Evaluation of Cytokine Release Assay for T-Cell Responses in Tick-Borne Encephalitis Using FSME-IMMUN Vuxen and Encepur Vaccines in Healthy and Immunosuppressed Subjects

The primary objective of this clinical trial is to evaluate the **magnitude** and **durability** of T-cell responses against the **tick-borne encephalitis virus (TBEV)**. This is assessed using a novel cytokine release assay and compared with antibody levels measured by commercially available IgM assays (Euroimmun and ReaScan) and/or a quantitative IgG assay (Euroimmun). The evaluation is conducted among patients hospitalized for tick-borne encephalitis (TBE) at the time of or within less than six months of enrollment, as well as those previously hospitalized for TBE six months or more prior to enrollment. Additionally, the study includes healthy or immunosuppressed participants initiating or having undergone past vaccination against TBEV, with assessments related to the number of previous vaccine doses, time elapsed since the most recent dose, and the vaccine manufacturer.

Secondary objectives include evaluating:

• The magnitude and durability of T- and B-cell responses directed against TBEV after TBE vaccination or natural infection.
• The impact of baseline characteristics such as body mass index (BMI), weight, gender, age, and host genetic factors (e.g., IFNL4 genotype) on the magnitude and durability of T- and B-cell responses against TBEV.
• The impact of concomitant medication on the magnitude and durability of T- and B-cell responses against TBEV.
• The impact of adverse events temporally associated with vaccination on the magnitude and durability of T- and B-cell responses against TBEV.
• The magnitude and durability of cross-reactive T-cell and B-cell responses directed against TBEV after recent (≤6 months) vaccination against Yellow Fever and Japanese Encephalitis among healthy participants, serving as negative controls using live attenuated and formalin-inactivated flavivirus vaccines, respectively.

The clinical trial focuses on **tick-borne encephalitis** and includes both male and female participants aged 18 years and older. The study population comprises individuals who have been hospitalized for tick-borne encephalitis, as well as healthy or immunosuppressed participants who are either initiating or have previously undergone vaccination against the tick-borne encephalitis virus. The trial includes a vulnerable population, and participants were selected based on their consent to participate. The sponsor has not provided information regarding the total number of participants. The trial does not specify any particular lifestyle considerations such as diet or physical activity. Key inclusion criteria include the requirement for participants to provide written consent and be of legal adult age. The study aims to evaluate the magnitude and durability of T-cell responses against the tick-borne encephalitis virus using a novel cytokine release assay in comparison with commercially available antibody assays.

The clinical trial is designed to evaluate the efficacy of a novel **cytokine release assay** for assessing T-cell responses against the **tick-borne encephalitis virus** (TBEV) in comparison to existing serological tests. This trial is a low-intervention, Phase IV study involving healthy and immunosuppressed participants. The trial employs a randomized, double-blind, controlled design to ensure the reliability and validity of the results. The estimated duration of the trial is from April 2024 to December 2026, with participant involvement expected to last up to 8 months.

Participants will undergo a series of study visits, beginning with an inclusion (screening) visit to confirm eligibility based on criteria such as age (18 years or older) and informed consent. Following the screening, participants will receive the TBE vaccine (FSME-IMMUN Vuxen) at specified intervals: dose 1 on day 0, dose 2 on day 28, dose 3 around week 13, and dose 4 around week 34. These visits are designed to monitor the immune response and ensure participant safety. Follow-up visits will be scheduled to assess the magnitude and durability of T-cell responses using the cytokine release assay, compared to antibody levels measured by commercially available assays.

The end-of-study visit will conclude the participant's involvement, during which final assessments will be conducted to evaluate the primary and secondary endpoints. The primary endpoint focuses on the concentrations of cytokines detected by the novel assay in relation to antibody responses. Secondary endpoints include the duration of detectable immune responses and the potential cross-reactivity with other flavivirus vaccinations. Participants may be withdrawn from the study early if they experience adverse reactions, withdraw consent, or fail to comply with study procedures. The trial aims to provide valuable insights into the immune response to TBEV and the potential utility of the cytokine release assay in clinical settings.

The clinical trial involves the administration of **FSME-IMMUN Vuxen**, a vaccine formulated as a **suspension for injection**. This vaccine is designed to protect against **tick-borne encephalitis** and contains the **tick-borne encephalitis virus Neudoerfl strain adsorbed on aluminium hydroxide, hydrated**, produced in chick embryo cells. The vaccine is provided in a pre-filled syringe and is administered via **intramuscular injection**. The dosing schedule for participants without prior vaccination history includes a total of four doses: the first dose on study day 0, the second dose on day 28, the third dose during month 3 (approximately week 13 ± 10 days), and the fourth dose during month 8. Each dose consists of 0.5 mL, with a maximum total dose of 2 mL over the treatment period. The vaccine is manufactured by Pfizer AB and is not a pediatric formulation.

Another vaccine used in the trial is **Encepur**, also formulated as a **suspension for injection**. This vaccine contains the **tick-borne encephalitis virus, strain K23, adsorbed on hydrogenated aluminum hydroxide, inactivated**. Like FSME-IMMUN Vuxen, Encepur is administered via **intramuscular injection** and is provided in a pre-filled syringe. The dosage for Encepur is similarly 0.5 mL per injection, with a maximum total dose of 2 mL over the treatment period. The vaccine is produced by Bavarian Nordic A/S and is also not a pediatric formulation. Both vaccines are classified under the ATC code J07BA01, indicating their use for inactivated whole virus vaccines against tick-borne encephalitis.

Efficacy in this clinical trial will be assessed by evaluating the magnitude and durability of T-cell responses against the **tick-borne encephalitis virus (TBEV)**. This will be measured using a novel cytokine release assay, which will be compared to antibody levels evaluated through commercially available IgM assays (Euroimmun and ReaScan) and a quantitative IgG assay (Euroimmun). The primary endpoint is the concentration of cytokines detected using the novel rapid release assay in relation to antibody responses measured by these commercially available assays.

Secondary endpoints include the duration of detectable T- and B-cell immune responses directed against TBEV after infection or vaccination. Additionally, the trial will assess whether cross-reactive T- and B-cell immune responses are detectable after prior vaccination against other flaviviruses, such as Yellow Fever and Japanese Encephalitis, and whether these responses are transient or persistent.

The trial will involve research participants who are either initiating vaccination against TBEV or have previously undergone vaccination or hospitalization for TBE. Participants initiating vaccination will receive the FSME-IMMUN Vuxen vaccine (Pfizer) during the study. The schedule for vaccination includes dose 1 on study day 0, dose 2 on day 28, dose 3 during month 3, and dose 4 during month 8. The efficacy assessments will be conducted in relation to the time elapsed from hospital admission for TBE or the number of previous vaccine doses received.