here the pathogenic process of systemic EV71 infection in rhesus monkeys after inoculation via intracerebral, intravenous, respiratory and digestive routes. Infection with EV71 via these routes resulted in different rates of targeting to and injury of the CNS. Intracerebral inoculation resulted in pulmonary edema and hemorrhage, along with impairment of neurons. However, intravenous and respiratory inoculations resulted in a direct infection of the CNS, accompanied by obvious inflammation of lung tissue, as shown by impairment of the alveoli structure and massive cellular infiltration around the terminal bronchioles and small vessels. These pathological changes were associated with a peak of viremia and dynamic viral distribution in organs over time in the infected monkeys. Our results suggest that the rhesus monkey model may be used to study not only the basic pathogenesis of EV71 viral infections, but also to examine clinical features, such as neurological lesions, in the CNS and pathological changes in associated organs.

Preclinical safety assessment of a combined vaccine against Hepatitis a virus and enterovirus 71.

Since 2007, Hepatitis A (HAV) vaccination has been a part of the National Immunization Program of China. Recognizing enterovirus 71 (EV71) as the most important pathogen in severe hand, foot and mouth disease, an inactivated EV71 vaccine was successfully marketed in 2015. Based on the concept of one vaccine preventing two diseases and owing to similarities in vaccine preparation and the overlap of the eligible population, a combination of the inactivated HAV vaccine and inactivated EV71 vaccine is theoretically feasible and desirable. However, the optimal vaccinationschedule for this combination vaccine has yet to be optimized. Use of this combined vaccine would not only decrease the number of vaccinations, but also lower associated cost. This study aimed to investigate the toxicity and adverse reactions of the combined HAV-EV71 vaccine under Good Laboratory Practice conditions to provide a reference for clinical studies/applications in the future. CD®(Sprague Dawley) IGS rats were employed for single-dose toxicity testing using a high dose, and repeated-dose toxicity testing using high, as well as low doses. Animals that received only a single dose showed no obvious clinical symptoms nor abnormal body weight, and no significant gross pathological change at the experimental endpoint at necropsy. In the rats injected with three doses, phagocytosis of basophilic granules by macrophages was observed in the inguinal, mesenteric, and local lymph nodes, besides irritation at the administration site. At 56 days after the last dose, no significant histopathological change was observed in the lymph nodes, and local irritation gradually faded. Further, systematic allergy testing was performed in guinea pigs. After systemic sensitization and challenge with the HAV-EV71 vaccine, animals showed normal weight gain and no allergic reactions. This study, therefore, confirmed a good safety profile of the inactivated HAV and EV71 combined vaccine.

Proliferation characteristics of coxsackievirus A10 in mice and immune protection ability of experimental inactivated vaccine.

Coxsackievirus A10 (CVA10) is the main pathogen of hand, foot, and mouth disease in China. However, there are no CVA10-specific drugs and vaccines, and the pathogenesis and effects of this virus in the body are unknown. We investigated the effect of a clinically isolated CVA10 virus strain (CVA10-25) to investigate its effect in suckling mice through different infection routes. We observed the dynamic distribution and proliferation of the virus in mouse tissues by infecting suckling mice with different doses of the virus and mice of different ages with the same dose of the virus. We also analysed the pathological characteristics after infection. A formaldehyde-inactivated experimental vaccine was prepared to immunise 5-week-old BALB/c female mice three times, and newborn suckling mice were tested for the presence of maternally transmitted antibodies. The viral load in each organ after intracerebral administration was higher than that after intraperitoneal administration; the peroral administration route did not cause disease in mice. Mouse paralysis and death after infection were related to age. The skeletal muscles, heart, and lung showed histopathological changes after infection. We established a 2-day-old BALB/c suckling mouse model that could be infected intracranially to study the pathogenesis and pathology of CVA10. Maternally transmitted antibodies protected the mice against the virus. This study provides a reference for CVA10-related pathogenesis and vaccine research.

Safety and immunogenicity of an experimental live combination vaccine against enterovirus 71 and coxsackievirus A16 in rhesus monkeys.

Enterovirus 71 (EV-A71) and Coxsackievirus A16 (CV-A16) are the two most common pathogens causing hand, foot, and mouth disease (HFMD). Previously, we obtained one candidate live attenuated strain each for EV-A71 and CV-A16; here, we evaluated the safety and immunogenicity of a combinedlive vaccine against EV-A71 and CV-A16 generated from these two candidate strains. Rhesus monkeys were intramuscularly treated with a live combinationvaccine against both EV-A71 and CV-A16 or with either vaccine alone. No fever or atypical clinical signs were observed in any animals. Monkeys vaccinated with the combinationlive vaccine presented no notable pathological changes in the brain, spinal cord, lung, and liver; in contrast, these regions showed inflammatory cell infiltration in monkeys treated with EV-A71 alone or CV-A16 alone. Weak viremia was detected in plasma after inoculation with the combinationvaccine; however, the duration of viral shedding in feces was increased. Biochemical studies revealed a slight increase in aspartate aminotransferase levels in monkeys inoculated with the live combination vaccine; however, histopathological findings did not attribute this change to liver damage. We also found that the live combinationvaccine induced a dual humoral immune response. Cytokine analysis indicated that the combined EV-A71/CV-A16 vaccine significantly down-regulated interleukin-8 production. Here, we have demonstrated that the live attenuated EV-A71/CV-A16 vaccine was safe and could trigger a dual specific immune response. However, its immune protection efficacy requires further investigation.

Immune responses of a CV-A16 live attenuated candidate strain and its protective effects in rhesus monkeys.

Coxsackievirus A16 (CV-A16) is a major causative pathogen of hand, foot, and mouth diseases (HFMDs). The licensed HFMD vaccine targets EV-A71 without cross-protection against CV-A16. Thus, a CV-A16 vaccine is needed. In this study, the immunogenicity and protective efficacy of a live attenuated CV-A16 candidate, K168-8Ac, were evaluated in a rhesus monkey model. Four passages of this strain (P35, P50, P60, and P70) were administered to monkeys, and its protective effect was identified. The immunized monkeys were clinically asymptomatic, except for slight fever. Weak viraemia was observed, and two doses of vaccination were found to significantly reduce virus shedding. High levels of antibody responses were observed (1:1024-1:2048), along with a significant increase in plasma IL-8. The I.M. group showed a much stronger humoural immunity. Pathological damage was detected mainly in lung tissues, although thalamus, spinal cord, lymph nodes, and livers were involved. After the viral challenge, it was found that two doses of vaccine reduced virus shedding, and the degree of lung damage and the number of organs involved decreased as the passage number increased. Overall, a robust immune response and partial protection against CV-A16, triggered by the K168-8Ac strain, were demonstrated. This study provides valuable data for CV-A16 vaccine development.