Immunomodulatory Potentials Of Engineered Nanohydroxyapatite Adjuvants

Infectious diseases such as hepatitis B,hepatitis C,cervical cancer are still one of the main courses threatening human health.Currently,the pandemic of COVID-19 has caused more than3 million deaths globally.Vaccination is regarded as the most effective manner for the prevention of these infectious diseases.The antigen in vaccine elicits innate and adaptive immune responses.However,in inactivated viral vaccines and subunit vaccines,antigens alone induce mild immune response,and adjuvants are often used to achieve a stronger and more durable immunity.As a natural component of human tissues,calcium phosphate(CaP)is regarded as a promising adjuvant for human use due to biocompatibility.Moreover,several CaP-adjuvanted antigens has been shown to induce significant humoral and cell-mediated immune responses,thus providing more balanced protection.Yet the use of various calcium phosphate compounds makes it difficult for the comparative and systematic study of CaP adjuvants.Hydroxyapatite(HAP),the most stable CaP phase,possesses defined chemical composition and has been demonstrated to exhibit stronger immunostimulation potentials.Meanwhile,HAP nanoparticles have also been shown to induce both humoral and cellular immune responses.Thus,HAP may be a potential candidate adjuvant in human vaccines.As engineered nanomaterials,the shape,size,surface charge and other properties will significantly affect the adjuvant effect.So it’s critical to understand how the physicochemical properties of HAP nanoparticles could mechanistically impact their adjuvanticity on the nano-immune interface,which is important for the design and development of engineered nanomaterial-based vaccine adjuvants.In this study,a library of HAP nanorods and nanospheres are synthesized to explore the effect of particle shape and aspect ratio on the immune responses in vitro and adjuvanticity in vivo,as well as the underlying mechanisms.It is demonstrated that long aspect ratio HAP nanorods induce higher degree of cell membrane depolarization and subsequent uptake,and the internalized particles elicit significant cathepsin B release and mitochondrial ROS generation,which lead to high level of IL-1β production.Furthermore,the physicochemical propertydependent immunostimulation capacities are correlated with the humoral responses in a murine hepatitis B surface antigen(HBs Ag)immunization model.Importantly,long aspect ratio HAP nanorods significantly increase the frequency of IFN-γ-secreting CD8+ T cells and up-regulates the expression of CD107α on CD8+ T cells in immunized mice,showing the induced stronger cellular immune response.Further mechanistic studies demonstrate that HAP nanorods could exert the immunomodulatory effects by enhanced antigen persistence and immune cells recruitments on injection sites.Our results demonstrate the key role of high aspect ratio nanorods in enhancing humoral and cellular immune responses and potential mechanism,and provide theoretical and practical guidance for optimizing the adjuvant effect of engineered HAP nanoparticles.