Comparison of Immunogenicity and Skin-Resident Memory T Cell Induction by Intradermal Versus Intramuscular Rabies Virus (Inactivated) Strain Flury LEP Vaccination in Rabies Patients

The primary objective of this study is to compare the number and proportion of skin-resident memory **T cells** against rabies virus (**RABV-TRM**) following a booster dose, administered via different vaccination routes. This objective is clinically relevant as it aims to evaluate the effectiveness of intradermal versus intramuscular rabies vaccination in inducing a robust immune response, which is crucial for long-term protection against rabies.

Secondary objectives include:

• Comparing the number and proportion of effector-memory (**RABV-TEM**) and central-memory T cells (**RABV-TCM**) after a booster dose by vaccination route.
• Assessing the rate of increase in RABV-TRM, RABV-TEM, and RABV-TCM cells post-booster vaccination compared to primary vaccination, by vaccination route.
• Evaluating the number and proportion of RABV-TRM, RABV-TEM, and RABV-TCM cells and their rate of increase by CD4+ and CD8+ T cell differentiation subsets.
• Comparing the RABV neutralizing antibody titre (RABV-nAbs) and those with a titre ≥ 3.0 IU/ml and ≥ 10.0 IU/ml at D210+D14 by vaccination route.
• Analyzing the dynamics in generation and durability of vaccine-induced, circulating nAbs and poly-functional RABV-specific T cell response over time by vaccination route.
• Determining the correlation between the conventional RABV-nAb response and the RABV-TEM, RABV-TCM, and RABV-TRM responses.

The clinical trial involves participants diagnosed with **rabies**. The sponsor has not provided information regarding the total number of participants. The study population includes both male and female subjects, aged between 18 and 50 years, who are generally in good health. Participants were selected based on specific criteria, including a body mass index (BMI) of 30 kg/m² or less and the absence of acute illness at the time of recruitment. The trial does not involve a vulnerable population. Participants are required to have completed or partially completed a rabies vaccination schedule more than one month prior to recruitment. Lifestyle considerations such as the willingness to use contraception during the trial (for women of childbearing potential) and agreement to refrain from blood donation and other vaccinations 30 days following each vaccination are noted. Additionally, participants must be able and willing to provide written informed consent and agree to share and discuss their medical history and records when relevant.

The clinical trial is designed as a **randomized controlled trial** to evaluate the immunogenicity and skin imprinting of intradermal versus intramuscular rabies vaccination. The trial aims to compare the number and proportion of skin-resident memory T cells specific to the rabies virus after a booster dose administered through different vaccination routes. The study will involve participants aged 18 to 50 years, with specific inclusion criteria such as a body mass index (BMI) of ≤30 kg/m² and the ability to provide informed consent. The trial is expected to commence recruitment on January 1, 2024, and conclude by October 1, 2025, with a maximum treatment period of 123 days for each participant.

Participants will undergo a series of study visits, beginning with an inclusion (screening) visit to assess eligibility based on the inclusion criteria. Following the initial screening, participants will receive vaccinations and attend follow-up visits at specified intervals, including days 0, 7, 28, 120, 120+14, and 120+90. These visits are designed to monitor the immune response and collect data on primary and secondary endpoints, such as the percentage and absolute number of skin-resident and circulating T cells, as well as the geometric mean titres of neutralizing antibodies. The end-of-study visit will occur after the final follow-up to ensure comprehensive data collection and participant safety.

The expected length of participant involvement is approximately 10 months, with conditions for early termination including the development of acute illness or withdrawal of consent. Participants are required to refrain from blood donation and other vaccinations for 30 days following each vaccination to ensure the integrity of the study results. The trial will utilize a double-blind methodology to minimize bias and ensure the reliability of the findings. The primary endpoint will be assessed using flow cytometry to measure the percentage and absolute number of skin-resident and rabies virus-specific T cells within the CD3+ lymphocyte parent population after booster vaccination.

The clinical trial involves the administration of **Rabipur**, a rabies vaccine formulated as a **solution for injection**. The active substance in this experimental medication is the **rabies virus (inactivated) strain Flury LEP**. The pharmaceutical form is a powder and solvent for solution for injection, provided in a pre-filled syringe. The vaccine is manufactured by Bavarian Nordic A/S. The trial will compare two routes of administration: intradermal and intramuscular. The intradermal route involves a test dosage of 2 x 0.1 mL, while the comparator dosage for the intramuscular route is 1 mL. The maximum daily dose is 1 mL, with a total maximum dose of 4 mL over a treatment period of 123 days. The trial aims to evaluate the immunogenicity and skin imprinting of the vaccine by assessing the number and proportion of skin-resident memory T cells against the rabies virus after a booster dose.

Efficacy in this clinical trial will be assessed by evaluating the immunogenicity and skin imprinting of intradermal versus intramuscular rabies vaccination. The primary endpoint for efficacy assessment is the percentage and absolute number of skin-resident and **RABV**-specific T cells within the CD3+ lymphocyte parent population after booster vaccination, measured at D120+14 using flow cytometry. Secondary endpoints include the percentage and absolute number of circulating and **RABV**-specific TCM and TEM cells within the CD3+ lymphocyte parent population at D120+14, as well as the increase in **RABV**-specific TRM, TCM, and TEM cells from D28 to D120+14. Additionally, the geometric mean titres (GMTs) of neutralizing RRFIT test at D210+14, and the nAbs GMT titres, IFNy+ spot forming units (SFU), and IFNy/TNF-a/IL-2+ SFU at various timepoints (D0, D7, D28, D120, D120+14, D120+90) will be measured using RRFIT and elispot/fluorospot assays. These assessments will provide comprehensive data on the immunogenic response elicited by the different vaccination routes.