Immunopotentiating Effects And Antitumor Effects Of Ophiopogonin D Self-nanoemulsion Adjuvant

Research background:Vaccination remains the most cost effective means of controlling and preventing infectious diseases.Recombinant subunit vaccines are widely accepted because of their safety and efficiency.However,recombinant protein vaccines do not elicit a strong enough immune response and require vaccine adjuvants to enhance the immune activity of the antigen to achieve the desired immune effect.Currently,there are only a limited number of human vaccine adjuvants approved for marketing overseas,mainly including aluminium adjuvant,MF59 adjuvant and AS series adjuvants and so on,while only aluminium adjuvant has been widely used in vaccine development and production in China so far.Aluminium adjuvant is the longest used human vaccine adjuvant,but it has weak ability to induce cellular immune response.Many vaccines,especially tumour vaccines,urgently require novel adjuvants that can elicit an effective cellular immune response in the immunosuppressed tumour microenvironment.Therefore,the search for novel vaccine adjuvants that can enhance antigens to generate efficient humoral and cellular immune responses is the goal of researchers.Saponins have gained increasing interest from researchers for their biodegradability and wide range of sources,mainly focusing on QS-21,Ophiopogonin D and ginsenoside.When used as adjuvants,these saponins can enhance cytophagy,stimulate cytokine production by immune cells and activate the cyclic adenosine monophosphate-protein kinase A(c AMP/PKA)signalling pathway in lymphocytes to enhance the vaccine cellular and humoral immune response.For example,QS-21 induces a balanced Th1/Th2 immune response by acting on antigen-presenting cells and T cells.AS01,AS02 and Matrix-M complex adjuvants,of which QS-21 is a core component,promote cross-presentation of antigens by dendritic cells and induce a strong humoral immune response in inflammatory vesicles.Despite the unique potency and promise of QS-21 in vaccine clinical trials,its inherent shortcomings in terms of scarcity,heterogeneity,dose-limiting toxicity and chemical instability have hindered its adoption into the community.Therefore,the development of novel naturally derived saponin adjuvants that can overcome(or partially overcome)the shortcomings of QS-21 is of great interest.Ophiopogonin is a triterpenoid saponin from the lily family Ophiopogon japonicus.Previous studies have found that it has biological activities such as cell proliferation promotion,antibacterial and antitumour activity,but its use as a vaccine adjuvant has not been reported.Our team found that maitake saponin D(OPD)has good adjuvant activity and has the ability to enhance cellular and humoral immune responses.However,the water solubility of OPD is poor(<20μg/m L)and additional technical means are required to effectively load the antigen for adjuvant activity.Studies have shown that nano-based drug delivery systems(NDDS)can be applied to load antigenic proteins and exhibit outstanding properties such as lymphocyte targeting,tumour microenvironmental response and site-specific release,making them ideal delivery systems for vaccine antigens and adjuvants.The self-emulsifying delivery system is a mixture of oil,surfactant and co-surfactant,which has the advantages of good biocompatibility,high thermodynamic stability,high protein loading capacity and ease of preparation,and has more applications in vaccines.It not only improves the solubility of the vaccine adjuvant and the bioavailability of the antigen,but also increases the accuracy of antigen delivery to dendritic cells after administration.Therefore,based on the OPD with adjuvant activity obtained from our team’s previous research,we used the self-emulsification technology to prepare a novel self-nanoemulsion of ophiopogonin D(SND)adjuvant that can greatly improve the solubility of OPD,enhance the cellular and humoral immune response of vaccines and have anti-tumour immunoprotective effects,laying a solid experimental foundation and providing a scientific and theoretical basis for the development of excellent saponin-based adjuvants in China.Research objectives:In order to overcome the shortcomings of OPD such as poor water solubility and haemolytic toxicity,SND adjuvant with good solubility and particle size of 25-40 nm was prepared by low energy emulsification method based on our team’s patented nano-technology and formulation.After studying the appearance,physicochemical properties,stability and in vitro cytotoxicity of SND adjuvant,the vaccine was prepared using chicken ovalbumin(OVA)as the model antigen and its immunobiological effects were investigated.Then the subcutaneous E.G7-OVA cell model of C57BL/6 mice was established to observe the preventive and therapeutic protective effects of SND adjuvant.It will provides experimental techniques and theoretical support for the development of saponin-based adjuvants.Research methods:1.Preparation and quality characterisation of SND adjuvants(1)SND adjuvant preparation research methodBased on the previous work of the group,an oil-in-water self-nanoemulsion adjuvant loaded with OPD(SND)using Tween-80 as surfactant,glycerol as co-surfactant and Caprylic/Capric Triglyceride(GTCC)as the oil phase was designed and prepared,and its external characteristics as well as the particle size,potential and polymer dispersibility index(PDI)values of the nanoparticles were evaluated.(2)Quality characterisation method for SND adjuvantsThe SND adjuvants were examined by transmission electron microscopy(TEM),scanning electron microscopy(SEM),atomic force microscopy(AFM),Nano ZS dynamic light scattering nanoparticle sizer,Q600 thermogravimetric-differential calorimetry,Lambda950 spectrometer,sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE),western blot(WB)and methylthiazolyldiphenyl-tetrazolium bromide(MTT)colourimetric assay to determine the quality characteristics of SND adjuvant in terms of particle size,potential,PDI value,structure,weight loss,stability,IR absorption,stability of OVA protein in the delivery system and toxicity to dendritic cells(DCs).2.Evaluation of the immunological enhancing effect of SND adjuvant with OVA antigenThe SND adjuvant combined with mode antigen OVA-induced mouse-specific serum antibody levels,serum cytokine levels,splenocyte-secreted cytokine levels and the proportion of OVA-specific secreted IL-17A and IFN-γcells in splenocytes were measured using ELISA,ELISpot and grpⅠPanel 23-plex liquid-phase suspension microarray microarrays,respectively.3.Effect of SND adjuvant on E.G7-OVA tumor protection and preliminary mechanism study(1)Method for evaluating the immunoprotective effect of subcutaneous vaccination with SND adjuvant combined with OVA antigenA stable subcutaneous E.G7-OVA cell-loaded tumour(murine T lymphoma)model was established in C57BL/6 mice.Mice were immunised with SND adjuvant combined with OVA antigen subcutaneously before and after implantation,and the prophylactic and therapeutic anti-tumour protective effects were evaluated based on body weight,tumour volume,mouse survival rate and HE-stained pathological sections.(2)A method for studying the mechanism of immune protection by subcutaneous vaccination with SND adjuvant combined with OVA antigenDetection of subcutaneous retention time of SND adjuvant-enhanced OVA antigen in mice using the Small Animal In Vivo Imaging System(IVIS).Research results:1.Preparation and quality characterisation of SND adjuvantsSND adjuvant with an average particle size of 25.27±0.1931 nm,an average zeta potential of-12.9±2.17 m V and a PDI value of 0.274±0.018 was successfully prepared using Tween-80,glycerol and GTCC using a low energy emulsification method,which was clear and transparent and stable for 28 days at room temperature.The SND particles were observed by TEM,SEM and AFM to be in the range of 25-40 nm in size,with a smooth spherical surface and good dispersion.The results of SDS-PAGE and WB showed that the protein bands were neat,uniform and bright in all groups,confirming that the OVA protein was stable in OPD,SND and blank self-nanoemulsion(BSN).In addition,the MTT assay demonstrated that SND adjuvant had lower DCs cytotoxicity and higher cell viability(P<0.05)than OPD adjuvant.2.Evaluation of the immunological enhancing effect of SND adjuvant with OVA antigenELISA results for the immunological effect of SND adjuvant combined with OVA antigen in mice vaccinated intramuscularly showed that the OVA/SND group induced higher production of IgG(P<0.01),IgG1(P<0.05),IgG2a(P<0.05)and IgG2b(P<0.001)at day 35compared to the OVA/OPD group serum antibody titres,and expression levels of the Th1-type cytokines IFN-γ(P<0.01)and IL-1β(P<0.001),Th2-type cytokine IL-4(P<0.05)and Th17-type cytokine IL-17A(P<0.001).ELISpot assays showed that SND adjuvant significantly increased splenocyte expression of OVA-specific IFN-γ-T(P<0.01)and IL-17A-T(P<0.01)cell activation ratios,suggesting that SND adjuvant could enhance the activation of CD8~+helper T lymphocytes and CD4~+helper T lymphocytes.The results of the23 cytokines produced by the helper T cell(Th)subpopulation in the body fluids of immunized mice as measured by grpⅠPanel 23-plex liquid-phase suspension microarray microarrays showed that SND adjuvant significantly enhanced the OVA-specific Th1-type,Th2-type and Th17-type cell immune responses.This was specifically seen for Th1-type cytokines(IL-1α,IL-2,IL-12p40,IL-13,Eotaxin,KC,MCP-1,TNF-α,IFN-γ,IL-1β,IL-3,IL-12p70,MIP-1α,MIP-1β,RANTES,C-GSF and GM-CSF),Th2(IL-4,IL-5,IL-6,IL-9and IL-10)and Th17-type cytokines(IL-17A)levels were significantly increased.3.Effect of SND adjuvant on E.G7-OVA tumor protection and preliminary mechanism studyIn E.G7-OVA tumour-bearing mice,SND adjuvant combined with OVA antigen subcutaneously immunised mice could exert effective prophylactic and therapeutic anti-tumour protection,effectively inhibiting normal tumour growth and prolonging the survival time of mice.The results of HE-stained pathological sections of prophylactic and therapeutic tumour tissues showed that the OVA/SND group had a large infiltration of inflammatory cells at×100,×200 and×400 magnification compared to the OVA,OVA/BSN and OVA/OPD groups,and the tumour tissues did not grow compactly.The fluorescence intensity of Cy5.0 almost completely disappeared within 1 h and 24 h after immunisation of mice,whereas it was maintained for 384 h after inoculation with Cy5.0-OVA/SND.The results of the IVIS assay showed that SND adjuvant significantly prolonged the retention time of OVA antigen subcutaneously in mice(P<0.01).Research conclusions:1.SND adjuvant with good quality characteristics,stability and low cytotoxicity was successfully prepared.2.SND adjuvant is effective in enhancing the dual humoral and cellular immune response to OVA antigens,including the production of specific serum antibodies(IgG,IgG1,IgG2a and IgG2b),the secretion of different types of cytokines(Th1,Th2 and Th17),and the proliferation activation of IFN-γ-specific CD8~+T cells and IL-17A-specific CD4~+T cells.3.SND adjuvant has a better preventive and therapeutic effect on E.G7-OVA tumour-bearing mice and significantly prolongs the survival time of the mice.4.SND adjuvant delays the rapid release of OVA antigen and significantly prolongs its retention time subcutaneously in mice.