Abstract
BACKGROUND/OBJECTIVES: Developing durable cellular immunity remains a critical challenge for HIV vaccine development. METHODS: We evaluated a sequential and repeated heterologous prime-boost vaccination regimen using four distinct vector-based vaccines (DNA, rAd5, rSeV, and rMVA) expressing HIV-1 gag in rhesus macaques over a decade-long observation period. RESULTS: Compared to the two-vector and control groups, the four-vector regimen elicited potent gag-specific cellular immune responses, as evidenced by IFN-γ ELISPOT assays showing sustained responses exceeding 500 SFCs/10(6) PBMCs for up to 52 or 69 weeks post-vaccination. Intracellular cytokine staining revealed multifunctional CD4+ and CD8+ T-cell responses, while humoral immunity against Ad5 vectors remained manageable despite repeated administrations. CONCLUSIONS: These findings demonstrate that sequential and repeated heterologous vaccination effectively induces and maintains durable cellular immunity, providing a strategic framework for HIV vaccine design.