Preparation And Immunogenicity Of Alum-stabilized Pickering Emulsion

Combating against the emerging pandemics,designing safe,effective and immune-enhanced vaccine adjuvant of the existing materials is regarded as the optimal strategy for rapid clinical translation.To date,the strict clinical requirements for the safety of adjuvants leads aluminum hydroxide adjuvant(termed as“alum”)has been used as the sole licensed adjuvant approved in China.Unfortunately,alum tends to attach on the membrane rather than entering the dendritic cells(DCs),leading to the absence of intracellular transfer and process of the antigens,and thus antigens are processed by lysosomal pathway and presented by the major histocompatibility complexⅡ,which leads to an enhanced humoral immune response and a limited T-cell-mediated immunity.Because of this disadvantage,alum alone cannot provide comprehensive protection in practical application,and cannot meet the increasing demand of adjuvants.Therefore,how to rationalize the modification of aluminum adjuvant,so that the vaccine adjuvant obtained after the modification can inherit the safety of aluminum adjuvant and effectively induce cellular immune response is an urgent problem to be solved.To address this,alum was packed on the squalene/water interphase,formed a Particulate Alum-stabilized Pickering Emulsion(PAPE),which combined the advantages of emulsion and particulate adjuvants.Intriguingly,with the dense array of alum on the oil/water interphase,PAPE demonstrated better hydrophobicity and membrane affinity compared with the alum alone,thus harbors a higher affinity for DCs uptake,which provoked the uptake and cross-presentation of antigens.At the same time,the surface positive charge property of aluminum adjuvant granulated emulsion is conducive to the realization of antigen lysosome escape into the cytoplasm,promote the cross-presentation of antigen in the cytoplasm,and effectively activate the cellular immune response.The specific research contents are as follows:(1)The Food and Drug Administration(FDA)-approved squalene as oil,alum was closely packed on the oil-water interface by one-step ultrasonic and formed PAPE.Conducted a series of optimizations included alum concentrations,buffer types,aqueous phase p H,the smallest emulsion droplets were prepared and with an adequate shelf life and stability for the distribution of PAPE.SEM results demonstrated a raspberry-like morphology of PAPE.(2)RBD of COVID-19 was selected as antigens to explore the potential of PAPE for the development of COVID-19 vaccines.First of all,PAPE adsorbed more than 90%of the fluidic antigens within 30 min,indicating that it contained a dense array of antigens to replicate SARS-Co V-2 and effectively trigger an immune response.Secondly,alum is closely packed at the oil-water interface,which can promote the interaction between PAPE and DCs membrane,and promote the uptake of antigens.PAPE,which contained surface-packed alum,experienced enhanced protonation within the acidic endosomes,which might have stimulated the rapid inflow of H~+(proton sponge effect),boosted the rupture of the lysosomes,and helped the RBD escape into the cytoplasm.Higher antibody titers indicated the immense potential for stronger antibody secretion to bind with the infecting SARS-Co V-2.At the same time,higher RBD-specific Ig G2a titers indicated a mixed Th1 and Th2 immune responses of PAPE.Being based on clinically approved alum and squalene,PAPE exhibited a good biosafety profile.Considering the accessibility of alum adjuvants,the feasibility of microgel adsorptions on the oil/water interphase,as well as the bedside mixing of antigens,PAPE may provide insights for the development of a safe,accessible,and efficient adjuvant strategy for COVID-19 vaccines.