HIV-1 prime-boost vaccination shapes distinct clonal trajectories and memory precursor states of Env- and Gag-specific T cells.

Despite decades of HIV-1 vaccine development, the clonal and cellular determinants of durable vaccine-induced T cell memory remain incompletely understood. Here, we combined antigen- specific T cell receptor (TCR) identification, longitudinal TCR sequencing, and single-cell multi- omics to characterize Env- and Gag-specific memory precursor T cells elicited by the HIV Vaccine Trials Network (HVTN) 505 DNA prime-recombinant adenovirus serotype 5 (rAd5) boost (DNA/rAd5) vaccine regimen. We developed a generalizable high-throughput approach to identify HIV-1 Env- and Gag-specific TCRs and found distinct patterns of clonal expansion, persistence, and contribution to memory between Env- and Gag-specific CD8⁺ T cell responses. The DNA prime and rAd5 boost differentially shaped these repertoires, with rAd5-induced clones contributing proportionally more to the Gag-specific than to the Env-specific memory precursor compartment. Single-cell immune profiling further revealed distinct memory precursor states, with Env-specific responses enriched for GZMB + PRF1 + cytotoxic effector-memory (EM) CD8⁺ T cells and Gag-specific responses containing a larger cycling/proliferative population. Together, these findings demonstrate that heterologous DNA/rAd5 vaccination generates antigen-specific CD8⁺ T cell memory with distinct clonal trajectories and cellular programs, providing new insights into how vaccine platform and antigen-specificity shape the durability and functional properties of HIV-1 specific cellular immunity.