Table 1 Study Objectives and Endpoints (Selected)

Primary

Evaluate the safety of monovalent DENV3 vaccination in those with distinct natural DENV infection histories living in non-endemic areas, and how prior DENV immunity influences protection against vaccine strain infection evaluated by the change in GMT and mean peak viremia.

The frequency and severity of local and systemic reactogenicity signs and symptoms during the 28 day period after each vaccination, unexpected AEs up to 28 days after each vaccination, and SAEs through day 180.

Change in the DENV1-4 neutralizing antibody GMT between days 0 and 28.

Mean peak viremia among groups as measured by viral qRT-PCR between days 3 and 15.

Secondary

Further evaluate how DENV infection history impacts the immunogenicity of the vaccine.

Change in DENV1-4 neutralizing antibody GMT between days 0 and 57.

DENV1-4 neutralizing antibody GMT at days 0, 28, and 57.

Magnitude of the CD8⁺ T-cell response at day 15 among groups as measured by AIM assays.

Tertiary/Exploratory

Further evaluate how DENV infection history impacts the immune response to rDEN3Δ30/31-7164 (all potential timepoints are listed; exploratory analyses will be conducted at selective timepoints depending on the results of primary and secondary endpoints).

Change in DENV3 neutralizing antibody titer between day 0 and peak titer (measured at days 28, 57, or 90).

Characterize the broadly neutralizing antibodies observed in each group at screening and may be performed at days 15, 28, 57, 90, 180, and 365 after vaccination using ELISAs and Blockade-of-Binding assays with previously isolated potently neutralizing antibodies.

Assess whether ADE assays are associated with increased rDEN3Δ30/31-7164 viremia.

Immunophenotyping at days 0, 1, 3, 6, 9, 15, and 28 post-vaccination in each group.

Characterize the phenotype, frequency, and magnitude of CD4⁺ and CD8⁺ T cells and T follicular helper (Tfh) cells in each group. These analyses may be performed at days 0, 3, 6, 9, 15, 28, 57, 90, and 180 using AIM assays.

Compare rash frequency among groups during the first 28 days post-vaccination.

Characterize the transcriptome and cell surface proteins in each group using single-cell RNA sequencing (RNAseq) and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq). These analyses may be performed at days 0, 1, 3, 6, 9, 15, 28, and 57.

Characterize serum cytokine responses using Luminex assays.

Evaluate how DENV infection history impacts the immune response to rDEN3Δ30/31-7164 in the LN.

Characterize the B and T cell phenotypes in peripheral blood versus the lymph node in each group by surface protein staining, AIM assays, and single-cell RNA-seq, BCR and TCR sequencing, and CITE-seq pre-vaccine and at days 15, 28, and 57.

Assess the nucleotide mutation frequencies in the immunoglobulin heavy chain (IGHV) genes of germinal center B cells at days 15, 28, and 57 in each group using BCR-seq.

Assess whether the nucleotide mutation frequencies in the IGHV genes of germinal center B cells at days 15, 28, and 57 are associated with the presence of potent, cross-serotypic antibodies.

Evaluate the cells involved in viral replication.

Assess PBMCs for DENV viral protein expression using intracellular staining for non-structural protein 3 (NS3).