Zika Virus Recombinant Protein Vaccine Based On Modification With β-glucan And Mannan

Zika virus(ZIKV)is a single-stranded positive-stranded RNA virus that has received widespread attention for its serious threat to global public health,ZIKV can lead to microcephaly in the fetus and serious neurological disease such as Guillain-Barre syndrome in adults.Because ZIKV is transmitted through both mosquito-borne and nonvector-borne routes,it is difficult to control the prevalence of Zika disease.Also,there are high risks and serious health threats after infection with ZIKV.Therefore,it is critically important and urgent to develop safe and effective vaccines against ZIKV infection.Recombinant protein vaccine is a new type of vaccine containing virus antigen protein,which is widely favored by researchers because of its good safety.However,due to the weak immunogenicity of the virus antigen itself,it is necessary to utilize adjuvants and select a suitable delivery system to induce strong humoral and cellular immunity.As a key viral element of ZIKV,E protein was an ideal antigen for vaccine development.In order to enhance the immunogenicity of E protein,the adjuvant is needed for formulation of E protein.As a polysaccharide adjuvant,β-glucan could activate macrophages and trigger intracellular processes.Thus,conjugation withβ-glucan was a strategy to improve the immunogenicity of E protein.However,the antigenic epitopes of E protein and the immunomodulatory sites of β-glucan were shielded in the conjugate.Moreover,the conjugate might elicit the undesired immune response to β-glucan.Thus,the acidic-labile hydrazone and the thiol-sensitive disulfide bonds were used as the linkers between E protein and β-glucan to obtain the conjugate E-PS-4.When the conjugate was uptaken into immune cells,the hydrazone hydrolysis and disulfide reduction could sufficiently detach E protein and β-glucan under the conditions of low pH in the lysosome and high glutathione in the cytoplasm,which could overcome the disadvantages of shielding antigen epitopes with each other.As compared with the conjugate without hydrazone and/or disulfide bond,the E-PS-4 could elicit robust E protein-specific IgG titers that was 27-fold higher than E protein,and 8-fold decrease in β-glucan-specific IgG titers.The E-PS-4 could also elicited high levels of IFN-y,TNF-α,IL-2 and IL-10.Moreover,E-PS-4 could facilitate the activation of dendritic cells without significant toxicity to the organs.A harmacokinetic study revealed that the half-life of E-PS-4 in serum was 3.3-fold longer than that of E protein,and the clearance rate was reduced to 1/5.Accordingly,conjugation of E protein with β-glucan by the hydrazone and disulfide linkers could promote a potent cellular and humoral immune response to E protein.The EDⅢ protein is the domain Ⅲ of ZIKV E protein,which contains most of the neutralizing antibody epitopes without any antibody-dependent enhancement(ADE)effect.However,EDⅢ also necessitates to be formulated with adjuvants and an antigen delivery system to improve its immunogenicity.Hemoglobin(Hb)could regulate inflammation,cytokine levels and activate macrophage.Mannan is a polysaccharide of the fungal cell wall with an immunomodulatory activity.Therefore,EDⅢ was conjugated with Hb and mannan.Both Hb and mannan functioned as the adjuvant to form a carrier HM as the delivery system for EDⅢ.The disulfide bond was used as the linker to combine with EDⅢ to obtain the conjugate HM-EDⅢ-2.HM and EDⅢ can be effectively detached by the reduction of disulfide bond in the high glutathione condition in the immune cell.Structural analysis showed that the secondary and tertiary structure of EDⅢ was essentially maintained upon conjugation of HM.The HM-EDⅢ-2 elicited robust EDⅢ-specific IgG titers that was 29-fold higher than EDⅢ protein,and could also elicit high levels of IFN-γ,IL-2,IL-5 and IL-10,along with no apparent toxicity to the organs.Moreover,the pharmacokinetic study revealed that the half-life of HM-EDⅢ-2 in serum was 2-fold longer than that of EDⅢ protein,and the clearance rate was reduced to 1/10.Thus,HM-EDⅢ-2 could boost a strong humoral and cellular immune response to EDⅢ.Our study was expected to provide a feasibility approach to develop a robust and potentially safe ZIKV vaccine.