Rhamnose-Modified Carrier Protein For Construction Of Redox-Responsive β-1,3-Glucan Antifungal Vaccines

With the increasing morbidity of systemic fungal infections worldwide,fungal treatment faces a serious challenge.Therefore,the development of effective antifungal vaccines is of great urgency and relevance.Most antifungal vaccines mainly target fungal surface polysaccharide antigens.These polysaccharides,such as β-1,3-glucan glycoantigens,are highly conserved in all growth stages of the fungus and have specific chemical structures,making them ideal targets for vaccine design.The traditional vaccine construction strategy is to couple the glycoantigens with carrier proteins,however,the carrier proteins have strong immune responses that are inhibitory to the glycoantigens.In a previous study,it was found that a less immunogenic carrier protein could recruit endogenous antibodies and target antigen-presenting cells(APCs)by modifying small hapten molecules such as rhamnose(Rha),thus effectively enhancing the immune response to glycoantigens.In this paper,a redox-responsive linker was designed to modify rhamnose Rha on the carrier protein BSA for the construction of a novel β-1,3-glucan fungal vaccine.In vitro experiments and in vivo mouse immunoreactivity assessment revealed that the redox-responsive linker could promote lysosomal escape and antigen release,and effectively enhance the immune response to β-1,3-glucan glycoantigens.The main findings were as follows:(1)A redox-responsive β-1,3-glucan glycoantigens fungal vaccine was constructed.The compounds such as rigid linker L1,redox-responsive linker L2 and Rha hapten derivatives were prepared by chemical synthesis,and their structures were verified by NMR and mass spectrometry.The glycoantigen β-1,3-glucan was obtained by curdlan acid hydrolysis,and the degree of polymerization(DP)was verified by MAIDI-TOF-MS to be 5-13.β-glucan-BSA was obtained by coupling the glycoantigen to a carrier protein via rigid linker L1 with a glycoantigen loading of 7.8%.Then Rha was coupled to β-glucan-BSA by two different linker to obtain two glycoconjugates,β-glucan-BSA-L1-Rha and β-glucan-BSA-L2-Rha,with Rha loading of 2.2%and 2.4%,respectively.(2)The redox-responsive linker was verified to be able to break in response to reducing conditions and promote lysosomal escape of vaccine molecules.In vitro kinetic experiments of the redox-responsive linker verified that the redox-responsive linker can break disulfide bonds rapidly in the presence of reducing agents.Analysis by lysosomal co-localization experiments demonstrated that the redox-responsive β-glucan-BSA-L2-Rha vaccine molecule was able to undergo lysosomal escape,while the rigid linker L1-modified β-glucan-BSA-L1-Rha was unable to undergo lysosomal escape.(3)The redox-responsive linker further enhanced the immune activity of the rhamnose modified carrier protein-constructed β-glucan vaccine.The in vivo immunization experiments in mice revealed that,in the previous study,the antibody titers of the Rha-modified glycoconjugated fungal vaccine were significantly enhanced with the aid of the endogenous antibody Rha.Moreover,the redox-responsive glycocalyx fungal vaccine β-glucan-BSA-L2-Rha elicited the strongest immune response,demonstrating that the redox-linked promoted the lysosomal escape of the vaccine molecule and effectively improved the immune efficiency of the vaccine.Meanwhile,immunofluorescence experiments demonstrated that the β-glucan antibodies produced by the vaccine could effectively recognize heat inactivated Candida albicans(HKCA)hyphae,indicating that the immune response provoked by the vaccine had fungal-specific recognition ability.