Evaluation of Immunogenicity and Skin-Resident Memory T Cell Induction by Intradermal, Subcutaneous, and Intramuscular Yellow Fever Virus Strain 17D-204 Vaccination

The primary objective of this study is to compare the number and proportion of skin-resident memory **T cells** against yellow fever virus (YFV-TRM) following yellow fever vaccination by different vaccination routes at day 28. This is clinically relevant as it may inform optimal vaccination strategies to enhance immunogenicity and protection against yellow fever.

Secondary objectives include:

• Comparing the number and proportion of circulating effector (YFV-TEM) and central-memory T cells (YFV-TCM) by vaccination route.
• Comparing the number/proportion of YFV-TRM, YFV-TEM, and YFV-TCM cells by vaccination route, stratified by CD4+ and CD8+ T cell differentiation subsets.
• Comparing the YFV neutralizing antibody titre (YFV-nAbs) by vaccination route.
• Comparing the dynamics in generation and durability of vaccine-induced circulating nAbs (YFV-nAbs) and poly-functional YFV-specific T cell responses (YFV-TEM, and YFV-TCM) over time by vaccination route.
• Determining the correlation between the conventional YFV-nAb response and the YFV-TEM, YFV-TCM, and YFV-TRM responses.
• Describing and reporting the safety by occurrence of serious adverse events by study arm.
• Estimating the association of the level of YFV-(n)Ab, YFV-TEM, YFV-TCM, and YFV-TRM responses with local and systemic adverse events after vaccination.

The clinical trial focuses on participants diagnosed with **yellow fever**. The study population includes both male and female subjects, aged between 18 and 50 years, with a **Body Mass Index (BMI)** ranging from 18.5 to 30 kg/m². Participants are required to be in general good health, as indicated by their ability to provide written informed consent and their agreement to share relevant medical history and records. The trial does not involve a vulnerable population. Participants are expected to refrain from blood donation and other vaccinations for 30 days following the study vaccination. Women of childbearing potential must agree to use a highly effective method of contraception up to 30 days after the study vaccination. The sponsor has not provided information regarding the total number of participants in the trial.

The clinical trial is designed as a **randomized, controlled** study to evaluate the immunogenicity and skin imprinting of different routes of **yellow fever** vaccination, specifically intradermal, subcutaneous, and intramuscular administration. The trial aims to compare the number and proportion of skin-resident memory T cells against the yellow fever virus (YFV) following vaccination by different routes at Day 28. The study will involve participants aged 18 to 50 years, with specific inclusion criteria such as a BMI between 18.5 and 30 kg/m², agreement to refrain from blood donation and other vaccinations for 30 days post-vaccination, and the ability to provide informed consent. The trial is expected to commence recruitment on September 25, 2024, and conclude by July 31, 2026.

Participants will be involved in the study for a maximum treatment period of one day, with follow-up visits extending up to 120 days post-vaccination. The sequence of study visits includes an initial screening visit to assess eligibility, followed by the administration of the vaccine. Subsequent visits will occur at Day 0, Day 14, Day 28, and Day 120 to monitor the primary and secondary endpoints, which include the percentage and absolute number of YFV-specific T cells, serum neutralization titers, and the occurrence of adverse events. The primary endpoints focus on the percentage and absolute number of YFV-specific T cells within the skin-resident lymphocyte population at Day 28, measured by flow cytometry. Secondary endpoints include the assessment of circulating T cell populations, serum neutralization titers, and the occurrence of adverse events.

Participant involvement is expected to last up to 120 days, with conditions for early termination including withdrawal of consent, non-compliance with study procedures, or the occurrence of serious adverse events. The trial will utilize the **STAMARIL®** vaccine, containing the **yellow fever virus strain 17D-204 (live, attenuated)**, administered via different routes to evaluate the immunogenic response. The study is not categorized as low intervention due to the comparison of a different route of administration against the already approved routes. The trial is conducted under the authorization of the relevant regulatory bodies, ensuring adherence to ethical and scientific standards.

The clinical trial involves the administration of **STAMARIL®**, a yellow fever vaccine containing the **yellow fever virus strain 17D-204 (live, attenuated)**. This vaccine is provided as a **suspension for injection** in a pre-filled syringe. The trial evaluates three different administration routes: subcutaneous, intradermal, and intramuscular. Each route is associated with a specific dosage and administration method.

For the subcutaneous injection, **STAMARIL®** is administered at a dosage of 0.5 mL. This method involves injecting the vaccine into the subcutaneous tissue, typically in the upper arm. The maximum treatment period for this administration is one day, with a single dose being sufficient for the study's requirements.

The intradermal administration of **STAMARIL®** involves a lower dosage of 0.1 mL. This method requires precise injection into the dermal layer of the skin, which is a deviation from the standard intramuscular or subcutaneous routes. The adjustment in dosage and route is intended to assess the immunogenicity and skin imprinting effects of the vaccine. The treatment period remains one day, with a single dose administered.

For the intramuscular injection, **STAMARIL®** is also administered at a dosage of 0.5 mL. This route involves injecting the vaccine into the muscle tissue, typically in the deltoid muscle of the upper arm. Similar to the other methods, the treatment period is limited to one day, with a single dose administered.

Throughout the trial, participant compliance with the dosing schedule is monitored to ensure accurate assessment of the vaccine's effects. The trial aims to compare the number and proportion of skin-resident memory T cells against the yellow fever virus following vaccination by different routes at day 28. No non-experimental treatments, such as standard-of-care therapy or placebo, are utilized in this study. The trial is conducted under the authorization of the relevant regulatory bodies, with **STAMARIL®** being marketed by Sanofi Pasteur Europe.

Efficacy in this clinical trial will be assessed by evaluating the immunogenicity and skin imprinting of yellow fever vaccination administered via different routes: intradermal, subcutaneous, and intramuscular. The primary endpoints for efficacy evaluation include the percentage and absolute number of **YFV-specific T cells** within the skin-resident lymphocyte parent population at Day 28, measured using flow cytometry. Secondary endpoints will assess the percentage and absolute number of circulating and YFV-specific TCM and TEM cells within the CD3+ lymphocyte parent population at Day 28, as well as the increase in their percentage and absolute number from Day 0 to Day 28.

Additional secondary endpoints include the serum dilutions at which 50% viral neutralization occurs (NT50), measured by certified virus neutralization test (VNT) assays at Day 28, and the increase in their percentage and absolute number from Day 0 to Day 28. The nAbs NT50 titres, IFNy+ spot forming units (SFU), and IFNy/TNF-a/IL-2+ SFU will be measured at Days 0, 14, 28, and 120 using VNT and ELISpot/fluorospot assays. The occurrence of solicited and unsolicited local and systemic adverse events (AEs) will be monitored up to Day 28, and serious adverse events (SAEs) will be tracked up to Day 120. These assessments will provide comprehensive data on the immunogenic response and safety profile of the yellow fever vaccine across different administration routes.