The Study On Immunological Enhancement Mechanism Induced By Autophagy Mediated DNA Vaccine Against Japanese Encephalitis

Objective:To probe the immune effect and protective mechanism of DNA vaccine mediated by autophagy,thus provide novel ideas for the research and development of Japanese encephalitis?JE?DNA vaccine.Methods:1.Construction and identification of pJME-LC3 plasmidLC3B coding gene fragment was amplified and synthesized by RT-PCR according to the sequence retrieved from GenBank and then recombined to the pJME plasmid which contained JEV prM-E protein coding gene for constructing the autophagy-mediated pJME-LC3 DNA vaccine followed by identifying with double enzyme digestion by BamH I/EcoR I or sequencing.The pJME-LC3 plasmid was transfected into CHO-K1 cells by liposome assay at different time or concentration,and the plasmid encoding protein was detected by western blot.G418 was used to screen positive cell strain while resistant clones was observed by optical microscope.Plasmid encoding protein in positive strain was identified after several generations of collection.pJME-LC3 plasmid was transfected into CHO-K1 cells for observing autophagic cell colony by optical microscope or autophagosome by transmission electron microscopy.Autophagy marker protein LC3?and substrates protein p62 were then detected through western blot method.Cells were sequentially treated with GFP-LC3 adenovirus and pJME/pJME-LC3 plasmid followed by labeling through indirect immunofluorescence method,and then co-localization of plasmid coding proteins and autophagosomes was observed by laser confocal microscopy.2.Animal grouping and immunization120 BALB/c mice were randomly divided into 6 groups.Those in pcDNA3.1?+?,pJME,pJME-LC3 and pJME-LC3+3-MA group were respectively intramuscular?i.m?injected with different plasmids?100?g/100?L?while the other two groups received PBS?100?L,i.m?or intraperitoneal?i.p?injection with 100?L JE live attenuated vaccine?JEV-L?.The immunization was performed three times,at two-week intervals.Mice in pJME-LC3+3-MA group received intraperitoneal injection of 60?g/d autophagy activity inhibitor 3-MA from the initial immunization until the final.3.Viral challengeFor the first bunch:15 mice/group were immunized with different vaccines as previous;For the second bunch:120 BALB/c mice were were randomly divided into 6 groups.Mice in pcDNA3.1?+?,pJME,pJME-LC3 and pJME-LC3+3-MA group were respectively injected with different plasmids?200?g/100?L,i.m?while the other two groups received PBS?100?L,i.m?or 100?L JEV-L?i.p?.The immunization was performed three times,at two-week intervals.Mice in pJME-LC3+3-MA group received60?g/d autophagy activity inhibitor 3-MA?i.p?from initial immunization until the final.Three weeks after the final immunization,mice in each group were intraperitoneally injected with JEV virulent strong strain?10⁵ PFU/500?L?for challenge.4.Study on immune effect and mechanism of pJME-LC3 DNA vaccineThree weeks after the final immunization,25 mice in each group were sacrificed and spleen were taken aseptically to separate splenic lymphocytes and DCs.Indirect immunofluorescence method was used to observe the expression of plasmid coding protein in spleen DCs of immunized mice.DCs uptake of FITC-Dextran,CD3⁺CD4⁺/CD3⁺CD8⁺T lymphocyte subsets were analyzed by flow cytometry.JEV specific CTL activity of immunized mice was detected with LDH released assay.The serum was isolated from peripheral blood one week after each immunization and three consecutive weeks after the final immunization?1,3,5,6 and 7w?.JEV specific IgG1/IgG2a antibody in serum samples and cytokines?IL-4,IL-10,IFN-?,IL-12?secreted by splenic T lymphocytes of immunized mice were detected by ELISA.Western blot method was used to detect JAK2/STAT1 signaling pathway proteins?pJAK2,pSTAT1?and the downstream transcription factor T-bet.3.Study on immunological memory and protective immunity of pJME-LC3 DNA vaccineFlow cytometry was used to analyze CD4+/CD8+memory T lymphocyte subsets in spleen of immunized mice.The condition and survival number of each group were observed within 3 weeks after challenge,and the survival rate was calculated.80%plaque reduction neutralization test?PRNT80?was used to detect neutralizing antibody titer in serum of immunized mice.Results:1.Recombinant plasmid pJME-LC3 was successfully constructedThe sequencing results showed that the gene sequences of JEV prM,E protein and LC3B were consistent with those retrieved from GenBank.Agarose gel electrophoresis indicated that:After double enzyme digestion with BamH?/EcoR?,pJME-LC3 plasmid released a DNA fragment which has the same size as LC3?390bp?.The other two DNA fragments have the same size as pcDNA3.1?+??5428bp?and prME?2001bp?,which released after after digesting by the same enzyme.2.pJME-LC3 plasmid coding protein expressed rapidly and stably in vitropJME-LC3 plasmid coding protein expressed only 12h after transfection and increased with the increase of treated time?12-36h?or plasmid concentration?1-4mg/mL?.G418 was used to screen the resistant clones strain of CHO-K1 cells transfected with pJME-LC3,and it was found that pJME-LC3 plasmid encoding protein expressed stably in resistant clones strain without attenuation.3.pJME-LC3 promoted autophagy activity of CHO-K1 cellsObserved by optical microscope,autophagic colonies in pJME-LC3 group was significantly more than that in pJME group,and the effect was similar to rapamycin treated group.pJME-LC3 induced more autophagosomes than pJME under transmission electron microscope.The autophagy marker protein LC3 II in pJME-LC3 group was significantly higher than that in pJME group,while the autophagy substrate protein p62showed an opposite trend.4.pJME-LC3 plasmid encoding protein co-located with LC3 in autophagosomeLaser confocal microscopy showed that the green puncta of LC3 in pJME-LC3plasmid transfected cells aggregated in the cytoplasm and membrane,and was merged with plasmid coding protein which labeled with red fluorescence in the autophagosome as yellow puncta.The green fluorescence in pJME plasmid transfected cells was dispersed in the cytoplasm without merging with plasmid coding protein.5.Expression of plasmid coding protein in splenic DCs of immuned murineFluorescence microscopy revealed that plasmid coding protein expressed only in minority DCs of pJME as conventional JE DNA vaccine control group.In pJME-LC3group,plasmid encoding protein expressed in majority DCs.However,in pJME-LC3+3MA group which autophagy activity was inhibited,plasmid coding protein expressed only in few DCs.No plasmid coding protein expression was found in DCs of empty vector group pcDNA3.1?+?as negative control.6.pJME-LC3 promotes endocytosis of splenic DCs in immunized miceThe ability of DCs of pJME-LC3 group to uptake FITC-Dextran was significantly stronger than that of JEV-L group as attenuated live vaccine control?P<0.05?,pJME as conventional DNA vaccine control group?P<0.05?and pJME-LC3+3MA group which autophagy activity was inhibited?P<0.05?.There was no significant difference among the later three groups.7.pJME-LC3 enhances proportion of CD3⁺CD4⁺/CD3⁺CD8⁺T lymphocytesThe percentage of CD3⁺CD4⁺T lymphocytes in pJME-LC3 group was?45.66±5.04?%,significantly higher than that in other groups?P<0.05?.The proportion of CD3⁺CD4⁺T lymphocytes in pJME as conventional DNA vaccine control group was?37.40±5.23?%,which was not significantly different from that in JEV-L control group?35.84±2.75?%and pJME-LC3+3-MA group which autophagy activity was inhibited?34.46±3.87?%.The proportion of CD3⁺CD4⁺T lymphocytes in pcDNA3.1?+?negative group was?26.38±4.69?%,which was consistent with?25.25±3.82?%in PBS as blank control group,both were lower than other groups?P<0.05?.The proportion of CD3+CD8+T lymphocytes in pJME-LC3 group was?30.23±1.43?%,significantly higher than that in other groups?P<0.05?.The proportion of CD3⁺CD8⁺T lymphocytes in pJME as conventional DNA vaccine control group was?15.89±1.45?%,which has not statistically significant comparing with JEV-L control group?15.07±0.96?%and the pJME-LC3+3-MA group which autophagy activity was inhibited?13.42±1.58?%.The proportion of CD3⁺CD8⁺T lymphocytes in pcDNA3.1?+?negative group was?9.67±1.17?%,which was basically consistent with?9.98±1.80?%in PBS as blank control group,both were significantly lower than that in other groups?P<0.05?.8.pJME-LC3 enhances JEV specific CTL activityWhen the ratio of effector and target cell was set as 10:1,JEV specific CTL activity of mice in different immune groups showed that:The CTL activity of pJME-LC3 group was?55.72±2.34?%,obviously higher than that of any other groups?P<0.05?.There was no significant difference among the groups of JEV-L control group?39.43±2.85?%,pJME as conventional DNA vaccine control group?37.88±2.47?%and pJME-LC3+3-MA group which autophagy activity was inhibited?31.96±3.21?%.9.pJME-LC3 enhances JEV specific IgG2a antibodyThe IgG1 antibody titer in PBS as blank control group and Vector groups did not change significantly from 1 week after the initial immunization to 3 weeks after the final immunization,were lower than other immune groups,and there was no significant difference between the two groups at 1,3,5,6 or 7w.The titers of IgG1 antibody at 1,3,5,6 or 7w in the pJME-LC3,pJME,JEV-L or pJME-LC3+3-MA group had no significant difference.IgG2a antibody titer was lower in the PBS as blank control group and the pcDNA3.1?+?as negative control group from 1 week to 3 weeks after the first immunization,and there was no significant difference at 1,3,5,6 or 7w.The titer of IgG2a antibody in the pJME-LC3 group gradually increased and reached the maximum 3weeks after the final immunization,while the titer and rising speed of IgG2a antibody in JEV-L control group,the pJME as conventional DNA vaccine control group and the pJME-LC3+3-MA group which autophagy was inhibited were all lower than those in pJME-LC3 group at 1,3,5,6 or 7w,and attenuated 3 weeks after the final immunization.10.pJME-LC3 promotes the secretion of Th1 cytokines by splenic lymphocytesIFN-?secreted by T lymphocyte in pJME-LC3 immunized mic was?48.26±5.47?pg/mL,significantly higher than pJME as conventional DNA vaccine control group?35.09±7.16?pg/m L?P<0.01?,pJME-LC3+3-MA group which autophagy was inhibited?33.64±4.80?pg/mL?P<0.05?or JEV-L control group?19.45±5.76?pg/m L?P<0.01?,while JEV-L group was significantly lower than pJME group and pJME-LC3+3-MA group,the differences were statistically significant?P<0.05?.IFN-?in PBS as blank control group,pcDNA3.1?+?as negative control group were?5.65±1.28?pg/mL or?7.82±1.94?pg/mL,either was lower than all other groups?P<0.05?.Il-12 in pJME-LC3 group was?62.19±9.75?pg/mL,significantly higher than that in pJME as conventional DNA vaccine control group?44.61±7.49?pg/mL?P<0.05?,pJME-LC3+3-MA group which autophagy was inhibited?39.88±9.04?pg/mL?P<0.05?and JEV-L control group?29.64±6.32?pg/mL?P<0.01?.IL-12 in PBS as blank control group,pcDNA3.1?+?as negative control group were?15.24±4.21?pg/mL and?16.90±6.46?pg/mL,either was lower than other groups?P<0.05?.There was no significant difference in the levels of cytokines IL-4 and IL-10 among all groups?P>0.05?.11.pJME-LC3 promotes the expression of signalling pathway protein pJAK2,pSTAT1 and the transcription factor t-bet in T lymphocytesThe expression of pJAK2,pSTAT1 and the downstream transcription factor T-bet in splenic T lymphocytes of immunized mice in pJME-LC3 group were significantly higher than that in JEV-L control group,the pJME as conventional DNA vaccine control group or pJME-LC3+3-MA group in which autophagy activity was inhibited,with statistically significant differences?P<0.05?.There was no significant difference among the later three groups.The differences in the expression of these three proteins between PBS as blank control group and pcDNA3.1?+?as negative control group was no statistical significance.12.pJME-LC3 enhances the ratio of CD4⁺,CD8⁺T_CM/T_EMM of BALB/c miceThe ratio of CD4⁺T_CM/T_EMM in pJME-LC3 group was?2.34±0.79?,higher than that in pJME as conventional DNA vaccine control group?1.93±0.64,P<0.05?,JEV-L control group?1.58±0.27,P<0.05?or pJME-LC3+3-MA group in which autophagy activity was inhibited?1.56±2.02,P<0.05?.The ratio of CD4⁺T_CM/T_EMM in PBS as blank control group?0.94±0.59?was consistent with that in pcDNA3.1?+?as negative control group?0.99±0.37?,both of which were lower than other groups?P<0.05?.The ratio of CD8⁺T_CM/T_EMM in pJME-LC3 group was?0.62±0.30?,higher than that in pJME as conventional DNA vaccine control group?0.40±0.29,P<0.05?,JEV-L group?0.36±0.13,P<0.05?or pJME-LC3+3-MA group in which autophagy activity was inhibited?0.36±0.21,P<0.05?.The ratio of CD8⁺T_CM/T_EMM in PBS as blank control group?0.28±0.17?was consistent with that in pcDNA3.1?+?as negative control group?0.27±0.05?,both of which were lower than other groups?P<0.05?.13.pJME-LC3 promotes rapid and continuous production of neutralizing antibodiesThe neutralizing antibody titer in pJME-LC3 group increased only 2 days before challenge?1:20?,and reached 1:160 at 14 days after challenge.In addition,neutralizing antibody titers in other groups decreased 21 days after challenge,while no attenuation was observed in the pJME-LC3 group.These results suggested that the production speed,growth rate of antibody titer,absolute value and duration of mice in pJME-LC3immunized group were better than those in the pJME as conventional DNA vaccine control group,JEV-L control group or pJME-LC3+3-MA group in which autophagy activity was inhibited.14.pJME-LC3 enhances protective immunity of BALB/c mice of JETwo days after challenging with JEV strong strain?YN2016-1?,most of the mice began to show damp hair,loss of appetite,weight loss and other symptoms,or death.On day 21 after challenge,for the first bunch:the survival rate of pJME-LC3 group was?13/15,86.67%?,higher than that of JEV-L control group?12/15,80%?,pJME as conventional DNA vaccine control group?8/15,53.33%?and pJME-LC3+3-MA group in which autophagy activity was inhibited?7/15,46.67%?.For the second bunch:The survival rate of pJME-LC3 group was?91.67±2.36?%,significantly higher than that of JEV-L group?81.67±2.36?%,pJME group?61.67±2.36?%and pJME-LC3+3-MA group?56.67±2.36?%.Conclusions:1.pJME-LC3 can expressed rapidly and stably in vitro to activate autophagy and transfer prME-LC3 fusion protein to autophagy pathway.2.pJME-LC3 promotes the uptake of prME-LC3 fusion protein by DCs,and enhances JEV specific CTL activity by increasing the number of CD3+CD4+/CD3+CD8+T lymphocytes in the spleen of immunized mice.3.pJME-LC3 induces Th1 cellular immune response by increasing the secretion of Th1 cytokines IFN-?and IL-12,thus promoting humoral immune response with IgG2a as main antibody.4.pJME-LC3 promotes the differentiation of initial CD4⁺T lymphocytes into Th1cells through the IFN-?-JAK2/STAT1-T-bet pathway,and thus further serves as one of immunological enhancement mechanisms of autophagy mediated JE DNA vaccine.5.pJME-LC3 induce long-term immune memory by increasing the ratio of JEV specific CD4⁺,CD8⁺T_CM/T_EM,6.pJME-LC3 induces strong protective immunity by promoting rapid,continuous generation of JEV specific neutralizing antibodies.