| Background Immunosuppressive tumor microenvironment significantly suppresses anti-tumor immune responses, promotes tumor occurrence and progression, and hinders the effectiveness of tumor immunotherapies. Interleukine (IL)-10 is an important suppressive cytokine which significantly suppresses the functions of anti-tumor effector cells such as CTLs and Thl and thus promotes the tumor immune escape and tumor growth. Vascular endothelial growth factor(VEGF) plays an important role in the formation of new blood vessels and the proliferation and migration of endothelial cells, and is strongly associated with tumor invasion and metastasis. Further, VEGF stimulates abnormal blood vessels and hypoxia in tumor, which induces the proliferation and infiltration of suppressive immune cells and thus contributes significantly to the formation of immunosuppressive tumor microenvironment.Purpose The study aimed to induce persistent VEGF and IL-10 specific antibodies through active immunization with anti-cytokine vaccines, and intervene in their immunosuppressive and tumor promoting effects. And, the work will assess the potentials of anti-cytokine vaccine on modulating tumor microenvironment and promoting anti-tumor immunityMethods Two potential antigenic peptides of mouse IL-10 (peptide A and peptide B) and one antigenic peptide of mouse VEGF were obtained by bioinformatic analyses, and then were chemically synthesized. The three peptides were chemically coupled to HBcAg VLPs presenting three continious lysine(KKK) on the surface (HBcAg-KKK). The peptide from mVEGF was also inserted into immunodominant domain of HBcAg through recombinant methods. Further, a recombinant protein with IL-10 fused to C-terminal of thiorexion was constructed. C57 mice were immunized with the vaccine candidates which were mixed with Freund’s adjuvants. Specific antibody responses were detected with ELISA. After the antibodies were fully induced, TC-1 tumor cells were grafted subcutaneously into mice. The tumor growth was monitored dynamically. At endpoint of the experiment, the mice were sacrificed, and the tumor mass was weighed. Further, splenocytes were isolated and stimulated with HPV16 E7 antigenic peptide and IFN-γ expressing cells were detected by ELISPOT, the level of Th2 cells were checked with flow cytometry, the concentration of IFN-γ in serum was measured with ELISA, and histology of tumor sections were assessed with H&E staining.Results Antigenic peptides of IL-10 and VEGF were presented on the surface of HBcAg VLPs through chmical coupling or in the form of fusion protein by gene recombinant technology. Mice immunized with the constructed vaccine candidates produced specific antibody responses. In a TC-1 cells grafted mouse tumor model, preventive immunization with HBcAg-VEGF, HBcAg-KKK-IL-10/A, and HBcAg-KKK-IL-10/F suppressed the growth of tumor. Comparing with the control mice, the immunized mice showed elevated IFN-γ expressing splenocytes level, increased serum IFN-γ concentration, lowered Th2 cells level in splenocytes, and reduced tumor angiogenesis.Conclusion The ani-cytokine vaccine targeting IL-10 or VEGF holds the potential to effectively modulate tumor microenvironment and promote tumor antigen-specific cellular immune responses, and thus could be a promising approach of tumor immunotherapy.Background Over the years, cervical cancer caused by HPV is a severe threat to human health. Although prophylactic HPV vaccines are now available commercially, the cost of vaccination is quite expensive. In addition, due to the lack of crossing protection between HPV genotypes, multivalent vaccine is necessarily needed, which requires the costs of vaccine production to be lowered. And thus, the technology of producing vaccine waits to be further improved.Purpose This study aimed to investigate the influence of the utilization of different carbon and nitrogen sources on the growth of Pichia pastoris in highly dense fermentation, and also the influence on HPV16_L1 yield under the guidance of methanol oxidase 1 (AOX1) promoter, constitutive translation elongation factor-1 (TEF-1) promoter or formaldehyde dehydrogenase (FLD) promoter, and provide a optional approach for producing HPV vaccine at a low cost and in an easily operated way.Methods Four carbon sources including glycerol, methanol, glucose, and sorbitol, and two different carbon sources including methylamine and ammonium sulfate were used for high-density fermentation and HPV16_L1 expression in Pichia pastoris. According to the curve of dissolved oxygen (DO), the carbon source fed-time and flow rate were controled. Sampling was performed once every 3 hours, and fermentation broth was collected at 144h later. The OD600 value was read and cell wet weight was weighed. The dynamic protein expression of HPV16_L1 was measured by Western blot. The expressed HPV L1 was purified with POROS 501+S cationic exchange chromatography, and virus-like particles were visualized with microscopy.Results In recombinat yeast cells with the use of a constitutive promoter PTEF-1, using glycerol as a carbon source, the wet weight of cells could reach to 510g/L, and the expression of HPV16_L1 maintain at a high level before 72h; as for using methanolor glucose as carbon soource, the wet weight of cells reached to about 220g/L, and the expression of HPV L1 remained at a high level after 144 hours using methanol and the expression was significantly decreased in 60 hours using glucose; in contrast, yeast cells failed to obtain a high-density in a fermenter using sorbitol as a solo carbon source. As a whole, using glycerol as the carbon source the maximum yield of protein could be obtained in about 72 hours. In recombinat yeast cells with the use of promoter PFLd, the influence of different combinations of carbon and nitrogen sources for fermentation were investigated. The wet weights of yeast cells were 320 g/L,300g/L,430g/L, and 400g/L respectively for methylamine+methanol, methanol+ammonium sulfate, methylamine+glycerin, and glycerine+ammonium sulfate. The combination of glycerol and methylamine got the largest amount of biomass, and the highest expression of HPV L1 was obtained by the combination of methanol and ammonium sulfate. In recombinat yeast cells with the use of promoter PAOX1, yeast cells were grew in glycerol and then induced by methanol, and the cell wet weight could reach to 417 g/L. The expressed HPV L1 was purified preliminarily, and virus like particles were observed.Conclusion The differnet combination of a carbon source and nitrogen source significantly affects the growth of P. pastoris cells in high density fermentation and also the expression of HPV16_L1 directed by different promoters, and thus finding the optimal balance between biomass and the expression level of protein of interest is important for lowering the production costs of HPV vaccines. |
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