Data Sheet 1_Poly(allylamine)/tripolyphosphate nanocomplex coacervate as a NLRP3-dependent systemic and mucosal adjuvant for vaccines.docx

Nanotechnology plays a crucial role in vaccine development, enabling the design of functional nanoparticles (NPs) that act as both antigen carriers and adjuvants to enhance immune responses. In this study, we evaluated complex coacervate-like NPs composed of poly(allylamine hydrochloride) (PAH) and tripolyphosphate (TPP) as a biocompatible and biosafe platform for systemic and mucosal subunit vaccines. We assessed NP-induced activation of antigen-presenting cells and their adjuvanticity in BALB/c and knockout mice immunized intraperitoneally and intranasally with NP-OVA. In vitro, NPs increased CD86 and MHC II expression and promoted interleukin-1β (IL-1β) and IL-18 secretion via NLRP3 inflammasome activation in macrophages and dendritic cells co-incubated with LPS. Cytokine release occurred through an unconventional autophagosome-dependent pathway, as inhibition of autophagy with 3-methyladenine reduced LPS/NP-induced IL-1β secretion. In vivo, NP-OVA administration induced robust OVA-specific IgG and IgG2a responses, increased IFN-γ secretion by splenocytes, and elevated frequencies of CD4⁺IFN-γ⁺ and CD8⁺IFN-γ⁺ T cells. Comparable responses were observed following intranasal immunization, highlighting the versatility of these NPs as vaccine platforms. Overall, PAH/TPP NPs activate the NLRP3 inflammasome in innate immune cells, promote antigen-presenting cell maturation, enhance cytokine secretion, and induce strong Th1-biased humoral and cellular immune responses. These findings support their potential as a safe dual-function nanoplatform for preventive and therapeutic vaccines against infectious and non-infectious diseases.