Optimizing Immunization Regimens Of Tuberculosis Subunit Vaccine Boosting BCG To Induce Long-lived Memory T Cell-mediated Immune Responses

BackgroundTuberculosis（TB）is a chronic infectious disease caused by Mycobacterium tuberculosis（M.tuberculosis）infection.T cell-mediated immune responses are important for host defense against M.tuberculosis infection.Bacillus Calmette-Guerin（BCG）is the only TB vaccine licensed for use in human beings,and is effective in protecting children against severe miliary and is variable in adults.The possible reason might be that BCG vaccination mainly induces effector memory T cell-mediated immunity,which is not sufficient to provide long-term anti-TB immune protection.BCG priming and subunit vaccine boosting is considered as a way to improve immunity in adults.However,the immunization regimens of boosting BCG with subunit vaccine did not induce long-term immunity in animal experiments or clinical trials.Therefore,how to boost BCG-primed immune memory with subunit vaccine to induce long-lived memory T cells is urgently needed to be investigated.The composition of the antigen,the number of stimulations,and the interval between immunizations all affect the generation of memory T cells.Our previous work found that compared with the traditional immunization program of 0-3-6 weeks,prolonging the intervals of immunization,the schedule of 0-4-12 weeks,could increase the number and function of long-lived memory T cells and improve the protective efficacy.And then we mainly studied optimizing the boosting schedule of subunit vaccines consisting of BCG and "non-BCG" antigens to induce long-term immune memory.In addition,interleukin 7（IL-7）and interleukin 15（IL-15）was used as vaccine adjuvant to enhance T cell immune memory.Our previous work found that recombinant adenovirus rAd-IL-7-Linker-IL-15 and recombinant adeno-associated virus rAAV-IL-7 helped tuberculosis subunit vaccine promote the generation of long-lived memory T cells.Therefore,we mainly investigated whether IL-7 and IL-7-Linker-IL-15 could help subunit vaccine enhance BCG-primed immune memory.The regulatory effects of IL-7 on T cell activation were further explored.Objective1.Following BCG priming,to study the effect of immunization regimens（boosting times and intervals）of BCG and "non-BCG" antigens on T cell immune memory.2.Following BCG priming,to investigate whether rAAV-IL-7 and rAAV-IL-7-Linker-IL-15 could help subunit vaccine enhance BCG-primed immune memory.3.To study the regulation of cytokine IL-7 on the ubiquitin system in T cell activation.Methods1.Following BCG priming,the effect of boosting times and intervals of BCG and "non-BCG" antigens on T cell long-term immune memory.（1）Vaccine preparation.Construction and purification of recombinant M.tuberculosis fusion protein ESAT6-CFP10（EC）.Mtb10.4-HspX（MH）fusion protein was constructed by our laboratory in previous study.The adjuvant（DP）was the mixture of Dimethyl dioctadecyl ammoniumbromide（DDA）and Polyriboinosinic polyribocytidylic acid（PolyI:C）.MH and EC were mixed with DDA and PolyI:C to form MH/DP and EC/DP tuberculosis subunit vaccines,respectively,representing the subunit vaccine consisting of BCG and "non-BCG" antigens,respectively.（2）Immunization schedule.BCG-primed mice were boosted with MH and EC twice or thrice subcutaneously with a long interval:MH/EC immunizations at 12-24 weeks groups;MH/EC immunizations at 12-16-24 weeks groups.The BCG-primed mice were revaccinated subcutaneously with BCG（5 × 105 CFU in 100 μl per mouse）at 24 weeks to be consistent with other boosters on immunoassays.PBS and BCG without boosting groups were used as control.Short interval immunization schedules:the BCG-immunized mice were boosted with MH at 8-14 weeks or EC at 8-10-14 weeks subcutaneously（SC）.PBS and BCG without boosting groups were used as control.（3）Immunodetection.The immune memory was evaluated at 12 weeks and 28 weeks after the last immunization.① The proportions of IL-2 and IFN-γ producing CD4+and CD8+T cells were detected by flow cytometry.②The number of central memory T cells（TCM）was detected by the cultured ELISPOT.③After antigen restimulation,IFN-y producing CD4+and CD8+T cells were detected by flow cytometry,reflecting the function of long-lived memory T cells.④The proportion of TB10.4-specific CD8+T cells were detected by Pentamer.⑤EdU method was used to detect the proliferation ability of CD4+T cells.⑥The levels of antigen-specific antibodies IgG,IgGl and IgG2c in serum were detected by ELISA.⑦H37Ra strain challenge experiment evaluated the immune protection effect of vaccine-induced memory T cells.2.Following BCG priming,the effect of recombinant adeno-associated virus rAAV-IL-7 helped tuberculosis subunit vaccine MH/DP boost BCG on T cell long-term immune memory.（1）Construction of recombinant adeno-associated virus.Adeno-associated virus rAAV-EGFP,rAAV-IL-7 and rAAV-IL-7-Linker-IL-15 were constructed and packaged using the adeno-associated virus helper system.（2）Immunization schedule.6-8 weeks C57BL/6 mice were subcutaneously immunized with BCG（5 × 105 CFU/mice）and boosted with the fusion protein Mtb 10.4-HspX（MH）in adjuvant of liposome DDA and Toll-like receptor 3 agonist Poly I:C（DP）combined with recombinant adeno-associated virus rAAV-IL-7 and rAAV-IL-7-Linker-IL-15 respectively at 12 and 24 weeks.rAAV-EGFP was the control group.（3）Immunodetection.The short-term immune response induced by the vaccine was detected at 1 week after vaccine boosting.Vaccine-induced long-term immune responses were detected at 12 to 26 weeks after the last immunization.①Flow cytometry was analyzed to central memory（CD44+CD62L+）and effector memory（CD44+CD62L-）T cells in CD4+T cells and CD8+T cells.②The evaluation method was the same as the immunodetection above.（4）Molecular mechanism of IL-7 affecting T cell immune memory.①Specific recognition of OVA257-264 antigen OT-I mouse model:OT-I mice were immunized with rAAV-IL-7 combined with OVA257-264 antigen for 7 days.Spleen CD8+T cells were sorted by magnetic bead sorting method.The changes in signaling molecules and transcription factors of Akt,Foxol,IL-7r and c-Myc in spleen CD8+T cells were analyzed by western blot and RT-PCR,respectively.②Murine T cell lymphoma EL4 model:After IL-7 and antigen PMA stimulation,western blot was used to analyzed the changes in signaling molecules of Akt,Foxol and IL-7R;③IL-7 knockout（IL-7 Knock out,IL-7KO）mouse model:Differences in signaling molecules between IL-7 KO and（wild-type,WT）mice were analyzed by RT-PCR.3.IL-7 regulated ubiquitin systerm in T cell activation.（1）OT-I mouse model.OT-I mice were immunized with OVA257-264 antigen and rAAV-IL-7.On 7 days after immunization,spleen CD8+T cells were sorted by magnetic beads.RT-PCR and Western blot were used to detect the transcription and protein levels changes of ubiquitinase and deubiquitinase.（2）EL4 cells model.EL4 cells were co-stimulated with IL-7 and PMA for 1 or 3 hours,and the changes of ubiquitinase and deubiquitinase were detected by western blot.（3）IL-7 KO model.The spleen CD4+T cells and CD8+T cells of IL-7 KO mice and WT mice were sorted by magnetic beads.The changes of ubiquitinase and deubiquitinase transcription levels were detected by RT-PCR.（4）Effects of ubiquitin editing enzyme A20 on T cell immune memory.rAAV-A20 was constructed and packaged using the adeno-associated virus helper system.C57BL/6 mice were immunized with rAAV-A20 combined with subunit vaccine.On 3 days and 7 days after immunization,transcription factors change of regulating T cell differentiation in CD4+T and CD8+T cells were detected by RT-PCR and western blot.Results1.Following BCG priming,BCG and "non-BCG" antigens required different immunization schedules to induce T cell long-term immune memory.Following BCG priming,MH boosting twice at 12-24 weeks,and EC immunizations thrice at 12-16-24 weeks could produce long-term immune responses and improved the BCG-primed protective efficiency.①Proportion of IFN-γ producing CD4+and CD8+T cells:at 12 weeks after the last immunization,compared with MH/DP boosted at 12-16-24 weeks group（0.82 ±0.46）and EC/DP immunizations at 12-24 weeks group（0.61 ± 0.21）,the proportion of IFN-γ producing CD4+T cells in MH/DP boosted at 12-24 weeks group significantly increased（1.97 ± 0.70,P<0.05）.Compared with PBS group（0.23 ±0.12）and BCG boosted group（0.36 ± 0.15）,MH/DP boosted at 12-24 weeks group（0.88 ± 0.32）and EC/DP immunizations at 12-16-24 weeks group（1.33 ± 0.56）cells produced more IFN-γ producing CD8+T cells（P<0.05）.②Number of IFN-γ producing T cells:at 28 weeks after the last immunization,the results of the cultured ELISPOT found that compared with the EC/DP immunizations at 12-24 weeks group（86.2 ± 33.7 SFCs/5 × 106 cells）,MH/DP boosted at 12-24 weeks group induced a higher number of antigen-specific IFN-γsecreting long-lived memory T cells（166 ± 22.4 SFCs/5 × 106 cells）（P<0.05）.Compared with MH/DP boosted at 12-16-24 weeks group（73 ± 52 SFCs/5 × 106 cells）,EC/DP immunizations at 12-16-24 weeks group（162.25 ± 18.3 SFCs/5 × 106 cells）significantly increased the number of antigen-specific IFN-γ secreting long-lived memory T cells（P<0.05）.③Proportion of IFN-γ producing CD4+T cells:at 28 weeks after the last immunization,after antigen restimulation,compared with EC/DP immunizations at 12-16-24 weeks group（1.59±0.34）and MH/DP boosted at 12-16-24 weeks group（1.00 ± 0.38）,MH/DP boosted at 12-24 weeks group（3.58 ± 0.37）increased the proportion of IFN-γ secreting CD4+T cells（P<0.05）.Compared with MH/DP boosted at 12-16-24 weeks group（1.00 ± 0.38）,EC/DP immunizations at 12-16-24 weeks group increased the proportion of IFN-γ secreting CD4+T cells（3.27±1.21）（P<0.05）.④Proportion of TB10.4-specific CD8+T cells:at 19 weeks after the last immunization,compared with the BCG group,MH/DP boosted at 12-24 weeks group induced more TB10.4-specific CD8+T cells（P<0.05）.⑤Proportion of EdU+CD4+T cells:at 12 weeks after the last immunization,compared with MH/DP boosted at 12-16-24 weeks group（1.42 ± 0.63）,MH/DP boosted at 12-24 weeks group（2.18 ± 0.32）and EC/DP immunizations at 12-16-24 weeks group（2.00 ± 0.36）significantly increased the proportion of EdU+CD4+T cells.At 28 weeks after the last immunization,compared MH/DP boosted at 12-16-24 weeks group（1.20 ± 0.78）and EC/DP immunizations at 12-24 weeks group（1.89 ±0.82）,MH/DP boosted at 12-24 weeks group（2.83 ± 0.57）had a higher proportion of EdU+cells（P<0.05）.⑥Bacterial load in the lungs:The immunized mice were challenged intranasally with avirulent M.tuberculosis H37Ra at 19 weeks after the last immunization.The results showed that compared with BCG group,MH/DP boosted at 12-24 weeks group and EC/DP immunizations at 12-16-24 weeks group reduced the bacterial load in lung tissue,declining approximately 0.1 logio CFU（P<0.05）.When the immunization interval was shortened,compared with BCG group,MH/DP boosted at 8-14 weeks group and EC/DP immunizations at 8-10-14 weeks group reduced vaccine-induced T cell proliferation and anti-tuberculosis protection.In addition,compared with MH/DP or EC/DP boosted twice at 12-24 weeks groups,the levels of antigen-specific IgG,IgG1,and IgG2c in serum were increased in the MH/DP or EC/DP boosted three times at 12-16-24 weeks groups,and were maintained until 28 weeks after the last immunization.2.Following BCG priming,rAAV-IL-7 helped tuberculosis subunit vaccine MH/DP enhance T cell immune memory.Following BCG priming,rAAV-IL-7 promoted the generation of TCM and TEM cells,rAAV-IL-7-Linker-IL-15 promoted the generation of TEM cells.The results showed that 12 weeks after immunization,compared with BCG+MH/rAAV-EGFP group,the groups of BCG+MH/rAAV-IL-7 increased the frequency of CD4+TCM cells and CD4+TEM cells（P<0.05）.Compared with the control of BCG+MH/rAAV-EGFP（38.8 ± 2.9）,BCG+MH/rAAV-IL-7-Linker-IL-15 increased the frequency of CD8+TEM cells（43.34 ± 5.1,P=0.03）.Following BCG priming,rAAV-IL-7,rather than rAAV-IL-7-Linker-IL-15,helped TB subunit vaccine MH/DP enhance the T cell short-term immune response.The results showed that on 7 days after first and second boosting,compared with the control of rAAV-EGFP,rAAV-IL-7 promoted the proportion of vaccine-induced IFN-γproducing CD4+T cells（0.8 ± 0.1,0.3 ± 0.07）and CD8+T cells（1.1 ± 0.2,0.35 ±0.06）（P<0.05）.Following BCG priming,rAAV-IL-7,rather than rAAV-IL-7-Linker-IL-15,helped subunit vaccine MH/DP enhance the T cell long-term immune response.① Proportion of IFN-γ or IL-2 producing CD4+T cells:12 weeks after last immunization,compared with the groups of BCG+MH/rAAV-EGFP（0.24 ± 0.05）and BCG+MH/rAAV-IL-7-Linker-IL-15（0.33 ± 0.03）,the group of BCG+MH/rAAV-IL-7（0.63±0.08）produced higher frequency of IFN-γ producing CD4+T cells（P<0.05）.BCG+MH/rAAV-IL-7 group produced higher frequency of IL-2 producing CD4+T cells（1.72 ± 0.14）than the group of BCG（1.23 ± 0.27,P<0.05）.②Proportion of IFN-γ producing CD4+and CD8+T cells:at 12 weeks after the last immunization,compared with the groups of BCG and BCG+MH/rAAV-EGFP and BCG+MH/rAAV-IL-7-Linker-IL-15,the group of BCG+MH/rAAV-IL-7 facilitated frequencies of IFN-γ producing CD4+T cells and CD8+T cells（P<0.05）.At 25 weeks after the last immunization,compared with the groups of BCG+MH/rAAV-EGFP（0.5 ± 0.09,1.07 ± 0.3 for CD4+T and CD8+T cells,respectively）,the group of BCG+MH/rAAV-IL-7 enhanced frequencies of IFN-γ producing CD4+T cells（0.7±0.05,P=0.04）and CD8+T cells（1.73±0.27,P=0.04）.③ Proportion of TB10.4-specific CD8+T cells:at 12 weeks after the last immunization,compared with the BCG+MH/rAAV-EGFP group,the BCG+MH/rAAV-IL-7 group could increase the proportion of TB10.4-specific CD8+T cells（P<0.05）.④Proportion of EdU+CD4+T cells:at 12 weeks after the last immunization,compared with the groups of BCG+MH/rAAV-EGFP（1.96 ± 0.13）and BCG+MH/rAAV-IL-7-Linker-IL-15（1.67±0.16）,the proliferation of antigen-specific CD4+T cells enhanced significantly in the group of BCG+MH/rAAV-IL-7（2.57 ± 0.33）（P<0.05）.⑤Bacterial load in the lungs:The immunized mice were challenged intranasally with avirulent M.tuberculosis H37Ra at 26 weeks after the last immunization.The results showed that rAAV-IL-7 helped subunit vaccine MH/DP reduce bacteria load in lung tissues,declining approximately 0.2 log10 CFU compared with BCG group（P<0.05）,and declining approximately 0.1 logio CFU compared with BCG+MH/rAAV-EGFP group（P<0.05）.rAAV-IL-7 activated Akt-Foxol signaling and increased the transcriptional level expression of Eomes,Tcf7,Id3,and Bach2,which control memory differentiation in spleen CD8+T cells of OT-I mice.In addition,IL-7 promoted Akt-Foxol-IL-7R expression in EL4 cells.Compared with WT mice,the expression of IL-7 receptor downstream signaling and transcription factors controlling memory T cell differentiation decreased in spleen CD4+and CD8+T cells of IL-7 KO mice.3.IL-7 regulated ubiquitin system in T cells activation.①OT-I mice were immunized with rAAV-IL-7 and OVA257-264.On 7 days after stimulation,compared with rAAV-EGFP,rAAV-IL-7 promoted the ubiquitination of intracellular proteins in CD8+T cells and increased the transcription levels of ubiquitinase Cbl-b,Fbxo38 and decreased the transcription levels of deubiquitinase Usp15,Uspl8 and increased the transcription and protein levels of ubiquitinase TRAF6 and deubiquitinase A20.②EL4 cells were stimulated with PMA and IL-7 alone or co-stimulated with PMA and IL-7 for 1 h or 3 h.Compared with EL4 cell without stimulation,PMA and IL-7 alone or co-stimulation increased the protein levels of ubiquitinase TRAF6 and ubiquitin editing enzyme A20.Compared with PMA and IL-7 co-stimulation,the expression of A20 was higher in the presence of proteasome inhibitor for 3 hours.③Compared with WT mice,transcription levels of A20 were decreased in spleen CD4+T and CD8+T cells of IL-7 KO mice.④rAAV-A20 regulated the expression of transcriptional levels that control T cell differentiation,as shown in up-regulation of memory differentiation transcription factors Foxol and IL-7r and down-regulation of effector differentiation transcription factor Blimp 1.Conclusion1.Following BCG priming,BCG antigen MH/DP boosted twice at 12-24 weeks and "non-BCG" antigen EC/DP immunizations three times at 12-16-24 weeks,could promote the vaccine-induced T cell long-term immune memory.2.Following BCG priming,recombinant adeno-associated virus rAAV-IL-7 combined with tuberculosis vaccine MH/DP boosted at 12-24 weeks promoted vaccine-induced short-term immune response and TCM cell-mediated long-term immune memory.3.IL-7 regulated ubiquitin system in T cell activation,promoted the expression of ubiquitinase TRAF6 and ubiquitin editing enzyme A20 in T cells.