Pharmacology: Pharmacodynamics: A randomized, double-blind, placebo-controlled and multi-center Phase III clinical trial for new drug registration in China was performed to evaluate the protective efficacy, safety and immunogenicity of the vaccine among 12,000 healthy children aged from 6 to 71 months, who are administrated with the vaccine on the day 0 and 28.
Efficacy test: The primary end point of the clinical trial was incidence rate against HFMD caused by EV71 in the participants administrated with the vaccine or placebo during active surveillance (within one year). The case determination criteria: both the HFMD clinical diagnosis and pathogenic detection (give priority to the enterovirus nucleic acid detection) were positive. All cases were confirmed by etiology retest as well as the confirmation of the clinical expert group. The results of clinical trials: the vaccine efficacy against EV71-associated hand, foot, and mouth disease was 97.3% (95% CI: 92.7, 99.0) using FAS analysis (12000 participants), and the vaccine efficacy was 97.3% (95% CI: 92.6, 99.0) using PPS analysis (10980 participants) after primary immunization in one year. At the site of 7 sub-centers, the incidence rate of the vaccine group is less than 0.18%, and that of the placebo group is 0.72%-4.40%. The vaccine efficacy, effect index and incidence rate among the all age groups and different age subgroups were shown in Tables 2-5. No efficacy prevent for HFMD caused by other enteroviruses (such as Coxsackievirus A16) infection was investigated in present study. (See Tables 2, 3, 4 and 5.)
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The protective efficacy was also observed during the second year after the primary immunization. It is conservatively estimated that the protective rate is 93.77% (95% CI: 88.34, 96.67).
Immunogenicity and immune persistent study: A persistent immunogenicity has been carried out in an immunogenicity subgroup after immunization in two years in order to evaluate the neutralization antibody positive rate against EV71 (For susceptible participants with negative antibody before immunization, the neutralization antibody should be ≥1:8 after immunization; for participants with positive antibody before immunization, the neutralization antibody should be increased quadruple or over) and geometric mean titer (GMT) of anti-EV71. The results were listed on Table 6. (See Table 6.)
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Safety evaluation: The Phase III clinical efficacy test was carrying out among 12,000 healthy children aged from 6 to 71 months, who were randomized to receive two doses of the product or placebo on day 0 and 28. Its active surveillance period for safety lasted 1 year. The occurrence rate of systemic adverse reactions in the vaccine group and the placebo group is 33.75% and 24.92%, respectively. The symptoms include pyrexia, decreased appetite, irritability postvaccinal, diarrhea, nausea, vomiting, fatigue, hypersensitivity, abdominal discomfort, constipation, oral inflammation and so on. Mild fever is the most common clinical manifestations, which appears transiently. As for the local adverse reactions, the occurrence rate of the vaccine group is 5.87% and of the placebo group is 2.25%. The symptoms include pain, erythema, pruritus, swell, induration on inoculation site and so on. The typical manifestations are local pain and erythema, which lasts no more than 3 days and can be recovered by its own. There was no intergroup difference in all symptoms, no evidence of serious adverse reaction related to the vaccine was observed, and the adverse reaction related to the vaccine was observed, and the adverse reaction reached level-3 or more showed no significant difference in vaccine group and placebo group. All adverse reactions were most apparent at the first dose and do not show a tendency of increase along inoculation times.
Cross neutralization: The cross neutralization test against 9 strains of EV71 virus was also performed using the serum samples collected from the participants at the first and the twelfth month after vaccine administration. The strains include Genotype A (BrCr strain), Genotype C (subtype C1, C2, C3, C4, C5) and Genotype B (subtype B3, B4, B5). The results show that the serum induced by the vaccine has cross-neutralizing reactivity to all the other EV71 genotypes.
Pharmacokinetics: Not applicable.
Toxicology: Preclinical safety data: Pharmacology: Mouse and rhesus macaque models were used to study the pharmacodynamics of Enterovirus Type 71 Vaccine, Inactivated (Human Diploid Cell).
Primary pharmacodynamics (protective effects) studies of the Enterovirus Type 71 Vaccine, Inactivated (Human Diploid Cell) showed that after 4-week-old Balb/C mice were immunized twice (0, 28 days) intramuscularly, their offspring were completely able to be resistant to the wild type virus (no death case occurred). Similarly, one month after two intramuscular injections (0, 28 days) to rhesus macaque infants aged 1 to 2 months, they also could resist the challenge of wild type virus (no death case occurred). Data from these two experimental animals showed that the protective rate of the vaccine reached 100%.
Secondary pharmacodynamics (immunogenicity) studies of the Enterovirus Type 71 Vaccine, Inactivated (Human Diploid Cell) showed that after 4-week-old Balb/C mice were immunized twice (0, 28 days) intramuscularly, neutralizing antibody titers in the vaccine group peaked 2 weeks after the second immunization, and the neutralizing antibody titers were dose-dependent. Besides, four weeks after the second immunization, the vaccine could induce a significant T cell immune response secreting IFN-γ. Similarly, when rhesus macaque infants aged 1 to 2 months were immunized twice (0, 28 days) intramuscularly, the peak time of neutralizing antibody titer in the vaccine group was also 2 weeks after the second immunization.
Toxicology: Repeat-dose Toxicity Studies in Rhesus Macaques: The Enterovirus Type 71 Vaccine, Inactivated (Human Diploid Cell) was administrated at doses of 8 µg/animal (low dose) and 32 µg/animal (high dose) to rhesus macaques at 0, 4, 8 and 12 weeks, respectively. During the administration period and recovery period (8 weeks after the administration), both low and high doses of the vaccine could cause local extreme-mild inflammation at the injection site, and the lesions was alleviated at the end of the recovery period. There was no statistically significant difference in lymphocytes absolute count and bone marrow cell count between the vaccine high dose group and the solvent control group. The antibody appeared in the vaccine group after the first immunization to rhesus macaques, and the antibody level gradually increased after the booster immunization, and began to decrease after 56 days of the fourth immunization. There were no significant differences in the cytokine levels between the vaccine group and the adjuvant control group compared with the solvent control group. The IFN-γ secreting level in the vaccine group was increased compared with both the solvent control group and the adjuvant control group. However, there was abnormalities relating to liver of some animal which was administrated at low doses of the vaccine but there was no abnormalities of animal which was administrated at high doses of the vaccine.
Single-dose toxicity (Acute Toxicity Studies in Rats): The Enterovirus Type 71 Vaccine, Inactivated (Human Diploid Cell) was injected subcutaneously to SD rats aged 5 to 6 weeks at dose of 16 µg/animal. A total of 10 animals were used as the solvent control group, and 20 animals as the vaccine group (half in male and half in female in both groups). The lethality caused by the test vaccine, toxicity and its severity of rats were observed. On the day of administration, Day 1 and 14 after the administration, there was no statistical difference in body weight between the vaccine group and the solvent control group. No significant abnormality in clinical symptoms and body weights were observed, and no significant anatomical changes were observed in all tissue and organs of animals.
Antinuclear Antibody Studies in Rats: A total of 40 rats were used in the experiment, and 4 groups were set up: single administration low dose group (4 µg/animal), single administration high dose group (8 µg/animal), repeated administration low dose group (4 µg/animal) and repeated administration high dose group (8 µg/animal). Each group was consisted of 10 animals, and half in male, half in female. The SD rats were injected with Enterovirus Type 71 Vaccine, Inactivated (Human Diploid Cell) subcutaneously, and no antinuclear antibodies were detected in all groups.
Systemic Acute Allergy Studies in Guinea Pigs: A total of 20 male guinea pigs aged 6 to 8 weeks were used in the experiment and 4 groups were set up: solvent control (PBS) group, positive control group (bovine serum albumin, BSA), vaccine group (Enterovirus Type 71 Vaccine, Inactivated (Human Diploid Cell) and spare group (Enterovirus Type 71 Vaccine, Inactivated (Human Diploid Cell)). Each experimental group was consisted of 5 animals. The Enterovirus Type 71 Vaccine, Inactivated (Human Diploid Cell) was administered by subcutaneous injection for sensitization. Anaphylaxis symptoms and incidence indicated that death cases occurred in the positive control group, i.e., the anaphylaxis was extremely-strong positive. The solvent control group and vaccine group were negative for anaphylaxis.
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