Poly-γ-Glutamic Acid-Modified Carbon Dots as an Enhanced Immune-Activating and Antigen-Delivering Nano-adjuvant for Improved Influenza Vaccine Efficacy

Vaccine adjuvants are essential components that enhance immune responses to antigens with limited intrinsic immunogenicity and shape the magnitude and quality of adaptive immunity. Although conventional adjuvants effectively stimulate innate immunity, their use is often limited by safety concerns or inefficient antigen delivery. Consequently, biologically derived and biodegradable biomaterials have gained attention as potent vaccine adjuvants. Among them, poly-γ-glutamic acid (γ-PGA) is a naturally derived biopolymer with intrinsic immunostimulatory activity that activates innate immune signaling and promotes cellular immune responses. However, γ-PGA alone shows limited antigen delivery efficiency, which can restrict formulation reproducibility and immune potency. Here, we report γ-PGA-modified carbon dots (PGA-CDs) as a rationally designed nano-adjuvant integrating γ-PGA-mediated immune activation with efficient antigen delivery. Compared with γ-PGA, PGA-CDs exhibited faster and more sustained cellular association, thereby inducing stronger dendritic cell maturation and proinflammatory cytokine production. They readily formed stable nanocomplexes with protein antigens, enhancing cellular uptake and antigen cross-presentation. In vivo, PGA-CDs formulated with ovalbumin elicited strong antigen-specific humoral and cellular immune responses. When combined with hemagglutinin (HA) from pandemic influenza A/California/04/2009 (pH1N1), PGA-CDs induced HA-specific humoral responses comparable to alum, while eliciting significantly stronger antibody and T cell responses than γ-PGA alone, resulting in complete protection against lethal viral challenge. Notably, HA/PGA-CDs also elicited more robust homosubtypic cross-reactive immunity than alum against influenza A/Puerto Rico/8/1934 (H1N1), conferring complete protection. Furthermore, PGA-CDs were well tolerated in vivo, with no detectable hematological or histopathological abnormalities. Collectively, these findings establish PGA-CDs as a promising vaccine adjuvant platform that enhances antigen delivery, amplifies immune activation, and broadens protective efficacy.