The Research On T Cell Immune Memory Induced By Different Vaccination Schedules Of Mycobacterium Tuberculosis Subunit Vaccine,and Coadministration Of IL-7 And IL-15

Part Ⅰ:The research on T cell immune memory induced by different vaccination schedules of Mycobacterium tuberculosis subunit vaccine,and coadministration of IL-7 and IL-15Tuberculosis(TB)is one of the most serious infectious diseases in the world caused by Mycobacterium tuberculosis(M.tuberculosis)infection.TB ranks as the first leading cause of death from infectious disease and the ninth leading cause of death in the world.The effective way to prevent and control infectious diseases is to vaccination.The main reason that why TB is difficult to control,is the lack of effective TB vaccine.Attenuated Mycobacterium bovis Bacillus Calmette-Guerin(BCG)is the only licensed TB vaccine in clinical currently.It could protect children from severe TB,but provide variable protection in adult,which waned with time and almost lost protection.It is reported that BCG vaccination mainly induces effector memory T cells(TEM)and effector T cells(Teff)rather than long-lived central memory T cells(TCM)due to persistent bacteria.Thus,it is extremely urgent to develop novel vaccines and vaccination strategies to induce sufficient quantity and quality of TCM to provide long-term protection against M.tuberculosis.infection.Protein subunit vaccines have the capability to induce TCM and provide long-term protection against M.tuberculosis.Subunit vaccines typically require multiple boosting to confer high quality and quantity of immune memory responses.Some researches believed that the vaccination intervals for boosting have a effect on the types of T cells,which is critical to induce long term immune memory against infection.Therefore,optimal vaccination intervals for boosting are critical to generate long term immune memory.As for TB protein-subunit vaccines,most studies traditionally applied a vaccination schedule of 3 doses at 2-week or 3-week intervals.However,there is no investigation on the correlates of vaccination schedule and T cell immune memory,which is to be helpful to optimize TB subunit vaccine immunization schedule to induce ideal immune memory.At the same time,subunit vaccines require adjuvant to induce a stronger immune response.Because a suitable adjuvant may increase the level of immune response.However,adjuvants currently in clinical used are few and mainly promote the humoral immune response.Therefore,new adjuvants which induce cell-mediated immune response are essential to the development of M.tuberculosis vaccines.Some studies showed that IL-7 and IL-15 are essential for development and maintenance of memory T cells.So,in this study,applying M.tuberculosis subunit vaccine ESAT6-Ag85B-MPT64<190-198>-Mtb8.4-Rv2626c(LT70 for short)and Mtb10.4-HspX(MH for short),which showed stronger protective efficacy in mice,we explored the T cell immune memory induced by different vaccination schedules,and coadministration of IL-7 and IL-15.Objective 1,To investigate the correlates of immune memory and vaccination schedules of TB subunit vaccine.In this study,applying M.tuberculosis subunit vaccine LT70,we explored the momory responses of three different vaccination schedules of 0-3-6w,0-4-12w and 0-4-24w.2,To investigate the role of IL-7 and IL-15 as adjuvants in enhancing the immune memory and protective efficacy,we comboned them with TB subunit vaccines.Methods 1,The subunit vaccine LT70 was prepared and mice were immunized with LT70 three times according to the following schedules:① 0,3rd and 6th week respectively(0-3-6w),②0,4th and 12th week(0-4-12w)and ③ 0,4th and 24th week(0-4-24w).2,Construction and packaging of recombinant adenoviruses rAd-IL-7,rAd-IL-15,rAd-IL-7-IL-15 and rAd-IL-7-IL-15(Linker)by adenovirus packaging system AdMax.Mice were immunized by TB subunit vaccine LT70+MH with cytokines IL-7 and IL-15 at 0,2nd and 4th week.3,The production of Ag85B-specific IgGl was detected by ELISA;The production of antigen specific IL-2 and IFN-y were detected by ELISA in spleen;Cytokine frequencies of IFN-γ,IL-2 and TNF-α on the CD4+ and CD8+ T cells were evaluated by flow cytometry for intracellular cytokine analysis;Antigen-specific central memory-like T cells(TCM like)were evaluated by cultured ELISPOT assay;IFN-γ secretion on the CD4+ and CD8+T cells following twice-stimulation was performed by intracellular cytokine analysis staining to analyze the numbers of antigen specific memory T cells to reflect the quality of Tcm like;The cell proliferation was detected by EdU;The number of TB10.4 specific CD8+memory T cells was evaluated by TB10.44-12pentamer;The protective efficacy was evaluated by BCG challenge.Results 1,We found that 0-3-6w(1.84±0.42)and 0-4-12w(1.42±0.19)produced significantly higher levels of Ag85B-specific IgG1 than 0-4-24w group(0.91±0.29)(P<0.001 and P<0.05,respectively).Vaccination schedules of 0-4-12w and 0-4-24w induced higher level of antigen specific IL-2,IFN-γ and TNF-α than 0-3-6w immunization.Among them,0-4-12w induced the highest level of IL-2.Moreover,by cultured IFN-γ ELISPOT,IFN-γ secretion following twice-stimulation with antigens and proliferation of memory T cells,we found that the vaccination schedule of 0-4-12w(5.01 ±0.14 Log 10 CFU)elicited higher numbers of central memory-like T cells,stronger proliferation of memory T cells.Moreover,the vaccination schedule of 0-4-12w(5.01+0.14 Log 10 CFU)provided higher protective efficacy against M.bovis BCG challenge than 0-3-6w(5.42+0.17 Log10 CFU)did.2,Recombinant adenovirus vector containing IL-7,IL-15,IL-7-IL-15,IL-7-IL-15(Linker)and vector have been constructed successfully and obtained high viral titer,8 ×109PFU/ml,1×1010PFU/ml,9×109PFU/ml,1 ×1010PFU/ml and 1 ×1010PFU/ml,respectively.The mice were immunized with LT70 and MH combined with IL-7 IL-15,IL-7-IL-15 and IL-7-IL-15(Linker)at 0,2nd and 4th week.At 20 weeks post vaccination,TCM like were detected by cultured ELISPOT IFN-γ secretion on the CD4+ and CD8+ T cells following twice-stimulation was performed by intracellular cytokine analysis staining;The cell proliferation was detected by EdU;The number of TB 10.4 specific CD8+memory T cells was evaluated by TB10.44-12pentamer.The results showed:①The number of long-term antigen-specific IFN-γ producing cells in the groups of LT70+MH+rAd-IL-7(299.3±20.9 SFC/1×106cells),LT70+MH+rAd-IL-15(273.3±25.2 SFC/1×1O6 cells),LT70+MH+r Ad-IL-7-IL-15(325.6±19.7 SFC/1×106cells),LT70+MH+rAd-IL-7-IL-15(Linker)(382.2±32.9 SFC/1×106cells)and LT70+MH+rAd-IL-7+rAd-IL-15(361.4± 17.2 SFC/1×106cells)induced a larger increase than the control groups of LT70+MH(186.0±30.5 SFC/1×106cells)and LT70+MH+rAd-vector(211.6±22.4 SFC/1×106cells).In the meantime,the group of LT70+MH+rAd-IL-7-IL-15(Linker)(382.2±32.9 SFC/1×106cells)induced a larger number of long-term antigen-specific IFN-γ producing cells than the groups of LT70+MH+rAd-IL-7,LT70+MH+rAd-IL-15 and LT70+MH+rAd-IL-7-IL-15.②In the spleen,the groups of LT70+MH+rAd-IL-7,LT70+MH+rAd-IL-7-IL-15,LT70+MH+rAd-IL-7-IL-15(Linker)and LT70+MH+rAd-IL-7+rAd-IL-15 produced significantly higher levels of IFN-γ secretion following twice-stimulation with antigens than that of the control groups of LT70+MH and LT70+MH+rAd-vector.In the bone marrow,compared with the control groups of LT70+MH and LT70+MH+rAd-vector,the levels of IFN-γ secretion following twice-stimulation with antigens in the groups of LT70+MH+rAd-IL-7,LT70+MH+rAd-IL-7-IL-15(Linker)and LT70+MH+rAd-IL-7+rAd-IL-15 were obviously higher.③In the spleen,for CD4+ T cells,the frequency of cells incorporated with EdU in the groups of LT70+MH+rAd-IL-7-IL-15(Linker)(1.57%),LT70+MH+rAd-IL-7+rAd-IL-15(1.13%)and LT70+MH+rAd-IL-7(1.05%)were obviously higher than that of the control groups of LT70+MH(0.23%)and LT70+MH+rAd-vector(0.27%);The LT70+MH+rAd-IL-7-IL-15(Linker)group showed an enhanced incorporation of 1.15%and 0.67%than the groups of LT70+MH+rAd-IL-15 and LT70+MH+rAd-IL-7-IL-15(P<0.001 and P<0.05),respectively.For CD8+ T cells and total lyphocytes,the tendency was consistent with that of CD4+ T cells.④The frequency of TB 10.4-specific CD8+ memory T cells in the group of LT70+MH+rAd-IL-7-IL-15(Linker)(0.6%)was highest,followed by LT70+MH+rAd-IL-7(0.40%).LT70+MH+rAd-IL-7-IL-15(0.31%).LT70+MH+rAd-IL-15(0.29%),LT70+MH+rAd-vector(0.22%),LT70+MH(0.15%),respectively.At 24 weeks post vaccination,the protective efficacy was evaluated by BCG challenge.The results showed:⑤Against BCG infection,in the lung,compared with the control groups of LT70+MH and LT70+MH+rAd-vector,the bacterial load of LT70+MH+rAd-IL-7.LT70+MH+rAd-IL-15,LT70+MH+rAd-IL-7-IL-15,LT70+MH+rAd-IL-7-IL-15(Linker)and LT70+MH+rAd-IL-7+rAd-IL-15 all had a significant decrease;Among them.the group of LT70+MH+rAd-IL-7-IL-15(Linker)was the lowest,declining 0.381og10 CFU and 0.501og10 CFU compared with the groups of LT70+MH+rAd-IL-7 and LT70+MH+rAd-IL-15,respectively.In the spleen,the bacterial load in the groups of LT70+MH+rAd-IL-7(4.73±0.95Logl0 CFU),LT70+MH+rAd-IL-15(4.74±0.14Log10 CFU),LT70+MH+rAd-IL-7-IL-15(4.75±0.12Log10 CFU),LT70+MH+rAd-IL-7-IL-15(Linker)(4.58±0.12Log10 CFU)and LT70+MH+rAd-IL-7+rAd-IL-15(4.66±0.12Log10 CFU)were significantly lower than the control groups of LT70+MH(5.02+0.15Log10 CFU)and LT70+MH+rAd-vector(5.02+0.15Log 10 CFU).Conclusion 1,The vacination schedule of 0-4-12w generated more Tcm like and provided stronger immune protection against BCG.The results suggest that prolonging the immunization interval of TB subunit vaccine to some extent contributes to inducing more abundant Tcm like and providing stronger immune protection against mycobacteria infection.2,Our study demonstrates that supplementation of subunit vaccine LT70+MH with IL-7,IL-7-IL-15(Linker)and IL-7+IL-15 would improve the efficacy of subunit vaccine by augmenting Tcm like and provide higher protective efficacy against M.tuberculosis challenge.Part Ⅱ:The immunotherapeutic effectiveness of IL-7 and IL-15 against cervical cancerCervical cancer(CC)is one of the most common malignant tumors affecting women.It was reported that CC was both the second-most common cause of cancer and death from cancer in women.CC is an important public health problem in low income countries,where over 85%of the global deaths occur annually.At present,main therapies of cancer are surgery,radiotherapy and chemotherapy.However,because of CC’s high case fatality and lack of effective therapy,it was aroused widely attention.In recent years,many scientists have increasingly focused on immunotherapy.This research focuses on the immunotherapy role of immunostimulatory cytokines IL-7 and IL-15.Objective:To investigate adenovirus mediated IL-7 and IL-15 on anti-tumor effect of mice U14 cervical subcutaneous transplantation tumor and analyze their mechanism.Methods:Establishing mice U14 cervical cancer ascites model and subcutaneous transplantation tumor model;Measuring the tumor size after treatment of rAd-IL-7 and rAd-IL-15;Detecting CD4+CD25+Foxp3+Treg in the spleen after rAd-IL-7 and rAd-IL-15 treatment by FCM;Detecting the expression of programmed death 1(PD-1)on the CD4+ and CD8+T cells were evaluated by FCM;IFN-γ were detected in sera by ELISA;the activity of lymphocytes by CCK-8.Results:1,We successfully established U14 cervical cancer ascites model and subcutaneous transplantation tumor model.2,Compared with rAd-vector and PBS group,the treatment of rAd-IL-15 significantly down-regulated the frequency of CD4+CD25+Foxp3+Treg(P<0.05);The tumor volume of mice with an additional treatment of rAd-IL-7,rAd-IL-15 and rAd-IL-7+rAd-IL-15 significantly decreased;The treatment of rAd-IL-15 significantly down-regulated the frequency of PD-1 in CD4+ and CD8+T cells(P<0.05);The activity of lyphocytes in the group of rAd-IL-15 was higher(P<0.001).Conclusion:1,rAd-IL-7,rAd-IL-15 and rAd-IL-7+ rAd-IL-15 all inhibited growth of U14 cervical subcutaneous transplantation tumor.2,rAd-IL-15 could reduce the expression level of CD4+CD25+Foxp3+Treg and PD-1 on CD4+T cell and CD8+T cells.rAd-IL-15 could inhibit the growth of U14 cervical subcutaneous transplantation tumor by down-regulating the expression of CD4+CD25+Foxp3+Treg and PD-1 on CD4+ and CD8+T cells;Moreover,it could induce more production of IFN-y and enhance the activity of lymphocytes.