Efficacy, Safety, and Immunogenicity of the Bivalent Inactivated Enterovirus Vaccine (Vero Cell)

A Multicenter, Randomized, Double-blind, Controlled Phase III Clinical Trial on the Efficacy, Safety, and Immunogenicity of the Bivalent Enterovirus Inactivated Vaccine (Vero Cell) in Children Aged 6 to 71 Months.

This multicenter, randomized, double-blind, controlled Phase III clinical trial aims to evaluate the efficacy, safety, and immunogenicity of the bivalent enterovirus-inactivated vaccine (Vero cell) in healthy children aged 6 to 71 months.

The main questions it aims to answer are:

• The primary vaccine efficacy of the investigational vaccine against Hand, Foot, and Mouth Disease（HFMD) caused by CA16 infection compared to the control vaccine.
• The neutralizing antibody levels against EV71 in the trial group are non-inferior to those in the control group after two doses of vaccination.

Researchers will compare the bivalent enterovirus-inactivated vaccine (Vero cell) to the EV71-inactivated vaccine (Vero cell) to prevent HFMD and Herpangina(HA).

Participants will be randomly assigned to the trial group and the control group in a 1:1 ratio to receive two doses of the investigational vaccine or the control EV71 vaccine, with a one-month interval between doses.

This trial employs a multicenter, randomized, double-blind, controlled design. With informed consent, 8000 healthy children participants aged 6 to 71 months will be enrolled, with 4000 participants aged 6 to 23 months and 4000 participants aged 24 to 71 months, maintaining a balanced gender ratio. Among them, 4000 participants aged 6 to 23 months and 3600 participants aged 24 to 71 months have no history of EV71 vaccination, while 400 participants aged 24 to 71 months have a history of EV71 vaccination. All participants will be randomly assigned to the trial and control groups at a 1:1 ratio, receiving two doses of the experimental vaccine or the control EV71 vaccine, with a one-month interval between doses.

Each participant enters the case monitoring period after the first dose, which lasts until the end of two consecutive epidemic seasons. The effective monitoring period begins 14 days after the second dose, with the period before being the monitoring window period. During the case monitoring period, if a participant meets the criteria for a suspected case of enterovirus infection through active follow-up by the researcher or self-reporting by the participant's guardian, a throat swab and/or rectal swab will be collected for RT-PCR nucleic acid testing within three days of the first appearance of symptoms, followed by subsequent follow-up/sampling based on the test results. The protective efficacy of the investigational vaccine will be analyzed based on cases caused by laboratory-confirmed CA16 infections. In addition, enterovirus infection cases collected in this study will be subjected to viral isolation and sequencing analysis to understand their genetic subtypes.

Researchers collect all adverse events (AEs) within 30 minutes after each dose for all participants and serious adverse events (SAEs) from the first vaccination dose to six months after the second dose. Furthermore, any AEs reported by the participant's guardian or known through any other means from the first dose to 30 days after the second dose will be paid attention to, recorded truthfully, and included in the analysis.

A subgroup of 2000 participants will be selected for reactogenicity, including 1000 participants aged 6 to 23 months with no history of EV71 vaccination, 800 participants aged 24 to 71 months with no history of EV71 vaccination, and 200 participants aged 24 to 71 months with a history of EV71 vaccination. For the reactogenicity subgroup, diary cards will be used to collect solicited and unsolicited AEs within 0 to 7 days after each dose, and contact cards will be used to collect AEs from 8 to 30 days after each dose.

A subgroup of 1200 participants from the reactogenicity subgroup will be selected for immunogenicity evaluation, including humoral and cellular immunogenicity subgroups.

The humoral immunogenicity subgroup includes 1160 participants aged 6 to 71 months, with 580 participants aged 6 to 23 months with no history of EV71 vaccination, 380 participants aged 24 to 71 months with no history of EV71 vaccination, and 200 participants aged 24 to 71 months with a history of EV71 vaccination. Venous blood of approximately 3.0ml will be collected from the humoral immunogenicity subgroup before the first dose and at 30 days, 6 months, and 12 months after the second dose.

Serum collected from all participants in the humoral immunogenicity subgroup at the above four time points will be used for neutralizing antibody testing against EV71 and CA16, to evaluate the immunogenicity and duration of immunity against EV71 and CA16. Additionally, the first 100 participants with a history of EV71 vaccination and the first 100 participants without a history of EV71 vaccination from the humoral immunogenicity subgroup will be selected for neutralizing antibody testing against CA10 and CA6 using their serum before the first dose and at 30 days after the second dose; the serum of the remaining 960 participants in the humoral immunogenicity subgroup will be used for neutralizing antibody testing against different serotypes of enteroviruses, including EV71 and CA16, before the first dose and at 30 days after the second dose.

From the humoral immunogenicity subgroup, 50 participants aged less than 12 months with no history of EV71 vaccination will be selected to complete the full vaccination through intramuscular injection on the anterolateral thigh, while the rest of the participants complete the full vaccination through intramuscular injection on the deltoid muscle of the upper arm, for analysis of immunogenicity at different injection sites.

The cellular immunogenicity subgroup will include 40 participants aged 24 to 71 months with no history of EV71 vaccination. Venous blood of approximately 3.0ml will be collected before each dose and on the 14th day after each dose for the detection and analysis of mononuclear cell INF-γ and IL-4 expression.

Inclusion Criteria:

• Healthy children aged 6 to 71 months with no history of EV71 vaccination; or healthy children aged 24 to 71 months who have completed two doses of EV71 vaccine at least 6 months prior.
• Guardians who can understand and voluntarily sign the informed consent form.
• Willing and able to comply with all visit schedules, sample collections, vaccinations, and other research procedures.
• Provide proof of identity documents.

Exclusion Criteria:

• A known history of HFMD/HA.
• Uncontrolled chronic diseases or a history of severe illnesses, including but not limited to cardiovascular diseases, blood disorders, liver or kidney diseases, digestive system diseases, respiratory system diseases, malignant tumors, or a history of major functional organ transplantation.
• Autoimmune diseases, immunodeficiency diseases (including but not limited to systemic lupus erythematosus, ankylosing spondylitis, autoimmune thyroid diseases, asplenia, functional asplenia, and HIV infection).
• Abnormal coagulation function (such as coagulation factor deficiencies, and platelet abnormalities).
• Suffering from/having a history of severe neurological diseases (epilepsy, convulsions, or seizures [excluding a history of febrile seizures]) psychiatric disorders, or a family history of psychiatric disorders.
• Various acute diseases or exacerbations of chronic diseases within the last 3 days, or known or suspected active infections.
• Received a vaccine containing CA16 components.
• Received immunosuppressive or other immunomodulatory treatments for ≥14 days within the past 6 months (prednisone ≥2mg/kg/day, or its equivalent; local or inhaled corticosteroids excluded), cytotoxic therapy, or planning to receive such treatment during the trial.
• Received immunoglobulin or other blood products within the past 6 months, or planning to receive such treatment during the trial.
• Received other investigational drugs or vaccines within the past 30 days, or planning to receive such drugs or vaccines during the trial.
• Received live attenuated vaccines or nucleic acid vaccines within the past 14 days, or subunit or inactivated vaccines within the past 7 days.
• Known allergy to any component of the investigational vaccine (inactivated EV71 virus, inactivated CA16 virus, aluminum hydroxide, sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, injectable water).
• On the day of the planned vaccination with the trial vaccine, there is a fever, with axillary temperature > 37.0°C before vaccination;
• On the day of the planned vaccination with the trial vaccine, the physical examination is not qualified.
• Skin damage, inflammation, ulcers, rash, or scars at the target injection site that may interfere with vaccination or observation of local reactions.
• According to the investigator's judgment, participants have any other factors that make them unsuitable for participation in the clinical trial.