Experimental Treatment Of AIM-2 Peptide Tumor Vaccine Based On Adenovirus Vector In Malignant Glioma

Malignant glioma is characteristic of invasive growth,no clear boundaries with brain tissue,difficulty in complete removal and easy to relapse.Currently,traditional treatments,such as surgery,radiotherapy and chemotherapy,have limited effect to malignant glioma.Immunotherapy provided a new treatment for malignant glioma due to its advantages of high specificity,safety,and less side-effect.Tumor vaccine is an important method of cancer immunotherapy.Tumor vaccine activates the patient’s own immune system to produce cell immune response and humoral immune response by introducing the tumor-associated antigens(TAAs)to the body of the patient in various forms,which could inhibit the growth of the tumor or remove the tumor eventually.Identification and selection of tumor antigens would play an important role in the preparation of tumor vaccine and its therapeutic effect.AIM-2(Antigen isolated from Immunoselected Melanoma-2)was a mutant protein that was initially discovered in human melanoma.The amino acid sequence of the antigenic epitope of AIM-2 is RSDSGQQARY,which was named AIM-2 peptide.Previous studies indicated that AIM-2 was specifically expressed in a number of tumors such as glioblastoma,breast cancer,ovarian cancer,lung cancer and colon carcinomas,but not expressed in normal cells.Therefore,AIM-2 epitope peptide can be used as a therapy target of tumor immunotherapy,which could be applied to the experimental therapeutics of malignant gliomas and other tumors.Chemical synthesized small peptides have low immunogenicity and are susceptible to be degraded in the body.Therefore,it is very crucial for improving the immunogenicity of tumor peptide and increasing the numbers of tumor peptide by choosing a suitable immune carrier for presenting tumor antigen peptide.Currently,commonly used immune carriers include viral vector and virus-like particles(VLPs).Common viral vector include adenovirus,retrovirus,lentivirus,and adeno-associated virus and so on.As a widely used immune carrier,adenovirus has some advantages of strong immunogenicity,insertion of multiple foreign genes,high safety and no integration into the genome.Adenovirus capsid contains 240 hexon proteins.Small exogenous peptide could be presented at the surface of viral particle when it is inserted into HVR5 of Hexon.Therefore,when the peptide AIM-2 was inserted into HVR5 region,the adenovirus with high copies of AIM-2 peptide on the capsid could induce effective immune response to AIM-2 peptide.Common virus-like particles include papilloma virus,hepatitis B virus,hepatitis C virus and other virus-like particles.180 or 240 Hepatitis B virus core protein(HBc)monomers could be self-assembled into Hepatitis B virus-like particles(HBc-VLPs),which have strong immunogenicity.High density of epitope peptides could be displayed on the surface of HBc-VLPs when epitope peptide was inserted into the major immune region(MIR)at the top of HBc-VLPs,which could induce strong humoral or cellular immune response against epitope peptide.Therefore,HBc-VLPs can be used as AIM-2 peptide immune carrier,and enhance the immune effect of AIM-2 peptide vaccine.In summary,five tumor vaccines were designed in this study by means of adenoviral vector and hepatitis B virus-like particles,then tumor vaccines were evaluated for its therapeutic effect for malignant glioma therapy in tumor-bearing animal model.The results will provide new ideas and new strategies for vaccine designing and immunotherapy protocol of malignant glioma.The research project includes the following aspects.1.The construction,preparation and purification of the adenovirus carrying the epitope AIM-2.By means of molecular biology methods,the AIM-2 peptide was inserted into the corresponding adenovirus.Then the adenoviral plasmid vectors based AIM-2 peptide were digested by Pac I,and were transfected into HEK 293 cells.Finally,adenoviral vector based AIM-2 peptide tumor vaccines were achieved by CsCl density gradient centrifugation.2.The study of immunotherapy in the malignant glioma tumor-bearing animal model with adenovirus vector based AIM-2 peptide tumor vaccines.(1)The evaluation of immune effect of the five tumor vaccines in vivo.Normal female mice were grouped randomly and intramuscularly injected with five different adenoviruses respectively for 3 times with an interval of two weeks,the immunizing dose was 2×1010 vp each mouse for one time.Then the level of antibodies against AIM-2 in the serum was evaluated by ELISA at different time points after immunization.(2)According to the amino acid sequence of AIM-2 protein(pAIM-2),the oligos for pAIM-2 fragment was synthesized.The oligos were annealed and inserted into the lentiviral vector.The produced lentivirus was used to construct stable mouse glioma cell line(pAIM-2/G422)expressing exogenous pAIM-2 protein.The pAIM-2/G422 cells are used target cells for evaluating immunotherapy effect of tumor vaccines.(3)Experimental therapy of adenoviral vector based AIM-2 peptide tumor vaccine in malignant glioma bearing mouse.The animal tumor model was established by s.c inoculation with pAIM-2/G422 cell in the right hind leg of mice after three immunizations by different tumor vaccine,the inoculation dose was 1×106 each mouse.Then the tumor volume was measured and tumor growth curve was drawn,which was used for evaluation of the effect of tumor vaccine on malignant glioma tumor growth.The results of this study are as follows:1.Five stains of adenovirus based AIM-2 peptide tumor vaccine were successfully constructed and prepared as follows.(1)Adenovirus expressing HBc-AIM-2 fusion protein in the El region:Ad5-E1-CMV-HBc-AIM-2/E3-CMV-luciferase-T2A-eGFP.The virus titer-was 1.13 ×1012vp/ml.(2)Adenovirus with the insertion of AIM-2 peptide into Hexon:Ad5-HVR5-AIM-2/E3-CMV-Iuciferase-T2A-eGFP.The virus titer was 0.7 x 1012vp/ml.(3)Adenovirus expressing HBc-AIM-2 fusion protein in the El region and the insertion of AIM-2 peptide into Hexon:Ad5-E1-CMV-HBc-AIM-2/HVR5-AIM-2/E3-CMV-Luciferase-T2A-eGFP.The virus titer was 1.43 × 1012vp/ml.(4)Adenovirus carrying an expression cassette secreting the fusion protein HBc-AIM-2.(5)Ad5-E1-CMV-H1H2-HBc-AIM-2/E3-CMV-luciferase-T2A-eGFP.The virus titer was 1.3 × 1012vp/ml.(6)Adenovirus carrying an expression cassette in the El region secreting the fusion protein HBc-AIM-2 and the insertion of AIM-2 peptide into Hexon:Ad5-El-CMV-H1H2-HBc-AIM-2/HVR5-AIM-2/E3-CMV-Luciferase-T2A-eG FP.The virus titer was 1.2 x 1012vp/ml.2.After immunization by each adenovirus for three times,humoral immune response was induced by each adenovirus,the antibodies against AIM-2 could be detected in serum.The level of anti-AIM-2 was the highest in the mice stimulated by adenovirus with an expression cassette in the El region secreting the fusion protein HBc-AIM-2 and the insertion of AIM-2 peptide into Hexon.3.The five strains of adenovirus based AIM-2 peptide tumor vaccines could inhibit the growth of malignant glioma,but theirinhibitory effect on tumor growth were different.The best one showing the inhibitory effect on the growth of glioblastoma is the adenovirus carrying an expression cassette secreting the fusion protein HBc-AIM-2 and the insertion of AIM-2 peptide into Hexon.The second one is the adenovirus expressing HBc-AIM-2 fusion protein and the insertion of AIM-2 peptide into Hexon.The third one is the adenovirus with the insertion of AIM-2 peptide into Hexon.The fourth one is the adenovirus carrying an expression cassette secreting the fusion protein HBc-AIM-2.The last one is the adenovirus expressing HBc-AIM-2 adenovirus.In summary,five adenovirus based AIM-2 peptide tumor vaccines were designed and produced in this study.All tumor vaccines could induce a humoral immune response against AIM-2 peptide and inhibit the growth of malignant glioma of tumor-bearing mice.Among them,the adenovirus carrying an expression cassette secreting the fusion protein HBc-AIM-2 and the insertion of AIM-2 peptide into Hexon showed the best the inhibitory effect on the growth of the tumor.These results laid the foundation for the research of immunotherapy and clinical therapy for AIM-2 based vaccine in malignant glioma.