Preparation And Evaluation Of Non-covalent Glycosylated Peptides Conjugated Nanoparticles Vaccine

ObjectiveAs a member of the immunotherapy,vaccines have been widely used in prevention and treatment of diseases,which activate the immune system,and the antibodies and cytokines against antigens are induced.In addition,when the same antigens invade again,the memory cells are induced rapidly then differentiate into plasma cells and effector T cell.The recognition process is an important part of the immune response process;since many antigens including certain peptide antigens are T cell-independent antigens,which express slightly in vivo,thus they cant’t be recognized as a foreign component and fail to elicit the immune response in vivo.Therefore,how to improve the immunogenicity of these antigens in order to be recognized and ingested by the immune system is the main challenge for immunotherapy.The host-guest system of β-cyclodextrin has been widely used as drug delivery systems.Studies have shown that the host-guest system of(3-cyclodextrin can improve the efficacy and targeting of drugs,moreover,the gold nanoparticle carriers introduced by covalent glycosidic bonds display outstanding effects on improving antigen immunogenicity.As far as we have known,there has been no application of host-guest system of P-cyclodextrin on vaccines.In order to improve the immunogenicity of the antigens and enable the antigen to be effectively recognized by the immune system,we developed a novel vaccine carrier based on host-guest system of β-cyclodextrin.The reversible combination between the antigen and the carrier is relied on non-covalent interaction.Next,we chose a model antigen for evaluation of immunological activity.Then,based on this,in order to explore whether the vaccine carrier can be applied on the field of tumor prevention and therapy,another peptide antigen(NY-ESO-1157-165)was chosen to and tumor challenge were performed on melanoma model of C57BL6/J mice.The results from tumor challenge showed that the nanovaccine shoued a significant effect on melanoma therapy,which suggested this vaccine carriercan not only improve the immunogenicity of pepetide antigens but also provide a new choice for novel vaccine carrier development.Method1.Preparation of non-covalent glycosylated peptides conjugated nanoparticles vaccine The β-cyclodextrin was attached onto the gold nanoparticle by chemical modification as the host,while the antigen was modified to the adamantane carboxylic acid as the guest,the two are interacted by the recognization of the host-guest system of β-cyclodextrin;the obtained reaction solution was mixed with Freund’s adjuvant to prepare an emulsifier to immunize mice.The structures of compounds were identified by 1H-NMR,Thermogravimetric Analysis(TG),High Resolution Transmission Electron Microscopy(HTEM),Infrared spectrum,Mass spectrometry and Dynamic Light Scattering Analysis(DLS)et.al.2.Immunological evaluation of the nanovaccineFemale C57BL6/J mice were injected with the nano-vaccines(with 15.5 nmol FFRKSIINFEKL as antigen)subcutaneously in the 7-day intervals for 4 injections,the group of mice received Adamantanecarbox ylic acid-Acp-FFRKSIINFEKL was set as control.Peripheral blood of mice was collected on days 14,21,and 28,and serum was obtained by centrifugation.Antibody titers in serum were determined by ELISA.3.Investigate the relationship between dose and immune effectThe nanovaccine group and the peptide control group were injected with high-dose vaccine at the same time(with 31 nmol FFRKSIINFEKL as antigen).Antibody titers in the serum of mice were analysised by ELISA and compared with the low dose group.4.Prevention of melanoma by non-covalent glycosylated peptides conjugated nanoparticles vaccineFemale C57BL6/J mice were injected subcutaneously on 0,7,14,21 days with nano-vaccine(with 31 nmol SLLMWITQC as antigen)and peptide vaccine.On day 35,mice were injected with B16-F10 cells subcutaneously.Tumors size,body weight,and survival of each mouse were recorded.5.Therapy against melanoma of non-covalent glycosylated peptides conjugated nanoparticles vaccineFemale C57BL6/J mice were subcutaneously injected with B16-F10 cells on day 0,the nano-vaccine(with 31 nmol SLLMWITQC as antigen)and peptide vaccine were injected subcutaneously on day 7,14,21.Tumors size,body weight,and survival of each mouse were recorded.Results1.Preparation of non-covalent glycosylated peptides conjugated nanoparticles vaccine The non-covalent glycosylated peptides conjugated nanoparticles vaccine was prepared successfully and a sries of characteristic were performed.From 1H-NMR,the characteristic peaks of Ar-H and-SH confirmed that the intermediate compounds Tosyl-βCD and P-CD-SH have been successfully synthesized.The size of β-CD-SH modified gold nanoparticles(β-CD-Au)confirmed by electron microscopic analysis was around 10nm,while the size of nanoparticles is 300 nm after loading the FFRKSIINFEKL linked to the adamantanecarboxylic acid.2.Immunological evaluation of non-covalent glycosylated peptides conjugated nanoparticles vaccineHigh-affinity and long-term IgG antibodies were induced after injection of the nanovaccine.The mice from nano-vaccine group induced higher titers of IgG,IgM and Kappa antibodies than those from peptide control group.3.Investigate the relationship between dose and immune effectThe high dose vaccine induced higher levels of IgG,IgM,IgG1,IgG2b and Kappa antibodies compared to low dose vaccine.4.Prevention of melanoma by non-covalent glycosylated peptides conjugated nanoparticles vaccineIn the tumor prevention model,although the tumor of mice from the nanovaccine group and the peptide group grew more slowly than that of the PBS group,the difference between the two groups was not significant.In the survival analysis,mice from the nano vaccine group and the peptide group survived longer than mice from PBS group.However,there was no significant difference between the peptide group and the nanovaccine group.No obvious change of body weight was found in the nanovaccine group and the peptide group and the body weight of the PBS group decreased slightly early after injection of B16-F10 cells;however,the body weight of the three groups increased after the 11th day.Overall,the difference in body weight between the three groups of mice was not significant.5.Therapy against melanoma of non-covalent glycosylated peptides conjugated nanoparticles vaccineIn the tumor treatment model,there was no significant difference in the volume growth between the peptide group and the nanovaccine group in the early stage after inoculation of tumor cells.From the 14th day,the difference between the two groups gradually became obvious.Compared with the PBS group,the mice in the nanovaccine group had a significantly slower volume growth.However,compared with the tumor prevention model,the nanovaccine had a less inhibitory effect on melanoma in the treatment model,while the survival time of the tumor treatment model was similar to that in the tumor prevention model.The overall weight difference of the three groups of mice was not obvious.ConclusionsThis paper reports a novel peptide nanovaccine based on the host-guest system ofβ-cyclodextrin for the first time.Studies have shown that the size of nanoparticles is closely related to the abilities to be recognized by antigen-presenting cells(APCs).If the size is below 10 nm,antigens directly enter the lymph node,and fail to induce immune response;when the nanoparticle size is larger than 500 nm,the antigen is difficult to be taken up by the antigen-presenting cells,but only stay at the injection site[1,2]The size of nanoparticles after being loaded with FFRKSIINFEKL linked to adamantanecarboxylic acid is around 300 nm,thus is easily taken up by phagocytic cells or dendritic cells.According to the immunological activity assays,we found that mice that received the nano vaccine induced high titers and high-affinity antibodies,which were higher than the peptide group.It was found that higher titers of antibodies were induced in the mice received the high dose vaccine,demonstrating that the non-covalent glycosylated peptides conjugated nanoparticles vaccine of high dose is more effective.In the analysis of antibodies of IgG subtypes,we found that IgGl antibody titers were the highest among IgG1,IgG2b and IgG3 antibody titers,indicating that the FFRKSIINFEKL specific immune response was induced.In the exploration of the prevention and therapy against melanoma,we found that nanovaccine has a certain inhibitory effect on the growth of melanoma,and the therapeutic effect on tumor is better than prevention.